Synthesis of Monoterpenoid Derivatives and Evaluation for Biocatalytic Transformations

Synthesis of Monoterpenoid Derivatives and Evaluation for

Biocatalytic Transformations

A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy

in the Faculty of Engineering and Physical Sciences

2016

Issa Sulaiman Issa

School of Chemistry

University of Manchester

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Contents

List of Figures .................................................................................................. 5

List of Tables .................................................................................................... 8

List of Schemes .............................................................................................. 10

Abbreviations ................................................................................................. 14

Abstract .......................................................................................................... 17

Declaration ..................................................................................................... 18

Copyright Statement ...................................................................................... 19

Acknowledgement .......................................................................................... 20

1 Overview of terpenoids ................................................................................ 21 1.1 Definition and classification ..................................................................................... 21

1.2 The isoprene rule ....................................................................................................... 22

1.3 The effect and use of terpenoids in the life ................................................................ 22

1.4 Biosynthesis of terpenoids ......................................................................................... 23

1.4.1 Biosynthesis of mevalonic acid ....................................................................... 23

1.4.2 1-Deoxy-D-xylulose 5-phosphate (DXP) pathway .......................................... 24

1.4.3 Bioconversion of mevalonic acid and/or DXP to isopentenyl pyrophosphate

(IPP) ......................................................................................................................... 24

1.4.4 Conversion of IPP and DMADP to terpenoids ............................................... 25

1.5 Monoterpenoids ......................................................................................................... 28

1.5.1 Acyclic monoterpenoids .................................................................................. 28

1.5.2 Cyclic monoterpenoids .................................................................................... 28

1.6 Objectives and approach ........................................................................................... 31

2 Synthesis of modified monoterpenoids ...................................................... 33 2.1 Overview of modified monoterpenoids in synthesis, and initial synthetic objectives 33

2.2 Precedent examples of alkylations of terpenone enolates ......................................... 34

2.3 Alkylations of R-(–)-carvone, (+)-isomenthone and (–)-isopinocamphone .............. 36

2.3.1 Alkylation of R-(–)-carvone ............................................................................. 38

2.3.2 Alkylation of (2R,5R)-(+)-isomenthone ........................................................... 41

2.3.3 Alkylation of (1S,2S,5R)-(–)-isopinocamphone ............................................... 43

3

2.4 Precedent examples of aldol additions to monoterpenoid enolates .......................... 45

2.5 Aldol addition to (–)-carvone, (+)-isomenthone, (–)-isopinocamphone and (+)-

dihydrocarvone ................................................................................................................ 51

2.5.1 Aldol additions to R-(–)-carvone ..................................................................... 51

2.5.2 Aldol addition to (+) isomenthone .................................................................. 62

2.5.3 Aldol addition to (1S,2S,5R)-(–)-isopinocamphone ........................................ 69

2.5.4 Aldol addition to diastereomeric mixture of (+)-dihydrocarvone .................. 74

2.6 Reduction of β-ketoalcohol adduct ............................................................................ 79

2.7 Synthesis of azides ..................................................................................................... 80

2.7.1 Azidation of β-hydroxyketones ........................................................................ 82

2.8 Addition of enolate/enol isomenthone to imine ......................................................... 82

3 Synthesis of alkylidenes from terpenone-derived β-keto alcohols ............ 84 3.1 Introduction ............................................................................................................... 84

3.2 Synthesis of alkylidenes from precursor β-ketoalcohols ........................................... 86

3.2.1 Synthesis of carvone-derived alkylidenes bearing aryl ring ........................... 87

3.2.2 Synthesis of carvone-derived alkylidenes alkyl-bearing ................................. 89

3.2.3 Synthesis of isomenthone-derived alkylidenes alkyl-bearing .......................... 92

3.3 Conclusions ............................................................................................................... 93

4 Biocatalysis of monoterpenoids .................................................................. 95 4.1 Introduction of biocatalysis ....................................................................................... 95

4.2 Objectives of monoterpenoid biotransformations ..................................................... 96

4.3 Bioreduction of monoterpenoids ............................................................................... 96

4.4 Biocatalytic reduction evaluations of carvone and synthetic derivatives ............... 103

4.4.1 First attempt for bioreduction of endo and exo double bond ........................ 103

4.4.2 Asymmetric biocatalytic hydrogenation of R-(–)-carvone ............................ 104

4.4.3 Asymmetric biocatalytic hydrogenation of non-natural 6-methyl carvone ... 108

4.4.4 Asymmetric biocatalytic hydrogenation of 6-hydroxycarvone ...................... 115

4.4.5 Asymmetric biocatalytic hydrogenation of 3-methylcarvone ........................ 123

4.4.6 Computational modelling of 6-methylcarvone with PETNR ......................... 126

4.4.7 Biocatalytic reduction mechanism ................................................................ 127

4.4.8 Conclusions ................................................................................................... 128

4.5 Carbonyl bioreduction ............................................................................................ 128

4

5 Expansion rings of cyclic monoterpenoids via Baeyer−Villiger reaction

...................................................................................................................... 132 5.1 Overview of Baeyer−Villiger reaction .................................................................... 132

5.2 Mechanism of Baeyer−Villiger reaction ................................................................. 133

5.3 Baeyer−Villiger reaction of monoterpenoids .......................................................... 135

5.4 Results and discussion ............................................................................................. 140

5.4.1 Chemical Baeyer−Villiger reactions of (+)-isomenthone and (–)-isopinocamphone

................................................................................................................................ 140

5.4.2 Direct oxidative cleavage of (+)-isomenthone and (–)-isopinocamphone with

Oxone® ................................................................................................................... 142

5.4.3 Investigating Baeyer–Villiger monooxygenase activity towards synthetically-

modified terpenones ...................................................................................................... 145

5.5 Conclusions ............................................................................................................. 148

6 Conclusions and future work ................................................................... 150 6.1 Conclusions ............................................................................................................. 150

6.2 Future work ............................................................................................................. 152

7 Experimental ............................................................................................. 154 7.1 General techniques .................................................................................................. 154

7.2 Experimental procedures and data ......................................................................... 155

7.2.1 Experimental procedures and data: Chapter 2 ............................................. 155


7.2.3 Experimental procedures and data: Chapter 4………......……………………213


8 References ................................................................................................. 241

9. Appendix .................................................................................................. 249

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List of Figures

Figure 1.1 Unit of isoprene .................................................................................................. 21

Figure 1.2 Chemical structures of DMADP 3 and IPP 4 .................................................... 23

Figure 1.3 Regular 22 and irregular 23 and 24 acyclic monoterpenoids skeletons ............ 28

Figure 1.4 Isomers of p-menthane ....................................................................................... 29

Figure 1.5 Compounds belonging to the p-menthane sub-class .......................................... 29

Figure 1.6 Bicyclic monoterpenoid skeletons ...................................................................... 29

Figure 1.7 Generation of new chirons from 36, 38 and 39 .................................................. 31

Figure 1.8 Applications of new substrates generated from R-(–)-carvone .......................... 31

Figure 2.1 X-ray crystal structure and stereochemistry of 44 ............................................. 39

Figure 2.2 1H NMR spectra of carvone 36 and spectra of a mixture of 44 and 45 and

separated 44 and 45 ............................................................................................................. 40

Figure 2.3 2D-COSY spectrum of 57 diastereomer ............................................................. 41

Figure 2.4 Stereochemistry of 59 and 60 ............................................................................. 41

Figure 2.5 Chromatogram of reverse phase HPLC column of 63 and 64 ........................... 42

Figure 2.6 1H NMR spectrum of the mixture of 63 and 64 .................................................. 43

Figure 2.7 Chromatogram from reverse-phase HPLC of 68 and 69 ................................... 44

Figure 2.8 Anti 73 and syn 74 aldol adducts of cycloheximide analogues .......................... 46

Figure 2.9 Chemical structures of 83, 84 and 85 ................................................................ 47

Figure 2.10 Chemical structures of tricyclic 97, 98 and 99 ................................................ 50

Figure 2.11 Stereochemistry of (1`R) diastereomer 103, and its acetate 104 ..................... 53

Figure 2.12 HPLC Chromatogram of diastereomeric mixture of aldol adducts 105 .......... 54

Figure 2.13 Configurations of aldol adducts 105, 106, 107 and 108 .................................. 54

Figure 2.14 Conformers of R configuration at C6 and C1` of aldol adduct ....................... 55

Figure 2.15 X-ray crystal structure of 109, and configurations of 109, 110 and the 4-

nitrobenzoate ester 111 ........................................................................................................ 55

Figure 2.16 Stereochemistry of 112 and 113 and their esters 114 and 115 ........................ 57

Figure 2.17 HPLC-MS chromatogram of diastereomeric mixture of aldol adducts of 116

and 117 ................................................................................................................................. 57

Figure 2.18 The structures of 120, 121, 122 and 123, and X-ray crystallography of 123 .. 59

Figure 2.19 The potential stereostructures of 122 ............................................................... 59

Figure 2.20 Stereochemistry of both isomers (124 and 125) and X-ray crystal structure of

125 ........................................................................................................................................ 60

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Figure 2.21 Zimmerman−Traxler transition state of cis (blue) and trans fused decalin (red)

.............................................................................................................................................. 62

Figure 2.22 Possible conformations of aldol products of 38 ............................................... 63

Figure 2.23 Conformers of S configuration (syn) at C2 and C1` of isomenthone aldol

adduct ................................................................................................................................... 64

Figure 2.24 X-ray crystal structure of 128, and configurations of 128 and 129 ................. 65

Figure 2.25 Configurations of 130 and 131 isomers and X-ray crystal structure of 130

aldol adduct .......................................................................................................................... 66

Figure 2.26 Conformers of R (anti) of aldol adduct between C2 and C1` protons ............. 67

Figure 2.27 Zimmerman−Traxler transition state of (+)-isomenthone aldol adducts cis

(blue) and trans fused (red) .................................................................................................. 69

Figure 2.28 Configurations of 136, 137, 138 and 139 ......................................................... 70

Figure 2.29 Conformation and X-ray crystal structure of 142 ............................................ 72

Figure 2.30 X-ray crystal structure of 148, and conformations of 148 and 149 ................. 73

Figure 2.31 Correlations of coupling constant values among adjacent protons of major

aldol adduct to 88 ................................................................................................................. 75

Figure 2.32 Possible configurations of aldol adducts of 154, 155 and 156 ........................ 76

Figure 2.33 Possible conformers of aldol adduct of (+)-dihydrocarvone ........................... 76

Figure 2.34 X-ray crystal structure, and stereostructure of 159 adduct ............................. 78

Figure 2.35 Possible configurations of aldol adduct of 159 ................................................ 78

Figure 2.36 Zimmerman−Traxler transition state of 88 cis (blue) and trans (red) fused .... 79

Figure 2.37 Possible conformations of diols 160 and 161 .................................................. 80

Figure 2.38 Structures of DPPA 162, diazidobis(pyridine)zinc 163, and DBU 164 ........... 81

Figure 3.1 Structures of intermediate 192 and product 193 of aldol condensation of 191 86

Figure 3.2 X-ray structure, 2D-NOESY correlations and 13C NMR shifts for 196 ............. 88

Figure 3.3 2D-NOESY spectrum of 172 ............................................................................... 90

Figure 4.1 Different binding of 34 with amino acids of OYE enzyme during site

mutagenesis process to access to different outcome ......................................................... 103

Figure 4.2 Substrates candidates screened against Nt-DBR enzyme ................................ 104

Figure 4.3 Chemical structures of octalones, thujosene and dihydromyurone ................. 104

Figure 4.4 The effect of time on bioconversion of 36 via OYE2 with respect to conversion,

yield and diastereomeric excess. ........................................................................................ 106

Figure 4.5 The structures of 1α,25-dihydroxyvitamin D3 analogues ................................. 109

Figure 4.6 Optimisation of 44 bioreduction via OYE2 ...................................................... 109

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Figure 4.7 The effect of pH buffer solution parameter on biotransformation of 44 via OYE2

enzyme ................................................................................................................................ 111

Figure 4.8 The correlation of coupling constants of adjacent protons of 248 and 250 .... 118

Figure 4.9 Optimisation pH buffer solution of KP of bioconversion of 243 and 244 ........ 118

Figure 4.10 Optimisation time (h) of bioconversion of 243 and 244 .................................. 119

Figure 4.11 (A) Proton NMR spectra of methyl substitutions of mixture of four isomers of

3-methyldihy-drocarvones 262−265. (B) Proton NMR spectrum of methyl substitutions of

two diasteromers of 3-(R)-methyldihydrocarvone (262 and 263) ...................................... 125

Figure 4.12 Selected conformations from molecular docking of (A,B) 45 and (C,D) 44 in

PETNR. Structures B and C are consistent with observed stereoselectivity. ..................... 126

Figure 4.13 Representative structures from simulations for each substrate 44 and 45 .... 127

Figure 4.14 Substrates were attempted to bioreduce via ADHs ........................................ 129

Figure 5.1 Transition state explains sequence of migratory group aptitude suggested by

Hawthorne et.al .................................................................................................................. 135

Figure 5.2 Structures of cyclobutane monoterpenoids 321, 322, 323 and 324 ................ 142

Figure 5.3 Biooxidation of 38 with CHMOs from Rhodococcus sp. Phi1…………………145

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List of Tables

Table 1. 1 Classification of terpenoids ................................................................................ 21

Table 2.1 Yields and diastereomeric ratios of alkylated 36, 38, and 39 .............................. 38

Table 2.2 1H NMR spectra data of Me substituted groups of 2-methyl isopinocamphone .. 44

Table 2.3 Isolated yields and diastereomeric ratios of hydroxy alkylation of R-(–)-carvone

36 with a variety of aldehydes .............................................................................................. 52

Table 2.4 Yields and diastereomeric ratios of aldol addition to (+)-isomenthone .............. 63

Table 2.5 Yields and diastereomeric ratios of aldol addition to (–)-isopinocamphone ...... 70

Table 3.1 Yields and by-products percentages of β-hydroxyketone 116 and 117 dehydration

using PTSA at various temperatures .................................................................................... 88

Table 3.2 Synthesis of alkylidene 196 and 197 from diastereomers mixture 116 and 117

using Tf2O with four different bases ..................................................................................... 89

Table 3.3 Chemical shifts (δ, ppm) and coupling constants (J, Hz) for E isomer 201 ........ 91

Table 3.4 Yields and diastereomeric ratios of alkylidenes derived from β-ketol ................. 93

Table 4.1 The effect of temperature and co-factor on bioreduction of 36 by OYE2 .......... 105

Table 4.2 The influence of enzyme and NADP+ concentrations on bioreduction of 36 using

OYE2 .................................................................................................................................. 107

Table 4.3 Biotransformation of 36 via OYE2, OYE3 and PETNR enzymes ...................... 107

Table 4.4 Bioconversion of 44 using OYE2 as a function of time (h) ................................ 110

Table 4.5 Bioconversion optimisation of 44 using OYE2 enzyme and NADP+ concentration

parameters .......................................................................................................................... 110

Table 4.6 Biotransformation of 44 and 45 against PETNR and OYE2 enzymes ............... 112

Table 4.7 Yields of bioreduction of a mixture of 44 and 45 by OYE2 and PETNR ........... 113

Table 4.8 1H NMR spectra data of four diastereomers of 2-methyldihydrocarvone 236,

237, 238 and 239 collected from reference 117 and current study ................................... 114

Table 4.9 Relevant 1H NMR spectra data of diastereomers 243 and 244 ......................... 117

Table 4.10 Optimisation [NADP+] and [OYE2] on outcomes of bioreduction of 243 and

244 ...................................................................................................................................... 119

Table 4.11 Bioconversion of 243 and 244 by PETNR, OYE2 and OYE3 within 2 and 24 h .... 120

Table 4.12 Bioreduction of 252 and 253 by PETNR, OYE2 within 2 and 24 h ................. 122

Table 4.13 Yields of bioreduction of 257 by OYE2 and PETNR ........................................ 126

Table 4.14 Bioreduction of various substrates using three different enzymes from

Ketoreductase ..................................................................................................................... 130

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Table 5.1 BV Oxidation of monoterpenoids with different reagents, yield and regioselectivity

............................................................................................................................................ 135

Table 5.2 Summary of lactones syntheses by BVMOs ........................................................ 137

Table 5.3 Optimisation of [CHMOs] and [NADP+] on biooxidation of 38 ...................... 146

Table 7.1 Temperature methods utilised to determine conversion and yield% of substrates

............................................................................................................................................ 214

Table 7.2 Details of the three OYE gene constructs and expression strains ..................... 214

Table 9.1 UV Absorbance of isomeric mixture of (–)-carveol with KT-to go plate at 492 nm.

............................................................................................................................................ 249

Table 9.2 UV Absorbance of (+)-isomenthol with KT-to go plate at 492 nm. .................. 249

Table 9.3 UV Absorbance of (+)-isopinocampheol with KT-to go plate at 492 nm……..258

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List of Schemes

Scheme 1.1 Formation of myrcene via head-to-tail joining of two isoprene units .............. 22

Scheme 1.2 Biosynthesis pathway of mevalonic acid ........................................................... 23

Scheme 1.3 Biosynthesis pathway of DXP 10 ...................................................................... 24

Scheme 1.4 Biosynthesis pathways of 4: (A) from 9 and (B) from 10 .................................. 25

Scheme 1.5 Bioconversion of IPP 4 to DMADP 3 catalysed with IPP isomerase ............... 25

Scheme 1.6 Biosynthesis of plant terpenoids from IPP 3 and DMADP 4 ............................ 26

Scheme 1.7 A) Bioconversion of FFP 20 to germacrene isomers in leaf oil of tomato B)

Biosynthesis of taxol in yew (Taxus) species ........................................................................ 27

Scheme 1.8 Pathways of biosynthesis of monocyclic, bicyclic and tricyclic terpenes from

GPP 19 ................................................................................................................................. 30

Scheme 1.9 Biosynthesis pathways of monoterpenes using Cytochrome P450s ................. 30

Scheme 1.10 Objectives of A) chemo−enzymatic B) enzymatic−chemical reaction ............ 32

Scheme 2.1 Synthesis of chiral 41, and multisteps to total synthesis of 43 .......................... 33

Scheme 2.2 Alkylation of R-(–)-carvone 36 under kinetic conditions .................................. 34

Scheme 2.3 Synthesis of pinguisenol 48 from R-(–)-carvone 36 .......................................... 35

Scheme 2.4 Alkylation of (−)-menthone 49 and silyloxymenthone 50 ................................ 35

Scheme 2.5 Alkylation of (+)-nopinone 51 in presence of NaI and/or DMPU .................... 36

Scheme 2.6 1) The mechanism proposed for oxidation of 54 via A) Pyridine sulphur

trioxide complex B) IBX 55 2) Oxidation of 56 using formation of 55 in situ ..................... 37

Scheme 2.7 Mechanism of R-(–)-carvone 36 methylation under kinetic conditions ............ 38

Scheme 2.8 Alkylation of 36 with benzyl bromide under kinetic control ............................. 40

Scheme 2.9 Methylation of 38 to afford 2-methylisomenthone 61 and 62 ........................... 42

Scheme 2.10 Methylation of 39 under kinetic control .......................................................... 43

Scheme 2.11 Benzylation of 39 in presence of DMPU ......................................................... 44

Scheme 2.12 Aldol addition to 49 using benzaldehyde and reduction of 70 ........................ 45

Scheme 2.13 Diels−Alder reaction catalysed by aluminium chiral menthone derivative 72

.............................................................................................................................................. 45

Scheme 2.14 Aldol addition to enolate lithium of 38 ........................................................... 46

Scheme 2.15 Aldol reaction of 77 ........................................................................................ 46

Scheme 2.16 Aldol addition to 34 tusing formaldehyde and 81 ........................................... 47

Scheme 2.17 Aldol addition to (S)-carvone and/or its hydrogenated derivatives ................ 48

Scheme 2.18 Aldol addition to 36, and multi-step to synthesis of 86 ................................... 48

Scheme 2.19 Aldol addition to 87 and 88 ............................................................................ 49

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Scheme 2.20 Nucleophilic addition to aldol adducts 91, 92 and 94 ................................... 50

Scheme 2.21 Aldol addition to 100 ...................................................................................... 51

Scheme 2.22 Outcomes of aldol addition to 36 .................................................................... 52

Scheme 2.23 Possible structures of 116 and 117 diastereomers and their acetates 118 and

119 ........................................................................................................................................ 58

Scheme 2.24 Addition of aldehydes to (+)-isomenthone 38 in the presence of LDA ........... 62

Scheme 2.25 Aldol addition of acetaldehyde to 38 under kinetic control ............................ 64

Scheme 2.26 Aldol addtion of salicylaldehde to 38 ............................................................. 66

Scheme 2.27 Aldol addition of acetone to 38 ....................................................................... 68

Scheme 2.29 Aldol addition to (–)-isopinocamphone 39 .................................................... 69

Scheme 2.30 Aldol addition of benzaldehyde to 39 ............................................................. 71

Scheme 2.31 Esterification of 140 and 141 .......................................................................... 72

Scheme 2.32 Mukaiyama aldol reaction of 39 ..................................................................... 74

Scheme 2.33 Aldol addition to 88 ........................................................................................ 77

Scheme 2.34 Reduction of 103 with NaHB4 in presence of CeCl3.7H2O ............................. 79

Scheme 2.35 Mitsunobu reaction using A) hydrazoic acid, B) DPPA, C) DBU .................. 81

Scheme 2.36 Attempting azidation of 103 using Thompson conditions ............................... 82

Scheme 2.37 Synthesis of 163 ............................................................................................... 82

Scheme 2.38 Mannich reaction A) using Zn(BF4)2 B) under kinetic control ....................... 83

Scheme 2.39 Treatment 38 with benzalaniline under kinetic effect ..................................... 83

Scheme 3.1 Aldol condensation of 49 and 87 with benzaldehyd .......................................... 84

Scheme 3.2 Aldol addition to 181 and dehydration of the product using PTSA .................. 85

Scheme 3.3 Dehydration process of 185 .............................................................................. 85

Scheme 3.4 Total synthesis of 187 ........................................................................................ 85

Scheme 3.5 Final steps for synthesis of 188 ......................................................................... 86

Scheme 3.6 Dehydration of ketol 194 .................................................................................. 86

Scheme 3.7 Proposed elimination strategy to new terpenoid exo-alkylidine/arylidine targets

.............................................................................................................................................. 87

Scheme 3.8 Synthesis of alkylidene bearing alkyl derived of (–)-carvone 172, 198, 199 and

200 ........................................................................................................................................ 89

Scheme 3.9 Synthesis of alkylidene bearing alkyl derived from (–)-carvone (201, 202, 203

and 204) ............................................................................................................................... 91

Scheme 3.10 Synthesis of alkylidene bearing alkyl derived from (+)-isomenthone (205,

206, 207 and 208) ................................................................................................................ 92

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Scheme 4.1 Bioreduction of 34 and 36 ................................................................................. 97

Scheme 4.2 Bioreduction of 29, 49 and 214 ......................................................................... 97

Scheme 4.3 Bioreduction of 34 using strains of yeasts under different physiological states

.............................................................................................................................................. 98

Scheme 4.4 Bioreduction of 36, 29 and 187 using rPRase .................................................. 99

Scheme 4.5 Bioreduction pathway of 36 via NCYs whole cell .......................................... 100

Scheme 4.6 Bireduction pathway of 221 via NCYs whole cell ........................................... 100

Scheme 4.7 Reduction of 36 and 34 using enoate and carbonyl reducatase ..................... 102

Scheme 4.8 Bioconversion of 34 via two types of OYEs .................................................... 103

Scheme 4.9 Bioreduction of 36 with OYE2 from Saccharomyces cerevisiae .................... 105

Scheme 4.10 Bioreduction of 44 using OYE2 from Saccharomyces cerevisiae ................. 109

Scheme 4.11 Chemical structures, ratios, and retention times of products resulted from

reduction of 44 and 45 diastereomeric mixture with sodium dithionite ............................ 113

Scheme 4.12 Possible explanation for the preferred formation 236 and 237 and upon

conjugate reduction of 44 and 45 ....................................................................................... 115

Scheme 4.13 Rubottom oxidation 36 with m-CPBA ........................................................... 116

Scheme 4.14 Synthesis of 247 pheromone from 83 ............................................................ 116

Scheme 4.15 Outcomes of reduction of both 6-hydroxycarvone isomers 243 and 244 ..... 117

Scheme 4.16 Synthesis of 252 and 253 from 34 via Rubottom oxidation .......................... 121

Scheme 4.17 1,2-Nucleophilic addition of MeMgI to 34, and 1,3 oxidative transportation

mechanism postulated by Dauben, and Michno ................................................................ 124

Scheme 4.18 Outcomes from methylation of 36 with TMA ................................................ 124

Scheme 4.19 Radical ring-opening of 266 ......................................................................... 125

Scheme 4.20 Reduction of enzyme−FMN by NAD(P)H (reductive half reaction) ............. 127

Scheme 4.21 Asymmetric bioreduction of 44 and 45 via PETNR enzyme (oxidative half

reaction) ............................................................................................................................. 128

Scheme 5.1 First attempts Baeyer−Villiger oxidation of terpenoids ................................. 132

Scheme 5.2 Proposed mechanism of ketone oxidation by Baeyer and Villiger ................. 133

Scheme 5.3 Proposed pathways of Criegee intermediate formation, and possible outcomes

from BV oxidation, and introduce experimentally proof via labelled 18O ......................... 134

Scheme 5.4 Baeyer−Villiger biooxidation of 36 using trichosporumcutaneum CCT 1903 ... 138

Scheme 5.5 Baeyer−Villiger oxidation of 210, 100, and 276 ............................................ 139

Scheme 5.6 Baeyer−Villiger oxidation of 219, 210, and 100 ............................................ 139

Scheme 5.7 Baeyer−Villiger oxidation of 83, 100, 276 and 313 ....................................... 140

13

Scheme 5.8 BV reaction of 38 and 39 via m-CPBA ........................................................... 141

Scheme 5.9 BV reaction of 325 using m-CPBA in EtOH and/or MeOH ........................... 142

Scheme 5.10 Esterification of isopinocamphone 39 .......................................................... 143

Scheme 5.11 Pathways of 38 oxidation via Oxone® in MeOH. ......................................... 144

Scheme 5.12 Biooxidation of 38 via CHMOs from Rhodococcus sp. Phi1 ........................ 145

Scheme 5.13 Biooxidation of 237 by CHMO_Phi1 from Rhodococcus sp. Phi1 ............... 147

Scheme 5.14 Biooxidation of 262 and 263 mixture by CHMO_Phi1 from Rhodococcus sp.

Phi1 .................................................................................................................................... 148

Scheme 6.1 General scheme of aldol reaction and synthesis of alkylidene of 36, 38 and 39

............................................................................................................................................ 150

Scheme 6.2 Chemo-enzymatic reactions of (–)-carvone derivatives .................................. 151

Scheme 6.3 BV reaction of 38, 39 and 237, and opening rings of 38 and 39 .................... 152

14

Abbreviations

BV Baeyer–Villiger reaction

BVMOs Baeyer–Villiger monooxygenase

nBuLi Butyl lithium

13C NMR Carbon Nuclear Magnetic Resonance

δ Chemical shift

COSY Correlated spectroscopy

J Coupling constant reported in Hz

CHMOs Cyclohexanone monooxygenase

d Doublet

d.e. Diastereomeric excess

DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene

DCM Dichloromethane

DEAD Diethyl azodicarboxylate

Et2O Diethyl ether

DEPT Distortionless enhancement by polarisation transfer

dd Doublet of doublets

DIAD Diisopropyl azodicarboxylate

DIPA Diisopropylamine

DMADP Dimethylallyl diphosphate

DMAP 4-Dimethylaminopyridine

DMPU 1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone

DMSO Dimethyl sulfoxide

DPPA Diphenylphosphoryl azide

15

dq Doublet of quartets

DXP 1-Deoxy-D-xylulose 5-phosphate

e.e. Enantiomeric excess

Equiv. Equivalents

EtOAc Ethyl acetate

FMN Flavin mononucleotide

h Hours

HMPA Hexamethylphosphoramide

HMQC Heteronuclear Multiple Quantum Coherence

HPLC High Performance Liquid Chromatography

HRMS High Resolution Mass Spectrometry

Hz Hertz

IBX 2-Iodoxybenzoic acid

IPP Isopentenyl pyrophosphate

IR Infrared

LDA Lithium diisopropylamide

MS Mass Spectrometry

m-CPBA meta-Chloroperoxybenzoic acid

m.p. Melting point

mg Milligram

MgSO4 Magnesium sulfate

mL Millilitres

m Multiplet

NAD(P)H Nicotinamide adenine dinucleotide phosphate

16

NADH Nicotinamide adenine dinucleotide

NOESY Nuclear Overhauser Effect Spectroscopy

OYEs Old yellow enzymes

PETNR Pentaerythritol tetranitrate reductase

ppm Parts per million

PTSA para-Toluenesulfonic acid

1H NMR Proton Nuclear Magnetic Resonance

q quartet

quint quintet

Rf Retention factor

ROP Ring-Opening Polymerization

s Singlet

t Triplet

THF Tetrahydrofuran

TLC Thin layer chromatography

TMA Trimethylaluminium

TMS Tetramethylsilane

TMSCl Trimethylsilyl chloride

17

Abstract

Monoterpenoid derivatives are some of the most effective asymmetric controllers

used in organic synthesis. They are also precursors in target syntheses, especially in

synthesis of natural products with useful biological properties. However, there are still

significant opportunities to develop new structural synthetic modifications. This project

target focuses on employing commercially available chiral pool cyclic ketones, such as R-

(–)-carvone, (+)-isomenthone, and (–)-isopinocamphone to create new potential substrates

for biocatalytic modifications, via terpenone enolate alkylations, aldol additions, and

formation of alkylidenes.

Evaluation of these substrates has been carried out using isolated enzymes as

biocatalysts to reduce the double bond and/or carbonyl group, as well expansion of six

membered rings by Baeyer–Villiger monooxygenase to generate lactone derivatives,

consequently resulting in new high-value terpenone, terpenol and lactone derivatives.

Bioreduction of R-(–)-carvone substituted (with Me or OH) at C6 and/or C3 via

OYEs afforded with highly diastereoselectivity in most cases with varied yields; and there

was no activity observed toward substrates with substituents bigger than Me. Biooxidation

of dihydrocarvone substituted (Me) at C6 or C3 via cyclohexanone monooxygenase

(CHMO_Phi1) was selective, and oxidised only one diastereomer. For instance, (2R,3R,6R)-

methyldihydrocarvone was completely converted to lactone with high regio- and

enantioselectivity, while for the (2S,3R,6R)-diastereomer no lactone was produced, and

starting material was recovered. (+)-Isomenthone, R-(–)-carvone, (–)-isopinocamphone and

their derivatives were treated with carbonyl reductase, and only (+)-isomenthone, R-(–)-

carvone and anti (5S,6S)-hydroxycarvone showed reaction, with varied yields and

selectivities.

The bioreduction and oxidation of substrates were scaled up to 50–100 mg as part

of chemo-enzymatic reactions. The simulation of substrates with PETNR enzyme was

studied, and docking was modelled.

18

Declaration

No portion of the work referred to in the thesis has been submitted in support of an

application for another degree or qualification of this or any other university or other

institute of learning

Issa Sulaiman Issa

19

Copyright Statement

The following four notes on copyright and the ownership of intellectual property rights

must be included as written below:

i. The author of this thesis (including any appendices and/or schedules to this thesis) owns

certain copyright or related rights in it (the “Copyright”) and s/he has given The University

of Manchester certain rights to use such Copyright, including for administrative purposes.

ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy,

may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as

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thesis, may not be owned by the author and may be owned by third parties. Such

Intellectual Property and Reproductions cannot and must not be made available for use

without the prior written permission of the owner(s) of the relevant Intellectual Property

and/or Reproductions.

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http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=487), in any relevant Thesis

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university’s policy, and in the presentation of thesis.

20

Acknowledgement

Foremost, I would like to sincerely thank my supervisor, Dr. John M. Gardiner, for giving

me the opportunity to work in his group, and for his valuable guidance, his patience, and

for his supporting me during the course of this research. Also, I would like to thank my co-

supervisor Professor Nigel S. Scrutton to offered me the chance to work in the enzymology

lab. A huge thank goes to Dr. Helen Toogood, for instructions in all aspects of

biotransformations and expression of enzymes. I would like to thank Mrs Rehana Sung, a

HPLC technician, and Dr. James Raftery (X-Ray). An enormous amount of thanks go to

my colleague Charlotte Dalton for reading my thesis. Many thanks to Dr. Linus Johannisen

(molecular docking).

I have extremely lucky to work with some fantastic people in the Gardiner group, Post

docs and research students, and I had great time with them in the lab, thanks to all.

Deepest respect to the soul of my dear parents, for their encouraging me to the last

moment in their life. Finally, my love and appreciation to my wife Rafah and my sons

Dara and Diyar.

Issa Sulaiman Issa

1. Overview of terpenoids

21

1 Overview of terpenoids

1.1 Definition and classification Natural products consist of many different types of organic compounds, and terpenoids

(isoprenoids/isopentenoids) are the major class of all known plant metabolites with

thousands of chemical structures known.1-3

Traditionally, humans have used terpenoids in the form of complex mixtures

without purification since ancient times. Kekule was the first researcher who reported the

term ‘terpene’ in 1866, to mean the volatile liquid extracted from pine trees. Terpene is

originally derived from turpentine (lat. Balsamum terebinthinate), and the name turpentine

has been employed to describe C10H16 hydrocarbons in turpentine oil. Thence the term

terpenoids has gained a generic significance throughout the years and refers to (C5H8)n

hydrocarbons and their oxygenated derivatives and their glycosides, containing ketones,

aldehydes, ethers, ester and carboxylic acid groups.

The immense variety of terpenoids compounds is biosynthesized in nature during

the successive combination of two or more isoprene units (2-methyl-1,3-butadiene 1).4,5

Figure 1.1 Unit of isoprene

Hence, the general formula of terpenoids is (C5H8)n, and they are classified basically

depending on number of isoprene units in the molecule.

Table 1. 1 Classification of terpenoids Class Number of carbon

atoms

n Value Example Hemiterpenoids 5 1 Isoprene Monoterpenoids 10 2 Limonene Sesquiterpenoids 15 3 Artemisinin

Diterpenoids 20 4 Forskolin Sesterterpenoids 25 5 Setsterol

Triterpenoids 30 6 α-Amylin Tetraterpenoids 40 8 β-Carotene Polyterpenoids >40 >8 Rubber

However, the empirical formula of some terpenoids does not follow the isoprene

rule due to the degradation or rearrangement processes, which leads to a change in number

of carbons in the backbone of terpenoids.6

1


22

1.2 The isoprene rule Terpenoids are divided into classes and sub-classes according to their biosynthetic

pathways, as the backbone of terpenoid structures arises directly from the biosynthesis.

Wallach was the first researcher who identified monoterpenoids constructed from the

isoprene unit in 1887. Three decades later, Robinson envisaged that monoterpenoids built

up in a head to tail fashion joining of isoprene units. Comprehensively and precisely, the

biogenetic isoprene rule has been suggested by Ruzicka,7 who referred to isoprene units

(C5)n as the basic carbon skeleton of terpenoids. The structure of five-carbon isoprene

contains branched end as the “head” and the “tail” is the other end, so the linkage between

two isoprene units could occur head-to-head, tail-to-tail or in the common fashion, head-to-

tail. For instance, myrcene 2 consists of two units of isoprene head-to-tail to form the

simplest terpenoid (Scheme 1.1).

Scheme 1.1 Formation of myrcene via head-to-tail joining of two isoprene units

Conversely, the distinctive feature of steroids and carotenoids is joining in tail-to-

tail fashion in the centre of the skeleton.6,8 Ruzicka also predicted the stereospecific

cyclizations, dimerizations and rearrangements from acyclic C-10, C-15, C-20, and C-30

precursors geraniol, farnesol, geranylgeraniol, and squalene, respectively.9

1.3 The effect and use of terpenoids in the life There is a wide structural diversity of terpenoids produced as second metabolites in

plants, animals and living organisms. For instance, terpenoids represent 55% of the total

natural products in plants with a wide variety of chemical components.10

Many interesting terpenoids are important in several industries due to their own

biological, physical and chemical properties like perfumery, flavour, cosmetics, pesticides

and disinfectants.4 Also, terpenoids are a useful source of precursors, and play a pivotal

role in the pharmaceutical and drug industries, for example, the anticancer agent paclitaxel

taxol and antimalarial drug artemisinin.11 Recently; the abundance and diversity of these

compounds have become widely recognized, conceptually and/or empirically, for their

ecosystem influences in plant biology. There are several plants, which produce terpenoids

as a defence mechanism. Eudesmane derivatives, for instance, are found in resin produced

by the shrub chmmiphoraabyssinca and are antibacterial and antifungal compounds.6 Also,

Tail

Head Myrcene2


23

there are a number of monoterpenoid derivatives which are toxic to insects, for example,

the leaves and flowers of Chrysanthemum species produce the monoterpenoid esters

(pyrethroids). The latter has become a good source of commercial insecticides because it

affects on the nervous system of several insects such as beetles, moths, bees, etc.,

meanwhile, pyrethroids easily decompose in nature and are not toxic for mammals.12

1.4 Biosynthesis of terpenoids All terpenoids originate from two isomers of C5 precursors, dimethylallyl

diphosphate (DMADP) 3 and isopentenyl pyrophosphate (IPP) 4, and both are generated in

the mevalonic acid 10 and 1-deoxy-d-xylulose 5-phosphate (DXP) 11 pathways in

secondary metabolic pathways in plants.

DMADP is responsible for formation of the smallest unit of terpenoids,

hemiterpenoids C5, by terpenoids synthase (TPS). Alternatively, the active prenyl synthase

catalyses gathering of two, three and four C5 units to yield geranyldiphosphate (C10),

farnesyl diphosphate (C15) and geranylgeranyl diphosphate (C20).13−17

Figure 1.2 Chemical structures of DMADP 3 and IPP 4

1.4.1 Biosynthesis of mevalonic acid

Two molecules of biogeneric precursor of terpenoids (acetyl-CoA) 5 undergo

Claisen condensation to yield acetoacetyl-CoA 6, which reacts with 5, to afford 3-hydroxy-

3-methylglutaryl-CoA (HMG-CoA) 7. The next step is reduction of the latter with HMG-

CoA reductase to give mevaldic acid 8, which has reduced again to mevalonic acid 9. The

source of hydride in the reduction steps is nicotinamide adenine dinucleotide phosphate

(NADPH).18

Scheme 1.2 Biosynthesis pathway of mevalonic acid 18

OPPOPP

Isopentenyl pyrophosphateDimethylallyl diphosphateDMADP 3 IPP 4

SCoA

O

SCoA

O O

OHCoAS

O OOH

OHHO

OOH

Acetoacetyl CoAthiolase

Claisencondensation

HMG-synthase

Acetyl-CoA 5 OHO

OOHHMGreductase

NADPH NADP+

Acetyl-CoA 5 Acetoacetyl-CoA 6

HMG-CoA 7 Mevaldic acid 8

Mevalonic acid 9

NADPH NADP+

HMGreductase

2


24

1.4.2 1-Deoxy-D-xylulose 5-phosphate (DXP) pathway

Biosynthesis of isopentenyl pyrophosphate (IPP) 4 from DXP (non-mevalonate)

pathway has been reported recently.18

Scheme 1.3 Biosynthesis pathway of DXP 10 18

DXP 10 forms as the product of condensation of pyruvate 11 and glyceraldehyde-3-

phosphate 12. The reaction steps involve synthesis of active acetaldehyde ((hydroxye-

thyl)thiamine diphosphate TPP-C2) 13 from 11 and thiamine diphosphate (TPP) 14, and

then 13 reacts with 12 to generate DXP (Scheme 1.3).

1.4.3 Bioconversion of mevalonic acid and/or DXP to isopentenyl pyrophosphate (IPP)

Three consecutive molecules of ATP are consumed in the steps of phosphorylation to

covert mevalonic acid 9 to 3-phospho-5-pyrophosphomevalonate 15, followed by

elimination of CO2 and PO4 to give 3-isopentenyl pyrophosphate (IPP) 4.

DXP 10 yields 2C-methyl-D-erythritol-4-phosphate 16 through a pinacol rearrangement,

and the product reacts with cytidine triphosphate (CTP) 17 in a phosphorylation step. The

triphosphate intermediate undergoes an internal rearrangement 2-C-Methyl-D-erthritol-2,4-

cyclodipho-sphate 18. Finally, IPP is obtained after a series of elucidated steps (Scheme

1.4).13,15

HOOC

O S

NR

OPPSNR

OPP

HOO

OH

SNR

OPP

OH H

O

OPOH

H

SNR

OPP

OOH

OPOH

H OPO

OH

OHDXP synthase

1-Deoxy-D-xylulose- 5-phosphate (DXP) 10

Pyruvate 11

12

Thiamine diphosphate 14

(Hydroxyethyl)thiamine- diphosphate 13


25

Scheme 1.4 Biosynthesis pathways of 4: (A) from 9 and (B) from 10 13,15

1.4.4 Conversion of IPP and DMADP to terpenoids

The rearrangement reaction of IPP 4 catalysed by IPP isomerase, forms DMADP 3.

The process includes generation of a stable tertiary carbocation by adding a proton to the

double bond, and eliminating a proton from Re face to form 4. For example, in eukaryotes

and yeast, the pro-R proton is removed from C2 of 4 (forward reaction), and delivered

from water to Re-face of C2-C3 double bond (reverse reaction).18

Scheme 1.5 Bioconversion of IPP 4 to DMADP 3 catalysed with IPP isomerase 18

In the first step, the nucleophile 4 attacks the ion of 3 to yield geranylpyrophosphate

(GPP) 19, a precursor of monoterpenoids, and the reaction is catalysed by prenyl

transferase (Scheme 1.6). Then a second molecule of 4 reacts with 19 to generate farnesyl

pyrophosphate (FPP) 20.17,18

OPP

O OHO

OPP

IPP 4

O OPO

O OPPO

MVA kinaseATP

ADP

Mevalonate5-phosphate

kinase

O OPPO

Mevalonate-5-diphosphatedecarboxylaseCO2

OPOH OH

OH

OPO

OH

OH

DXPR

OPP-CytidineOH OH

OH

CMK

OPP-CytidineOH OH

OP

OOH OH

O

MECPS

DXP 10

2C-Methyl-D-erythritol-4-phosphate 16

2-C-Methyl-D-erthritol-2,4-cyclodiphosphate 18

Mevalonic acid 9

Dimethylallyl diphosphateDMADP 3

Isopentenyl diphosphate

Cytidine triphosphate (CTP) 17

Mevalonate-5-phosphate

Mevalonate-5-diphosphate 4-(Cytidine 5'-diphospho)-2C-methyl-D-erytheritol

2-Phospho-4-(cytidine 5'-diphospho)-2C-methyl-D-erytheritol

OH

OH

OH

OP

3-Phospho-5-pyrophospho-mevalonate 15

Biosynthesis of IPP 4 via mevalonic acid pathway 9

ABiosynthesis of IPP 4 via

DXP pathway 10

B

PO43-

ATP

ADP

ATP

ADP

NADPH

P OP O

OH

O OH

PPOHR

BH

HS

PPOHRHS

B:

PPOHS HEHZ

H

*+

* *HEHZ HEHZ

H

B: B:

BHR

IPP isomerase

4 3


26

Scheme 1.6 Biosynthesis of plant terpenoids from IPP 3 and DMADP 4 17,18

FPP 20 and GGPP 21 are precursors to a wide variety of sesquiterpenoids and diterpen-

oids, respectively. Biologically active germacrenes, and their lactone derivatives such as

eudesmanolides, germacrnolides and guaianolides are examples of sesquiterpenoids. Taxol

(paclitaxel) is an important example of a diterpenoids (Scheme 1.7).18

OPP

Geranylgeranyl pyrophosphate GGPP(Diterpenes) 21

23

OPPHSHR

OPPHS

OPP

20

Gerany lpyrophosphate GPP 19 (Monoterpene)

IPP 4

IPP 4

4

OPP 3

Prenyl transferase

Prenyl transferase

Prenyl transferase

Farnesyl pyrophosphate FPP(Sesquiterpenes)


27

Scheme 1.7 A) Bioconversion of FFP 20 to germacrene isomers in leaf oil of tomato B)

Biosynthesis of taxol in yew (Taxus) species 18

GGPP

HH

D

HD

H

D

HHH

H

D

H H HOH

H HOAc

HOO O

OH

OO

Ph O

OOO

OPh

O

HO

NH

Ph

O

Verticillyl cation

Taxenyl cation

A BC

Taxa-4(5),11(12)-diene(Taxadiene)

Taxa-4(20),11(12)-dien-5α-ylacetate

Taxa-4(20),11(12)-dien-5α-ol

Taxol (Paclitaxel)

1 2

3

4 5

6

78

910

111213

14

15

16

17

18

19

20

i

ii

iii

i = Taxadiene synthase; ii = Taxadiene hydroxylase; iii = Taxadienol O−acetyltransferase

B

−OPP

Germacrene A

Germacrene B

Germacrene CGermacrene D

−OPP

FPP

20Nerolidyl diphosphate

Germacryl cation1,2-Hydride

shift1,3-Hydride

shift

A

21

OPP


28

1.5 Monoterpenoids Many monoterpenoids have been reported over the last hundred years and many

have proved to be fragrant oils, which are extracted from flowers, leaves and fruits of

many plants. In spite of the fact that many monoterpenoids exist as isomeric mixtures, and

can be difficult to separate, there are a few monoterpenoids that occur as single isomers

and this allows crystallization, for example camphor.5

Monoterpenoids are part of the terpenoid family, which contain 10 carbon atoms,

and they are generated from biosynthesis via geranyl pyrophosphate (see scheme 1.6). The

C10 series of monoterpenoids involve a broad range of hydrocarbons, aldehydes, ketones

and alcohols. Although, there are only a few skeletal themes for monoterpenoids, a large

number exist as simple derivatives, isomers and stereochemical variants in which cyclohe-

xanoids represent the major type of monoterpenoids.19 Monoterpenoids are classified in to

three sub-groups:

1.5.1 Acyclic monoterpenoids

There is huge number of monoterpenoid volatile liquids known, which are saturated

hydrocarbons, originally derived from 2,6-dimethyloctane, called regular acyclic

monoterpenoids. Acyclic monoterpenoids are used in the perfumery industry due to their

distinguished odours.8 The vast majority of acyclic monoterpenoids are made of head-to-

tail joined isoprene units 22. Meanwhile, irregular compounds are linked by head-to-head

rearrangement 23 or made by cleavage of cyclic monoterpenoids 24 20 (Figure 1.3).

Figure 1.3 Regular 22 and irregular 23 and 24 acyclic monoterpenoids skeletons

1.5.2 Cyclic monoterpenoids

This type comprises monocyclic and bicyclic monoterpenoids. Monocyclic examples

originate from cis–trans isomers of p-menthane 25 and 26 (saturated hydrocarbon).

22 23 24Head to tail fashion Head to head fashion Cyclomonoterpenoids cleavage


29

Figure 1.4 Isomers of p-menthane

Also, there are a variety of compounds belonging to the p-menthane sub-class like

(+)-limonene 27 (unsaturated), (–)-menthol 28 and (+)-pulegone 29.

Figure 1.5 Compounds belonging to the p-menthane sub-class

Meanwhile, there are many diverse structures of bicyclic monoterpenoids with

constrained rings.21

Figure 1.6 Bicyclic monoterpenoid skeletons

The cyclic monoterpenoids are biosynthesized via isomerization and cyclisation

processes. The first process results through binding of geranyl diphosphate (GPP) 19 with

a single enzyme in presence of divalent metal, and 19 converts to (+)-(3S)-linalyl

diphosphate (LPP) 30 or to (–)-(3R)-LPP 31. The ionization of allylic linalyl cation

produces the LPP cation, and allows the opportunity for cyclization via the stereo-

chemically favorable linalyl cation (Scheme 1.8).18

= =

cis-p-Methane25

trans-p-Methane26

OH O

(+)-Limonene 27

(−)-Isopulegol28

(+)-Pulegone29

=

Pinane

Carane Thujane

Camphane, Bornane or Isobornane Isocamphane Fenchane

Isobornylane


30

Scheme 1.8 Pathways of biosynthesis of monocyclic, bicyclic and tricyclic terpenes from

GPP 19 18

Biosynthesis pathways of oxygenated monoterpenoids with specific stereostructures

have been extensively reported, for example, bioconversion of limonene enantiomers 27 and

32 to (–)-menthol 28 and (–)-carveol 37 using cytochrome P450s (Scheme 1.9). 22−24

Scheme 1.9 Biosynthesis pathways of monoterpenes using cytochrome P450s 22−24

OPPOPP

OPP

OPPOPP

H

H

H1 1

2

3

1

2

3

1931

30

2-Carene

Tricyclene

Limonene

OPP

≡

≡

(+)-Limonene 27

OHOH

HOO

OH O

(+)-Carvone 34

(−)-Carvone 36

(−)-Limonene 32 (+)-trans-Carveol 33

(−)-trans-Carveol 37

(−)-trans-Isopiperitenol 35(−)-Menthol 28

Biosynthesis pathways of monoterpene in A) Oxpeppermint by (-)-4S-limonene-3-hydroxylaseB) Spearmint by (-)-4S- limonene-6-hydroxylase C) Caraway by (+)-limonene-6-hydroxylase* The dotted arraow represents five enzymatic steps.

B

AC


31

1.6 Objectives and approach Several monoterpenoids are commercially available, inexpensive and renewable

natural precursors and their derivatives include some of the most effective asymmetric

controllers used in organic synthesis, with particular utility in ligand-based25 and, more

recently, organocatalysis.26,27 They are also precursors in target syntheses and there are

gaps in synthetic modifications. An objective of the research was to synthesize new chirons

with evaluation of biocatalysis for modifications, either after or before chemical

modifications, which could yield functionalized derivatives that many have synthetic

uses.28−30 Two main areas were experimentally carried out:

First area: Generation of new types of substrates based on R-(–)-carvone 36, (+)-

isomenthone 38 and (–)-isopinocamphone 39 using short chemical routes providing

potentially versatile novel substrate diversity to novel terpenoid scaffolds.

Figure 1.7 Generation of new chirons from 36, 38 and 39

All routes in the research involve installing new stereogenic centres via α-

substitution of enolate (and/or cyclohexanol) derivatives.

Second area: One of the applications of biocatalysis is synthesis of intermediates or fine

organic products with high chemo- and regioselectivity.

Figure 1.8 Applications of new substrates generated from R-(–)-carvone

Thus, the chirons were to be evaluated for biocatalysis in chemo-enzymatic

processes, and cascade enzymatic reactions. For instance, non-natural monoterpenoids 6-

methylcarvone diastereomers were chemically synthesised, and treated with ene-reductase

O O

O *Alkylation*Aldol addition*Elimination of aldol adduct

36 38 39

Asymmetric controllerEvalution for biocatalysis

Precursor for natural and non-natural biological molecules

O O

R

R

O

RR

OH

H

H


32

(OYEs). R-(–)-Carvone 36, (2R,5R)-(+)-isomenthone 38, and (1S,2S,5R)-(–)-isopinocamphone

39 were selected as starting materials.

Scheme 1.10 Objectives of A) chemo−enzymatic B) enzymatic−chemical reaction

O O

O O

OMethylation

OYEs

Separable isomers

OO

O

OYEs

Aldol addition

R

OHH

A Chemo-enzymatic

BEnzymatic-chemical reaction

H

2. Synthesis of modified monoterpenoids

33

2 Synthesis of modified monoterpenoids

2.1 Overview of modified monoterpenoids in synthesis, and initial synthetic

objectives Terpenoid derivatives are known as a chiral pool source,31 supplying a number of

chiral auxiliaries,32−36 reagents37,38 and catalysts.39,40 For instance, (+)-dimenthyl fumarate

40 has been utilised for enantioselective synthesis of chiral bicyclic enone 41 with yield

80%. Firstly, Diels-Alder reaction of 40 with butadiene was smoothly proceeded to afford

diester 42 (yield 88%), which subsequently converted to 41 under kinetic control. 41 was

effectively employed in multisteps synthesis for enantioselective totally of C15 polycyclic

lactone bilobalide 43. 41

Scheme 2.1 Synthesis of chiral 41, and multisteps to total synthesis of 43 41

Both natural and modified monoterpenoids are in some cases important targets for

themselves (see chapter 1) and many are precursors in target syntheses.28−30 There are gaps

in synthetic modifications, so the aim of the research is to employ inexpensive chiral pool

cyclic ketones to generate useful synthons or chiral templates relevant to the synthesis of

natural compounds and analogues. These would serve to provide a new range of chirality-

elaborated structures and also as substrates for evaluating biocatalytic modifications of

synthetically modified terpenones. Thus, the concept is to provide these towards potential

chemo-enzymatic applications, but also, to effect some initial enzymatic modifications to

afford opportunities for enzymatic-chemo approaches to modified new terpenoids (See

Scheme 1.10).

O

OO

O

(+)-Dimenthyl fumarate 40

CO2Men

CO2Men

O

MenO2C

MenO2C

t-Bu

O

O

t-BuOH

OHO

O H

O

(−)-Bilobalide 43

i,ii iii,iv,v

42

41

i = Butadiene, ii = (i-Bu)2AlCl, iii = LDA/THF, −78 °C,

t-Bu CO2Phiv = v = KHMDS/THF, −78 °C

Yield 88% of R,R isomer

Yield 80% of S,R isomer


34

2.2 Precedent examples of alkylations of terpenone enolates

α-Alkylation of carbonyl compounds is one of the most valuable reactions to

construct C-C bonds in organic synthesis.42 Installing enolates from carbonyl compounds is

extensively applied to alkylate cyclic and acyclic carbonyl compounds due to the high

nucleophilicity of enolates, which consequently undergo various reactions with electrop-

hiles such as alkyl halides, aldehydes and ketones.43 In this section the alkylations of these

terpenones will be described.

Alkylation of R-(−)carvone 36 has been demonstrated under kinetic conditions.44

The result of methylation was previously reported to lead to formation of diastereomers

(3:1) identified by two doublets at δ 1.07 and 0.92 ppm in the 1H NMR spectrum.

Scheme 2.2 Alkylation of R-(–)-carvone 36 under kinetic conditions 44

The product mixture of 6-methylcarvone (44 and 45) reacted with several alkyl

halides in presence of LDA affording diastereoselective products 46 with the alkyl group

attacking cis to the isopropenyl group. The sequence of the two steps are reversed by

alkylation of carvone with different alkyl groups, followed by methylation of the

monoalkylated product to yield a mixture of disubstituted carvone 47, for example,

treatment of the starting material with benzyl and methyl halide respectively resulted in a

O

H

O

H

R2R1

H

O

O

H

MeR

O

H

R

O

H

R

O

H

RMe

46a-g

a : R = C2H5 b : allyl

c : R = CH2-CBr=CH2

d : R = CH2-CCl=CH2

e : R = -CH2C6H5

f : R = CH2CH=CCl (Z + E)

g : R = CH2-CH=CBr (Z + E)

i) LDAii) CH3I

i) LDA

ii) R-X44 R1= CH3, R2= H45 R1= H, R2= CH3

i) LDAii) R-X

i) LDA

CH3I

47a-e

46a-g

36


35

75:25 isomers mixture ratio, with the benzyl group mainly positioned trans to the

isopropenyl group.

Srikrishna and Vijaykumar 45 have reported that the mixture of 6-methylcarvone

diastereomers (3:2) was equilibrated with DBU to afford a (3:1) diastereomeric ratio. The

mixture was recrystallized with hexane at −10 °C to yield pure trans 6-methylcarvone 44.

The latter was the starting point to enantiospecific total synthesis of (+)-pinguisenol 48.

Scheme 2.3 Synthesis of pinguisenol 48 from R-(–)-carvone 36 45

Alkylation of (−)-menthone 49 and silyloxymethone analogue 50 using LDA was

the first step to synthesize (+)-dihydro-epi-deoxyarteannuin B (artemisinin-derived

antimalarial compounds).46 The products 2-allylsilyloxymethone (49a and 50a) were only

obtained in less than 45% yield. The issue was solved by adding diethyl zincate and

HMPA, and the yields increased to over 80%.

Scheme 2.4 Alkylation of (−)-menthone 49 and silyloxymenthone 50 46

A general procedure for kinetically-controlled monoalkylation of (+)-nopinone 51

with various electrophiles including even heteroatoms, has been described, reporting that

use of 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU) and/or NaI at −45 °C

improved the diastereoselectivity. For example, the alkylation of 51 with PhCH2Br using

NaI/DMPU system gave 78% yield and 99:1 diastereoselectivity of kinetically preferred

monoalkylated product 52, with no epimerisation (Scheme 2.5).47

O O O+i,ii

44 453 : 23 : 1

ii

HO44

48

i = LDA/THF, MeI, −78 °C; ii = DBU, recrystallization

ZO

ZOLi

ZO

i) LDA/THF, ii) allyl bromide, (C2H5)2Zn, HMPA

i ii

49a and 50a, Yield >80%Z in 49 = HZ in 50 = OTIPS


36

Scheme 2.5 Alkylation of (+)-nopinone 51 in presence of NaI and/or DMPU 47

2.3 Alkylations of R-(–)-carvone, (+)-isomenthone and (–)-isopinocamphone

In this research, some of the terpenone substrates for alkylations and other

modifications are not commercially available and thus required developing suitable

oxidation methods from precursor terpenols to corresponding ketones. This work therefore

commenced with such oxidations to synthesise staring terpenones. Whilst R-(–)-carvone is

commercially available at reasonable cost, the same is not true for (+)-isomenthone 38 and

(–)-isopinocamphone 39, however, precursor terpenols are available. Therefore, oxidation

was required to provide these two targets. The first approach employed use of sulphur

trioxide; conveniently in the form of its pyridine complex, and dimethyl sulfoxide (DMSO)

as a solvent in presence of triethylamine. This was evaluated for oxidation of (+)-

isomenthol 54 to the corresponding ketone 38.48 This method is attractive due to the mild

conditions, as it proceeds at RT. However, TLC revealed that the starting material was not

totally consumed, and there was difficulty in purifying the product due to impurities.

Moreover, oxidation using pyridine sulphur trioxide complex reagent afforded modest

conversion (53%) to 38. Thus, the oxidation of 54 was instead attempted using a more

powerful oxidant reagent, 2-iodoxybenzoic acid (IBX) 55.49,50 Utilizing IBX to produce 38

was effective and has several advantages. For example, the reaction reaches completion

and gives a pure product without formation of side products, with a good yield of 38 was

obtained (70%). Moreover, IBX 55 is easily and smoothly synthesized by an accepted

environmentally friendly method with H2O as the solvent.51 The suggested mechanism for

oxidation of the alcohol includes an equilibrium between 55 and alcohol with iodinane

oxide, where increasing water concentration leads to decease in the concentration of the

reactive intermediate (scheme 2.6).49 The 13C NMR spectrum clearly shows formation of

38 through presence of C=O signal at δ 214.6 and disappearance of CH-OH at δ 67 ppm of

alcohol starting material.

O

Kinetically favoured 52 Thermodynamically favoured 53

i, ii

i) LDA/THF, –10 °C ii) PhCH2Br, NaI/DMPU,–45 °C

51 O

H

Yield 78%, d.r 99:1

OH


37

Scheme 2.6 1) The mechanism proposed for oxidation of 54 via A) Pyridine sulphur

trioxide complex B) IBX 55 2) Oxidation of 56 using formation of 55 in situ

Successful oxidation of 54 using 55 encouraged the use of 55 to oxidise (+)-

isopinocampheol 56 (scheme 2.6). The reaction proceeded smoothly, and the yield was

good (67%). Attention then was turned to synthesis of IBX 55 in situ with acetone-

trile/water as solvent, where reports indicate this type of process can use 0.3 eq. of 2-

iodobenzoic acid with 0.8 eq. Oxone®, in comparison to the 8 eq. of IBX.52 Heating at 70

°C activated the reaction, and acetonitrile was easily removed under high vacuum, raising

the yield to 78%. A cursory evaluation of this process reveals there would be several

advantages over oxidation of 54 with two steps (IBX synthesis and oxidation). Formation

of 39 was confirmed using 1H NMR data, which revealed disappearance of alcohol OH

proton, and reduction of the number of protons by two. In addition, appearance of the C=O

signal at 215 ppm in the 13C NMR spectrum.

In conclusion, both ketones 38 and 39 were synthesised from their corresponding

secondary alcohols in good yields, and under mild conditions.

With viable routes to syntheses of 38 and 39, and commercial supply of carvone 36,

the synthetic work now had objectives to evaluate enolate alkylation, enolate reaction

56

O

OH

39

OH

OI

HO O

O

OI

O O

O

H2O

O

O

OH

IH2O

54

55

+

+

SO

SO S

O

O OSO

SO

O

O

N

OS N

H

SO42-

Triethylamine

H

pyHSO4

-Me2S

OH

54

A B

OSH

38

+

Formation of 55 in situ

21


38

through aldol reactions and also oxidation. The following sections describe work to

develop these reactions, and the stereochemical outcomes obtained.

In this work, the kinetic lithium enolate of R-(–)-carvone 36, (+)-isomenthone 38

and (–)-isopinocamphone 39 have been synthesised, and then reacted with alkyl halide to

afford two possible epimers at new stereogenic of monoalkylation. Several alkyl halides

were tested in this reaction (Table 2.1), and the reactions with CH3I and C6H5CH2Br

proceeded with high diastereoselectivity except 36 to generate α-substituted products (due

to less steric hindrance), favouring formation of the (S) configuration at the new

stereocentre added to (C6) of 36 (Scheme 2.7).

Scheme 2.7 Mechanism of R-(–)-carvone 36 methylation under kinetic conditions

However, no products were detected using isopropyl and cyclohexyl halide, even

after 6 h at −78 °C.

2.1 Yields and diastereomeric ratios of alkylated 36, 38, and 39 Subs.

no. CH3I d.r. A C2H5

I d.r. PhCH2Br d.r.

36 85% 3:1B 64% 1:1B 71% 5:1B 38 40% 9:1C N/A N/A 36% 97.5:2.5C 39 41% 99.5:0.5D N/A N/A 37% 99:1D

A = diastereomeric ratio were calculated form 1H NMR spectra and/or from HPLC chromograms. B = (R,S):(R,R), C = (S,R,R):(R,R,R), D = (R,S,S,S):(R,R,S,S). N/A = The reaction was not applied.

2.3.1 Alkylation of R-(–)-carvone

Addition of methyl iodide (MeI) to enolate lithium of 36 afforded a diastereomeric

mixture of 6-methylcarvone 44 and 45, purified using flash column chromatography to

yield a pure product (85%), which was readily identified by 1H NMR from observation of

two doublets of the new methyl group at the C6 position at δ 0.98 ppm for the major 44

and δ 0.85 ppm for the minor isomer 45.

1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) at RT for 24 h was employed to

catalyse an equilibration of the diastereomeric mixture of 44/45, converting the ratio of

epimeric mixture from 3:2 to 3:1 of (44:45). This increases the proportion of the equatorial

orientation of methyl group (trans isomer). The diastereomeric mixture was a pale yellow

(R)(R)

(Z) O

(R)(R)

(Z) O

CH3-I(R)(R)(S)(S)

(Z) O

(R)(R)(R)(R)

(Z) Oi ii

i) LDA/ THF, −78 °C ii) CH3I, 0 °C−RT

36 44 45d.r 3:2


39

oil, and it was crystalized from hexane, after storage at −10 °C for several days to afford

the major diastereomer (trans-6-methylcarvone, 44) with yield 38%. Assignment of the 1H

NMR spectrum (obtained for the crystals dissolved in CDCl3) identified a doublet peak of

the methyl group at δ 1.05 ppm with J value (6.6 Hz) at new stereocentre at C6, as well as

two peaks of two methyl groups at δ 1.71 and 1.78 ppm. Moreover, four multiplets of CH

and CH2 groups were observed, including the peaks of H-6 and H-5. The configuration of

44 was established with X-ray crystallography, and revealed the stereochemistry at α

position to be anti at C6 to isopropenyl.

Figure 2.1 X-ray crystal structure and stereochemistry of 44

13C NMR showed 11 non-equivalent peaks, including three signals at δ 21.9, 16.2

and 12.5 ppm of three methyl groups, along with a C=O at δ 203.6, and 51 ppm peak for

C5.

The minor diastereomer 45 was partially separated with column chromatography

using hexane:EtOAc 40:1, and full characterised via NMR, MS and IR spectroscopies. For

instance, the 1H NMR spectrum exhibited three methyl signals; a doublet (J = 7.2 Hz) at

0.92 ppm for axial Me at C6, and signals at 1.70 and 1.78 ppm of two olefinic methyls. 13C

NMR showed the C=O peak at 203.7 ppm, and three signals of three substituted methyls at

22.0, 16.1 and 10.6 ppm.

(R)(R)(S)(S)

(Z) O

44


40

Figure 2.2 1H NMR spectra of carvone 36 and spectra of a mixture of 44 and 45 and

separated 44 and 45

In case of connecting benzyl group at C6, the 1H NMR of 57 showed two peaks for

two methyl groups at δ 1.66 and 1.78 ppm, and a multiplet signal with integration equal to

5H at δ 7.27−7.07 ppm. Furthermore, two crucial peaks were identified, first a doublet of

triplets (J = 10.1, 6.9 Hz) at δ 2.57 ppm for H-5, and a ddd at δ 2.72 ppm for H-6 with J

values 10.1, 6.9, 5.1 Hz. The correlation of J values of H-5 and H-6 showed the

stereochemistry of this diastereomer is (S) at the new stereogenic center (C6 position). The

2D-COSY spectrum indicated the correlation between adjacent nuclei, for instance, (Figure

2.3) shows H-6 correlated to H-5, and also to CH2-Ph.

Scheme 2.8 Alkylation of 36 with benzyl bromide under kinetic control

Me-11

(R)(R)(S)(S)

(Z) O

(R)(R)(R)(R)

(Z) O

57 58

(R)(R)

(Z) Oi,ii

i) LDA/ THF, −78 °C ii) PhCH2Br, 0 °C-RT


41

Figure 2.3 2D-COSY spectrum of 57 diastereomer

Establishing the configuration of 58 via 1H NMR of a mixture was difficult because

of overlapping of CH, and CH2 signals arising from both diastereomers. However, it was

assumed that configuration would be R at the C6 stereocentre.

The reaction of ethyl iodide (1.2 eq.) with the lithium enolate of 36 gave pure

diastereomers (R,S) 59 and (R,R) 60 in a 1:1 diastereomeric ratio and yield 64%. The

proton NMR spectrum showed three peaks for three methyl groups at δ 0.84, 1.68 and 1.73

ppm, and methylene group at C11 was assigned at δ (1.49−1.19) ppm as a multiplet peak.

The signal of H-5 was distinguished as a multiplet peak at δ 2.75−2.65 ppm, and H-6

overlapped with CH2 at δ 2.48−2.22 ppm. More confirmation was obtained via 13C NMR

showing 12 pairs of peaks, for instance, two peaks of C=O at δ 202.9 and 201.3 ppm, and

CH2 peaks at δ 22.1 and 20.2 ppm.

Figure 2.4 Stereochemistry of 59 and 60

2.3.2 Alkylation of (2R,5R)-(+)-isomenthone

Alkylation of isomenthone 38 has been attempted previously, using a variety of

electrophiles and conditions. For example addition of iodomethane to lithium hexamethyl-

disilizane in presence of 5 equivalents of co-solvent HMPA, was not effective and only

(R)(R)(S)(S)

(Z) O

(R)(R)(R)(R)

(Z) O

59 60

Hz500 5001000100015001500200020002500250030003000

500

1000

1500

2000

2500

3000

H-3 H-4a,bH-5H-6CH2-Ph

H-5H-4a,b

H-6CH2-Ph

MeMeH-9a,b

H-9a,b

H-5H-6CH2-Ph

H-4a,b

OH

H

HH

H

HH

1234 5

6


42

starting material was recovered.53 Our work was re-evaluating the conditions of the

methylation utilising LDA/DMPU, and the temperature was kept at –45 °C. Under these

conditions, isomenthone gave 40% of mono-methyl isomenthone 61 and 62 with high

diastereoselectivity (d.r. 9:1) in favour of the kinetically-preferred 2S-methylisomenthone

61.

Scheme 2.9 Methylation of 38 to afford 2-methylisomenthone 61 and 62

The isomer 61 was a colorless oil, and proton NMR showed three peaks for the four

Me groups at δ 0.78 (d, J = 6.4 Hz), 0.93 (d, J = 6.4 Hz), 1.01 (t, J = 6.3 Hz, 2 x Me).

Unfortunately, the signal for H-2 is a multiplet, and the peak for H-6 is overlapping with

CH2 peaks. The C=O functional group was evidenced by 13C NMR, where it appeared at

216.7 ppm. In addition, the signals for Me groups were evident at 21.0, 20.8, 20.4 and 12.4

ppm. Mass spectrometry showed the expected peak of the product at 191.1 (MNa)+. The

second isomer 62 represents only 10%, and it was not assigned as its peaks overlapped

with 61.

The kinetic lithium enolate of isomenthone was reacted with benzyl bromide (1.2

eq.) and 1 equivalent of DMPU at strictly −45 °C. Two diastereomers (63 and 64) were

indicated by proton NMR, and the yield of pale yellow oil was 36%. Reversed phase

HPLC exhibited two peaks of the isomers with ratio of 97.5:2.5.

Figure 2.5 Chromatogram of reverse phase HPLC column of 63 and 64

(S)(S)O (R)(R)O6261

Oi, ii

Yield 40%, d.r. 9:1

i = LDA/THF, −10 °C, ii = MeI, DMPU, −45 °C

38

O

O


43

Examination of the 1H NMR of 63 revealed two doublet of doublet peaks for the

two protons of CH2-Ph with J values 13.8, 8.4 and 13.8, 4.7 Hz at δ 3.05 and 2.74 ppm.

The value of 13.8 Hz coupling constant represents the correlation of CH2 protons with each

other, while, second values 8.4 Hz and 4.7 Hz represent the correlation of CH2 protons and

the next proton (H-2) of cyclohexanone ring.

Figure 2.6 1H NMR spectrum of the mixture of 63 and 64

2.3.3 Alkylation of (1S,2S,5R)-(–)-isopinocamphone

Addition of methyl iodide to the lithium enolate of (–)-isopinocamphone 39 in an

ice-acetone bath afforded mainly dimethyl-substituted product 65 in 66% yield, and

approximately 5% of monosubstituted product 66. 1H NMR of the dimethyl isopinoca-

mphone major product presented five peaks for the five methyl groups between δ 1.31 and

0.87 ppm, and the product was also confirmed by MS, where the main peak was 181

(MH)+.

Scheme 2.10 Methylation of 39 under kinetic control

The reaction was repeated using DMPU, to give 41% yield as a diastereomeric

mixture of 2-methylisopincamphone 66 and 67. The ratio of isomers was investigated with

reverse-phase HPLC using (MeCN:H2O 50:50), and shown to be 99:1. The 1H NMR of the

non-natural bicyclic monoterpenoid revealed four Me peaks (Table 2.2).

65

i = LDA/THF, −10 °C, ii = MeI, −78 °C

O O (S)(S)O+

66Yield 66% Yield 5%

66

(S)(S)O (R)(R)O+

67Yield 41% d.r. 99:1

i,iia,b

a = LDA/THF, −10 °C, ii = MeI/DMPU, −45 °C

39


44

Table 2.2 1H NMR spectra data of Me substituted groups of 2-methyl isopinocamphone

13C NMR spectroscopy showed 11 non-equivalent carbons, with the C=O signal

present at 219.6 ppm. The reaction is highly diastereo- and regioselective with α-methyl

substituent at C2 positioned in an axial position to avoid steric hindrance.

The reaction of benzyl bromide with lithium enolate 39 was also then conducted in

presence of DMPU, giving a 99.5:0.5 diastereomeric mixture 68 and 69.

Figure 2.7 Chromatogram from reverse-phase HPLC of 68 and 69

The product was confirmed by NMR, IR and HRMS. For instance, the most crucial

peaks in 1H NMR were two doublet of doublet peaks for two protons of CH2-Ph at δ 3.20

ppm (J = 13.2, 3.8 Hz) and 2.66 ppm with coupling constants of 13.2, 11.7 Hz. Also,

multiplet signal for H-2 was indicated at δ 2.81−2.67 ppm. 13C NMR spectrum showed the

peak of CH2-Ph at 36.2 ppm, and a signal of C-2 was shown at 51.1 ppm.

Scheme 2.11 Benzylation of 39 in presence of DMPU

O1234

5 67

89

10

1166

O

67

(S)(S)O (R)(R)O

68 69

i, ii

i = LDA/THF, −10 °C, ii = PhCH2Br/ DMPU, −45 °C

O +

Yield 37% d.r 99:1

Me groups 1H δ (ppm) Mutiplicity and J (Hz) CH3 at C7 1.33 Single CH3 at C4 1.21 Doublet (J = 7.4 Hz) CH3 at C2 1.15 Doublet (J = 7.4 Hz) CH3 at C7 0.89 Single


45

To continue with alkylation process, this study investigated yield, diastereo-

selectivities and stereostructures of aldol addition outcomes for monoterpenoids. Also, the

aim was to prepare novel substrates for biotransformation evaluation.

2.4 Precedent examples of aldol additions to monoterpenoid enolates Undoubtedly, the aldol reaction is one of the most important reactions to construct

C-C bonds in organic synthesis, and it was discovered in 1872 by Wurtz.54 Aldol reactions

of natural product cyclic ketones, generating new stereo-centres in the β-ketol product,

have been widely reported. For example, Go et al.55 studied the effect of solvent on the

enantioface selectivity of Diels-Alder reaction catalysed by aluminium complex of a chiral

menthol derivative (Scheme 2.12). The synthetic steps for the chiral menthol derivative

were: firstly, (–)-menthone 49 treated with benzaldehyde at −78 ºC to form diastereomeric

mixture of β-hydroxyketone, and the major diastereomer was successively isolated through

flash column chromatography. Secondly, the major diastereomer 70 of β-hydroxyketone

was regioselectivity reduced to afford a single diastereomer 71, which subsequently

reacted with several metal ions (M) to form a 6-membered chelate 72 (Scheme 2.12).

Scheme 2.12 Aldol addition to 49 using benzaldehyde and reduction of 70 55

The Diels–Alder reaction was carried out as described in Scheme 2.13.

Scheme 2.13 Diels−Alder reaction catalysed by aluminium chiral menthone derivative 72 55

Also, menthone 49 was reacted with 4-(2-oxo-ethyl)-2,6-piperidinedione to

synthesise cycloheximide analogues, a powerful antimicrobial activator, under kinetic

O O

Ph

OH

HOH

Ph

OH

H O MO

Complex of chiral menthol derivative

i = LDA/THF, ii = benzaldehyde, iii = TMSCl, imidazole,iv = diisobutylalminium hydridev = tetrabutylammonium floride, yield 93% two steps

i, ii iii, iv, v

49 70 71

72

X

O

COX

exo HN O

O

X =+ +

i, ii+

i = diol 71, ii = EtAlCl2endo ent-endo

Solvent Enantiomeric ratio Overall yield%

THF

DCM 91:9 89%14:86 87%

COX


46

conditions. The 1H NMR of the product distinguished diastereomers anti-aldol 73 and syn-

aldol 74 using chemical shifts of the ´2 position signals.56

Figure 2.8 Anti 73 and syn 74 aldol adducts of cycloheximide analogues56

Prior work in our group showed that the lithium enolate of (+)-isomenthone 38

underwent aldol addition57 with alkyl and aryl aldehydes to yield two diastereomers 75 and

76. However, the ratio of diastereomers was interestingly reversed by quenching the

reaction at 0 ºC instead of −78 ºC. For example, addition of benzaldehyde to the lithium

enolate of (+)-isomenthone and quenching the reaction at −78 ºC afforded diastereomeric

ratio 62:38 of 75:76, while the ratio is 22:78 of 75:76 when the reaction quenched at 0 ºC.

The study was confirmed by X-ray crystallography which proved that the predominant

diastereomer is the threo product through Si face addition to the aldehyde, whereas,

quenching the reaction at 0 ºC lead to addition on the Re face of aldehyde.

Scheme 2.14 Aldol addition to enolate lithium of 38 57

Enantioselective deprotonation of tropinone 77 with the chiral lithium amide 78, in

the presence of LiCl, was employed to generate the lithium enolate.58 Addition of

benzaldehyde gave high diastereoselectivity and yield (91%), and only the anti aldol

adduct 79 observed.

Scheme 2.15 Aldol reaction of 77 58

Aldol addition to (S)-(+)-carvone 34 was successfully carried out twice to obtain

new chirons which are necessary to construct a pentacyclic intermediate for target

molecules simalikalactone D and quassimarin.29 Treatment of 34 with LDA in 3:1

O

H

ROH

HO

OH

RH

H R =NH

CH2

OO

73 74

O OHO R

OHO R

i, ii

i = LDA, ii = RCHO75 76

38

NMe

O

NMe

O

Ph

OHH

78 = N

PhNtBuLi

i, ii

i = 78, ii = PhCHO

77 79Yield 91%


47

THF/DMPU at −78 ºC to form the lithium enolate, followed by addition of formaldehyde

gave 90% of a single diastereomer of β-ketoalcohol 80 (Scheme 2.16) The high value of

the J coupling constants of H-6 and H-5 indicated the aldol reaction had proceeded at the

less hindered α-face. Addition of aldehyde 81 to the enolate of 34 provided 62% of the

alcohol 82 (Scheme 2.16).

Scheme 2.16 Aldol addition to 34 using formaldehyde and 81 29

Schöttner and co-workers59 suggested that the aldol adduct of tetrahydrocarvone 83

with geraniol-derived aldehydes affords access to synthesis of diterpenoid allyl phosphates

84, and cyclization of the latter using SmI2 gives the marine diterpenoid eunicellane

(ANSA diterpenoid) 85. Surprisingly, the aldol addition to 83 yielded recovery of the

starting materials (Figure 2.9).

Figure 2.9 Chemical structures of 83, 84 and 85

However, testing the aldol addition to (S)-carvone 34 and its hydrogenated

derivatives using five aldehydes was carried out, and the results were analysed by 2D-

NOESY, 1H NMR, and X-ray crystallography. It was shown that hydroxyalkylation

connected to (S)-carvone 34 from the sterically less hindered side except in hydroxyl

benzylation formed two minor (3R) diastereomers. However, the researchers decided to

treat (S)-carvone 34 with geraniol-derived aldehydes, thence, reduce the double bond of

cyclohexenone ring, in order to obtain the required hydroxyalkylated cyclohexanone

(Scheme 2.17).

O

O

OH5 6

11i,ii

i = LDA/THF, ii = CH2O, iii = 81

34

80

O

SO2

H

OH SO2

OH

81 =

82

i,iii

Yield 62%

Yield 90%

85

OTBSO

HHH

OP

OTBSH

O

OCH2CH3

OCH2CH3

84

O

83


48

Scheme 2.17 Aldol addition to (S)-carvone and/or its hydrogenated derivatives59

Addition of acetaldehyde to the lithium enolate of R-(–)-carvone 36 was the first

step for preparation of the intermediate 86, and consequently multiple steps were

completed for synthesis of the polycyclic compound. The latter is the key to synthesis of four

biogenetically related polycyclic diterpenes: trachylobane, beyerane, atisane, and kaurane.

An excellent yield (92%) of β-ketol was obtained and the 1HNMR spectrum showed

generation of four diastereomers (4:3:1:1 ratio), and no further separation was achieved.60

Scheme 2.18 Aldol addition to 36, and multi-step to synthesis of 86 60

Adducts of aldol addition to camphor 87 and to (+)-dihydrocarvone 88 have been

reported in the synthesis of enantiomerically-enriched alcohols via the retroaldol

reaction.61 The ketones were treated with LDA to generate the lithium enolate (89 and 90)

followed by adding benzaldehyde to give the ketoalcohol (yield 42% with d.e. >98% of 91

and yield 78% with d.e. 60% of 92). While, the silylenol ether of dihydrocarvone 92 was

cleaved via potassium tert-butoxide, and K-Li exchanged to furnish the lithium enolate of

dihydrocarvone 93. Addition of benzaldehyde, and crystallization the product subsequently

gave a diastereomeric mixture (51% yield with d.e. 98% with respect to 94) (Scheme 2.19).

O O

OH

RH

O

OH

RH

i, iiR =

OAc

OAci = LDA/ THF, ii = RCHO

O O

OH

H

H

i = LDA/THF, CH3CHO

i

36 86Yield 92%, d.r 4:3:1:1

O


49

Scheme 2.19 Aldol addition to 87 and 88 61

The aldol products were treated with nucleophilic species like organolithiums and

Grignard reagents. For instance, aldol adduct of camphor 91 gave an enone 95, while no

products derived from 92 were observed. However, treatment 94 with methyllithium and

methylmagnesium bromide gave a single isomer of diol 95, whilst, addition of a bigger

nucleophile drove the retroaldol reaction to obtain enantiomerically enriched alcohols with

e.e. up to 35% (Scheme 2.20).

The crux of the work is that the metal coordinates to fragmentations via oxygen

atoms of both enolate and aldehyde, where the enolate facial selectivity of the aldehyde is

masked, and nucleophile selectively attacks other face (transition state 96 ).

OH

O O

OH

Ph

i

i = LDA, ii = Benzaldehyde/THF, −78 °C, iii = Crystallisation, a = TMSCl, b = Potassium t-butoxide, c = LiBr

O

HPhOH

87OLi

ii,iii

91

iOLi

ii

92

OTMS OM

M = KM = Li

a b

89

90

cAB

94

ii,iii

93

Yield 42%, d.e. >98%

Yield 78%, d.e. 60%

Yield 51%, d.e. 98%

HPhHO

O

88

O

88


50

Scheme 2.20 Nucleophilic addition to aldol adducts 91, 92 and 94 61

Mondal and co-workers reported an expedient synthesis of 97, tricyclic skeleton 98,

and of a biologically active norditerpene called caribenol A 99 with domino metathesis

rings opening and closing steps, with terpenone enolate alkylation as key initial step.62

Figure 2.10 Chemical structures of tricyclic 97, 98 and 99

Initially, aldol addition of R-(–)-carvone 36 to aldehyde 5-norbornene-2-carboxald-

ehyde failed to afford the product due to steric hindrance effect. The strategy was altered to

set up aldol addition of (+)-dihydrocarvone 100 to the less sterically-demanding aldehyde

acrolein, giving anti aldol adduct 101 as a single diastereomer. Analogous aldol addition of

100 (derived from 36) with aldehyde (5-norbornene-2-carboxaldehyde) under kinetic

control showed formation of an epimeric mixture (2:1) at the new stereocentre of the

carbon bearing OH, in 70% yield. A domino process of reactions finally furnished 97, so

the researchers made the decision to rebuild 98 from dihydrocarvone derived from S-(+)-

carvone 34 (Scheme 2.21).

94

HPhHO

O

O

Ph

95

PhHO

HOH

Ph OH

RO

i

ii

i = MeLi, MeMgBr ii = EtLi, EtMgBr

95

OH

HPhOH

91

O

OH

Ph92

No producti i

88

+

OLiO

HPh

Transition state 96

e.e. >35%

HOO

HH

H

97

H

H

99

H

HO O

O

HOO

H

HH

R

98


51

Scheme 2.21 Aldol addition to 100 62

As noticed from literature review above, there are a variety applications of

monoterpenoid aldol adducts including catalyst in asymmetric synthesis, and precursors of

several natural and non-natural products. Studies and investigations of aldol adducts, from

both sides yield, and stereoselectivities, are the key for the synthesis. Also, many of

dihydrocarvone aldol adducts afforded poor yield, and in some cases low stereoselectivity.

This background led us to interest in preparing aldol adducts such as those derived from

carvone for biotransformation evaluation.

2.5 Aldol addition to (–)-carvone, (+)-isomenthone, (–)-isopinocamphone

and (+)-dihydrocarvone

2.5.1 Aldol additions to R-(–)-carvone

Given the prior literature and our interest in modified carvones, this work then

assessed reactivity of carbonyl substrates (aldehydes) with the enolate of 36 (generated

using LDA). The latter was generated in situ by slowly adding a hexane solution of nBuLi

to DIPA, under cold conditions (0 °C), then 36 was added at −78 °C in order to form the

kinetic lithium enolate. The aldehyde was added in one portion to produce β-ketoalcohol

within 15–60 minutes reaction times. The reaction mixture was quenched with saturated

NH4Cl, and the crude oil was purified using flash column chromatography. The

diastereomeric mixture ratios were determined via reverse phase HPLC-MS. The data

indicate formation of two to four diastereomers in each reaction. In most cases, the major

diastereomer was successfully separated, and data analysis of the major diastereomers

showed formation of R,R and R,S configurations at the new stereogenic centres (C6 and

C1`) when enolate added to aliphatic aldehydes, while in the case of aromatic aldehydes,

O

OOH

H

Hi

i= LDA, THF, −78 °C, Acrolein, ii = LDA, THF, −78 °C, 5-Norbornene-2-carboxaldehyde

ii OOH

H97

H

H100

101

102

Yield 75%, one isomer

Yield 70%, d.r. 2:1


52

the stereochemistry would be speculated to be R,S and R,R. The table below represents the

crude yield and percentage of combined diastereomers, also diastereomer ratios as

determined from HPLC-MS and/or 1H NMR.

Table 2.3 Isolated yields and diastereomeric ratios of hydroxy alkylation of R-(–)-carvone 36 with a variety of aldehydes

Entry Aldehyde Crude Yield

%

Yield % Separation Diastereomeric Ratio*

1 Ethanal 98% 75% Yes 68:18:11:3

2 Propanal 98% 64% Yes 53:40:6:1

3 Isobutyraldehyde 89% 72% Yes 79:19:1:1

4 Cyclohexane-carboxyaldehyde

88% 61% Yes 60:34:6

5 Benzaldehyde 97% 67% Yes 69:31

6 p-Nitro benzaldehyde

94% 54% No 45:44:7:4

7 p-Methoxy benzaldehyde

94% 45% No 50:44:6

8 Naphthaldehyde 96% 67% Yes 63:34:2:1

9 2-Pyridine- carboxyaldehyde

96% 85% Yes 72:26:2

*Diastereomeric ratios were reported from HPLC (reverse-phase, 40% CH3CN:60% H2O, 0.5 mL/min., Inj. Volume 1 µl, Phenomenex Sphereclone 5u ODS(2), UV 220 nm.

As shown by the results above the stereoselectivity was observed to be high with

six aldehydes, namely: ethanal, isobutyraldehyde, benzaldehyde, naphthaldehyde,

cyclohexanecarboxyaldehyde and 2-pyridinecarboxyaldehyde. Scheme 2.22 shows all

possibilities of diastereomers of aldol adducts of 36. Two major isomers formed with same

stereochemistry at new stereocentre C6, and would be anti to isopropenyl substituted,

while, both isomers have different configuration at C1` with R or S configuration.

However, minor isomers are expected to have the R group at C6 syn to isopropenyl.

Scheme 2.22 Outcomes of aldol addition to 36

R

OH

R

OH

H

O OH

OH

major minor

HOH

R

OH

major

i, ii

i = LDA/THF, −10 °C, ii = RCOH, −78 °C

OH

R

OH

minor36


53

2.5.1.1 Aldol addition to R-(–)-carvone using aliphatic aldehydes

Aldol reaction of (–)-carvone 36 with acetaldehyde afforded a yellow oil mixture of

four diastereomers (yield 75%), which was proved with LC-MS to have ratio 68:18:11:3.

Figure 2.11 Stereochemistry of (1`R) diastereomer 103, and its acetate 104

The major diastereomer (1`R) 103 and a mixture of three isomers were obtained via

flash column chromatography using hexane:EtOAc 20:1. IR Spectroscopy of 103

confirmed the presence of (OH) and (C=O) stretches at 3450 and 1655 cm-1 respectively.

Meanwhile, 1H and 13C NMR indicated that the product was a single diastereomer. For

instance, one doublet for (CH3CHOH) was present at δ 1.19 ppm, whereas as three

doublets identified for the same group at δ 1.13, 1.22 and 1.35 ppm were detected in the

crude of diastereomers mixture. In addition, the presence of one signal as qd (J = 6.7, 3.8

Hz) for (CHOH) at δ 3.70 ppm indicated a pure diastereomer. Assignment of proton NMR

of 103 shows anti stereo-correlation between H-5 and H-6 with large J value 12.8 Hz.

Meanwhile, the correlation of stereochemistry of H-6 and H-1` would be expected with

small J value (3.8 Hz), in gauche conformation. The 13C NMR spectrum of 103 showed 12

signals with C=O at δ 203.2 ppm and δ 67.6 ppm for CHOH differing for four signals for

each C=O and CHOH in the diastereomers mixture.

The crude product and separated major diastereomer 103 were oils and attempts to

crystallize them using cold hexane and also, hexane-Et2O at room temperature were

unsuccessful. So conversion of the ketol to corresponding acetate 104 was carried out, and

it was also an oil. The most important signal in the 1H NMR spectrum of the 104 was

CHOAc at δ 6.00 (d, J = 3.5 Hz). Crystallising the acetate at −10 °C was ultimately

successful using hexane-Et2O, but the crystals were not good enough quality to obtain X-

ray data.

Addition of propanal to the kinetic enolate of (–)-carvone resulted in four

diastereomers (HPLC-MS showed four peaks for diastereomers in ratio 53:40:6:1 Figure

2.12).

103 104

(R)(R)(R)(R)

(Z) O

(R)(R)HO HH

H

(R)(R)(R)(R)

(Z) O

(R)(R)HO C

H

H O

CH31

23

4

5

6

7

89 10

12

1 `


54

Figure 2.12 HPLC Chromatogram of diastereomeric mixture of aldol adducts 105

Further separation of the diastereomeric mixture was attempted using normal phase

HPLC, and two separable isomers were collected, and confirmed by 1H and 13C NMR. For

example, the proton NMR spectrum of (1`R) isomer 105 indicated overlapping of H-5 peak

with OH at δ 2.50−2.42 ppm, and ddd for H-6 at 3.01 ppm with coupling constants of 11.7,

10.0 and 5.1 Hz. Moreover, multiplet signal for the proton at the carbon bearing OH was

appeared at δ 3.61−3.56 ppm. The second major diastereomer (1`S) proton NMR spectrum

revealed presence of a CHOH signal as a td at δ 3.41 ppm (J = 9.6, 3.6 Hz). Meanwhile,

H-6 appeared as a ddd at δ 2.74 ppm with J = 13.0, 3.7, 1.2 Hz, and H-5 was identified at δ

2.68 ppm (ddd, J = 13.3, 9.8, 3.9 Hz).

The reaction of (–)-carvone enolate with isobutyraldehyde provided a good yield of

aldol product (72%), and four diastereomers (79:19:1:1), confirmed by LC-MS. Purification

and separation of the crude oil was successively achieved using hexane:EtOAc (10:1) to

afford the two main diastereomers (1`R) 105 and (1`S) 106. Both isomers were

characterised by 1H and 13C NMR, IR and mass spectroscopies.

Figure 2.13 Configurations of aldol adducts 105, 106, 107 and 108

The proton NMR of 107 indicated overlapping of CHOH and OH as a single peak

at δ 3.56 ppm. Using D2O, clear signals were obtained, most specifically, a doublet of

doublets (J = 5.8, 4.1 Hz) for CHOH at δ 3.55 ppm, and a doublet of triplets for H-5 at δ

2.79 ppm changed to a ddd (J = 9.5, 8.6, 5.9 Hz). Additionally, the dd (J = 9.7, 5.8 Hz) for

H-6 appeared at 2.50 ppm, which is a crucial peak to determine the stereochemistry of this

sample. It is clear from Figure 2.13 that S configuration was formed at C6.

(R)(R)(R)(R)

(Z) OH

(R)(R)H

O HH

(R)(R)(R)(R)

(Z) O

(S)(S) HO HH

H

(R)(R)(R)(R)

(Z) O

(S)(S) HO HH

H(R)(R)

(R)(R)

(Z) O

(R)(R) HO HH

HJ =5.8 Hz

105 108107106

J = 9.5–9.7 Hz


55

The 1H NMR of 108 showed a triplet signal for CHOH at δ 3.17 (J = 8.1 Hz) and

dd (J = 11.4, 2.4 Hz) peak for H-6 at δ 2.60 ppm. Also, H-5 was characterized as a

multiplet at δ 2.40−2.35 ppm. However, the sample was resolved by D2O, and the triplet

peak for CHOH converted to a double doublet (J = 8.7, 2.0 Hz). Aldol addition of R-(–)-carvone enolate to 2-cyclohexanecarboxyaldehyde gave

three diastereomers, which was then were proved via reverse-phase LC-MS analysis to

have been formed in a ratio of 60:34:6. The mixture of isomers was readily separated via

flash column chromatography using hexane:EtOAc (10:1) as eluent.

The major diastereomer (1`R) 109 was obtained as a white crystals, and was

successfully recrystallized using hexane. Proton NMR showed H-6 appeared as a dd at δ

2.70 ppm with coupling between H-5, H-6 (J = 9.9 Hz), and from H-6 and H-1` (J = 5.5

Hz). This coupling constant J is consistent with conformer C, where the conformer B

requires higher J value than (5.5 Hz) for the anti-periplanar alignment of the coupling

protons at C6 and C1`. Conformer A is not possible because the X-ray structure showed no

intramolecular hydrogen bond had formed between OH and C=O.

Figure 2.14 Conformers of R configuration at C6 and C1` of aldol adduct

The stereostructure of 109 was established by X-ray crystallography, and proved to

be anti orientation between protons of C5 and C6 (R), and also to be (R) at C1` stereocentre.

Figure 2.15 X-ray crystal structure of 109, and configurations of 109, 110 and the 4-

nitrobenzoate ester 111

The β-hydroxyketone of 109 was esterified using 4-nitrobenzoate, and the reaction

completed within 1 h to afford pure ester 111 as crystals. The analysis of 1H NMR showed

a doublet of doublets (J = 8.0, 4.1 Hz) at δ 5.26 ppm for H-1`, and also, dd at δ 2.92 ppm

for H-6 with J value (8.8, 4.1 Hz) arising from coupling between H-6, H-5 and H-6, H-1`.

HaHbCy

OHO

HaCyHO

HbO

HaOHHb

CyO

A B C

1 `

(Z) O

(R)(R)H

OH

H O

(Z) OH

(S)(S)H

OH

H

NO2

(R)(R)

(Z) OHa

H

OHb

H

109 111 110

J = 5.5 Hz

109

65


56

The major diastereomer 110 was separated (Rf 0.36 in 4:1 hex:EtOAc), and the 1H

NMR spectrum indicated a triplet signal for CHOH proton at δ 3.24 ppm with J coupling

8.6 Hz, additionally the doublet peak (J = 10.1 Hz) at δ 2.21 ppm for OH. Confirmation of

R stereochemistry at C6 was proved via the ddd (J = 11.8, 10.3, 5.1 Hz) at δ 3.05 ppm for

H-5, and H-6 was assigned as a doublet of doublets (J = 11.9, 1.9 Hz) at δ 2.64 ppm.

2.5.1.2 Aldol addition to R-(–)-carvone using aromatic aldehydes

Reaction of benzaldehyde with carvone enolate led to formation of two

diastereomers as indicated by NMR and confirmed by HPLC-MS. The latter showed two

peaks integrating 69:31. The most important peaks characterised in the 1H NMR of the

crude were two doublet peaks for CHOH at δ 4.84 and 5.17 ppm, respectively. 13C NMR

indicated two peaks for C=O group at δ 204 and 203 ppm of 112 and 113, and two signals

for the CHOH at δ 77.4 and 77.1 ppm were also assigned. Separation of diastereomers was

successful using flash column chromatography (20:1−10:1 hex:EtOAc) providing the two

separate isomers.

The 1H NMR spectrum of the isolated major diastereomer 112 showed a double

doublet at δ 4.84 ppm for the CHOH (J = 9.5, 4.8 Hz). Also, a doublet signal for OH was

shown at δ 5.11 ppm with J = 9.5 Hz from coupling OH, H-1`. The doublet of doublets

signal at δ 3.02 ppm (J = 11.2, 4.8 Hz) was assigned as the H-6 with coupling constant

arising from H-6, H-5 and H-6, H-1` correlations respectively. It assumed that the

stereochemistry at C6 to be R, and the configuration at C1` would be S.

The 1H NMR spectrum of the major diastereomer 113 clearly showed a doublet of

doublets signal at δ 2.90 ppm (J = 8.3, 2.4 Hz) for the H-6 from coupling H-6, H-5 and H-

6, H-1` respectively. It was thus assigned that the configuration is R at C6. The 13C NMR

spectrum showed 15 signals, as two groups of symmetrical carbon atoms in aromatic

system exist.

Unfortunately, both isomers were oils, and attempting to grow crystals failed at

room and low temperature. The diastereomeric mixture was converted to the corresponding

acetates (acetic anhydride, pyridine) and highly viscous pale oil was afforded. Precipitation

of the acetate was attempted by addition of hexane. However, this process also failed.


57

Figure 2.16 Stereochemistry of 112 and 113 and their esters 114 and 115

The 1H NMR of the isomeric mixture revealed overlapping signals for both 114 and

115 diastereomers, and two doublet peaks for (CHOAc) at δ 6.30 ppm (J = 8.8 Hz) of the

114 and δ 6.50 ppm (J = 6.8 Hz) of the 115 were observed. Moreover, the doublet of

doublets for H-6 was assigned at δ 2.97 (J = 8.7, 4.3 Hz) for 114, and δ 2.92 ppm (J = 6.9,

2.5 Hz) of 115. The value of the J coupling (6.9 Hz) could result from an equilibrium

between the conformers.

Aldol addition to p-nitrobenzaldehyde afforded a diastereomeric mixture, and NMR

spectrum showed the evidence of this, which was supported by HPLC–MS (ratio

45:44:7:4).

Figure 2.17 HPLC-MS chromatogram of diastereomeric mixture of aldol adducts of 116

and 117

The 1H NMR spectrum of the diastereomeric mixture of 116 showed multiplet

peaks at δ 3.09−2.98 and 2.55−2.51 ppm for H-6 and H-5, respectively. The spectrum of

the second diastereomer 117 revealed a doublet of doublets at 3.01 ppm (J = 12.1, 4.4 Hz)

at H-6 and multiplet signal for H-5 at δ 2.62−2.58 ppm.

(R)(R)(R)(R)

(Z) O

(R)(R) HO HH

H(R)(R)(R)(R)

(Z) O

(S)(S) HOH

H

H(R)(R)(R)(R)

(Z) O

(S)(S) HOH

H O

(R)(R)(R)(R)

(Z) O

(R)(R)HOH

H O

112 113114 115

1 `65


58

Scheme 2.23 Possible structures of 116 and 117 diastereomers and their acetates 118 and

119

The mixture of isomers was a sticky oil and attempting to crystallise this was

unsuccessful. In order to obtain crystals, the diastereomeric mixture was converted to the

corresponding acetates; however, the isomeric acetates were also oils and proved difficult

to crystallise. Further confirmation of structures was provided by conversion of the aldol

product to corresponding acetates. Proton NMR of the acetates exhibited two separated

doublets of (CHOAc) at δ 6.30 (J = 6.0 Hz) and 6.12 ppm (J = 4.6 Hz) of 118 and 119

respectively.

Addition of p-methoxybenzaldehyde to the enolate of (–)-carvone generated a

diastereomeric mixture, and LC-MS analysis revealed three diastereomers were produced

with ratios 50:44:6. Inspection of 1H NMR spectrum of the isomeric mixture showed a

doublet signal for CHOH at δ 4.76 ppm (coupling constant 4.9 Hz) of major isomer (1`S)

120, while, the same proton for 121 was overlapped with an olefinic proton at 4.79−4.78

ppm. The peak of H-6 was clearly characterised as a dd at δ 3.00 ppm (J = 11.4, 4.9 Hz)

for 120, and it indicated at 2.88 ppm as a dd peak for isomer 121 with J = 7.4, 5.8 Hz.

Meanwhile, the ddd signal for H-5 appeared at 2.54 ppm (J = 11.4, 9.5, 5.6 Hz) for 120,

and quartet peak at 2.65 ppm with J = 6.4 Hz for 121. It was challenging to hypothesise the

stereochemistry at C6. Although, there is a possibility of stereostructure of 121 to be S at

C6 stereocentre, this is unlikely (ratio (50 + 44 = 94):6 of R:S). Also, precedent examples

of numerous carvone, dihydrocarvone and isomenthone aldol derivatives from this and

previous work of our group53 revealed formation of anti stereostructure at C6. Therefore, it

expected that the stereochemistry of 121 will be anti at the new stereogenic C6, and would

be R configuration at C1`.

(R)(R)(R)(R)

(Z) O

(R)(R)H

O HH

H

(R)(R)(R)(R)

(Z) O

(S)(S)H

O HH

H

NO2 NO2

(R)(R)(R)(R)

(Z) O

(R)(R)H

OH

H O(R)(R)

(R)(R)

(Z) O

(S)(S)H

OH

H O

NO2NO2116 118117 119

(R)(R)

(Z) O

36

i,ii iii

i = LDA/THF, −10 °C, ii = 4-nitrobenzaldehyde, −78 °C, iii = acetic anhydride, pyridine, 50 °C


59

Figure 2.18 The structures of 120, 121, 122 and 123, and X-ray crystallography of 123

Addition of naphthaldehyde afforded a diastereomeric mixture in a ratio 63:34:2:1.

The proton NMR of the crude oil showed a mixture of two major diastereomers. Flash

chromatography using hexane:EtOAc 10:1 was used for separation of the isomers. The 1H

NMR spectrum of 122 showed a dd peak at δ 3.25 ppm (J = 7.0, 6.0 Hz) for H-6, and an

apparent quartet signal at δ 2.64 ppm with J = 6.3 Hz for H-5. Furthermore, the CHOH

peak appeared as a doublet (J = 5.6 Hz) at 5.56 ppm from coupling of H-1` and OH. A

broad signal at δ 3.35 ppm was indicated for the OH. The 13C NMR spectrum resolves all

21 signals for the 21 carbon atoms. Key indicative peaks include the C=O peak at δ 201.7

ppm, the CHOH at δ 70.9 ppm, as well as two olefinic CH3 groups at δ 20.9 and 16 ppm.

The diastereomer was also characterised by HRMS and IR spectroscopies and the latter

revealed O-H and C=O peaks at 3382 and 1642 cm-1 respectively. Its difficult to provide

absolute proof of the stereostructure of this isomer based on 1H NMR because there is a

chance the structure of 122 is S at C6, and thus could in theory be B or C (Figure 2.19).

Figure 2.19 The potential stereostructures of 122

However, such a stereochemical outcome for the aldol affording 122 as B or C

would be very uncommon. The 2D-NOESY NMR spectrum revealed no correlation

between H-1` and protons of olefinic CH2 at C8. Moreover, the crystal structures (X-ray)

of precedent examples of numerous carvone, dihydrocarvone and isomenthone aldol

derivatives from this and previous work of our group,53 all confirmed anti stereo structure

at C6. Therefore, it proposed with some confidence that the stereochemistry of 122 is anti

at the new stereogenic C6, and would be S configuration at C1`.

1 `

6

5

(R)(R)(R)(R)

(Z) O

(R)(R)HO HH

H(R)(R)

(R)(R)

(Z) O

(S)(S)H

O

OMe

H

120 121 122 123

(R)(R)(R)(R)

(Z) O

(S)(S)HO HH

H

(R)(R)(R)(R)

(Z) O

(R)(R)H

OH

OMe

H HH

(Z)(R)(R)

(R)(R)(S)(S)O

HHO

(Z)(R)(R)

(S)(S)

(S)(S)

O HOH

(Z)(R)(R)

(S)(S)

(R)(R)

O OHH

A B C

1`

H-8a,b


60

The 1H NMR spectrum of the second diastereomer (1`R) as indicated by X-ray

structure) 123 showed a doublet peak for CHOH at δ 6.11 ppm with J coupling equal to

(3.3 Hz), and dd (J = 8.8, 3.5 Hz) at δ 3.08 ppm for H-6. The X-ray crystallography studies

of the substrate revealed R,R stereochemistry at new stereocentres C6 and C1`.

As with the aldol addition to naphthaldehyde, the electrophile (2-pyridinecar-

boxaldehyde) was reacted with carvone enolate, and 1H NMR of the crude oil showed two

diastereomers had been formed an a 72:26:2 ratio. The crude oil was purified and the two

diastereomers were separated using hexane:EtOAc 5:1. The first isomer was a highly

viscous oil 124, and the second isomer 125 was obtained as a white crystals, and high

quality crystals were obtained after the recrystallization using hexane at room temperature.

The 1H NMR of major diastereomer (1`S) 124 was studied via proton NMR, which

exhibited a doublet peak (J = 3.6 Hz) at δ 5.16 ppm for the CHOH (C1`), meanwhile the

H-6 appeared at δ 2.94 ppm as a doublet of doublets (J = 9.6, 4.0 Hz), additionally a

multiplet signal for H-5 was observed at 3.12−3.06 ppm.

Similarly to the (1`S) isomer, (1`R) diastereomer 125 revealed a doublet peak for H-1`

at δ 4.76 ppm with J value 2.0 Hz. The most important peaks were a doublet of doublets (J

= 12.4, 2.2 Hz) at δ 3.53 ppm for H-6, and a doublet of doublet of doublets (J = 12.4, 10.6,

5.0 Hz) at δ 3.15 ppm of H-5.

Figure 2.20 Stereochemistry of both isomers (124 and 125) and X-ray crystal structure of

125

The R,R configuration of 125 was confirmed using X-ray crystallographic studies, which

proved the anticipated α,α’-geometry about the C=O and established the stereochemistry at the β-

stereocentre. The carbon bearing the hydroxyl group has R configuration, expected from Re face

addition to 2-pyridinecarboxaldehyde.

2.5.1.3 Conclusions

Aldol addition of R-(–)-carvone 36 to various aldehydes was carried out, and the

stereochemistry of the products proved using X-ray crystallographic and NMR spectra. It

established the stereostructure of the major diastereomer 109 of the cyclohexanecarbox-

yaldehyde, and revealed anti (R) conformation at C6 stereocentre, as well as determining

(R)(R)

(Z) O

HO HH

HN

125

(R)(R)

(Z) O

HH

HN

O H

124 125


61

the stereochemistry at the new stereogenic centre C1` as R, arising from Si face addition to

aldehyde of cis fused decalin. Similarly, the 125 isomer of pyridinecarboxyaldehyde has R

stereochemistry at C6, and R at C1àrising from Si face addition to the aldehyde. An X-ray

crystal structure of the diastereomer 123 of the naphthaldehyde derivative showed R,R as

the new stereocentres C6 and C1` arising from Si face addition to naphthaldehyde of trans

fused decalin.

In general, it is assumed that addition of lithium enolates of R-carvone to aldehydes

gave two stereostructures (R,R and R,S) in case of aliphatic aldehydes, or (R,S and R,R) in

case of aromatic aldehydes, at new stereocentres (C6 and C1`). Both isomers (1`R and 1`S)

predicted to have arisen from Si face addition of the enolate to the aldehyde of trans fused

decalin for 1`R and cis fused decalin for 1`S.

2.5.1.4 Zimmerman−Traxler transition states for R-(–)-carvone aldol addition reaction

It is hypothesised that Z (cis) and E (trans) enolates formed from deprotonation of

the α-position of carbonyl compounds, and the geometry of coordinating the enolate with a

metal ion, primarily determines the stereochemistry of these aldol addition outcomes.

Although several types of transition state models have been reported, the six-membered

ring Zimmerman−Traxler model has gained most attention to explain the stereochemistry

of aldol addition adducts, in which the two oxygen atoms of the enolate and electrophile

(aldehyde) coordinate with the metal (Li) ion.63

Explanations of aldol addition adducts of R-(–)-carvone 36 according to

Zimmerman−Traxler would be more complex than cyclohexanone chair conformation due

to the endo double bond causing a half chair conformation.

In the cis fused decalin-like isomer (blue), although Req is eclipsed with a proton in

the carvone ring, Si face enolate attack would be more preferred than Re face, because of

steric interactions between Rax and the Me substituent. In the trans isomer (red), Si face

addition is anticipated to be not favored due to eclipsing (pseudo-axial) Req and H in the

carvone ring, while Re face addition to aldehyde would be more unfavorable because of 1,3

diaxial steric interaction of Rax and the ring proton.


62

Figure 2.21 Zimmerman−Traxler transition state of cis (blue) and trans fused decalin (red)

As shown in the above results, aldol addition of 36 showed high selectivity at Si

face, and it might be Zimmerman−Traxler transition state comprises A and C.

2.5.2 Aldol addition to (+)-isomenthone

These are novel materials with the exception of the ethanol adduct (prepared in a

previous study in our group).57 These β-hydroxyketones were prepared by aldol reactions

of the relevant aldehyde, which was added in one portion to the kinetic enolate of (+)-

isomenthone 38 at −78 °C. All reactions were quenched by addition of NH4Cl at the same

temperature. All reactions yielded what were assumed to be a mixture of diastereomers as

crude oils, which were purified using flash column chromatography to give separated

diastereomers or in some case, isomeric mixtures. The diastereomer ratios were determined

via reverse phase HPLC-MS or 1H NMR, and the results showed high stereoselectivity in

formation of the aldol products using this low temperature reaction and quenching. The β-

ketol products were revealed to be epimeric at the carbon bearing the hydroxyl group only,

to afford R or S configurations at new stereocentre (C1`), arising from Si and Re additions

of isomenthone enolate to the aldehydes.

Scheme 2.24 Addition of aldehydes to (+)-isomenthone 38 in the presence of LDA

All diastereomers were characterised using NMR, IR and HRMS spectroscopies.

The stereochemistry of the isomers was confirmed using 1HNMR and X-ray crystallography;

for example, J coupling constant determined the correlation between two neighbouring

protons on adjacent carbons at C2, C3, and C2, C1`.

O COLi

ReqH

O COLi

HRax

A Si B Re

O LiO

H

ReqO Li

O

H

Rax

D Re

cis–fused decalin trans–fused decalin

C Si

O OH

O

RO

RHO

ORHO

i ii

i = LDA, ii = RCHO

38


63

Figure 2.22 Possible conformations of aldol products of 38

However, It is clear that the data of NMR spectroscopy were insufficient to

determine the stereostructure of the aldol product, especially as the aldol product can adopt

different conformers (Fig. 2.22). Diastereomer formation was examined with 1H, 13C NMR

assisted by DEPT-135, 2D-COSY, 2D-HMQC and 2D-HMBC, as well by HRMS and IR.

Diastereoselectivity of the aldol reaction was investigated with reverse-phase HPLC and/or

proton NMR, and determined to be 30−80% depending on the electrophile, for example,

addition of (+)-isomenthone enolate to ethanal was confirmed to be 56% selective (Table

2.4).

Table 2.4 Yields and diastereomeric ratios of aldol addition to (+)-isomenthone Entry Aldehyde Yield% Si:Re 1 Ethanal 67% 78:22 2 Isovaleraldehyde 37% 65:35 3 2-Pyridinecarboxyaldehyde 55% 76:24 4 Salicylaldehyde 65% 66:34 5 Acetone 18−22% 99:1

2.5.2.1 Aldol addition to (+)-isomenthone with aliphatic aldehydes

Addition of acetaldehyde to 38 afforded two diastereomers, which were separated

with flash column chromatography. The 1H NMR spectrum of major diastereomer 126

showed a singlet signal at δ 2.60 ppm for (CHOH) and four multiplet signals of 8 protons

from δ 2.10 to 1.43 ppm were observed. Although the H-2 peak overlapped with other

signals, a crucial peak was assigned as a quartet of doublets for CHOH at δ 3.92 (J = 6.6,

3.0 Hz). The J value (6.6, 3.0 Hz) indicates that conformer 1 would be favourable (Figure

2.22). This conformer was suggested, and proved via single crystal X-ray in the previous

study.53,57 13C NMR data from current study was completely consistent with the published

data,53,57 and showed twelve signals for 12 carbon atoms, including C=O signal at δ 219

and CHOH at δ 66.1 ppm. The β-stereocentre of carbon bearing hydroxyl group is assigned

to be R configuration (anti) arising from Si face addition to the acetaldehyde.

1 `1 `

O

HH

RHO HO

H

H

HO RH

2

3

2 3 large Jsmall J

Conformer 1 Conformer 2


64

Scheme 2.25 Aldol addition of acetaldehyde to 38 under kinetic control

IR and 13C NMR spectra of 127 were similar to 126, however, 1H NMR spectrum

was slightly different and the signals were clearer than those of the major diastereomer.

The NMR of 127 showed a doublet of doublets signal for H-2 at δ 2.36 (J = 9.5, 5.5 Hz)

from coupling of C2, C3 and C2, C1` protons, additionally a quartet of doublets at δ 3.98

(J = 6.6, 5.5 Hz) of (CHOH). Compound 127 would appears to favour conformer 2

(Figure 2.22) due to high J value (9.5 Hz) from correlation between H-2 and H-3. The

coupling constant from CHOH and H-2 (5.5 Hz) is consistent with conformers A and B,

and there is probability to form an intramolecular hydrogen bond OH...C=O. However, it is

difficult to assume this due to the rotation of the carbon-carbon single bond (C1`).

However, conformer C should show higher coupling constant than 5.5 Hz for the anti-

periplanar arrangement of the coupling protons at C2 and C3. Compound 127 would be S

configuration at β-stereocentre of the carbon bearing the hydroxyl group arising from Re

(syn) face addition of enolate to acetaldehyde.

Figure 2.23 Conformers of S configuration (syn) at C2 and C1` of isomenthone aldol

adduct

Addition of isovaleraldehyde to the enolate of isomenthone generated an isomeric

mixture (diastereomeric ratio 65:35 of major isomer 128 and minor 129). Both isomers

were oils and adding cold hexane lead to growth of crystals of 128, which proved to be

unstable at room temperature and rapidly melted. 1H NMR spectrum of 128 indicated a

doublet of doublet of doublet peak at δ 1.19 (J = 13.5, 8.6, 4.7 Hz), and this signal was not

identified, even using 2D-NMR (COSY and HMQC), because it correlated with multiple

peaks of CH and CH2 protons. The most useful peaks were a doublet of doublet of doublets

at δ 3.79 (J = 9.1, 4.4, 2.0 Hz) for CHOH proton, and a singlet at δ 2.84 ppm for OH

1 `1 Ò

HH

(R)(R)HO H dq, J = 6.6, 3.0 Hz

O

H

H

(S)(S)HO

H

2 3

J = 5.5 Hz

J = 9.5 Hz

126 127

2

3

O

i,ii

i = LDA, ii = acetaldehyde/THF, −78 °C

38

HH

OHO

HHHO

O

HOH

HO

small J large Jsmall J

A B C


65

proton. The configuration of the substrate was established with X-ray crystallography

studies, and proved as expected to be α,α’ geometry about the C=O bond, the stereochemistry

of β-stereocentre of carbon bearing hydroxyl group is R configuration (anti) arising from Si

face addition to the isovaleraldehyde.

Figure 2.24 X-ray crystal structure of 128, and configurations of 128 and 129

The 1H NMR of 129 exhibited a multiplet signal for CHOH at δ 3.91−3.82 ppm and

a single peak for OH at δ 2.62 ppm. Also, a doublet of doublets (J = 9.2, 5.6 Hz) at δ 2.36

ppm for H-2. This isomer was characterised by IR spectroscopy and the spectrum showed

a broad peak at 3430 cm-1 for O-H absorbance and peaks at 2954, 2929 and 2869 cm-1 for

aliphatic methyl groups, as well as a carbonyl peak at 1691 cm-1.

2.5.2.2 Aldol addition to (+)-isomenthone with aromatic aldehydes

The crude oil of the diastereomeric mixture (76:24) resulted from addition of 2-

pyridylcarboxyaldehyde to lithium enolate of isomenthone was partially separated (by

using 5:1 hexane:Et2O chromatography) to yield one separated isomer and the rest was a

diastereomeric mixture. Major isomer 130 was obtained as white crystals, and assigned as

a pure diastereomer. 1H NMR spectrum showed H-2 appeared as a doublet of doublets at δ

2.72 ppm with J coupling 7.4, 4.1 Hz from coupling of H-2, H-3 and H-2, H-1`.

Additionally the OH signal was a doublet peak (J = 9.5 Hz) at 4.10 ppm from coupling to

CHOH. Also, H-1` showed as a single peak at δ 5.25 ppm. However, addition of one drop

of D2O converted it to a doublet peak (J = 4.0 Hz).

The 1H NMR spectrum of minor diastereomer 131 clearly showed three crucial

peaks, firstly, a doublet of doublets signal of H-2 at δ 3.27 ppm with J value 10.8, 2.8 Hz

from correlation of C2, C3 and C2, C1` protons. The second peak was also a doublet of

doublets (J = 10.8, 2.4 Hz) at δ 4.91 ppm of CHOH from coupling of H-1`, OH and H-1`,

H-2. Finally, the OH peak was a doublet (10.8 Hz) at δ 4.07 ppm.

1 `1 `

O

HH

HO HO

H

H

HOH

2 3

dd, J = 9.2, 5.6 Hz128 129

2

3


66

Figure 2.25 Configurations and conformations of 130 and 131 isomers and X-ray crystal

structure of 130 aldol adduct

X-ray analysis of the selected crystal showed a structure consistent with major

adduct 130, and as anticipated to be α,α’ trans geometry, and confirmed the configuration

to be S at C1` stereocentre. The stereochemistry of 130 and 131 was concluded from the

above information, and the major isomer is S configuration at new stereogenic centre of

carbon bearing hydroxyl group, while 131 has R configuration.

Salicylaldehyde was added to the lithium enolate of isomenthone, and formed crude

of diastereomeric mixture (66:34 132:133). Attempting to purify it using flash column

chromatography (10:1 hexane:Et2O) was not successful. The next step was to add cold

hexane and keep the solution at −10 °C for multiple days.

Scheme 2.26 Aldol addtion of salicylaldehde to 38

Crystals of only one diastereomer would be predicated to grow; however, 1H NMR

of the filtered crystals revealed a mixture of two diastereomers was formed, and also

showed a triplet (J = 7.0 Hz) for CHOH of major isomer 132 at δ 5.10 ppm, and a doublet

of doublets (J = 7.0, 3.9 Hz) of minor isomer 133 at δ 4.77 ppm. Meanwhile, other peaks

were identified, for example, H-2 was a doublet of doublets at δ 2.83 ppm (J = 7.2, 6.7 Hz)

for 132, and δ 3.05 ppm (J = 7.6, 3.8 Hz) for 133 (Scheme 2.26). It is clear that the

correlation between H-2 and H-3 of both diastereomers could refer to an equilibrium

between conformer 1 and 2 (Figure 2.22), whilst the J coupling (7.2 Hz) between H-2 and

H-1` would represent equilibrium between conformers (Figure2.26).

O

H

H

HOH

NO

HH

HO HN

J = 9.5 Hz

J = 7.4 HzJ = 4.1 Hz

J = 10.8Hz

J = 10.8 Hz

J = 2.8 Hz

130 131

1 Ò

HH

(S)(S)

HO HJ = 7.0 Hz

J = 6.7 Hz

J = 7.2 HzOH O

HH

(R)(R)

HO HJ = 7.0 Hz

J = 7.6 Hz

J = 3.8 HzOH2

3

132133

O

38

i,ii

i = LDA/THF, −10 °C, ii = Salicylaldehyde,−78 °C


67

Figure 2.26 Conformers of R (anti) of aldol adduct between C2 and C1` protons

The 13C NMR was complicated in the aromatic area, but showed two carbonyl

groups at δ 218.5 and 220.9 ppm, also, two signals of CHOH at δ 74 and 64.5 ppm.

It assumed that S configuration at the new stereogenic of CHOH arising from Si

face addition to the salicylaldehyde would be the predominant diastereomer 132, while, the

minor isomer would be R configuration at β-stereocentre arising from Re face addition to

the aldehyde.

2.5.2.3 Aldol addition to (+)-isomenthone using acetone

Aldol reaction of (+)-isomenthone 38 with dry acetone was carried out firstly

without catalyst, and no product was observed. Addition of Lewis acid (dried ZnCl2)

afforded just 5% of product as a diastereomeric mixture (10:1). In order to improve the

yield anhydrous CeCl3 was used in this reaction, and following the literature an equimolar

amount of CeCl3 and lithium enolate were used.74 It was rationalised that CeCl3 generates

cerium enolate as intermediate. The yield of product was slightly raised (from 5% to 11%).

The final attempt was generating silylenol ether from isomenthone via LDA and

chlorotrimethylsilane (TMSCl) in acetone-dry ice bath, followed by addition of acetone

and TiCl4, which gave 18% of the β-ketalcohol.64

The 1H NMR spectrum showed a diastereomeric mixture (10:1) of 134 and 135,

with five signals for five methyl groups (three signals were doublets and other two were

singlet peaks). Furthermore, the OH proton was positioned at δ 2.70 ppm as a singlet, and a

doublet peak for H-2 appeared at δ 2.48 ppm with J = 3.6 Hz. 13C NMR showed 12 peaks

of 13 carbon atoms, for examples, C=O signal was at δ 218.4 ppm, and CHOH was at δ

71.6 ppm. IR spectroscopy showed O-H and C=O at 3452 and 1647 cm-1 respectively.

H

HO H

small J large Jsmall J

A B C

OOH

HH

OOHH

HO


68

Scheme 2.27 Aldol addition of acetone to 38

2.5.2.4 Conclusions

Aldol addition of the kinetic enolate of (+)-isomenthone 38 to aldehyde, such as

ethanal, isovaleraldehyde, salicylaldehyde and pyridinecarboxyaldehyde gave single

substituents at C2, with two diastereomeric outcomes from Si and Re face addition to the

aldehyde. X-ray crystal structures of 128, 130 as well as 126 (in previous study)57

confirmed that the predominant adduct is generated from Si addition to furnish R

conformation at β-stereogenic in case of aliphatic aldehydes and S with aromatic aldehyde.

However, the reaction afforded two isomers at C2 substituents with high diastereoselectivity

when (+)-isomenthone enolate was added to the acetone in the presence of Lewis acid, for

example TiCl4.

2.5.2.5 Zimmerman−Traxler transition states for (+)-isomenthone aldol addition reaction

There are two possibilities for the lithium enolate to attack the carbonyl of the

aldehyde, from Si or Re face. The former would be more expected due to less steric

interactions of the aldehyde alkyl/aryl and substituted methyls at the isomenthone ring,

whilst the latter would cause unfavorable steric hindrance resulting from steric interactions

with the aldehyde group and alkyl substituents at the ring.

There are two fused decalin types of Zimmerman−Traxler transition state, cis and

trans. In the cis form, attack of the aldehyde from Si face seems to be reasonable and

possible, since the R group of the aldehyde would be in an equatorial-like position with

little interaction with the ring. But Re face addition would undergo steric interaction,

whereas, R is positioned in an axial-like position, and interaction with H axial is placed at

isopropyl substituents.

In the alternative trans enolate Zimmerman−Traxler transition state form, it is

assumed that Si face addition would be unfavorable due to eclipsing of an equatorial R

aldehyde with methyl substituent of the isomenthone ring. However, Re face addition

seems to be more unfavorable because of 1,3 diaxial steric interaction of R aldehyde and

isopropyl group of the ring.

O(R)(R)

O

O

O

OH

i iia b

i = LDA/ THF, −78 °C, ii = Acetone, ZnCl2 or CeCl3.a = LDA, TMSCl/ THF, −78 °C, b = Acetone, TiCl4

O OH

38134 135


69

Figure 2.27 Zimmerman−Traxler transition state of (+)-isomenthone aldol adducts cis

(blue) and trans fused (red)

Addition of acetone to the silylenolether of (+)-isomenthone should be catalysed by

Lewis acid, since the silylenolether is inactive, and the mechanism involves binding Lewis

acid with the oxygen of C=O in acetone. The positive charge on the titanium complex

makes acetone activated to be attacked by the silylenolether, then SiMe3 would be removed

by Cl−, and trapped with TiCl3-O complex, hydrolysis at work up would form the ketol.64

Scheme 2.28 Mechanism of Mukaiyama aldol reaction of 38, and proposed transition state

However, Mukaiyama proposed that the intermediate would be generated from

coordination of enol ether and acetone oxygen atoms with the metal.

2.5.3 Aldol addition to (1S,2S,5R)-(–)-isopinocamphone

Various novel substrates were synthesised from aldol addition of electrophiles to

the enolate of bicyclic monoterpenoids (–)-isopinocamphone 39, and the reaction mixture

was quenched at low temperature (−78 °C) with NH4Cl.

Scheme 2.29 Aldol addition to (–)-isopinocamphone 39


O

HH

LiHRax

O

O

HH

LiReqH

O

A Si B Re

OLi HO

Rax

OLi ReqO

H

A Si B Re

Me3SiO

HH

OTiCl3

O

HH

OTiCl3

Me3Si

Cl

O

HH

OTiCl3

ClMe3Si

ClTiCl4

O

HH

OSiMe3

O

HH

Cl3Ti OMe3Si

Transition state

1 ` ORHO

1

234

5 6

789

10H

ORHO

H

O39

i,ii

i = LDA, ii = RCHO/THF, −78 °C


70

The resulting β-ketoalcohol was purified with flash column chromatography using

hexane:Et2O, and isolated product was analysed with NMR, IR, HRMS, as well as reverse-

phase HPLC.

Table 2.5 Yields and diastereomeric ratios of aldol addition to (–)-isopinocamphone 39 Entry Aldehyde Yield% Si:Re* 1 Acetaldehyde 91% 90:10 2 Isobutyraldehyde 65% 98:2 3 Benzaldehyde 89% 98:2 4 2-Nitrobenzaldehyde 73% 63:37 5 Acetone 82% 99:1

*Diastereomeric ratios were reported from HPLC (reverse-phase, 60% CH3CN:40% H2O, 0.5 mL/min., Inj. Volume 5 µl, Kinetex 5u C18, UV 220 nm.

The data for the aldol product revealed high diastereoselectivity with respect to OH

to afford R or S configurations at new stereocentre (C1`), and complete control at

stereogenic centre C2 to be trans to the bridging carbon at C7.

2.5.3.1 Aldol addition to (–)-isopinocamphone with aliphatic aldehydes

Addition of ethanal afforded two diastereomers in the ratio 90:10 of 136:137,

which was shown using reverse-phase HPLC. Proton NMR spectrum of 136 showed

doublet of quartet of doublet signal at δ 3.99 ppm (J = 9.2, 6.2, 1.0 Hz) for CHOH, and the

9.2 Hz coupling represents correlation of H-1`, H-2 and shows an anti conformation. Also,

single peak for OH at δ 4.11 ppm, and a doublet signal at δ 2.40 ppm for H-2 with J

coupling constant 9.2 Hz. More information was acquired from the 13C NMR spectrum

which showed 12 non-equivalents peaks, the carbonyl signal appears at 221.2 ppm, the

CHOH peak at 67.9 ppm and four peaks at 27.7, 24.3, 22.3 and 17.7 ppm for four Me

groups.

The stereochemistry at the new stereogenic centre of carbon bearing hydroxyl

group would be R, with respect to stereocentre R configuration at C2. However, there was

no other supporting data, and unfortunately, no crystals were formed with crystallization

process with hexane or hexane/Et2O even at low temperatures.

Figure 2.28 Configurations of 136, 137, 138 and 139

1 ` 1 `J = 9.2 Hz

O (R)(R)

HOH

HO (S)(S)

HOH

H2

136 137J = 1.4 Hz

J = 9.6 HzO (R)(R)

HOH

HO (S)(S)

HOH

H2

138 139


71

The reaction of isopinocamphone enolate with isobutyraldehyde is highly selective,

and afforded a diastereomeric mixture with d.e. 96% of 138. The major diastereomer was

analysed with NMR, IR, MS and LC-MS. For example the 1H NMR spectrum showed a

triplet peak (J = 1.4 Hz) at δ 3.72 ppm for OH, and a doublet of triplet signal at 3.64 ppm

(J = 9.2, 2.0 Hz) for CHOH. In addition, a multiplet peak at δ 2.50−2.52 ppm for H-2 was

assigned. The stereostructure was predicted from J coupling constants, for instance the

correlation of protons at C1` and C2 was concluded from J = 9.2 Hz, where both adjacent

protons gave J = 9.2 Hz (Figure 2.28).

2.5.3.2 Aldol addition to (–)-isopinocamphone with aromatic aldehydes

Addition of benzaldehyde to the lithium enolate of isopinocamphone gave aldol

adducts in excellent yield of 89% and 96% d.e. 1H NMR of the major diastereomer 140

exhibited a doublet peak at 4.86 ppm for CHOH with J = 9.6 Hz, and a singlet single was

assigned for the OH at 4.65 ppm. The peak for H-2 appeared as a multiplet at 2.67−2.52

Hz. The stereostructure of this diastereomer expected to be S configuration of new stereogenic

centre of carbon bearing hydroxyl group, with respect to stereocentre R configuration at C2

Scheme 2.30 Aldol addition of benzaldehyde to 39

The diastereomer was also analysed with 13C NMR, which showed 15 signals for

17 carbon atoms. For instance, the C=O, CHOH and C2 were characterised at 220.4 ppm

73.9 and 58.4 ppm respectively.

Attempts to grow crystals from the diastereomeric mixture failed, and no crystals

were formed at ambient or low temperature. Thus, the yellow oil of diastereomeric mixture

was converted to the corresponding acetates, 4-nitrobenzoates, and trans-cinnamate.

1

234

56

789

10

O (R)(R)

HOH

HO (S)(S)

HOH

H

140 141

1 Ò

39

i = LDA, ii = Benzaldehyde/THF, −78 °C

i,ii


72

Scheme 2.31 Esterification of 140 and 141

Addition of acetic anhydride to the diastereomeric mixture of α-hydroxybicyclic

ketones afforded 78% yield of corresponding crystals acetates 142 and 141 within 2 h. 1H

NMR spectrum of acetates showed a doublet signal (J = 9.3 Hz) at 6.10 ppm for CHOAc,

and a multiplet peak for H-2 at 2.95−2.90 ppm. The stereostructure of 142 determined with

X-ray crystallography, and proved to be trans at stereogenic centre C2 to the bridge carbon

at C7, and to be S configurations at new stereocentre (C1`).

Figure 2.29 Conformation and X-ray crystal structure of 142

The p-nitrobenzoate ester was generated as a diastereomeric mixture (ratio 97:3) in

61%, yield as a white solid, but attempting to crash the product(s) out as crystals failed

even at low temperature. 1H NMR and 13C NMR supported acylation with multiplet peaks

for two phenyl rings, doublets for CHO-4NB at δ 6.30 ppm (J = 9.6 Hz) and the H-2 with

J = 9.5 Hz.

O OH

O OO

OO O

OO

NO2O

i ii iii

i = Ac2O, pyridine/DCM, 40 °C, ii = 4-nitrobenzyl choride, pyridine/DCM, 40 °Ciii =trans-cinnamic acid, DCC,DMAP, DCM, reflux, overnight

142 and143144 and 145

146 and 147

140 and 141

(S)(S)

(S)(S) (R)(R)

(R)(R)

O (S)(S)

OH

H

O


73

The diastereomeric mixture of β-ketol 140 and 141 was treated with trans-cinnamic

acid in presence of coupling reagent dicyclohexylcarbodiimide (DCC) and catalytic 4-

dimethylaminopyridine (DMAP) to give 146 and 147 in 75% yield as a white solid with a

98:2 (d.r.). The 1H NMR showed two doublet peaks for the double bond protons, with the

same J value 16.0 Hz, at 7.70 and δ 6.52 ppm of CH=CHCAr and CH=CHCAr respectively.

Signals for CHO and H-6 were assigned at δ 6.25 ppm (J = 8.8 Hz) and 3.06 ppm (J = 8.8

Hz). The white solid was recrystallized with hexane, petroleum ether, hexane:Et2O and

hexane:EtOAc at room temperature, but the crystals were not suitable to obtain an X-ray

structure.

The isopinocamphone enolate was also reacted with 2-nitrobenzaldehyde to afford

a diastereomeric mixture of 63:37 and yield 73%. Separation of the isomers was achieved

using flash column chromatography using hexane:Et2O to give crystals of major 148 and

minor 149 isomers. X-ray crystallography of 148 diastereomer was established the

stereostructure (Figure 2.30). 1H NMR of 148 was also analysed with, the spectrum

showed doublet peak for CHOH and at 5.54 ppm (J = 9.2 Hz) and single peak for OH at

4.61 ppm.

Figure 2.30 X-ray crystal structure of 148, and conformations of 148 and 149

Diastereomer 149 was also yellowish-brown crystals, but unfortunately, these were

found to not be suitable for X-ray crystallography. The 1H NMR spectrum showed two

doublet peaks at 5.40 ppm (J = 9.2 Hz) for CHOH and 5.20 ppm (J = 1.0 Hz) for OH.

Also, the H-2 peak was confirmed at 2.81 ppm (d, J = 9.1 Hz).

2.5.3.3 Aldol reaction to (–)-isopinocamphone with acetone

Synthesis of the tertiary alcohol via addition of acetone to the lithium enolate gave

a low yield, thus the reaction was carried out using the silylenolether. Addition of dry

acetone to the trimethylsilyl ether was catalysed with TiCl4 at −78 °C according to Mukaiyama

method.64 The reaction was regioselective, and smoothly proceeded to yield 82% of

isopinocamphone tertiary alcohol adduct as a colourless oil, but this was not crystallisable.

1 `

1

24

5 6

789

10

O (R)(R)

HOH

HO (S)(S)

HOH

H

148 149

NO2NO23

148


74

1H NMR spectrum of the tertiary alcohol adduct 151 showed single peaks for OH at

4.57 ppm and H-2 at 2.70 ppm. Proton signals of five methyl groups also being key. A by-

product 153 resulting from deprotonating H-2 (CHCCHCH2) was also isolated from the

reaction mixture with only 5% yield but as a single isomer, where the Me substituent

occupied the equatorial position, and the new C-C was generated axial. The proton NMR

spectrum of 153 showed overlapping of the OH peak with two protons of C4, which

appeared as a multiplet at δ 2.64−2.57 ppm, with disappearance of the C2 proton. The 13C

NMR spectrum revealed three tertiary carbon atoms at 57.5, 49.2 and 41.6 ppm for C-OH,

C2 and CH3CCH3 respectively. Furthermore, two CH2 were indicated at 46.1 for C4 and

28.0 for C6

Scheme 2.32 Mukaiyama aldol reaction of 39

2.5.3.4 Conclusions

Several aliphatic and aryl aldehydes as well as acetone were added to the lithium

enolate of (–)-isopinocamphone 39 under kinetic control in good to excellent yield, and

good diastereomeric excess (80−98%), except for 2-nitrobenzaldehyde, which gave only

26% d.e. Establishing the stereostructure of major diastereomer of aldol adduct 148 and the

acetate derivate 142 via X-ray crystallography revealed complete control at stereocentre at

C2 to be trans to the bridge carbon at C7, and to be S at carbon bearing OH (C1`).

The stereochemistry of the rest of substrates was predicted from 1H NMR spectra. For

example, it would be R,R and R,S at C2, C1`stereocentres of major and minor diastereo-

mers resulting from addition of aliphatic aldehydes. Meanwhile, the same stereogenic

centres would be R,S and R,R of major and minor isomers in case of aromatic aldehydes.

2.5.4 Aldol addition to diastereomeric mixture of (+)-dihydrocarvone

(+)-Dihydrocarvone 88 is an important structure to build biologically active natural

products, and the mixture of dihydrocarvone diastereomers chemically results from

reduction of R-(–)-carvone 36 with various chemical reagents. These reduction reactions

i ii

i = LDA, TMSCl/THF, −10 °C, ii = Acetone, TiCl4/THF, −78 °C

Yield 82%, d.r. 99:1

O TMSO (R)(R)O

OH

(S)(S)OOH+

151 152

TMSO O

OH

12

3 4

56

11

153 Yield 5%

i

ii

15039


75

are accompanied with multiple issues, for example, low diastereoselectivity and in some

cases, low yield. Also these methods do not comply with green chemistry, in another

words, environmentally non-accepted reagents and solvents have been utilised in these

reactions. Biosynthesis of dihydrocarvone has been reported in many articles, including

isolated and whole cell enzymes from different sources (see chapter 4).

Diverse aldehydes were added to the lithium enolate of (+)-dihydrocarvone

mixture, and the crude was purified with flash column chromatography. The number of

isomers present and ratios of diastereomeric mixtures have been confirmed with LC-MS

purification of isomers afforded one major isomer and mixture of minor isomers. The

results show the yield to be 41−64%, with modest to excellent diastereoselectivity. In the

NMR analysis, the most crucial signals of protons and their J values at C3, C2, C1` and

OH are very similar, except substrate 114, which exhibits completely different J values

(Figure 2.31).

Figure 2.31 Correlations of coupling constant values among adjacent protons of major

aldol adduct to 88

This proposed conformation of the cyclohexanone adduct is total agreement with 1H NMR spectra for five substrates out of six, and this suggests Si addition to the aldehyde

is favoured. The Zimmerman−Traxler transition state proposes R group would be

equatorial with partial interaction with the cyclohexanone ring of dihydrocarvone.

2.5.4.1 Aldol addition to (+)-dihydrocarvone with aliphatic aldehydes

Acetaldehyde has been added to the lithium enolate of (+)-dihydrocarvone

diastereomers mixture, and the 1H NMR spectrum of crude product showed a mixture of

diastereomers as well as starting material. The major isomer 154 was successfully purified

with flash column chromatography using hexane:EtOAc (10:1) to afford 64% of colourless

oil. The 1H NMR spectrum revealed a double of quartet of doublets at 3.70 ppm for CHOH

(J = 11.6, 6.7, 1.7 Hz) and a doublet peak at 3.25 ppm for OH with J = 11.3 Hz. Signals

for H-2 and H-6 were multiplets at 2.76−2.68 and 2.52−2.43 ppm. The 13C NMR spectrum

1 `

J = 12-12.5Hz

J = 11.2- 11.7 Hz

OOHH

R

H

HH

J = 1-2 Hz

2 3

456

1


76

showed 12 peaks for 12 non-equivalent carbon atoms, with the C=O peak at 216.6 ppm,

and CHOH 66.6 ppm.

The geometry of this adduct according to the proton NMR spectrum showed that

anti conformation would exist at new stereogenic centre C2, and the proton on carbon

bearing OH would be R configuration arising from Si face addition to aldehyde.

Figure 2.32 Possible configurations of aldol adducts of 154, 155 and 156

Addition of isobutyraldehyde to dihydrocarvone enolate gave a diastereomeric

mixture of β-ketol in 43% yield and the diastereomeric ratio was 95:2.5:2.5, shown by

proton NMR. This mixture is an oil, and attempting to grow crystals failed at ambient and

low temperature. The 1H NMR spectrum of 155 revealed a doublet peak for OH at 3.09

ppm, with J = 11.7 Hz, and a ddd signal for CHOH was shown at 2.93 ppm with J = 11.5,

9.9, 1.4 Hz. Also, the H-3 was assigned at 2.57 ppm as a doublet of triplets (J = 12.0, 1.2),

while, H-2 was a multiplet at 2.51−2.44 ppm. Assignment the peaks of minor isomers were

complicated because they were too small and overlapped with major diastereomer signals.

Although, the proposed stereochemistry of 155 adduct is consistent with proton NMR

spectrum, the atoms free rotation around single bonds is problematic. The J coupling

constant between protons of C2 and C1` (1.2 Hz) refers to tetrahedral angle (θ) to be 90°

between Ha and Hb, and consistent with conformer B and C with possibility to form

intramolecular hydrogen bond OH...C=O, since the conformer C requires high J coupling

to be anti-periplanar arrangement. However, establishing this configuration requires X-ray,

in this case, due to atoms free rotation around single bond.

Figure 2.33 Possible conformers of aldol adduct of (+)-dihydrocarvone

The TLC analysis of the reaction resulting from addition of cyclohexanecarbox-

yaldehyde to the lithium enolate of dihydrocarvone showed accumulated spots and flash

column chromatography failed, furnishing a diastereomeric mixture in 41% yield as a

colourless oil. The LC-MS showed a mixture of three diastereomers with ratio 95:2.5:2.5.

1 `

1

2

34

5

(R)(R)(R)(R)

(R)(R)O

(R)(R)

H

H HOH

(R)(R)(R)(R)

(R)(R)O

(R)(R)

H

H HOH

(R)(R)(R)(R)

(R)(R)O

(R)(R)

H

H HOH6

154 155 156

HaHbHO

HaHb

OHO Ha

OH

HbOO

A B C


77

The 1H NMR spectrum of 156 showed a doublet signal for CHOH at 3.07 ppm (J = 3.9

Hz), and peaks for H-2 and H-3 appeared as multiplet at 2.84−2.77 and 2.20−2.15 ppm in

sequence. Further confirmation was also proved with MS, with a peak of 265.2 (MH+).

Based on proton NMR assignment results, it is presumed that stereochemistry of the

predominant diastereomer would be R configuration at the new stereocentre of C1`, with

respect to C2 to be anti (R) resulted from Si addition to aldehyde.

2.5.4.2 Aldol addition to (+)-dihydrocarvone with aromatic aldehydes

The reaction of 88 with benzaldehyde gave six isomers with 92% d.e. of major one

157, and separation of fractions via column chromatography furnished major separated

isomer 157, with yield 56%. The spectrum of the 1H NMR of 157 showed a doublet peak

at 4.70 ppm for CHOH (J = 11.6 Hz), and d signal at 4.27 ppm for OH with J = 11.6 Hz.

Peaks of H-2 and H-3 were assigned as multiplet. Diastereomer 157 was a colorless oil,

and attempting to crystallize it out was not successful, thus, determining the chirality of

these substrates has to be made according to 1H NMR assignment. From the J constant

coupling of C3, C2, C1`, and OH the predominant diastereomer was provisionally assigned

as possessing S configuration at the β-stereocentre arising from Si face addition to the

benzaldehyde.

This reaction of dihydrocarvone enolate with naphthaldehyde furnished a mixture

of diastereomers (d.r. 77:17:3:3) containing starting materials, and this was purified

through flash column chromatography, which afforded pure predominant isomer 158 of

highly viscous oil in 51% yield, and also a minor diastereomeric mixture.

Scheme 2.33 Aldol addition to 88

The NMR of 158 exhibited OH and CHOH signals as doublets at 5.53 ppm (J =

11.2 Hz), and 4.77 ppm with J = 11.2 Hz. The protons of C2 and C3 were observed as a

multiplets at 2.99−2.94 ppm.

White crystals of 159 were crystallised from mixture of aldol adduct using 2-

pyridinecarboxyaldehyde, in 63% yield. The proton NMR spectrum indicated some J

coupling constants were different form previous substrates, for instance, CHOH was

assigned as double doublet at 5.33 ppm with J = 6.0, 2.0 Hz, and also a doublet was shown

1 `

O

(R)(R)

H

HR

HOH

1

234

56O

i,ii R = R =

157 158

i = LDA/THF, ii = benzaldehyde or naphthaldehyde

88 Major isomer


78

for OH at 3.85 ppm (J = 6.0 Hz). In addition, a doublet of doublet of doublets for H-2 was

suggested at 3.21 ppm with J = 11.9, 2.1, 1.3 Hz. X-Ray crystallographic of 159 confirmed

the anticipated α,α’trans geometry and identified the configuration at the β-stereocentre as

S.

Figure 2.34 X-ray crystal structure, and stereostructure of 159 adduct

The J coupling constant of C2 and C1` protons (4.1 Hz) is consistent with

conformer C, since conformer B is expected to introduce larger J than 4.1 Hz for the anti-

periplanar arrangement of the coupling protons Ha-Hb. The solid state conformer A shows

an intramolecular hydrogen bond between hydroxyl H and C=O, and X-ray crystallography

exhibited no hydrogen bond formed in the plane of C=O.

Figure 2.35 Possible configurations of aldol adduct of 159

2.5.4.3 Zimmerman−Traxler transition states for (+)-dihydrocarvone aldol Addition

reaction

X-Ray crystallography studies proved that (+)-dihydrocarvone 76 aldol addition to

pyridinecarboxyaldehyde resulted in isomer 119 with S configuration at new stereocentre

of carbon bearing OH arising from Si addition to aldehyde, with respect to R configuration

at C2. (+)-Dihydrocarvone 88 aldol adducts to acetaldehyde, isobutyraldehyde and

cyclohexanecarboxyaldehyde generated R conformation at new stereogenic center of

carbon bearing OH of the predominant diastereomer, while benzaldehyde and naphthalene

adducts anticipated to be S configuration.

The stereochemistry of aldol adducts of (+)-dihydrocarvone 88 was also controlled

by Zimmerman−Traxler transition state. Like aldol addition to (+)-isomenthone 38, there

are two fused decalin type transition states, cis or trans. The former would be more

favorable than the trans isomer due to less steric interactions of R aldehyde with R

substituents of (+)-dihydrocarvone.

OHa

(R)(R)H

HbO

N

J = 6.0 Hz

J = 2.0 Hz

H

159

HaHbPy

OHO

HaPyHO

HbO

HaOHHb

PyO

A B C


79

Figure 2.36 Zimmerman−Traxler transition state of 88 cis (blue) and trans (red) fused

In the cis isomer, in spite of R being pseudo equatorial, Si face addition is expected

to be favoured, to avoid 1,3 diaxial steric interactions of Rax aldehyde with H at

dihydrocarvone ring. In the trans isomer, Si face addition is expected to be not favoured

due to eclipsing isopropenyl substituent at dihydrocarvone ring with Req, and also Re face

addition of enolate to aldehyde is more unfavourable due to 1,3 diaxial steric interactions

of Rax aldehyde with H at dihydrocarvone ring.

2.6 Reduction of β-ketoalcohol adduct Introduction of non-natural cyclohexene-1,3 diol monoterpenoid with four

stereocentres (two fixed, two variable) would provide valuable ligands, for example

coordination of the diols with Et2AlCl has been published as a catalyst in the Diels-Alder

reaction55 and also the catalyst has been used in the Mukaiyama aldol reaction.65

An attempt to reduce 103 was implemented with sodium borohydride (NaBH4) in

presence of CeCl3.7H2O. The reaction was run in THF:MeOH (12:1) solvent system, at

room temperature. The reaction totally completed with full starting material consumption,

to afford two isomers with Rf 0.20 and 0.22 (hexane:EtOAc 4:1). The mixture was purified

with flash column chromatography to afford diol isomers (145 mg of 160 and 190 mg of

161, overall 66%).

Scheme 2.34 Reduction of 103 with NaHB4 in presence of CeCl3.7H2O

The 1H NMR spectrum of 1R isomer 160 showed a doublet signal (J = 8.8 Hz) at

4.39 ppm for CHOH at C1, and qd peak (J = 6.4, 3.4 Hz) at 4.00 ppm for CHOH at C1` as

well as a ddd for H-6 at 2.06 ppm with J value 12.0, 8.8, 3.4 Hz represent correlation with


OLi

H

O

ReqO

LiRax

O

H

A Si B Re

OLi ReqO

H

OLi HO

Rax

A Si B Re

1 `

(R)(R)(R)(R)

(Z) OH

(R)(R)H

OHH i

i = NaBH4, CeCl3.7H2O/ THF-MeOH, RT

103

1

23

45

6

7

89 10

(R)(R)OHH

H

OHH

H

160

(S)(S)

(Z)OHH

H

OHH

H

161

+

d.r. 3:4


80

protons at H-5, H-1, and H-1` respectively. These values supported that H-1 would be in

the axial position of the carveol ring, and consequently, OH at C1 would be at equatorial

position, and syn with the isopropenyl substituent (Figure 2.37 conformer A). However,

both OH appeared as a singlet signal at 3.09 ppm with integration equal to 2. The second

half chair possible would be the high-energy triaxial conformer, with high steric hindrance

(conformer B). The two hydrogen atoms at C1 and C2 are positioned as equatorial, but the

observed value of H-1 J constant (8.8 Hz) contradict this.

Figure 2.37 Possible conformations of diols 160 and 161

The 1H NMR of 1S 161 revealed a multiplet peak at 4.09−4.06 ppm for H-1`,

while, the H-1 is doublet with J coupling constant 3.6 Hz at 4.05 ppm. The H-6 was

appeared at 1.80 ppm as ddd with J = 12.0, 5.7, 3.5 Hz. In additional to, two protons of two

hydroxyl groups were singlet peaks showed at 2.10 ppm. The stereochemistry assumed

was that OH at C1 would be anti to the isopropenyl substituted (conformer C would be

more favorable than D). Based on assigning the 13 C NMR spectrum, the peak of carbon C1

of 160 isomer appeared at 69.9 ppm, while, its of counterpart of the 161 isomer was

positioned at 70.7 ppm, as the carbon shifts to lower field for an equatorial than axial OH

functional group.66

2.7 Synthesis of azides Formation of azides from alcohols can be achieved by Mitsunobu reaction,67 and it

is widely applied to produce esters through the condensation of alcohols and carboxylic

acids. It can be used to generate carbon-halogen68, carbon-nitrogen69 and carbon- sulphur

compounds70 as well as to form carbon-carbon linkages,71 via reaction of alcohols with a

range of acidic hydrogen compounds using Mitsunobu reagents.72 Subsequent studies have

introduced C-N bond by Mitsunobu reaction, in which hydrazoic acid has been

(R)(R)

(Z)OHH

H

OHH

H HHO

H

HO

HH

=OH

HH

H

HHO

A B

(S)(S)

(Z)OHH

H

OHH

H HHO

H

H

HHO

=

160

161 DC

H

OHH

H

HOH


81

employed,69 as it contains sufficiently acidic hydrogen to react instead of carboxylic acid.

However, diphenylphosphorazidate (DPPA) 162 and diazidobis(pyridine)zinc 163 has

been reported as azide source instead of explosive HN3. For example, DPPA has

introduced to convert an alcohol to an azide using the same reagents as the Mitsunobu

reaction (PPh3 and diethyl azodicarboxylate DEAD).73

Figure 2.38 Structures of DPPA 162, diazidobis(pyridine)zinc 163, and DBU 164

DPPA 162 and DBU 164 were suggested to convert alcohols to the corresponding

azides by Thompson et al.,74 and it as found that mixing alcohol with DEAD/PPh3 before

DPPA produces racemic product and olefin, which was attributed the substrates favouring

SN1 mechanism over usual SN2. However, it was rationalised that to using the base 164

with 162 suppresses the SN1 process.

Scheme 2.35 Mitsunobu reaction using A) hydrazoic acid,73 B) DPPA,74 C) DBU 75

PO

N3PhOPhO Zn N

NN

NNN

N

N

N

N

162 164163

HO N3

i

i = PPh3, DEAD, HN3

Ar

OH

Ar

OP(OP)2

N

NH N3

Ar

N3

i

i = DPPA 162, DBU 164

O

OHO

O

OO

O

N3O

O

OO

i

i = DBU 164, DIAD, PPh3

A

B

C

165 166

167 169

170 171

O

168


82

Compound 162 has been introduced as an azide source by Rollin and Viaud, and

the procedure involved mixing 162 with alcohol in presence of DIAD, PPh3 and toluene as

a solvent for 2 h to afford 82% of azide.75

2.7.1 Azidation of β-hydroxyketones

Following Thompson’s procedure substrate 103 and DPPA (1.2 eq.) 162 were

dissolved in dry toluene to afford a final concentration of alcohol of 0.5−1.0 M, and then

DBU was added at 0 ºC and the mixture allowed to stir for 2 h at same temperature, then

left stirring overnight. TLC indicated formation of two spots and incomplete reaction. The

higher spot (Rf 0.76 hexane 3:1 EtOAc) was just 2% crude yield, and could not be

identified. The second one was 29% (Rf 0.72), and its 1H NMR spectrum revealed

formation of alkylidene (enone) 172 in preference to the azide, as observed in the total

integration and the quartet signal at δ 6.60 ppm of the exo-double bond proton.

Furthermore, desired azide signal was not observed in IR spectrum, and finally, mass

spectroscopy gave a main peak for the enone component (M+ = 177.2). It could be DBU

caused elimination of phosphonate, and enone formed.

Scheme 2.36 Attempting azidation of 103 using Thompson conditions

Attempting to use Zn(N3)2.2Py 163 as a source of azide was carried out, and 163

was synthesised by heating a mixture of sodium azide, hydrate zinc nitrate and pyridine at

50 ºC for 5 min. The diastereomer 103 was reacted with 163 in presence of DIAD and

PPh3 but led to an intractable mixture.

Scheme 2.37 Synthesis of 163

2.8 Addition of enolate/enol isomenthone to imine The idea of using C=N instead of C=O as an electrophilic acceptor for the enolate

and/or enol of (+)-isomenthone 38 in a Mannich reaction faced a problem in that the imine

O

HOHH

H O

O

HN3

H

Hi,ii

i,ii

i =DPPA, ii = DBU/toluene

103

172

163Zn(NO3)2.6H2O 2C5H5N 2NaNO3 Zn(N3)2.2Py+ +


83

is less electrophilic. Addition of ketone to Schiff bases like benzalaniline 173 in presence

of a small amount of HCl has been reported.76 Treatment of 38 with benzalaniline in

presence of HCl did not yield the product even after stirring overnight.

Zinc tetraflouroborate in water has been introduced to catalyse the reaction of

benzalaniline 173 with silylenol ether 174 at room temperature in THF. The reaction was

applied on varieties of silylenol ether of acyclo and cyclo ketones, with high to excellent

yield, and high diastereoselectivity for cycloketones 175.77

The lithium enolate of organometallic acyl η5-CpFe(PPh3)(CO)COCH 176 was

successfully reacted with benzalaniline 173 under the effect of kinetic conditions, to yield

79% and high stereoselectivity (92%) of 177.78

Scheme 2.38 Mannich reaction A) using Zn(BF4)2

77 B) under kinetic control78

The lithium enolate of 38 was added to benzalaniline at −78 ºC using fresh distilled THF as

solvent, and intractable mixtures were formed.

Scheme 2.39 Treatment 38 with benzalaniline under kinetic effect

177

N+ (CH2)n

OTMS

i,ii(CH2)n

O

NHPh

Ph

i = Zn(BF4)2, ii = THF/RT

Fe

O

PPh3OCFe

O

PPh3OC

i,iiNHPh

Ph

i = LDA/THF, ii = benzalaniline 173 / −78 °C

A

B

173 174

176

175

O O

PhN Ph

OPhPhHN

i

i = LDA/THF, −78 °C

38

3. Synthesis of alkylidenes from terpenone-derived β-keto alcohols

84

3 Synthesis of alkylidenes from terpenone-derived β-keto alcohols

3.1 Introduction Elimination of a water molecule from β-ketoalcohol to form α,β-unsaturated

compounds is very useful process in synthesis of natural products. For instance, 2-

alkylidenecyclohexanone derivatives exist in the backbone of biological natural product

structures.79 Several papers reported the preparation of 2-alkylidenecycloalkanone

derivatives and cycloterpenoids from precursor alcohols.

A new strategy of Claisen–Schmidt condensation has been documented.80 The

tactic involves synthesis of arylidene moiety via condensing sterically hindered ketones,

like (–)-menthone 49 and camphor 87 with aromatic aldehydes in strong alkaline media

such as KOH, CsOH, Bu4NOH and t-BuOK in aprotic solvents like DMSO, DMF or

DMA. The effect of base type and solvent type on yield percentage of product has been

studied. The authors referred to use of t-BuOK/DMSO combination as the best effective

medium to obtain chiral α,β-unsaturated ketones.

Scheme 3.1 Aldol condensation of 49 and 87 with benzaldehyde 80

The aldol reaction of 2,4-dimethyl cyclohexanone 181 with a variety of aldehydes

was reported by Reuvers et al. in 1982.81 The results showed formation of threo and

erythreo diastereomers of ketoalcohol 182. These were ultimately converted to E (183) and

Z (184) isomers of enone through dehydration of product using a benzene solution of p-

toluenesulfonic acid PTSA. Purification and separation using flash column chromate-

graphy afforded predominant isomer 183 and minor isomer 184, which represent erythreo

and threo isomers of the aldol products, respectively. The researchers employed the 184

isomer of enone for synthesis δ-substituted δ-lactones (Scheme 3.2).

O +

O

O

Ph

O

Ph

+i

i = stronge base

O

+

O

R

i = stronge base R = H, Br, OMe

O

R

H

49

87

i

178 179

180


85

Scheme 3.2 Aldol addition to 181 and dehydration of the product using PTSA81

Bartoli and co-workers79 introduced a new methodology for the dehydration

process. The novel annulation uses a mixture of CeCl3.7H2O/NaI to catalyse β-elimination

reaction of alcohols 185. The reaction was found to be highly regio-and diastereoselective

to yield only (E)-2-alkylidenecycloalkanones 186.

Scheme 3.3 Dehydration process of 185 79

Moreover, the research demonstrated a new method for synthesis of monoterpenoid (S)-(–

)-pulegone 187 from cyclohexenone under mild conditions (Scheme 3.4).

Scheme 3.4 Total synthesis of 187 79

Synthesis of the sesquiterpenoid natural product (+)-fukinone 188 was proposed in

1970 by Piers and Smillie.82 They suggested mixing ketoalcohol intermediate 189 with

thionyl chloride SOCl2, followed by reaction of the olefin product with p-toluenesulfonic

acid PTSA to afford 70% of the desired product. However, by-products were also

produced, such as 20% of the decalone and 10% of an unrecognised component (Scheme

3.5). Prasad and Chan 83 also reported preparation of, eremophilane-type, (+)-fukinone, but

the intermediate β-ketoalcohol 190 mixed with SOCl2, then the product was eluted from

O O OH

R

O(E)

R

O

(Z)

R

i,ii iii

i = LDA, ii = RCHO when R = CH3, n-C6H9, iso-C3H7, C6H5, iii = PTSA

183181 182 184

O OH Oi

i = CeCl3.7H2O/NaI, CH3CN, reflux

185 186

O O OH O OTMS

O OTMS O

i ii

iii iv

NPPh2

Ot-Bu

i = LDA, Acetone, CeCl3, ii = TMSCl, Et3N, iii = MeLi,CuCN, LiBr, Pivaloylamidophosphine A, iv = CeCl3.7H2O/NaI, CH3CN

187

=A


86

alumina with 30% EtOAc-hexane solvent system and purified by column chromatography

to obtain 81% of the natural product (Scheme 3.5).

Scheme 3.5 Final steps for synthesis of 188 82, 83

Bicyclo[2.2.2]octan-2-one 191, a norbornane derivative, is a valuable compound in

fragrance chemistry. It was treated with a variety of aldehydes to obtain 40–60%

ketoalcohols 192, followed by dehydrating the latter via SOCl2 to form corresponding

enones 193. The olfactory properties of ketol and alkylidene products were evaluated due

to relationship of their odour to chemical structure.84

Figure 3.1 Structures of intermediate 192 and product 193 of aldol condensation of 191 84

Formation and elimination of mesylates has been carried out85 to a 4:1 diastereomeric

mixture of aldol product of disubstituted cycloketone 194, an intermediate for synthesis of the

spirocyclic system of marine natural product Vannusal A, giving a 95% yield of isomeric

enone (E/Z 20:1) 195.

Scheme 3.6 Dehydration of ketol 194 85

3.2 Synthesis of alkylidenes from precursor β-ketoalcohols Formation of cyclomonoterpenoid enones (alkylidenes) from their corresponding β-

hydroxyketones is a synthetically useful method to produce α,β-unsaturated derivatives,

OH

OH

OH O

H

OH

OH

i ii

a,b

i = SOCl2, pyridine ii = PTSA a = SOCl2, b = Al2O3

188189

190

O O

R

OH H

R

O191 192 193

OHOH

C6H13

O

C6H13

i

194 195

i = MsCl,Et3N, Al2O3


87

and offers a valuable procedure related to synthesis of biologically activity of natural

products, which have alkylidene cyclohexanones in their structures. This project therefore

set out to evaluate and optimize methodology for versatile synthesis of such defined

alkylidines.

Scheme 3.7 Proposed elimination strategy to new terpenoid exo-alkylidine/arylidine

targets

3.2.1 Synthesis of carvone-derived alkylidenes bearing aryl ring

The R-(–)-carvone aldol adduct of a diastereomeric mixture of nitroaromatic-

bearing 116 and 117 (see Scheme 2.23 for synthesis) was selected to optimise the

conditions of this reaction, and several reagents were evaluated for dehydration of the β-

ketol. An equimolar amounts of reagent and a mixture of 116 and 117 were refluxed in

toluene with p-toluenesulfonic acid (PTSA) with TLC showing that three spots were

formed, and there was no starting material detectable. The fractions were isolated using

flash column chromatography (20:1 hexane:EtOAc), and analysed. 1H NMR of upper spot

(Rf 0.34 hexane 5:1 EtOAc) revealed generation of alkylidene as an isomeric mixture (95:5),

and the other two spots were R-(–)-carvone and p-nitrobenzaldehyde produced as a result of

the retro-aldol reaction. Occurrence of the retro-aldol reaction was rationalised due to use

of excess of p-toluenesulfonic acid, and/or high temperature. Due to the modest yield and

by-products formation, the experiment was repeated at different temperatures using 5%

PTSA. Table 3.1 shows the ratio of product and by products under different conditions and

that using 5% of PTSA at 40 ºC gave a good yield of alkylidene, with minimum by-

products.

O

OH

R

O

R

O

R

and/or

O

OH

R and/or

carvone-derived

isomenthone-derived

O

R

O

R


88

Table 3.1 Yields and by-products percentages of β-hydroxyketone 116 and 117 dehydration using PTSA at various temperatures

Entry PTSA%

Temp. Time Alkylidene %

R-carvone% Impurities% 1 100 Reflux 1.5 h 15 18 38 2 5 80 ºC 1.0 h 41 13 19 3 5 40 ºC ON 64 9 5 4 5 RT ON 11 ----- -----

It was difficult to identify the impurities due to intractable mixture formation. The 1H NMR clearly identified two isomers (ratio 99.5:0.05), and assignment of the 196 was

established though a single peak at δ 7.62 ppm for exo-double bond proton at C-11. In

addition, two aromatic resonances were evident at δ 7.49−7.48 ppm as a multiplet of and a

doublet of triplets at δ 8.10 ppm.13 C NMR spectrum showed 15 peaks assigned as below

(Figure 3.2).

Figure 3.2 X-ray structure, 2D-NOESY correlations and 13C NMR shifts for 196

The assignment of E stereochemistry was supported using 2D-NOESY NMR,

which showed correlation between the proton of the exo-double bond and aromatic

protons, also with a strong signal correlating H-5 with aromatic protons. Moreover, the H-3

showed correlation with the H-5 (Figure 3.2). Attempts to crystallise the oily mixture of

isomers 196 and 197 eventually succeeded, and the structure of the E isomer was

established with X-ray crystallography (Figure 3.2).

In order to evaluate optimisation of dehydration of β-hydroxyketone 116 and 117,

methanesulfonyl chloride (MsCl) was also utilised. Refluxing mixture of 116 and 117 with

MsCl and DMAP in THF solution overnight showed incomplete reaction with two new

spots. Separation of the fractions revealed generation of enone in 46% yield, along with

6% impurities. Alternatively, the mesylate was generated by adding MsCl (3 eq.) and

triethylamine TEA (6.0 eq.) to a DCM solution of 116 and 117 at 0 ºC. Then DBU (2.0 eq.)

was added (when mesylate had formed), and the reaction allowed to stir overnight. The

results showed formation of alkylidene products 196 and 197 in 32% yield with and E:Z

ratio of 77:23, and the rest of the unreacted mesylate was recovered. With the relatively

O

NO2

HH

H

H1

2

345

6

7

89 10

11

12

13

1415

1617

O

NO2

16.6

141.6136.6

44.229.1

123.9

114.122.0

145.0

147.5

189

142.7

130.1

133.4141.4

133.4130.1


89

poor outcomes using mesylation, mixture of 116 and 117 was converted into an intermediate

triflate (using triflic anhydride) and four different bases were tested using DCM was the solvent

in all cases (Table 3.2).

Table 3.2 Synthesis of alkylidene 196 and 197 from diastereomers mixture 116 and 117 using Tf2O with four different bases

Entry Base Temp. Time of reaction Yield% 1 DMAP (3 eq.) 0 °C 2 h 26 2 DIPA (3 eq.) 0 °C 2 h 8 3 TEA (3 eq.) 0 °C 2 h 10 4 Pyridine (3 eq.) 0 °C 2 h 26 5 Pyridine (6 eq.) RT Overnight 14 6 Pyridine (6 eq.) 0 °C 4 h at 0 °C, overnight at RT 34

3.2.2 Synthesis of carvone-derived alkylidenes alkyl-bearing

With conditions developed for an arylidine target, similar conditions were pursued

for alkyidine precursors. The mixture of diastereomers 103 was dissolved in toluene and

heated to 40 ºC which showed total consumption of the starting materials within two hours,

and the appearance of two products (Rf 0.77 and 0.73; hexane:EtOAc 5:1). 1H NMR of the

mixture proved formation of a 20:1 mixture of E:Z enone isomers 172 and 198. The exo-

double bond proton of the E isomer appeared as a quartet at δ 6.67 ppm with J value of 7.4

Hz. Moreover, three signals for three methyl groups were identified at δ 1.62 (dt), 1.70 (d)

and 1.72 ppm (dt) for 172. The stereochemistry of 172 was supported with 2D-NOESY

NMR, showing correlation between the exo-double bond proton H-11 and CH3 at C12, as

well protons of CH3 at C12 and H-9 (Scheme 3.8).

Scheme 3.8 Synthesis of alkylidene bearing alkyl derived of (–)-carvone 172, 198, 199 and

200

O

OH

R

(Z) O

R(Z)

(Z) O

R

(E)

H

H +i

R = Me, d.r 95:05 E:Z 172:198, yield 86%R = Ethyl, d.r 100:00 E:Z 199:200, yield 30%

i = PTSA/toluene, 40 °C

12

3

4 56

7

89 10

11


90

Figure 3.3 2D-NOESY spectrum of 172

Similarly to the elimination of 102, a mixture of diastereomers of the homologue

105 and 106 was mixed with PTSA in toluene, and the reaction stirred overnight at 40 ºC.

TLC revealed incomplete reaction, and a single product spot was seen with Rf 0.52 (hexane

5:1 EtOAc). Flash column chromatography afforded the E-alkylidene 199 in 30% yield,

along with 52% recovery of starting material. There were no by-products of retro-aldol

reaction or other impurities detectable. 1H NMR confirmed a single isomer 199 was

formed, as indicated by a triplet at δ 6.70 ppm for the exo-double bond proton with J value

7.3 Hz and three signals at δ 1.03, 1.68 and 1.78 ppm for the methyl groups. 2D-NOESY

NMR spectra indicated a strong correlation between the exo-double bond proton and

adjacent CH2, and the latter was correlated with C10 CH3 protons. 13C NMR spectrum

exhibited all anticipated 13 signals with both of the exo-double bond carbon atoms

identified at δ 135.5 and 136.7 ppm, as well as three peaks (δ 21.7, 16.7, and 13.8 ppm) for

the methyl groups. IR showed a carbonyl group absorbance at 1665 cm-1 and confirmed the

absence of any OH group.

Although PTSA was effective in catalysing dehydration of the methyl and ethyl

substrates, these conditions were ineffective with the analogous isopropyl substrate 107.

Reaction using 5% PTSA at 40 ºC in toluene for 2 h led to a complex mix of at least six

spots by TLC and there were no starting materials remaining. Fortuitously, three fractions

were successively isolated and 1H NMR analysis showed products of retro-aldol reaction

as well 5% of the alkylidene product 201 and 202 (Scheme 3.9). The other three spots were

isolated as a mixture and were not identified by NMR or MS. Due to the modest yield of

ppm0 011223344556677

0

1

2

3

4

5

6

7

H-11 H-12

1234

56

7

89 10

11

12

O

HH

H-9a,b

H-9a,b

H-12

H-11


91

the alkylidene product, the mesylate was generated via dissolving MsCl and DMAP in

freshly distilled THF at room temperature and the reaction stirred overnight. Initially, TLC

showed three spots, with no starting materials, and two spots of 201 and 202 and with Rf

0.76 and 0.77 (hexane 3:1 EtOAc), and the third spot was mesylate (Rf 0.45). However, the

yield of enone was just 8%, and the rest of the yield was the mesylate ester, which was

easily recovered. In order to increase the yield, tetrabutylammonium fluoride TBAF was

employed to eliminate the ester, and was added to a THF solution of mesylate, and the

reaction stirred overnight at room temperature.

Scheme 3.9 Synthesis of alkylidene bearing alkyl derived from (–)-carvone (201, 202, 203

and 204)

A 37% yield of enone 201 and 202 was obtained, and the remainder was mesylate,

which was also recovered. Flash column chromatography partially separated the isomers,

and pure E isomer 201 was collected as well to an isomeric mixture of E and Z (Scheme

3.9).

Table 3.3 Chemical shifts (δ, ppm) and coupling constants (J, Hz) for E isomer 201 Entry Integration δ (ppm) Splitting J (Hz) Protons no.

1 6 0.98 dd 7.2, 6.7 2 x CH3, 13 and 14 2 3 1.67 dt 1.4, 0.7 1 x CH3, 10 3 3 1.77 dt 2.4, 0.8 1 x CH3, 7 4 3 2.59−2.52 m ------- 1 x CH and 1 x CH2, 12, 4 5 1 3.62 d 3.6 1 x CH, 5 6 1 4.57−4.56 m ------- ½ x CH2, 9 7 1 4.74−4.73 m ------- ½ x CH2, 9 8 1 6.50 d 10.3 1 x CH, 11 9 1 6.56−6.54 m ------- 1 x CH, 3

As with the previous substrates, 5% of PTSA was used to dehydrate the major

diastereomer 109, however, the reaction was not completed within 24 h. The fractions

contained by-products of retro-aldol reaction and just 4% of the enone 203 and 204. The

elimination reaction was repeated using MsCl and DMAP, but also an unsatisfactory result

O

OH

R

(Z) O

R(Z)

(Z) O

R

(E)

H

H +

R = Isopropyl, d.r. 91:9 E:Z 201:202, yield 37%R = Cyclohexyl, d.r. 89:11 E:Z 203:204, yield 38%

i =MsCl, DMAP/THF, RT, ii = TBAF

i,ii


92

was obtained (7.5%), and mesylate was recovered. Eventually, TBAF was mixed with

mesylate at room temperature overnight, and the yield was slightly improved (38%). The E

isomer 203 was isolated from the 10:1 E:Z mixture, and its proton NMR confirmed

formation of alkylidene. For example, a doublet signal (J = 10.4 Hz) was assigned at δ

6.50 ppm for the exo-double bond proton, moreover, detection of 11 protons for cyclohexyl

also proved production of substrate. Also, proton NMR of the minor isomer (Z) 204

showed a doublet of doublets (J = 9.6, 1.2 Hz) peak at δ 5.52 ppm for H-11.

3.2.3 Synthesis of isomenthone-derived alkylidenes alkyl-bearing

Aldol addition to (+)–isomenthone 38 using ethanal afforded two diastereomers

126 and 127 as mentioned previously, and 126 was treated with PTSA (5%) to synthesise

the valuable enone substrate. Indeed, this reaction was completed within 2 h, to give a

good yield (76%) of the isomers mixture E and Z with ratio 3:2 205:206. Flash column

chromatography partially resolved the isomers and afforded pure 206 isomer and a mixture

of both (Scheme 3.10).

Scheme 3.10 Synthesis of alkylidene bearing alkyl derived from (+)-isomenthone (205,

206, 207 and 208)

However, the structure of 205 was easily identified from the 1H NMR spectrum, and

revealed a quartet of doublets (J = 7.2, 1.0 Hz) at δ 6.43 ppm for the exo-double bond

carbon. The 13C NMR spectrum also proved formation of the alkylidene, for instance, the

carbon atoms of exo-double bond indicated at δ 146.6 and 133.9 ppm, also, four peaks at δ

22, 20.4, 19.3 and 15.1 ppm were assigned for the four methyl groups. Compound 206 was

also confirmed using NMR. The 1H NMR spectrum showed a quartet of doublets (J = 7.2,

1.5 Hz) of the exo-double bound proton, as well as four signals of four methyl groups:

doublet at δ 0.76, doublet at 0.82, doublet at 0.97 and doublet of doublets at 1.67 ppm.

The isomer 128 was stirred with PTSA in toluene, and reaction did not complete

within 4 h, so it was stirred overnight. TLC detected four new spots with Rf 0.17, 0.31,

0.55 and 0.77 (hexane:EtOAc 5:1), while the spot for the starting material disappeared.

The lower two spots were separated and analysed, and found to be products of retro-aldol

R = Me, d.r. 60:40 E:Z 205:206, yield 76%R = Isobutyl, d.r. 83:17 E:Z 207:208, yield 43%

i = PTSA/toluene, 40 °C

O

OH

R

O

(Z)R

O

(E)

R

i,ii +


93

reaction (isomenthone and isovaleraldehyde). Separation of the two upper spots failed, and

they were collected as an isomeric mixture (5:1). Inspection of the 1H NMR spectrum of

the mixture indicated two signals for the exo-double bond proton, with the first peak at δ

6.37 ppm (t, J = 7.6 Hz) for major isomer (E) 207, and the second one at δ 6.17 ppm (td, J

= 7.6, 1.4 Hz) for Z isomer 208. Furthermore, three peaks for five methyl groups were

characterised for 207, whereas, it was difficult to assign methyl peaks of the minor isomer

due to overlapping of those of 208 with their counterparts in 207.

3.3 Conclusions Synthesis of alkylidenes from aldol adducts of carvone 36 and isomenthone 38 was

successfully achieved with modest to very good yield under various conditions of reagents,

temperature and reaction medium. Although alkylidene formation from both carvone and

isomenthone proved to be highly diastereoselective, except for 205 and 206, the substrate

199 was indicated to be only the E isomer, and was thus a completely diastereospecific

reaction. The stereostructure of 196 was established with X-ray crystallography, and

proved to be E isomer.

Table 3.4 Yields and diastereomeric ratios of alkylidenes derived from β-ketol Entry Products Yield% Diastereomeric ratio

172 and 198

86

95:05

199 and 200

30

100:00

201 and 202

37

91:09

203 and 204

38

89:11

(S)(S)

(Z) O

Me(Z)

(S)(S)

(Z) O

Me

(E)

(S)(S)

(Z) O

Et

(E)

(S)(S)

(Z) O

iPr(Z)

(S)(S)

(Z) O

iPr

(E)

(S)(S)

(Z) O

Cy(Z)

(S)(S)

(Z) O

Cy

(E)


94

196 and 197

64

95:05

205 and 206

76

60:40

207 and 208

43

83:17

(S)(S)

(Z) O

PNBA(Z)

(S)(S)

(Z) O

PNBA

(E)

(R)(R)(R)(R) (E)

Me

O(R)(R)(R)(R)

(Z)

O

Me

(R)(R)(R)(R) (E)

i-Bu

O(R)(R)(R)(R)

(Z)

O

i-Bu

4. Biocatalysis of monoterpenoids

95

4 Biocatalysis of monoterpenoids

4.1 Introduction of biocatalysis In the last few decades, chemical transformations using biocatalysts such as isolated

enzymes or whole cells have become an efficient alternative method to standard chemical

synthesis in production of new synthons in the drug, chemical materials, and agrochemical

industries.86 For instance, biocatalysts have used in preparation of almost one in ten

drugs.87

Utilizing enzymes in biocatalysis has become of interest for many synthetic

chemists and biologists due to their remarkable advantages. Primarily, they can be very

efficient, because most enzymatic reactions have high chemo-, regio-, and stereo-

selectivity. Also, biocatalysis are used under relatively mild conditions (pH, temperature)

as well as avoiding organic solvents which are environmentally unfriendly.88, 89 However,

instability is still the essential problem in biotransformation, through deactivating the

enzymes under harsh conditions of pH, temperature and physical forces. Moreover,

although there are an excess of 4000 known biocatalysts, only a few enzymes were well

identified and commercially available.90 However, enzyme properties have been enhanced

with applying the technology of bioengineering processes such as routine site-directed

mutagenesis, or random or focused evolution methods, and this has led to increasing

enzyme stability at specific temperature and solvent, better product enantiopurity and

alteration of the specificity of substrate.91

Isolated enzymes and whole cells can be utilized in synthetic chemistry and both

have pros and cons. Isolated enzymes can be used to obtain high enantiomeric excess or

enantiopure synthons and prevent the competition of catalysts, to afford different and high

stereoselectivity. Consequently, the simplicity of purifying the product makes this

technique attractive in some biotransformations. However, there are limitations of applying

isolated enzymes method including expense of reduced cofactors, which must be

regenerated. Fortunately, exogenous cofactors do not need to be added using whole living

cells, but presence of more than one enzyme to catalyse the substrate gives low

enantiomeric excesses.92−94 Baker’s yeast alcohol dehydrogenase (YADH), for example,

has been used to reduce ketones to secondary alcohols, unfortunately, by-product

formation and low product e.e. occurred due to the presence of at least four reductases

which have different enantiopreference.95,96


96

4.2 Objectives of monoterpenoid biotransformations A number of terpenoids are low cost, available in large quantities, with more than

400 structures of monoterpenoids known, and renewable natural precursors.97 They play a

vital role in the flavour, fragrance and food industries, and have biological and

pharmalogical properties making them ideal precursors to produce natural aroma chemicals

in the biotechnology production field. The importance of terpenoids has noticeably

increased and the investigation to prepare new derivatives has interestingly grown with

desirable organoleptic and pharmaceutical properties.98

This study aimed to screen isolated enzymes, available in the lab, to bio-reduce

double bonds of natural and synthesised monoterpenoid substrates in order to prepare

valuable intermediates with high regio-, and stereoselectivity. Moreover, investigations of

new non-natural monoterpenoids as substrates for Baeyer−Villager bio-oxidation using

CHMOs was attempted. Eventually, scaling up the reactions will be the next goal to

translate screening process to lab applications.

4.3 Bioreduction of monoterpenoids Translation of global biocatalysis research to manufacturing routes has been

strongly supported with the on-going trends to process modification such as pollution

reduction, low-cost process and quality enhancement.99 Asymmetric syntheses with

biological microorganisms have been extensively reported, and have allowed the industry

access to produce fine chemicals, pharmaceuticals, and agrochemical products. Surely,

asymmetric hydrogenation of activated double bonds is an essential reaction in industrial

synthesis due to generation of up to two stereogenic centers with often-unmatched regio-,

stereo-, and enantioselectivity of enzymes catalysed bio-reductions. Indeed, “green” or

sustainable chemistry is the core of current trends and requires broad application of

enzymes in the industry. Here, the reports of monoterpenoids bio-reduction, with micro-

organisms as whole cells and isolated enzymes, are reviewed.

Reduction of several monoterpenoids has been carried out through screening a large

number of yeasts.100 In this research, (–) and (+)-carvone (36 and 34) were reduced using

several yeasts with different activities, and some yeasts reduced double bond in 36, with

yield up to 90% within incubation for 2 h, to produce R configuration at C1 of

dihydrocarvone isomer 100, while, other yeasts reduced the carbonyl group and double

bond to yield dihydrocarveol 209 with stereochemistry S at C2 and R at C1 (Scheme 4.1).

Obviously, bioreduction of S-(+)-carvone 34 compromised C=C with up to 90% within 24

h (210), and other microorganisms reduced both C=O and C=C to yield dihydrocarveol


97

isomers 211 and 212 with stereochemistry sometimes S and sometimes R at C2 and always

R at C2, depending on the type of strain (Scheme 4.1).

Scheme 4.1 Bioreduction of 34 and 36 100

Also, (–)-menthone 49 and (+)-pulegone 29 were reduced to (+)-neomenthol 213

by Hormonema isolate (UOFS Y-0067), whilst (4S)-isopiperitenone 214 was reduced to

(3R,4S)-isopiperitenol 215(Scheme 4.2).

Scheme 4.2 Bioreduction of 29, 49 and 214 100

(R)(R)

(Z) O

(R)(R)(R)(R)

O

(R)(R)(S)(S)(R)(R)

OH

(S)(S)

(Z) O

(S)(S)(R)(R)

O

(S)(S)(R)(R)(R)(R)

OH

(S)(S)(S)(S)

(R)(R)OH

i i

i

i = Plant/algae/yeast

36

34

100 209

210

211

212

(+)-Dihydrocarvone (+)-Neodihydrocarveol(–)-Carvone

(+)-Carvone (–)-Isodihydrocarvone

(–)-Isodihydrocarveol

(–)-Neoisodihydrocarveol

Yeast H.guilliermondii CSIR Y8997 Yield 95% Yield 3%

Yeast Y.lipolytica KBP 3364 Not detected Yield 100%

[Yeast] = 0.7–0.9 g/l[monoterpenoid] = 0.5 mL/l,25 °C, pH = 7.0, 2 h

Conditions

(S)(S)(R)(R)

O

(R)(R)

O

i i

(S)(S)

(Z)

O(S)(S)

(R)(R)

(Z)

OH

i = Hormonema sp.

49 29213

214 215

(+)-Neomenthol(−)-Menthone (+)-Pulegone

Isopiperitenone

i

IsopiperitenolConditions: [Yeast extract] = 3 g/l, pH = 7.0, [monoterpenoid] = 0.5 mL/l, 25 °C, 48 h

(S)(S)(R)(R)(R)(R)

OH


98

Biotransformation of S-(+)-carvone 34 has been implemented by 146 strains of

yeasts under different physiological states (growing, resting and lyophilised cells). Only 16

strains exhibited good activity to bioreduce 34 to 1S,4S- and 1R,4S-dihydrocarvone (210

and 83), (1S,2R,4S)-, (1S,2S,4S)- and (1R,2S,4S)-dihydrocarveol (216, 217 and 212), and

also, a small quantity of (1R,2R,4S)-dihydrocarveol (211) was detected in a few strains.

Yields of product varied from 0.14 to 30.04% depending on both the yeast strain and

organism physiological state. In this work, glucose was added to lyophilised cells, as

auxiliary substrate for the cofactor recycling system, to enhance the activity of bioreduction

process (Scheme 4.3).101

Scheme 4.3 Bioreduction of 34 using strains of yeasts under different physiological

states101,102

Bioreduction of double bond and carbonyl functions of (+) and (–)-carvone (34 and

36) was investigated by Pseudomonas ovalis, to produce a variety of products, for

example, bio-conversion of 34 produced (–)-dihydrocarvone (83), (–)-neodihydrocarveol

216, (+)-dihydrocarveol 217, (–)-isodihydrocarvone 210, (–)-isodihydrocarveol 211 and (–

)-neoisodihydrocarveol 212, while the pathway of bio-reduction of R-(–)-carvone 36 has to

be (+)-dihydrocarvone (100) and further to (–)-dihydrocarveol 218 (Scheme 4.3).102

Interestingly, in contrast to carvone reduction, pulegone stereoisomers were reduced using

recombinant Escherichia coli expressing Nicotianatabacum pulegone reductases rPRase to

yield opposite stereoselectivity,103 in other words, (R)-pulegone 29 produces (1R,4R)-

isomenthone 38 and (1S,4R)-menthone 219, while, (1R,4S)–menthone 49 and (1S,4S)–

isomenthone 220 are generated from (S)-pulegone 187 (Scheme 4.4).

Conditions[Lyophilised yeast] = 30 mg/25 mL, [(+)-carvone 34],= 10 mMphosphate buffer pH = 6.5, T = 25 °C, rpm = 125,Time = 24 h

(S)(S)(R)(R)

O

(S)(S)(R)(R)

(R)(R)OH

(S)(S)(S)(S)

(R)(R)OH210

211

212

(S)(S)(S)(S)

O

(S)(S)(R)(R)

(S)(S)OH

(S)(S)(S)(S)

(S)(S)OH

(S)(S)

(Z) O

3483

216

217

(+)-Carvone(–)-Isodihydrocarvone



(–)-Dihydrocarvone


(+)-Dihydrocarveol


99

Scheme 4.4 Bioreduction of 36, 29 and 187 using rPRase103

Bio-reduction of R-(–)-carvone 36 and R-(–)-myrtenal 221 104 via whole-cells of non-

conventional yeast (NCYs) belonging to the genera Candida, Cryptococcus, Debaryomyces,

Hanseniaspora, Kazachstania, Kluyveromyces, Lindnera, Nakaseomyces, Vanderwaltozyma and

Wickerhamomyces has been reported by Goretti and co-workers. The collected data revealed

conversion of 36 to both 100 and 222 with variable diastereoselectivities up to 95% of 100,

and maximum conversion 62% with ERs of H. guilliermondii. In some cases, both 100 and

222 products underwent 1,2 reduction, and afforded a trace of four isomers of (1R,2S,4R)-,

(1R,2R,4R)-, (1S,2S,4R)- and (1S,2R,4R)-dihydrocarveol (209, 218, 223 and 224). Also, the

researchers reported that addition of co-substrate glucose to the reaction mixture

surprisingly raised the aptitude of some strains to reduce 36 (Scheme 4.5).

(R)(R)

(Z) O

(R)(R)(R)(R)

O

(R)(R)(R)(R)

(R)(R)OH

i i

36 100 218(+)-Dihydrocarvone (–)-Dihydrocarveol(–)-Carvone

(R)(R)

O(R)(R)(R)(R)

(S)(S)(R)(R)

OO

(S)(S)

O(R)(R)(S)(S)

(S)(S)(S)(S)

OO

29 38

219

49

220187

i

i

i = rPRase

(+)-Pulegone (+)-Isomenthone (–)-Menthone

(–)-Pulegone (+)-Menthone (–)-Isomenthone

+

+

Conditions: [Enzyme] = 50 µg, [Substrate],= 1 mM, NADPH 4.4 mM,phosphate buffer pH = 7.0, T = 37 °C,Time = 12 h

Yield 4–14% Yield 4–18%

Yield 0.6–16% Yield 0.6–5.2%


100

Scheme 4.5 Bioreduction pathway of 36 via NCYs whole cell 104

In contrast, bio-conversion of R-(–)-myrtenal 221 showed that α,β-unsaturated

carbonyl substrate converted in almost all cases to unsaturated alcohol 225 with conversion

≥95% in third strains, for instance, Candida freyschussii DBVPG 6208 and Kazachstania

spencerorum DBVPG 6746 exhibited ability to convert the precursor in 100% yield. However,

dihydromyrtenals 226 were apparently observed with few strains prevalently related to ERs,

and further reduction of CRs was noticed in some cases to give dihydromyrtenols 227 and

228 (Scheme 4.6).104

Scheme 4.6 Bioreduction pathway of 221 via NCYs whole cell104

Baker yeast has also been documented as a biocatalyst to reduce the endo-double

bond of R-(–)-carvone 36 in in aqueous mono- and biphasic systems, with variations of,

enzyme and substrate concentrations, temperature, pH, the effect of organic solvents and

additives. It was concluded that 100 g L−1 of BY, 16.6 mM of substrate, and pH 7.5 at 26 °C in the presence of DMSO, and additives trehalose, or sucrose were the ideal conditions

of R-(–)-carvone bio-reduction to afford 70–74% of (1R,4R) and (1S,4R) dihydrocarvone

100 and 222 with d.e. of 92–99%. In addition, it was confirmed that (1R,2S,4R)-

dihydrocarveol 209 was produced, as well as traces of (1R,2R,4R)- 218 and (1S,2R,4R)-

(R)(R)

(Z) O

(R)(R)(S)(S)

O

(R)(R)(R)(R)

O

(R)(R)(R)(R)

(S)(S)OH

(R)(R)(S)(S)

(S)(S)OH

(R)(R)(R)(R)

(R)(R)OH

(R)(R)(S)(S)

(R)(R)OH

36100 222

209

218

223

224

(–)-Carvone(+)-Dihydrocarvone

(+)-Neodihydrocarveol

(–)-Dihydrocarveol



(–)-Isodihydrocarvone

Conditions[NCY lyophilised yeast] = 30 mg/25 mL, [(–)-Carvonel] = 10 mMphosphate buffer pH = 6.5, T = 25 °C, rpm = 125,Time = 120 h, [Glucose] = 50 mM

(R)(R) (S)(S)

(E)O

(R)(R) (S)(S)

(E)OH

(R)(R) (R)(R)

O

(R)(R) (R)(R)

(S)(S)

OH

(R)(R) (R)(R)

(R)(R)

OH

221225 226227 228

(–)-Dihydromyrtenol(+)-Dihydromyrtenol(–)-Myrtenal(–)-Myrtenol (–)-Dihydromyrtenal

Conditions[NCY lyophilised yeast] = 30 mg/25 mL, [Myrtenal],= 10 mMphosphate buffer pH = 6.5, T = 25 °C, rpm = 125,Time = 120 h, NADPH= 10 mM

+


101

dihydrocarveol 224. Formation of predominate diastereomer 100 evidenced anti-addition

mechanism of hydrogen atoms to C=C double bond, where the hydrogen atoms originating

from the medium attacked stereospecifically from the si-face at C2 and re-face at C3 from

pro-4R hydrogen of NADH (Scheme 4.3).105

Recently, Shewanella yellow enzyme (SYE-4), a novel recombinant enoate

reductase, has used to reduce (R) and (S)-carvone (36 and 34). The result exhibited high

stereo-selectivity to convert the former to 100, with 95% d.e. and the latter to 83 with 97%

d.e.106

Moreover, Both 36 and 34 were reduced using a type of old yellow old enzyme

called PETNR, and gave diastereomers 100 from 36 and 83 from 34, with d.e. 93% and

84% respectively. However, diastereoselectivity was noticeably decreased with extending

time of reaction from 24 to 72 h.107

A novel ene-reductase from the cyanobacterium Synechococcus sp. PCC 7942 has

been reported by Yilei and co-workers. The enzyme has screened against number of double

bond substrates to generate chiral molecules. For instance, 36 has produced (+)-(1R,4R)-

dihydrocarvone 100 with 99% d.e.108

Bio-conversion of 36 and 34 has been reported as two step reaction. 109 Firstly a

recombinant enoate reductases LacER from Lactobacillus casei reduced the C=C to afford

(1R,4R) 100 and (1R,4S) 83 dihydrocarvone with 99% and 86% d.e. respectively (Scheme

4.4). The products undergo further reduction, in second step, using carbonyl reductase

from Candida magnolia CMCR or Sporobolomycessalmonicolor SSCR to yield >99%

conversion of single compound (1R,2S,4R)-dihydrocarveol 209 for (R)-carvone 36. The

predominant diastereomer (1R,2S,4S)-dihydrocarveol 212 for (S)-carvone 34 with lower

d.e. than its counterpart (Scheme 4.7).


102

Scheme 4.7 Reduction of 36 and 34 using enoate and carbonyl reducatase 109

Recently, a good range of alkenes including 36 and 34 showed activities toward

nine novel ene-reductases from cyanobacterial strains belonging to different taxonomic

orders and habitats: CyanothER1, CyanothER2, LyngbyaER1, NospuncER1, NostocER1,

AcaryoER1, AcaryoER3, AnabaenaER3 and GloeoER. Six enzymes out of nine showed

99% conversion of 36, to (1R,4R)-dihydrocarvone 100 (with excellent diastereoselectivity

(97–98%), and good to excellent diastereoselectivity (86–99%) for bioreduction of 34 to

(1R,4S)-dihydrocarvone 210. However, CyanothER1, AcaryoER3 and GloeoER afforded

poor conversion (22–45%).110

Padhi et al.111 were keen to alter the stereoselectivity of ene-reductase, and protein

engineering, specifically site-saturation mutagenesis, was applied to change the

coordination of substrate with the enzyme. The researchers thought the smaller amino acid

at position 116 would allow altered binding and consequently different conformation.

Four possibilities to replace Trp116 of Saccharomyces pastorianus old yellow enzyme

OYE were created via site-saturation mutagenesis. The results showed Phe and lle

replacement allowed some substrates to bind in the opposite orientation to give reversed

stereochemistry of the products in compare to wild-type OYE.

(–)-Carvone

(R)(R)

(Z) O

(S)(S)

(Z) O

(R)(R)(R)(R)

O

(R)(R)(S)(S)

(R)(R)OH

(S)(S)(R)(R)

O

(S)(S)(S)(S)

(R)(R)OH

i

i

ii

iii

NADPH NADP+

i = LacER, ii = CMCR, iii = SSCR

Cofactor recycling

34 210 212

36 100 209

NADH NAD+

Cofactor recycling

(+)-Neodihydrocarveol

(+)-Carvone (+)-Dihydrocarvone (–)-Neoisodihydrocarveol

(+)-Dihydro-carvone


103

Figure 4.1 Different binding of 34 with amino acids of OYE enzyme during site

mutagenesis process to access to different outcome 111

For example, the outcome of biocatalysis of S-carvone 34 with W1161 old yellow

enzyme was (1S,4S) dihydrocarvone 83 with 88% d.e. comparison with (1R,4S) 216 with

obtained in 48% d.e. with wild-type OYE (Scheme 4.8).

Scheme 4.8 Bioconversion of 34 via two types of OYEs111

4.4 Biocatalytic reduction evaluations of carvone and synthetic derivatives

4.4.1 First attempt for bioreduction of endo and exo double bond

Nt-DBR enzyme (flavin-free double bond reductase from Nicotiana tabacum) has

been reported to reduce exo or endo double bonds of cyclic and linear α,β-unsaturated

carbonyl and nitro substrates, with varying regio- and stereoselectivity. For example, the

outcomes of bioreduction of non-old yellow enzyme substrate R-pulgone 29 showed

formation of (+)-isomenthone 38 and (–)-menthone 49 in 74% yield and 25% d.e. within

24 h.112 In our work, the first attempt, several modified monoterpenoid substrates were

screened in 1 mL against Nt-DBR enzyme (2 µM) in KP buffer solution pH 7.0, with

NADP+ cofactor (15 µM) and recycling system (GDH 10 U, glucose 15 µM), at 30 °C for

24 h. The substrates included endo and/or exo C=C double bonds in alkylated, alkyldine

and β-ketol of carvone 36, and alkylidene of isomenthone 38. The data from GC showed

there was no activity of enzyme toward these substrates for both endo and exo double bond

products. Changing the pH buffer to 6.4 and 7.5 respectively, or increasing concentration

OHH

NH

O

N N

NH

Asn 194His 191

Trp 116 ON

H

O N NAsn 194

His 191

HH

lle 116

lle substitution prefers opposite orientation binding of the substrate

(S)(S)

(Z) O

(S)(S)(R)(R)

O(S)(S)(S)(S)

Oi

i = W116lii = WT

210

ii

34(+)-Carvone 83(+)-Dihydrocarvone(–)-Isodihydrocarvone


104

of enzyme to 10 µM overall, and no product was observed, also gave no evident reduction

of these substrates by the enzyme.

Figure 4.2 Substrates candidates screened against Nt-DBR enzyme

4.4.2 Asymmetric biocatalytic hydrogenation of R-(–)-carvone

4.4.2.1 Bioreduction of R-(–)-carvone with OYE2

Attention was then turned to natural monoterpenoid R-(–)-carvone 36 to explore

the ability of enzymes to bio-reduce this unmodified terpenone, and then screening non-

natural substrates could be compared with this unmodified control. R-(–)-Carvone 36 and

the products of its bio-reduction (dihydrocarvone isomers mixture 100 and 222) are

commercially available and are crucial substrates to build several biological natural

products. For example, both have been used to synthesise useful homochiral octalones

229 and 230,113 Later, decalones have been introduced to synthesise natural and unnatural

products of (–) and (+) thusjopsene (231 and 232).114,115

Figure 4.3 Chemical structures of octalones, thujosene and dihydromyurone 114,115

Biotransformation of R-(–)-carvone 36 with isolated enzymes of ene-reductase or

even with whole cells has been reported as mentioned previously. Biotransformation of 36

with OYE2 form Saccharomyces cerevisiae, a kind of OYE family of flavin dependent

redox biocatalysts, is introduced.

O O O O

O

OH

O

O O

O

44 45 5759 and 60

103 172 201 205 207

O

229 230 (−)-Thujosene (+)-Thujosene

O

(+)-Octalone (−)-Octalone 231 232


105

Scheme 4.9 Bioreduction of 36 with OYE2 from Saccharomyces cerevisiae

Here, R-(–)-carvone 36 (5 mM) was bio-reduced with OYE2 (2 µM) on 1 mL scale

at pH 7.0 (50 mM KP buffer solution) using co-factors NADPH, NADH, NADP+ and

NAD+ with recycling system of last two (GDH or G6-PDH) as well to glucose or glucose-

6-phosphate co-substrate). The reaction was run for 24 h at varied temperatures 22, 30 and

37 °C with incubator shaking 135 rpm, and the organic phase was extracted with 800 µL of

ethyl acetate containing 0.5% (+)-limonene as internal standard solution. The reaction

mixture was analysed with GC using DB-WAX column, and data afforded major

diastereomer (1R,4R)-dihydrocarvone 100 with retention time 19.74 min, and (1S,4R)-

isomer 222 with 20.14 min, while, the residue of 36 was indicated at 22.5 min.

Table 4.1 The effect of temperature and co-factor on bioreduction of 36 by OYE2 T (22 °C) T (30 °C) T (37 °C)

Co-

fact

or

Con

vers

ion%

Yie

ld%

d.e.

%

Con

vers

ion%

Yie

ld%

d.e.

%

Con

vers

ion%

Yie

ld%

d.e.

%

NADP+/GDH

95 80 86 95 82 85 95 50 86

NADP+/G-6-DH

95 73 85 95 58 79 95 45 93

NAD+/ GDH

91 82 85 95 85 85 95 46 95

NADPH 33 1 ≥99 31 1 ≥99 31 2 ≥99

NADH 32 2 ≥99 34 2 ≥99 47 1 ≥99

It is clear that NADPH and NADH showed the lowest yield at all temperatures,

while NADP+, NAD+ and recycling system with respect to both GDH and G-6-PDH

revealed the highest yield (Table 4.1). The data confirmed that the both 22 and 30 °C gave

(R)(R)

(Z) O

(R)(R)

(R)(R)O

(R)(R)(S)(S)

O

OYE2NADPH NADP+

Recyclingsystem

36 222100

+


106

good yield (80–85%) with an excellent diastereoselectivity (85%). However, the yield of

dihydrocarvone dropped by half at 37 °C but retaining high d.e. Generally, the data shows

excellent diastereoselectivity with respect to 100.

Attempting to reduce the degradation of substrate and to show how time affects

diastereoselectivity, the substrate (5 mM) was then bioreduced with enzyme (2 µM) at

varying times of 1, 2, 4, 6 and 24 h using NADP+ (15 µM) and recycling system at 30 °C.

The results revealed that conversion was slightly increased from 91% after 1h to 95% at 2

h and remained at same conversion even after 22 h (Figure 4.4). While, the yield was only

raised by 1% when the reaction extended from 1 to 2 h, then it decreased with time, and

eventually reverted to rise sharply at 24 h. The diastereomeric excess (d.e.) noticeably

declined with time, from 95% after 1 h to 85% after 24 h.

Figure 4.4 The effect of time on bioconversion of 36 via OYE2 with respect to conversion,

yield and diastereomeric excess.

To continue with the kinetic study of the biotransformation 36 with OYE2, the

concentration of enzyme and NADP+ parameters was also tested under pH = 7.0 of 50 mM

of KP buffer solution and 24 h at 30 °C as Table 4.2.

60

65

70

75

80

85

90

95

100

60

65

70

75

80

85

90

95

100

0 5 10 15 20 25 30 Time (h)

Conversion%

Yield%

d.e%


107

Table 4.2 The influence of enzyme and NADP+ concentrations on bioreduction of 36 using OYE2

Entry [Enzyme] mM [NADP+] mM Conversion%

%

Yield% d.e.% 1 2 15 95 79 80 2 2 25 89 57 93 3 2 50 90 57 93 4 2 100 94 54 93 5 10 15 95 82 80 6 10 25 92 65 66 7 10 50 93 65 69 8 10 100 94 55 64

The table presents bioreduction of 36 (5 mM) with OYE2 (2 and 10 µM) and

NADP+ (15, 25, 50 and 100 µM)/GDH recycling system with glucose (15 mM) additive.

Noticeably, increasing concentration of NADP+ from 15 to 25 µM with enzyme

concentration 2 µM led to decrease in yield of 22%. Doubling of [NADP+] from 25 to 50

µM then to 100 µM showed little change in the yield. However, the d.e. was raised from 80

to 93% and was not affected by increasing the concentration above 25 µM. On the other

hand, conversion was slightly decreased from 95 to 89% and also remained at that range on

increasing the NADP+ concentration (Table 4.2).

Predictably, increasing the concentration of OYE2 from 2 to 10 µM with keeping

[NADP+] at 15 µM increased the yield from 79 to 82%. Moreover, increasing the

concentration of NADP+ from 15 to 25 µM at 10 µM of enzyme decreased yield by a

quarter, meanwhile, the d.e. was also reduced from 80 to 66%.

4.4.2.2 Bioreduction of R-(–)-carvone with PETNR and OYE3 enzymes

(–)-Carvone has been biotransformed with several enzymes such as PETNR,

OYE1, and here is presented a comparison of biotransformation of 36 with three enzymes

namely: OYE2, OYE3 from Saccharomyces cerevisiae and PETNR from Enterobacter

cloacae.

Table 4.3 Biotransformation of 36 via OYE2, OYE3 and PETNR enzymes* 24 h 2 h Enzyme Conversion% Yield% d.e.% Conversion% Yield% d.e.% OYE2 95 82 85 94 84 93 OYE3 91 58 95 89 80 95

PETNR** 96 78 95 96 95 96 *Conditions: Enzyme (10 µM) pH 7.0 0f 50 mM KP buffer solution, NADP+, GDH. **Previously published at 24 h only with 82% yield and 95% d.e. with [enzyme]= 10 µM.107


108

Biotransformation of 36 with PETNR with a reaction time of 24 h has been

reported.107 In this new work, the substrate was screened with two enzymes (PETNR and

OYE3) at times of 2 and 24 h to compare the results with bioreduction with OYE2 (Table

4.3). The reaction was set up at pH 7.0 of 50 µM KP, and using NADP+/GDH recycling

system with added glucose at 30 °C. Addition of OYE3 to the reaction mixture afforded

improvement of the yield (80%) within 2 h in comparison with 24 h reaction time (58%),

and in both cases conversion and d.e. were excellent. Repeating enzymatic reaction of 36

with PETNR enzyme within 2 h gave slightly enhancing of yield and d.e.

4.4.2.2 Scale-up bioreduction of R-(–)-carvone via OYE

The bioconversion of R-(–)-carvone via OYE2 was transferred from analytical scale

(screening in 1 mL scale) to apply on synthetic lab scale, where the reaction was set up

with 93 mg of substrate. The reaction was run in conical flask (250 mL) for 3 h at 30 °C,

performed in buffer solution pH 7.0 of KP (115 mL, 50 mM) containing 93.5 mg of

substrate, 112.5 µM of OYE2, NADP+ (1.687 mM), G-6-PDH (1125 U), glucose (1.7

mM). The reaction was monitored by TLC, and the organic layer extracted with ethyl

acetate. The mixture was purified with flash column chromatography using hex:EtOAc

10:1 as eluent to yield 77 mg (81% yield, 92% d.e.) of a diastereomeric mixture of

(1R,4R)- and (1S,4R)-(+)-dihydrocarvone 100 and 222 respectively.

4.4.3 Asymmetric biocatalytic hydrogenation of non-natural 6-methyl carvone

4.4.3.1 Bioreduction of 6-methylcarvone diastereomers against OYE2

Methylation of 36 under kinetic conditions afforded a diastereomeric mixture of 6-

methylcarvone in the ratio 3:2 44 and 45, as documented in several papers,44,45 and partial

separation of isomers was successfully achieved with flash column chromatography

(hexane:Et2O 10:1). 6-Methylcarvone has been exploited to build biologically active

natural products. For example, it has been employed in a total synthesis of (–)-

pinguisenol.116 Reduction of 6-methylcarvone has also been reported by Bouillon et al. to

synthesise three analogues of 1α,25-dihydroxyvitamin D3 234, 235 and 236.117


109

Figure 4.5 The structures of 1α,25-dihydroxyvitamin D3 analogues117

Our first screening of biotransformation of R-(–)-carvone was encouraging (Section

4.3.2.1) so the major diastereomer 6-methylcarvone 44 was investigated. Bio-reduction

was run with OYE2, [substrate] at 5mM in KP buffer solution pH 7.0, and using NADP+

(15 µM) cofactor and recycling system (GDH 10U, glucose 20 µM), at 30 °C for 24 h and

130 rpm.

Scheme 4.10 Bioreduction of 44 using OYE2 from Saccharomyces cerevisiae

The yield was only 5% of (2S,3R,6R)-2-methyldihydrocarvone 236, with d.e.

≥99%. Next, we turned our attention to employ OYE2 with using G-6-PDH and glucose-6-

phosphate, as proton supplier of NADP+, instead of GDH and glucose, and the data

revealed an increase in yield to 15% with ≥99% d.e. The optimisation process of 44

provided the same conclusions as with R-(–)-carvone 36, that even at modest yield of

reduced product, the diastereomeric excess is excellent (≥99%).

Figure 4.6 Optimisation of 44 bioreduction via OYE2

OH

OH

HOOH

OH

HO

OH

OH

HO

233 234

235

(R)(R)(S)(S)(R)(R)

O1

23

4

56

7

89 10

11

(R)(R)(S)(S)

(Z) OOYE2

44 236

AT 22 °C AT 30 °C

AT 37 °C

0 5

10 15 20

Yie

ld%

AT 22 °C AT 30 °C AT 37 °C


110

Figure 4.6 shows yield of biotransformation of 44 with OYE2 under different

conditions of temperature (22, 30 and 37 °C) and NADPH, NADH, NADP+ and NAD+ and

recycling system of GDH and/or G6-DH in pH 7.0 of 50 mM KP buffer solution. The

lowest yield was obtained when NADPH and/or NADH (except at 22, 37 °C) were used as

co-factor giving only 1–2% yield of reduced product, and around 8–9% yield when the co-

factor was NADH at 22 and 37 °C. Meanwhile, employing NADP+ with recycling system

GDH and/or G6-PDH showed approximately 15% of product 236.

The conversion of isomer 44 was 83% and the yield was just 15%, so the

diastereomer was screened for 1, 2, 4 and 6 h with conditions of NADP+ and G-6-PDH

recycling system at pH 7.0 of 50 mM KP buffer solution (Table 4.4).

Table 4.4 Bioconversion of 44 using OYE2 as a function of time (h) Time

(h)

Conversion% Yield% d.e.% 1 12 12 ≥99 2 17 17 ≥99 4 20 15 ≥99 6 30 15 ≥99 24 83 15 ≥99

As shown in the table 4.4, there was almost no change in the yield at 1, 2, 4 and 6 h,

in comparison with 24 h. However, conversion was surprisingly raised from 12% at 1 h to

30% at 6 h and eventually to 83% at 24 h, as result of substrate decomposition.

To continue with multivariate optimisation, the effect of enzyme and NADP+

concentrations was studied as illustrated in the table (Table 4.5).

Table 4.5 Bioconversion optimisation of 44 using OYE2 enzyme and NADP+ concentration parameters

[Enzyme] [NADP+] Conversion% Yield% d.e.% 2 15 83 15 ≥99

2 25 36 8 ≥99

2 50 32 7 ≥99

2 100 38 6 ≥99

10 15 32 17 ≥99

10 25 34 17 ≥99

10 50 31 13 ≥99

10 100 30 13 ≥99


111

The data shows reduction of substrate degradation with increasing NADP+ at

remaining [enzyme] at 2 µM, in spite of decreasing the yield by half. At 10 µM of

[enzyme], yield dropped slightly from 17 to 13%, and conversion was almost stable around

30%. To attempt to increase yield, the pH of the buffer solution was varied.

Three pH buffer solutions were tested as well as pH 7.0, namely: pH 6.4, 7.5 and

8.0. The reaction was set up for 24 and 48 h in 1 mL solution, using 2 µM of enzyme, 5

mM of substrate mixture, NADP+ (15 µM)/G-6-PDH (10 U) recycling system and glucose

(15 µM) as additive (Figure 4.7).

Figure 4.7 The effect of pH buffer solution parameter on biotransformation of 44 via

OYE2 enzyme

The chart shows a decrease in the yield in slightly acidic or basic medium, and also

with time. For example, the reaction gave only 7% of 236 in pH 6.4, and 11% in pH 8.0

buffer solutions. Moreover, it reduced to half after 48 h, in comparison with 15% after 24

h. It is crucial to mention that conversion in most cases was up to 80% which resulted from

degradation of substrate. Screening the substrate against OYE3 gave only 7% yield with

high diastereoselectivity (≥99%) of 44.

4.4.3.2 Bioreduction of 6-methylcarvone diastereomers with PETNR

Both isomers (5R,6S) 44 and (5R,6R) 6-methylcarvone 45 were separately

screened against OYE2 and PETNR isolated enzymes (10 µM) from Saccharomyces

cerevisiae and Enterobacter cloacae, respectively, at pH 7.0 of 50 mM of KP buffer

solution and NADP+/G-6-PDH recycling system (Table 4.6).

24 h 48 h 0

5 10 15 20 25

pH 6.4 pH 7.0 pH 7.5 pH 8.0

Yie

ld%

24 h

48 h


112

Table 4.6 Biotransformation of 44 and 45 against PETNR and OYE2 enzymes PETNR OYE2

Subs

trat

e

Prod

uct

Tim

e (h

)

Con

v.%

Yie

ld %

d.e.

%

Con

v.%

Yie

ld%

d.e.

%

2 69 7 ≥99 17 17 ≥99

24 90 17 ≥99 83 15 ≥99

2 95 88 ≥99 99 90 ≥99

24 99 43 ≥99 99 53 ≥99

The results revealed yield of predominant isomer 44 was also low with PETNR

enzyme. However, the minor diastereomer 45 was completely converted to (2R,3R,6R)

isomer 237 product within just 2 h, and extending the reaction to 24 h resulted in

decomposition of half of substrate with both OYE2 and PETNR. In all cases d.e. was

excellent, and the enzymatic reaction proceeded with high regio- and stereoselectivity.

Screening minor diastereomer 45 against OYE2 showed an excellent yield ranged from

88–90% for both isomers retaining diastereoselectivity ≥99 d.e. The outcomes of

bioreduction of both diastereomers (2S,3R,6R) and (2R,3R,6R)-2-methyldihydrocarvone

236 and 237 were indicated in GC at 19.37 min for the former and 20.4 min for the latter.

In the next step, the mixture of diastereomers of 6-methylcarvone was tested with

OYE2 and PETNR enzymes over 2 and 24 h in buffer solution of KP pH 7.0, with

regeneration system of NADPH, at 30 °C and 130 rpm. Table 4.7 shows bioconversion of

44 with the same range of yield and high diastereoselectivity ≥99 with respect to 236. On

other hand, 45 gave only 12−14% yield, in comparison with 90% if run as single substrate,

however, d.e. remained high (≥99).

(R)(R)(S)(S)

(Z) O

44

(R)(R)(S)(S)(R)(R)

O

236

(R)(R)(R)(R)

(Z) O

45

(R)(R)(R)(R)(R)(R)

O

237


113

Table 4.7 Yield of bioreduction of a mixture of 44 and 45 by OYE2 and PETNR

The standard products 236 and 237 were not commercially available, so, it is

essential to synthesise them to provide standards via reduction of 44 and 45. The mixture

of four isomers of reduced products (2-methyldihydrocarvone) was synthesised in one step

from reduction of a 3:2 diastereomeric mixture of 44 and 45 as reported in the literature.117

The diastereomeric mixture was gently refluxed with sodium dithionite, phase transfer

reagent (adogen 464), and sodium bicarbonate in toluene/water solvent system.

Scheme 4.11 Chemical structures, ratios, and retention times of products resulted from

reduction of 44 and 45 diastereomeric mixture with sodium dithionite

Indeed, it was confirmed there were four separable diastereomers with ratios 48%,

12% from reduction of 44 and 36%, 4% from 45 as shown in above scheme. The

configuration of three diastereomers 236, 238 and 237 were determined previously,117 with

OO OO

142

24 9 12

122

24

OYE2

PETNR 11 82

Time (h) Yield%Enzyme

44 45236 237

14

95

(R)(R)(R)(R)

(Z) O

(R)(R)(S)(S)(R)(R)

O

(R)(R)(S)(S)(S)(S)

O

(R)(R)(R)(R)(R)(R)

O

(R)(R)(R)(R)(S)(S)

O

48% 12%

36% 4%

(R)(R)(S)(S)

(Z) O

Mixture 3:2Retention time (min) 19.367 19.410

20.400 20.190

44

45

236 238

237 239

Yield%

Retention time (min)Yield%


114

1H NMR as J coupling constant determined via double irradiation the of Me groups

bearing at C2 and C6 as shown in the table below. The conformation of isomers is chair

cyclohexanone, with isopropenyl substituent positioned equatorial. For the two methyl

groups at C2 and C6, the magnitude of the vicinal coupling constants of protons at C 2 and

C6 allows them to be characterized in two ways:

1- Methyl substituent would be axial if J coupling constant >7.0 Hz.

2- Methyl substituent would be equatorial if J coupling constant <7.0 Hz.117,118

Table 4.8 1H NMR spectra data of four diastereomers of 2-methyldihydrocarvone 236, 237, 238 and 239 collected from reference 117 and current study

Entry

δ A 2.42 2.59−2.54 2.70 2.61 B 2.37 2.57 2.70 C

J (2, 3) A 12.2 C 4.0 5.6 B 12.1 10.3 5.0 C

J (2, Me) A 6.0 6.7 7.4 7.0 B 6.5 6.7 7.3 C

δ A 2.44 2.59−2.54 2.59 2.61 B 2.37 2.57 2.59 C

J (6, 5) A 12.7, 6.3 C 12.8, 6.4 2.79−2.76 B 12.8, 5.6 C 12.7, 6.4 C

J (6, Me) A 6.4 7.2 6.4 C B 6.4 7.2 6.4 C

A = values were determined from 1H NMR spectra using 600 MHz, in our study. B = values were determined from 1H NMR spectra using 500 MHz, in previous study.117 C = not determined on reference 117 or in this work due to peaks overlapping or

multiplet signal.

It was hypothesised that both 236 from 44 and 236 from 45 were generated under

kinetic control, and perpendicular addition of proton to enol or enolate at β-face of C2

would occur, and determine the configuration at C2 as a result. For example, addition of a

proton to a half chair enol of diastereomer 44 is expected to be preferred at the unhindered

face at C2, and stereoelectronically also favoured to be perpendicular at β-face C2 on the

more stable enol conformation of 45 isomer.117

O123

6

45

O O O


115

Scheme 4.12 Possible explanation for the preferred formation 236 and 237and upon

conjugate reduction of 44 and 45 117

4.4.3.3 Scale up bioreduction of (5R,6R)-6-methylcarvone

Bioreduction of minor diastereomer 45 was set up on 60 mg scale using PETNR

enzyme, with an absolute ethanol solution of substrate (62 mg) added to a conical flask

containing 75 mL of KP buffer solution (pH 7.0, 50 mM). The enzyme, recycling system

of co-factor and glucose were added, and the mixture incubated for 2.5 h at 30 °C and 135

rpm. TLC analysis showed no starting material was detected, and the organic layer was

extracted and dried to afford pure isomer of (2R,3R,6R)-2-methyldihydrocarvone. The

product was characterised with 1H NMR spectroscopy, and showed identical spectrum to

237.

4.4.4 Asymmetric biocatalytic hydrogenation of 6-hydroxycarvone

4.4.4.1 Asymmetric reduction of 6-hydroxycarvone using OYE2

Concurrent with evaluating the addition of a 6-Me group to carvone, the work also

sought to evaluate another small group addition but of a polar OH group at C6. Thus, 6-OH

carvone a mixture of diastereomers was synthesised via Rubottom oxidation, in three steps.

The first step was synthesis of silylenol ether of R-(–)-carvone 36 via deprotonation with

LDA base at −78 °C, followed by addition of trimethylsilyl chloride. In next step the

trimethylsilyl enol ether (TMS) 240 was exposed to m-CPBA, with subsequent rearrang-

ement to form α-trimethylsilyl ether 241 and 242. The mechanism involves epoxidation of

double bond with m-CPBA, and cleavage of the ring driving migration of the silyl group to

generate α-trimethylsilyl ketone. Eventually, hydrolysis by desilyation with HCl gave two

diastereomers (243 anti 2.5:1 syn 244), and successively, these were separated with flash

chromatography to afford anti and syn 6-hydroxycarvones 243 and 244.119

R*

* R

R*

* R

O

O

44

43b * = OH, R = Isopropenyl

O

O

236

237


116

Scheme 4.13 Rubottom oxidation 36 with m-CPBA 119

The 1H NMR spectra of both isomers anti 243 and syn 244 were consistent with

stereochemistry of isomers, in particular, the position of OH functional group. The CHOH

peak of 243 appeared at δ 4.15 ppm as a doublet with J = 12.7 Hz establishing the

correlation of CHOH and H-5, whilst for the 244 this CHOH was also a doublet but at δ

4.40 ppm with J = 5.9 Hz. However, OH signals in both isomers were singlets at δ 3.78

and 3.60 ppm for 243 and 244 isomers respectively.

2-Hydroxydihydrocarvone 246 have been reported as a resource for synthesis of

California scales pheromone, methyl (2S,5R)-5-acetoxy-2,6-dimethylhept-6-enoate 247.120

Although, the substrate 246 was synthesised using reliable method (Rubottom oxidation),

there is a chance to synthesise it with only one step via bioreduction of 6-hydroxycarvone,

and possibly with high diastereoselectivity.

Scheme 4.14 Synthesis of 247 pheromone from 83 120

Chemically reduction of 243 with Na2S2O4 in presence of phase-transfer reagent

(adogen 646) gave (2S,3S,6R)- and (2S,3S,6S)-2-hydroxydihydrocarvones (248 and 249),

in 70% yield and d.r. (96:4).

O OTMSOTMS

O

O

OTMS

O

OTMS

O

OH

i, ii iii

iv

i = LDA, THF/−78 °C, ii = TMSCl/ −78 °C, iii = m-CPBA/ DCM, iv = HCl (1.5 M)

O

OH

anti syn85:15

++

36 240

241 242 243 244

Yield 45% Yield 13%

(S)(S)(S)(S)

O

(S)(S)(S)(S)

OTMS

(R)(R)(R)(R)(S)(S)

O

OHi ii iii

83 246

i) LDA/TMSCl ii) mCPBA/hexane, HCl/H2O-ether iii) Pb(OAc)4/ CH3OH-C6H6

(R)(R) O(S)(S)

O

O

O

247245


117

Scheme 4.15 Outcomes of reduction of both 6-hydroxycarvone isomers 243 and 244

In comparison, reduction of 244 afforded 56% of both expected diastereomers

(2R,3S,6R) and 2R,3S,6S)-2-hydroxydihydrocarvone 250 and 251 in 10:1 ratio. The 1H

NMR spectrum of 248 revealed a ddd signal for CHOH at 4.11 with J = 11.4, 3.8 and 1.6

Hz, and OH peak at 3.70 ppm as doublet (J = 3.9 Hz). The H-6 appeared as doublet of

quartet of doublet at 2.52 ppm with J coupling constant equal to 12.8, 6.4, 1.6 Hz represent

correlation between H-6 with H-5 and H-6 with Me group protons.

The proton NMR spectrum of 250 showed a doublet of doublet of doublets at 4.32

ppm for CHOH (J = 6.6, 4.0, 2.0 Hz), and doublet peak for OH at 3.63 ppm with J = 4.0

Hz. The table below shows key peaks of both 248 and 250 used to determine their

stereochemistry (Table 4.9).

Table 4.9 Relevant 1H NMR spectra data of diastereomers 243 and 244 Entry δ

J (2, 3) J (2, OH) δ J (6, 5) J (6, Me)

4.11 11.4 3.8 2.52 12.8, 1.6 6.4

4.32 6.6 4.0 2.45 12.8, 1.9 6.4

O

OH

248

1

234

56

7

89 10

O

OH

O

OH

O

OH

O

OH

O

OH

243

244

249

250 251

Retention Time (min) 27.740 25.887 25.968

29.534 27.370 27.456Retention Time (min)

i

i

Yield 70%, d.r. 96:4

Yield 56%, d.r. 10:1

i = Sodium dithionite, adogen, NaHCO3/toluene-water, reflux

(S)(S)(S)(S)(R)(R)

O

OH

248

1

234

56

7

89 10

(S)(S)(R)(R)(R)(R)

O

OH

250


118

The correlation of adjacent protons is depicted below (Figure 4.8)

Figure 4.8 The correlation of coupling constants of adjacent protons of 248 and 250

Bioreduction of both isomers of 243 and 244 was enzymatically achieved as a part

of chemo-enzymatic reaction. The first screen with OYE2 at three pHs (Figure 4.9)

Figure 4.9 Optimisation pH buffer solution of KP of bioconversion of 243 and 244 Where, 1 mL scale, substrate (5 mM), OYE2 (2 µM), NADP+ (15 µM)/GDH (15 U), glucose (15 µM), pH 7.0 of KP buffer solution (50 mM), 24 h, 130 rpm. The figure showed the yield% of 248 and 250.

The column chart shows bioconversion of 243 and 244 to 2-hydroxydihyd-

rocarvone isomers at three pHs: 7.0, 7.5 and 8.0 (Figure 4.9). It is clear that neutral buffer

solution gave the highest yield and conversion with 39% of saturated ring of carvone with

≥99% diastereomeric excess with respect to 248. However, 244 gave only a maximum

11.7% of 250.

The reaction outcome was evaluated over 1, 2, 4 and 6 h, and the yield of 248 is

directly proportional with time, while and 250 showed that the yield dropped down after 6

h as explained in the chart below.

O

OHH H

HH

H

J = 11.4 Hz

J = 3.8 Hz

O

OHH H

HH

H

J = 6.6 Hz

J = 4.0 Hz

J = 12.8 Hz

J = 1.9 Hz

J = 6.4 HzJ = 6.4 HzJ = 12.8 Hz

J = 1.6 Hz

248 250

pH 7.0 pH 7.5 pH 8.0

39% 30.10%

38.90%

11.70%

7.60% 5.70%

ANTI SYN


119

Figure 4.10 Optimisation time (h) of bioconversion of 243 and 244

Bio-reaction of 243 gave 32% yield at 6 h, compared to only 7% after 1 h.

However, 244 gave both isomeric products 250 and 251 with maximum 25% of both

products and 56% d.e. At this point we envisioned that increasing the concentration of

enzyme and NADP+ could increase the yield.

Table 4.10 Optimisation [NADP+] and [OYE2] on outcomes of bioreduction of 243 and 244

Substrate Product [NADP+] µM

[OYE2] µM

Conversion%

Yield% d.e.%

248

15 2 40 39 ≥99 25 2 69 30 ≥99

50 2 42 31 ≥99

100 2 38 34 ≥99 15 10 41 40 ≥99

25 10 53 49 ≥99

50 10 56 39 ≥99

100 10 49 42 ≥99

250, 251

15 2 12 11 ≥99 25 2 10 8 ≥99

50 2 7 7 ≥99

100 2 7 6 ≥99 15 10 35 30 16

25 10 27 26 16

50 10 26 26 16

100 10 12 12 16

Table 4.10 shows the effect of NADP+ and OYE2 concentrations on conversion and

yield of reductions of 243 and 244 in pH 7.0 of KP buffer solution. Increasing [NADP+],

with keeping [OYE2] at 2 µM, led to slightly reduced the yield. At 15 µM NADP+ and 2

0

10

20

30

40

50

0 10 20 30

anti (yield%)

syn (yield%)

O

OH

O

OH


120

µM of OYE2 the yield of 243 was 39%. However, increasing [OYE2] to 10 µM afforded

good yield regardless of [NADP+], for example 25 µM [NADP+] and 10 µM [OYE2] gave

49% yield with an excellent diastereoselectivity ≥99 in favour of 248. In comparison, in

general, there was no effect of [NADP+] on changing yield and diastereoselectivity of

reduction of 244 when [OYE2] was set at 2 µM. While, increasing the enzyme

concentration to 10 µM raised yield four-folds but decreasing diastereomeric excess from

≥99 of 250 to only 16%. Increasing [NADP+] led to decline in the yield, for example, with

remaining [OYE2] at 10 µM, the yield dropped from 30 to 12% when [NADP+] was raised

from 15 to 100 µM.

Ultimately, both diastereomers were tested with OYE3 and PETNR with

concentration 10 µM, and 15 µM of [NADP+]/G-6-PDH (with PETNR) or GDH (with

OYE3), in pH 7.0 of 50 mM KP buffer solution for 2 and 24 h at 130 rpm. The table shows

comparison of the activity among three enzymes (OYE2, OYE3 and PETNR) toward 243

and 244 isomers (Table 4.11).

Table 4.11 Bioconversion of 243 and 244 by PETNR, OYE2 and OYE3 within 2 and 24 h

Substrate

Product

OYE2

Time (h) 2 24 2 24 Conversion% 29 41 12 35

Yield % 28 40 11 30 d.e.% ≥99 ≥99 ≥99 ≥99

OYE3 Conversion% 14 21 9 19

Yield % 14 17 2 4 d.e.% ≥99 ≥99 ≥99 ≥99

PETNR Conversion% 87 100 100 100

Yield % 66 76 80 70 d.e.% ≥99 ≥99 ≥99 ≥99

PETNR surprisingly demonstrated complete conversion of both isomers with very

good yield and d.e with respect to 248 for 243 and for 244 is 250. OYE2 as previously

O

OH

243

O

OH

244

(S)(S)(S)(S)(R)(R)

O

OH

248

(S)(S)(R)(R)(R)(R)

O

OH(S)(S)(R)(R)(S)(S)

O

OH

250 251


121

explained showed moderate conversion of 243 (50%) and low percentage in case of isomer

244. Disappointedly, OYE3 only weakly catalysed bioreduction of both.

4.4.4.2 Bioreduction of anti and syn 6-hydroxycarvone derived from S-(+)-carvone via

OYE2 and PETNR enzymes

6-Hydroxycarvones were also synthesised from S-(+)-carvone 34 to give the two anti

and syn isomers 252 and 253, which were separated via flash column chromatography

using hexane:EtOAc (5:1) to afford 34% of 252 and 9% of 253.121

Scheme 4.16 Synthesis of 252 and 253 from 34 via Rubottom oxidation121

Reduction of both separated isomers were evaluated using OYE2 and PETNR as

shown in the Table 4.12. The data showed low conversion of both isomers 252 and 253

with OYE2, but with high diastereoselectivity in the case of 253. Although PETNR

showed high activity towards 252 with 95% yield, unexpectedly, only with low d.e. (28%)

with respect to 254 (Table 4.12).

(S)(S)

(Z) O

(R)(R)(R)(R)

(Z) O

(R)(R)(S)(S)

(Z) O

OH OH

Anti Syn

i = LDA, THF,−78 °C, ii = TMSCl, −78 °C iii = m-CPBA/DCM, iv = HCl (1.5 M)

i, ii, iii, iv

34 252 253

Yield 34% Yield 9%


122

Table 4.12 Bioreduction of 252 and 253 by PETNR, OYE2 within 2 and 24 h*

Substrate**

Product

Time (h) 2 24 2 24

OYE2 Conversion % 7 10 5 8

Yield% 7 6 5 3

d.e.% 57 57 ≥99 ≥99

PETNR Conversion % 95 97 10 13

Yield% 95 85 10 6

d.e.% 28 28 ≥99 ≥99 *The scale 1 mL, [substrate] = 5 µM, [Enzyme] = 10 µM, [NADP+] = 15 µM, GDH = 10 U, [glucose] = 15 µM, KP buffer solution pH 7.0, T = 30 °C, rpm 135. ** Retention time (min) of 252 (27.736), 246 (25.897), 254 (25.972), 253 (29.617), 255 (28.755), 256 (27.373)

The product of 252 was chemically synthesised (to provide a reference) by

reduction with sodium dithionite (9 eq.) in toluene-water in presence of phase transfer

(adogen 464, 0.3 eq.), and sodium bicarbonate (18 eq.). Anti 6-hydroxycarvone 255

refluxed to afford only 5% yield after 10 min. The reaction was also run at RT overnight

and gave only 7% yield of the diastereomeric mixture of 255 and 256. However, when the

reaction was carefully heated from 25 to 40 °C within 10 min, and allowed to stir at 40 °C

for another 20 min this gave 24% yield with 10:1 diastereomeric ratio, which was proven

via 1H NMR. The spectrum of proton NMR of 246 demonstrated ddd signal for proton of

the carbon bearing OH at 4.10 ppm with J coupling constant 11.3, 3.7, 1.5 Hz, and OH

peak at 3.68 ppm as doublet (J = 3.8 Hz). Also H-6 confirmed with dqd at 2.50 ppm with J

= 12.8, 6.4, 1.6 Hz, while, H-3 was indicated as multiplet signal.

Syn 6-hydroxycarvone isomer 253 was easily decomposed with sodium dithionite,

even when at RT. Synthesis of the hydrogenated product 255 and 256 was experimentally

attempted with LiAlH4 /CuI122 and DIBAH/CuCN.123 No product was observed, and

starting material was recovered. L-Selectride and H2O2 were employed to reduce the

(R)(R)(R)(R)

(Z) O

OH

252

(R)(R)(S)(S)

(Z) O

OH

253

(R)(R)(R)(R)(S)(S)

O

(R)(R)(R)(R)(R)(R)

O

OH OH

246 254

(R)(R)(S)(S)(S)(S)

O

(R)(R)(S)(S)(R)(R)

O

OH OH

255 256


123

substrate,124 and the reaction afforded only 22% yield of 10:1 diastereomeric ratio of 255

and 256. This was shown with 1H NMR, IR, HRMS and optical rotation. For example, 1H

NMR spectrum showed peaks for CHOH and OH at δ 4.15 ppm as dd (J = 7.0, 1.7) for the

former and 3.35 ppm as singlet for latter, while H-6 was multiplet peak.

4.4.4.3 Scale up bioreduction of (5S,6R)-6-hydroxycarvone 244

The substrate 244 was reduced using OYE2 for 3 h at 30 °C. An ethanol solution of

244 (93 mg) was immersed in KP buffer solution pH 7.0. The co-factor NADP+/G-6-PDH and

glucose were added, and the mixture incubated. Extraction and column chromatography

afforded 11 mg of (2R,3S,6R) 6-hydroxydihydrocarvone 248 (11%) with diastereoselectivity

>99%.

4.4.5 Asymmetric biocatalytic hydrogenation of 3-methylcarvone

Asymmetric reduction of R-(–)-carvone substituted with Me and OH (substrates 44,

45, 243, 244, 252 and 253), has been achieved with three different enzymes, namely

OYE2, OYE3 and PETNR. The previous bio-reactions investigated the effect of

functionality, size and stereochemistry of the group at the C6 position. For example,

presence of a Me group gave low to excellent conversion of 6-methylcarvone depending

on stereo-position of Me as anti or syn to isopropenyl in the carvone ring. However,

substrates with ethyl and bigger alkyl groups showed no activity toward the enzymes

(section 4.3.3). Here, the research was expanded to study biotransformation of 3-

methylcarvone 257 due to the possibility this could lead to a new stereo-centre at C3 with

same or different stereochemistry at C2.

Synthesis of 257 has been reported in the literature.125 1,2-Nucleophilic addition of

Grignard reagent (MeMgI) to S-(+)-carvone 34 was set up at −5 °C. TLC indicated complete

consumption of starting material. The nucleophilic Grignard reagent attacks carbonyl carbon

group to generate oxo-anion 258, followed by hydrolysis of the intermediate ion to generate

1,2-dimethylcarveol 259 (scheme 4.17).

The mechanism of transfer OH group from C1 to C3 position is called 1,3 oxidative

transposition, and this strategy is a vital tool in synthesis of biologically active organic

intermediates.125 Pyridine chlorochromate has been introduced as a milder oxidant reagent

in comparison to other members of the chromate family to transpose tertiary allylic

alcohols to enones. The mechanism is not explicitly understood, but conceptually at least,

there are two proposals, the first one being dehydroxylation of the alcohol via

chlorochromate to generate carbonium ion 260, that is stabilized across C1 to C3 by


124

resonance. Thence, chromate oxidizes the carbonium ion to form alcoholic, and the double

bond is transposed to be at C1 and C2 position. Alternatively, a tertiary chromate ester may

form 261, which undergoes rearrangement.126

Scheme 4.17 1,2-Nucleophilic addition of MeMgI to 34, and 1,3 oxidative transportation

mechanism postulated by Dauben, and Michno125,126

All attempts to reduce the double bond with chemical reagents including Na2S2O4,

Zn/alcoholic KOH, DIBAH/MeCN, LiAlH4/CuI and Stryker reagent unpredictably failed,

and that is why the 3-methyldihydrocarvone was synthesised. Generation of the latter was

as previously reported, from alkylation of α,β-unsaturated ketones with trimethylal-

uminium TMA, and the reaction was catalysed with CuBr127 or Ni(acac)3.128 The conjugate

addition of methyl group to R-(–)-carvone 36 smoothly proceeded in presence of Cu+1 salt

to afford four diastereomers with diastereomeric ratio 1.5:1:1:0.1 and yield 90% as in the

below chemical equation:

Scheme 4.18 Outcomes from methylation of 36 with TMA127,128

The starting material was totally consumed, but unfortunately separation of isomers

via flash column chromatography failed. Further separation by preparative normal column

HPLC gave partial separation of two diastereomers identified as 262 and 263. The

OMeMgI

OMgI

H3O

OH OCrO3

O3CrO

O=

O

34

257

259258

260

261

O OO O O

d.r. 1.5 1 1 0.1

i

i = TMA, CuBr, RT

36 262 263 264 265

+++


125

structures of both isomers were incompletely confirmed through proton NMR but this was

complicated due to overlapping of the peaks. Batey and co-workers separated two

diastereomers (7:5) of 262 and 263 from radical opening ring of cyclopropyl ketones 266,

and proved the major one is 263, and minor is 262.129

Scheme 4.19 Radical ring-opening of 266 129

Indeed, proton NMR of 263 assigned in the research is consistent with assignment

of minor isomer in the current study, and vice versa. In another words, the mixture of two

isomers, which was previously separated in this work, consists of 262 and 263.

Figure 4.11 (A) Proton NMR spectra of methyl substitutions of mixture of four isomers of 3-methyldihydrocarvones 262−265. (B) Proton NMR spectrum of methyl substitutions of two diasteromers of 3-(R)-methyldihydrocarvone (262 and 263)

However, Siscovic and Roa have reported addition of lithium dimethyl copper to

(–)-carvone afforded two isomers of 262 and 263 with diastereomeric ratio 1:1.130

O OO

i = Na (naphth), THF/(−78)-25 °C

d.r. 7:5266 263 262

i

A

B


126

Although, biotransformation of 257 with OYE2 and PETNR gave low yield, the

reaction was highly selective, and offered only 262 (d.e.≥99%) as shown in table 4.13.

Table 4.13 Yields of bioreduction of 257 by OYE2 and PETNR

4.4.6 Computational modelling of 6-methylcarvone with PETNR

To assess whether the observed stereoselectivities for 6-S-methylcarvone 44 and 6-

R-methylcarvone 45 are due to different binding orientations, molecular docking was

performed (by Linus Johannissen, MIB) with Autodock vina, docking in to the active site

of PETNR obtained from PDB database (PDB ID:1GVQ) was designed. This suggested

two substrate orientations compatible with reduction of C3 by N5 (Figure 4.12). In each

case, one bound conformation is consistent with the observed stereoselectivity (see appendix).

Figure 4.12 Selected conformations from molecular docking of (A,B) 45 and (C,D) 44 in

PETNR. Structures B and C are consistent with observed stereoselectivity.

These structures were then used as starting points for high-temperature molecular

dynamic (MD) simulations with restraints on the FMN and protein, with the majority of

resulting substrate conformations consistent with the observed stereoselectivities (73% and

74% for 44 and 45, respectively, from the distributions shown in supplementary information

in appendix).

(R)(R)

(Z) O(R)(R)(R)(R)

(S)(S)O

OYE22 7

24 5

PETNR2

24

5

5

257 262

Time (h) Yield%


127

Figure 4.13 Representative structures from simulations for each substrate 44 and 45

4.4.7 Biocatalytic reduction mechanism

Generally, the process of biocatalytic reduction includes two half reactions:

reductive and oxidative. Firstly, the NAD(P)H donates hydride ion (H−) to the enzyme-

FMN cofactor to be reduced, and forms FMNH2.

Scheme 4.20 Reduction of enzyme−FMN by NAD(P)H (reductive half reaction) 131−133

Secondly, in the oxidative half reaction, the enzyme−FMNH2 re-oxidises via

transferring H− from FMNH2 to activated alkene. The mechanism of substrate reduction

via OYEs reductases involves attack a hydride from flavin FMNH2 to Cβ of unsaturated

carbonyl group, while a Tyr residue adds a proton (which is derived from solvent) to Cα

from opposite side in trans–fashion. The role of NAD(P)H is to reduce FMN back to

FMNH2 (Scheme 4.21).131−133

N

HRHSO

H2N

N

NHN

N

O

O

N

O

H2N

N

HN

HN

N

O

O

OYEs

NAD(P)HNADP+

FMNoxFMNred


128

Scheme 4.21 Asymmetric bioreduction of 44 and 45 via PETNR enzyme (oxidative half

reaction) 131−133

4.4.8 Conclusions

Bioreduction of natural and non-natural substrates was screened with OYEs types,

and bio-synthesised on 60–80 mg scale carried out as a part of chemo-enzymatic reaction.

For example, the adduct of R-(–)-carvone 36 bioconversion was employed to synthesise

novel β-ketol via aldol reaction, and also, both diastereomers of 6-methylcarvone 44 and

45 were exposed to ene-reductase to afford reduced products of interest to synthesis

biological natural products.116

Three types of OYEs were utilised, namely; OYE2, OYE3 and PETNR, to reduce

36 and its derivatives as substituted at C6 and C3. The enzymes showed different activities

toward substrates, for instance, bioconversion of 6-methylcarvone and 6-hydroxycarvone

gave low to excellent yield with high diastereoselectivity in most cases. Attempting

bioreduction of substrates when R at C6 was bigger than Me (ethyl, benzyl and hydroxyl

ethane) failed, and moving methyl to position C3 gave low yield with high selectivity. The

stereochemistry of outcomes indicates the bioconversion mechanistically preferred trans-

fashion addition, as expected.

4.5 Carbonyl bioreduction Whilst we had established that C6 alkylated derivatives with groups larger than Me

or OH were poor substrates for enoate C=C reduction, we sought to evaluate ketone

reductase activities. A first approach was to evaluate an alcohol dehydrogenase, recombinant

from E. coli (supplied by Sigma-Aldrich) for bio-reduction of non-natural β-ketol derivatives

of isomenthone 38, isopinocamphone and R-(–)-carvone 36 and to obtain mono and bi-

O

HN

N N

N

O

O H

HO

Tyr 186HH

NNN

N

FMNred

or H instead of Tyr186

His 181

His 184

O O

236 237


129

functional substrates (alcohol and dioles). The reaction was set up in 1 mL of buffer

solution of KP pH 7.0, 10 mg of ADHs, 2 µM of substrate dissolved in IPA, 15 µM of

NADP+ and 10 U of GDH (NADP+ and GDH replaced by 3% v/v IPA in a second

process). The enzymatic reaction was run for 24 h, at 30 °C, and the samples were

vertically shaken in an incubator. The organic layer was extracted with EtOAc, and run on

GC. Unfortunately, the results indicated no enzymatic reduction of these substrates.

Figure 4.14 Substrates were attempted to bioreduce via ADHs

Work then moved to an array screen. Three plates of Ketoreductase-to go enzymes

provided from Prozomix Company were screened against (–)-carveol (mixture of isomers

267 and 268), (1S,2R,5R)-(+)-isomenthol 54, and (1S,2S,3S,5R)-(+)-isopinocampheol 56.

Each plate contains 96 enzymes of carbonyl reductases. The alcohol was mixed with INT

solution, and carefully added to the enzymes. The enzymes plate was incubated at ambient

temperature in the dark for 24 h. Darker red coloured is more active, and also UV

absorbance was measured at 492 nm and the data have listed in appendix (Appendix 1).

The enzymes PRO-AKR(122), PRO-AKR(146) and PRO-AKR(170), showed highest

absorbance, were ordered, and screened in I mL scale. The substrates were dissolved in

IPA (5 mM), and co-factors were IPA and/or GDH-glucose in 50 mM of KP buffer

solution pH 7.0. Biotransformation of 36 and its derivatives were catalyzed with AKR

(122) and PRO-AKR (170), and 38 and its derivatives with PRO-AKR (122), and (–)-

isopinocamphone 39 and its derivatives with PRO-AKR (170).

O

OH

O

O

OH O OH

36 243 126 136 and 137

O

38

O

39


130

Table 4.14 Bioreduction of various substrates using three different enzymes from Ketoreductase

Subs

trat

e

Prod

uct

Enz

yme

Co-

fact

or

Con

vers

ion

%

Yie

ld%

Isom

ers r

atio

PRO-AKR (146)

NADP+

/ GDH 96 95 95:5

PRO-AKR (146)

NADP+

/ IPA 44 44 71:29

PRO-AKR (122)

NAD+/ GDH 29 29 54:46

PRO-AKR (146)

NADP+

/ GDH 10 9 80:20

PRO-AKR (122)

NAD+/

GDH 81 80 59:41

PRO-AKR (122)

NAD+/ IPA n.d n.d n.d

PRO-AKR (122)

NAD+/ GDH n.d n.d n.d

PRO-AKR (170)


PRO-AKR (170)

NAD+/ IPA n.d n.d n.d

PRO-AKR (170)


*n.d = not detected.

O

36

OH OH

37 267

O

OH

243

OH OH

OH OH

268 269

O

38

OH OH

54 270

O

OH

126 271 272

OH OH

OH OH

H H

O

39

OH OH

56 273

136 and 137

O OH

274 275

OH OH OHOH

H H


131

The results showed bioconversion of 36 by PRO-AKR (146) to a mixture of 37 and

267 with an excellent yield (95%) and 95:5 ratio, when GDH/glucose have used for

NADP+ regeneration. While, employing IPA (3%) instead of GDH gave moderate yield

reduced to more than half. However, the bioconversion process of 36 by PRO-AKR(122)

yielded only 29% with ratio 54:46. The isomers produced from keto-reduction were 37 and

267 with retention time 17.75 min for the former, and 18.05 min for the latter. Bio-

reduction of anti 6-hydroxycarvone 243 with PRO-AKR(146) and NADP+/GDH co-factor

afforded only 9% yield as 268 and 269 (with retention time 20.65 and 20.92 min respectively)

and ratio is 80:20. (+)-Isomenthone 38 was also reduced to mixture of (1S,2R,5R)-(+)-

isomenthol 54 and (1R,2R,5R)-(+)-neomenthol 270, by PRO-AKR(122) enzyme and co-

factor NADP+/GDH to give 80% yield of both isomers with retention time 19.90 min of 54

and 19.74 min of (+)-270. Attempting to run the reaction to use IPA as H+ donor to

regenerate NADH showed no product was detected. Diastereomer 126 was treated with

PRO-AKR(122) enzyme but no diol product was detected. (–)-Isopinocamphone 39 and

mixture of (136 and 137) also showed no product from bioreduction with keto-reductase

PRO-AKR(122).

4.5.1 Conclusions

Carbonyl bioreduction of 36 was successfully achieved with three enzymes of

PRO-AKR, and afforded an excellent yield and isomeric ratio, while 243 exhibited low

yield with modest selectivity. Compound 38 was also screened, and gave good yield.

However, aldol adducts of isomenthone 126, and 39 showed no products.

5. Expansion rings of cyclic monoterpenoids via Baeyer−Villiger reaction

132

5 Expansion rings of cyclic monoterpenoids via Baeyer−Villiger reaction

5.1 Overview of Baeyer−Villiger reaction Baeyer–Villiger reaction is an effective approach to synthesise of various organic

products such as esters and lactones by inserting an oxygen atom in to C-C bond next to

carbonyl of cyclic and acyclic ketones respectively.134 It was suggested by Adolf von

Baeyer and Victor Villiger in 1899, and they proposed peracid oxidant reagent KHSO5 to

convert monoterpenoids to their conjugated lactones with 40–50% yield.135

Scheme 5.1 First attempts Baeyer−Villiger oxidation of terpenoids135

More than one hundred years has elapsed since the exploration of Baeyer−Villiger

oxidation, and still there is potential to enhance this synthesis method. The modification

comprises to control regio-, stereo- and enantioselectivity of the reactions, and to avoid

generating racemic mixture. In addition, health and safety requirements and environmental

concerns mean is good to avoid peracid reagents that generate carboxylic acid salt and

require recycling. Also, there are risks resulting from transportation and utilisation,

consequentially; this gives a motive for reduced use or even preventing use.136-138

Unsurprisingly, a wide range of modifications such as new oxidant reagents and conditions

were developed.139,140 However, the advantages of BV reaction has increased, and the

reaction has become more widely applicable in organic synthesis due to:

1) Ability to oxidize carbonyl compounds, such as acyclic and cyclic ketones to esters and

lactones respectively, and also, broad ranges of functional groups is tolerated within the

process such as sulfoxide.

2) The reaction would be highly stereo- and regioselective because that the migrating

group retains its configuration (highly stereoselective reaction). Also, highly regioselective

OO

O

OO

O

OO

49 276 87

277 279278

iii

i = KHSO5, RT, Yield 40−50%

O


133

reaction due to migratory aptitude following the sequence:137 tertiary alkyl > cyclohexyl >

secondary alkyl > benzyl > phenyl > primary alkyl > CH3

5.2 Mechanism of Baeyer−Villiger reaction Baeyer and Villiger explained the mechanism to be similar to the Beckmann

rearrangement, with generation of an intermediate dioxirane 280. The explanation was 280

rearranges to a lactone for a cyclic ketone, and to a dimer 281 or even polymer for acyclic

ketones. However, this dioxirane intermediate was not proven, and there was no explaining

of chemo- and stereo-selectivity of the reaction.141

Scheme 5.2 Proposed mechanism of ketone oxidation by Baeyer and Villiger141

Later, BV mechanism has been comprehensively reported, with many suggestions.

For example, Doering and Dorfman reported the experiment of benzophenone esterification

with perbenzoic acid (PBA). Substrate labeled with 18O was employed to determine the

pathway of BV reaction. Indeed, there were three possibilities for the intermediate; 1)

Dioxirane 280 as Baeyer and Villiger documented. 2) Transfer of OH+ from perbenzoic

acid to oxygen atom of the ketone. 3) Criegee intermediate generated from nucleophilic

attack of PBA to the substrate, with the mechanism involving formation of C=O again, and

migration of one substituent to partially positive oxygen atom accompanied with leaving of

carboxylate ion (Scheme 5.3).

The ester product was reduced with LiAlH4 to afford non-labeled phenol 282 and

93% labeled 18O benzol 283, which confirmed the origin of the O atom from ketone

carbonyl. This result agrees with Criegee suggestion (third possibility).142

R1 R2

O

R1 R2

O O

R1 R2

O OOO

R1 R2

R1 O R2

O

R1 OR2

O280

281


134

Scheme 5.3 Proposed pathways of Criegee intermediate formation, and possible outcomes from BV oxidation, and introduce experimentally proof via labelled 18O 142

The sequence of migratory group aptitude (section 5.1, point 2) was presented by

Doering and Speers in 1950.143 Later, Hawthorne and co-workers, explained that large

substituents preferentially migrate due to the stability caused by partial positive charge of

transition state, they have also proposed more substituted alkyl groups to be trans to the

leaving group carboxylic acid, while smaller groups would be “staggered” to the

carboxylic group.144 After decades, the stereoelectronic effect has returned to be discussed

in several papers, where they referred to two terms; A) Primary stereoelectronic effect in

which the migrating alkyl group (Rm) should be antiperiplanar to carboxylate leaving

group and B) Secondary stereoelectronic effect which indicates Rm has to be antiperiplanar

to a lone pair of electrons on OH.145,146

O

ClCl

H2O

O

OOH

HO OOO

O

O

O OOOH

O

O

CriegeeBV-PhOOH

-PhCOO-

-PhCOOH50%50%

-H+

O

OHO

HO

0% 18O100% 18OHypothesis

Experimentalresult

LiAlH4 285286

284283 282

287

288

284

289

Third possibleSecond possible

First possible


135

Figure 5.1 Transition state explains sequence of migratory group aptitude suggested by

Hawthorne et.al144

5.3 Baeyer−Villiger reaction of monoterpenoids Oxidation of natural product substrates to obtain valuable lactones has been

reported in several papers, with various oxidising agents. For example, peracids are widely

used in oxidation reactions, and m-CPBA is the most commonly oxidising agent. This

reagent is low reactivity with ketones, but it demands prolonged reaction time to achieve

good conversion.147 Table 5.1 shows various examples of expanding natural cyclic ketones

with several oxidising agents, yield, regioselectivity and the solvent of the reaction.148−158

Table 5.1 BV Oxidation of monoterpenoids with different reagents, yield and regioselectivity 148–158

Lac

tone

Rea

gent

s

Yie

ld %

Rat

io

(nor

:abn

)*

Solv

ent

Ref

.

m-CPBA/ NaHCO3

90 100:00 DCM 149

m-CPBA/ [bmim]BF4

90 100:00 [bmim]BF4 150

H2O2/AlCl3 100 100:00 EtOH 151 H2O2/AlCl3 on

silica 97 81:19 EtOH 152

H2O2/Sn complex 67 100:00 DCE 153

Decaneper-sulfanic acid 98 100:00 ACN 154

m-CPBA 97 96:04 DCM 155 m-CPBA/

[bmim]BF4 85 90:10 [bmim]BF4 150

KHSO5:silica 95 100:00 DCM 156

RL= large alkyl group, RS = small alkyl group

Favour due to trans betweenRL and carboxylate

Unfavour due to steric hindrancebetween RL and carboxylate

RS OH

RL

OO

O

R

RL OH

RS

OO

O

R

OO O

O

277 290

O

OOO

291 292


136

m-CPBA 94 100:00 DCM 156

H2O2/Sn complex 43 100:00 DCE 153

bis(trimethyl-silyl) peroxide/

bmimNTf2

89 53:47 bmimNTf2 157

CH3COOH 94 100:00 CH3COOH/ CH3COONa

148

CH3CO3H

42 100:00

CH3COOH/ CH3COONa 148

35 55:45

m-CPBA 37 90:10 DCM 158

* (nor:abn) = ratio of normal:abnormal lactones

Although a wide range of reagents has been reported, still the selectivity is low in

some cases, in additional, to utilise unaccepted environmentally peracid reagents and/or

chlorinated solvents. However, catalysing the reaction using enzymes called Baeyer–

Villiger mono-oxygenase (BVMOs) has had more attention due to generation of single

stereoisomers159 and superior degree of enantio- and regioselectivity as well to use

environmentally attractive solvent such as water.160

Here, biooxidation of monoterpenoids will be reviewed; cyclohexanone oxygenase

(EC l.l4.l3) isolated and purified from Acinetobacter NCIB 9871 has been utilized to

expand the ring of cyclic ketone. Immobilized enzymes have been used for

biotransformation of 2-norbornanone 300, L-fenchone 301, D-fenchone 302, (+)-camphor

87, and (+)-dihydrocarvone 100 on scale 30–80 mmol in pH 8.0 of glycine-NaOH buffer

solution. The study aimed to establish the substrate specificity, regioselectivity, and

enantioselectivity of the enzyme.161 The results is depicted in table 5.2:

O

OOO

279 293

OO O

O

294 295

OOO

O

296 297

OO

OO

298 299


137

Table 5.2 Summary of lactones syntheses by BVMOs161 Sub Product Yield% Comments

81 High

regioselectivity

76 Low selectivity

79 Low selectivity

89 High

regioselectivity

75 High

regioselectivity

Pinheiro and co-workers162 have reported bioconversion of six substrates of

fragrances with trichosporumcutaneum CCT 1903 whole cells namely: cis-jasmone, R-(–)-

carvone, α and β-ionones and (R)-(+)-limonene. For example, analysis of the spectroscopic

study of (R)-(–)-carvone 36 biotransformation characterized formation of four products

namely: (1R,2S,4R)-neoisodihydrocarveol 252 (from partial reduction of double bond and

carbonyl function group), (6R)-isopropenyl-(3R)-methyl-2-oxo-oxepanone 308 (unusual

lactone from bio-oxidation of trans-dihydrocarvone 100), (3R)-isopropenyl-6-oxoheptanoic

O

300

OO

2922-Oxabicyclo[3.2.1]octan-3-one

301 OO O

O

O303 304

2.3-Fencholide1.2-Fencholide

302

O OO O

305 3062.3-Fencholide1.2-Fencholide

O

87

O279

OO

1,8,8-Trimethyl-2-oxabicyclo-[3.2.1]octan-3-one

100

O

307

OO

7-Methyl-4-(prop-1-en-2-yl)oxepan-2-one


138

acid 309 (from opening ring of 308) and 2,3-epoxy-(5R)-isopropenyl-2-methylcyclohexenol

310 from (oxidation of endo-double bond of carvone 36). Apparently, the substrate

underwent both 1,2 and 1,4-reduction and BV oxidation, with prevailing bio-oxidation

(Scheme 5.4).

Scheme 5.4 Baeyer−Villiger biooxidation of 36 using trichosporumcutaneum CCT 1903 162

A recombinant whole-cell Baeyer−Villiger monooxygenase (BVMO) from

Xanthobacter sp. ZL5 has been screened against diverse sets of monoterpenoids, to

highlight the remarkably wide-range of substrates accepted by the enzyme. The research

was also focused on kinetic resolutions; regiodivergent conversions and phylogenetic

comparison with other studies. The results agree with literature reports of CHMOs in both

substrate acceptance as well as in stereo- and regio-preference. For example, conversion of

(–) and (+) dihydrocarvone (210 and 100) to normal 310 and abnormal lactone 308

respectively, with yield up to 90%. (–)-Carvomenthone 278 also produced normal 278 and

abnormal lactone 311 (50−90%). However, (±)-menthone was unreacted (Scheme 4.19).163

Also, same conversion and regioisomers of menthones (49 and 219), and dihydrocarvones

(210 and 100) has been reported with cyclopentadecanone monooxygenase (CPDMOs)

whole-cell biocatalyst.164

O OHO COOH

OOH

O

+++

(−)-Carvone 251 308 309 310

Isodihydrocarveol36

Abnormal lactone

Heptanoic derivative Epoxide

i = trichosporumcutaneum CCT 1903a = pathway of lactone formation

ConditionsSubstrate = 20 µL, enzyme = 0.5–2 g,125 mL of phosphate buffer pH 6.5,25 °C, 96–120 h.

i

O

a a

3.8% 31% 5.0% 2.2%Yield (mixture)

100

O


139

Scheme 5.5 Baeyer–Villiger oxidation of 210, 100, and 276 163

Alphand and Furstoss have described biotransformation of isomers of two di-

substituted cyclohexanone monoterpenoids; menthone (49 and 219) and dihydrocarvone

(210 and 100)) via Acinetobacter NCIEI 9871 and Acinetobacter TD63 whole cell. The

outcomes referred to stoichiometrically conversion of (+)-menthone 219 to normal lactone

312, but there was no activity of bacteria towards the (–)-isomer. On the other hand, both

isomers of dihydrocarvone were more surprising, and underwent bio-oxidation as (–)-

isomer 210 transformed to normal lactone 310, and 100 to abnormal one 308 (Scheme 5.6).

Attempting to bio-oxidise (+)-isomenthone and (+)-isodihydrocarvone failed.165

Scheme 5.6 Baeyer−Villiger oxidation of 219, 210, and 100 165

Bio-oxidation of cis and trans dihydrocarvone (83 and 100), carvomenthone (276

and 313) and menthone (49 and 219) with two types of BVMOs was reported.166 The paper

has studied the regio-preference of (CH) and (CP) monooxygenase with terpenone

precursors, and afforded good access to all regioisomeric lactones. For instance,

bioconversion of (–)-trans-dihydrocarvone 83 and (–)-carvomenthone 313 with “CHMOs”

O

O

O

O

O

OO

O

100 Abnormal lactone 308

+

(+)-Dihydrocarvone

Cavomenthone 276 Normal lactone 278 Abnormal lactone 311

O

210

OO

310Normal lactone(−)-Dihydrocarvone

i

ii

i = BVMO) from Xanthobacter sp. ZL5

ConditionsCulture 25 mL with OD590 = 0.6–0.7,substrate = 10 mg, 24 °C, 24 h

Yield >90% Yield >90%

Yield (mixture) 50−90%

A and/or B

OO

O

(+)-Menthone 219 Normal lactone 312

O O

210 Normal lactone 310

O

O

100

OO

307Normal lactone(–)-DihydrocarvoneYield A B90% 61%

Yield A B80% 95%

Yield A B73% 66%

A and/or B

A and/or B

A = Acinetobacter NCIEI 9871 B = Acinetobacter TD63

ConditionsCulture 1L, [ketone] = 1 g/ 5 mL EtOH cis/trans 1,2-cyclohexanediol 2.25 g as co-substrate, tetraethylpyrophosphate 200 mg as hydrolase inhibitor, 25 °C, 2-6 h.


140

type enzymes (CHMOAcineto, CHMOArthro, CHMOBrachy, CHMORhodo1, CHMORhodo2) gave

normal lactones 314 and 315 respectively with good to excellent yield (> 80%).

Meanwhile, (+)-trans-dihydrocarvone 100 and (+)-carvomenthone 276 were exclusively

catalysed to abnormal lactones 308 and 311 respectively, but with poor conversion

(<50%). “CPMO” enzyme types (CHMOBrevi2, CPMOComa) converted both isomers to

abnormal lactones with low conversion. (Scheme 5.7).

Scheme 5.7 Baeyer−Villiger oxidation of 83, 100, 276 and 313 166

(2R,5R)-(+)-Isomenthone 38 was formed from oxidation of counterpart of alcohol

54, and it has typical and specific structure, with equatorial isopropyl and axial methyl. As

previous reported, bioconversion of 38 by Acinetobacter NCIEI 9871 and Acinetobacter

TD63 using whole-cell failed.165 Below, 38 was evaluated via cyclohexanone monooxygenase

(CHMO_Phi1).

5.4 Results and discussion

5.4.1 Chemical Baeyer−Villiger reactions of (+)-isomenthone and (–)-isopinocamphone

Oxidation of (2R,5R)-(+)-isomenthone 38 using m-CPBA in DCM was evaluated.

The reaction mixture was monitored via TLC over 5 h and this showed a spot with Rf 0.35

for lactone (hexane:EtOAc, 5:1). The lactone was purified using flash column

chromatography to afford a volatile pale yellow oil of only normal lactone in 45% yield,

and the rest of the unreacted ketone was recovered. 1H NMR and chiral GC showed

formation of exclusively normal lactone due to migration of the most positive charge of

substituted carbon according to the conventional migratory aptitude. 1H NMR spectrum

O O

OO

OO

314

O

O

O

O

i iii

i = !CHMO! type enzymes

83 313 100 276

Normal lactone Normal lactone Abnormal lactone Abnormal lactoneConditionsCultural medium OD590 0.6,substrate 3–6 mM, β-cyclodextrin 1eq.,19−72 h, RT

308315 311

O O


141

revealed a multiplet peak at δ 4.02−3.99 for CHOC=O, and two peaks of a double doublet

for CH2C=O at 2.89 ppm with J = 13.6, 3.2 Hz and at 2.54 ppm with J = 13.6, 5.9 Hz.

More information was acquired from the 13C NMR spectrum with a C=O at δ 174.5 ppm,

and signal for CHOC=O at δ 85.1 ppm.

Scheme 5.8 BV reaction of 38 and 39 via m-CPBA

Analogous conditions were evaluated for ring expansion of (1S,2S,5R)-(–)-

isopinocamphone 39. After 5 h a mixture of two lactones was obtained in 38% yield with

ratio 90:10 normal:abnormal (317:318). The structures of 317 and 318 were established via 1H NMR spectroscopy, and the spectrum of the 317 showed deshielding of proton α to the

oxygen to 4.69−4.66 ppm. Also, signal for CH2C=O was shifted to downfield at 3.02−2.85

ppm. In the 13C NMR spectrum the C=O was shifted to 174.6 ppm, and the α-carbon to

82.9 ppm.

The colourless oil lactones mixture was totally hydrolysed to form a solid at RT. 1H

NMR spectroscopy indicated that the lactone rings were opened to yield a 90:10 mixture of

Pinolic acid and cyclobutane derivative 319:320. Formation of 319 was confirmed with

proton NMR, where one broad signal with integration to two protons was deshielded at

4.78 ppm, and determined as two protons for acid and alcohol functional groups.

Meanwhile, the signal of CHOH (H-1`) was shifted to 3.74 ppm, and seen as dq signal

with J = 9.8, 6.2 Hz. Moreover, α protons of CH2 (CH2COOH) were assigned as multiplet

(R)(R)(R)(R)

O(R)(R) O

(R)(R) Oi

i = m-CPBA, NaHCO3/DCM, RT

38 316

(S)(S)(S)(S)

(S)(S)

O

O

(S)(S)(R)(R)

(R)(R)

OO

(S)(S)

(S)(S) (R)(R)

O

39

317

318

i

Yield 45%

Yield 38%, ratio 90:10 normal:abnormal

HO(S)(S)

OH

O(R)(R)

(R)(R)

(S)(S)

OH

(S)(S)(S)(S)O

OH

319

320

Quantitative, ratio 90:10 (319:320)

1

2

31`

4


142

at 2.42−2.31 and 2.28−2.18 ppm. The 13C NMR spectrum showed a peak for C=O at 178.8

ppm, and also, the carbon bearing OH at 69.4 ppm. Mass spectrum demonstrated a highest

mass peak of 187.1 (MH+). Also, the IR spectrum showed a broad peak for the OH at 3400

cm cm–1, and strong peak at 1704 cm cm–1of carbonyl group stretch.

5.4.2 Direct oxidative cleavage of (+)-isomenthone and (–)-isopinocamphone with Oxone®

Pinanones (bicyclic[3.1.1]heptanones) have been expanded to lactones using m-

CPBA, but some of the reactions were difficult. The lactones were esterified with methanol

and ethanol. These reactions give access to cyclobutane monoterpenoids such as grandisol

321, fraqranol 322, junionone 323 and citrus mealybug pheromone, cis- planococcyl

acetate 324.158

Figure 5.2 Structures of cyclobutane monoterpenoids 321, 322, 323 and 324 158

Cyclobutane monoterpenoids were synthesised from nopinone, methylnopinone in

low yield, while, no product was observed from oxidation of dimethylnopinone

Scheme 5.9 BV reaction of 325 using m-CPBA in EtOH and/or MeOH158

Here, the aim of this reaction was to produce a cyclobutane monoterpenoid from

oxidation and esterification of 39 using an environmental-friendly reagent such as Oxone®,

and avoid use of chlorinated solvent.167 The substrate 39 was mixed with Oxone® in

methanol at RT, and TLC showed no product even after two days, the reaction was heated

to 50 °C and allowed to stir overnight. TLC revealed multiple spots as well 25% of

recovered starting material. Flash column chromatography separated the products whose

structures were mainly confirmed with 1H NMR as:

(R)(R)

(R)(R)

OH

(S)(S)

(R)(R)

H

OH

H(E) O

(S)(S)

(S)(S)

AcOH2C

321 322 323 324

(R)(R)

(S)(S)

O(R)(R)

(S)(S)

OO

(R)(R)

(R)(R)

O

O

(R)(R)(S)(S)

HOCOOR

(R)(R)(S)(S)

RO2CCH2OH

i

i) m-CPBA, EtOH and/or MeOH, R= C2H5, CH3

325

298

299

326

327


143

Scheme 5.10 Esterification of isopinocamphone 39

1) First fraction: mixture of normal and abnormal lactones (317 and 318). It was only

2% converted and was difficult to fully assign using 1H NMR due to being partially mixed

with cyclobutanone derivative. However, the mass spectrum showed mainly peaks at 168

for the lactone (M+), and small peak at 200 (M+) for the alcohol ester.

2) The second fraction was a mixture of cyclobutane derivatives (328 and 329), in a

combined 12% yield and ratio 90:10, as indicated by proton, 13C NMRs and MS. For

instance, 1H NMR revealed an OH signal peak at 3.40 ppm, and two multiplet peaks for

CH2OH at δ 2.34−2.27 and 2.17−2.14 ppm. Moreover, the spectrum showed four peaks for

four Me groups. Meanwhile, the 13C NMR spectrum gave a peak at δ 173.5 for ester C=O.

Also, formation of the product was consistent with MS, where showed a peak at 200 (M+).

It is important to note that the proton NMR of 328 and 329 exhibited small peaks expected

to belong to 320 resulting from opening the ring of the abnormal lactone 318, however, it

is difficult to confirm because of overlapping peaks with their counterparts in the major

product 319 (Scheme 5.10).

3) Third fraction: acid ester of cyclobutane derivatives (330 and 331) as well impurities.

The yield was 4% and the products were confirmed via HPLC-MS, but unfortunately, it

was problematic to purify with column chromatography, so, further purification was by

prep TLC but did not afford pure acid ester of cyclobutanone derivative (Scheme 5.10).

(+)-Isomenthone 38 was oxidized using 8 eq. of Oxone® in methanol at RT, and

TLC showed no lactone or hydroxyl ester was produced, even after 48 h.168 Activation of

the reaction was carried out via heating to 40 °C, and after 2 h. TLC showed two spots

(with Rf 0.29 and 0.27 of first and second spots respectively in hexane:EtOAc 5:1), with

(S)(S)(S)(S)

(S)(S)

O

O

(S)(S)(R)(R)

(R)(R)

OO

(S)(S)

(S)(S) (R)(R)

O

39

317

318

i +

HO

(S)(S) OMeO

(R)(R)

(R)(R)

OMeO

HO

(S)(S) (S)(S)

(S)(S)

329

OMeO

MeOOC(S)(S)

+

O OMeO

HO

(S)(S)

(S)(S) (S)(S)

+

328

330

331i) Oxone®, MeOH, 40 °C

Yield 317+318 = 2%328+329 = 12%, ratio 90:10 (328:329)

330+331 = 4%

+ (S)(S)

(S)(S)


144

total consumption of starting material. More information was acquired from reverse

HPLC-MS (50:50 ACN:H2O), and the chromatogram exhibited two peaks with the same

molecular mass with 255.1 (MNa)+ and integrated area ratio of 65:35. Purification and

separation of products was carried out by flash column chromatography, and gave two

fractions of pale yellow oil with overall yield 47%.

Proton and 13C NMR of the major fraction revealed two components (333 and

334), with the same general structure, but different stereochemistry. For example, 1H NMR

revealed six doublet signals for six Me groups partially overlapping at δ 0.95−0.88 ppm,

and two singlets for two CH3O groups at δ 0.37 and 0.36 ppm. In addition, the 13C NMR

spectrum showed two peaks at δ 173.9 and 173.8 ppm for C=O, two signals for CHOH at δ

77.0 and 76.7 ppm. The NMR spectra also showed ca. 3% impurities, and the possibility

that the product was a lactone was enhanced by observation of a signal at δ 85 ppm for

CHOCO.

Scheme 5.11 Pathways of 38 oxidation via Oxone® in MeOH.

The minor fraction (335 and 336) was produced from esterification of lactones (227

and 290), and represents only 17% of the yield. The ratio of two structures was confirmed

to be 1:1 via 1H NMR spectrum. The latter was assigned as a multiplet peak for CH2OH at

δ 3.50−3.38 ppm, and three doublet peaks with J = 6.8 Hz at δ 0.92, 0.91 and 0.89 ppm of

the three methyls as well as a singlet for the methyl ester at δ 3.67 ppm.

It is clear that some of 38 was epimerized to the more stable enantiomer menthone

49 during the activation of reaction, and both (38 and 49) were oxidized to normal and

abnormal lactones. Esterification of four lactones with methanol leads to formation of two

kinds of open chain alcohol ester, each one with two isomer patterns.

(R)(R)(R)(R)

O

OHO

(S)(S)(R)(R)

O

OHO

(R)(R) O

(R)(R) O(R)(R)(R)(R)

O

(S)(S)(R)(R)

O

(S)(S) O

(R)(R) O

(R)(R)O(R)(R)

(S)(S)O(R)(R)

i

i

i) Oxone®, MeOH, 40 °C

38

49

316 332

277 290

333

334

+

+

+

+

Yield 333+334 = 42%, ratio 1:1Yield 335+336 = 5%, ratio 1:1

335

336

(S)(S)O

(R)(R)+

O

O

HO

OMe

(R)(R)O

(R)(R)

HO

OMe


145

5.4.3 Investigating Baeyer–Villiger monooxygenase activity towards

synthetically-modified terpenones 5.4.3.1 Expansion of ring of (+)-isomenthone

Initial biotransformation of 38 by cyclohexanone monooxygenase (CHMO_Phi1)

from Rhodococcus sp. Phi1.was set up at variable pH (6.4, 7.0, 7.5 and 8.0) of KP buffer

solution (50 mM) at 30 °C and 130 rpm. At this experiments, the co-factor NADPH and/or

NADP+ with recycling system were utilised as proton donor. Analysis on GC chiral

revealed only 2–10% converted lactone with high regioselectivity, and the formed lactone

was identical to normal lactone (retention time 12.227 min), which was synthesised via m-

CPBA/TFA reaction.

Scheme 5.12 Biooxidation of 38 via CHMOs from Rhodococcus sp. Phi1

However, (–)-isopinocamphone 39 was not metabolised and remained in the buffer

solution without undergoing further transformation.

Figure 5.3 Biooxidation of 38 with CHMOs from Rhodococcus sp. Phi1

The chart shows the bio-oxidation of isomenthone to normal lactone at different

pH, and it showed maximum 10% of the yield at pH 7.5 of Tris.HCl buffer solution,

whilst, pH 6.4, 7.0 and 8.0 indicated less activity of enzyme toward the substrate.

Moreover, the experiment displays the efficiency of co-factor NADPH on the yield% in

comparison with NADP+/GDH and/or NADP+/G-6-PDH. From these results, the

conclusion is using pH 7.5 of buffer solution of tris.HCl, and NADP+/GDH allows the

CHMOs to be more active with isomenthone.

O OO

38 316(+)-Isomenthone 7-Methyl-4-(prop-1-en-2-yl)oxepan-2-one

i = CHMO_Phi1 Yield 2- 25%

i

0

2

4

6

8

10

pH 6.4 pH 7.0 pH 7.5 pH.8.0

NADP+/G-6-PDH

NADP+/GDH

NADPH


146

The scope of optimisation also included the influence of temperature, concentration

of enzyme and/or NADP+. Table 5.3 shows the conversion and yield% of bio-conversion

of isomenthone at 25, 30 and 37 °C at 2 or 10 µM of enzyme concentration at variable

concentration of NADP+ 15, 50, 100 and 250 µM using buffer solution at pH 7.5 of

Tris.HCl.

Table 5.3 Optimisation of [CHMOs] and [NADP+] on biooxidation of 38

[CHMs]

µM

[NADP+]

µM Conv.% Yield% Conv.% Yield% Conv.% Yield%

25 °C 30 °C 37 °C

2 15 25 11 38 8 56 5 2 50 30 10 38 8 58 5

2 100 25 11 37 9 61 7

2 250 23 9 40 19 39 5 10 15 36 25 46 19 61 15

10 50 38 22 46 18 61 15

10 100 37 21 46 18 62 16

10 250 35 20 30 19 61 15

The yield of lactone produced at 25 °C increased with [CHMOs] equal to 10 µM

compared to using [enzyme] of 2µM, for instance, utilizing 15 µM of NADP+ at 10 µM of

[CHMOs] gave 25% of yield in compare with 2 µM afforded only 11%. The data also

show reduction in the yield with raise in temperature to 30 °C then to 37 °C, with

increasing the conversion of isomenthone due to the volatility of isomenthone.

5.4.3.2 Baeyer−Villiger oxidation of 2-methyldihydrocarvone

(+)-Dihydrocarvone 100 has been reported in many papers as a good substrate for a

number of BVMOs (Section 5.3), and most likely abnormal lactone was formed. However,

dihydrocarvone derivatives, such as 2-methyldihydrocarvones, are introduced in this

section as new examples of non-natural substrates for BVMOs. (2S,5R,6R), and

(2R,5R,6R)-2-methyldihydrocarvone isomers (236 and 237) were screened against

CHMO_Phi1 on 1 mL scale. The concentrations involved 5 mM of [substrate], 2 µM of

[enzyme] with NADP+ 15 µM/G-6-PDH (10 U), and Tris.HCl buffer solution pH 7.5. The

reaction was incubated for 24 h at 25 °C, and the organic layer extracted with 0.8 mL ethyl

acetate, and run on GC. 237 was completely consumed, and converted to lactone 337 with


147

high regioselectivity (100:00 normal to abnormal lactone), with retention time 13.868 min

of 337. However, there was no observed lactone produced from 236, and starting material

was totally recovered.

The bio-expansion of 237 was scaled up 50 mg scale, and the TLC analysis showed

no starting material and no by-product have formed, so that, the lactone solution was

concentrated without further purification, dried and characterised with NMR, HRMS, and

IR as well as optical rotation.

Scheme 5.13 Biooxidation of 237 by CHMO_Phi1 from Rhodococcus sp. Phi1

The 1H NMR spectrum of 337 revealed that the oxygen atom was inserted between

C=O and CH with R configuration. For example, the proton on the carbon next to the

oxygen atom (CH-O, H-7) appeared at δ 4.66 ppm as doublet of quartet with J coupling

constant 7.2, and 3.3 Hz represent correlation of CH-O, CH3 and CH-O, H-6. In addition

the signal for H-3 was dqd at δ 2.81 ppm (J = 10.6, 6.8, 5.0 Hz), and H-6 was seen at δ

2.40 ppm as a ddd (J = 8.7, 7.1, 3.3 Hz). 13C NMR spectrum gave 11 peaks of 11 carbon

atoms, with the most important signals being C=O at δ 177.1 ppm, C-7 at 75.4 and C-3 at

50.7 ppm. IR spectroscopy was also utilised to confirm lactone formation, for example

C=O signal was at 1717 cm–1, and C-O stretches at 1208 and 1168 cm–1.

5.4.3.3 Biooxidation of 3-methyldihydrocarvone

3-Methyldihydrocarvone is mixture of four isomers (262, 263, 264 and 265) with

diastereomeric ratios (1.5:1:1:0.1), and 262 and 263 were partially separated. Both

mixtures of four and two isomers were screened to ring expand with CHMOs to obtain

novel trisubstituted lactone. The reaction was set at same conditions with 2-methyldihyd-

rocarvone 257. Both 262 and 263 isomers were totally converted to their corresponding

lactones with 98% yield of both isomers. However, no lactones were observed from

biooxidation of diastereomer 264 and 265. In the line of outgoing trends, the biooxidation

O CHMO_Phi1

Yield 90%

(R)(R)(R)(R)(R)(R)

237337

12

3

4

56

7

8

9

(S)(S)(R)(R)O(R)(R)

O


148

of 262 and 263 mixture was scaled up to 50 mg, and no starting materials were indicated

via TLC after 24 h. The organic layer was extracted with ethyl acetate, concentrated, dried

and afforded 90% of a mixture of the two lactones with retention times 15.942 and 16.188

min for abnormal lactones 338 and 339, respectively.

Scheme 5.14 Biooxidation of 262 and 263 mixture by CHMO_Phi1 from Rhodococcus sp.

Phi1

The lactone mixture was fully characterised with 1H NMR, HRMS and IR

spectroscopies. For example, the 1H NMR spectrum of 338 showed a multiplet peak at δ

4.15−4.12 ppm for (2 x H-7), and doublet of quartet signal at δ 2.60 ppm (J = 10.1, 6.8 Hz)

for H-3. The peak for H-4 appeared at δ 2.35 ppm as doublet of doublet of triplet (J = 12.0,

8.8, 3.1 Hz).

Lactone 339 was also established to be abnormal lactone, where protons H-7 are

deshielded to δ 4.20−4.16 ppm and seen as a multiplet. Moreover, H-3 was assigned as a

doublet of quartet at δ 3.02 ppm with J = 6.8, 1.5 Hz.

5.5 Conclusions Oxidation of (2R,5R)-(+)-isomenthone 38 and (1S,2S,5R)-(–)-isopinocamphone 39

was successfully achieved with m-CPBA oxidant reagent, with good yield and high

regioselectivity of normal lactones. Direct oxidative cleavage of 38 with Oxone® failed at

RT, but afforded fractions of four structures at 40 °C. 39 was converted to cyclobutane

ester at 40 °C in only 12% yield when heated with Oxone® reagent.

Also, cyclohexanone monooxygenase (CHMO_Phi1) from Rhodococcus sp. Phi1

was screened in various conditions against 38, and the enzyme showed high

regioselectivity towards substrate, although, the yield is not more 25% of pure normal

lactone.

Interestingly, the enzyme exhibited excellent activity toward non-natural

trisubstituted cyclohexanone of 236 with 90% yield However, biooxidation of 235 via

CHMOs showed no lactone was produced, and starting material was recovered. Extending

1

23

4

56

7

8

9 (S)(S)(R)(R)(S)(S)

O (R)(R)(R)(R)(S)(S)

O

O O

(R)(R)(R)(R)(R)(R)

O(R)(R)(R)(R)(S)(S)

O

Yield 90%

CHMO_Phi1

+

262 263 338 339


149

to BVMOs of synthesised trisubstituted cyclohexanone, 3-methyldihydrocarvone (four

diastereomers) were screened and showed 262 and 263 were converted to lactones with

90% yield, while 264 and 265 were totally unreacted and starting materials recovered.

BVMO was scaled up to 50−60 mg, and no further purification was required due to full

conversion and no by-product being detected.

6. Conclusions and future work

150

6 Conclusions and future work

6.1 Conclusions The project aimed to prepare new monoterpenoid derivatives for evaluation as

substrates for biocatalysis. Alkylated and alkylidene derivatives of R-(–)-carvone, (+)-

isomenthone and (–)-isopinocamphone were synthesised via enolate alkylation and aldol

reactions to functionalize alpha to the terpenone C=O, most of which derivatives were

novel (Scheme 6.1). The stereostructures of a number of these novel derivatives were

established via X-ray crystallography, and the all were investigated via NMR spectra to

assist with stereo-assignments. The stereochemistry of aldol adducts were rationalized

according to a Zimmerman-Traxler transition state.

Scheme 6.1 General scheme of aldol reaction and synthesis of alkylidene of 36, 38 and 39

These extended terpenone substrates were evaluated in a number of biocatalytic

assays. Substrates (alkylated and alkylidene derivatives) with at least 2-carbon or larger

groups added, did not undergo reductions with ene-reductases (OYE2, OYE3 and PETNR)

and alcohol dehydrogenases (96 enzymes of ‘Ketoreductase-to go’ provided from

Prozomix Company).

O

R

O

R

Yield30-86%, d.e = 20−100%No further bioreduction was occurred

5 E

xam

ples

OBio-reduction

OAldol reaction6 examples

O

OH

RH

H

Aldol reaction

OH

H R

OH9 examples

No further bioreduction was occurred

41−64%

3 different enzymes

Major aldol adduct

Yield 58-82%,d.e. 93-96%

36 88

ORHO

ORHO

O39

Aldol reaction

5 examples

Yield = 65−91%

O ORHO

ORHO38

Aldol reaction

5 examples

Yield = 18−67%

Yield = 45−85%

OR

OR

Yield = 43−76%

2 Examples

No further bioreduction was occurred

Dehydration

Deh

ydra

tion

OH

H R

OH


151

In contrast, monoterpenoids with small additional substitutions, specifically methyl

and hydroxyl groups (also introduced by chemical modification), proved to be effective

substrates in several biocatalysis. For example, bioreduction of 6-methylcarvone afforded

products with good conversions and high diastereoselectivity (Scheme 6.2).

Scheme 6.2 Chemo-enzymatic reactions of (–)-carvone derivatives

Moreover, (+)-isomenthone 38, (–)-isopinocamphone 39 and non-natural

dihydrocarvone derivatives such as 2-methyldihydrocarvone 237 were biocatalytically

oxidised to form lactones via BVMOs transformation, which were compared to the

chemical Baeyer-Villiger reaction using m-CPBA. Isomenthone 38 converted to only

normal lactone via BVMOs or m-CPBA, with higher yield regarding to chemical reagent.

Biooxidation of (–)-isopinocamphone 39 did not afforded lactone, while m-CPBA gave

both normal and abnormal lactones with ratio 90:10, and yield 38%. However,

Biooxidation of 2-methyldihydrocarvone 237 furnished only normal lactone, with an

excellent yield 90%, and no oxidation via chemical reagents was attempted.

Chemical ring-opening of some of these lactones to form open chain esters was

demonstrated. This is of interest for potential future applications to Ring-Opening

Polymerization (ROP) to novel polyesters.

OAlkylation

O

R

Bio

-red

uctio

n

O

R

O

R

4 examoples

OxidationO

OHB

io-r

educ

tion

O

OH

O

OH

64–85%

3 di

ffere

nt

enzy

mes

3 di

ffere

nt

enzy

mes

2 examples

Only when R = MeYield = 17–90%, d.e. ≥99.9

Yield 15−80%,d.e. ≥99.9

56–58%


152

Scheme 6.3 BV reaction of 38, 39 and 237, and opening rings of 38 and 39

6.2 Future work These outcomes (section 6.1) offer potential for developing scale-up of the most

effective biotransformations, and thereby evaluation of new further chemical transform-

ations into new high-value derivatives, or intermediates. Furthermore, the outcomes will

help inform applications of the existing enzymes evaluated but also could direct potential

mutations to address substrate range limits.

Future work could relate to both redox biocatalysis and Baeyer−Villiger transfor-

mations of further substrates for conversion to new lactones.

(R)(R)(R)(R)

O

OHO

O

O

OO

O38

+

O

O

OO

O

39

Yield 38% ratio 90:10 normal:abnormal

HO OH

O

OH

O

OH

Quantitative, ratio 90:10

+ +

CHMOsor m-CPBA

CHMOs, yield 25%or m-CPBA yield 45% Yield 47%, ratio 8:1

O

Yield 90%

O

O

CHMOs

237

m-CPBA

(R)(R)O

(R)(R)

HO

OMe


153

Aldol-derived novel terpenones could be evaluated as substrates for other redox

biocatalysts, including other isolated enzymes such as OYE1 but also other classes of

whole cell biocatalysts, for example, Bakers yeast.

Bio-production of new substituted lactones by Baeyer–Villiger monooxygenase

from (CHMO_Phi1), can be further evaluated by assessing substrates with a larger

substituent than methyl, including isomenthones and isopinocamphones, and different

substituted dihydrocarvones are also significant as potential substrates. Moreover, these

substrates could be screened against other new type of BVMOs such as CHMOAcineto,

CHMOArthro, CHMOBrachy, and CHMORhodo1, CHMORhodo2.

A number of monoterpenoid derivatives have been found to be effective

asymmetric controllers used in modern organic synthesis, including with utility in

organocatalysis. There is significant potential to utilise terpenoid derivatives in

organocatalytic approaches and as precursors to new ligands. Exploiting new biocatalytic

routes to modified terpenoids thus offers possible applications to these areas of asymmetric

synthesis.

The potential exploitation of new lactones for ROP is also important, as the

evaluation of new ROP-based polyesters is a topic of current interest, and providing new

biotransformation-derived feedstocks offers prospects for evaluating new polymer

backbones.

7. Experimental

154

7 Experimental

7.1 General techniques Chemical reagents were purchased from different commercial sources with

different purities. Calcium hydride was used to dry THF by distillation process. Molecular

sieves (4Å) were used to dry the rest of solvents such as DCM and MeOH. Thin Layer

Chromatography (TLC) employed aluminium-backed plates (silica gel 60 F254), and

visualised under UV light. Flash column chromatography was utilised to purify and

separate organic substrates via normal silica gel 60. NMR spectra were recorded at 400 and

100 MHz for 1H and 13C respectively on a Bruker AC400 spectrometer. The chemical

shifts (δ) in ppm and the coupling constants (J in Hz) were recorded in the standard fashion

with tetramethylsilane TMS as internal reference (for 1H) or the central line (77.1 ppm) of

CDCl3 (for 13C). Full assignment of spectra based on 1H, and 13C NMR with aid of DEPT-

135, 2D-COSY, 2D-HSQC, 2D-HMBC and 2D-NOESY NMR. A Bruker Alpha FT-IR

spectrometer was used to obtain infrared spectra. High resolution and electrospray mass

spectra were obtained using a Micromass Platform II Spectrometer. Stuart Scientific SMPI

apparatus was utilised to measure the melting points, which are uncorrected. Optical

rotations were determined at 589 nm in a 2 cm3 cell using an AA-1C00 optical activity

polarmeter, and the latter was calibrated with R-carvone. X-Ray structure determinations

were obtained using a Nonius kCCD or a Nonius Mach3 machine. nBuLi was standardised

against N-pivaloyl-o-toluidine or diphenylacetic acid.

7. Experimental

155

7.2 Experimental procedures and data

7.2.1 Experimental procedures and data: Chapter 2

1-Hydroxy-1,2-benziodoxol-3-(1H)-one1-oxide (2-iodoxybenzoic acid), IBX, 55 51

To a solution of Oxone® (45.25 g, 72.5 mmol, 1.3 equiv., dissolved in 200 mL deionised

water in a 1L flask), was added 2-iodobenzoic acid (12.5 g, 50 mmol) in one portion. The

reaction mixture was heated to 70 ºC and mechanically stirred for 3 h. Initially, thick slurry

coated the walls of the flask, which subsequently dispersed. The reaction was cooled to 5

ºC for 1 h. A sintered-glass funnel was used to filter the mixture and solid IBX washed

with cold water (6 x 25 mL) and acetone (2 x 25 mL). The crystalline solid was dried

overnight at RT to afford white powder of IBX 55 (11.22 g, 79%); (lit. 232−234 ºC)51; m.p

231−233 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.15 (d, J = 8.0 Hz, 1H), 8.08–8.03 (m,

2H), 7.84 (t, J = 7.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 167.5 (C=O), 146.5 (CH),

133.4 (C), 132.9 (C), 131.4 (CH), 130.0 (CH), 125.0 (CH).

(2R,5R)-(+)-Isomenthone 38 57

Method (a) Pyridine sulphur trioxide 48

To a magnetically-stirred mixture of (1S,2R,5R)-(+)-isomenthol 54 (3.06 g, 19.6 mmol),

triethylamine (20 mL, 143 mmol) and DMSO (5 mL) at 0 °C, was added pyridine sulphur

trioxide complex (9.3 g, 59.4 mmol), dissolved in DMSO (10 mL). The mixture was

allowed to stir for 4 h at RT. The contents of the round-bottomed flask were poured into a

mixture of saturated ammonium chloride (25 mL) and diethyl ether (25 mL), transferred to

separatory funnel and washed with saturated brine (3 x 5 mL), dried (MgSO4) and filtered.

IOHO

O

OO

OH

I

55

(R)(R)(R)(R)

O(R)(R)(S)(S)(R)(R)

OH

54 38

7. Experimental

156

The crude product 38 was concentrated in vacuo, and purified by flash column

chromatography (10:1−8:1 hex:Et2O) to afford 38 (1.61 g, 53% yield as a colourless oil)

Method (b): Iodoxybenzoic acid (IBX)/DMSO 53

(1S,2R,5R)-(+)-Isomenthol 54 (3.0 g, 19.2 mmol) was added in one portion to a solution of

IBX (8 g, 28.5 mmol dissolved in DMSO (20 mL). A milky white solution was formed and

the mixture allowed to stir for 24 h at RT. Water was added to quench the reaction and the

precipitate filtered off and washed with ether (2 x 100 mL). The organic layer was

separated via separatory funnel and the aqueous layer washed with ether (30 mL) The total

organic extracts were dried over (MgSO4), filtered and concentrated via reduced pressure

to afford crude ketone of 38. The crude was purified via column chromatography using

hex:Et2O (10:1−8:1) to furnish (2R,5R)-(+)-isomenthone 38 (2.1 g, 70%) as a colourless

oil. 1H NMR (400 MHz, CDCl3) δ 2.23 (ddt, J = 13.0, 4.5, 1.3 Hz, 1H), 2.03 (dd, J = 12.8,

10.0 Hz, 1H), 1.91−1.83 (m, 4H), 1.70−1.57 (m, 2H), 1.45−1.36 (m, 1H), 0.92 (d, J = 6.4

Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H, CH3CHCH3), 0.77 (d, J = 6.8 Hz, 3H, CH3CHCH3). 13C NMR (100 MHz, CDCl3) δ 214.6 (C=O), 57.2 (CH), 48.0 (CH2), 34.4 (CH), 29.4

(CH2), 26.9 (CH), 26.8 (CH2), 21.2 (CH3), 21.0 (CH3), 19.7 (CH3).

HRMS (ES) m/z for C10H18ONa, (Calc.: 177.1255, Found: 177.1248); colourless oil; Anal.

Calc. for C10H18O, Expected C, 77.9; H, 11.8; Found C, 77.8; H, 11.7; IR νmax 2955, 2912,

2837, 1704, 1520, 1340, 1125, 1020 cm–1; lit [α]D + 92° (c 0.46, CH2Cl2, 24 °C)53, [α]D +

94.4° (c 0.12, CH2Cl2, 24 °C).

(1S,2S,5R)-(–)-Isopinocamphone 39 169

Method (a): Iodoxybenzoic acid (IBX)/DMSO

(1S,2S,3S,5R)-(+)-Isopinocampheol 56 (6.0 g, 3.9 mmol) was added in one portion to a

solution of IBX (16.4 g, 5.7 mmol, in 40 mL DMSO). The mixture was allowed to stir for

24 h at RT, and the reaction then quenched by addition of water (30 mL). The precipitate

was filtered off and washed with ether (2 x 150 mL). The organic layer was separated and

12

3 4

56

789

10(S)(S)

(S)(S) (R)(R)

O

(S)(S)

(S)(S)

(R)(R)

O≡

(S)(S)

(S)(S) (R)(R)

(S)(S)

OH

56 39

7. Experimental

157

the aqueous layer washed with ether (30 mL). The total organic extracts were dried

(MgSO4), filtered and solvents removed via reduced pressure to give the crude of 39.

DMSO was removed via distillation to afford (–)-isopinocamphone 39 (4.0 g, 67% yield)

as a colourless oil.

Method (b): Formation of IBX in situ

2-Iodobenzoic acid (0.3 eq., 3.5 gm) and Oxone® (1.0 eq., 11.1 gm) were added to

acetonitrile/water (500 mL, 2:1 v/v) solution of 56 (45 mmol, 9 g), and the temperature

elevated to 70 °C. The reaction was stirred for (6 h), and cooled via ice bath to precipitate

hypervalent iodine by-product. The precipitate was filtered off and washed with H2O (2 x

100 mL), and DCM (2 x 100 mL). The combined organic layer washed with DCM (150

mL) and aq. Na2CO3 (150 mL, 15% solution). The organic layer was dried (MgSO4), and

purified via column chromatography (10:1 hex:Et2O) to obtain 39 (5.1 g, 78%) as a

colourless oil along with recovered 56 (1.8 g, 20%). 1H NMR (400 MHz, CDCl3) δ 2.63−2.54 (m, 2H, CHCH2C=O, 2 x H-4), 2.49 (m, 1H,

CH3CHCH, H-2), 2.44−2.38 (m, 1H, CHCH2CH, H-6), 2.07 (td, J = 6.0, 3.0 Hz, 1H,

CHCH2CH, H-5), 2.01 (m, 1H, CHCH2CH, H-1), 1.27 (s, 3H, CH3CCH3), 1.16 (d, J = 7.4

Hz, 3H, CH3CHCH), 1.15 (m, 1H, CHCH2CH, H-6), 0.83 (s, 3H, CH3CCH3). 13C NMR (100 MHz, CDCl3) δ 215.2 (C=O), 51.4 (CH3CCH3, C-7), 45.1 (CH3CHCH, C-

2), 44.9 (CHCH2C=O, C-4), 39.3 (CHCH2CH, C-1), 39.1 (CHCH2CH, C-5), 34.5 (CHCH2CH,

C-6), 27.2 (CH3CHCH, C-10), 22.1 (CH3CCH3), 17.0 (CH3CCH3).

HRMS (ES) m/z for C10H16ONa, [MNa]+, (Calc.: 175.1099, Found: 175.1092); colourless

oil; Anal. Calc. for C10H16O, Expected C, 78.9; H, 10.6; Found C, 78.8; H, 10.4; IR νmax

2977, 2917, 1707 (C=O), 1469, 1370, 1179, 1086, 1049 cm–1; lit [α]D −15.2º, (c 0.42,

MeOH, 25 °C)169, [α]D −16º (c 0.90, CH2Cl2, 24 ºC).

(5R,6S)-6-Benzyl-2-methyl-5-(prop-1-en-2-yl) cyclohexane-2-enone, 57, and (5R,6R)-

6-Benzyl-2-methyl-5-(prop-1-en-2-yl) cyclohexane-2-enone, 58 30,44,45

(R)(R)(S)(S)

(Z) O

(R)(R)(R)(R)

(Z) O1

23

45

6

7

810

11

9

(R)(R)

(Z) O

36 44 45

7. Experimental

158

A hexane solution of nBuLi (59.44 mL, 83.20 mmol, 1.4 M) was added dropwise to a cold

solution (−10 °C under nitrogen gas) of DIPA (11.7 mL, 83.20 mmol) in anhydrous THF

(60 mL) over a period of 10 min. The mixture was continuously stirred for 10 min to allow

LDA formation. (R)-carvone (9.6 g, 63.8 mmol) in anhydrous THF (90 mL) was slowly

added and the mixture stirred for 45 min at the same temperature. The enolate was then

reacted with an excess of methyl iodide CH3I (20 mL, 321.3 mmol), and the reaction

mixture allowed stirring overnight at RT. It was extracted with diethyl ether (3 x 30 mL).

The organic layer was acidified with 3M aq. HCl and then washed with NaHCO3 and

brine. The combined organic extracts were dried and filtered, followed by evaporating the

solvent. The residue was chromatographed (eluent: EtOAc:hexane 40:1–20:1) to give a 3:2

epimeric mixture of 6-methyl carvone (pale yellow oil, 9.63 g, 85% yield).

To a diastereomeric mixture of 6-methylcarvone (1 g, 6.09 mmol) in CH2Cl2 (10 mL), was

added DBU (0.9 mL, 6.09 mmol) and the reaction mixture was allowed to stir for 24 h at

room temperature. The reaction mixture was washed with 1 M HCl (1 x 30 mL), water (1 x

30 mL) and brine (1 x 30 mL). The organic layer was dried (Na2SO4), solvents removed in

vacuo and column chromatography eluting with hexane:EtOAc (40:1–50:1) afforded a 3:1

epimeric mixture of 6-methylcarvone. Partial separation of diastereomers (250 mg),

providing major diastereomer 44 (80 mg, 32%, Rf 0.54) as white crystals, and, minor

diastereomer 45, (20 mg, 8%, Rf 0.52) as a colourless oil, along with 150 mg of a

diastereomeric mixture (60%).

The mixture of isomers (200 mg) was also recrystallized using hexane (−10 °C) to yield

white crystals of 44 (76 mg, 38%).

(6S)-Diastereomer (44): 1H NMR (400 MHz, CDCl3) δ 6.69−6.65 (m, 1H, CH=CCH3, H-

3), 4.81−4.79 (m, 2H, CH2=C, H-9), 2.52−2.39 (m, 2H, CH2CHCH and CH2CHCH, H-6

and H-5), 2.37−2.32 (m, 1H, CH2CHCH, H-5), 2.29−2.23 (m, 1H, CH2CHCH, H-4), 1.78

(dt, J = 2.5, 1.3 Hz, 3H, COCCH3), 1.71 (dd, J = 1.4, 0.8 Hz, 3H, CH2=CCH3), 1.05 (d, J

= 6.6 Hz, 3H, CH3CHC=O). 13C NMR (100 MHz, CDCl3) δ 203.6 (C=O), 145.6 (CH3C=CH2, C-8), 144.1 (CH3C=CH,

C-3), 134.7 (CH3C=CH, C-2), 113.2 (CH3C=CH2, C-9), 50.6 (CH2CHCH, C-5), 44.2

(CH2CHCH, C-6), 31.2 (CH2CHCH, C-4), 21.9 (CH2=CCH3, C-10), 16.2 (COCCH3, C-7),

12.5 (CH3CHC=O, C-11).

HRMS (ES) m/z for C11H16ONa, [MNa]+, (Calc.: 187.1099, Found: 187.1096); oil; Anal.


7. Experimental

159

2924, 1664 (C=O), 1444, 1365, 1290, 1134, 1045. 951, 846 cm–1; lit [α]D +12.5º (c 1.1,

CH2Cl2, 24 ºC)30; [α]D +12.9º (c 1.7, CH2Cl2, 24 ºC); (lit m.p 38−40 ºC)30; m.p 37−38 ºC.

(6R)-Diastereomer (45): 1H NMR (400 MHz, CDCl3) δ 6.72 (ddt, J = 5.7, 2.7, 1.4 Hz, 1H,

CH=CCH3, H-3), 4.91 (dq, J = 2.7, 1.4 Hz, 1H, CH2=C, H-9), 4.73 (s, 1H, CH2=C, H-9),

2.74−2.64 (m, 2H, CH2CHCH and CH2CHCH, H-6 and H-5), 2.50−2.45 (m, 1H,

CH2CHCH, H-4), 2.33−2.26 (m, 1H, CH2CHCH, H-4), 1.78 (dt, J = 2.5, 1.3 Hz, 3H,

COCCH3), 1.70 (dt, J = 1.4, 0.7 Hz, 3H, CH2=CCH3), 0.92 (d, J = 7.2 Hz, 3H,

CH3CHC=O). 13C NMR (100 MHz; CDCl3) δ 203.7 (C=O), 145.0 (CH3C=CH2, C-8), 144.2 (CH3C=CH,

C-3), 133.7 (CH3C=CH, C-2), 111.5 (CH3C=CH2, C-9), 44.8 (CH2CHCH, C-5), 43.1

(CH2CHCH, C-6), 26.4 (CH2CHCH, C-4), 22.0 (CH2=CCH3, C-10), 16.1 (COCCH3, C-7),

10.6 (CH3CHC=O, C-11).

HRMS (ES) m/z for C11H16ONa, [MNa]+, (Calc.: 187.1099, Found: 187.1105); oil; IR νmax

2969, 2923, 1667 (C=O), 1450, 1365, 1249, 1130, 1061, 989, 888 cm–1; [α]D +28.5º (c 0.7,

CH2Cl2, 24 ºC).

(5R,6S) 6-Benzyl-2-methyl-5-(prop-1-en-2-yl) cyclohexane-2-enone, 57, and (5R,6R) 6-

Benzyl-2-methyl-5-(prop-1-en-2-yl) cyclohexane-2-enone, 58 44

A solution of hexane of nBuLi (2.2 mL, 2.05 M) was added over 10 min to a solution of

DIPA (0.6 mL, 4.2 mmol) in freshly distilled THF at (−10 ºC) under nitrogen atmosphere.

The reaction mixture was cooled to (−78 ºC), and R-carvone (0.61 g, 4.0 mmol) in

anhydrous THF was slowly added. The mixture was allowed to stir for 45 min, and then,

benzyl bromide (1.2 eq., 4.8 mmol, 0.59 mL) in THF was added one portion. The reaction

mixture was vigorously stirred for further 50 min at same temperature, thence, the reaction

was quenched with saturated ammonium chloride (10 mL). The crude was transferred to

(250 mL) separatory funnel, and diluted with diethyl ether (100 mL). The organic layer

was acidified with 3M aq. HCl, and then washed with saturated NaHCO3 and brine. The

crude was purified via flash column chromatography (hexane:EtOAc 10:1), to afford major

(R)(R)

(Z) O

36

(S)(S)

(Z) O(R)(R)

(Z) O

1213

109 8

7

654

2

11

57 58

7. Experimental

160

diastereomer 57 (colourless oil, 227 mg, 23%, Rf 0.54 in 1:1 hexane:EtOAc), and mixture

of both isomers 57 and 58, (455 mg, 48%). The total yield was 71% with a diastereomeric

ratio 5:1.

(6S)-Diastereomer (57): 1H NMR (400 MHz, CDCl3) δ 7.27−7.07 (m, 5H, 5 x HAr),

6.66−6.62 (m, 1H, CH=CCH3, H-3), 4.88 (quint, J = 1.5 Hz, 1H, CH2=C, H-9), 4.79−4.78

(m, 1H, CH2=C, H-9), 2.95 (dd, J = 12.3, 5.8 Hz, 2H, CH2-Ph, 2 x H-11), 2.72 (ddd, J =

10.1, 6.9, 5.1 Hz, CH2CHCH, H-6), 2.57 (dt, J = 10.1, 6.9 Hz, CH2CHCH, H-5),

2.42−2.38 (m, 2H, CH2CHCH, H-4), 1.78 (q, J = 1.7 Hz, 3H, COCCH3), 1.66 (dd, J = 1.3,

0.7 Hz, 3H, CH2=CCH3). 13C NMR (100 MHz, CDCl3) δ 201.4 (C=O), 146.1 (CH3C=CH2, C-8), 143.3 (CH3C=CH,

C-3), 140.5 (CAr, C-12), 135.5 (CH3C=CH, C-2), 131.3 (CHAr), 129.9 (CHAr), 128.5

(CHAr), 128.2 (CHAr), 126.3 (CHAr), 114.0 (CH3C=CH2, C-9), 54.0 (CH2CHCH, C-5), 51.7

(CH2CHCH, C-6), 47.0 (CH2CHCH, C-4), 34.3 (CH2-Ph, C-11), 20.0 (CH2=CCH3, C-10),

16.6 (COCCH3, C-7).

HRMS (ES) m/z for C17H20ONa, [MNa]+, (Calc.: 263.1412, Found: 263.1412); oil; Anal.


2970, 2920, 1665 (C=O), 1451, 1365, 1166, 1044 cm–1; [α]D +23º (c 0.60, CH2Cl2, 24 ºC).

(5R,6S)-6-Ethyl-2-methyl-5-(prop-1-en-2-yl)cyclohexane-2-enone, 59, and (5R,6R)-6-Ethyl-

2-methyl-5-(prop-1-en-2-yl)cyclohexane-2-enone, 60 (mixture of isomers) 44

The title compounds were prepared from 36 according to the procedure used for 57 and 58.

Ethyl iodide (10 eq., 3.0 g, 39 mmol) in THF (10 mL) was added to the lithium enolate of

carvone, and the reaction stirred for 1 h. The combined organic extracts were dried and

filtered, followed by evaporating the solvent in vacuo. The residue was purified with flash

column chromatography (hexane:EtOAc 10:1) to afford (1:1) diastereomeric mixture (pale

yellow oil, 456 mg, 64%).

(6R and 6S) Diastereomeric mixture was assigned based on 1H, 13C, DEPT-135, 2D-

COSY, 2D-HSQC, and 2D-HMBC NMR spectra.

(R)(R)

(Z) O

36

(S)(S)

(Z) O

(R)(R)

(Z) O12

13

109 8

7

654

2

11

59 60

7. Experimental

161

1H NMR (400 MHz, CDCl3) δ 6.65−6.60 (m, 1H, CH=CCH3, H-3), 4.90−4.80 (m, 1H,

CH2=C, H-9), 4.77−4.70 (m, 1H, CH2=C, H-9), 2.75−2.65 (m, 1H CH2CHCH, H-5),

2.48−2.22 (m, 3H, CH2CHCH, (2 x H-4) and H-6), 1.73 (dt, J = 2.5, 1.3 Hz, 3H,

COCCH3), 1.68 (dd, J = 1.3, 0.7 Hz, 3H, CH2=CCH3, H-7), 1.49−1.19 (m, 2H, CH3CH2, 2

x H-11), 0.84 (t, J = 7.3 Hz, 3H, CH3CH2, H-12). 13C NMR (100 MHz, CDCl3) δ 202.9, 201.3 (C=O), 145.5, 145.2 (CH3C=CH2, C-8),

143.3, 142.9 (CH3C=CH, C-3), 135.2, 133.6 (CH3C=CH, C-2), 113.1, 111.7 (CH3C=CH2,

C-8), 50.3, 50.1 (CH2CHCH, C-5), 46.6, 45.9 (CH2CHCH, C-6), 30.6, 27.1 (CH2CHCH,

C-4), 22.1, 20.2 (CH3CH2, C-11), 18.8, 18.0 (CH2=CCH3, C-10), 16.1, 16.0 (COCCH3, C-

7), 12.1, 10.8 (CH3CH2, C-12).

(6R and 6S) Diastereomers mixture: HRMS (ES) m/z for C12H19O, [MH]+, (Calc.:

179.1436, Found: 179.1436); oil mixture; Anal. Calc. for C12H18O, Expected C, 80.8; H,

10.1; Found C, 80.3, H, 9.8; IR νmax 2968, 2922, 1666 (C=O), 1438, 1378, 1233, 1085,

1046, 1033, 893, 851, 843 cm–1.

(2S,3R,6R)-6-Isopropyl-2,3-dimethylcyclohexan-1-one, 61, and (2R,3R,6R)-6-Isopropyl-

2,3-dimethylcyclohexan-1-one 62 (mixture of isomers)

A solution of n-BuLi in hexane (1.6 mL, 3.26 mmol, 2.04 M) was added dropwise to

solution of DIPA (3.36 mmol, 0.5 mL) in THF (0.8 M) at −10 °C, and the mixture allowed

to stir for 15 min. Then, (+)-isomenthone 38 (3.25 mmol, 500 mg) was added dropwise to

the LDA solution and stirred for 25 min. The vessel was cooled to −50 °C and DMPU

(3.26 mmol, 0.4 mL) was added in one portion to the reaction mixture. Iodomethane (5 eq.,

11.25 mmol, 0.73 mL) was slowly added at a temperature strictly maintained at −45 °C.

The reaction was monitored by TLC plate and quenched after 40 min with saturated NH4Cl

(10 mL):Et2O (100 mL). The organic layer was extracted and aqueous layer washed with

diethyl ether (3 x 25 mL) and the combination of organic layer was dried (MgSO4),

evaporated and purified by flash column chromatography using hex:Et2O 20:1 to afford a

diastereomeric mixture of 61 and 62 as a colourless oil (219 mg, 40% yield, d.r. 9:1, Rf

0.76 in 7:1 hexane:Et2O).

(S)(S)

H

OH

(R)(R)H

H

O1 2

345

67

8

9

10

H

O

38 61 62

7. Experimental

162

(2S)-Diastereomer (61): 1H NMR (400 MHz; CDCl3) δ 2.45−2.37 (m, 1H), 2.09−1.94 (m,

3H), 1.74−1.41 (m, 4H), 1.01 (t, J = 6.3 Hz, 6H, 2 x CH3), 0.93 (d, J = 6.4 Hz, 3H, CH3), 0.78

(d, J = 6.4 Hz, 3H, CH3). 13C NMR (100 MHz; CDCl3) δ 216.7 (C=O), 57.6 (CH), 49.0 (CH), 41.5 (CH2), 29.5

(CH2), 27.9 (CH), 27.5 (CH2), 21.0 (CH3), 20.8 (CH3), 20.4 (CH3), 12.4 (CH3).

HRMS (ES) m/z for C11H20ONa, [MNa]+, (Calc.: 191.1412, Found: 191.1417); colourless

oil; IR νmax 2955, 2926, 2870, 1703, 1455, 1370, 1186, 1036 cm–1.

(2R,3R,6R)-2-Benzyl-6-isopropyl-3-methylcyclohexan-1-one, 63, and (2S,3R,6R)-2-

Benzyl-6-isopropyl-3-methylcyclohexan-1-one, 64 (mixture of isomers)

The title compounds were synthesised from (+)-isomenthone according to the procedure

used for 61 and 62. Benzyl bromide (1.2 eq., 3.9 mmol, 0.48 mL) was added to the lithium

enolate of isomenthone, and the reaction stirred for 1 h. The crude product was purified via

flash column chromatography (hex:Et2O 20:1) to give a diastereomeric mixture as a yellow

oil with d.r. (63:64, 97.5:2.5) and yield (286 mg, 36%, Rf 0.62 , 7:1; hexane:Et2O).

(2S)-Diastereomer (63): 1H NMR (400 MHz; CDCl3) δ 7.55-7.12 (m, 5H, 5 x HAr), 3.05

(dd, J = 13.8, 8.4 Hz, 1H, CH2-Ph, H-11), 2.74 (dd, J = 13.8, 4.7 Hz, 1H, CH2-Ph, H-11),

2.48−2.44 (m, 1H, CHCH2-Ph, H-2), 2.08−1.98 (m, 2H), 1.92−1.42 (m, 5H), 1.07 (d, J =

6.5 Hz, 3H, CH3), 0.87 (d, J = 6.4 Hz, 3H, CH3), 0.60 (d, J = 6.4, 3H, CH3). 13C NMR (100 MHz; CDCl3) δ 215.9 (C=O), 141.4 (CAr, C-12), 130.2 (2 x CHAr), 128.8 (2

x CHAr), 126.5 (CHAr), 58.5 (CHCH2-Ph, C-2), 57.3 (CH), 39.1 (CH), 34.3 (CH), 29.7

(CH), 27.7 (CH2, CH2-Ph), 27.6 (CH3), 21.3 (CH3), 20.4 (2 x CH3).

HRMS (ES) m/z for C17H24ONa, [MNa]+, (Calc.: 267.1725, Found: 227.1733); yellow oil;

IR νmax 2955, 2926, 2869, 1703, 1494, 1368, 1031 cm–1.

H

O38

63 64

1 2

34

5

67

8

9

10

1112

(S)(S)

H

O (R)(R)H

H

OH

7. Experimental

163

(1S,4S,5S)-2,2,4,6,6-Pentamethylbicyclo[3.1.1]heptan-3-one, 65

A solution of n-BuLi (2.3 M, 2.3 mL, 3.3 mmol in hexane) was dropwise added to round-

bottomed flask containing DIPA (0.49 mL, 3.45 mmol) in dry THF (10 mL) under nitrogen

gas at 0 °C. The mixture was allowed to stir for 15 min, thence, the solution cooled to −78

°C, and a THF solution of isopinocamphone (477 mg, 3.14 mmol) was added and stirred

for 45 min. An excess of methyl iodide (1 mL, 5.0 eq.) in THF was added in one portion to

the lithium enolate solution and the mixture stirred for (1 h) at the same temperature. Then,

the mixture was allowed to stir overnight at RT, and extracted with (50 mL) diethyl ether

using a separatory funnel. The reaction mixture was neutralised by washing with (3 x 30

mL) of 1M HCl and (3 x 30 mL) of water, and ultimately treated with NaHCO3 (30 mL)

and brine (50 mL). The organic layer was dried over (MgSO4), solvents removed in vacuo,

and purified using flash column chromatography using hexane:Et2O 30:1−20:1 to afford

(350 mg, 66%, Rf 0.44 in hex:Et2O 4:1) of 65 and (23 mg, 5% of 66) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 2.67−2.60 (m, 1H, CH3CH, H-4), 2.50−2.36 (m, 2H, CHCH2CH,

H-6), 2.01 (td, J = 6.3, 2.0 Hz, 1H, CHCH2CH, H-5), 1.90 (td, J = 6.2, 2.8 Hz, 1H, CHCH2CH,

H-1), 1.31 (s, 3H, CH3), 1.25 (d, J = 10.8 Hz, 3H, CH3), 1.19 (d, J = 7.8 Hz, 3H, CH3), 1.13 (d, J

= 7.8 Hz, 3H, CH3), 0.87 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 215.2 (C=O), 51.4 (C), 45.3 (CH), 44.9 (CH2), 39.3 (CH),

39.1 (CH), 34.5 (CH2), 27.2 (CH3), 22.1 (2 x CH3), 17.0 (2 x CH3).

MS (ES) m/z 180 (M+, 30%), 181 (MH+, 90%); oil; IR νmax 2976, 2907, 1707 (C=O), 1370,

1087, 976 cm–1; [α]D +39º (c 0.36, CH2Cl2, 24 ºC).

(1R,2R,4S,5S)-2,4,6,6-Tetramethylbicyclo[3.1.1]heptan-3-one, 66, and (1R,2S,4S,5S)-

2,4,6,6-Tetramethylbicyclo[3.1.1]heptan-3-one, 67 (mixture of isomers)

O

39 65

O

1

234

5 6

7

8

10

9

11

12

1

234

5 6

789

10

(R)(R)O(S)(S)O11

O

39 66 67

7. Experimental

164

The title compounds were synthesised from (–)-isopinocamphone 39 according to the

procedure used for 61 and 62. Iodomethane (0.73 mL, 11.3 mmol, 5eq.) was added to the

lithium enolate of isopinocamphone, and the reaction stirred for 30 min. The crude product

was purified via flash column chromatography (hex:Et2O 20:1) to afford a diastereomeric

mixture (d.r. 99.5:0.5, 66:67) as a colourless oil (224 mg, 41%, Rf 0.7 in 4:1 hex:Et2O).

(2S)-Diastereomer (66): 1H NMR (400 MHz, CDCl3) δ 2.68−2.61 (m, 1H, CHCHCH2, H-

2), 2.51−2.45 (m, 1H, CHCH2CH, H-6), 2.43−2.38 (m, 1H, CH2CHCH, H-4), 2.03 (td, J =

6.2, 2.8 Hz, 1H, CHCH2CH, H-1), 1.93 (td, J = 6.2, 2.8 Hz, 1H, CHCH2CH, H-5), 1.33 (s,

3H, CH3CCH3), 1.28 (m, 1H, CHCH2CH, H-6), 1.21 (d, J = 7.4 Hz, 3H, CH3CHCH), 1.15

(d, J = 7.4 Hz, 3H, CH2CHCHCH3), 0.89 (s, 3H, CH3CCH3). 13C NMR (100 MHz, CDCl3) δ 219.6 (C=O), 51.2 (CHCHCH2, C-4), 47.2 (CH2CHCH, C-2),

45.9 (CHCH2CH, C-1), 45.8 (CHCH2CH, C-5), 40.4 (CH3CCH3, C-7), 31.0 (CHCH2CH, C-

6), 27.9 (CH3CCH3), 22.2 (CH3CCH3), 17.6 (CH3CH, C-10), 16.3 (CH3CC=O, C-11).

HRMS (ES) m/z for C11H18ONa, [MNa]+, (Calc.: 189.1255, Found:189.1254); colourless

oil; IR νmax 2976, 2907, 1708 (C=O), 1470, 1458, 1387, 1326, 1087, 975 cm–1.

(1R,2R,4S,5S)-2-Benzyl-4,6,6-trimethylbicyclo[3.1.1]heptan-3-one, 68, and (1R,2S,4S,5S)-2-

Benzyl-4,6,6-trimethylbicyclo[3.1.1]heptan-3-one, 69 (mixture of isomers)

The title compounds were synthesised from (–)-isopinocamphone according to the

procedure used for 61 and 62. Benzyl bromide (1.2 eq., 3.9 mmol, 0.48 mL) was added to

the lithium enolate of isopinocamphone, and the reaction stirred for 45 min. The crude

product was purified via flash column chromatography (hex:Et2O 20:1) to afford a

diastereomeric mixture of 68 and 69 (291 mg, 37%, Rf 0.71 in 4:1 hex:Et2O, d.r. 99:1) as a

colourless oil.

(2S)-Diastereomer (68): 1H NMR (400 MHz, CDCl3) δ 7.33−7.28 (m, 2H, Ar), 7.24−7.21

(m, 1H, Ar), 7.18−7.16 (m, 2H, Ar), 3.20 (dd, J = 13.2, 3.8 Hz, 1H, CH2-Ph), 2.81−2.76

(m, 1H, CHCHC=O, H-2), 2.66 (dd, J = 13.2, 11.7 Hz, 1H, CH2-Ph), 2.54−2.46 (m, 2H,

O

39 68 69

1

234

5 6

789

10

(S)(S)O (R)(R)O11

12

14

15

1716

13

7. Experimental

165

CH3CH and CHCH2CH, H-4 and H-6), 2.06 (td, J = 6.2, 2.0 Hz, 1H, CHCH2CH, H-1),

1.84 (td, J = 6.2, 2.7 Hz, 1H, CHCH2CH, H-5), 1.42 (m, 1H, CHCH2CH, H-6), 1.27 (d, J =

7.3 Hz, 6H, 2 x CH3, CH3CH, and CH3CCH3), 0.86 (s, 3H, CH3CCH3). 13C NMR (100 MHz; CDCl3) δ 217.3 (C=O), 139.8 (CAr), 129.1 (2 x CHAr), 128.5 (2 x

CHAr), 126.2 (CHAr), 54.1 (CHCHCH2, C-4), 51.1 (CHCH2-Ph, C-2), 45.1 (CHCH2CH, C-

1), 41.1 (CHCH2CH, C-5), 39.4 (CH3CCH3, C-7), 36.2 (CH2-Ph, C-11), 30.1 (CHCH2CH,

C-6), 27.3 (CH3CCH3), 21.6 (CH3CCH3), 17.1 (CH3CH, C-10).

HRMS (ES) m/z for C17H22ONa, [MNa]+ (Calc.: 265.1568, Found: 265.1574); colourless

oil; IR νmax 3338, 2960, 1744 (C=O), 1524, 1431, 1367, 1208, 962 cm–1.

(1`R,5R,6R)-6-(1-Hydroxyethyl)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-one, 103

Under nitrogen atmosphere, to a solution of DIPA (1.2 mL, 8.4 mmol) in anhydrous THF

(15 mL) was slowly added nBuLi (6.3 mL, 1.3 M) over 10 min at 0 °C. The basic solution

was cooled to (−78 °C) and R-carvone 36 (1.168 mg, 7.8 mmol) in freshly distilled THF

was added dropwise. The reaction mixture was magnetically stirred for 45 min and ethanal

(348 mg, 7.8 mmol) was added in one portion. The reaction was allowed to stir for 20 min

at the same temperature, followed by adding saturated ammonium chloride NH4Cl (15 mL)

to quench the reaction. The mixture was transferred to a separatory funnel, and diluted with

diethyl ether (150 mL). The contents of flask were washed with saturated ammonium chloride

(3 x 20 mL) and water (20 mL). The organic layer was dried (MgSO4), filtered and solvents

removed in vacuo to afford crude β-hydroxyketone (1.48 g, 98%, diastereomeric ratio

68:18:11:3). Diastereomers were separated using flash column chromatography (10:1

hex:EtOAc) to give a combined yield of diastereomers (1.13 g, 75%) consists of (5R,6R)

major diastereomer (103, 1.01 g) as a yellow oil (Rf 0.29, 6:1 hex:EtOAc, and a mixture of

three isomers (0.12 g).

(1`R)-Diastereomer (103): 1H NMR (400 MHz, CDCl3) δ 6.75−6.71 (m, 1H, CH=CCH3,

H-3), 4.83 (q, J = 1.1 Hz, 2H, CH2=CCH3, 2 x H-9), 3.72 (s, 1H, CHOH), 3.70 (qd, J =

6.7, 3.8 Hz, 1H, CHOH, H-1`), 2.64 (ddd, J = 12.8, 3.8, 0.9 Hz, 1H, CH2CHCH, H-6),

1 `1

23

45 6

7

89 10

(R)(R)

(Z) O

(R)(R)HO HH

H

(Z) O

36 103

7. Experimental

166

2.57 (ddd, J = 12.8, 10.3, 4.3 Hz, 1H, CH2CHCH, H-5), 2.46−2.36 (m, 1H, CH2CHCH, H-

4), 2.27−2.20 (m, 1H, CH2CHCH, H-4), 1.74 (dt, J = 2.6, 1.3 Hz, 3H, COCCH3), 1.73 (t, J

= 1.1 Hz, 3H, CH2=CCH3), 1.19 (d, J = 6.4 Hz, 3H, CH3CHOH). 13C NMR (100 MHz, CDCl3) δ 203.2 (C=O), 145.4 (CH3C=CH2, C-8), 144.2 (CH3C=CH, C-

3), 135.7 (CH3C=CH, C-2), 114.0 (CH3C=CH2, C-9), 67.6 (CHOH, C-1`), 53.2 (CH2CHCH,

C-5), 46.2 (CH2CHCH, C-6), 31.2 (CH2CHCH, C-4), 17.9 (CH2=CCH3, C-10), 17.8

(COCCH3, C-7), 15.5 (CH3CHOH).

HRMS (ES) m/z for C12H18O2Na, [MNa]+, (Calc.: 217.1199, Found: 217.1196); oil; IR νmax

3450 (OH), 2974, 2938, 1655 (C=O), 1453, 1433, 1361, 1267, 1169, 1056, 1015 cm–1; [α]D

–10.9º (c 0.70, CH2Cl2, 24 ºC).

(1`R,5R,6R)-3-Methyl-2-oxo-6-(prop-1-en-2-yl)cyclohex-3-en-1-yl)ethyl acetate, 104

Acetic anhydride (0.40 mL, 4.27 mmol, 10 eq.) and pyridine (0.17 mL, 2.14 mmol, 5.0 eq.)

were added to DCM solution of diastereomer 103 (83 mg, 0.427 mmol), and the reaction

stirred at (50 ºC) till no starting materials were detectable on TLC. The mixture was diluted

with DCM (30 mL), and then washed with CuSO4 (3 x 5 mL) and water (3 x 5 mL). The

organic layer was dried, concentrated and filtered over a small pad of silica gel

(hexane:EtOAc 5:1) to yield 104 (85 mg, 79%) as a yellow oil.

(1`R)-Diastereomer (104): 1H NMR (400 MHz, CDCl3) 6.64−6.62 (m, 1H, CH=C, H-3),

6.00 (d, J = 3.5 Hz, 1H, CHOAc, H-1`), 4.78−4.58 (m, 2H, CH2=CCH3, 2 x H-9),

2.99−2.92 (m, 1H, CH2CHCH, H-6), 2.59−2.56 (m, 1H, CH2CHCH, H-5), 2.30−2.25 (m,

2H, CH2CHCH, 2 x H-4), 1.95 (s, 3H, COOCH3), 1.70−1.66 (m, 3H, CH3CCO), 1.65 (dd,

J = 1.4, 0.7 Hz, 3H, CH2=CCH3), 1.60 (d, J = 6.4 Hz, 3H, CH3CHOAc). 13C NMR (100 MHz, CDCl3) δ 196.2 (C=O), 171.5 (COOCH3), 145.0 (CH3=CCH2, C-8),

144.0 (CH3C=CH, C-3), 143.2 (CH3C=CH, C-2), 113.1 (CH3C=CH2, C-9), 75.4

(CHCHOAc, C-1`), 55.9 (CH3CHCH, C-6), 43.0 (CH2CHCH, C-5), 29.9 (CH2CHCH, C-

4), 21.5 (COOCH3), 21.4 (CH2=CCH3, C-10), 21.2 (COCCH3, C-7) 16.5 (CH3CHOAc).

1 `

O

HOH

H

12

3

4

5

6

7

89 10

12

O(R)(R)

(Z) O

(R)(R)HO HH

H

103 104

7. Experimental

167

HRMS (ES) m/z for C14H21O3, [MH]+, (Calc.: 237.1485, Found: 237.1480); IR νmax 2985,

2926, 1766 (C=O ester), 1661 (C=O), 1470, 1357, 1222, 1106, 855, 762 cm–1; [α]D –45º (c

0.42, CH2Cl2, 24 ºC).

(1`R,5R,6R)-6-(1-Hydroxypropyl)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-one, 105, and

(1`S,5R,6R)-6-(1-Hydroxypropyl)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-one, 106

The title compounds were prepared from 36 according to the procedure used for 103.

Propanal (458 mg, 7.8 mmol) in THF (10 mL) was added to the lithium enolate of carvone,

and the reaction stirred for 45 min. The organic layer was dried (MgSO4), filtered and

solvents removed in vacuo to give crude product mixture (1.62 g, 98%). The crude product

was purified with flash column chromatography (10:1 hex:EtOAc) to afford diastereomeric

product 64.3% (1.04 g, Rf 0.28 hexane:EtOAc 5:1, diastereomeric ratio 53:40:6:1), and

further separation with reverse-column HPLC (hex:EtOAc 90:10) afforded separation of

the major diastereomers as oils (0.57 g of 105 and 0.47 g of 106).

(1`R)-Diastereomer (105): 1H NMR (400 MHz, CDCl3) δ 6.69 (ddd, J = 5.5, 2.9, 1.4 Hz,

1H, CH=CCH3, H-3), 4.92−4.91 (m, 1H, CH2=CCH3, H-9), 4.90 (quint, J = 1.5 Hz, 1H,

CH2=CCH3, H-9), 3.61−3.56 (m, 1H, CHOH, H-1`), 3.01 (ddd, J = 11.7, 10.0, 5.1 Hz, 1H,

CH2CHCH, H-6), 2.50−2.42 (m, 2H, CH2CHCH and OH), 2.38−2.32 (m, 1H, CH2CHCH,

H-4), 1.95−1.87 (m, 2H, CH2CHCH and CH3CH2CHOH), 1.77 (dt, J = 2.5, 1.3 Hz, 3H,

COCCH3), 1.72 (dd, J = 1.4, 0.7 Hz, 3H, CH2=CCH3), 1.70−1.62 (m, 1H, CH3CH2), 0.97

(t, J = 7.5 Hz, 3H, CH3CH2). 13C NMR (100 MHz; CDCl3) δ 202.4 (C=O), 145.5 (CH3C=CH2, C-8), 144.1 (CH3C=CH,

C-3), 136.5 (CH3C=CH, C-2), 114.2 (CH3C=CH2, C-9), 72.3 (CHOH, C-1`), 53.7

(CH2CHCH, C-5), 46.0 (CH2CHCH, C-6), 31.4 (CH2CHCH, C-4), 30.1 (CH3CH2CHOH),

18.5 (CH2=CCH3, C-10), 15.7 (COCHCH3, C-7), 11.4 (CH3CH2CHOH).


3455 (OH), 2966, 2921, 2875, 1659 (C=O), 1456, 1449, 1351, 1309, 1175, 1110, 971 cm–1;

[α]D −32º (c 0.20, CH2Cl2, 24 ºC).

1 `1

23

45 6

7

89 10

(R)(R)

(Z) O

(R)(R)HO HH

H

(Z) O

36 105

(R)(R)

(Z) O

(S)(S) HO HH

H

106

7. Experimental

168

(1`S)-Diastereomer (106): 1H NMR (400 MHz, CDCl3) δ 6.78 (ddq, J = 6.1, 2.5, 1.3 Hz,

1H, CH=CCH3, H-3), 4.85−4.82 (m, 2H, CH2=CCH3, 2 x H-9), 3.41 (td, J = 9.6, 3.6 Hz,

1H, CHOH, H-1`), 2.74 (ddd, J = 13.0, 3.7, 1.2 Hz, 1H, CH2CHCH, H-6), 2.68 (ddd, J =

13.3, 9.8, 3.9 Hz, 1H, CH2CHCH, H-5), 2.52−2.41 (m, 2H, CH2CHCH, and OH, H-4 and

OH), 2.34−2.24 (m, 1H, CH2CHCH, H-4), 1.77 (dt, J = 2.6, 1.3 Hz, 3H, COCCH3), 1.74

(dd, J = 1.4, 0.7 Hz, 3H, CH2=CCH3), 1.51−1.41 (m, 2H, CH3CH2CHOH), 1.00 (t, J = 7.4

Hz, 3H, CH3CH2). 13C NMR (100 MHz; CDCl3) δ 203.3 (C=O), 145.6 (CH3C=CH2, C-8), 144.3 (CH3C=CH,


(CH2CHCH, C-5), 46.7 (CH2CHCH, C-6), 31.5 (CH2CHCH, C-4), 24.6 (CH3CH2CHOH),

18.1 (CH2=CCH3, C-10), 15.7 (COCHCH3, C-7), 11.0 (CH3CH2CHOH).


3452 (OH), 2968, 2924, 2876, 1661 (C=O), 1454, 1355, 1301, 1166, 1099, 959 cm–1; [α]D

−52º (c 0.30, CH2Cl2, 24 ºC).

(1`R,5R,6R)-6-(1-Hydroxy-2-methylpropyl)-2-methyl-5-(prop-1-en-2yl)cyclohexane-2-

enone, 107, and (1`S,5R,6R)-6-(1-Hydroxy-2-methylpropyl)-2-methyl-5-(prop-1-en-

2yl)cyclohexane-2-enone, 108


Isobutyraldehyde (560 mg, 7.8 mmol) in THF (10 mL) was added to the lithium enolate of

carvone, and the reaction stirred for 50 min. The organic layer was dried (MgSO4), filtered

and solvents removed in vacuo to afford crude β-hydroxyketone (89%, d.r. 79:19:1:1). The

crude product was purified via flash column chromatography (hexane:EtOAc10:1) to

afford a highly viscous mixture of separable diastereomers in a combined yield (1.127 g,

72 %; 1.056 g Rf 0.44 of 107, 0.053 g of 108), Rf 0.31 and diastereomeric mixture 0.018 g,

(4:1 hexane:EtOAc).


H-3), 4.77 (quint, J = 1.5 Hz, 1H, CH2=CCH3, H-9), 4.73−4.72 (m, 1H, CH2=CCH3, H-9),

1 `

1

23

45 6

7

89 10

(R)(R)

(Z) O

(R)(R)HO HH

H

(Z) O

36 107

(R)(R)

(Z) O

(S)(S) HO HH

H

108

7. Experimental

169

3.56 (s, 2H, CHOH, H-1ànd OH, after resolving with D2O afforded dd with J coupling

5.8, 4.1 Hz, H-1`), 2.79 (dt, J = 8.5, 7.0 Hz, 1H, CH2CHCH and gave ddd (J = 9.5, 8.6, 5.9

Hz) after resolving with D2O, H-5), 2.50 (dd, J = 9.7, 5.8 Hz, 1H, CH2CHCH, H-6),

2.39−2.34 (m, 2H, CH2CHCH, 2 x H-4), 1.75−1.72 (m, 1H, (CH3) 2CH), 1.70 (q, J = 1.8

Hz, 3H, COCCH3), 1.67 (dd, J = 1.4, 0.7 Hz, 3H, CH2=CCH3), 0.88 (d, J = 6.9 Hz, 3H,

(CH3)2CH), 0.77 (d, J = 6.8 Hz, 3H, (CH3)2CH). 13C NMR (100 MHz, CDCl3) δ 202.8 (C=O), 145.4 (CH3C=CH2, C-8), 144.5 (CH3C=CH,

C-3), 135.7 (CH3C=CH, C-2), 113.3 (CH3C=CH2, C-9), 76.1 (CHOH, C-1`), 52.4 (CH2CHCH,

C-5), 43.9 (CH2CHCH, C-6), 30.3 ((CH3)2CH), 30.1 (CH2CHCH, C-4), 20.7 (CH2=CCH3,

C-10), 19.6 (COCCH3, C-7), 16.9 ((CH3) 2CH), 15.7 ((CH3) 2CH).


3455 (OH), 2961, 2925, 2874, 1645 (C=O), 1448, 1365, 1012, 891 cm–1; [α]D −114º (c

0.07, CH2Cl2, 24 ºC).

(1`S)-Diastereomer (108): 1H NMR (400 MHz, CDCl3) δ 6.64 (m, 1H, CH=CCH3, H-3),

4.87−4.84 (m, 2H, CH2=CCH3, 2 x H-9), 3.17 (t, J = 8.1 Hz, 1H, CHOH, changed to dd (J

= 8.7, 2.0 Hz) after resolving by D2O), 2.98 (ddd, J = 11.3, 9.9, 5.1 Hz, 1H, CH2CHCH,

H-5), 2.60 (dd, J = 11.4, 2.4 Hz, 1H, CH2CHCH, H-6), 2.40−2.35 (m, 2 H, CH2CHCH, 2 x

H-4), 2.25−2.17 (m, 2H, (CH3)2CH, CHOH), 1.72 (dt, J = 2.4, 0.8 Hz, 3H, COCCH3), 1.70

(dd, J = 1.4, 0.8 Hz, 3H, CH2=CCH3), 0.97 (d, J = 6.6 Hz, 3H, (CH3) 2CH), 0.84 (d, J =

6.7 Hz, 3H, (CH3)2CH). 13C NMR (100 MHz, CDCl3) δ 202.3 (C=O), 145.4 (CH3C=CH2, C-8), 144.5 (CH3C=CH,


(CH2CHCH, C-5), 46.3 (CH2CHCH, C-6), 32.7 ((CH3)2CH), 30.9 (CH2CHCH, C-4), 20.2

(CH2=CCH3, C-10), 19.3 (COCCH3, C-7), 19.0 ((CH3)2CH), 15.5 ((CH3)2CH)).

HRMS (ES) m/z for C14H22O2Na, [MNa]+, (Calc.: 245.1512, Found: 245.1506); oil; Anal.

Calc. for C14H22O2, Expected C, 75.6; H, 10.0; Found C, 75.5; H, 9.9; IR νmax 3517 (OH),

3075, 2959, 2923, 2871, 1655 (C=O), 1449, 1434, 1366, 111, 972, 892 cm–1; [α]D −111º (c

0.09, CH2Cl2, 24 ºC).

(1`R,5R,6R)-6-(Cyclohexyl(hydroxy)methyl)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-

en-1-one, 109, and (1`S,5R,6R)-6-(Cyclohexyl(hydroxy)methyl)-2-methyl-5-(prop-1-

en-2-yl)cyclohex-2-en-1-one, 110

7. Experimental

170


Cyclohexanecarboxyaldehyde (872 mg, 7.8 mmol) in THF (10 mL) was added to the

lithium enolate of carvone, and the reaction stirred for 45 min. The organic layer was dried

(MgSO4), filtered and solvents removed in vacuo to afford crude β-hydroxyketone (1.8 g,

88%, diastereomeric ratio 60:34:6). The crude product was chromatographed using flash

column (10:1 hexane:EtOAc) to give two separable isomers in a combined yield 60.5 %

(1273 mg combined, 165 mg of isomers mixture, 1034 mg of 109 as a white crystals-Rf

0.43, 74 mg of 110 as a white crystals-Rf 0.36, 4:1 hexane:EtOAc).


H-3), 4.86 (quint, J = 1.5 Hz, 1H, CH2=CCH3, H-9), 4.80−4.79 (m, 1H, CH2=CCH3, H-9),

3.73 (d, 7.6 Hz, 1H, CHOH), 3.60 (s, 1H, CHOH), 2.84 (ddd, 9.8, 8.2, 5.5 Hz, CH2CHCH,

H-5), 2.70 (dd, J = 9.9, 5.5 Hz, CH2CHCH, H-6), 2.44−2.34 (m, 2H, HCy), 1.76 (q, J = 1.6

Hz, 3H, COCCH3), 1.74−1.73 (m, 1H, HCy), 1.72 (dd, J = 1.4, 0.7 Hz, 3H, CH2=CCH3),

170−1.68 (m, 2H, HCy), 1.62−1.52 (m, 2H, HCy), 1.51−1.41 (m, 3H, HCy), 1.26−1.13 (m,

3H, HCy) 13C NMR (100 MHz, CDCl3) δ 203.1 (C=O), 145.5 (CH3C=CH2, C-8), 144.8 (CH3C=CH,

C-3), 135.8 (CH3C=CH, C-2), 113.5 (CH3C=CH2, C-9), 76.3 (CHOH, C-1`), 52.0 (CH2CHCH,

C-5), 44.2 (CH2CHCH, C-6), 40.6 (CH2, CCy), 30.8 (CH2, CCy), 30.5 (CH2, CCy), 29.7 (CH,

CCy), 26.5 (CH2, CCy), 26.0 (CH2CHCH, C-4), 19.5 (CH2, CCy), 18.8 (CH2=CCH3, C-10),

16.1 (COCCH3, C-7).

HRMS (ES) m/z for C17H27O2, [MH]+ (Calc.: 263.2006, Found: 262.2009); white crystals;

Anal. Calc. for C17H26O2, Expected C, 77.8; H, 10.0; Found C, 77.6; H, 9.7; IR νmax 3442

(OH), 2921, 2850, 1646 (C=O), 1447, 1365, 1088, 1003, 1022, 987, 891 cm–1; [α]D +67º (c

0.45, CH2Cl2, 24 ºC); m.p 41–42 ºC.

(1`S)-Diastereomer (110): 1H NMR (400 MHz, CDCl3) δ 6.69 (ddd, J = 5.6, 2.8, 1.4 Hz,

1H, CH=CCH3, H-3), 4.94−4.91 (m, 1H, CH2=CCH3, H-9), 4.90 (quint, J = 1.5 Hz, 1H,

CH2=CCH3, H-9), 3.24 (t, J = 8.6 Hz, 1H, CHOH, H-1`), 3.05 (ddd, J = 11.8, 10.3, 5.1 Hz,

1 `

1

23

45 6

7

89 10

(R)(R)

(Z) O

(R)(R) HO HH

H

(Z) O

36 109

(R)(R)

(Z) O

(S)(S) HO HH

H

110

7. Experimental

171

1H, CH2CHCH, H-5), 2.64 (dd, J = 11.9, 1.9 Hz, 1H, CH2CHCH, H-6), 2.54−2.40 (m, 1H,

CH2CHCH, H-4), 2.39−2.32 (m, 1H, CH2CHCH, H-4), 2.21 (d, J = 10.1 Hz, CHOH),

2.15−2.10 (m, 1H, HCy-12), 2.01−1.93 (m, 1H, HCy), 1.81−1.79 (m, 1H, HCy), 1.77 (dt, J =

2.5, 1.3 Hz, 3H, COCCH3), 1.76−1.73 (m, 1H, HCy), 1.71 (dd, J = 1.4, 0.7 Hz, 3H,

CH2=CCH3), 1.69−1.65 (m, 2H, HCy), 1.33-1.20 (m, 2H, HCy), 1.15−1.11 (m, 1H, HCy),

0.97−0.84 (m, 2H, HCy). 13C NMR (100 MHz, CDCl3) δ 202.4 (C=O), 145.1 (CH3C=CH2, C-8), 143.9 (CH3C=CH,


(CH2CHCH, C-5), 46.5 (CH2CHCH, C-6), 42.3 (CH2, CCy), 31.1 (CH2, CCy), 30.5 (CH2, CCy), 29.8 (CH, CCy), 26.4 (CH2, CCy), 26.0 (CH2, CCy), 19.9 (CH2CHCH, C-4), 18.8

(CH2=CCH3, C-10), 15.8 (COCCH3, C-7).

HRMS (ES) m/z for C17H27O2, [MH]+ (Calc.: 263.2006, Found: 262.2006); white crystals;


(OH), 2933, 2918, 2841, 1661 (C=O), 1439, 1362, 1209, 1003, 984, 893 cm–1; [α]D +29º (c

0.34, CH2Cl2, 24 ºC); m.p 43–44 ºC.

(1`R)-Cyclohexyl((1R,6R)-3-methyl-2-oxo-6-(prop-1-en-2-yl)cyclohex-3-en-1-yl)

methyl 4-nitrobenzoate, 111

4-Nitrobenzoyl chloride (113 mg, 0.61 mmol.) and pyridine (2 mL) were added to a DCM

solution of (1`R) diastereomer 109 (105 mg, 0.40 mmol), and the reaction stirred at 50 ºC

till no starting materials were detectable on TLC. The mixture was diluted with DCM (30

mL), and then washed with CuSO4 (3 x 5 mL) and water (3 x 5 mL). The organic layer was

dried, concentrated and filtered through a small pad of silica gel (hexane:EtOAc 5:1) to

yield diastereomer 4-nitrobenzoate 111 (140 mg, 85%) as a viscous oil.

(1`R)-Diastereomer (111): 1H NMR (400 MHz, CDCl3) δ 8.27−8.24 (m, 2H, HAr),

8.14−8.11 (m, 2H, HAr), 6.64−6.62 (m, 1H, CH=CCH3, H-3), 5.26 (dd, J = 8.0, 4.1 Hz,1H,

CH-4NB, H-1`), 4.90 (quint, J = 1.2 Hz, 1H, CH2=CCH3, H-9), 4.76 (s, 1H, CH2=CCH3,

1 `

(Z) OH

(R)(R)H

OHO

NO2

1

23

456

7

89 10

(Z) O

(R)(R) HO HH

H

109 111

7. Experimental

172

H-9), 2.92 (dd, J = 8.8, 4.1 Hz, 1H, CH2CHCH, H-6), 2.42−2.39 (m, 2H, CH2CHCH, 2 x

H-4), 2.75−2.70 (m, 1H, CH2CHCH, H-5), 2.30−2.20 (m, 1H, HCy), 1.76−1.63 (m, 11 x H,

2 x CH3 and 5 x HCy), 1.34−0.98 (m, 5H, 5 x HCy). 13C NMR (100 MHz, CDCl3) δ 199.3 (C=O), 165.3 (COO), 151.7 (C-NO2, CAr), 145.9

(CH3C=CH2, C-8), 143.8 (CH3C=CH, C-3), 137.0 (CH3C=CH, C-2), 136.8 (C-COO, CAr),

132.2 (CH, CAr), 130.0 (CH, CAr), 129.4 (CH, CAr), 124.7 (CH, CAr), 115.0 (CH3C=CH2,

C-9), 80.1 (CH-4NB, C-1`), 50.9 (CH2CHCH, C-5), 45.8 (CH2CHCH, C-6), 41.1 (CH2,

CCy), 31.1 (CH2, CCy), 30.5 (CH2, CCy), 29.6 (CH, CCy), 27.3 (CH2, CCy), 27.2 (CH2CHCH,

C-4), 26.9 (CH2, CCy), 21.3 (CH2=CCH3, C-7), 17.3 (COCCH3, C-10).

HRMS (ES) m/z for C24H30NO5, [MH]+, (Calc.: M, 412.2118, Found: 412.2118); viscous

oil; IR νmax 2924, 2850, 1724 (C=O ester), 1646 (C=O), 1524, 1344, 1224, 1118, 1049,

967, 870 cm–1; [α]D −10º (c 0.40, CH2Cl2, 24 ºC); m.p 46−47 ºC.

(1`R,5R,6R)-6-(Hydroxy(phenyl)methyl)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-

one, 112, and (1`S,5R,6R)-6-(Hydroxy(phenyl)methyl)-2-methyl-5-(prop-1-en-2-

yl)cyclohex-2-en-1-one, 113


Benzaldehyde (0.83 g, 7.8 mmol) in THF (10 mL) was added to the lithium enolate of

carvone, and the reaction stirred for 30 min. The organic layer was dried over (MgSO4),

filtered and solvents removed in vacuo to afford crude β-hydroxyketone (1.94 g, 97%, d.r.

69:31). The crude product was purified via flash column chromatography (hexane:EtOAc

20:1−10:1) to obtain two separated diastereomers in a combined yield 67.4 % (oil, 1.35 g

combined, mixture 634 mg, 442 mg of 112 with Rf 0.51, 270 mg of 113 with Rf 0.42, 1:1

hexane:EtOAc).

(1`S)-Diastereomer (112): 1H NMR (400 MHz, CDCl3) δ 7.29-7.25 (m, 5H, HAr), 6.70

(ddd, J = 5.6, 3.2, 1.6 Hz, 1H, CH=C, H-3), 5.11 (d, J = 9.5 Hz, 1H, CHOH), 4.94 (quint,

J = 1.5 Hz, 1H, CH2=C, H-9), 4.84 (dd, J = 9.5, 4.8 Hz, 1H, CHOH, H-1`), 4.79 (dt, J =

1 `

1

23

45 6

7

89 10

(R)(R)

(Z) O

(S)(S) HO HH

H

(Z) O

36 112

(R)(R)

(Z) O

(R)(R) HO HH

H

113

7. Experimental

173

1.5, 0.7 Hz, 1H, CH2=C, H-9), 3.02 (dd, J =11.2, 4.8 Hz, 1H, CH2CHCH, H-6), 2.54 (ddd,

J = 11.2, 9.2, 5.8 Hz, CH2CHCH, H-5), 2.41−2.26 (m, 2H, CH2CHCH, 2 x H-4),

1.75−1.74 (m, 3H, COCCH3), 1.73 (dd, J = 1.4, 0.7 Hz, 3H, CH2=CCH3). 13C NMR (100 MHz, CDCl3) δ 203.0 (C=O), 145.7 (CH3C=CH2, C-8), 145.0 (CH-CAr ),

144.1 (CH3C=CH, C-3), 136.0 (CH3C=CH, C-2), 128.1 (2 x CH, CAr), 127.4 (CH, CAr),

127.0 (2 x CH, CAr), 114.3 (CH2=CCH3, C-9), 74.8 (CHCHOH, C-1`), 53.9 (CH2CHCH,

C-5), 44.7 (CH2CHCH, C-6), 31.1 (CH2CHCH, C-4), 19.3 (CH2=CCH3, C-10), 15.8 (COCCH3,

C-7).



2971, 2921, 1640 (C=O), 1450, 1368, 1040, 892, 747, 699 cm–1; [α]D −10º (c 0.60, CH2Cl2,

24 ºC).


7.33−7.17 (m, 4H, HAr), 6.70−6.67 (m, 1H, CH=C, H-3), 4.80 (quint, J = 1.4 Hz, 1H,

CH2=C, H-9), 4.77−4.75 (m, 2H, CH2=C, H-9 and CHOH, H-1`), 3.21 (s, 1H, CHOH),

2.90 (dd, J = 8.3, 2.4 Hz, 1H, CH2CHCH, H-6), 2.76−2.71 (m, 1H, CH2CHCH, H-5),

2.49−2.44 (m, 2H, CH2CHCH, 2 x H-4), 1.76 (q, J = 1.6 Hz, 3H, COCCH3), 1.70 (dd, J =

1.4, 0.7 Hz, 3H, CH2=CCH3). 13C NMR (100 MHz, CDCl3) δ 201.4 (C=O), 145.3 (CH3C=CH2, C-8), 143.7 (CH-CAr),

143.3 (CH3C=CH, C-3 ), 135.4 (CH3C=CH, C-2), 128.3 (2 x CH, CAr), 127.5 (CH, CAr),

126.1 (2 x CH, CAr), 113.7 (CH3C=CH2, C-9), 72.8 (CHCHOH, C-1`), 56.1 (CH2CHCH,

C-5), 44.7 (CH2CHCH, C-6), 29.5 (CH2CHCH, C-4), 20.1 (CH2=CCH3, C-10), 15.9

(COCCH3, C-7).


3441 (OH), 3064, 3027, 2970, 2921, 1654 (C=O), 1449, 1367, 1043, 1026, 1022, 892, 746,

698 cm–1; [α]D +52º (c 0.54, CH2Cl2, 24 ºC).

(1`S)-((1R,6R)-3-Methyl-2-oxo-6-(prop-1-en-2-yl)cyclohex-3-en-1yl)(phenyl)methyl

acetate, 114, and (1`R)-((1R,6R)-3-Methyl-2-oxo-6-(prop-1-en-2-yl)cyclohex-3-en-

1yl)(phenyl)methyl acetate, 115 (mixture of isomers)

7. Experimental

174

Acetic anhydride (0.40 mL, 4.27 mmol, 10 eq.) and pyridine (0.17 mL, 2.14 mmol, 5.0 eq.)

were added to DCM solution of diastereomers 112 and 113 (110 mg, 0.427 mmol), and the

reaction stirred at (50 ºC) till no starting materials were detectable on TLC. The mixture

was diluted with DCM (30 mL), and then washed with CuSO4 (3 x 5 mL) and water (3 x 5

mL). The organic layer was dried, concentrated and filtered over a small pad of silica gel

(hexane:EtOAc 5:1) to yield a mixture of diastereomers 114 and 115 (89 mg, 82%,

diastereomeric ratio 5:1) as a yellow oil.

The mixture of (1`S) and (1`R)-diastereomers (114 and 115) was assigned based on 1H, 13C, DEPT-135, 2D-COSY, 2D-HSQC, and 2D-HMBC NMR spectra.

(1`S)-Diastereomer (114): 1H NMR (400 MHz, CDCl3) δ 7.34−7.16 (m, 5H, HAr), 6.58−6.53

(m, 1H, CH=C, H-3), 6.30 (d, J = 8.8 Hz, 1H, CHOAc, H-1`), 4.76−4.74 (m, 1H, CH2=CCH3,

H-9), 4.57−4.55 (m, 1H, CH2=CCH3, H-9), 2.97 (dd, J = 8.7, 4.3 Hz, 1H, CH2CHCH, H-6),

2.57−2.55 (m, 1H, CH2CHCH, H-5), 2.28−2.24 (m, 2H, CH2CHCH, 2 x H-4), 1.95 (s, 3H,

COOCH3), 1.65 (q, J = 1.8 Hz, 3H, COCCH3), 1.55 (s, 3H, CH2=CCH3). 13C NMR (100 MHz, CDCl3) δ 198.7 (C=O), 170.3 (COOCH3), 145.8 (C-CHOAc, CAr),

145.0 (CH3=CCH2, C-8), 144.0 (CH3C=CH, C-3), 143.2 (CH3C=CH, C-2 ), 138.7 (2 x

CHAr), 135.9 (2 x CHAr), 128.6 (CHAr), 113.1 (CH3C=CH2, C-9), 75.4 (CHCHOAc, C-1`),

55.9 (CH3CHCH, C-6), 43.0 (CH2CHCH, C-5), 29.9 (CH2CHCH, C-4), 21.5 (COOCH3),

21.4 (CH2=CCH3, C-10), 16.5 (COCCH3, C-7).

(1`R)-Diastereomer (115) was partially assigned due to overlapping of its peaks with

signals of major isomer (114).

(1`R)-Diastereomer (115): 1H NMR (400 MHz, CDCl3) δ 6.50 (d, J = 6.8 Hz, 1H, CHOAc,

H-1`), 2.92 (dd, J = 6.9, 2.5 Hz, 1H, CH2CHCH, H-6), 2.47−2.38 (m, 1H, CH2CHCH, H-5),

2.03 (s, 3H, COOCH3), 1.62 (q, J = 1.7 Hz, 3H, COCCH3), 1.53 (s, 3H, COCCH3). 13C NMR (100 MHz, CDCl3) δ 197.8 (C=O), 170.3 (COOCH3), 145.1 (C-CHOAc, CAr,),

144.9 (CH3=CCH2, C-8), 144.5 (CH3C=CH, C-3), 142.9 (CH, CH3C=CH, C-2), 135.4 (2 x

CH, CAr), 128.8 (2 x CH, CAr), 128.4 (CHAr), 113.0 (CH3C=CH2, C-9), 75.1 (CH,

1 `

(R)(R)

(Z) O

(S)(S) HO HH

H

112

(R)(R)

(Z) O

(R)(R) HO HH

H

113

(R)(R)

(Z) O

(S)(S)

OH

H

114

(R)(R)

(Z) O

(R)(R)

OH

H

115

O O

1

2

3

456

7

89 10

7. Experimental

175

CHCHOAc, C-1`), 55.3 (CH3CHCH, C-6), 42.6 (CH2CHCH, C-5), 27.8 (CH2CHCH, C-4),

21.4 (COOCH3), 21.2 (CH2=CCH3, C-10), 16.4 (COCCH3, C-7).

(1`S and 1`R) Diastereomers mixture: HRMS (ES) m/z for C19H26O3N1, [MNH4]+;

(Calc.: 316.1913, Found: 316.1914); oil; IR νmax 3020, 2922, 1736 (C=O ester), 1671

(C=O), 1433, 1367, 1224, 1119, 834, 767 cm–1.

(1`S,5R,6R)-6-(Hydroxy(4-nitrophenyl)methyl)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2en-

1-one, 116, and (1`R,5R,6R)-6-(Hydroxy(4-nitrophenyl)methyl)-2-methyl-5-(prop-1-en-2-

yl)cyclohex-2-en-1-one, 117 (mixture of isomers)

The title compounds were synthesised from 36 according to the procedure used for 103. p-

Nitrobenzaldehyde (1.18 g, 7.8 mmol) in THF (10 mL) was added to the lithium enolate of


off and solvents removed in vacuo to give β-hydroxyketone crude (2.22 g, 94%,

diastereomeric ratio 45:44:7:4). A pure sticky yellow diastereomers mixture (1.28 g, 54%,

Rf 0.22 and 0.15 in hexane:EtOAc 1:1) was collected via flash column chromatography.

The mixture of (1`S) and (1`R)-diastereomers (116 and 117) was assigned based on 1H, 13C, DEPT, 2D-COSY, HSQC, and HMBC NMR spectra.

(1`S)-Diastereomer (116): 1H NMR (400 MHz, CDCl3) δ 8.18−8.16 (m, 2H, HAr), δ 7.60−7.53

(m, 2H, HAr), 6.76−6.72 (m, 1H, CH=C, H-3), 5.11 (s, 1H, CHOH), 4.99−4.95 (m, 3H,

CH2=C and CHOH, 2 x H-9 and H-1`), 3.09−2.98 (m, 1H, CH2CHCH, H-6), 2.55−2.51

(m, 1H, CH2CHCH, H-5), 2.48−2.28 (m, 2H, CH2CHCH, 2 x H-4), 1.80−1.72 (m, 6H, 2 x

CH3). 13C NMR (100 MHz, CDCl3) δ 201.0 (C=O), 152.0 (C-NO2, CAr), 147.2 (CH3=CCH2, C-

8), 146.2 (C, CH3C=CH, C-3), 144.2 (CH3=CCH, C-2), 135.8 (CAr), 126.4 (2 x CHAr),

123.2 (2 x CHAr), 114.9 (CH3C=CH2, C-9), 71.4 (CHCHOH, C-1`), 55.6 (CH3CHCH, C-

6), 44.4 (CH2CHCH, C-5), 31.1 (CH2CHCH, C-4), 19.1 (CH3,), 15.8 (CH3).

1 `

1

23

45 6

7

89 10

(R)(R)

(Z) O

(S)(S) HO HH

H

(Z) O

36 116

(R)(R)

(Z) O

(R)(R) HO HH

H

117NO2 NO2

7. Experimental

176


7.49−7.47 (m, 2H, HAr), 6.74 (ddd, J = 5.4, 2.8, 1.4 Hz, 1H, CH=C, H-3), , 4.94 (quint, J =

1.5 Hz, 1H, CH2=CCH3, H-9), 4.91 (s, 1H, CHOH, H-1`), 4.82−4.81 (m, 1H, CH2=CCH3,

H-9), 3.13 (s, 1H, CHOH), 3.01 (dd, J = 12.1, 4.4 Hz, 1H, CH2CHCH, H-6), 2.62−2.58

(m, 1H, CH2CHCH, H-5), 2.49−2.28 (m, 2H, CH2CHCH, 2 x H-4), 1.78 (dt, J = 2.4, 1.3

Hz, 3H, COCCH3), 1.74−1.71 (m, 3H, CH2=CCH3). 13C NMR (100 MHz, CDCl3) δ 202.0 (C=O), 149.0 (C-NO2, CAr), 146.7 (CH3=CCH2, C-

8), 146.2 (CH3C=CH, C-3), 144.7 (CH3=CCH, C-2), 135.6 (CAr), 128.6 (2 x CHAr), 123.5

(2 x CHAr), 115.0 (CH3C=CH2, C-9), 73.4 (CHCHOH, C-1`), 55.7 (CH3CHCH, C-11),

46.1 (CH2CHCH, C-5), 31.5 (CH2CHCH, C-4), 19.5 (CH2=CCH3, C-10), 15.8 (COCCH3,

C-7).

(1`R and 1`S)-Diastereomers mixture: HRMS (ES) m/z for C17H19NO4Na, [MNa]+,

(Calc.: 324.1212, Found: 324.1214); viscos oil; Anal. Calc. for C17H19NO4, Expected C,

67.7; H, 6.4; N 4.7, Found C, 66.9; H, 7.0; N 4.6; IR νmax 3444 (OH), 2982, 2921, 1665

(C=O), 1460, 1361, 1112, 1049, 1030, 902, 760 cm–1.

(1`S)-((1R,6R)-3-Methyl-2-oxo-6-(prop-1-en-2-yl)cyclohex-3-en-1-yl)(4-nitrophenyl)methyl

acetate, 118, and (1`R)-((1R,6R)-3-Methyl-2-oxo-6-(prop-1-en-2-yl)cyclohex-3-en-1-yl)(4-

nitrophenyl)methyl acetate, 119 (mixture of isomers)

A mixture of diastereomers 116 and 117 (78 mg, 0.3 mmol) was dissolved in (3 mL) DCM

in round flask, and under N2 gas. Pyridine (122 µL, 5.0 eq.) and acetic anhydride (287 µL,

10 eq.) were added to this solution of both diastereomers, and the reaction allowed stirring

overnight. The mixture was extracted with DCM (30 mL), and then washed with saturated

copper sulfate solution (3 x 5 mL) and water (3 x 5 mL). The organic layer was over

(MgSO4) and concentrated in vacuo to afford crude oil, which was filtered through a small

1 `

(R)(R)

(Z) O

(S)(S) HO HH

H

116

(R)(R)

(Z) O

(R)(R) HO HH

H

117

(R)(R)

(Z) O

(S)(S)

OH

H

118

(R)(R)

(Z) O

(R)(R)

OH

H

119

O O

1

2

3

456

7

89 10

NO2 NO2 NO2 NO2

7. Experimental

177

pad of silica (hexane:EtOAc 5:1) to give a diastereomeric mixture of 118 and 119 (65 mg,

83%) as a yellow oil.


(1`S)-Diastereomer (118): 1H NMR (400 MHz, CDCl3) δ 8.17−8.13 (m, 2H, HAr), 7.46−7.43

(m, 2H, HAr), 6.70−6.68 (m, 1H, CH=C, H-3), 6.30 (d, J = 6.0 Hz, CHCHOAc, H-1`), 4.91

(quint, J = 1.4 Hz, 1H, CH2=CCH3, H-9), 4.76 (s, 1H, CH2=C-CH3, H-9), 2.98 (dd, J =

7.2, 6.0 Hz, 1H, CH2CHCH, H-6), 2.49−2.41 (m, 3H, CH2CHCH, 2 x H-4 and H-5), 2.13

(s, 3H, CH3COO), 1.74−172 (m, 3H, COCCH3), 1.54 (dd, J = 1.4, 0.7 Hz, 3H, CH2=CCH3). 13C NMR (100 MHz, CDCl3) δ 196.8 (C=O), 169.8 (COOCH3), 147.5 (CCHOAc, CAr),

146.3 (C-NO2, CAr), 144.9 (CH3C=CH2, C-8), 143.5 (CH3=CCH, C-3), 135.5 (CH3C=CH,

C-2), 127.9 (2 x CHAr), 123.5 (2 x CHAr), 114 (CH3C=CH2, C-9), 73.3 (CHCHOAc, C-1`),

55.1 (CH3CHCH, C-6), 43.3 (CH2CHCH, C-5), 29.5 (CH2CHCH, C-4), 21.0 (COOCH3), 20.1

(CH2=CCH3, C-10), 16.1 (COCCH3, C-7).


7.50−7.47 (m, 2H, HAr), 6.70−6.68 (m, 1H, CH=C, H-3), 6.12 (d, J = 4.6 Hz, CHCHOAc,

H-1`), 4.78 (quint, J = 1.4 Hz, CH2=CCH3, 1H, H-9), 4.76 (s, 1H, CH2=CCH3, 1H, H-9),

2.99−2.97 (m, 1H, CH2CHCH, H-6), 2.86 (q, J = 6.4 Hz, CH2CHCH, H-5), 2.49−2.41 (m,

2H, CH2CHCH, 2 x H-4), 2.13 (s, 3H, CH3COO), 1.74 (s, 3H, COCCH3), 1.77−1.75 (m,

3H, CH2=CCH3). 13C NMR (100 MHz, CDCl3) δ 196.9 (C=O), 169.9 (COOCH3), 147.3 (CCHOAc, CAr),

147.0 (C-NO2, CAr), 144.3 (CH3C=CH2, C-8), 143.6 (CH3=CCH, C-3), 135.6 (CH3C=CH,

C-2), 127.2 (2 x CHAr), 123.6 (2 x CHAr), 114.5 (CH3C=CH2, C-9), 73.3 (CHCHOAc, C-1`),

55.1 (CH3CHCH, C-6), 44.9 (CH2CHCH, C-5), 29.9 (CH2CHCH, C-4), 21.1 (COOCH3), 19.6

(CH2=CCH3, C-10), 16.2 (COCCH3, C-7).

(1`S and 1`R)-Diastereomer mixture: HRMS (ES) m/z for C19H21NO5Na, [MNa]+,

(Calc.: 366.1317, Found: 366.1319); oil; Anal. Calc. for C19H21NO5; IR νmax 2923, 1740

(C=O ester), 1668 (C=O), 1517, 1433, 1343, 1225, 1108, 1049, 967, 853, 730 cm–1.

(1`S,5R,6R)-6-(Hydroxy(4-methoxyphenyl)methyl)-2-methyl-5-(prop-1-en-2-yl)cyclohex-

2-en-1-one, 120, and (1`R,5R,6R)-6-(Hydroxy(4-methoxyphenyl)methyl)-2-methyl-5-

(prop-1-en-2-yl)cyclohex-2-en-1-one, 121 (mixture of isomers)

7. Experimental

178

The title compounds were synthesised from 36 according to the procedure used for 103. p-

Methoxybenzaldehyde (1.06 g, 7.8 mmol) in THF (10 mL) was added to the lithium

enolate of carvone, and the reaction stirred for 40 min. The organic layer was dried over

(MgSO4), filtered off and evaporated via reduced pressure to give crude β-hydroxyketone

crude (2.1 g, 94%, d.r. 50:44:6). A pure pale yellow oil mixture of diastereomers was

collected via flash column chromatography (1.0 g, 45%, Rf 0.32 and 0.25 in hexane 4:1

EtOAc).


(1`S)-Diastereomer (120): 1H NMR (400 MHz, CDCl3) δ 7.23 (t, J = 2.4 Hz, 1H, HAr),

7.12 (t, J = 2.5 Hz, 1H, HAr), 6.83 (t, J = 2.5 Hz, 1H, HAr), 6.75 (t, J = 2.5 Hz, 1H, HAr),

6.71 (ddt, J = 5.6, 2.9, 1.4 Hz, 1H, CH=C, H-3), 4.92 (quint, J = 1.5 Hz, 1H, CH2=C, H-9),

4.78−4.77 (m, 1H, CH2=C, H-9), 4.76 (d, J = 4.9 Hz, 1H, CHOH, H-1`), 4.66 (d, J = 2.8

Hz, 1H, CHOH) 3.78 (s, 3H, OCH3), 3.00 (dd, J = 11.4, 4.9 Hz, 1H, CH2CHCH, H-6),

2.54 (ddd, J = 11.4, 9.5, 5.6 Hz, 1H, CH2CHCH, H-5), 2.37−2.27 (m, 2H, CH2CHCH, 2 x

H-4), 1.74−1.73 (m, 6H, 2 x CH3). 13C NMR (100 MHz, CDCl3) δ 203.0 (C=O), 159.0 (C-OMe, CAr), 145.7 (CH3=CCH2, C-

8), 145.0 (CH3C=CH, C-2), 136.0 (CH3C=CH, C-3), 133.8 (CCHOH, CAr), 128.9 (2 x

CHAr), 114.4 (2 x CHAr), 113.7 (CH3C=CH2, C-9), 74.4 (CHOH, C-1`), 55.5 (C-OCH3),

55.3 (CH2CHCH, C-6), 44.3 (CH2CHCH, C-5), 31.0 (CH2CHCH, C-4), 19.3 (CH3), 15.8

(CH3).

(1`R)-Diastereomer (121): 1H NMR (400 MHz, CDCl3) δ 7.32 (t, J = 2.3 Hz, 1H, HAr),

7.30 (t, J = 2.3 Hz, 1H, HAr), 6.90 (t, J = 2.5 Hz, 1H, HAr), 6.88 (t, J = 2.5 Hz, 1H, HAr),

6.71−6.66 (m, 1H, CH=C, H-3), 4.85 (quint, J = 1.4 Hz, 1H, CH2=C, H-9), 4.79−4.78 (m,

2H, CH2=C and CHOH, H-9 and H-1`), 3.80 (s, 3H, OCH3), 2.88 (dd, J = 7.4, 5.8 Hz, 1H,

CH2CHCH, H-6), 2.65 (q, J = 6.4 Hz, 1H, CH2CHCH, H-5), 2.47−2.42 (m, 3H,

1 `

(R)(R)(R)(R)

(Z) OH

(S)(S)H

OH

H

OMe

(R)(R)(R)(R)

(Z) O

(R)(R)H

OH

OMe

(Z) O

36

120 121

H

H

7. Experimental

179

CH2CHCH and OH, 2 x H-4 and OH), 1.78 (q, J = 1.7 Hz, 3H, COCCH3), 1.67 (dd, J =

1.4, 0.7 Hz, 3H, CH3C=CH2). 13C NMR (100 MHz, CDCl3) δ 201.5 (C=O), 159.0 (C-OMe, CAr), 145.3 (CH3=CCH2),

143.5 (CH3C=CH), 135.2 (CH3=CCH, C-2), 135.1 (CCHOH, CAr), 127.4 (2 x CHAr), 114.8

(2 x CHAr), 113.4 (CH3C=CH2, C-9), 73.1 (CHOH, C-1`), 55.7 (OCH3), 55.3 (CH3CHCH,

C-6), 44.4 (CH2CHCH, C-5), 29.1 (CH2CHCH, C-4), 20.3 (CH2=CCH3, C10), 16.0

(COCCH3, C-7).

(1`R and second isomer 120 and 121): HRMS (ES) m/z for C18H22O3Na, [MNa]+, (Calc.:

309.1461, Found: 309.1449); oil; IR νmax 3447 (OH), 3047, 2970, 2922, 2836, 1649 (C=O),

1440, 1367, 1244, 1174, 1032, 892, 831 cm–1.

(1`S,5R,6R)-6-(Hydroxy(naphthalen-1-yl)methyl)-2-methyl-5-(prop-1-en-2yl)cyclohex-2-en-

1-one, 122, and (1`R,5R,6R)-6-Hydroxy(naphthalen-1-yl)methyl)-2-methyl-5-(prop-1-en-2-

yl)cyclohex-2-en-1-one, 123

The title compounds were synthesised from 36 according to the procedure used for 103.

Naphthaldehyde (1.2 g, 7.8 mmol) in THF (10 mL) was added to the lithium enolate of


and solvents removed in vacuo to afford crude β-hydroxyketone (2.3 g, 96%, d.r.

63:34:7:4). The crude was chromatographed using flash column (10:1 hexane:EtOAc) to

give two separable isomers in a combined yield 67.4% (1610 mg combined, 890 mg of 122

as a viscous oil-Rf 0.42, and 720 mg of 123 as a pale yellow crystals with Rf 0.31, 4:1

hexane:EtOAc).

(1`S)-Diastereomer (122): 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 8.3 Hz, 1H, HAr),

7.87 (d, J = 7.6 Hz, 1H, HAr), 7.81 (d, J = 7.6 Hz, 1H, HAr), 7.61 (d, J = 8.2 Hz, 1H, HAr),

7.54−7.46 (m, 3H, HAr), 6.72 (td, J = 4.1, 1.3 Hz, 1H, CH=CCH3, H-3), 5.56 (d, J = 5.6

Hz, 1H,CHOH, H-1`), 4.85−4.84 (m, 1H, CH2=CCH3, H-9), 4.79 (s, 1H, CH2=CCH3, H-9)

3.35 (s, br., 1H, CHOH), 3.25 (dd, J = 7.0, 6.0 Hz, 1H, CH2CHCH, H-6), 2.64 (q, J = 6.3

1 `

1

23

45 6

7

89 10

(R)(R)

(Z) O

(S)(S)

O H

H

(Z) O

36 122

(R)(R)

(Z) O

(R)(R) HO HH

H

123

HH

7. Experimental

180

Hz, 1H, CH2CHCH, H-5), 2.50−2.30 (m, 2H, CH2CHCH, 2 x H-4), 1.83 (q, J = 1.5 Hz,

3H, COCCH3), 1.60 (s, 3H, CH2=CCH3). 13C NMR (100 MHz, CDCl3) δ 201.7 (C=O), 145.4 (CH3C=CH2, C-8), 143.6 (CH3C=CH),

137.6 (CH3C=CH, C-2), 135.4 (CAr), 134.0 (CAr), 131.2 (CAr), 129.0 (CHAr), 128.6 (CHAr), 126.1

(CHAr), 125.6 (CHAr), 125.3 (CHAr), 125.1 (CHAr), 123.7 (CHAr), 113.2 (CH2=CCH3, C-9), 70.9

(CHCHOH, C-1`), 54.9 (CH2CHCH, C-6), 44.5 (CH2CHCH, C-5), 29.2 (CH2CHCH, C-4), 20.9

(CH2=CCH3, C-10), 16.0 (COCCH3, C-7).



3071, 2966, 2922, 1642 (C=O), 1427, 1318, 1119, 980, 801, 697 cm–1; [α]D +29º (c 0.34,

CH2Cl2, 24 ºC).

(1`R)-Diastereomer (123): 1H NMR (400 MHz, CDCl3): δ 8.09 (d, J = 8.3 Hz, 1H, HAr),

7.85 (d, J = 7.7 Hz, 1H, HAr), 7.76 (d, J = 8.2 Hz, 1H, HAr), 7.54−7.45 (m, 3H, HAr), 7.41

(d, J = 7.7 Hz, 1H, HAr), 6.81−6.78 (m, 1H, CH=CCH3, H-3), 6.11 (d, J = 3.3 Hz, 1H,

CHOH, H-1`), 4.55 (quint, J = 1.4 Hz, 1H, CH2=CCH3, H-9), 4.51−4.50 (m, 1H,

CH2=CCH3, H-9), 3.27 (s, 1H, CHOH), 3.08 (dd, J = 8.8, 3.5 Hz, 1H, CH2CHCH, H-6),

3.04−2.98 (m, 1H, CH2CHCH, H-5), 2.57−2.32 (m, 2H, CH2CHCH, 2 x H-4), 1.85 (q, J =

1.7 Hz, 3H, COCCH3), 1.23−1.21 (m, 3H, CH2=CCH3). 13C NMR (100 MHz, CDCl3) δ 201.3 (C=O), 145.7 (CH3C=CH2, C-8), 145.2 (CH3C=CH,

C-3), 137.2 (CH3C=CH, C-2), 135.6 (CAr), 133.7 (CAr), 130.8 (CAr), 128.9 (CHAr), 128.1

(CHAr), 126.3 (CHAr), 125.5 (CHAr), 124.8 (CHAr), 124.4 (CHAr), 123.2 (CHAr), 112.9

(CH2=CCH3, C-9), 70.5 (CHOH, C-1`), 53.8 (CH2CHCH, C-6), 42.9 (CH2CHCH, C-5),

30.1 (CH2CHCH, C-4), 19.3 (CH2=CCH3, C-10), 16.1 (COCCH3, C-7).

HRMS (ES) m/z for C21H22O2Na, [MNa]+, (Calc.: 329.1512, Found: 329.1507); crystals;.


(OH), 3048, 2969, 2920, 1648 (C=O), 1433, 1360, 1042, 966, 866, 776 cm–1; [α]D −62º (c

0.26, CH2Cl2, 24 ºC); m.p 48–49 ºC.

(1`S,5R,6R)-6-(Hydroxy(pyridin-2-yl)methyl)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-

en-1-one, 124, and(1`R,5R,6R)-6-(Hydroxy(pyridin-2-yl)methyl)-2-methyl-5-(prop-1-

en-2-yl)cyclohex-2-en-1-one, 125

7. Experimental

181

The title compounds were synthesised from 36 according to the procedure used for 103. 2-

Pyridinecarboxyaldyhde (0.8 g, 7.8 mmol) in THF (10 mL) was added to the lithium

enolate of carvone, and the reaction stirred for 30 min. The organic layer was dried

(MgSO4), filtered and solvents removed in vacuo to afford crude β-hydroxyketone (1.93

gm, 96%, d.r. 72:26:2). The crude product was chromatographed to separate diastereomers

and gave a combined yield of diastereomers (85%, 1.7 g), and in details (0.24 g of isomers

mixture, 0.86 g of 124 as an oil with Rf 0.34, and 0.605 g of 125 as a white crystals with Rf 0.21

in 4:1 hexane:EtOAc).

(1`S)-Diastereomer(124): 1H NMR (400 MHz, CDCl3) δ 7.47 (ddd, J = 4.9, 1.7, 0.9 Hz,

1H, HAr), 7.65 (td, J = 7.7, 1.8 Hz, 1H, HAr), 7.33−7.30 (m, 1H, HAr), 7.18−7.15 (m, 1H,

HAr), 6.76−6.74 (m, 1H, CH=C, H-3), 5.16 (d, J = 3.6 Hz, 1H, CHOH, H-1`), 4.90 (s, 1H,

CHOH), 4.69 (quint, J = 1.5 Hz, 1H, CH2=CCH3, H-9), 4.62−4.61 (m, CH2=CCH3, 1H, H-

9), 3.12−3.06 (m, CH2CHCH, H-5), 2.94 (dd, J = 9.6, 4.0 Hz, 1H, CH2CHCH, H-6),

2.50−2.35 (m, 2H, CH2CHCH, 2 x H-4), 1.83−1.82 (m, 3H, COCCH3), 1.57 (dd, J = 1.3,

0.7 Hz, 3H, CH2=CCH3). 13C NMR (100 MHz, CDCl3) δ 201.3 (C=O), 160.6 (CAr), 147.7 (CHAr), 145.4 (CH3C=CH2,

C-8), 144.2 (CH=CCH, C-3), 136.2 (CHAr), 135.7 (CH3C=CH, C-2), 122.2 (CHAr), 122.1

(CHAr), 113.2 (CH3C=CH2, C-9), 73.5 (CHOH, C-1`), 54.7 (CH2CHCH, C-6), 43.4

(CH2CHCH, C-5), 30.4 (CH2CHCH, C-4), 19.6 (CH2=CCH3, C-10), 16.0 (COCCH3, C-7).

HRMS (ES) m/z for C16H19NO2Na, [MNa]+, (Calc.: 280.1308 Found: 280.1315); oil; IR

νmax 3466 (OH), 3072, 2967, 2919, 1655 (C=O), 1433, 1368, 1123, 1048, 995, 896, 761

cm–1; [α]D +151º (c 0.66, CH2Cl2, 24 ºC).

(1`R)-Diastereomers (125): 1H NMR (400 MHz, CDCl3) δ 8.51 (ddd, J = 4.9, 1.7, 0.9 Hz,

1H, HAr), 7.72 (td, J = 7.7, 1.8 Hz, 1H, HAr), 7.54 (dq, J = 8.0, 0.9 Hz, 1H, HAr), 7.16−7.13

(m, 1H, HAr), 6.71 (ddd, J = 5.8, 2.7, 1.4 Hz, 1H, CH=CCH3, H-3), 4.99−4.98 (m, 1H,

CH2=CCH3, H-9), 4.94 (quint, J = 1.6 Hz, 1H, CH2=CCH3, H-9), 4.76 (d, J = 2.0 Hz, 1H,

CHOH, H-1`), 3.92 (d, J = 8.8 Hz, 1H, CHOH), 3.53 (dd, J = 12.4, 2.2 Hz, 1H,

1 `

1

23

45 6

7

89 10

(R)(R)

(Z) O

(S)(S)HO HH

H

(R)(R)

(Z) O

36

124

(R)(R)

(Z) O

(R)(R) HO HH

HN N

125

7. Experimental

182

CH2CHCH, H-6), 3.15 (ddd, J = 12.4, 10.6, 5.0 Hz, 1H, CH2CHCH, H-5), 2.64−2.52 (m,

1H, CH2CHCH, H-4), 2.44−2.40 (m, 1H, CH2CHCH, H-4) 1.86 (dd, J = 1.4, 0.8 Hz, 3H,

CH2=CCH3), 1.70 (dt, J = 2.5, 1.3 Hz, 3H, COCCH3). 13C NMR (100 MHz, CDCl3) δ 200.8 (C=O), 162.8 (CAr), 147.9 (CHAr), 145.2 (CH3C=CH2,

C-8 ), 144.3 (CH3C=CH, C-3), 136.5 (CHAr), 135.5 (CH=CCH3, C-2), 121.5 (CHAr), 119.6

(CHAr), 114.3 (CH3C=CH2, C-9), 71.9 (CHOH, C-1`), 54.4 (CH2CHCH, C-6), 46.1

(CH2CHCH, C-5), 31.1 (CH2CHCH, C-4), 19.0 (CH2=CCH3, C-10), 15.7 (COCCH3, C-7).

HRMS (ES) m/z for C16H19NO2Na, [MNa]+, (Calc.: 280.1308 Found: 280.1310); crystals ;

Anal. Calc. for C16H19NO2, Expected C, 73.8; H, 7.3; N 5.4, Found C, 74.1; H, 7.3; N 5.4;

IR νmax 3204 (OH), 3071, 2961, 2922, 1660 (C=O), 1433, 1371, 1045, 888, 750 cm–1; [α]D

+120º (c 0.78, CH2Cl2, 24 ºC).

(1`R,2R,3R,6R)-2-(1-Hydroxyethyl)-6-isopropyl-3-methylcyclohexanone, 126, and

(1`S,2R,3R,6R)-2-(1-Hydroxyethyl)-6-isopropyl-3-methylcyclohexanone. 12757

To a solution of DIPA (1.31 mL, 9.14 mmol) in anhydrous THF (20 mL) at 0 ºC, under

nitrogen gas, a hexane solution of nBuLi (4.47 mL, 2.04 M) was added slowly. The LDA

solution was cooled to −78 ºC and (+)-isomenthone 38 (1.27 g, 8.29 mmol) in THF was

added dropwise. The enolate solution was stirred for 45 min and ethanal (183 mg, 8.29

mmol) in anhydrous THF (5 mL) added at once. The solution was allowed stirring for 15

min and the reaction quenched by addition saturated ammonium chloride (20 mL). The

mixture has been transferred to separatory funnel and diluted with diethyl ether (150 mL),

washed with saturated ammonium chloride (3 x 30 mL) and water (30 mL). The organic

extracts were dried (MgSO4), filtered and solvents removed in vacuo to afford the crude β–

hydroxyketone. The separation of diastereomers was carried out with flash column

chromatography (10:1 hexane:Et2O) to afford the two diastereomers 126 and 127 as a

yellow oils with overall yield 67.4% and d.r. 78:22 (mixture 0.08 g, 0.78 g with Rf 0.40 of

126, and 0.14 mg with Rf 0.22 of 127, 1:1 hexane:Et2O).

(1`R)-Diastereomer (126): 1H NMR (400 MHz, CDCl3) δ 3.92 (qd, J = 6.6, 3.0 Hz, 1H,

CHOH, H-1`), 2.60 (s, 1H, CHOH), 2.10−2.00 (m, 3H), 1.99−1.90 (m, 2H), 1.76−1.62 (m,

1 `1 Ò

38

1 2

345

67

8

9

10

O

HH(R)(R)

HO HO

H

H

(S)(S)HO

H126 127

12

3

7. Experimental

183

2H), 1.55−1.43 (m, 1H), 1.29 (d, J = 6.4 Hz, 3H, CH3CHOH), 1.04 (d, J = 6.0 Hz, 3H,

CH3), 0.90 (d, J = 6.4 Hz, 3H, CH3), 0.81 (d, J = 6.4 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 219.0 (C=O), 66.1 (CHOH), 59.8 (CH), 58.7 (CH), 37.1

(CH3), 29.4 (CH2), 27.9 (CH2), 27.3 (CH), 22.4 (CH), 20.7 (CH3), 20.3 (CH3), 20.0 (CH3).

HRMS (ES) m/z for C12H23O2, [MH]+, (Calc.: 199.1693, Found: 199.1693); oil; IR νmax

3435 (OH), 2956, 2896, 1714 (C=O), 1448, 1419, 1372, 1170 cm–1; lit [α]D +139º (c 0.55,

CH2Cl2, 24 ºC) 57, [α]D +139.6° (c 0.50, CH2Cl2, 24 °C).

(1`S)-Diastereomer (127): 1H NMR (400 MHz, CDCl3) δ 3.98 (qd, J = 6.6, 5.5 Hz, 1H,

CHOH, H-1`), 3.07 (s, 1H, CHOH), 2.36 (dd, J = 9.5, 5.5 Hz, 1H, CHCHCH2, H-2),

2.06−2.00 (m, 1H), 1.95−1.88 (m, 3H), 1.81−1.70 (m, 2H), 1.61−1.52 (m, 1H), 1.27 (d, J =

6.4 Hz, 3H, CH3CHOH), 1.02 (d, J = 6.0 Hz, 3H, CH3), 0.93 (d, J = 6.4 Hz, 3H, CH3),

0.85 (d, J = 6.4 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 217.2 (C=O), 66.9 (CHOH, C-1`), 58.9 (CH), 57.1 (CH),

35.5 (CH3), 28.9 (CH2), 28.0 (CH2), 27.0 (CH), 21.3 (CH), 20.7 (CH3), 20.0 (CH3), 19.8

(CH3).


3424 (OH), 2957, 2929, 2870, 1691 (C=O), 1456, 1376, 1212 cm–1; lit [α]D +79º (c 0.35,

CH2Cl2, 24 ºC) 57, [α]D +77° (c 0.32, CH2Cl2, 24 °C).

(1`R,2R,3R,6R)-2-(1-Hydroxy-3-methylbutyl)-6-isopropyl-3-methylcyclohexanone, 128, and

(1`S,2R,3R,6R)-2-(1-Hydroxy-3-methylbutyl)-6-isopropyl-3-methylcyclohexanone, 129

The title compounds were synthesised from 38 according to the procedure used for 126 and

127. Isovaleraldehyde (713 mg, 0.91 mL, 8.29 mmol) was added to the lithium enolate of

isomenthone, and the reaction stirred for 30 min. The crude was purified via flash column

chromatography (hex:Et2O 10:1) to afford a combined diastereomers product 36.6% ( 203

mg as mixture of isomers, 500 mg of 128 as oil with Rf 0.51, and 25 mg of 129 as oil with

0.43 hexane: 1:1 Et2O, diastereomeric ratio 65:35).

1 Ò

38

128 129

O

HH

HO HO

H

H

HOH

2

3

1

45

6

7

8

9 1 23

1 `

7. Experimental

184

(1`R)-Diastereomer (128): 1H NMR (400 MHz, CDCl3) δ 3.79 (ddd, J = 9.1, 4.4, 2.0 Hz,

1H, CHOH, H-1`), 2.84 (s, 1H OH), 2.16−2.00 (m, 3H), 1.99−1.92 (m, 2H), 1.83−1.64 (m,

4H), 1.57−1.48 (m, 1H), 1.19 (ddd, J = 13.5, 8.6, 4.7 Hz, 1H), 1.08 (d, J = 6.2 Hz, 3H,

CH3), 0.93−0.88 (m, 9H, 3 x CH3), 0.83 (d, J = 6.5 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 219.5 (C=O), 68.2 (CHOH, C-1`), 59.8 (CH), 48.0 (CH2),

37.2 (CH), 29.5 (CH2), 28.2 (CH2), 27.5 (CH), 25.0 (CH), 23.3 (CH3), 22.1 (CH3), 20.9

(CH3), 20.4 (CH3), 20.1 (CH3).

HRMS (ES) m/z for C15H28O2Na, [MNa]+, (Calc.: 263.1987, Found: 263.1974); oil at RT

and crystals at −10 ºC; Anal. Calc. for C15H28O2, Expected C, 74.9; H, 11.7; Found C,

74.6; H, 11.6; IR νmax 3444 (OH), 2953, 2928, 2869, 1693 (C=O), 1464, 1366, 1248, 1163,

1080, 968 cm–1; [α]D +146° (c 0.30, CH2Cl2, 24 °C).

(1`S)-Diastereomer (129): 1H NMR (400 MHz, CDCl3) δ 3.91−3.82 (m, 1H, CHOH, H-

1`), 2.62 (s, 1H, OH), 2.36 (dd, J = 9.2, 5.6 Hz, 1H, CHCHCH2, H-2), 2.26−1.86 (m, 5H),

1.82−1.72 (m, 2H), 1.70−1.52 (m, 2H), 1.12−1.06 (m, 1H), 1.01 (d, J = 6.6 Hz, 3H, CH3),

0.96−0.90 (m, 9H, 3 x CH3), 0.86 (d, J = 6.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 218.1 (C=O), 68.1 (CHOH), 59.3 (CH), 58.7 (CH), 47.7

(CH2), 37.0 (CH), 29.4 (CH2), 28.6 (CH2), 27.4 (CH), 24.6 (CH), 23.9 (CH3), 22.0 (CH3),

20.8 (CH3), 20.3 (CH3), 19.7 (CH3).


3429 (OH), 2954, 2929, 2869, 1691 (C=O), 1465, 1383, 1267, 1141, 1068, 987 cm–1; [α]D

+26° (c 0.15, CH2Cl2, 24 °C).

(1`S,2R,3R,6R)-2-(1-Hydroxy(pyridin-2-yl)methyl)-6-isopropyl-3-methylcyclohexan-

1-one, 130, and (1`R,2R,3R,6R)-2-(1-Hydroxy(pyridin-2-yl)methyl)-6-isopropyl-3-

methylcyclohexan-1-one, 131 (mixture of isomers)


127. 2-Pyridine carboxyaldehyde in THF (885 mg, 8.29 mmol) was added to the lithium

enolate of isomenthone, and the reaction stirred for 40 min. The crude product was purified

1 ` 1 Ò

38

130 131

O

HH

HO HO

H

H

HOH

2

3

1

45

6

7

8

9

N N12

3

7. Experimental

185

via flash column chromatography (hex:Et2O 5:1) to give one separated isomer (130) as a

white crystals, and the remainder was mixture of isomers (combined yield 54.7%, 1.18 g,

with Rf 0.24, 1:1 hexane:Et2O, d.r. 76:24, pale yellow oil).

(1`S)-Diastereomer (130): 1H NMR (400 MHz, CDCl3) δ 8.45−8.43 (m, 1H, HAr),

7.71−7.66 (m, 2H, 2 x HAr), 7.12−7.10 (m, 1H, HAr), 5.25 (s, 1H, CHOH, resolved to d (J =

4.0 Hz) after D2O), 4.10 (d, J = 9.5 Hz, 1H, OH, disappeared after adding D2O), 2.72 (dd, J =

7.4, 4.1 Hz. 1H, CHCHCH2, H-2), 2.25−2.17 (m, 3H), 2.16−2.08 (m, 1H), 1.88−1.81 (m, 3H),

0.93 (d, J = 6.6 Hz, 3H, CH3), 0.90 (d, J = 6.6 Hz, 6H, 2 x CH3). 13C NMR (100 MHz, CDCl3) δ 216.3 (C=O), 161.3 (CAr, C-12), 148.0 (CHAr), 136.5

(CHAr), 121.6 (CHAr), 120.5 (CHAr), 71.9 (CHOH, C-1`), 58.7 (CH), 56.8 (CH), 33.1

(CH2), 29.7 (CH2), 27.6 (CH2), 22.4 (CH), 21.7 (CH3), 20.7 (CH3), 19.9 (CH3).

HRMS (ES) m/z for C16H24NO2, [MH]+, (Calc.: 262.1807; Found: 262.1798); white

crystals; IR νmax 3400 (OH), 2955, 2928, 2870, 1691 (C=O), 1465, 1383, 1267, 1189, 856,

771 cm-1.

(1`R)-Diastereomer (131) was assigned based on 1H, 13C, DEPT-135, 2D-COSY, 2D-

HSQC, and 2D-HMBC NMR spectra of diastereomers mixture and 130.


7.66−7.43 (m, 2H, 2 x HAr), 7.40 (d, 1H, 8.0 Hz, HAr), 4.91 (dd, J = 10.8, 2.4 Hz, 1H,

CHOH, H-1`), 4.07 (d, J = 10.8 Hz, 1H, CHOH), 3.27 (dd, J = 10.8, 2.8 Hz, 1H,

CHCHCH2, H-2), 2.26−2.04 (m, 3H), 2.02−1.97 (m, 1H), 1.77−1.60 (m, 3H), 1.23 (d, J =

6.6 Hz, 3H, CH3), 0.89 (d, J = 6.6 Hz, 3H, CH3), 0.75 (d, J = 6.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 219.2 (C=O), 163.2 (CAr, C12), 148.1 (CHAr), 136.5

(CHAr), 122.2 (CHAr), 120.6 (CHAr), 72.1 (CHOH, C−1`), 61.6 (CH), 58.4 (CH), 37.1

(CH), 29.4 (CH2), 27.9 (CH2), 27.3 (CH), 21.5 (CH3), 20.8 (CH3), 20.6 (CH3).

(1`S) and (1`R)-Diastereomers mixture: HRMS (ES) m/z for C16H23NO2Na, [MNa]+,

(Calc.: 282.1621, Found: 284.1621); oil; Anal. Calc. for C16H23NO2, Expected C, 73.5; H,

8.8; N, 5.3, Found C, 73.1; H, 8.6; N, 5.0; IR νmax 3391 (OH), 2956, 2927, 2868, 1701

(C=O), 1468, 1383, 1236, 1173, 860, 780 cm–1.

(1`S,2R,3R,6R)-2-(1-Hydroxy((2-hydroxyphenyl)methyl)-6-isopropyl-3-methylcycl-

hexananone, 132, and (1`R,2R,3R,6R)-2-(1-Hydroxy(2-hydroxyphenyl)methyl)-6-

isopropyl-3-methylcyclohexananone, 133 (mixture of isomers)

7. Experimental

186


127. Salicylaldehyde (1.01 g, 5.85 mmol) was added to the lithium enolate of isomenthone,

and the reaction stirred for 45 min. The crude product was purified via flash column

chromatography (hex:Et2O 5:1) to afford a diastereomeric mixture (2:1) of 132 and 133

(1.49 g, 65% with Rf 0.22, 1:1 hexane:Et2O) as a colourless oil.

(1`S) and (1`R)-Diastereomers mixture were assigned based on 1H, 13C, DEPT-135, 2D-

COSY, 2D-HSQC, and 2D-HMBC NMR spectra.

(1`S)-Diastereomer (132): 1H NMR (400 MHz, CDCl3) δ 7.90 (s, Phenolic OH, and

disappeared with resolving OH), 7.17 (td, J = 7.2, 1.3 Hz, 1H, HAr), 7.02 (dd, J = 7.6, 1.6

Hz, 1H, HAr), 6.87 (dd, J = 7.4, 1.6 Hz, 1H, HAr), 6.84 (dd, J = 7.4, 1.2 Hz, 1H, HAr), 5.10

(t, J = 7.0 Hz, CHOH, changed to d (J = 7.2 Hz) after addition of D2O), 4.05 (d, J = 7.0

Hz, 1H, OH, disappeared on addition of D2O, a single signal of HOD appeared at 4.79

ppm), 2.83 (dd, J = 7.2, 6.7 Hz, 1H, CHCHCH2, H−2), 2.24−2.07 (m, 2H), 2.01−1.88 (m,

3H), 1.65−1.50 (m, 1H), 1.49−1.30 (m, 1H), 0.99 (d, J = 6.6 Hz, 3H, CH3), 0.88 (d, J = 6.6

Hz, 3H, CH3), 0.84 (d, J = 6.8 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 218.5 (C=O), 155.4 (CAr-OH), 130.3 (CAr), 129.3 (CHAr),

126.3 (CHAr), 121.1 (CHAr), 117.4 (CHAr), 73.6 (CHOH, C-1`), 59.1 (CH), 50.9 (CH), 34.9

(CH), 29.1 (CH2), 27.6 (CH2), 26.1 (CH), 24.7 (CH3), 21.1 (CH3), 19.5 (CH3).

(1`R)-Diastereomer (133): 1H NMR (400 MHz, CDCl3) δ 7.90 (Phenolic OH, disappeared

with resolving OH), 7.32 (dd, J = 7.6, 1.7 Hz, 1H, HAr), 7.24−7.22 (m, 1H, HAr), 6.96 (td, J

= 7.4, 1.7 Hz, 1H, HAr), 6.94−6.90 (m, 1H, HAr), 4.77 (dd, J = 7.0, 3.9 Hz, CHOH,

changed to d (J = 3.8 Hz) after addition of D2O, H-1`), 4.00 (d, J = 7.0 Hz, 1H, OH),

disappeared on addition of D2O and a single signal for HOD appeared at 4.79), 3.05 (dd, J

= 7.6, 3.8 Hz, 1H, CHCHCH2, H-2), 2.24−2.07 (m, 2H), 2.01−1.88 (m, 3H), 1.65−1.50 (m,

1H), 1.49−1.30 (m, 1H), 1.15 (d, J = 6.5 Hz, 6H, CH3), 1.10 (d, J = 6.5 Hz, 3H, CH3), 0.97

(dd, J = 6.9, 2.0 Hz, 6H, CH3).

1 Ò

38

132 133

O

HH

HO H

2

3

1

45

6

7

8

9 O

HH

HO H

OHOH

7. Experimental

187

13C NMR (100 MHz, CDCl3) δ 220.9 (C=O), 150.7 (CAr-OH), 129.9 (CAr), 127.6 (CHAr),

124.3 (CHAr), 119.7 (CHAr), 117.0 (CHAr), 64.5 (CHOH, C-1`), 56.6 (CH), 44.9 (CH), 30.3

(CH), 28.7 (CH2), 27.1 (CH2), 24.9 (CH), 21.8 (CH3), 20.2 (CH3), 19.4 (CH3).

Diastereomers mixture HRMS (APCI) m/z for C17H24O3, [M]+, (Calc.: 276.1725, Found:

276.1720); oil; Anal. Calc. for C17H24O3, Expected C, 73.5; H, 8.8; N, 5.3, Found C, 73.1;

H, 8.6; N, 5.0; IR νmax 3391 (OH), 2956, 2927, 2868, 1701 (C=O), 1468, 1383, 1236, 1173,

860, 780 cm–1.

(2R,3R,6R)-2-(2-Hydroxypropan-2-yl)-6-isopropyl-3-methylcyclohexan-1-one, 134, and

(2S,3R,6R)-2-(2-Hydroxypropan-2-yl)-6-isopropyl-3-methylcyclohexan-1-one, 135, (mixture

of isomers)

Method (a): Using CeCl3

A suspension solution of anhydrous CeCl3 (0.246 g, 1 mmol) in THF (15 mL) was

vigorously stirred under (0 °C) and allowed to stir overnight at RT. The suspended solution

was cooled to (−78 °C) and acetone (0.01 mL, 1.9 mmol) in THF (1 mL) was added in one

portion and the solution was stirred for further 1 h at the same temperature. In a separate

flask, DIPA (0.15 mL, 1.05 mmol) in THF (2 mL) was injected, cooled to 0 °C, and nBuLi (0.84 mL, 1.22 M) was added dropwise. The solution was allowed to stir for 20 min,

thence the solution was cooled to −78 °C. Isomenthone 38 (0.16 g, 1.04 mmol) in THF (3

mL) was added, and the mixture allowed to stir for 30 min. The solution of the lithium

enolate of isomenthone was transferred via cannula to a white suspension. The reaction

mixture was stirred at −78 °C and monitored by TLC for 3 h. The mixture was quenched

with NH4Cl solution and the organic layer was separated and solvent removed in vacuo.

The aqueous layer was washed with diethyl ether (3 x 10 mL) and then the combined

organic layer was washed with water and brine, dried (MgSO4) and solvent removed in

vacuo. The residue was purified by flash chromatography using hexane:EtOAc (8:1) as

eluent to afford a mixture of diastereomers (10:1, 23 mg, 11%) as a pale yellow oil.

Method (b): Using ZnCl2

O

38

134 135

1 2

345

6

7

8

9

10

(R)(R)H

O

OH

(S)(S)H

O OH

7. Experimental

188

To DIPA (0.3 mL, 2.1 mmol) in freshly distilled THF (5 mL) 0 ºC, nBuLi (1.01 mL, 2.02

M) was slowly added over 5 min. The solution was cooled to −78 ºC and stirring continued

for 1 h. (+)-Isomenthone 38 (300 mg, 1.95 mmol) in THF was injected dropwise, and the

mixture allowed stirring for 45 min. The suspension of ZnCl2 (26.2 mg, 0.195 mmol) in

THF (10 mL) was transferred by cannula from a separate flask into the reaction mixture.

After 10 min, a solution of THF containing acetone (127 mg, 2.2 mmol) was added in one

portion, and the reaction was monitored using TLC for 4 h at −78 ºC. The reaction was

quenched by adding saturated NH4Cl (5 mL). The mixture was transferred to a separatory

funnel (250 mL), and diluted with diethyl ether (100 mL). The contents of flask were

washed with saturated ammonium chloride (3 x 10 mL) and water (10 mL). The organic

layer was dried (MgSO4), filtered and evaporated via reduced pressure. The crude product

was purified via flash column chromatography to yield 5% (22 mg) of a diastereomeric

mixture (10:1) as a pale yellow oil.

C) Using silylenol ether

DIPA (21 mmol, 3 mL) was added into a round-bottomed flask containing dry THF (20

mL) under N2 gas, cooled to 0 °C. A hexane solution of nBuLi (10 mL, 20.4 mmol) was

slowly added over 5 min and the reaction allowed to stir for a further 15 min. The reaction

mixture was cooled to −78 °C, and (+)-isomenthone 38 (3 g, 20.0 mmol in 15 mL THF)

was slowly added over a 10 min period, and the reaction was left for further 30 min.

TCMS (25.2 mmol, 3.2 mL) was added via dropping funnel, and product formation was

indicated by TLC. The mixture was diluted with petroleum ether (200 mL), and washed

with saturated NaHCO3 (2 x 30 mL) and water (1 x 30 mL). The organic layer was

evaporated with reduced pressure vacuum to give silyenol ether of isomenthone.

In a three-neck round-bottomed flask, fresh DCM (28 mL) was charged under N2

atmosphere, and a toluene solution of TiCl4 (23.3 mmol, 1M) added at 0 °C. Dry acetone

in DCM (23.3 mmol, 1.71 mL) was dropwise over a 15 min period via cannula. Once

completed, a DCM solution of trimethylsilylenol ether of isomenthone (21.3 mmol, 4.5 g)

was slowly added. The reaction was monitored using TLC, and the reaction mixture

poured into ice-water (100 mL). The organic layer was extracted with DCM (3 x 50 mL)

and washed with saturated sodium bicarbonate (2 x 50 mL) and water (1 x 50 mL). The

organic mixture was dried (MgSO4), evaporated, and purified by flash column

chromatography using hexane:Et2O to afford products (0.78 g, 18%) as a pale yellow

mixture of diastereomers (d.r. 99:1), and Rf 0.14 and 0.2, (4:1 hexane:Et2O)

7. Experimental

189

(2R)-Diastereomer (134): 1H NMR (400 MHz, CDCl3 δ 2.70 (s, 1H, OH), 2.48 (d, J = 3.6

Hz, 1H, CHCHCH2, H-2), 2.17−2.00 (m, 4H), 1.92−1.89 (m, 2H), 1.70−1.64 (m, 1H), 1.18

(s, 3H, (CH3)2C-OH), 1.08 (s, 3H, (CH3)2C-OH), 0.94 (d, J = 7.2 Hz, 3H, CH3), 0.87 (d, J

= 6.8 Hz, 3H, CH3), 0.84 (d, J = 6.8 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 218.4 (C=O), 71.6 ((CH3)2C-OH), 62.0 (CH), 59.2 (CH),

36.0 (CH2), 33.7 (CH), 31.5 (CH), 26.8 (CH2), 26.7 (CH3), 21.6 (CH3), 18.8 (CH3), 14.8

((CH3)2C-OH).

HRMS (ES) m/z for C13H24O2Na, [MNa]+, (Calc.: 235.1674, Found: 235.1677); oil; IR

νmax 3505 (OH), 2958, 2927, 1689 (C=O), 1455, 1386, 1257, 1151, 1049, 1017, 954 cm–1.

(1`R,1R,2R,4S,5S)-2-(Hydroxyethyl)-4,6,6-trimethylbicyclo[3.1.1]heptan-3-one, 136,

and (1`S,1R,2R,4S,5S)-2-(Hydroxyethyl)-4,6,6-trimethylbicyclo[3.1.1]heptan-3-one,

137 (mixture of isomers)

A hexane solution of n-BuLi (2.0 mL, 1.48 M) was slowly added to DIPA (0.43 mL, 3.1

mmol) in THF (6 mL) at 0 °C, and the reaction stirred for 15 min. The reaction flask was

cooled to −78 °C, then, a THF solution of (1S,2S,5R)-(–)-isopinocamphone 39 (0.45 g, 2.9

mmol) was dropwise added, and reaction allowed stirring for 45 min at same temperature.

Acetaldehyde (175 µl, 3.1 mmol) in THF (2 mL) was injected in one portion via syringe,

and the reaction monitored via TLC. The mixture was quenched with NH4Cl (5 mL) and

diethyl ether (30 mL), and organic layer separated with ether (3 x 10 mL). Thence, the

combined organic layer washed with NH4Cl (1 x 10 mL) and water (1 x 10 mL), dried

(MgSO4) and solvents removed in vacuo. The product was purified using flash column

chromatography using hex:Et2O (10:1) to give a diastereomeric mixture (90:10, 518 mg,

91% yield, Rf 0.64 in hex:Et2O) as a colourless oil.

(1`R)-Diastereomer (136): 1H NMR (400 MHz, CDCl3) δ 4.11 (s, 1H, OH), 3.99 (dqd, J =

9.2, 6.2, 1.0 Hz, 1H, CHOH, H-1`), 2.60−2.49 (m, 2H, CHCH2CH and CHCHCH2, H-6

and H-4), 2.40 (d, J = 9.2 Hz, 1H, CH2CHCH, H-2), 2.14 (td, J = 6.2, 2.9 Hz, 1H,

CHCH2CH, H-1), 2.07 (td, 6.2, 1.9 Hz, 1H, CHCH2CH, H-5), 1.37 (s, 3H, CH3CCH3),

1 ÒHO

H

1

234

5 6

789

10H

OHO

H

HO39

136 137

7. Experimental

190

1.30 (d, J = 10.9 Hz, 1H, CH2CHCH), 1.27 (d, J = 7.8 Hz, 3H, CH3CH), 1.18 (dd, J = 6.2,

1.0 Hz, 3H, CH3CHOH), 0.92 (s, 3H, CH3CCH3). 13C NMR (100 MHz, CDCl3) δ 221.2 (C=O), 67.9 (CHOH, C-1`), 59.1 (CH2CHCH, C-2),

51.7 (CHCHCH2, C-4), 45.4 (CHCH2CH, C-1), 41.7 (CHCH2CH, C-5), 40.4 (CH3CCH3,

C-7), 30.8 (CHCH2CH, C-6), 27.7 (CH3CCH3), 22.3 (CH3CHOH), 21.4 (CH3CH, C-10),

17.7 (CH3CCH3).

(136) and (137) Diastereomers mixture: HRMS (ES) m/z for C12H20O2Na, [MNa]+

(Calc.: 219.1361, Found: 219.1369); colourless oil; Anal. Calc. for C12H20O2, Expected C,

73.4; H, 10.3; Found C, 72.5; H, 10.6; IR νmax 3560 (OH), 2955, 2923, 1711 (C=O), 1466,

1445, 1376, 1322, 1078, 988 cm–1.

(1`R,1R,2R,4S,5S)-2-(1-Hydroxy-2-methylpropyl)-4,6,6-trimethylbicyclo[3.1.1]heptan-

3-one, 138, and (1`S,1R,2R,4S,5S)-2-(1-Hydroxy-2-methylpropyl)-4,6,6-trimethylbicy-

clo[3.1.1]heptan-3-one, 139 (mixture of isomers)

The title compounds were prepared from isopinocamphone 39 according to the procedure

used for 136 and 137. Isobutyraldehyde (283 µl, 3.1 mmol) in THF (6 mL) was added to

the lithium enolate of isopinocamphone, and the reaction stirred for 25 min. The product

was purified via flash column chromatography (10:1 hexane:Et2O) to furnish

diastereomeric mixture of 138 and 139 (449 mg, 65%, d.r. 98:2, Rf 0.25 in hexane:Et2O

4:1) as a viscous oil.

(1`R)-Diastereomer (138): 1H NMR (400 MHz, CDCl3) δ 3.72 (t, J = 1.4 Hz, 1H, OH),

3.64 (dt, J = 9.2, 2.0 Hz, 1H, CHOH, H-1` converted to dd with J = 9.2, 2.1 Hz after

resolving by addition of D2O), 2.52−2.50 (m, 1H, CH2CHCH, H-2), 2.47−2.41 (m, 2H,

CHCH2CH and CHCHCH2, H-6 and H-4), 2.24−2.00 (m, 2H, CHCH2CH and CHCH2CH,

H-1 and H-5), 1.70−1.62 (m, 1H, CH(CH3)2), 1.29 (s, 3H, CH3CCH3), 1.26−1.22 (m, 1H,

CHCH2CH, H-6), 1.19 (d, J = 7.8 Hz, 3H, CH3CH, H-10), 0.97 (d, J = 6.9 Hz, 3H,

CH(CH3)2), 0.84 (s, 3H, CH3CCH3), 0.77 (d, J = 6.8 Hz, 3H, CH(CH3)2).

1 ÒHO

H

1

234

5 6

789

10H

OHO

H

HO39

138 139

7. Experimental

191

13C NMR (100 MHz, CDCl3) δ 221.1 (C=O), 73.9 (CHOH, C-1`), 55.0 (CH2CHCH, C-2),


C-7), 31.6 (CHCH2CH, C-6), 29.3 (CH(CH3)2, C-12), 27.0 (CH3CCH3), 21.6 (CH3CH, C-

10), 20.1 (CH3, CH(CH3)2), 17.0 (CH3, CH3CCH3), 13.8 (CH(CH3)2).

(138) and (139) Diastereomers mixture: HRMS (ES) m/z for C14H24O2Na, [MNa]+,

(Calc.: 247.1674, Found: 247.1674); yellow viscos oil; Anal. Calc. for C14H24O2, Expected

C, 75.0; H, 10.8; Found C, 74.5; H, 10.5; IR νmax 3494 (OH), 2972, 2956, 2935, 2873,

1689 (C=O), 1468, 1371, 1253, 1198, 1076, 993 cm–1.

(1`S,1R,2R,4S,5S)-2-(Hydroxy(phenyl)methyl)-4,6,6-trimethylbicyclo[3.1.1]heptan-3-

one, 140, and (1`R,1R,2R,4S,5S)-2-(Hydroxy(phenyl)methyl)-4,6,6-trimethylbicyclo-

[3.1.1]heptan-3-one, 141 (mixture of isomers)

The title compound was prepared from isopinocamphone 39 according to the procedure

used for 140 and 141. Benzaldehyde (321 µl, 3.1 mmol) in THF (6 mL) was added to the

lithium enolate of isopinocamphone, and the reaction stirred for 30 min. The combined

organic layer was separated with flash column chromatography using hexane:Et2O (10:1)

to afford diastereomers mixture (98:2) with yield (666 mg, 89%, Rf 0.28 in 7:1

hexane:Et2O) as a yellow oil.

(1`S)-Diastereomer (141): 1H NMR (400 MHz; CDCl3) δ 7.38−7.28 (m, 5H), 4.86 (d, J =

9.6 Hz, 1H, CHOH, H-1`), 4.65 (s, 1H, OH), 2.67−2.52 (m, 2H, CH2CHCH and

CHCHCH2, H-2), 2.49−2.43 (m, 1H, CHCH2CH, H-6), 2.06 (td, J = 6.2, 1.3 Hz, 1H,

CHCH2CH, H-1), 1.49 (td, J = 6.1, 2.5 Hz, 1H, CHCH2CH, H-5), 1.45−1.41 (m, 1H,

CHCH2CH, H-6), 1.30 (d, J = 7.3 Hz, 3H, CH3CH), 1.21 (s, 3H, CH3CCH3), 0.86 (s, 3H,

CH3CCH3). 13C NMR (100 MHz, CDCl3) δ 220.4 (C=O), 140.9 (CAr), 128.4 (CH, CHAr), 128.0 (2 x

CHAr), 126.8 (CH, 2 x CHAr), 73.9 (CHOH, C-1`), 58.4 (CH2CHCH, C-2), 51.4 (CHCHCH2,

C-4), 44.6 (CHCH2CH, C-1), 40.9 (CHCH2CH, C-5), 39.5 (CH3CCH3, C-7), 30.5

(CHCH2CH, C-6), 26.8 (CH3CCH3), 21.6 (CH3CH), 17.1 (CH3CCH3).

1 ÒHO

H

1

234

5 6

789

10 HOHO

H

HO39

140 141

7. Experimental

192


(Calc.: 281.1517, Found: 281.1507); oil; IR νmax 3465 (OH), 2974, 2937, 2890, 1685

(C=O), 1494, 1371, 1252, 1197, 1081, 982 cm–1.

(S)-Phenyl((1R,2R,4S,5S)-4,6,6-trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl)methyl acetate, 142,

and (R)-Phenyl((1R,2R,4S,5S)-4,6,6-trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl)methyl acetate,

143 (mixture of isomers)

Acetic anhydride (0.45 µl, 3.8 mmol) was one-portion flashed to mixture of 140 and 141

(100 mg, 0.38 mmol), DCM (4 mL) and pyridine (1 mL) under nitrogen atmosphere. The

reaction stirred for 2 h at 40 °C, and once completion the reaction, the mixture diluted with

ether (10 mL). the organic layer was washed with CuSO4 (3 x 5 mL), water (3 x 5 mL),

dried and solvents removed in vacuo to afford 142 and 143 (90.4 mg, 78%, Rf 0.33 in

hex:Et2O 4:1, d.r. 98:2) as a pale yellow crystals.

(S)-Diastereomer (142): 1H NMR (400 MHz; CDCl3) δ 7.36−7.29 (m, 5H, Ar), 6.10 (d, J

= 9.3 Hz, 1H, CHOAc, H-1`), 2.95−2.90 (m, 1H, CH2CHCH, H-2), 2.58−2.54 (m, 1H,

CHCHCH2, H-4), 2.43−2.37 (m, 1H, CHCH2CH, H-6), 2.10 (s, 3H, CH3COO), 1.98 (td, J

= 6.3, 1.8 Hz, 1H, CHCH2CH, H-1), 1.66 (td, J = 6.2, 2.9 Hz, 1H, CHCH2CH, H-5),

1.49−1.46 (m, 1H, CHCH2CH, H-6), 1.20 (s, 3H, CH3CCH3), 1.19 (d, J = 7.3 Hz, 3H,

CH3CH), 0.82 (s, 3H, CH3CCH3). 13C NMR (100 MHz; CDCl3) δ 214.1 (C=O), 170.8 ( COO), 138.5 (CAr), 128.9 (2 x CHAr),

128.7 (2 x CHAr), 127.5 (CAr), 75.1 (CHOAc, C-1`), 56.7 (CH2CHCH, C-2), 51.4 (CH,

CHCHCH2, C-4), 45.5 (CH3CCH3, C-7), 42.0 (CHCH2CH, C-1), 40.0 (CHCH2CH, C-4),

30.6 (CHCH2CH, C-6), 22.6 (CH3COO), 21.5 (CH3CCH3), 21.3 (CH3CCH3), 17.3

(CH3CH).

(142) and (143) Diastereomers mixture: HRMS (ES) m/z for C19H24O3Na, [MNa]+;

(Calc.: 323.1623, Found: 323.1630); crystals; IR νmax 2988, 2943, 2927, 2855, 1732 (C=O

ester), 1703 (C=O), 1455, 1363, 1237, 1105, 1081, 966 cm–1.

1 ÒHO

H

4H

OHO

H

H

140 141

OOH

1

234

5 6

789

10 HOOH

H

142 143O O

7. Experimental

193

(S)-Phenyl((1R,2R,4S,5S)-4,6,6-trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl)methyl-

4-nitrobenzoate, 144, and (R)-Phenyl((1R,2R,4S,5S)-4,6,6-trimethyl-3-oxobicy-

clo[3.1.1]heptan-2-yl)methyl-4-nitrobenzoate, 145 (mixture of isomers)

Dry pyridine (2 mL) was flashed to dry DCM (4 mL) solution of 140 and 141 (100 mg,

0.38 mmol) under N2 gas, and 4-nitrobenzoyl chloride added (108 mg, 0.5 mmol). The

reaction was stirred for overnight at RT, and monitor via TLC plate. The organic layer was

extracted with ether (10 mL), and washed with CuSO4 (3 x 5 mL), water (3 x 5 mL).

Thence, the organic layer was dried over (MgSO4), solvents removed in vacuo and purified

with flash column chromatography to yield white solid ester (96 mg, 61%, d.r. 97:3).

(S)-Diastereomer (144): 1H NMR (400 MHz; CDCl3) δ 8.31−8.26 (m, 4H, HAr),

7.42−7.33 (m, 5H, HAr), 6.30 (d, J = 9.5 Hz, 1H, CHO-4-NB, H-1`), 3.12 (d, J = 9.6 Hz,

1H, CH2CHCH, H-2), 2.62−2.57 (m, 1H, CHCHCH2, H-4), 2.51−2.44 (m, 1H, CHCH2CH,

H-6), 2.03 (td, J = 6.3, 1.8 Hz, 1H, CHCH2CH, H-1), 1.73 (td, J = 6.2, 2.9 Hz, 1H,

CHCH2CH, H-5), 1.57−1.53 (m, 1H, CHCH2CH, H-6), 1.24 (s, 3H, CH3CCH3), 1.19 (d, J

= 7.2 Hz, 3H, CH3CH), 0.86 (s, 3H, CH3CCH3). 13C NMR (100MHz; CDCl3) δ 213.4 (C=O), 164.1 (OC=O), 150.5 (CAr), 137.7 (CAr),

135.9 (CAr), 130.9 (2 x CHAr), 128.8 (2 x CHAr), 128.7 (2 x CHAr), 127.1 (CHAr), 123.5 (2

x CHAr), 76.4 (CHO-4NB, C-1`), 56.3 (CH2CHCH, C-2), 51.2 (CHCHCH2, C-4), 45.2

(CH3CCH3, C-7), 41.7 (CHCH2CH, C-1), 39.7 (CHCH2CH, C-5), 30.4 (CHCH2CH, C-6),

27.0 (CH3CCH3), 21.4 (CH3CCH3), 16.9 (CH3CH).

(144) and (145) Diastereomers mixture: HRMS (ES) m/z for C24H29N2O5, [MNH4]+,

(Calc.: 425.2071, Found: 407.2070; white solid; Anal. Calc. for C24H25NO5, Expected C,

70.8; H, 6.2; N, 3.4 Found C, 71.0; H, 6.4; N 3.3; IR νmax 2976, 2956, 2913, 2853, 1701

(C=O ester), 1609 (C=O), 1470, 1344, 1235, 1116, 1073, 996 cm–1.

1 ÒHO

H

4H

OHO

H

H

140 141

OOH

1

234

5 6

789

10 HOOH

H

144 145

O O

O2N O2N

7. Experimental

194

(S)-Phenyl((1R,2R,4S,5S)-4,6,6-trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl)methyl cinnamate,

146, and (R)-Phenyl((1R,2R,4S,5S)-4,6,6-trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl)methyl

cinnamate, 147 (mixture of isomers)

Dry pyridine (2 mL) was added to a solution of 140 and 141 (100 mg, 0.38 mmol) in dry

DCM (4 mL) under N2 gas, and 4-nitrobenzoyl chloride added (108 mg, 0.5 mmol). The

reaction was stirred for overnight at RT, and monitored via TLC. The organic layer was

extracted with ether (10 mL), and washed with CuSO4 (3 x 5 mL), water (3 x 5 mL).

Thence, the organic layer was dried (MgSO4), concentrated and purified by flash column

chromatography to yield the esters as a white solid (96 mg, 61%, d.r. 97:3).

(S)-Diastereomer (146): 1H NMR (400 MHz; CDCl3) δ 7.70 (d, J = 16.0 Hz, 1H,

CH=CHCAr), 7.55−7.51 (m, 2 H, HAr), 7.39−7.29 (m, 8H, HAr), 6.52 (d, J = 16.0 Hz, 1H,

CH=CHCAr), 6.25 (d, J = 8.8 Hz, 1H, CHOCH=CH, H-1`), 3.06 (d, J = 8.8 Hz, 1H,

CHCHCH2, H-2), 2.63−2.56 (m, 1H, CHCHCH2, H-4), 2.45−2.39 (m, 1H, CHCH2CH, H-

6), 1.99 (td, J = 6.3, 1.8 Hz, 1H, CHCH2CH, H-1), 1.77 (td, J = 6.2, 2.9 Hz, 1H,

CHCH2CH, H-5), 1.49−1.44 (m, 1H, CHCH2CH), 1.23 (s, 3H, CH3CCH3), 1.20 (d, J = 7.2

Hz, 3H, CH3CH), 0.85 (s, 3H, CH3CCH3). 13C NMR (100 MHz; CDCl3) δ 213.5 (C=O), 166.3 (OC=O), 145.4 (CH=CHCAr), 138.6

(CAr), 134.6 (CAr), 130.4 (2 x CHAr), 128.9 (2 x CHAr), 128.7 (2 x CHAr), 128.4 (CHAr),

128.3 (2 x CHAr), 127.2 (CHAr), 118.2 (CH=CHCAr), 75.1 (CHOCH=CH, C-1`), 56.6

(CH2CHCH, C-2), 51.3 (CHCHCH2, C-4), 45.3 (CH3CCH3, C-7), 41.9 (CHCH2CH, C-1),

39.9 (CHCH2CH, C-5), 30.5 (CHCH2CH, C-6), 27.2 (CH3CCH3), 21.5 (CH3CCH3), 17.1

(CH3CH).

(146) and (147) Diastereomers mixture: HRMS (ES) m/z for C26H29O3, [MH]+, (Calc.:

389.2111, Found: 389.2116; white solid; IR νmax 2973, 2933, 1705 (C=O ester), 1636 (C=O

ester), 1449, 1327, 1280, 1102, 1052, 978 cm–1.

1 ÒHO

H

4H

OHO

H

H

140 141

OOH

1

234

5 6

789

10 HOOH

H

146 147

O O

7. Experimental

195

(1`R,1R,2R,4S,5S)-2-(Hydroxy(2-nitrophenyl)methyl)-4,6,6trimethylbicycl[3.1.1] heptan-

3-one, 148, and (1`S,1R,2R,4S,5S)-2-(Hydroxy(2-nitrophenyl)methyl)-4,6,6-trimethylbi-

cyclo[3.1.1] heptan -3-one, 149

The title compounds were prepared from isopinocamphone 39 according to the procedure

used for 140 and 141. 2-Nitrobenzaldehyde (468 mg, 3.1 mmol) in THF (6 mL) was added

to the lithium enolate of isopinocamphone, and the reaction stirred for 15 min. The crude

product was purified via flash column chromatography using hex:Et2O (10:1−5:1) as

eluent to afford separable diastereomers (combined 663 mg, 73% with Rf 0.25 of 148,

brown crystals and 0.17 of 149, viscous brown oil, in hex:Et2O 4:1, d.r. 63:37).

(1`S)-Diastereomer (148): 1H NMR (400 MHz; CDCl3) δ 7.77 (ddd, J = 8.0, 3.2, 1.3 Hz,

2H, 2 x CHAr), 7.64 (td, J = 7.6, 1.1 Hz, 1H, CHAr), 7.43 (td, J = 7.8, 1.3 Hz, 1H, CHAr),

5.54 (d, J = 9.2 Hz, 1H, CHOH, H-1`), 4.61 (s, 1H, OH), 2.80−2.75 (m, 1H, CH2CHCH,

H-2), 2.65 (m, 1H, CHCHCH2, H-4), 2.53−2.49 (m, 1H, CHCH2CH, H-6), 2.04 (td, J =

6.3, 2.0 Hz, 1H, CHCH2CH, H-1), 1.50 (td, J = 6.0, 2.7 Hz, 1H, CHCH2CH, H-5),

1.48−1.44 (m, 1H, CHCH2CH, H-6), 1.27 (d, J = 7.4 Hz, 3H, CH3CH), 1.22 (s, 3H,

CH3CCH3), 0.85 (s, 3H, CH3CCH3). 13C NMR (100 MHz; CDCl3) δ 219.9 (C=O), 149.3 (CAr), 136.1 (CAr), 133.2 (CHAr), 129.3

(CHAr), 128.7 CHAr), 123.9 (CHAr), 68.6 (CHOH, C-1`), 58.3 (CH2CHCH, C-2), 51.8

(CHCHCH2, C-4), 44.6 (CHCH2CH, C-1), 41.2 (CHCH2CH, C-5), 39.8 ( CH3CCH3, C-7),

31.4 (CHCH2CH, C-6), 27.0 (CH3CCH3), 21.8 (CH3CH, C-10), 17.2 (CH3CCH3).

HRMS (ES) m/z for C17H22NO4, [MH]+, (Calc.: 304.1543, Found: 304.1545); brown

crystals; Anal. Calc. for C17H21NO4, Expected C, 67.3; H, 7.0; N, 4.6 Found C, 67.4; H,

6.9; N 4.6; IR νmax 3429 (OH), 2945, 3024, 2935, 2922, 1682 (C=O), 1444, 1370, 1260,

1189, 1081, 963 cm–1; [α]D −67º (c 0.60, CH2Cl2, 24 ºC).

(1`R)-Diastereomer (149): 1H NMR (400 MHz; CDCl3) δ 7.76 (td, J = 7.8, 1.0 Hz, 2H, 2

x CHAr) 7.63 (td, J = 7.6, 1.0 Hz, 1H, CHAr), 7.42 (ddd, J = 8.1, 7.4, 1.4 Hz, 1H, CHAr),

5.40 (d, J = 9.2 Hz, 1H, CHOH, H-1`), 5.20 (d, J = 1.0 Hz, 1H, OH), 2.81 (d, J = 9.1 Hz,

1 ÒHO

H

1

234

5 6

789

10H

OHO

H

HO39

148 149

NO2 NO2

7. Experimental

196

1H, CH2CHCH, H-2), 2.76−2.72 (m, 1H, CHCHCH2, H-4), 2.44−2.37 (m, 1H, CHCH2CH,

H-6), 1.90 (td, J =6.0, 2.0 Hz, 1H, CHCH2CH, H-1), 1.47 (td, J = 6.0, 2.0 Hz, 1H,

CHCH2CH, H-4), 1.32−1.28 (m, 1H, CHCH2CH, H-6), 1.24 (s, 3H, CH3, CH3CCH3), 1.17

(d, J = 7.1 Hz, 3H, 3H, CH3CH), 0.87 (s, 3H, CH3CCH3). 13C NMR (100 MHz; CDCl3) δ 220.5 (C=O), 149.4 (CAr), 136.0 (CAr), 133.2 (CHAr), 129.2

(CHAr), 128.7 (CHAr), 123.8 (CHAr), 69.0 (CHOH, C-1`), 58.1 (CH2CHCH, C-2), 47.1

(CHCHCH2, C-4), 44.4 (CHCH2CH, C-1), 41.0 (CHCH2CH, C-4), 39.7 (CH3CCH3, C-7),

27.7 (CHCH2CH, C-6), 26.4 (CH3CCH3), 19.6 (CH3CH, C-10), 15.1 (CH3CCH3).

HRMS (ES) m/z for C17H22NO4, [MH]+, (Calc.: 304.1543 Found: 304.1545); viscous

brown oil; Anal. Calc. for C17H21NO4, Expected C, 67.3; H, 7.0; N, 4.6 Found C, 67.4; H,

6.8; N 4.5; IR νmax 3382 (OH), 3025, 2969, 2937, 2920, 1699 (C=O), 1454, 1368, 1262,

1186, 1085, 969 cm–1; [α]D −84º (c 0.62, CH2Cl2, 24 ºC).

Trimethyl(((1R,4S,5S)-4,6,6-trimethylbicyclo[3.1.1]hept-2-en-3-yl)oxy)silane, 150

DIPA (21 mmol, 3 mL) was purged to the round flask of dry THF under N2 gas, and n-

BuLi solution of hexane (10 mL, 20.4 mmol) was slowly added over 20 min at 0 °C. The

reaction allowed to stir for further 15 min, and cooled to −78 °C. (–)-Isopinocamphone 39

(3 g, 20.0 mmol in 15 mL THF) was slowly added over a 10 min period, and the reaction

was aged for 30 min. TMSCl (25.2 mmol, 3.2 mL) was dropped via dropping funnel, and

product formation was indicated with TLC plate. The mixture was diluted with petroleum

ether (150 mL), and washed with saturated NaHCO3 (2 x 30 mL) and water (1 x 30 mL).

The solvent was removed in vacuo to give silylenol ether of isopinocamphone (4.62 g,

88%) as a pale yellow oil. 1H NMR (400 MHz; CDCl3) δ 2.36−2.32 (m, 1H, CH2CHCH, H-2), 2.28−2.20 (m, 2H,

CHCHCH2 and CHCH2CH, H-4 and H-5), 2.09 (dt, J = 6.2, 3.1 Hz, 1H, CHCH2CH, H-1),

1.90 (dd, J = 6.7, 5.3 Hz, 1H, CHCH2CH, H-6), 1.58 (s, 3H, CH3CCH3), 1.26 (s, 3H,

CH3CH), 1.22−1.19 (m, 1H, CHCH2CH, H-6), 0.87 (s, 3H, CH3CCH3), 0.21 (s, 9H, 3 x

CH3, Me3Si).

TMSO

1

23 4

56

789

10

O39 150

7. Experimental

197

13C NMR (100 MHZ, CDCl3) δ 152.7 (C-O), 109.2 (OC=CH, C-4), 48.3 (CHCHCH2, C-2),

44.6 (CHCH2CH, C-5), 40.4 (CHCH2CH, C-1), 32.3 (CHCH2CH, C-7), 26.5 (CH3CH),

21.3 (CH3CCH3), 16.9 (CH3CCH3), 16.1 (CH3CC-O), 0.9 (3 x CH3, (CH3)3-O-Si) .

HRMS (ES) m/z for C13H25OSi, [MH]+, (Calc.: 225.1675, Found: 225.1675); oil; [α]D +28º

(c 0.05, CH2Cl2, 24 ºC).

(1R,2R,4S,5S)-2-(2-Hydroxypropan-2-yl)-4,6,6-trimethylbicyclo[3.1.1]heptan-3-one, 151, and

(1R,2S,4S,5S)-2-(2-Hydroxypropan-2-yl)-4,6,6-trimethylbicyclo[3.1.1]heptan-3-one, 152

(mixture of isomers)

In three neck round bottle flask, fresh DCM (28 mL) was charged under N2 atmosphere,

and a toluene solution of TiCl4 (23.3 mmol, 1M) added at 0 °C. Dry acetone in DCM (23.3

mmol, 1.71 mL) was dropped-wise over a 15 min period using cannula, once completion, a

DCM solution of trimethylsilyl enol ether of isopinocamphone 150 (21.3 mmol, 4.50 g)

was slowly added. The reaction was monitored with TLC plate, and the reaction mixture

poured in ice-water (100 mL). The organic layer was extracted with DCM (3 x 50 mL) and

washed with saturated sodium bicarbonate (2 x 50 mL) and water (1 x 50 mL). The organic

mixture was dried (MgSO4), concentrated, and purified with flash column chromatography

using hexane:Et2O to afford diastereomer mixture of 151 and 152 (3.45 g, 77% from 150

and 82% from isopinocamphone 39, d.e. 98%, Rf 0.21 in hex:Et2O 4:1) as a pale yellow

oil.

(2R)-Diastereomer (151): 1H NMR (400 MHz; CDCl3) δ 4.57 (s, 1H, OH), 2.70 (s, 1H,

CH2CHCH, H-2), 2.54−2.49 (m, 1H, CHCH2CH, H-6), 2.44−2.40 (m, 1H, CHCHCH2, H-

4), 2.16 (td, J = 6.2, 2.3 Hz, 1H, CHCH2CH, H-1), 2.01 (td, J = 6.2, 2.0 Hz, 1H,

CHCH2CH, H-5), 1.50−1.45 (m, 1H, CHCH2CH), 1.35 (s, 3H, CH3CCH3), 1.24 (d, J = 7.4

Hz, 3H, CH3CH), 1.23 (d, J = 5.6 Hz, 3H, CH3COHCH3), 1.21 (s, 3H, CH3COHCH3), 1.18

(s, 3H, CH3CCH3). 13C NMR (100 MHz; CDCl3) δ 221.3 (C=O), 73.9 (C-OH), 61.4 (CH, CH2CHCH, C-2),


TMSO

150

1

234

5 6

789

10

(R)(R)O

OH

(S)(S)OOH

151 152

7. Experimental

198

C-7), 30.9 (CHCH2CH, C-6), 29.5 (CH3COHCH3), 28.4 (CH3COHCH3), 27.5 (CH3,

CH3CCH3), 22.1 (CH3, CH3CCH3), 17.5 (CH3, CH3CH).


(Calc.: 233.1512, Found: 233.1507); oil; IR νmax 3467 (OH), 2975, 2936, 2874, 1703

(C=O), 1687, 1470, 1327, 1225, 1137, 1048, 969 cm–1.

(1R,2S,5R)-2-(2-Hydroxypropan-2-yl)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one, 153

1H NMR (400 MHz; CDCl3) δ 2.64−2.57 (m, 3H, OH, CH2CHCH2, H-4), 2.37 (m, 1H,

CHC, H-1), 2.07−2.02 (m, 2H, CHCH2CH, H-6 and H-5), 1.64 (s, 1H, CHCH2CH, H-6),

11.36 (s, 6H, 2 x CH3, CH3COHCH3 and CH3CCH3), 1.18 (s, 3H, CH3CCH3), 1.07 (s, 3H

CH3, CH3COHCH3), 0.93−0.90 (m, 3H, CH3C). 13C NMR (100 MHz; CDCl3) δ 217.8 (C=O), 57.5 (C-OH), 49.2 (CC-OH, C-2), 46.6

CH2CHCH2, C-4), 41.1 (CH3CCH3, C-7), 38.3 (CHCH2CH, C-1), 28.5 (CHCH2CH, C-5), 28.1

(CHCH2CH, C-6), 25.9 (2 x CH3, CH3COHCH3), 23.6 (CH3C), 21.7 (2 x CH3, CH3CCH3).

HRMS (ES) m/z for C13H22O2Na, [MNa]+, (Calc.: 233.1512, Found: 233.1509); oil.

(1`R,2R,3R,6R)-2-(Hydroxyethyl)-6-methyl-3-(prop-1-en-2-yl)cyclohexan-1-one, 154

A hexane solution of n-BuLi (0.2 mL, 2.04 M) was purged to DIPA (60 µl, 0.42 mmol) in

THF (1 mL) under N2 gas, and the solution allowed stirring for further 15 min at −10 °C.

Then, the reaction flask was cooled to −78 °C, and THF solution of (+)-dihydrocarvone

isomers mixture 88 (60 mg, 0.4 mmol) was dropwise added. The mixture was magnetically

stirred for further 15 min, followed by adding acetaldehyde (23 µl, 0.42 mmol). The

reaction was stirred for 15 min, quenched with saturated NH4Cl (2 mL), and diluted with

diethyl ether (10 mL). The organic layer was extracted, washed with NH4Cl (4 mL) and

TMSO

150

O

OH

1

2 3 4

56

78 9

10

153

1 `

(R)(R)(R)(R)

(R)(R)O

(R)(R)

H

H HOH1

23

4

5

6

7

89 10

(R)(R)

O

88 Major product 154

7. Experimental

199

water (4 mL), dried over (MgSO4) and solvent removed in vacuo to give a mixture of

diastereomers and starting materials. The product was purified via flash column

chromatography using hex:EtOAc (10:1) as eluent to afford major diastereomer 154 (50

mg, 64% as a colourless oil with Rf 0.28 in hex:EtOAc 7:1), and (10 mg, 13%) of a

diastereomeric mixture.

(1`R)-Diastereomer (154): 1H NMR (400 MHz; CDCl3) δ 4.89 (s, 1H, CH2=C, H-9), 4.86

(quint, J = 1.6 Hz, 1H, CH2=C, H-9), 3.70 (dqd, J = 11.4, 6.7, 1.7 Hz, 1H, CHOH, H-1`),

3.25 (d, J = 11.3 Hz, 1H, OH), 2.76−2.68 (m, 1H, CHCHCH2, H-2), 2.52−2.43 (m, 1H,

CH2CH2CH, H-6), 2.33 (dt, J = 12.2, 1.4 Hz, 1H, CHCHCH2, H-3), 2.13−2.07 (m, 1H,

CHCHCH2, H-4), 1.88−1.77 (m, 2H, CH2CH2CH, 2 x H-5), 1.71 (dd, J = 1.4, 0.8 Hz, 3H,

CH3C=CH2), 1.45−1.35 (m, 1H, CHCHCH2, H-4), 1.31 (d, J = 6.8 Hz, 3H, CH3CHOH),

1.00 (d, J = 6.4 Hz, 3H, CH3CHCO). 13C NMR (100 MHz; CDCl3) δ 216.6 (C=O), 145.8 (CH2=C, C-8), 113.2 (CH2=C, C-9),

66.6 (CHOH, C-1`), 57.7 (CH2CH2CH, C-6), 51.5 (CHCHCH2, C-2), 46.7 (CHCHCH2, C-

3), 36.0 (CHCHCH2, C-4), 31.5 (CH2CH2CH, C-5), 22.7 (CH3CHOH), 18.7 (CH3C=CH2,

C-10), 14.2 (CH3CHCO,C-7).

HRMS (ES) m/z for C12H21O2, [MH]+, (Calc.: 197.1542, Found: 197.1545); colourless oil;


(OH) 2969, 2930, 2858, 1693 (C=O), 1645, 1447, 1371, 1266, 1118, 1078, 987 cm–1; [α]D

+90º (c 0.40, CH2Cl2, 24 ºC).

(1`R,2R,3R,6R)-2-(Hydroxy-2-methylpropyl)-6-methyl-3-(prop-1-en-2-yl)cyclohexan-

1-one, 155

The title compound was synthesised from diastereomeric mixture 88 according to the

procedure used for 154. Isobutyraldehyde (29 mg, 38 µl, 0.4 mmol) in THF (1 mL) was

added to the lithium enolate of dihydrocarvone, and the reaction stirred for 25 min. The

crude involves six diastereomers mixture, and purified via flash column chromatography

1 `

(R)(R)(R)(R)

(R)(R)O

(R)(R)

H

H HOH1

23

4

5

6

7

89 10

(R)(R)

O

88 Major product 155

7. Experimental

200

using hex:EtOAc (10:1) as eluent to afford diastereomers mixture of three isomers (39 mg,

43%, Rf 0.40 in hex:EtOAc 7:1, diastereomers ratio 95:2.5:2.5) as a yellow oil.


(quint, J = 1.6 Hz, 1H, CH2=C, H-9), 3.09 (d, J = 11.7 Hz, 1H, OH), 2.93 (ddd, J = 11.5,

9.9, 1.4 Hz, 1H, CHOH, H-1`, converted to d with J = 9.6 Hz when drop of D2O added),

2.81−2.74 (m, 1H, CH2CH2CH, H-2), 2.57 (dt, J = 12.0, 1.2 Hz, 1H, CHCHCH2, H-3),

2.51−2.44 (m, 1H, CHCHCH2, H-6), 2.11−2.06 (m, 1H, CHCHCH2, H-4), 1.98-193 (m,

1H, CH(CH3)2), 1.85−1.83 (m, 2H, CHCH2CH2, 2 x H-5), 1.68 (dd, J = 1.4, 0.8 Hz, 3H,

CH3C=CH2), 1.45−1.34 (m, 1H, CHCHCH2, H-4), 0.98 (d, J = 6.5 Hz, 3H, CH(CH3)2),

0.99 (d, 6.8 Hz, 3H, CH(CH3)2), 0.76 (d, J = 6.7 Hz, 3H, CH3CHCO). 13C NMR (100 MHz; CDCl3) δ 217.1 (C=O), 145.4 (CH2=C, C-8), 113.4 (CH2=C, C-9),

76.6 (CHOH, C-1`), 53.7 (CHCHCH2, C-3), 51.8 (CHCHCH2, C-2), 46.7 (CH2CH2CH, C-

6), 36.3 (CHCHCH2, C-4), 32.6 (CH(CH3)2), 31.6 (CHCH2CH2, C-5), 20.3 (CH(CH3)2),

20.2 (CH(CH3)2), 18.6 (CH3C=CH2, C-10), 14.2 (CH3CHCO, C-7).

HRMS (ES) m/z for C14H25O2, [MH]+, (Calc.: 225.1855, Found: 225.1854); oil; Anal.

Calc. for C14H24O2, Expected C, 75.0; H, 10.8; Found C, 75.0; H, 11.0; IR νmax 3537 (OH)

2959, 2930, 2869, 1694 (C=O), 1646, 1456, 1376, 1282, 1120, 1085, 982 cm–1.

(1`R,2R,3R,6R)-2-(Cyclohexyl(hydroxy)methyl)-6-methyl-3-(prop-1-en-2-yl)cyclo

hexan-1-one, 156

The title compound was synthesised from diastereomeric mixture of 88 according to the

procedure used for 154. Cyclohexanecarboxyaldehyde (111 mg, 120 µl, 0.4 mmol) in THF

(1 mL) was added to the lithium enolate of dihydrocarvone, and the reaction stirred for 30

min to afford crude β-hydroxyketone. The crude was chromatographed using flash column

(5:1 hexane:EtOAc) to furnish (43 mg, 41% of diastereomers mixture with d.r. 95:2.5:2.5,

Rf 0.40 in hex:EtOAc 7:1) as a colourless oil.

1 `

(R)(R)(R)(R)

(R)(R)O

(R)(R)

H

HH

OH1

23

4

5

6

7

89 10

(R)(R)

O

88

Major product 156

7. Experimental

201


(quint, J = 1.6 Hz, 1H, CH2=C, H-9), 3.07 (d, J = 3.9 Hz, 2H, CHOH and OH, adding drop

of D2O converted to d with J = 9.5 Hz), 2.84−2.77 (m, 1H, CHCHCH2, H-2), 2.60−2.57

(m, 1H, CH2CH2CH, H-6 ), 2.53−2.46 (m, 1H, CHOHCH, HCy), 2.20−2.15 (m, 1H,

CHCHCH2, H-3), 2.14−2.07 (m, 2H, CH2, HCy and H-4), 1.86−1.82 (m, 2H, CHCH2CH2, 2

x H-5), 1.77−1.71 (m, 2H, CH2, HCy), 1.70 (dd, J = 1.4, 0.8 Hz, 3H, CH3C=CH2),

1.68−1.61 (m, 2H, CH2, 2 x HCy), 1.54−1.36 (m, 2H, CH2, HCy and H-4 ), 1.26−1.08 (m,

2H, CH2, HCy), 1.01 (d, J = 6.4 Hz, 3H, CH3CHCO), 0.92−0.74 (m, 2H, CH2, HCy). 13C NMR (100 MHz; CDCl3) δ 217.1 (C=O), 145.2 (CH2=C, C-8), 113.3 (CH2=C, C-9),

75.3 (CHOH, C-1`), 53.2 (CHCHOH, C-2), 51.6 (CCyHCHOH, CCy), 46.6 (CHCHCH2, C-

3), 41.9 (CH2CH2CH, C-6), 36.2 (CHCHCH2, C-4), 31.5 (CHCH2CH2, C-5), 30.4 (CH2,

CCy), 30.3 (CH2, CCy), 26.4 (CH2, CCy), 26.1 (CH2, CCy), 25.9 (CH2, CCy), 18.5

(CH3C=CH2, C-10), 14.1 (CH3CHCO, C-7).

HRMS (ES) m/z for C17H29O2, [MH]+, (Calc.: 265.2168, Found: 265.2181); colourless oil;


(OH) 2967, 2921, 2851, 1693 (C=O), 1646, 1447, 1376, 1282, 1139, 1078, 978 cm–1.

(1`S,2R,3R,6R)-2-(Hydroxy(phenyl)methyl)-6-methyl-3-(prop-1-en-2-yl)cyclohexan-1-

one, 157

The title compound was synthesised from a diastereomeric mixture of 88 according to the

procedure used for 154. Benzaldehyde (41 µl, 0.4 mmol) in THF (1 mL) was added to the

lithium enolate of dihydrocarvone, and the reaction stirred for 25 min to give a crude

mixture of diastereomers (six isomers, d.e. 92% with respect to 157). The crude was

purified via flash column chromatography using hex:EtOAc (10:1) as eluent to afford

major diastereomer 157 (58 mg, 56% with Rf 0.36 in hex:EtOAc 7:1) as a colourless oil.

(1`S)-Diastereomer (157): 1H NMR (400 MHz; CDCl3) δ 7.35−7.29 (m, 4H, HAr),

7.22−7.18 (m, 1H, HAr), 5.03 (s, 1H, CH2=C, H-9), 4.97 (quint, J = 1.5 Hz, 1H, CH2=C, H-

1 `

(R)(R)(R)(R)

(R)(R)O

(S)(S)

H

HH

OH1

23

4

5

6

7

89 10

(R)(R)

O

88

Major product 157

7. Experimental

202

9), 4.70 (d, J = 11.6 Hz, 1H, CHOH, H-1`), 4.27 (d, J = 11.6 Hz, 1H, OH), 2.94−2.84 (m,

2H, CHCHCH2 and CHCHCH2, H-2 and H-3), 2.47−2.37 (m, 1H, CH2CH2CH , H-6),

2.14−2.08 (m, 1H, CHCHCH2, H-4), 1.92−1.86 (m, 2H, CHCH2CH2, 2 x H-5), 1.84 (dd, J

= 1.4, 0.8 Hz, 3H, CH3C=CH2), 1.47−142 (m, 1H, CHCHCH2, H-4), 0.92 (d, J = 6.4 Hz,

3H, CH3CHCO). 13C NMR (100 MHz; CDCl3) δ 216.5 (C=O), 145.4 (CH2=C, C-8), 144.5 (CHOH-CAr,

CAr), 128.2 (2 x CHAr), 126.6 (2 x CHAr), 125.6 (CHAr), 113.8 (CH2=C, C-9), 71.9 (CHOH,

C-1`), 58.7 (CHCHCH2, C-2), 51.9 (CHCHCH2, C-3), 46.7 (CH2CH2CH, C-6), 36.2

(CH2CH2CH, C-4), 31.5 (CH2CH2CH, C-5), 19.0 (CH3C=CH2, C-10), 14.1 (CH3CH, C-7).


3466 (OH) 2969, 2930, 2886, 2858, 1669 (C=O), 1644, 1448, 1378, 1239, 1125, 1070, 969

cm–1; [α]D +100º (c 0.40, CH2C22, 24 ºC).

(1`S,2R,3R,6R)-2-(Hydroxy(naphthalen-1-yl)methyl)-6-methyl-3-(prop-1-en-2-yl)

cyclohexan-1-one, 158

The title compound was synthesised from a diastereomeric mixture of 88 according to the

procedure used for 154. Naphthaldehyde (59 mg, 0.4 mmol) in THF (1 mL) was added to

the lithium enolate of dihydrocarvone, and the reaction stirred for 40 min to give a crude

mixture of diastereomers (d.r. 77:17:3:3). The crude mixture was purified via flash column

chromatography (7:1 hexane:EtOAc) to furnish (65 mg, 51% of pure major diastereomer

158, Rf 0.28 in hexane:EtOAc 7:1) as a viscous oil.

(1`S)-Diastereomer (158): 1H NMR (400 MHz; CDCl3) δ 7.90−7.85 (m, 2H, HAr), 7.74 (d,

J = 8.2 Hz, 1H, HAr), 7.68 (d, J = 7.6 Hz, 1H, HAr), 7.50−7.44 (m, 3H, HAr), 5.53 (d, J =

11.2 Hz, 1H, CHOH, H-1`), 5.19−5.16 (m, 2H, CH2=C, H-9), 4.77 (d, J = 11.2 Hz, 1H,

OH), 2.99−2.94 (m, 2H, CHCHCH2 CHCHCH2 and, H-2 and H-3), 2.40 (dquint, J = 12.5,

6.2 Hz, 1H, CH2CH2CH, H-6), 2.13−2.05 (m, 1H, CHCHCH2, H-4), 1.96 (s, 3H,

1 `

(R)(R)(R)(R)

(R)(R)O

(S)(S)

H

HH

OH1

23

4

5

6

7

89 10

(R)(R)

O

88

Major product 158

7. Experimental

203

CH3C=CH2), 1.94−1.84 (m, 2H, CHCH2CH2, 2 x H-5), 1.48−1.38 (m, 1H, CHCHCH2, H-

4), 0.92 (d, J = 6.3 Hz, 3H, CH3CHCO). 13C NMR (100 MHz; CDCl3) δ 217.4 (C=O), 145.8 (C, CH2=C, C-8), 138.7 (CAr), 133.8

(CAr), 130.5 (CAr), 129.1 (CHAr), 127.7 (CHAr), 125.9 (CHAr), 125.5 (CHAr), 125.3 (CHAr),

124.6 (CHAr), 123.2 (CHAr), 114.0 (CH2=C, C-9), 69.5 (CHOH, C-1`), 57.4 (CHCHCH2,

C-2), 52.0 (CHCHCH2, C-3), 46.9 (CH2CH2CH, C-6), 36.5 (CHCHCH2, C-4), 31.5 (CH2,

CHCH2CH2, C-5), 20.8 (CH3C=CH2, C-10), 14.0 (CH3CHCO, C-7).

HRMS (ES) m/z for C21H24O2Na, [MNa]+, (Calc.: 331.1674, Found: 331.1682); viscous

oil; IR νmax 3487 (OH) 2966, 2954, 2886, 2865, 1676 (C=O), 1645, 1444, 1365, 1265,

1132, 1064, 982 cm–1; [α]D −67º (c 0.06, CH2Cl2, 24 ºC).

(1`R,2R,3R,6R)-2-(Hydroxy(pyridin-2-yl)methyl)-6-methyl-3-(prop-1-en-2-yl)

cyclohexan-1-one, 159

The title compound was synthesised from diastereomeric mixture of 88 according to the

procedure used for 154. 2-Pyridinecarboxyaldehyde (39 mg, 35 µl, 0.4 mmol) in THF (1

mL) was added to the lithium enolate of dihydrocarvone, and the reaction stirred for 20

min to yield crude β-hydroxyketone (d.r. 73:22:4:1). The crude was purified via flash

column chromatography (4:1 hexane:EtOAc) to furnish (65 mg, 63% of pure major

diastereomer, Rf 0.21 in hexane:EtOAc 3:1) as a white crystals.

(1`R)-Diastereomer (159): 1H NMR (400 MHz; CDCl3) δ 8.44 (ddd, J = 4.8, 1.7, 0.9 Hz,

1H, HAr), 7.61 (td, J = 7.7, 1.8 Hz, 1H, HAr), 7.44−7.42 (m, 1H, HAr), 7.14−7.10 (m, 1H,

HAr), 5.33 (dd, J = 6.0, 2.0 Hz, 1H, CHOH, H-1`), 4.64 (s, 1H, CH2=C, H-9), 4.44 (quint, J

= 1.6 Hz, 1H, CH2=C, H-9), 3.85 (d, J = 6.0 Hz, 1H, OH), 3.21 (ddd, J = 11.9, 2.1, 1.3 Hz,

1H, CHCHCH2, H-2), 2.83 (td, J = 11.9, 4.2 Hz, 1H, CHCHCH2, H-3), 2.54−2.46 (m, 1H,

CH CH2CH2, H-6), 2.11−2.05 (m, 1H, CHCH2CH2, H-5), 1.86−1.76 (m, 1H, CHCHCH2,

H-4), 1.74−1.69 (m, 1H, CHCH2CH2, H-5), 1.50 (qd, J = 12.9, 3.8 Hz, 1H, CHCHCH2, H-

4), 1.34 (dd, J = 1.3, 0.7 Hz, 3H, CH3C=CH2), 1.10 (d, J = 6.8 Hz, 3H, CH3CHCO).

1 `

(R)(R)(R)(R)

(R)(R)O

(S)(S)

H

HH

OH1

23

4

5

6

7

89 10

(R)(R)

O

88

Major product 159

N

7. Experimental

204

13C NMR (100 MHz; CDCl3) δ 215.1 (C=O), 161.3 (CAr), 147.4 (CHAr), 145.7 (CH2=C, C-

8), 136.2 (CHAr), 122.2 (CHAr), 121.6 (CHAr), 113.2 (CH2=C, C-9), 72.1 (CHOH, C-1`),

57.3 (CHCHCH2, C-2) 49.0 (CHCHCH2, C-3), 45.6 (CH2CH2CH, C-6), 34.0 (CHCHCH2,

C-4), 31.5 (CHCH2CH2, C-5), 18.6 (CH3C=CH2, C-10), 13.9 (CH3CH, C-7).

HRMS (ES) m/z for C16H21NO2Na, [MNa]+, (Calc.: 282.1470, Found: 282.1481); white

crystals; IR νmax 3364 (OH) 3289, 2976, 2961, 2871, 2852, 1696 (C=O), 1648, 1437, 1376,

1254, 1166, 1074, 999 cm–1; [α]D +40º (c 0.05, CH2C22, 24 ºC); m.p 41–42 ºC.

(1`R,1R,5R,6R)-6-(Hydroxyethyl)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-ol, 160, and

(1`R,1S,5R,6R)-6-(Hydroxyethyl)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-ol, 161

A substrates 103 (500 mg, 2.57 mmol) and CeCl3.7H2O (957 mg, 1 eq.) in MeOH (2.5 mL)

were added in one portion to a suspension of NaHB4 (390 mg, 4 eq.) in THF (30 mL) at

RT. The color of the reaction mixture changed from colorless to brown, and starting

material was totally consumed within 2 h. Thence, the reaction was quenched with water (5

mL), and extracted with Et2O (30 mL). The diastereomers mixture was purified with flash

column chromatography using hex:EtOAc (5:1) to afford (145 mg of 160, 0.22 and 190 mg

of 161, Rf 0.20 in hex:EtOAc 4:1, overall 335 mg, 66%) as a colourless oil.

(1R)-Isomer (160): 1H NMR (400 MHz, CDCl3) δ 5.47−5.39 (m, 1H, C=CH, H-3),

4.80−4.74 (m, 2H, CH2=C, 2 x H-9), 4.39 (d, J = 8.8 Hz, 1H, CHOH, H-1), 4.00 (qd, J = 6.4,

3.4 Hz, 1H, CHOHCH3, H-1`), 3.09 (s, 2H, 2 x OH), 2.30−2.10 (m, 2H, CHCH2CH, 2 x H-

4), 2.06 (ddd, J = 12.0, 8.8, 3.4 Hz, 1H, CH2CHCH, H-6), 1.90−1.83 (m, 1H, CH2CHCH, H-

5), 1.76−1.74 (m, 3H, CH3C=CH), 1.69 (dd, J = 1.4, 0.9 Hz, 3H, CH3C=CH2), 1.22 (d, J =

6.4 Hz, 3H, CH3CHOH). 13C NMR (100 MHz, CDCl3) δ 146.1 (C=CH2, C-8), 135.4 (C=CH, C-3), 123.0 (C=CH, C-

2), 113.1 (C=CH2, C-9), 69.9 (CHOH, C-1), 69.5 (CHOHCH3, C-1`), 47.6 (CH2CHCH, C-

6), 44.4 (CH2CHCH, C-5), 31.2 (CH2CHCH, C-4), 18.9 (CH2=CCH3, C-10), 17.8

(CH3CCH, C-7) 17.2 (CH3CHOH).

1 `

(R)(R)(R)(R)(R)(R)

(Z) OH

(R)(R) HOHH

H(R)(R)(R)(R)(S)(S)

(Z) OH

(R)(R) HOHH

H

12

3

456

7

89 10

(R)(R)(R)(R)

(Z) O

(R)(R) HOHH

H

103 160 161

7. Experimental

205


3278 (OH), 2971, 2906, 2892, 1435, 1379, 1296, 1117, 1037, 903 cm–1; [α]D +40º (c 0.2,

CH2Cl2, 24 ºC).

(1S)-Isomer (161): 1H NMR (400 MHz; CDCl3) δ 5.56−5.54 (m, 1H, m, C=CH, H-3),

4.91−4.86 (m, 2H, CH2=C, 2 x H-9), 4.09−4.06 (m, 1H, CHOHCH3, H-1`), 4.05 (d, J = 3.6

Hz, 1H, CHOH, H-1), 2.57 (dt, J = 11.5, 5.6 Hz, 1H, CH2CHCH, H-5), 2.10 (s, 2H, 2 x

OH), 2.05−1.94 (m, 2H, CH2CHCH, 2 x H-4), 1.80 (ddd, J = 12.0, 5.7, 3.5 Hz, 1H,

CH2CHCH, H-6), 1.80−1.78 (m, 3H, CH3C), 1.77 (dd, J = 1.4, 0.7 Hz, 3H, CH2=CCH3),

1.33 (d, J = 6.6 Hz, 3H, CH3CHOH). 13C NMR (100 MHz, CDCl3) δ 149.4 (C=CH2, C-8), 135.8 (C=CH, C-3), 123.0 (C=CH,C-

2), 113.1 (C=CH2, C-9), 71.4 (CHOH, C-1), 68.3 (CHOHCH3, C-1`), 52.3 (CH2CHCH, C-

6), 43.8 (CH2CHCH, C-5), 31.6 (CH2CHCH, C-4), 19.2 (CH2=CCH3, C-10), 18.2 (CCH3,

C-7) 17.4 (CH3CHOH).


3266 (OH), 2976, 2907, 1370, 1087, 976 cm–1; [α]D −84º (c 0.36, CH2Cl2, 24 ºC).


(S,E)-2-Methyl-6-(4-nitrobenzylidene)-5-(prop-1-en-2-yl)cyclohexane-2-enone, 196, and

(S,Z)-2-Methyl-6-(4-nitrobenzylidene)-5-(prop-1-en-2-yl)cyclohex-2-en-1-one, 197 (mixture

of isomers)

Method (a): Dehydration of 116 and 117 diastereomeric mixture with p-toluenesulfonic acid

(PTSA)

A sufficient amount of diastereomers mixture 116 and 117 (115 mg, 0.38 mmol) was

dissolved in 4 mL toluene, and allowed to stir for 5 min in round flask (10 mL). Thence, p-

toluenesulfonic acid monohydrate (PTSA, 3.7 mg, 5%) was added, and the reaction

mixture stirred at (40 ºC) over night. The crude was poured in saturated NaHCO3, and the

(S)(S)

(Z) O(E)

H

NO2

(S)(S)

(Z) O

(Z)H

NO21

234

5

6

7

89 10

11

1213

14

1516

17

(R)(R)

(Z) O

(S)(S)HOHH

H

116

(R)(R)

(Z) O

(R)(R) HOHH

H

117NO2

NO2

++

196 197

7. Experimental

206

organic layer extracted from mixture with diethyl ether (10 mL). The crude oil was washed

with brine (1 x 5 mL), and then, the residue dried over (MgSO4) and evaporated to afford

crude (104 mg, 96%). The fractions were separated by flash column chromatography

(hexane:EtOAc 10:1) to obtain (69 mg, 64%, Rf 0.20 hexane:Et2O 5:1) as a pale yellow oil

of 196 and 197 with ratio 95:5).

Method (b): Dehydration of 116 and 117 diastereomeric mixture with methanesulfonyl

chloride MsCl

Methanesulfonyl chloride MsCl (0.064 mL, 0.83 mmol, 2.5 equiv.) and 4-dimethylam-

inopyridine DMAP (202 mg, 5.0 equiv.) were added to THF solution of diastereomers 116

and 117 (100 mg, 0.33 mmol), and the mixture was refluxed for 48 h. The crude was

extracted with diethyl ether (30 mL), and washed with water (10 mL). The contents of

funnel were transferred to conical flask, and then dried over (MgSO4). The crude was

concentrated in vacuo, and separated by flash column chromatography on silica gel to give

mixture of isomers (3:1, 43 mg, 45.7%).

Method (c): Dehydration of 116 and 117 diastereomeric mixture with triflic anhydride

Under 0 ºC, N2 gas was flashed in (10 mL) round bottom flask and triflic anhydride (70

mg, 1.5 equiv.) was slowly added to DCM solution of diastereomers mixture (50 mg, 0.17

mmol, dissolved in 2 mL fresh DCM) and pyridine (40 µL, 3 equiv.). The mixture was

allowed to stir for 2 h, and then quenched with cold water (5 mL). The crude was extracted

with DCM (10 mL), and the combined organic layers dried over (MgSO4). The product

was purified and separated using column chromatography (hexane:EtOAc 10:1) to obtain

(12.7 mg, 27%) as a pale yellow oil.

(E)-Isomer 196): 1H NMR (400 MHz, CDCl3) δ 8.12 (dt, J = 8.8, 2.0 Hz, 2H, HAr), 7.62

(s, 1H, C=CH, H-11), 7.49−7.48 (m, 2H, HAr), 6.78−6.76 (m, 1H, CH=CCH3, H-3), 4.97

(dq, J = 2.6, 1.3 Hz, 1H, CH3C=CH2, H-9), 4.74−4.72 (m, 1H, CH3C=CH2, H-9), 3.72 (s,

1H, CH2CHC, H-5), 2.88−2.82 (m, 2H, CH2CHC, 2 x H-4), 1.87 (q, J = 1.7 Hz, 3H,

COCCH3), 1.81 (dt, J = 1.4, 0.7 Hz, 3H, CH2=CCH3). 13C NMR (100 MHz, CDCl3) δ 189.0 (C=O), 147.5 (C=CH-CAr), 145.0 (C=CH, C-11),

142.7 (CH2CHC, C-6), 141.6 (CH2=CCH3, C-8), 141.4 (CH=CCH3, C-3), 136.8 (CHC=CH3, C-

3,), 133.4 (C-NO2, CAr), 130.1 (2 x CHAr), 123.9 (2 x CHAr) 114.1 (CH2=CCH3, C-9), 44.2

(CH2CHC, C-5), 29.1 (CH2CHC, C-4), 22.0 (CH2=CCH3, C-10), 16.6 (COCCH3, C-7).

7. Experimental

207

(E and Z Isomers 196 and 197): HRMS (ES) m/z for C17H18NO3, [MH]+, (Calc.:

284.1281, Found: 284.1281); oil at RT and crystals at −10 °C; IR νmax 2963, 2921, 1667

(C=O), 1613, 1519,1341, 1148, 1013, 966, 867, 811, 745 cm–1.

(S)-6-Ethylidene-2-methyl-5-(prop-1-en-2-yl)cyclohexane-2-enone, 172, and (S)-6-

Ethylidene-2-methyl-5-(prop-1-en-2-yl)cyclohexane-2-enone, 198 (mixture of isomers)

Under N2 gas, PTSA (6 mg, 5%) was added in one portion to a solution of major

diastereomer 103 (125 mg, 0.64 mmol) in toluene (2 mL), and the reaction mixture was

stirred at 40 ºC for 2 h. The mixture was poured in saturated NaHCO3 (10 mL), and the

organic layer extracted with diethyl ether (10 mL) and washed with brine (10 mL). Then, it

was dried over (MgSO4) and evaporated in vacuo to afford crude oil (91 mg, 97%). The

latter was chromatographed via flash column (hexane:EtOAc 10:1) to give isomeric

mixture (98 mg, 86%, Rf 0.70 hexane 3:1 EtOAc, d.r. 20:1 E:Z) as a yellow oil.

(E)-Isomer (172): 1H NMR (400 MHz, CDCl3) δ 6.67 (q, J = 7.3 Hz, C=CH, H-11), 6.55−6.52

(m, 1H, CH=CCH3, H-3), 4.66−4.65 (m, 1H, CH2=CCH3, H-9), 4.50−4.49 (m, 1H, CH2=CCH3,

H-9), 3.57 (d, J = 5.7 Hz, 1H, CH2CHC, H-5), 2.52−2.47 (m, 2H, CH2CHC, 2 x H-4), 1.72

(dt, J = 2.6, 1.3 Hz, 3H, COCCH3), 1.70 (d, J = 7.4 Hz, 3H, CH3CH=C), 1.62 (dt, J = 1.4,

0.7 Hz, 3H, CH2=CCH3). 13C NMR (100 MHz, CDCl3) δ 189.6 (C=O), 146.3 (CH3C=CH2, C-8), 142.2 (C=CH, C-11),

138.8 (CH3C=CH, C-3), 137.3 (CH2CHC, C-6), 135.3 (CH3C=CH, C-2) 114.0 (CH3C=CH2,

C-9), 42.8 (CH2CHC, C-5), 30.1 (CH2CHC, C-4), 22.6 (CH2=CCH3, C-10), 17.4 (COCCH3,

C-7), 14.8 (CH3CH=C, C-12).

(E and Z) Isomers mixture: HRMS (ES) m/z for C12H17O, [MH]+, (Calc.: 177.1279,

Found: 177.1281); oil; IR νmax 2950, 2935, 2921, 1665 (C=O), 1617, 1434, 1362, 1174 cm–1.

(S,E)-2-Methy-5-(prop-1-en-2-yl) -6-propylidenecyclohexane-2-enone, 199

(S)(S)

(Z) O(E)

H

(S)(S)

(Z) O

(Z)H

12

34

5

6

7

89 10

11

12

(R)(R)

(Z) O

(R)(R)HOHH

H

103

+

172 198

7. Experimental

208

A mixture of diastereomers (105 and 106) (372 mg, 1.78 mmol) was dissolved in (4 mL)

toluene, and (17 mg, 5%) of PTSA has been added. The mixture was heated at (40 ºC) for

overnight, and poured in saturated NaHCO3 (30 mL). Thence, the mixture was washed

with brine and dried over (MgSO4). The crude was evaporated and separated by flash

column chromatography to yield pure E isomer (102 mg, 30%, Rf 0.51, hexane 5:1 EtOAc)

as a colorless oil.

(E)-Isomer (199): 1H NMR (400 MHz, CDCl3) δ 6.70 (t, J = 7.3 Hz, 1H, C=CHCH2, H-

11), 6.60−6.56 (m, 1H, CH=CCH3, H-3), 4.74−4.73 (m, 1H, CH2=CCH3, H-9), 4.58−4.57

(m, 1H, CH2=CCH3, H-9), 3.61 (d, J = 6.0 Hz, 1H, CH2CHCH, H-5), 2.61−2.47 (m, 2H,

CH2CHC, 2 x H-4), 2.23−2.04 (m, 2H, CH3CH2CH, 2 x H-12), 1.78 (dt, J = 2.6, 1.3 Hz,

3H, COCCH3), 1.68 (dt, J = 1.4, 0.7 Hz, 3H, CH2=CCH3), 1.03 (t, J = 7.5 Hz, 3H,

CH3CH2CH). 13C NMR (100 MHz, CDCl3) δ 189.2 (C=O), 145.7 (CH3C=CH2, C-8), 142.0 (CCHCH2, C-11),

140.4 (CH3C=CH, C-3), 136.7 (CH3C=CH, C-2), 135.5 (CH2CHC, C-6), 112.8 (CH3C=CH2, C-

9), 42.4 (CH2CHC, C-5), 29.5 (CH2CHC, C-4), 21.9 (CHCH2CH3, C-12), 21.7 (CH2=CCH3, C-

10), 16.7 (COCCH3, C-7), 13.8 (CH3CH2CH, C-13).

HRMS (ES) m/z for C13H19O, [MH]+, (Calc.: 191.1439, Found: 191.1436); IR νmax 2976,

2934, 2877, 1665 (C=O), 1642, 1449, 1362, 1119, 951 cm–1; [α]D −150º (c 0.10, CH2Cl2,

24 ºC).

(S,E)-2-Methyl-6-(2-methylpropylidene)-5-(prop-1-en-2-yl)cyclohexane-2-enone, 201, and

(S,Z)-2-Methyl-6-(2-methylpropylidene)-5-(prop-1-en-2-yl)cyclohexane-2-enone, 202

(mixture of isomers)

(S)(S)

(Z) O(E)

H

12

34

5

6

7

89 10

11

12

13

(R)(R)

(Z) O

(R)(R)HOHH

H

105

(R)(R)

(Z) O

(S)(S) HOHH

H

106

+

199

7. Experimental

209

MsCl (0.44 mL, 5.4 mmol, 2.5 equiv.) and DMAP (700 mg, 5.0 equiv.) were added to THF

solution of ketol diastereomers 107 and 108 (510 mg, 2.16 mmol), and the mixture was

refluxed for overnight. The crude was extracted with diethyl ether (150 mL), and washed

with water (50 mL). The contents of funnel were transferred to conical flask, and then

dried over (MgSO4). The crude product was concentrated in vacuo, and separated by flash

column chromatography on silica gel to give mesylate (Rf 0.44 hexane 3:1 EtOAc, 700

mg). TBAF (0.8 mL, 1.2 equiv.) was slowly added to THF solution of mesylate (700 mg,

2.31 mmol) at 0 ºC, and the mixture allowed stirring for overnight at RT. The crude was

diluted with NH4Cl, and extracted with ethyl acetate. The organic layer was concentrated

in vacuum, and purified using flash column chromatography (hexane:EtOAc 10:1) to

afford (5:1) of isomers mixture (165 mg, 37%, combined isomers, Rf 0.84 and 0.77, hexane

3:1 EtOAc) as a colourless oil.

(E)-isomer (201): 1H NMR (400 MHz, CDCl3) δ 6.56−6.54 (m, 1H, CH=CCH3, H-3), 6.50

(d, J = 10.3 Hz, 1H, CCHCH, H-11), 4.74−4.73 (m, 1H, CH2=CCH3, H-9), 4.57−4.56 (m,

1H, CH2=CCH3, H-9), 3.62 (d, J = 3.6 Hz, 1H, CH2CHC, H-5), 2.59−2.52 (m, 3H,

(CH3)2CH, CH2CHC, H-12 and 2 x H-4), 1.77 (dt, J = 2.4, 0.8 Hz, 3H, COCCH3), 1.67 (dt,

J = 1.4, 0.7 Hz, 3H, CH2=CCH3), 0.98 (dd, J = 7.2, 6.7 Hz, 6H, 2 x CH3, (CH3)2CH). 13CNMR (100 MHz, CDCl3) δ 190.2 (C=O), 146.8 (CH3C=CH2, C-8), 146.5 (CH=C, C-11),

142.4 (CH3C=CH, C-3), 137.5 (CH2CHC, C-6), 135.4 (CH3C=CH, C-2) 113.0 (CH3C=CH2,

C-9), 43.2 (CH2CHC, C-5), 30.3 (CH2CHC, C-4), 28.2 ((CH3)2CH, C-12), 23.0 (CH2=CCH3,

C10), 22.7 (COCCH3, C-7). 17.4 ((CH3) 2CH, C-13 and C-14).

(Z)-Isomer (202) was partially assigned based on 1H, 13C, DEPT-135, 2D-COSY, 2D-

HSQC, and 2D-HMBC NMR spectra.

(Z)-Isomer (202): 1H NMR (400 MHz, CDCl3): δ 6.61−6.60 (m, 1H, CH=CCH3, H-3),

5.51 (dd, J = 9.7, 1.6 Hz, CCHCH, H-11), 4.87−4.86 (m, 1H, CH2=CCH3, H-9), 4.71−4.70

(m, 1H, CH2=CCH3, H-9), 3.27−3.17 (m, 2H), Peaks of methyls and the rest of

cyclohexene ring overlapped with their counterpart signals of E isomer 201.

(S)(S)

(Z) O(E)

H

12

34

5

6

7

89 10

11

1213

(R)(R)

(Z) O

(R)(R)HOHH

H

107

(R)(R)

(Z) O

(S)(S) HOHH

H

108

+

201

(S)(S)

(Z) O

(Z)H

14

202

+

7. Experimental

210

13C NMR (100 MHz, CDCl3) δ 192.4 (C=O), 148.4 (CH3C=CH2, C-8), 146.0 (CCHC, C-

11), 142.7 (CH3C=CH, C-3), 138.0 (CH2CHC, C-6), 135.3 (CH3C=CH2, C-2) 113.7

(CH3C=CH2, C-9), 51.4 (CH2CHC, C-6), 31.7 (CH2CHC, C-4), 28.7 ((CH3) 2CH, C-12),

23.7 (CH2=CCH3, C-10), 22.3 (COCCH3, C-7), 16.7 (2 x CH3, (CH3)2CH).

(E and Z Isomers): HRMS (ES) m/z for C14H20ONa, [MNa]+, (Calc.:227.1416, Found:

227.1412); oil; IR νmax 2959, 2923, 2868, 1665 (C=O), 1641, 1434, 1361, 1166, 999, 897

cm–1.

(S,E)-6-(Cyclohexaneylmethylene)-2-methyl-5-(prop-1-en-2-yl)cyclohexane-2-enone, 203,

and (S,Z)-6-(Cyclohexaneylmethylene)-2-methyl-5-(prop-1-en-2-yl)cyclohexane-2-

enone, 204 (mixture of isomers)

In a round flask connected to condenser, a (600 mg, 2.29 mmol) of 109 dissolved in 35 mL

of fresh distilled THF, followed by adding MsCl (0.53 mL, 6.9 mmol, 2.5 equiv.) and

DMAP (1.040 g, 5.0 equiv.). The mixture was heated at reflux for 72 h and the organic

layer extracted with diethyl ether (200 mL). The organic layer was washed with water (60

mL), and dried over (MgSO4). Then, the crude product was purified using column

chromatography (10:1 hexane:EtOAc) to afford (42 mg, 7.5%) and mesylate (600 mg,

77%). Tetrabutylammonium fluoride solution TBAF (615 µL, 1.2 equiv.) was slowly

added to a THF solution of recovered mesylate (600 mg, 1.76 mmol) at 0 ºC, and the

mixture allowed stirring for 2 h at RT. The crude was diluted with NH4Cl, and extracted

with ethyl acetate, concentrated in vacuo, and purified using flash column chromatography

(hexane:EtOAc 10:1) to afford of the isomers mixture ( 50 mg of E isomer, and 34 mg of

isomers mixture, total 38%, Rf 0.8 hexane 3:1 EtOAc) as a colourless oil along with

recovering the rest of mesylate.

(E)-Isomer (203): 1H NMR (400 MHz, CDCl3) δ 6.55−6.51 (m, 1H, CH=CCH3, H-3),

6.50 (d, J = 10.4 Hz, 1H, H-11), 4.72−4.71 (m, 1H, CH2=CCH3, H-9), 4.56 (s, 1H,

CH2=CCH3, H-9), 3.61 (t, J = 4.3 Hz, 1H, CH2CHC, H-5), 2.54−2.50 (m, 2H, CH2CHC, 2

(S)(S)

(Z) O

(E)

H

12

34

5

6

7

89 10

11(R)(R)

(Z) O

(R)(R)HOHH

H

109 203

(S)(S)

(Z) O

(Z)H

204

+

7. Experimental

211

x H-4), 2.25−2.15 (m, 1H, HCy), 1.76 (q, J = 1.8 Hz, 3H, COCCH3), 1.69−1.67 (m, 1H,

HCy), 1.66 (s, 4H, CH2=CCH3 and 1H of cyclohexyl ring), 1.64−1.52 (m, 3H, HCy),

1.29−1.08 (m, 5H, HCy). 13C NMR (100 MHz, CDCl3) δ 189.6 (C=O), 145.9 (CH3C=CH, C-8), 145.5 (CH2CHC, C-

11), 141.6 (CH3C=CH, C-3), 136.9 (CCHC, C-6), 134.8 (CH3C=CH2, C-2) 114.4

(CH3C=CH2, C-9), 42.5 (CH2CHC, C-6), 37.4 (CH, CCy), 33.1 (CH2, CCy), 31.1 (CH2, CCy),

29.6 (CH2CHC, C-4), 26.1 (CH2, CCy), 26.0 (CH2, CCy), 25.9 (CH2, CCy), 21.9

(CH2=CCH3, C10), 16.6 (COCCH3, C-7).

HRMS (ES) m/z for C17H25O, [MH]+, (Calc.: 245.1900, Found: 245.1893); oil; IR νmax

3074, 2969, 2923, 2887, 1667 (C=O), 1449, 1372, 1285, 1130 1161, 1046 cm–1; [α]D −88º

(c 0.16, CH2Cl2, 24 ºC).

Z)-Isomer (204) was assigned based on 1H, 13C, DEPT-135, 2D-COSY, 2D-HSQC, and

2D-HMBC NMR spectra of E isomer (203) and isomers mixture.

(Z)-Isomer (204): 1H NMR (400 MHz, CDCl3) 6.62−6.59 (m, 1H, CH=CCH3, H-3), 5.52

(dd, J = 9.6, 1.2 Hz, 1H, H-11), 4.88−4.87 (m, 1H, CH2=CCH3, H-9), 4.72−4.71 (m, 1H,

CH2=CCH3, H-9), 3.19 (t, J = 5.4 Hz, 1H, CH2CHC, H-5), 2.99−2.89 (m, 1H, HCy),

2.55−2.51 (m, 2H, CH2CHC, 2 x H-4), 1.79 (q, J =1.8 Hz, 3H, COCCH3), 1.76−1.73 (m,

2H, HCy), 1.71−1.62 (m, 8H, HCy and CH2=CCH3), 1.32−1.27 (m, 3H, HCy). 13C NMR (100 MHz, CDCl3) δ 191.5 (C=O), 146.4 (CH3C=CH2, C-8), 144.6 (CH2CHC,

C-11), 142.0 (CH3C=CH, C-3), 137.4 (CCHC, C-6), 135.4 (CH3C=CH, C-2) 113.0

(CH3C=CH2, C-9), 50.9 (CH2CHC, C-5), 37.7 (CH, CCy), 33.3 (CH2, CCy), 32.1 (CH2,

CCy), 29.9 (CH2CHC, C-4), 26.3 (CH2, CCy), 25.9 (CH2, CCy), 25.8 (CH2, CCy), 22.0

(CH2=CCH3, C-10), 16.3 (COCCH3, C-7).

(3R,6R,E)-2-Ethylidene-6-isopropyl-3-methylcyclohexananone, 205, and (3R,6R,Z)-2-

Ethylidene-6-isopropyl-3-methylcyclohexananone, 206 (mixture of isomers)

Under N2 gas, PTSA (5 mg, 5%) was one portion added to a solution of major diastereomer

126 (100 mg, 0.5 mmol) in toluene (2 mL), and the reaction mixture stirred at (40 ºC) for 2

1

2

3

4

5

6

789

10

11

12

(R)(R)

(R)(R)

O

(E)(R)(R)

(R)(R)

O

(Z)

(R)(R)(R)(R)

(R)(R)

O

(R)(R)

OHH

126 205 206

7. Experimental

212

h. The mixture was poured in saturated NaHCO3 (10 mL), and the organic layer extracted

with diethyl ether (10 mL) and washed with brine (10 mL). The residue was dried over

(MgSO4) and solvent removed in vacuo to afford crude oil. The latter was purified via

flash column chromatography (hexane:EtOAc 10:1) to give partially separation of 206, and

mixture of isomers 205 and 206 (68 mg, 76%, Rf 0.66 and 0.54, hexane 3:1 EtOAc, yellow

oil, diastereomeric mixture 3:2).

(E)-Isomer (205): 1H NMR (400 MHz, CDCl3) δ 6.43 (qd, J = 7.2, 1.0 Hz, 1H, H-11),

3.07−3.00 (m, 1H), 2.14−1.70 (m, 3H), 1.67 (d, J = 7.2 Hz, 3H, CH3CH=C), 0.94 (d, J =

7.3 Hz, 3H, CH3), 0.92−0.86 (m, 3H), 0.84 (d, J = 7.0 Hz, 3H, CH3), 0.76 (d, J = 6.8 Hz,

3H, CH3). 13C NMR (100 MHz, CDCl3) δ 2.08.3 (C=O), 146.6 (C=CHCH3, C-6), 133.9 (C=CHCH3,

C-11), 56.6 (CH), 32.3 (CH), 29.4 (CH2), 28.2 (CH2), 22.6 (CH), 22.0 (CH3CH=C), 20.4

(CH3), 19.3 (CH3), 15.1 (CH3).

Z-Isomers was partially assigned based on 1H, 13C, DEPT-135, 2D-COSY, 2D-HSQC, and

2D-HMBC NMR spectra of isomers mixture and E isomer.

(Z)-Isomer (206): 1H NMR (400 MHz, CDCl3) δ 6.26 (qd, J = 7.2, 1.5 Hz, 1H, H-11),

2.94 (q, J = 3.2 Hz, 1H), 1.68 (dd, J = 7.2, 0.6 Hz, 3H, CH3CH=C), 1.59−1.19 (m, 1H),

0.97 (d, J = 7.1 Hz, 3H, CH3), 0.82 (d, J = 8.1 Hz, 3H, CH3), 0.79 (d, J = 10.8 Hz, 3H,

CH3). 13C NMR (100 MHz; CDCl3) δ 2.04.3 (C=O), 143.5 (C=CHCH3, C-6), 132.4 (C=CHCH3,

C-11), 59.3 (CH), 31.7 (CH), 31.6 (CH2), 26.2 (CH2), 20.5 (CH), 19.8 (CH3CH=C), 18.1

(CH3), 17.8 (CH3), 13.3 (CH3).

(E and Z) Isomers mixture: HRMS (ES) m/z for C12H20ONa, [MNa]+, (Calc.: 203.1412,

Found: 203.1416); oil; IR νmax 2957, 2934, 2870, 1685 (C=O), 1617, 1449, 1453, 1379,

1272, 1100 cm–1.

(3R,6R,E)-6-Isopropyl-3-methyl-2-(3-methylbutylidene)cyclohexane-none, 207, and

(3R,6R,Z)-6-Isopropyl-3-methyl-2-(3-methylbutylidene)cyclohexane-none, 208 (mixture

of isomers)

7. Experimental

213

The title compounds were synthesised from diastereomeric mixture of 130 and 131

according to the procedure used for 205 and 206, to afford crude (100 mg, 96%). The

fractions were separated by flash column chromatography (hexane:EtOAc 10:1) to obtain

isomeric mixture (45 mg, 43 %, Rf 0.77 and 0.55, hexane:EtOAc 5:1, d.r. 5:1) as a pale

yellow oil.

E isomer was assigned based on 1H, 13C, DEPT-135, 2D-COSY, 2D-HSQC, and 2D-

HMBC NMR spectra.

(E)-Isomer (207): 1H NMR (400 MHz, CDCl3): δ 6.37 (t, J = 7.6 Hz, 1H, H-11),

3.08−3.05 (m, 1H), 2.55−2.47 (m, 1H), 2.13−1.95 (m, 3H), 1.84−1.65 (m, 5H), 0.99 (d, J =

7.2 Hz, 3H), 0.93−0.89 (m, 9H, 3 x CH3), 0.83 (d, J = 6.8 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 204.3 (C=O), 143.9 (C=CHCH3, C-6), 137.1 (C=CHCH3, C-

6), 56.1 (CH), 37.3 (CH), 31.4 (CH), 30.9 (CH2), 29.0 (CH2), 27.3 (CH2), 23.5 (CH), 23.1

(CH3CH=C), 21.2 (CH3), 20.8 (CH3), 18.9 (CH3), 18.5 (CH3).

(E and Z) Isomers mixture: HRMS (ES) m/z for C15H26ONa, [MNa]+, (Calc.: 245.1881,

Found: 245.1883); oil; IR νmax 2956, 2931, 2870, 1685 (C=O), 1617, 1453, 1367, 1100 cm–1.


7.2.3.1 General procedure: Biocatalysis

Biohydrogenation substrates were prepared as stock solutions (250 mM) in absolute

ethanol. Glucose dehydrogenase from Pseudomonas sp. (GDH), glucose-6-phosphate

dehydrogenase from Leuconostoc mesenteroids (G-6-PDH), glucose-6-phosphate and

potassium phosphate buffers KH2PO4 and K2HPO4 were obtained from Sigma-Aldrich.

The oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate

(NADP+/NADPH), and nicotinamide adenine dinucleotide (NAD+/NADH) were obtained

from Melford. Ketoreductase-to-go plates, and the ketoreductases Pro-AKR 122, 146 and

170 were obtained from Prozomix Company. The results were examined with achiral DB-

Wax or HP-5 column GC and chiral Varianchiralsil-DEX CB column.

12

3

4

5

6

789

10

11

12

O

(E)

O

(Z)

(R)(R)(R)(R)

(R)(R)

O

(S)(S)

OHH

131 207 208

(R)(R)(R)(R)

(R)(R)

O

(R)(R)

OHH

130

++

7. Experimental

214

Table 7.1 Temperature methods utilised to determine conversion and yield% of substrates

Temperature Methods Method 1

(DB-Wax column)

Method 2

(HP-5 column)

Method 3

Varianchiralsil

-DEX CB column

Split injector ratio (1:x) 20: 1 20: 1 splitless

Flow rate (mL.min-1) 1.5 1.5 1.0

Injector Temp (°C) 220 220 200

Start Temp (°C) 40 40 70

Start Temp Hold (min) 2 2 2

End Temp (°C) 210 300 200

End Temp Hold (min) 3 3 3

Temp ramping (°C/ min) 5 5 2

Detector Temp (°C) 250 250 200

Purified enzyme stocks of pentaerythritol tetranitrate reductase (PETNR) from

Enterobacter cloacae170 and Old Yellow Enzyme 3 (OYE3) from Saccharomyces

cerevisiae171 were kindly provided by the Nigel Scrutton group. Additionally, the plasmid

encoding the gene sequence of Old Yellow Enzyme 2 (OYE2) from Saccharomyces

cerevisiae171 was also supplied by the Nigel Scrutton group. Details of the plasmid-gene

constructs and Escherichia coli expression strains of the three OYEs are found in Table

7.1. The protein sequences for OYE2 (UniProt: Q03558), OYE3 (UniProt: P41816) and

PETNR (UniProt: P71278) were obtained from the UniProt online database

(http://www.uniprot.org).

Table 7.2 Details of the three OYE gene constructs and expression strains

Enzyme Source Plasmid Resr. Sites E.coli Strain His6-tag Ref.

7. Experimental

215

OYE2 S. cerevisiae pET21b Ndel/Notl BL21(DE3) C-term 171

OYE3 S. cerevisiae pET21a NdeI/XhoI BL21(DE3) C-term 171

PETNR E. cloacae pET21a NdeI/XhoI BL21(DE3)

Star

C-term 170

Resr. Sites = restriction sites through which the gene was cloned into the plasmid.

Cyclohexanone monooxygenase (CHMO_Phi1) from Rhodococcus sp. Phi1. UniProt

amino acid sequence database ID = Q84H73. Purified recombinant C-terminally His6-

tagged CHMO_Phi1was supplied by the Nigel Scrutton group (unpublished results).172

7.2.3.2 Expression and purification of recombinant OYE2

The expression and purification of OYE2 was performed due to limited supplies of

purified enzyme within the Scrutton group. The method detailed below is based on the

production and purification of OYE2 as described previously.171,173

7.2.3.3 Medium and sterile reagents preparation

Sterile stock solutions (1000x) of ampicillin (100 mgmL-1) and isopropyl β-D-1-

thiogalactopyranoside (IPTG; 0.5 M) in distilled water were filter sterilised (0.2 mm

filtration) and stored at −20 °C prior to use. Super optimal broth (SOC) medium was

prepared by combining tryptone (20 gL-1), yeast extract (5 gL-1), NaCl (10 mM), KCl (2.5

mM), MgCl2 (10 mM) and MgSO4 (10 mM) in distilled water and sterilising by

autoclaving. A filter-sterilised stock of glucose in distilled water was prepared (1M; 0.2

mm filtration), and added aseptically to the sterile medium to a final concentration of 20

mM. Lysogeny broth-Miller (LB) was prepared by combining tryptone (10 gL-1), yeast

extract (5 gL-1) and NaCl (10 gL-1) followed by autoclaving. Lysogeny broth-Miller agar

(LB agar) was prepared as for LB, except agarose (15 gL-1) was added to the medium prior

to autoclaving. After sterilisation, the medium was cooled to ~ 50 °C, and a sterile

ampicillin solution was added (100 mgmL-1) before pouring into sterile agar plates.

7.2.3.4 Transformation of competent cells

Purified OYE2 plasmid DNA (0.4 mL; Table 6.2) was mixed with pre-chilled competent

E. coli BL21 (DE3) cells (50 mL) and incubated on ice for 30 minutes. The suspension was

heat shocked by incubating at 42 °C for 30 seconds, followed by cooling on ice for 2

minutes. Cell recovery and growth was initiated by the addition of SOC medium (0.45 mL)

7. Experimental

216

and the culture was incubated at 37 °C for 1 hour at 200 rpm agitation. The cell suspension

was spread aseptically on to LB agar, containing ampicillin (100 mgmL-1), and incubated

overnight at 37 °C.

7.2.3.5 Growth and expression of OYE2 clone

Starter cultures of OYE2 in E. coli BL21(DE3) were produced by inoculating LB media (5

mL) containing ampicillin (100 mgmL-1) with a colony from the transformation plate

(Section 7.2.3.2). Cultures were incubated for 1 hour at 37 °C with 200 rpm agitation.

Aliquots of starter cultures (0.5 mL) were used to inoculate (6 x 1L LB) containing

ampicillin (100 mgmL-1) and incubated at 37 °C with 200 rpm agitation until the OD 600

nm reached ~ 0.5. Protein expression was initiated by the addition of sterile IPTG (0.5

mM) and the cultures were incubated overnight at 37 °C with 200 rpm agitation. Cultures

were harvested by centrifugation (6,238 g, 5 °C, 10 minutes), and the cell pellet was frozen

in liquid nitrogen and stored at −80 °C.

7.2.3.6 Purification of OYE2

The OYE2 cell pellet was resuspended in lysis buffer (50 mM KH2PO4/K2HPO4 pH 8.0

containing 10 mgmL-1 DNAse and 1 x Complete EDTA-free protease inhibitor blend).

Lysozyme (10 mgmL-1) and flavin mononucleotide (FMN) were added, and the slurry was

stirred until uniform. The cells were disrupted by sonication (BANDELIN SONOPULS)

(20 minutes) and the cell debris was pelleted by centrifugation (4,3667 g, 1 hour, 4 °C).

The supernatant was retained, and NaCl (3.9 mL, 0.3 M) and imidazole (0.65 mL, 10 M)

were added.

The OYE2 supernatant was applied to a Ni-NTA affinity column (30 mL) pre-equilibrated

in wash buffer 1 (50 mM KH2PO4/K2HPO4 pH 8.0 containing 10 mM imidazole). The

column was washed with wash buffer 2 (50 mM KH2PO4/K2HPO4 pH 8.0 containing 300

mM NaCl and 25 mM imidazole). Protein was recovered from the column in a step to

elution buffer (50 mM KH2PO4/K2HPO4 pH 8.0 containing 300 mM NaCl and 200 mM

imidazole).

Eluted protein fractions were checked for protein content and purity by SDS-PAGE using

stain-free precast gels (Gel DOC EZ Imager BioRad) in standard running buffer (25 mM

Tris containing 192 mM glycine and 0.1 % sodium dodecyl sulfate). Samples were

prepared by combining protein fractions (5 mL) with water (5 mL) and 2 X SDS gel

loading buffer (10 mL; 100 mM Tris pH 6.8 containing 4 % SDS, 0.2 % bromophenol

7. Experimental

217

blue, 200 mM b-mercaptoethanol and 20 % glycerol). The samples were boiled at 95 °C

for 5 min to denature the proteins, and centrifuged at 4 °C for 1 minute (13,000 g) to pellet

any debris. Samples were loaded onto the precast gel, and run for 20 min at constant

voltage of 180 V, and scanned using transilluminator.

The purest protein fractions (Figure 7.1) were dialysed against a storage buffer (10 mM

Tris pH 7.0; 7 L) to remove the NaCl and imidazole prior to protein storage. The dialysis

was performed overnight in the cold room (4 °C). The final protein concentration was

determined by measuring the absorbance at 280 nm, and using the extinction coefficient of

10600 M–1 cm–1.

Figure 7.1 SDS-PAGE of the first OYE2 affinity chromatography purification with Ni2+ NTA Lanes 1-4 = elution fractions for first column and black box shows OYE2 bands

7.2.3.7 General enzymatic procedure for double bond reduction by PETNR, OYE2 and

OYE3

The standard enzymatic reaction (1 mL) was achieved in buffer solution of KH2PO4/

K2HPO4 (50 mM, pH 7.0), comprises [substrate] = 5 µM, [NADP+] = 15 µM, GDH or G-6-

PDH = 10 U, glucose or glucose-6-phosphate 15 µM, [Enzyme] = 2 or 10 µM. The

reaction mixture was shaken at required temperature 30 °C at 135 rpm for desired time.

The reaction was then terminated by extraction into an organic layer of 800 µL of

EtOAc:internal standard (+)-limonene (99.5:0.5). The organic mixture was dried over

MgSO4, and analysed with GC using DB-wax column to determine % of yield, conversion

and diastereomeric excess.

(2R,5R)-2-Methyl-5-(prop-1-en-2-yl)cyclohexan-1-one, 100, and (2S,5R)-2-Methyl-5-

(prop-1-en-2-yl)cyclohexan-1-one, 222 (mixture of isomers)174

7. Experimental

218

Sodium dithionite Na2S2O4 (10.1 g, 58 mmol, 9 eq.) was added in one-portion to a two-

necked round-bottomed flask containing R-(–)-carvone (1000 mg, 6.6 mmol, 1 eq.),

sodium bicarbonate (9.74 g, 120 mmol, 18 eq.) and phase transfer catalyst (adogen, 350

mg, 1.9 mmol, 0.3 eq.) in toluene-water solvent (75:75 mL). The mixture was gently

heated and refluxed for 1.5 h under nitrogen atmosphere. Then, the reaction was cooled,

the organic layer was separated, and the aqueous phase was washed with diethyl ether (2 x

50 mL). The combined organic layer was washed with water (1 x 50 mL), dried over

(MgSO4) and the solvent was removed in vacuo. The product was purified using flash

column chromatography (hexane:Et2O 20:1) to furnish a diastereomeric mixture of 100

and 122 (925 mg, 92%, d.r. 96:4) as a colorless oil.

(2R,5R)-Isomer (100): 1H NMR (400 MHz, CDCl3) δ 4.74−4.72 (m, 1H, C=CH2, H-9),

4.71−4.69 (m, 1H, C=CH2, H-9), 2.44−2.39 (m, 1H, CH2CHCH2, H-6), 2.37−2.30 (m, 2H,

CHCH2CH2, CH2CHCH2, H-2 and H-5), 2.28−2.21 (m, 1H, CH2CHCH2, H-6), 2.12−2.06

(m, 1H, CH2CH2CH, H-3) 1.94−1.89 (m, 1H, CH2CH2CH, H-4), 1.71−1.70 (m, 3H,

CH3C=CH2), 1.67−1.57 (m, 1H, CH2CH2CH, H-4), 1.34 (qd, 1H, J = 13.2, 3.5 Hz,

CH2CH2CH, H-3), 1.00 (d, J = 6.5 Hz, 3H, CH3CHCH2). 13C NMR (100 MHz, CDCl3) δ 213.1 (C=O), 148.1 (C=CH2, C-8), 110.1 (C=CH2, C-9),

47.5 (CH2CHCH2, C-5), 47.4 (CH2CHCH2, C-6), 45.2 (CHCH2CH2, C-2), 35.4 (CHCH2CH2, C-

3), 31.2 (CHCH2CH2, C-4), 21.0 (CH3C=CH2, C-10), 14.8 (CH3CH, C-7).

HRMS (ES) m/z C10H16ONa, [MNa]+, (Calc.: 175.1099, Found: 175.1101; colorless oil; IR

νmax 2964, 2939, 2875, 1690 (C=O), 1601, 1455, 1329, 1176, 927, 865 cm–1.

Scale up bioreduction of R-(–)-carvone 36 by OYE2

An absolute EtOH solution of substrate (93.5 mg in 2.5 mL) was added to buffer solution

of KP (pH 7.0, 50 mM), followed by adding 120 µM of OYE2, NADP+ (1.670 mM), G-6-

PDH (1125 U), glucose (320 mg) and the volume was completed to 115 mL with buffer

solution. The reaction was incubated for 24 h at 30 °C, and extracted with ethyl acetate (3

x 50 mL). The organic layer was dried, concentrated, and purified via flash column

(R)(R)

(R)(R)O

1

2

3

4

56

7

8910

(R)(R)

(Z) O

36 100 222

(R)(R)

(S)(S)O

7. Experimental

219

chromatography using hex:EtOAc 10:1 as eluent to yield 77 mg (81%) of a diastereomeric

mixture of 2R,5R (100) and 2S,5R (+)-dihydrocarvone (222). The product was confirmed

with 1H NMR, IR spectroscopies, and run on GC using DB-Wax column to give two peaks

at retention times 19.74 min for 100 and 20.14 min for 222 diastereomer.

(2S,3R,6R)-2,6-Dimethyl-3- (prop-1-en-2-yl)cyclohexan-1-one, 236

(2R,3R,6R)-2,6-Dimethyl-3-(prop-1-en-2-yl)cyclohexan-1-one, 237

(2S,3R,6S)-2,6-Dimethyl-3-(prop-1-en-2-yl)cyclohexan-1-one, 238, and

(2R,3R,6S)-2,6-Dimethyl-3-(prop-1-en-2-yl)cyclohexan-1-one, 239 117

The title compounds were synthesised from 6-methylcarvone diastereomeric mixture (44

and 44) according to the procedure of substrates (100 and 222). The yield was purified

with flash column chromatography (hexane:Et2O 20:1), and further separation was with

normal phase HPLC (hexane:Et2O 97:3) to furnish four diastereomers of product with

combined yield% (76%) and ratio (48%, 36%,12%, 4% 236:237:238:239) as colorless oil.

(2S,3R,6R)-Diastereomer (236): 1H NMR (600 MHz, CDCl3) δ 4.77 (quint, J = 1.6 Hz,

1H, C=CH2, H-9), 4.73 (s, 1H, C=CH2, H-9), 2.44 (dq, J = 12.7, 6.3 Hz, 1H, CH2CH2CH,

H-6), 2.42 (dq, J = 12.2, 6.0 Hz, 1H, CHCHCH2, H-2), 2.11−2.04 (m, 2H, CH2CH2CH and

CHCH CH2, H-5 and H-3), 1.85 (qd, J = 12.9, 3.6 Hz, 1H, CHCH2CH2, H-5), 1.76−1.72

(m, 1H, CHCH2CH2, H-4), 1.71−1.70 (m, 3H, CH3C=CH2), 1.37 (qd, J = 13.2, 3.6 Hz, 1H,

CHCH2CH2, H-4), 1.03 (d, J = 6.4 Hz, 3H, CH3CHCH2), 0.91 (d, J = 6.4 Hz, 3H,

CH3CHC=O). 13C NMR (100 MHz; CDCl3) δ 213.9 (C=O), 146.1 (C=CH2, C-8), 112.1 (C=CH2, C-9),

55.6 (CHCHCH2, C-3), 47.4 (CH2CH2CH, C-6), 45.0 (CHCHCH2, C-2), 35.5

(CH2CH2CH, C-5), 31.3 (CH2CH2CH, C-4), 17.9 (CH3C=CH2, C-10), 14.6 (CH3CHC=O,

C-7), 11.9 (CH3CHCH2, C-11).

HRMS (APCI) m/z for C11H18O, [M]+, (Calc.: 166.1358, Found: 166.1354); oil; Anal.


(R)(R)(S)(S)(R)(R)

O

(R)(R)(S)(S)(S)(S)

O

(R)(R)(R)(R)(R)(R)

O

(R)(R)(R)(R)(S)(S)

O1

23

4

56

7

89 10

11

(R)(R)(R)(R)

(Z) O

(R)(R)(S)(S)

(Z) O

44 45 236 237 238 239

7. Experimental

220

2859, 1709 (C=O), 1645, 1450, 1375, 1177, 979, 865 cm–1; [α]D +27º (c 0.22, CH2Cl2, 24

ºC).

(2R,3R,6R)-Diastereomer (237): 1H NMR (600 MHz; CDCl3) δ 4.88−4.86 (m, Hz 1H,

C=CH2, H-9), 4.73 (s, 1H, C=CH2, H-9), 2.70 (qdd, J = 7.4, 4.0, 1.0 Hz, 1H, CHCHCH2,

H-2), 2.59 (dq, J = 12.8, 6.4 Hz, 1H, CH2CH2CH, H-6), 2.36 (dt, J = 12.4, 4.0 Hz, 1H,

CHCHCH2, H-3), 2.06 (ddt, J = 13.2, 6.0, 3.4 Hz, 1H, CHCH2CH2, H-5), 1.90 (qd, J =

13.0, 3.7 Hz, 1H, CHCH2CH2, H-5), 1.69−1.68 (m, 1H, CHCH2CH2, H-4), 1.67 (dt, J =

1.4, 0.7 Hz 3H, CH3C=CH2), 1.29 (qd, J = 13.0, 3.7 Hz, 1H, CHCH2CH2, H-4), 1.01 (d, J

= 6.4 Hz, 3H, CH3CHCH), 0.94 (d, J = 7.4 Hz, 3H, CH3CHC=O). 13C NMR (100 MHz; CDCl3) δ 216.8 (C=O), 145.2 (C=CH2, C-8), 111.3 (C=CH2, C-9),

48.4 (CHCHCH2, C-3), 46.8 (CHCHCH2, C-2), 40.0 (CH2CH2CH, C-6), 34.4

(CHCH2CH2, C-4), 23.8 (CHCH2CH2, C-5), 22.1 (CH3C=CH2, C-10), 14.6 (CH3CHC=O,

C-7), 11.9 (CH3, CH3CHCH, C-11).

HRMS (APCI) m/z for C11H18O, [M]+, (Calc.: M, 166.1358, Found: 166.1354); Anal. Calc.

for C11H18O, Expected C, 79.5; H, 10.9; Found C, 78.8; H, 11.2; IR νmax 2969, 2931,

2869,1706 (C=O), 1645, 1451, 1375, 1129, 888 cm–1; [α]D −46º (c 0.26, CH2Cl2, 24 ºC).

(2S,3R,6S)-Diastereomer (238): 1H NMR (600 MHz; CDCl3) δ 4.78−4.76 (m, 1H,

C=CH2, H-9), 4.72 (s, 1H, C=CH2, H-9), 2.59−2.54 (m, 2H, CHCH2CH2 and CH2CHCH,

H-2 and H-6), 2.15−2.11 (m, 1H, CH2CH2CH, H-3), 1.92−1.84 (m, 2H, CHCH2CH2 and

CHCH2CH2, H-4 and H-5 ), 1.75−1.72 (m, 1H, CHCH2CH2, H-5), 1.71 (dd, J = 1.4, 0.70

Hz, 3H, CH3C=CH2), 1.65 (td, J = 8.4, 4.0 Hz, 1H, CHCH2CH2, H-4), 1.17 (d, J = 7.2 Hz,

3H, CH3CHCH), 0.96 (d, J = 6.7 Hz, 3H, CH3CHC=O). 13C NMR (100 MHz; CDCl3) δ 216.9 (C=O), 146.4 (C=CH2, C-8), 112.3 (C=CH2, C-9),

53.5 (CHCHCH2, C-3), 43.9 (CH2CH2CH, C-6), 43.5 (CHCHCH2, C-2), 31.5 (CH

CH2CH2, C-5), 25.5 (CHCH2CH2, C-4), 18.9 (CH3C=CH2, C-10), 16.8 (CH3CHC=O, C-7),

13.2 (CH3CHCH, C-11).



2858, 1708 (C=O), 1644, 1456, 1376, 1133, 896 cm–1; [α]D +18º (c 0.15, CH2Cl2, 24 ºC).

(2R,3R,6S)-Diastereomer (239): 1H NMR (600 MHz; CDCl3) δ 4.88−4.87 (m, 1H,

C=CH2, H-9), 4.38 (s, 1H, C=CH2, H-9), 2.79−2.76 (m 1H, CH2CH2CH, H-6), 2.61 (qdd, J

= 6.8, 5.6, 1.8 Hz, 1H, CHCHCH2, H-2), 2.41−2.40 (m, 1H, CHCHCH2, H-3), 2.01−1.94

7. Experimental

221

(m, 1H, CHCH2CH2, H-5), 1.93−1.89 (m, 1H, CHCH2CH2, H-4), 1.85 (dq, J = 13.6, 3.4

Hz, 1H, CHCH2CH2, H-5), 1.67−1.66 (m, 3H, CH3C=CH2), 1.64 (dq, J = 13.4, 3.8 Hz, 1H,

CHCH2CH2, H-4), 1.08 (d, J = 7.0 Hz, 3H, CH3CHCH), 1.05 (d, J = 6.6 Hz, 3H,

CH3CHC=O). 13C NMR (100 MHz; CDCl3) δ 215.1 (C=O), 143.9 (C=CH2, C-8), 113.0 (C=CH2, C-9),

50.0 (CH2CH2CH, C-6), 47.3 (CHCHCH2, C-2), 44.8 (CHCHCH2, C-3), 31.1

(CHCH2CH2, C-5), 28.7 (CHCH2CH2, C-4), 24.2 (CH3C=CH2, C-10), 15.0 (CH3CHC=O,

C-7), 12.3 (CH3CHCH, C-11).



2873, 1708 (C=O), 1645, 1453, 1375, 1132, 892 cm–1; [α]D +14º (c 0.10, CH2Cl2, 24 ºC).

Scale up bioreduction of (5R,6R)-6-methylcarvone 45 via PETNR

The enzyme (150 µM), NADP+ (1.2 mM), G-6-PDH (750 U), glucose (216 mg) were

added to 75 mL buffer solution of KP (pH 7.0, 50 mM), and the mixture incubated for 2.5

h at 30 °C at 135 rmp. TLC plate showed no starting material was detected, and the organic

layer extracted and dried to afford pure isomer of (2R,3R,6R)-2-methyldihydrocarvone 237

(53 mg, 84%). The product was characterised with 1H NMR spectroscopy, and showed

identical spectrum to 237. It was run on GC using DB-Wax column to yield one peak at

retention times 20.4 min.

(R)-Trimethyl((6-methyl-3-(prop-1-en-2-yl)cyclohexa-1,5-dien-1-yl)oxy)silane, 240 119

DIPA (5.7 mL, 40.4 mmol) was purged to dry THF (50 mL) under N2 gas, and a hexane

solution of n-BuLi (19.4 mL, 40.6 mmol, 2.09 M) was slowly dropped at 0 °C. The

reaction mixture was allowed to stir for further 2 h, and the vessel was cooled to −78 °C.

Thence, R-(–)-carvone (5 g, 33.3 mmol) in fresh THF (50 mL) was dropwise added via

cannula, and the reaction aged for 30 min. The reaction mixture was quenched with

TMSCl (8.3 mL, 65.7 mmol in 50 mL THF), and the ice-acetone removed to allow the

reaction to stir for an additional 60 min at ambient temperature. The solvent was removed

OTMS1

2

34

56

7

89 10

O

36 240

7. Experimental

222

in vacuo, and the concentrated organic layer was added to Celite®. The slurry was taken up

with pentane (200 mL), and more pentane was added to wash the precipitate. The organic

layer was washed with Na2S2O3 (2 x 30 mL), saturated NaHCO3 (2 x 30 mL) and water (2

x 50 mL), dried over (MgSO4) and concentrated to give silylenol ether (6.80 g, 92%) as a

yellow oil.

Compound (240): 1H NMR (400 MHz, CDCl3) 6.85−6.81 (m, 1H, C=CH, H-5), 5.65−5.62 (m,

1H, (CH3)3SiO-C=CH, H-2), 4.99−4.94 (m, 2H, C=CH2, 2 x H-9), 2.87−2.80 (m, 1H,

CHCHCH2, H-3), 2.18−2.10 (m, 2H, CH2CHC, H-4), 1.78−1.72 (m, 6H, CH3C=CH and

CH2=CCH3), 0.31 (s, 9H, 3 x CH3, CHOMe3). 13C NMR (100 MHz, CDCl3) δ 149.8 (CH=C-OTMS, C-1), 143.6 (C=CH, C-6), 141.6

(C=CH, C-5), 133.1 (C=CH2, C-8), 132.5 (CH=C-OTMS, C-2), 109.9 (C=CH2, C-9), 41.0

(CHCHCH2, C-3), 30.6 (CH2CHCH, C-4), 19.7 (CH3C=C), 15.6 (CH3C=CH), 0.31 (3 x

CH3, OSiMe3).

MS m/z 223 (MH+, 100%); IR νmax 2969, 2923, 1669 (C=O), 1579, 1247, 959 cm-1; [α]D

−12º (c 0.16, CH2Cl2, 24 ºC).

(5S,6R)-2-Methyl-5-(prop-1-en-2-yl)-6-((trimethylsilyl)oxy)cyclohex-2-en-1-one, 241,

and (5S,6S)-2-Methyl-5-(prop-1-en-2-yl)-6-((trimethylsilyl)oxy)cyclohex-2-en-1-one,

242 (mixture of isomers)119

Dry DCM was added to a round bottomed flask (70 mL) containing silylenol ether (6.50 g,

27.3 mmol) under nitrogen atmosphere, and m-CPBA (7 g, 40.6 mmol) in dry DCM (150

mL) was dropwise transferred from second round flask at 0 °C. The reaction mixture was

allowed to warm at RT, and stirred for further 60 min. Then, the mixture was washed with

NaS2O3 (1 x 30 mL), and neutralised to pH = 7.0 via adding saturated NaHCO3. The solid

was filtered off by through a pad of Celite®, and the filtrate was concentrated under

vacuum to yield a mixture of silylenol ether diastereomers (6.05 g, 76%) as a yellowish-

green oil.

OTMS

240

1

2

3

45 6

7

89 10

(S)(S)(S)(S)

(Z) O

OTMS(S)(S)(R)(R)

(Z) O

OTMS

241 242

7. Experimental

223

Both isomers (241 and 242) were assigned based on 1H, 13C, DEPT-135, 2D-COSY, 2D-

HSQC, and 2D-HMBC NMR spectra.

(6R)-Diastereomer (241): 1H NMR (400 MHz, CDCl3) 6.74−6.72 (m, 1H, C=CH, H-3),

4.84−4.83 (m, 1H, C=CH2, H-9), 4.81 (s, 1H, C=CH2, H-9), 4.13 (d, 1H, J = 12.2 Hz,

CHOSiMe3, H-6), 2.77 (td, J = 11.5, 5.0 Hz, 1H, CH2CHCH, H-5), 2.51−2.41 (m, 1H,

CH2CHCH, H-4) 2.30−2.24 (m, 1H, CH2CHCH, H-4), 1.77−1.76 (m, 3H, CH3C=CH),

1.75−1.74 (m, 3H, CH2=CCH3), 0.15 (s, 6H, 2 x CH3, CHOMe3), 0.07 (s, 3H, CH3,

CHOMe3). 13C NMR (100 MHz, CDCl3) δ 199.5 (C=O), 144.7 (C=CH2, C-8), 143.5 (C=CH, C-3),

134.2 (C=CH, C-2), 112.4 (C=CH2, C-9), 76.6 (CHOSiMe3, C-6), 51.0 (CH2CHCH, C-5),

30.6 (CH2CHCH, C-4), 19.7 (CH3C=C), 15.6 (CH3C=CH), 0.31 (3 x CH3, OSiMe3).

(6S)-Diastereomer (242): 1H NMR (400 MHz, CDCl3): 6.67−6.65 (m, 1H, C=CH, H-3),

4.90−4.89 (m, 1H, C=CH2, H-9), 4.87 (quint, J = 1.5 Hz, 1H, C=CH2, H-9), 4.09 (d, 1H, J

= 2.4 Hz, CHOSiMe3, H-6), 2.72−2.66 (m, 1H, CH2CHCH, H-5), 2.59−2.56 (m, 1H,

CH2CHCH, H-4), 2.23−2.17 (m, 1H, CH2CHCH, H-4), 1.77−1.76 (m, 3H, CH3C=CH),

1.75−1.74 (m, 3H, CH2=CCH3), 0.15 (s, 6H, 2 x CH3, CHOMe3), 0.07 (s, 3H, CH3,

CHOMe3). 13C NMR (100 MHz, CDCl3) δ 197.8 (C=O), 145.2 (C=CH, C-8), 143.9 (C=CH, C-3),

132.7 (C=CH, C-2), 112.9 (CH2, C=CH2, C-9), 74.4 (CHOSiMe3, C-6), 48.1 (CH2CHCH,

C-5), 26.7 (CH2CHCH, C-4), 22.6 (CH3C=CH), 15.7 (CH2=CCH3), 0.1 (3 x CH3,

OSiMe3).

(5R and 6S)-Diastereomers mixture: HRMS (ES) m/z for C13H22O2SiNa, [MNa]+, (Calc.:

261.1287, Found: 261.1286); oil; IR νmax 2955, 2923, 1684 (C=O), 1451, 1372, 1247, 1163,

884 cm–1.

(5S,6R)-6-Hydroxy-2-methyl-5- (prop-1-en-2-yl) cyclohex-2-en-1-one, 243, and

(5S,6S)-6-Hydroxy-2-methyl-5- (prop-1-en-2-yl) cyclohex-2-en-1-one, 244 119

(S)(S)(S)(S)

(Z) O

OTMS(S)(S)(R)(R)

(Z) O

OTMS

241 242 243 244

1234

56

7

89 10

(S)(S)(S)(S)

(Z) O

OH(S)(S)(R)(R)

(Z) O

OH

7. Experimental

224

HCl (50 mL, 1.5 M) was added to substrates mixture of 241 and 242 (25.38 mmol), and

allowed to stir overnight at RT. The mixture was neutralised with NaOH (0.1 M), and

washed with water (3 x 20 mL), brine (3 x 20 mL), dried over (MgSO4), and solvent

removed in vacuo. The mixture was purified with flash column chromatography using

hex:EtOAc (4:1) to afford separable diastereomers of anti isomer of 6-hydroxycarvone 243

(2.49 g, 59%) and syn 244 (0.7 g, 16.6%) as a yellow oil. The total yield from R-(–)-

carvone for 243 (2.49 g, 45%) and 244 (0.7 g, 13%).

(6S)-Diastereomer (243): 1H NMR (400 MHz, CDCl3) δ 6.76−6.74 (m, 1H, C=CH, H-3),

4.94−4.91 (m, 2H, CH2=C, 2 x H-9), 4.15 (d, 1H, J = 12.7 Hz, CHOH, H-6), 3.78 (s, 1H,

OH), 2.69 (ddd, J = 12.7, 11.0, 5.1 Hz, 1H, CH2CHCH, H-5), 2.53−2.43 (m, 1H,

CH2CHCH, H-4) 2.40−2.33 (m, 1H, CH2CHCH, H-4), 1.83−1.82 (m, 6H, 2 x CH3). 13C NMR (100 MHz, CDCl3) δ 200.6 (C=O), 145.7 (C=CH2, C-8), 144.2 (C=CH, C-3),

133.1 (C=CH, C-2), 113.6 (C=CH2, C-9), 74.4 (CHOH, C-9), 51.1 (CH2CHCH, C-5), 30.7

(CH2CHCH, C-4), 18.8 (CH3), 15.4 (CCH3).


3476 OH), 2971, 2922, 2892, 1670 (C=O), 1646, 1434, 1371, 1286, 1136, 931 cm–1; lit

[α]D –30.5° (c 1.0, MeOH, 23 ºC)119, [α]D −33º (c 1.4, CH2Cl2, 24 ºC).

(6R)-Diastereomer (244): 1H NMR (400 MHz, CDCl3) δ 6.65 (ddt, J = 4.1, 2.7, 1.4 Hz,

1H, C=CH, H-3), 4.82 (quint, J = 1.5 Hz, 1H, CH2=C, H-9), 4.68−4.67 (m, 1H, CH2=C, H-

9), 4.40 (d, 1H, J = 5.9 Hz, CHOH, H-6), 3.60 (s, 1H, OH), 3.15 (dt, J = 5.8, 2.6 Hz, 1H,

CHCHOH, H-5), 2.74−2.65 (m, Hz, 1H, CH2CHCH, H-4), 2.54−2.46 (m, 1H, CH2CHCH,

H-4) 1.80 (dt, J = 2.7, 1.4 Hz, 3H, COCCH3), 1.66−1.65 (m, 3H, CH2=CCH3). 13C NMR (100 MHz, CDCl3) δ 199.8 (C=O), 144.2 (C=CH2, C-8), 143.0 (C=CH, C-3),


(CH2CHCH, C-4), 23.8 (CH3C=CH2, C-10), 15.3 (COCCH3, C-7).


3463 OH), 2964, 2922, 1711(C=O), 1670, 1433, 1360, 1256, 1132, 870 cm–1; lit [α]D –

10.5° (c 1.0, MeOH, 23 ºC)119, [α]D −10.0º (c 0.88, CH2Cl2, 24 ºC).

(2S,3S,6R)-2-Hydroxy-6-methyl-3-(prop-1-en-2-yl)cyclohexan-1-one, 248, and (2S,3S,6S)-2-

Hydroxy-6-methyl-3-(prop-1-en-2-yl)cyclohexan-1-one, 249 (mixture of isomers)

7. Experimental

225

To a two-necked round bottomed flask containing anti 6-hydroxycarvone 243 (300 mg, 1.8

mmol, 1 eq.), sodium bicarbonate (42.63 g, 32.4 mmol, 18 eq.) and phase transfer catalyst

(adogen, 94.5 mg, 0.51 mmol, 0.3 eq.) in toluene-water solvent system (20:20 mL), sodium

dithionite (2.73 g, 15.6 mmol, 9 eq.) were added. The mixture was gently heated and

reflexed in oil–bath for 1 h under nitrogen atmosphere. Then, the reaction was cooled, and

the organic layer was separated, and the aqueous phase was washed with diethyl ether (1 x

20 mL). The combined organic layer was washed with water (1 x 10 mL), dried over

(MgSO4) and solvent removed in vacuo. The product was purified by flash column

chromatography (hexane:EtOAc 7:1) to furnish (96:4) diastereomers (212 mg, 70%, with

Rf 0.16 in hex: EtOAc 3:1) as a colourless oil.

(6R)-Diastereomer (248): 1H NMR (400 MHz; CDCl3) δ 4.92 (quint, J = 1.6, 1H, C=CH2,

H-9), 4.89−4.88 (m, 1H, C=CH2, H-9), 4.11 (ddd, J = 11.4, 3.8, 1.6 Hz, 1H, CHOH, H-2),

3.70 (d, J = 3.9 Hz, 1H, OH), 2.52 (dqd, J = 12.8, 6.4, 1.6 Hz, 1H, CH2CH2CH, H-6),

2.28−2.21 (m, 1H, CHCH2CH2, H-3), 2.13 (ddt, J = 13.2, 6.3, 3.2 Hz, 1H, CHCH2CH2, H-

5) 1.84 (dd, J = 1.5, 0.8 Hz, 3H, CH3=C-CH2), 1.81 (dd, J = 3.5, 1.7 Hz, 1H, CHCH2CH2,

H-5), 1.79 (d, J = 3.3 Hz, 1H, CHCH2CH2, H-4), 1.45−1.35 (m, 1H, CHCH2CH2, H-4),

1.13 (d, J = 6.5 Hz, 3H, CH3CHC=O). 13C NMR (100 MHz; CDCl3) δ 212.9 (C=O), 145.6 (C=CH2, C-8), 113.1 (C=CH2, C-9),

77.6 (CHOH, C-2), 56.7 (CH2CH2CH, C-6), 43.8 (CHCH2CH2, C-3), 35.5 (CHCH2CH2,

C-4), 29.5 (CHCH2CH2, C-5), 19.9 (CH3C=CH2, C-10), 14.6 (CH3CH, C-7).

(248 and 249 mixture): HRMS (APCI) m/z for C10H16O2, [M]+ (Calc.: 168.1145, Found:

168.1148); oil; IR νmax 3473 (OH), 2969, 2932, 2854, 1708 (C=O), 1666, 1448, 1375,

1233, 1126, 890 cm–1.

(2R,3S,6R)-2-Hydroxy-6-methyl-3-(prop-1-en-2-yl)cyclohexan-1-one, 250, and (2R,3S,6S)-2-


1

234

56

7

89 10

(S)(S)(S)(S)(R)(R)

O

OH(S)(S)(S)(S)(S)(S)

O

OH

O

OH

243 248 249

7. Experimental

226

The title compounds were prepared from syn 6-hydroxycarvone 244 according to the

procedure used for 248 and 249. The product (170.0 mg, 56% with Rf 0.13 in hex; EtOAc

3:1, oil) was obtained as a colourless mixture of diastereomers (d.r. 10:1).

(6R)-Diastereomer (250): 1H NMR (400 MHz; CDCl3) δ 4.95−4.94 (m, 1H, C=CH2, H-9),

4.41−4.40 (m, 1H, C=CH2, H-9), 4.32 (ddd, J = 6.6, 4.0, 2.0 Hz, 1H, CHOH, H-2), 3.63 (d, J =

4.0 Hz, 1H, OH), 3.19−3.13 (m, 1H, CHCH2CH2, H-3), 2.45 (dqd, J = 12.8, 6.4, 1.9 Hz, 1H,

CH2CH2CH, H-6), 2.01−1.87 (m, 3H, CHCH2CH2, and CHCHCH2, H-4 and 2x H-5), 1.73 (s,

3H, CH3=C-CH2), 1.68−1.58 (m, 1H, CHCH2CH2, H-4) 1.11 (d, J = 6.5 Hz, 3H, CH3CHC=O). 13C NMR (100 MHz; CDCl3) δ 213.6 (C=O), 143.8 (C=CH2, C-8), 113.3 (C=CH2, C-9),

75.0 (CHOH, C-2), 51.2 (CH2CH2CH, C-6), 40.4 (CHCHCH2, C-3), 33.5 (CHCH2CH2, C-

4), 23.0 (CHCH2CH2, C-5), 22.7 (CH3=C-CH2, C-10), 14.7 (CH3CHC=O, C-7)

(250 and 251 mixture): HRMS (APCI) m/z for C10H16O2, [M]+ (Calc.: 168.1145, Found:

168.1147); oil, IR νmax 3452 (OH), 2968, 2932, 2855, 1709 (C=O), 1646, 1449, 1376, 1231,

1180, 855 cm–1.

Scale up bioreduction of (5S,6R)-6-hydroxycarvone

An absolute EtOH solution of substrate 244 (93 mg in 2.5 mL) was added to buffer

solution of KP (PH 7.0, 50 mM), followed by addition of 112 µM of OYE2, NADP+ (1.7

mM), G-6-PDH (1200 U), glucose (320 mg) and the volume was completed to 115 mL

with buffer solution. The reaction was incubated for 2.5 h at 30 °C, and extracted with

ethyl acetate (3 x 50 mL). The organic layer was dried, concentrated, and purified via flash

column chromatography using hex:EtOAc (5:1−3:1) as eluent to yield 10 mg (11%), d.e.

>99%) of one diastereomer of (2R,3S,6R)-2-hydroxydihydrocarvone 250. The product was

confirmed using NMR, and IR spectroscopies, and run on GC using achiral DB-Wax

column to give one peak at retention time 30.974 min.

(5R,6R)-6-Hydroxy-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-one, 252, and (5R,6S)-6-

Hydroxy-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-one, 253 120

1

234

56

7

89 10

(S)(S)(R)(R)(R)(R)

O

OH(S)(S)(R)(R)(S)(S)

O

OH

O

OH

244 250 251

7. Experimental

227

The title compounds were prepared from S-(+)-carvone 34 according to the procedure used

for 252 and 253, to afford syn 253 (0.51 g, 9.2%), and anti 252 (1.88 g, 34%) isomers as a

colourless oil.

(6R)-Diastereomer (252): 1H NMR (400 MHz; CDCl3) δ 6.76−6.74 (m, 1H, C=CH, H-3),

4.94−4.91 m, 2H, CH2=C, 2 x H-9), 4.15 (dd, J = 12.7, 1.9 Hz, 1H, CHOH, H-6), 3.79 (d,

J = 1.8 Hz, 1H, OH), 2.69 (ddd, J = 12.7, 11.0, 5.1 Hz, 1H, CH2CHCH, H-5), 2.52−2.43

(m, 1H, CH2CHCH, H-4), 2.41−2.33 (m, 1H, CH2CHCH, H-4), 1.84−1.83 (m, 6H, 2 x

CH3). 13C NMR (100 MHz, CDCl3) δ 200.7 (C=O), 145.8 (C=CH2, C-8), 144.3 (C=CH, C-3),

133.2 (C=CH2, C-2), 113.7 (C=CH2, C-9), 74.5 (CHOH, C-6), 51.2 (CH2CHCH, C-5),

30.8 (CH2CHCH, C-4), 18.9 (CH3), 15.5 (CH3).


3477 OH), 2971, 2892, 1724 (C=O), 1670, 1434, 1371, 1242, 1136, 891 cm–1; [α]D +28º (c

0.50, CH2Cl2, 24 ºC).

(6S)-Diastereomer (253): 1H NMR (400 MHz; CDCl3) δ 6.67−6.66 (m, 1H, 1H, C=CH,

H-3), 4.86 (quint, J = 1.5 Hz, 1H, CH2=C, H-9), 4.71−4.70 (m, 1H, CH2=C, H-9), 4.43 (dd,

J = 6.0, 2.5 Hz, 1H, CHOH, H-6), 3.60 (d, J = 2.5 Hz, 1H, OH), 3.21−3.18 (m, 1H,

CH2CHCH, H-5), 2.78−2.69 (m, 1H, CH2CHCH, H-4), 2.57−2.50 (m, 1H, CH2CHCH, H-

4), 1.83 (dt, J = 2.7, 1.4 Hz, 3H, COCCH3), 1.69 (dd, J = 1.3, 0.7 Hz, 3H, CH3C=CH2). 13C NMR (100 MHz, CDCl3) δ 200.0 (C=O), 144.2 (C=CH2, C-8), 143.2 (C=CH, C-3),


(CH2CHCH, C-4), 23.2 (CH3C=CH2, C-10), 15.4 (COCCH3, C-7).


3477 (OH), 2971, 2892, 1724 (C=O), 1670, 1434, 1371, 1242, 1136, 891 cm–1; [α]D −90º

(c 0.2, CH2Cl2, 24 ºC).

(2R,3R,6S)-2-Hydroxy-6-methyl-3-(prop-1-en-2-yl)cyclohexan-1-one, 246, and (2R,3R,6R)-2-


1

23

456

(R)(R)(S)(S)

(Z) O

OH(R)(R)(R)(R)

(Z) O

OH

O

34 252 253

7. Experimental

228

The title compounds were prepared from anti 6-hydroxycarvone 252 according to the

procedure used for 248 and 249. The yielded was (72 mg, 24% with Rf 0.16 in hex; EtOAc

3:1) of diastereomers mixture (10:1) as a colourless oil.

(6S)-Diastereomer (246): 1H NMR (400 MHz; CDCl3) δ 4.91 (quint, J = 1.6 Hz, 1H,

C=CH2, H-9), 4.88−4.86 (m, 1H, C=CH2, H-9), 4.10 (ddd, J = 11.3, 3.7, 1.5 Hz, 1H,

CHOH, H-2), 3.68 (d, J = 3.8 Hz, 1H, OH), 2.50 (dqd, J = 12.8, 6.4, 1.6 Hz, 1H,

CH2CH2CH, H-6), 2.26−2.19 (m, 1H, CHCH2CH2, H-3), 2.12 (ddt, J = 13.3, 6.2, 3.2 Hz,

1H, CHCH2CH2, H-4), 2.09−1.98 (m, 2H, CHCH2CH2, H-5), 1.81 (dd, J = 1.4, 0.7 Hz, 3H,

CH3C=CH2), 1.44−1.33 (m, 1H, CHCH2CH2, H-4), 1.11 (d, J = 6.5 Hz, 3H, CH3CHC=O). 13C NMR (100 MHz; CDCl3) δ 212.3 (C=O), 145.0 (C=CH2, C-8), 112.6 (C=CH, C-3),


C-4), 29.0 (CHCH2CH2, C-5), 19.4 (CH3-C=CH2, C-10), 14.1 (CH3CHC=O, C-7).

(246 and 254 mixture): HRMS (APCI) m/z for C10H16O2, [M]+, (Calc.: 168.1145, Found:

168.1147); oil; IR νmax 3477 (OH), 2969, 2933, 2861, 1708 (C=O), 1647, 1448, 1375, 1272,

1180, 841 cm–1.

(2S,3R,6R)-2-Hydroxy-6-methyl-3-(prop-1-en-2-yl)cyclohexan-1-one, 255, and (2S,3R,6S)-2-


A THF solution of L-selectride® (0.94 mL, 1.0 M) was added dropwise to a THF solution

of syn 6-hydroxycarvone 253 (150 mg, 0.91 mmol) at −78 °C, and the reaction allowed to

stir at same temperature for 30 min. The acetone-dry ice bath was changed to ice, and a

solution of NaOH (2.5 mL, 10%) was added slowly. Thence, a solution of H2O2 (1.8 mL,

30%) was added dropwise to reaction mixture, and once addition completed, the reaction

1

234

56

7

89 10

(R)(R)(R)(R)(S)(S)

O

OH(R)(R)(R)(R)(R)(R)

O

OH

O

OH

252 246 254

1

234

56

7

89 10

(R)(R)(S)(S)(S)(S)

O

OH(R)(R)(S)(S)(R)(R)

O

OH

O

OH

253 255 256

7. Experimental

229

allowed to warm to RT. The reaction mixture was transferred to a separatory funnel, and

extracted with hexane (3 x 15 mL), and washed with water (2 x 10 mL), sodium bisulfite

(1 x 10 mL, 1.5 M), and brine (1 x 10 mL). The crude product was dried, concentrated and

purified by flash column chromatography (hex:EtOAc 6:1) to furnish diastereomeric

mixture of 255 and 256 (d.r.10:1) (57 mg, 38%, and Rf 0.13 in hex:EtOAc 3:1) as a

colorless oil.

(6S)-Diastereomer (255): 1H NMR (400 MHz; CDCl3) δ 4.86−4.85 (m, 1H, C=CH2, H-9),

4.70−4.69 (m, 1H, C=CH2, H-9), 4.15 (dd, J = 7.0, 1.7 Hz, 1H, CHOH, H-2), 3.35 (s, 1H,

OH), 2.77−2.72 (m, 1H, CH2CH2CH, H-6), 2.01−1.90 (m, 2H, CHCH2CH2, and CHCH2CH2,

H-3 and H-4), 1.87−1.76 (m, 3H, CHCH2CH2, and CHCH2CH2, H-4 and 2 x H-5), 1.72 (dt, J

= 1.4, 0.7 Hz, 3H, CH3C=CH2), 1.37 (d, J = 6.5 Hz, 3H, CH3CHC=O). 13C NMR (100 MHz; CDCl3) δ 213.9 (C=O), 146.1 (C=CH2, C-8), 112.3 (C=CH2, C-9),


C-4), 37.1 (CHCH2CH2, C-5), 25.4 (CH3C=CH2, C-10), 21.9 (CH3CHC=O, C-7).

HRMS (ES) m/z for C10H17O2, [MH]+, (Calc.:169.1229, Found: 169.1226); oil; IR νmax

3434 (OH), 2975, 2933, 2874, 1711 (C=O), 1643 1447, 1374, 1279, 1199, 840 cm–1.

(R)-2,3-Dimethyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-one, 257 125

Methylmagnesium iodide (2.9 mL, 3.0 M, 1.3 eq.) was added to diethyl ether (10 mL) in

round bottomed flask (250 mL) under N2 atmosphere. Then, S-(+)-carvone 34 (1 g, 6.65

mmol) in dry ether (7 mL) was slowly added over 30 min at −5 °C, and the colour of

mixture changed from brown to grey. The reaction was allowed to warm gradually, and

stirred for further 3 h at room temperature. The mixture was quenched with saturated

NH4Cl (20 mL), extracted with diethyl ether (100 mL), and dried over (MgSO4). The

solvent was removed under reduced pressure to afford alcohol intermediate product. A

DCM solution of the alcohol was added to an orange suspension of PCC (2.5 g, 11.7

mmol, 1.8 eq.) and silica gel (2.5 g) in dry DCM (10 mL). The colour of the reaction

mixture was altered to dark brown. The reaction mixture was allowed to stir for 3 h at

(R)(R)

(Z) O1

234

56

7

89 10

11

(S)(S)

O

34 257

7. Experimental

230

ambient temperature, then, filtered off via silica gel using DCM as eluent, and solvents

removed in vacuo. The product was purified via flash column chromatography using

hex:EtOAc (40:1), to give 3-methylcarvone (1.01 g, 84%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 4.79 (qd, J = 1.4, 0.7 Hz, 1H, C=CH2, H-9), 4.74−4.73 (m,

1H, C=CH2, H-9), 2.66−2.58 (m, 1H, CH2CHCH2, H-6), 2.57−2.52 (m, 1H, CH2CHCH2,

H-5), 2.37−2.34 (m, 2H, CH2CHCH2, 2 x H-4), 2.25−2.20 (m, 1H, CH2CHCH2, H-6), 1.95

(q, J = 0.8 Hz, 3H, 3 x H-7, CH3C=CCH3), 1.76 (td, J = 1.7, 0.9 Hz, 3H, 3 x H-11,

CH3C=CCH3), 1.74−1.73 (m, 3H, CH2=CCH3). 13C NMR (100 MHz; CDCl3) δ 199.1 (C=O), 154.3 (CH3C=CCH3, C-3), 146.9 (CH3C=CCH3,

C-2), 130.8 (C=CH2, C-8), 110.3 (C=CH2, C-9), 42.5 (CH2CHCH2, C-6), 41.4 (CH2CHCH2, C-

5), 38.0 (CH2CHCH2, C-4), 21.6 (CH3C=CCH3, C-7), 20.6 (CH3, CH3C=CCH3, C-11), 10.9

(CH3C=CH2, C-10).

MS (ES) m/z 165 (MH+, 100%); colourless oil; Anal. Calc. for C11H16O, Expected C, 80.4;

H, 9.8; Found C, 79.3; H, 10.0; IR νmax 2955, 2932, 2917, 1661 (C=O), 1433, 1376, 1257,

1142, 108, 889 cm–1; lit [α]D −100.6º (c 0.80, CHCl3, 25 ºC)125, [α]D −100.4º (c 0.56,

CH2Cl2, 24 ºC).

(2R,3R,5R)-2,3-Dimethyl-5-(prop-1-en-2-yl)cyclohexan-1-one, 262

(2R,3S,5R)-2,3-Dimethyl-5-(prop-1-en-2-yl)cyclohexan-1-one, 263

(2S,3R,5R)-2,3-Dimethyl-5-(prop-1-en-2-yl)cyclohexan-1-one, 264, and

(2S,3S,5R)-2,3-Dimethyl-5-(prop-1-en-2-yl)cyclohexan-1-one, 265 (mixture of isomers)127

To magnetically stirred suspension of cupper bromide (CuBr, 5% mole, 36 mg) in dry THF

(4 mL), TMA (5 mmol, 2M in hexane) was added under N2 atmosphere, and the mixture

allowed to stir for 5 min at RT. R-(–)-carvone 36 (5 mmol, 750 mg) was one-portion

added, and the reaction mixture stirred for further 30 min. The mixture of reaction was

carefully quenched with saturated NH4Cl (1 mL), and left for 10 min to re-cooling. The

solution was filtered with Duran-sintered disc filter, and the solid part washed with THF (3

x 10 mL). The combined organic layer was concentrated with reduced pressure evaporator,

(R)(R)

(Z) O

36

(R)(R)(R)(R)

(R)(R)O

(S)(S)(R)(R)

(R)(R)O

(R)(R)(R)(R)

(S)(S)O

(S)(S)(R)(R)

(S)(S)O

1

23

45

6

7

89 10

11

262 263 264 265

7. Experimental

231

and the mixture was purified with flash column chromatography using hexane:EtOAc

(10:1) to afford mixture of four diastereomers (1.5:1:1:0.1) as a colourless oil with yield

(740 mg, 90%), and further separation was run on normal phase HPLC.

(Mixture of isomers): 1H NMR (400 MHz, CDCl3) δ 4.85−4.70 (m, 2H), 2.71−2.57 (m,

1H), 2.40 (s, 3H), 2.07−1.80 (m, 2H), 1.76−1.71 (m, 3H), 1.53−1.43 (m, 1H), major isomer

(262): 1.11 (d, J = 6 Hz, 3H), 1.06 (d, J = 6.4 Hz, 3H); second isomer (263): 1.10 (d, J =

6.8 Hz, 3H ), 1.05 (d, J = 6.4 Hz, 3H); third isomer (264): 1.01 (d, J = 6.8 Hz, 3H), 0.85 (d,

J = 6.8 Hz, 3H). 13C NMR (100 MHz; CDCl3) δ 213.5, 212.9, 212.5 (C=O), 147.9, 147.7, 147.0 (C=CH2,

C-8), 111.7, 109.8, 109.6 (C=CH2, C-9), 51.6 (CH), 51.3 (CH), 48.5 (CH), 46.7 (CH2),

46.6 (CH2), 45.2 (CH), 44.1 (CH2), 40.8 (CH), 40.0 (CH), 39.9 (CH2), 37.8 (CH2), 36.3

(CH), 35.2 (CH2), 35.0 (CH), 21.9, 20.8, 20.7, 20.6, 20.5, 14.1, 13.2, 12.1, 11.6 (CH3).

MS m/z 189 (MNa+, 80%), 167 (MH+, 60%); colourless oil; IR νmax 2969, 2929, 2873,

1708 (C=O), 1645, 1451, 1375, 1213, 889 cm–1.

(1S,2R,6S)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol, 268, and (1S,2S,6S)-3-

Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol, 269 175

Dry THF (4 mL) was purged to round bottle flask (10 mL) of anti-6-hydroxycarvone 243

(100 mg, 0.6 mmol) under N2 gas, and the mixture was transferred via cannula to second

round flask of LiAlH4 (46 mg, 2 eq.) at 0 °C. The reaction was stirred at 0 °C for 2 h, and

ambient temperature for further 3 h. The solid residue was filtered off with silica pad; the

reaction mixture was diluted with EtOAc, washed with HCl (0.1 M), dried over (MgSO4),

and solvent removed in vacuo. The crude was purified via flash column chromatography

using hex:EtOAc as eluent to afford (35 mg, 34%, greasy oil, Rf 0.2 in hex:EtOAc 2:1) of

268 and (20 mg, 20%, yellow oil with Rf 0.29 in hex:EtOAc 2:1) of 269.

(1S,2R,6S)-Diastereomer (268): 1H NMR (400 MHz; CDCl3) δ 5.58−5.57 (m, 1H, CH=C,

H-4), 4.94−4.91 (m, 1H, CH2=C, H-9), 4.90−4.89 (m, 1H, CH2=C, H-9), 4.02 (d, J = 3.7

Hz, 1H, CHOH, H-2), 3.69 (dt, J = 11.4, 3.5 Hz, 1H, CH2CHCHOH, H-6), 2.52−2.47 (m,

(S)(S)(S)(S)(S)(S)

(Z) OH

OH(S)(S)(S)(S)(R)(R)

(Z) OH

OH1

2

3

4

56

7

89 10

(S)(S)(S)(S)

(Z) O

OH

268 269243

7. Experimental

232

2H, CH2CHCHOH, H-1 and OH), 2.25 (d, J = 3.7 Hz, 1H, OH), 2.13−2.05 (m, 2H,

CH2CHCHOH, 2 x H-5), 1.84−1.82 (m, 3H, CH3CCH), 1.75 (dd, J = 1.4, 0.7 Hz 3H,

CH2=CCH3). 13C NMR (100 MHz, CDCl3) δ 145.2 (C=CH2, C-8), 143.9 (C=CH, C-4), 132.5 (C=CH, C-

3), 113.4 (C=CH2, C-9), 74.9 (CHOH, C-1), 74.7 (CHOH, C-2), 45.7 (CHCHOH, C-6),

30.3 (CH2CHCHOH, C-5), 19.8 (CH3CCH, C-7), 17.3 (CH2=CCH3, C-10).

MS m/z 168 (M+, 40%), 169 (MH+, 70%), greasy oil ; IR νmax 3374 (OH), 3019, 2936,

2875, 1649, 1466, 1376, 1099, 1056 cm–1. [α]D +36º (c 0.4, CH2Cl2, 24 ºC).

(1S,2S,6S)-Diastereomer (269): 1H NMR (400 MHz; CDCl3) δ 6.76−6.74 (m, 1H, CH=C,

H-4), 4.94−4.91 (m, 2H, CH2=C, H-9), 4.10 (d, 1H, J = 7.6 Hz, CHOH, H-2), 3.67 (dd, J =

11.2, 7.6 Hz, 1H, CHOH, H-1), 2.62 (s, 1H, OH), 2.46 (ddd, J = 11.6, 10.8, 6.4 Hz, 1H,

CH2CHCHOH, H-6) 2.36 (s, 1H, OH), 2.23−2.07 (m, 2H, CH2CHCHOH, 2 x H-5),

1.82−1.80 (m, 6H, 2 x CH3). 13C NMR (100 MHz, CDCl3) δ 145.7 (C=CH2, C-8), 144.2 (C=CH, C-4), 133.1 (C=CH, C-

3), 113.6 (C=CH2, C-9), 76.2 (CHOH, C-1), 74.4 (CHOH, C-2), 51.1 (CHCHOH, C-6),

30.7 (CH2CHCHOH, C-5), 18.8 (CH3), 15.4 (CH3).


3372 (OH), 3011, 2923, 2881, 1642, 1451, 1384, 1102, 1048 cm-1; [α]D −70º (c 0.7, CH2Cl2,

24 ºC).


(4R,7R)-7-Isopropyl-4-methyloxepan-2-one, 316

A DCM:dichloroethane (3 mL, 1:1) solution of m-CPBA (0.44 mmol, 1.5eq.) was

transferred via cannula to second round flask containing suspension of (+)-isomenthone 38

(250 mg, 1.6 mmol) and NaHCO3 (1.65 mmol, 138 mg) in DCM (7 mL). The reaction was

allowed to stir for 5 h at RT in the dark, thence, the organic layer extracted with Et2O (3 x

15 mL), and washed one time with Na2SO4 (10%, 10 mL), water (10 mL) and brine (10

mL). The organic layer was dried (MgSO4), solvents removed in vacuo, and purified with

1

2

34

5

67

89

(R)(R) O

(R)(R) O

10

O

38 316

7. Experimental

233

flash column chromatography using hexane:EtOAc (10:1) to afford normal lactone 316

(114 mg, 45%, Rf 0.63, 1:1 hexane:EtOAc) as a volatile colourless oil.

Normal lactone (316) 1H NMR (400 MHz; CDCl3) δ 4.02−3.99 (m, 1H, CH-O, H-7), 2.89

(dd, J = 13.6, 3.2, 1H, CH2CHCH2, H-3), 2.54 (dd, J = 13.6, 5.9, 1H, CH2CHCH2, H-3),

2.17−2.15 (m, 1H, CHCH2CH2, H-6), 1.86−1.81 (m, 1H, CHCH2CH2, H-4), 1.75−1.72 (m,

4H, CHCH2CH2, CHCH2CH2 and CH(CH3)2, H-6, 2 x H-5 and H-8), 1.04 (d, J = 7.2, 3H,

CH3), 0.98 (d, J = 1.8, 3H, CH3 ), 0.96 (d, J = 1.8, 3H, CH3). 13C NMR (100 MHz; CDCl3) δ 174.5 (COO), 85.1 (CH-O-, C-7), 40.9 (CH2CHCH2, C-3),

34.5 (CH2CH2CH, C-6), 33.4 (CH(CH3)2, C-8), 29.4 (CH3CH, C-4) 26.9 (CH2CH2CH, C-

5), 18.6 (CH3) , 17.8 (CH3) , 17.4 (CH3).


2959, 2925, 2873, 1728 (C=O ester), 1461, 1365, 1265, 1186, 1030 cm–1; [α]D + 50° (c

0.20, CH2Cl2, 24 °C).

(1R,2S,6R)-2,7,7-Trimethyl-3-oxabicyclo[4.1.1]octan-4-one, 317, and (1S,5S,6S)-5,7,7-

Trimethyl-3-oxabicyclo[4.1.1]octan-4-one, 318 (mixture of normal and abnormal lactones)

A) A solution of (–)-isopinocamphone 39 (300 mg, 1.97 mmol) in dry DMF (4 mL) was

transferred to round flask containing Oxone® (1230 mg, 2 eq.) under N2 atmosphere. The

mixture was stirred at 50 °C for overnight, and the colour was changed from white to pink

then to orange. The reaction mixture was filtered through Celite®, and the organic layer

extracted with ether (3 x 5 mL), washed with water (3 x 10 mL), dried (MgSO4). The crude

was purified via flash column chromatography using hex:EtOAc (5:1) to afforded lactone

(16 mg, 5%, ratio of nornmal:abnormal lactones 95:05, Rf 0.53 in hex:EtOAc 5:1) as a

colourless oil.

B) m-CPBA (5 eq., 9.85 mmol, 2.2 g, 78%) was added to solution of (–)-isopinocamphone 39

(300 mg, 1.97 mmol) in dry DCM (0.05M), and the reaction magnetically stirred at room

temperature for 20 h. The reaction mixture was washed 10% sodium metabisulfite (1 x 20

mL), and extracted with DCM (30 mL). The organic layer washed with saturated sodium

bicarbonate (10 mL), brine (10 mL) and water (10 mL), dried over (MgSO4), and the

(S)(S)

(R)(R)(R)(R)

OO

(S)(S)

(S)(S)(S)(S)

O

O

1

23 4

5

67

89

10

(S)(S)

(S)(S) (R)(R)

O

39 317 318

11

7. Experimental

234

solvent removed in vacuo to give the crude product. The mixture of lactones was carefully

purified using flash column chromatography (5:1 hex:EtOAc) to obtained pure product

(126 mg, 38%, with ratio 90:10 normal:abnormal lactones) as a colourless oil.

Normal lactone (317): 1H NMR (400 MHz; CDCl3) δ 4.69−4.66 (m, 1H, CHCHCH3, H-

2), 3.02−2.85 (m, 2H, COCH2CH, 2 x H-5), 2.69−2.56 (m, 1H, CHCHCH2, H-7),

2.28−2.19 (m, 1H, CHCH-O, H-1), 1.95−1.90 (m, 1H, CH2CHCH2, H-6), 1.47−1.43 (m,

3H, CHCHCH3, H-11), 1.40−1.30 (m, 1H, CH2CHCH2, H-7), 1.34 (dd, J = 5.6, 2.5 Hz,

3H, CH3CCH3), 1.12 (dd, J = 5.6, 2.5 Hz, 3H, CH3CCH3). 13C NMR (100 MHz; CDCl3) δ 174.6 (C=O), 82.9 (CHCHCH3, C-2), 48.5 (CHCH-O, C-

1), 39.1 (CH3CCH3, C-8), 38.1 (CH2CO, C-5), 37.5 (CH2CHCH2, C-6), 30.2 (CH3CCH3),

28.8 (CH2CHCH2, C-7), 21.8 (CH3CH, C-11), 21.5 (CH3CCH3).


2935, 2865, 1707 (C=O ester), 1687, 1463, 1337, 1221, 1143, 1063, 983 cm–1.

(1`S,1R,3R)-3-(1-Hydroxyethyl)-2,2-dimethylcyclobutyl)acetic acid, 319, and (S)-2-((1S,3S)-3-

(Hydroxymethyl)-2,2-dimethylcyclobutyl)propanoic acid, 320

§

The mixture of lactones 317 and 318 was colorless oil, and totally converted to white solid

of pinolic acid 319 and cyclobutane derivative 320 within 48 h without further

modifications as hydrolyzed at RT to afford 319:320 with ratio 90:10.

(319) was assigned based on 1H, 13C, DEPT-135, 2D-COSY, 2D-HSQC, and 2D-HMBC

NMR spectra.

Compound (319): 1H NMR (400 MHz; CDCl3) δ 4.78 (s, br., 2H, 2 x OH), 3.74 (dq, J =

9.8, 6.2 Hz, 1H, CH3CHOH, H-1`), 2.42−2.31 (m, 1H, CHCH2COOH), 2.28−2.18 (m, 2H,

CHCH2COOH and CHCCH, H-3), 2.07−1.98 (m, 1H, CCHCH2, H-4), 1.84−1.75 (m, 1H,

(S)(S)

(R)(R)(R)(R)

OO

(S)(S)

(S)(S)(S)(S)

O

O

317

318

HO

(S)(S)

OHO

(R)(R)

(R)(R)

(S)(S)OH

(S)(S)

(S)(S)O

OH

1

2

3

4

++

319

320

1`

7. Experimental

235

CHCCH2, H-1), 1.24−1.21 (m, 1H, CCHCH2, H-4), 1.17 (s, 3H, CH3CCH3), 1.07 (d, J =

6.2 Hz, 3H, CH3CHOH), 1.05 (s, 3H, CH3CCH3). 13C NMR (100 MHz; CDCl3) δ 178.8 (C=O), 69.4 (CH3CHOH, C-1`), 50.4 (CHCCH2, C-

1), 40.0 (CH3CCH3), 37.8 (CHCCH, C-3), 35.0 (CH2COOH), 31.0 (CH3CCH3), 26.6

(CCHCH2, C-4), 21.3 (CH3CHOH), 16.9 (CH3CCH3).

HRMS (ES) m/z for C10H19O3, [MH]+, (Calc.: 187.1329, Found: 187.1328); solid; IR νmax

3430 (OH), 2922, 2873, 1704 (C=O), 1667, 1463, 1357, 1248, 1188, 1070, 957 cm–1.

Methyl (3R,6R)-6-hydroxy-3,7-dimethyloctanoate, 333, and Methyl (3R,6S)-6-hydroxy-3,7-

dimethyloctanoate, 334 (mixture of isomers)

Methyl (2R,5R)-6-hydroxy-2-isopropyl-5-methylhexanoate, 335, and Methyl (2S,5R)-

6-hydroxy-2-isopropyl-5-methylhexanoate, 336 (mixture of isomers)

Oxone® (4eq., 12.96 mmol, 7.95 g) was one portion added to solution of 38 (500 mg, 3.24

mmol) in dry methanol (16 mL), and the reaction heated for 50 °C under N2 gas. The

starting material was completely consumed within 2 h, and the crude diluted with diethyl

ether (10 mL), and filtered through Celite®. Solvents were removed in vacuo, and flash

column chromatography (hex:EtOAc 7:1) afforded two products as a colourless oils (major

fraction 333 and 334, 275 mg, 42%, Rf 0.38, ratio 1:1) and (minor fraction 335 and 336,

34 mg, 5%, Rf 0.27 hex:EtOAc 5:1, ratio 1:1).

Mixture of 333 and 334: 1H NMR (400 MHz; CDCl3) δ 3.65 (s, 3H, COOMe), 3.35−3.31

(m, 1H, CHOH, H-6), 2.30 (dd, J = 7.9, 6.4 Hz, 1H, CH2COO, H-2), 2.16 (dd, J = 12.5, 7.9

Hz, 1H, CH2COO, H-2), 2.01−1.92 (m, 1H), 1.70−1.59 (m, 2H), 1.55−1.16 (m, 4H), 0.95

(d, J = 6.6 Hz, 3H, CH3), 0.92−0.88 (m, 6H, 2 x CH3).

(R)(R)(R)(R)

O

OHO

(S)(S)(R)(R)

O

OHO

(R)(R)(R)(R)

O

(S)(S)(R)(R)

O

38

49

333

334

+

12

3

4

5

6

335

336

(S)(S)O

(R)(R)

+HO

OMe

(R)(R)O

(R)(R)

HO

OMe

12

3

45

6

7. Experimental

236

13C NMR (100 MHz; CDCl3) δ 173.9, 173.8 (C=O), 77.0, 76.6 (CHOH, C-6), 51.5, 51.4

(COOMe), 41.8, 41.5 (CH2COO, C-2), 33.7, 33.5 (CH), 33.1, 33.0 (CH2), 31.6, 31.4

(CH2), 30.6, 30.2 (CH), 20.0, 19.7 (CH3) , 19.0, 18.9 (CH3), 17.3, 17.0 (CH3).

Mixture of 333 and 334: HRMS (ES) m/z for C11H22O3Na, [MNa]+, (Calc.: 225.1467,

Found: 225.1460); oil, IR νmax 3435 (OH), 2955, 2930, 2873, 1736 (C=O ester), 1436,

1381, 1258, 1168, 1002, 856 cm–1.

Mixture of 335 and 336: 1H NMR (400 MHz; CDCl3) δ 3.65 (s, 3H, COOMe), 3.50−3.38

(m, 2H, CH2OH, 2 x H-6), 2.12−2.06 (m, 1H, CHCOOMe, H-2), 1.84−1.70 (m, 2H, CH2

and OH), 1.64−1.51 (m, 2H), 1.41−1.24 (m, 2H), 1.10−1.01 (m, 1H), 0.92 (d, J = 6.8 Hz,

3H, CH3), 0.91 (d, J = 6.7 Hz, 3H, CH3), 0.89 (d, J = 6.7, 3H, CH3). 13C NMR (100 MHz; CDCl3) δ 176.6, 176.5 (C=O), 68.2, 67.9 (CH2OH, C-6), 53.1, 53.0

(CHCOO, C-2), 51.2, 51.1 (COOMe), 35.9, 35.8 (CH), 31.4, 31.1 (CH2), 30.8, 30.7 (CH) ,

27.1, 27.0 (CH2) , 20.7, 20.5 (CH3) , 20.3, 20.2 (CH3), 16.9, 16.3 (CH3).

Mixture of 335 and 336: HRMS (ES) m/z for C11H22O3Na, [MNa]+, (Calc.: 225.1467;

Found: 225.1459); oil, Anal. Calc. for C11H22O3, Expected C, 65.3; H, 11.0, Found C, 65.1;

H, 11.0; IR νmax 3409 (OH), 2956, 2930, 2874, 1734 (C=O ester), 1435, 1373, 1264, 1163,

1036, 864 cm–1.

Methyl 2-((1`S,1R,3R)-3-(1-hydroxyethyl)-2,2-dimethylcyclobutyl)acetate, 328, and

Methyl (S)-2-((1S,3S)-3-(hydroxymethyl)-2,2-dimethylcyclobutyl)propanoate, 329 (mixture)

(–)-Isopinocamphone 39 (1 g, 6.6 mmol) was dissolved in dry methanol (34 mL, 0.2 M),

and Oxone® (20 g, 5 eq.) was one portion added under N2 gas. The suspension was

warmed to 50 °C, and stirred for 24 h. The mixture was diluted with diethyl ether (50 mL),

and unreacted KHSO4 and KHSO5 were filtered out through a pad of Celite®. The crude

was concentrated, and purified using flash column chromatography (hex:EtOAc 5:1) to

furnish the product (175 mg, 12%, Rf 0.51 in hex:EtOAc 7:3, ratio 90:10 of 328:329) as a

white solid.

Compound (228) was assigned based on 1H, 13C, DEPT-135, 2D-COSY, 2D-HSQC, and

2D-HMBC NMR spectra.

(S)(S)

(S)(S) (R)(R)

O

39

HO

(S)(S) OMeO

(R)(R)

(R)(R) (S)(S)OH

(S)(S)

(S)(S)O

OMe1

2

3

4

328 329

+1`

7. Experimental

237

Compound (228): 1H NMR (400 MHz, CDCl3) δ 3.72−3.64 (m, 1H, CH3CHOH, H-1`),

3.62 (s, 3H, COOCH3, OMe), 3.40 (s, 1H, OH), 2.34−2.27 (m, 1H, CH2COOMe), 2.22−2.16

(m, 2H, CH2COOMe and CHCH2CH, H-3), 1.99−1.92 (m, 1H, CHCH2CH, H-4), 1.73 (td,

J = 10.2, 8.0 Hz, 1H, CHCH2CH, H-4), 1.25−1.13 (m, 1H, CHCH2CH, H-1), 1.10 (s, 3H,

CH3CCH3), 1.02 (d, J = 6.2 Hz, 3H, CH3CHOH), 0.99 (s, 3H, CH3CCH3). 13C NMR (100 MHz, CDCl3) δ 173.5 (C=O), 69.1 (CH3CHOH, C-1`), 51.3 (CH2CO2CH3,

OMe), 50.1 (CHCH2CH, C-1), 39.7 (CH3CCH3, C-2), 37.7 (CHCH2CH, C-3), 34.9

(CH2COOMe), 30.7 (CH3CCH3), 26.4 (CHCH2CH, C-4), 21.3 (CH3CHOH), 16.6 (CH3CCH3).

HRMS (ES) m/z for C11H21O3, [MH]+, (Calc.: 201.1491 Found: 201.1495); solid; IR νmax

3434 (OH), 2955, 2873, 1735 (C=O ester), 1460, 1366, 1258, 1161, 1098, 889 cm–1.

General enzymatic procedure for Baeyer−Villiger oxidation using CHMOs

The standard enzymatic reaction (1 mL) was achieved in buffer solution of Tric.HCl (50

mM, pH 7.5), comprises [substrate] = 5 µM, [NADP+] = 15 µM, GDH = 10 U, glucose or

glucose-6-phosphate 15 µM, [Enzyme] = 2 or 10 µM. The reaction mixture was shaken at

required temperature (25 or 30 or 37 °C) at 135 rpm for the desired time. The reaction was

then terminated by extraction into an organic layer with 800 µL of EtOAc:internal standard

(+)-limonene (99.5:0.5). The organic mixture was dried over (MgSO4), and analysed with

Varianchiralsil-DEX CB column to determine the enatiomeric excess.

Scale up of BV oxidation of (2R,3R,6R)-2,6-dimethyl-3-(prop-1-en-2-yl)cyclohexan-1-one 237

by CHMOs

(3R,6S,7R)-3,7-Dimethyl-4-(prop-1-en-2-yl)oxepan-2-one, 337

In conical flask contains buffer solution of Tris.HCl pH 7.5, 60 mL, a solution of 237 (50

mg dissolved in 1.2 mL absolute EtOH), NADP+ (0.9 mM), GDH (600 U), glucose (1.0

mM) and CHMOs (120 µM) were rapidly mixed. The enzymatic reaction mixture was

incubated for 24 h at 25 °C, and monitored via TLC plate. The organic layer was extracted

12

3

4

56

7

8

10

9

(S)(S)(R)(R)O(R)(R)

O

1211

O

237 337

7. Experimental

238

with EtOAc (3 x 50 mL), dried over (MgSO4), and concentrated to afford a lactone 337

with yield (49 mg, 90%) as a colorless oil.

Lactone (337): 1H NMR (400 MHz; CDCl3) δ 4.88 (quint, J = 1.5 Hz, 1H, C=CH2, H-11),

4.77−4.75 (m, 1H, C=CH2, H-11), 4.66 (qd, J = 7.2, 3.3 Hz, 1H, CHCHOCO, H-7), 2.81

(dqd, J = 10.6, 6.8, 5.0 Hz, 1H, CHCH2CH2, H-3), 2.40 (ddd, J = 8.7, 7.1, 3.3 Hz, 1H,

CHCHOCO, H-6), 1.92−1.80 (m, 3H, CHCH2CH2 and CHCH2CH2, 2 x H-4 and H-5), 1.77

(dd, J = 1.4, 0.8 Hz, 3H, CH3C=CH2, 3 x H-12), 1.47−1.39 (m, 1H, CHCH2CH2, H-5),

1.34 (d, J = 7.2 Hz, 3H, CH3CHOCO, 3 x H-9), 1.28 (d, J = 6.8 Hz, 3H, CH3CHCH2, 3 x

H-8). 13C NMR (100 MHz; CDCl3) δ 177.1 (C=O), 144.9 (C=CH2, C-10), 113.6 (C=CH2, C-11),

75.4 (CH2CHCH, C-7), 50.7 (CH2CHCH, C-6), 41.2 (CHCH2CH2, C-3), 30.2 (CHCH2CH2, C-

4), 27.4 (CHCH2CH2, C-5), 22.2 (CH3C=CH2, C-12), 19.0 (CH3CHCH2, C-7), 16.1

(CH3CH, C-9).


2972, 2934, 2874, 1717 (C=O ester), 1644, 1456, 1378, 1261, 1208, 1095 cm–1; [α]D +50º

(c 0.04, CH2Cl2, 24 ºC), Retention time 13.868 min.

Scale up of BV oxidation of two isomers mixture of 3-methyldihydrocarvone 262 and

263 by CHMOs

(3R,4R,6S)-3,4-Dimethyl-6-(prop-1-en-2-yl)oxepan-2-one, 338, and (3S,4R,6S)-3,4-Dimethyl-

6-(prop-1-en-2-yl)oxepan-2-one, 339 (mixture of normal and abnormal lactones)

In conical flask contains buffer solution of Tris.HCl pH 7.5, 70 mL, a solution of 262 and

263 (63 mg dissolved in 1.5 mL absolute EtOH), NADP+ (1.2 mM), GDH (750 U), glucose

(1.2 mM) and CHMOs (150 µM) were rapidly mixed. The enzymatic reaction mixture was

incubated for 24 h at 25 °C, and monitored via TLC plate. The organic layer was extracted

with EtOAc (3 x 50 mL), dried over (MgSO4), and concentrated to afford a lactone 338 as

a colorless volatile oil (62 mg, 90%).

1

23

4

56

7

8

9

10

(S)(S)(R)(R)(S)(S)

O (R)(R)(R)(R)(S)(S)

O

1211

O OO O

262 263 338 339

7. Experimental

239

Abnormal lactone (338): 1H NMR (400 MHz; CDCl3) δ 4.83−4.81 (m, 2H, C=CH2, 2 x

H-12), 4.15−4.12 (m, 2H, CH2-O, 2 x H-7), 2.60 (dq, J = 10.1, 6.8 Hz, 1H, CH=O, H-3),

2.35 (ddt, J = 12.0, 8.8, 3.1 Hz, 1H, CHCH2-O, H-6), 1.89−1.85 (m 1H, CH2CHCH, H-5),

1.76−1.74 (m, 3H, CH3C=CH2, 3 x H-12), 1.71−1.64 (m, 1H, CHCHC=O, H-4), 1.48−1.46

(m, 1H, CHCHCH2, H-5), 1.17 (d, J = 6.8 Hz, 3H, CH3CHC=O, 3 x H-8), 1.02 (d, J = 6.8

Hz, 3H, CH3CHCHC=O, 3 x H-9). 13C NMR (100 MHz; CDCl3) δ 178.0 (C=O), 145.9 (C=CH2, C-10), 111.2 (C=CH2, C-11),

71.9 (CH2-O, C-7), 46.4 (CHCH2-O, C-6), 44.3 (CHCHCH2, C-5), 41.5 (CHCHC=O, C-3),

40.9 (CHCHC=O, C-4), 22.0 (CH3C=CH2, C-12), 16.7 (CH3CHCH, C-8), 13.7 (CH3CH,

C-9).

Normal lactone (339): 1H NMR (400 MHz; CDCl3) δ 4.71−4.69 (m, 2H, C=CH2, H-11),

4.20− 4.16 (m, 2H, CH2-O, 2 x H-7), 3.92−3.86 (m, 1H, CHCHCH2, H-4 ), 3.02 (qd, J =

6.8, 1.5 Hz, 1H, CHCHC=O, H-3), 2.49 (dq, J = 10.3, 5.4 Hz, 1H, CHCH2-O, H-6),

2.00−1.93 (m, 2H, CH2CHCH, 2 x H-5), 1.76−1.74 (m, 3H, CH3C=CH2, 3 x H-12), 1.21 (d, J =

6.8 Hz, 3H, CH3CHC=O, 3 x H-8), 0.96 (d, J = 7.2 Hz, 3H, CH3CHCHC=O, 3 x H-9). 13C NMR (100 MHz; CDCl3) δ 176.4 (C=O), 145.7 (C=CH2, C-10), 111.4 (C, C=CH2, C-11),

71.2 (CHCH2-O, C-7), 44.3 (CHCH2-O, C-6), 41.6 (CHCHCH2, C-3), 41.3 (CH2CHCH, C-

5), 34.1 (CHCHCH2, C-4), 21.8 (CH3C=CH2, C-12), 20.6 (CH3CHCH, C-8), 15.3

(CH3CH, C-9).

(338 and 339)-Lactones mixture: HRMS (ES) m/z for C11H19O2, [MH]+, (Calc.:

183.1385, Found: 183.1384); oil; IR νmax 2970, 2933, 2887, 1715 (C=O ester), 1640, 1458,

1391, 1278, 1234, 1111 cm–1. Retention time 15.942 and 16.165 min of 338 and 339

respectively.

Scope KT-to go plate (ketoreductase) against substrates

The target alcohol substrates (0.05 mL of (−)-carveol mixture, 0.05 g of (+)-isomenthol

and isopinocampheol) were to the INT solution and stir until fully dissolved. Carefully, the

INT/alcohol solution (0.10 mL) was added to each enzyme on microplate using multi-

channel pipette and mixed carefully to reconstitute the freeze-dried enzymes. The

microplates were incubated for 3 h at ambient temperature in the dark place. The color of

few enzymes was changed to red indicated activity of enzyme towards substrates. The UV

absorption was measured at 493 nm.

7. Experimental

240

General enzymatic procedure for bioreduction of carbonyl by keto-reductases (Pro-

AKR 122, 146 and 170)

The standard enzymatic reaction (1 mL) was achieved in buffer solution of KH2PO4/K2HPO4

(50 mM, pH 7.0), comprises [substrate] = 5 µM, [NADP+] or [NAD+] = 15 µM, GDH = 10 U

and/or Isopropyl alcohol (3%), glucose or G-6-P 15 µM, [Enzyme] = 10 mg. The reaction

mixture was shaken at 30 °C at 180 rpm for 24 h. The reaction was then terminated with

extraction organic layer with 800 µL of EtOAc. The organic mixture was dried over

(MgSO4), and analysed with GC using achiral HP−5 column to determine the yield,

conversion and enatiomeric excess.

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162 Pinheiro, L.; Marsaioli, A. J. J. M. Catal. B: Enzym. 2007, 44,78–86.

163 Rial, D. V.; Cernuchova, P.; Van-Beilen, J. B.; Mihovilovic, M. D. J. M. Catal. B: Enzym.

2008, 50, 61–68.

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M. Catal. B: Enzym. 2011, 73, 9–16.

165 Alphand, V.; Furstoss, R. Tetrahedron: Asymmetry 1992, 3, 379–382.

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2015, 13, 223–233.

174 Camps, F.; Coll, J.; Guitart, J. Tetrahedron 1986, 42, 4603−4609.

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9.Appendix

249

9. Appendix

9.1 UV absorbance of monoterpenoids alcohol with Ketoreductase-to go enzymes plates at

492 nm.

Table 9.1 UV Absorbance of isomeric mixture of (–)-carveol with KT-to go plate at 492 nm.

Table 9.2 UV Absorbance of (+)-isomenthol with K-to go plate at 492 nm.

Table 9.3 UV Absorbance of (+)-isopinocampheol with KT-to go plate at 492 nm.

<> 1 2 3 4 5 6 7 8 9 10 11 12A 0.0684 0.2359 0.0908 0.1045 0.097 0.0909 0.112 0.0724 0.0723 0.0758 0.0574 0.091B 0.0754 0.4717 1.1643 0.468 0.2531 0.4669 0.1016 0.0727 0.0904 0.9076 0.0705 0.0871C 0.0751 1.2411 0.0606 0.2221 0.0865 0.0772 0.1138 0.0447 0.0885 0.1234 0.1127 0.0977D 0.0954 0.1056 0.1517 0.2202 0.4607 0.1727 0.0691 0.0864 0.1434 0.1161 1.1817 0.1547E 0.0826 1.7669 0.1082 0.176 0.7314 0.513 0.6539 0.0468 0.0675 0.1156 0.0895 0.1026F 0.0635 0.0853 0.0897 0.1475 0.081 0.081 0.2755 0.4274 0.0832 1.3045 1.4718 0.0779G 0.3245 1.4459 0.0836 0.0644 0.0819 0.0881 0.0688 0.0779 0.098 1.8344 0.1128 0.1127H 0.0887 0.0961 0.1491 0.0607 0.08 0.1471 1.5613 0.1069 0.1968 0.2664 0.1365 0.1225

<> 1 2 3 4 5 6 7 8 9 10 11 12A 0.0997 0.0763 0.0794 0.0625 0.1083 0.0784 0.092 0.0803 0.043 0.0725 0.071 0.0776B 0.1081 0.2263 0.1374 0.0832 0.0871 0.0931 0.097 0.1218 0.0814 0.0858 0.0931 0.0647C 0.1443 0.6675 0.0779 0.0721 0.0638 0.1029 0.0633 0.117 0.1379 0.1878 0.1097 0.1123D 0.0968 0.0547 0.0951 0.18 0.165 0.121 0.0446 0.0528 0.1156 0.0603 0.2643 0.1166E 0.0906 0.1931 0.1932 0.2251 0.205 0.6651 0.1614 0.067 0.0808 0.1109 0.1151 0.0981F 0.086 0.1504 0.1938 0.1261 0.1188 0.1731 0.2514 0.0838 0.0693 0.1679 0.1158 0.0752G 0.0985 0.0958 0.0503 0.0907 0.0734 0.1076 0.1028 0.083 0.094 0.1127 0.102 0.1433H 0.0666 0.1538 0.1045 0.0882 0.0812 0.1434 0.2774 0.0874 0.1096 0.1185 0.0911 0.1867

<> 1 2 3 4 5 6 7 8 9 10 11 12A 0.2617 0.1214 0.099 0.1262 0.0847 0.1031 0.1061 0.109 0.08 0.0769 0.0839 0.1114B 0.1016 1.0821 0.9524 0.1197 0.1556 0.5853 0.1082 0.1861 0.0847 1.2117 0.3019 0.1C 0.1278 0.3252 0.0794 0.104 0.0823 0.0474 0.2015 0.1257 0.1336 0.2496 0.0786 0.1374D 0.0966 0.1397 0.1151 0.2232 0.2626 0.1341 0.1135 0.0767 0.1589 0.0921 1.0711 0.1172E 0.0984 0.2624 0.1434 0.1746 0.5502 0.649 0.1586 0.0778 0.0755 0.1059 0.6324 0.0927F 0.0969 0.095 0.2087 0.1636 0.1259 0.1746 0.3309 0.1899 0.075 0.2546 0.1547 0.0933G 0.0911 1.1151 0.0958 0.0794 0.154 0.1042 0.0609 0.0721 0.0832 0.2213 0.1054 0.2157H 0.1068 0.1078 0.1314 0.0816 0.0737 0.2302 0.5663 0.103 0.0931 0.138 0.1485 0.1631

9.Appendix

250

9.2 The Conformational parameters from simulated annealing of (5R,6R) and

(5R,6S) –methylcarvone diastereomers in PETNR

Figure 9.1 Conformational parameters from simulated annealing of 6 R-Me carvone and 6

S-Me carvone in PETNR. (A) Distribution of C1-N5 distances for 6-R (blue) and 6-S (red)

Me carvone; (B) definition of dihedral angle θ which describes the orientation of the

substrate relative to the FMN (the angle of the 6-R/S Me bond relative to the plane formed

by N5 and the atoms labelled 1 and 2); (C) distribution of θ for 6-R (blue) and 6-S (red) Me

carvone, for conformations with r(C1-N5) < 5 Å to ensure that only structures consistent

with nucleophilic attack on C1 by N5 were included. This leads to 73 % and 74 % of

structures consistent with the observed stereoselectivities for R- and S-Me carvone,

respectively. Including all structures changes these values to 68 % and 69 %.

9.Appendix

251

9.3 Crystal data and Structure Refinement for 44

Table 1. Crystal data and structure refinement for s4180ma

Identification code s4180ma

Empirical formula C11H16O

Formula weight 164.24

Temperature 100(2) K

Wavelength 1.54178 A

Crystal system, space group Orthorhombic,P2(1)2(1)2(1)

Unit cell dimensions a = 7.0550(3) A alpha = 90 deg.

b = 7.4694(2) A beta = 90 deg.

c = 18.8013(5) A gamma = 90 deg.

Volume 990.76(6) A^3

Z, Calculated density 4, 1.101 Mg/m^3

Absorption coefficient 0.526 mm^-1

F(000) 360

(R)(R)(S)(S)

(Z) O

9.Appendix

252

Crystal size 0.13 x 0.11 x 0.04 mm

Theta range for data collection 4.70 to 72.16 deg.

Limiting indices -8<=h<=7, -8<=k<=9, -20<=l<=23

Reflections collected / unique 5067 / 1922 [R(int) = 0.0298]

Completeness to theta = 67.00 99.7 %

Absorption correction Semi-empirical from equivalents

Max. and min. transmission 0.9793 and 0.825569

Refinement method Full-matrix least-squares on F^2

Data / restraints / parameters 1922 / 0 / 112

Goodness-of-fit on F^2 1.071

Final R indices [I>2sigma(I)] R1 = 0.0394, wR2 = 0.1022

R indices (all data) R1 = 0.0428, wR2 = 0.1044

Absolute structure parameter 0.0(3)

Largest diff. peak and hole 0.226 and -0.161 e.A^-3

Table 2. Atomic coordinates ( x 10^4) and equivalent isotropic displacement

parameters (A^2 x 10^3) for s4180ma.

x y z U(eq

C(1) 2488(3) 2210(2) 3297(1) 25(1)

C(2) 864(2) 2051(2) 2798(1) 23(1)

C(3) 1323(2) 1738(2) 2018(1) 22(1)

C(4) 2997(2) 2969(2) 1808(1) 20(1)

C(5) 4724(2) 2485(2) 2264(1) 22(1)

C(6) 4246(2) 2381(2) 3040(1) 24(1)

C(7) 2041(3) 2165(2) 4080(1) 32(1)

C(8) -417(2) 1968(2) 1550(1) 28(1)

9.Appendix

253

C(9) 3429(2) 2902(2) 1016(1) 26(1)

C(10) 4079(3) 1138(3) 710(1) 37(1)

C(11) 3204(3) 4343(3) 617(1) 34(1)

O(1) -766(2) 2131(2) 3016(1) 30(1)

U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.

Table 3. Bond lengths [A] and angles [deg] for s4180ma. C(1)-C(6) 1.337(2)

C(1)-C(2) 1.486(2)

C(1)-C(7) 1.506(2)

C(2)-O(1) 1.222(2)

C(2)-C(3) 1.521(2)

C(3)-C(8) 1.520(2)

C(3)-C(4) 1.5482(19)

C(3)-H(3) 1.0000

C(4)-C(9) 1.5214(19)

C(4)-C(5) 1.533(2)

C(4)-H(4) 1.0000

C(5)-C(6) 1.500(2)

C(5)-H(5A) 0.9900

C(5)-H(5B) 0.9900

C(6)-H(6) 0.9500

C(7)-H(7A) 0.9800

C(7)-H(7B) 0.9800

C(7)-H(7C) 0.9800

C(8)-H(8A) 0.9800

C(8)-H(8B) 0.9800

9.Appendix

254

C(8)-H(8C) 0.9800

C(9)-C(11) 1.322(3)

C(9)-C(10) 1.509(2)

C(10)-H(10A) 0.9800

C(10)-H(10B) 0.9800

C(10)-H(10C) 0.9800

C(11)-H(11A) 0.9500

C(11)-H(11B) 0.9500

C(6)-C(1)-C(2) 119.69(14)

C(6)-C(1)-C(7) 123.35(15)

C(2)-C(1)-C(7) 116.96(15)

O(1)-C(2)-C(1) 120.74(15)

O(1)-C(2)-C(3) 122.04(15)

C(1)-C(2)-C(3) 117.20(14)

C(8)-C(3)-C(2) 111.67(13)

C(8)-C(3)-C(4) 113.70(12)

C(2)-C(3)-C(4) 108.45(12)

C(8)-C(3)-H(3) 107.6

C(2)-C(3)-H(3) 107.6

C(4)-C(3)-H(3) 107.6

C(9)-C(4)-C(5) 112.36(13)

C(9)-C(4)-C(3) 112.45(13)

C(5)-C(4)-C(3) 108.91(12)

C(9)-C(4)-H(4) 107.6

9.Appendix

255

C(5)-C(4)-H(4) 107.6

C(3)-C(4)-H(4) 107.6

C(6)-C(5)-C(4) 112.17(13)

C(6)-C(5)-H(5A) 109.2

C(4)-C(5)-H(5A) 109.2

C(6)-C(5)-H(5B) 109.2

C(4)-C(5)-H(5B) 109.2

H(5A)-C(5)-H(5B) 107.9

C(1)-C(6)-C(5) 124.40(14)

C(1)-C(6)-H(6) 117.8

C(5)-C(6)-H(6) 117.8

C(1)-C(7)-H(7A) 109.5

C(1)-C(7)-H(7B) 109.5

H(7A)-C(7)-H(7B) 109.5

C(1)-C(7)-H(7C) 109.5

H(7A)-C(7)-H(7C) 109.5

H(7B)-C(7)-H(7C) 109.5

C(3)-C(8)-H(8A) 109.5

C(3)-C(8)-H(8B) 109.5

H(8A)-C(8)-H(8B) 109.5

C(3)-C(8)-H(8C) 109.5

H(8A)-C(8)-H(8C) 109.5

H(8B)-C(8)-H(8C) 109.5

C(11)-C(9)-C(10) 122.10(14)

C(11)-C(9)-C(4) 120.32(15)

C(10)-C(9)-C(4) 117.57(14)

9.Appendix

256

C(9)-C(10)-H(10A) 109.5

C(9)-C(10)-H(10B) 109.5

H(10A)-C(10)-H(10B) 109.5

C(9)-C(10)-H(10C) 109.5

H(10A)-C(10)-H(10C) 109.5

H(10B)-C(10)-H(10C) 109.5

C(9)-C(11)-H(11A) 120.0

C(9)-C(11)-H(11B) 120.0

H(11A)-C(11)-H(11B) 120.0

Symmetry transformations used to generate equivalent atoms:

Table 4. Anisotropic displacement parameters (A^2 x 10^3) for s4180ma. The anisotropic displacement factor exponent takes the form:-2 pi^2 [ h^2 a*^2 U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 U23 U13 U12 C(1) 35(1) 16(1) 23(1) 0(1) 2(1) 0(1)

C(2) 26(1) 15(1) 28(1) 2(1) 4(1) 0(1)

C(3) 22(1) 18(1) 25(1) -1(1) 1(1) -1(1)

C(4) 22(1) 18(1) 22(1) 0(1) 1(1) 0(1)

C(5) 20(1) 21(1) 26(1) 0(1) 1(1) 0(1)

C(6) 27(1) 20(1) 24(1) -1(1) -7(1) 2(1)

C(7) 44(1) 30(1) 24(1) 1(1) 3(1) -2(1)

C(8) 25(1) 30(1) 30(1) -1(1) -1(1) -3(1)

C(9) 22(1) 31(1) 24(1) -2(1) 0(1) -4(1)

C(10) 46(1) 38(1) 27(1) -5(1) 5(1) 6(1)

C(11) 43(1) 35(1) 23(1) 2(1) 1(1) -6(1)

O(1) 28(1) 28(1) 34(1) 2(1) 10(1) -2(1)

Table 5. Hydrogen coordinates ( x 10^4) and isotropic displacement parameters (A^2 x 10^3) for s4180ma. x y z U(eq)

9.Appendix

257

H(3) 1761 471 1967 26

H(4) 2629 4227 1928 25

H(5A) 5232 1315 2106 27

H(5B) 5725 3396 2192 27

H(6) 5260 2441 3371 28

H(7A) 3218 2268 4353 49

H(7B) 1414 1031 4196 49

H(7C) 1199 3164 4199 49

H(8A) -1416 1149 1709 43

H(8B) -86 1700 1055 43

H(8C) -872 3205 1584 43

H(10A) 4242 1257 195 56

H(10B) 3128 215 810 56

H(10C) 5289 797 927 56

H(11A) 3453 4288 121 41

H(11B) 2792 5433 826 41

Table 6. Torsion angles [deg] for s4180ma C(6)-C(1)-C(2)-O(1) -170.28(15)

C(7)-C(1)-C(2)-O(1) 9.8(2)

C(6)-C(1)-C(2)-C(3) 11.3(2)

C(7)-C(1)-C(2)-C(3) -168.62(14)

O(1)-C(2)-C(3)-C(8) 12.9(2)

C(1)-C(2)-C(3)-C(8) -168.68(12)

O(1)-C(2)-C(3)-C(4) 138.93(15)

C(1)-C(2)-C(3)-C(4) -42.63(17)

C(8)-C(3)-C(4)-C(9) -49.03(17)

C(2)-C(3)-C(4)-C(9) -173.89(13)

9.Appendix

258

C(8)-C(3)-C(4)-C(5) -174.25(12)

C(2)-C(3)-C(4)-C(5) 60.89(15)

C(9)-C(4)-C(5)-C(6) -174.52(13)

C(3)-C(4)-C(5)-C(6) -49.25(15)

C(2)-C(1)-C(6)-C(5) 2.0(2)

C(7)-C(1)-C(6)-C(5) -178.11(14)

C(4)-C(5)-C(6)-C(1) 18.2(2)

C(5)-C(4)-C(9)-C(11) -121.18(18)

C(3)-C(4)-C(9)-C(11) 115.51(19)

C(5)-C(4)-C(9)-C(10) 59.9(2)

C(3)-C(4)-C(9)-C(10) -63.4(2)


Table 7. Hydrogen bonds for s4180ma [A and deg.]. D-H...Ad(D-H) d(H...A) d(D...A) <(DHA)

9.Appendix

259


Table 1. Crystal data and structure refinement for s3868ma. Identification code s3868ma Empirical formula C16H19 NO2 Formula weight 257.32 Temperature 150(2) K Wavelength 1.54178 Å Crystal system Monoclinic Space group P2(1) Unit cell dimensions a = 9.1108(2) Å a= 90°. b = 8.4062(2) Å b= 99.667(2)°. c = 18.3436(4) Å g = 90°. Volume 1384.94(5) Å3 Z 4 Density (calculated) 1.234 Mg/m3 Absorption coefficient 0.645 mm-1 F(000) 552 Crystal size 0.19 x 0.17 x 0.02 mm3 Theta range for data collection 2.44 to 70.14°. Index ranges -10<=h<=11, -10<=k<=8, -22<=l<=20 Reflections collected 6468 Independent reflections 4067 [R(int) = 0.0404] Completeness to theta = 66.60° 98.5 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.9872 and 0.834789 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 4067 / 1 / 349

O

OHN

H

9.Appendix

260

Goodness-of-fit on F2 1.042 Final R indices [I>2sigma(I)] R1 = 0.0369, wR2 = 0.0850 R indices (all data) R1 = 0.0461, wR2 = 0.0890 Absolute structure parameter -0.2(2) Largest diff. peak and hole 0.196 and -0.168 e. Å-3

Table 2. Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103) for s3868ma. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x y z U(eq) C(1) 5678(2) 4335(3) 2288(1) 16(1) C(2) 4769(2) 5838(3) 2392(1) 16(1) C(3) 4136(2) 5690(3) 3112(1) 18(1) C(4) 2863(2) 6713(3) 3209(1) 20(1) C(5) 2275(2) 7697(3) 2670(1) 22(1) C(6) 2778(2) 7805(3) 1937(1) 21(1) C(7) 3509(2) 6248(3) 1743(1) 17(1) C(8) 2318(2) 6613(3) 3938(1) 25(1) C(9) 3946(2) 6358(3) 982(1) 18(1) C(10) 3298(2) 5389(3) 440(1) 25(1) C(11) 5013(2) 7625(3) 844(1) 24(1) C(12) 6809(2) 4614(3) 1775(1) 17(1) N(1) 7868(2) 5698(2) 2010(1) 18(1) C(13) 8886(2) 5997(3) 1574(1) 22(1) C(14) 8896(2) 5247(3) 905(1) 23(1) C(15) 7819(2) 4126(3) 673(1) 23(1) C(16) 6759(2) 3788(3) 1117(1) 19(1) C(17) 7623(2) -619(3) 2828(1) 17(1) C(18) 8469(2) 889(3) 2655(1) 16(1) C(19) 8966(2) 649(3) 1901(1) 20(1) C(20) 10310(2) 1488(3) 1747(1) 23(1) C(21) 11039(2) 2480(3) 2248(1) 25(1) C(22) 10596(2) 2798(3) 2982(1) 21(1) C(23) 9792(2) 1361(3) 3256(1) 16(1) C(24) 10735(3) 1234(3) 995(1) 34(1) C(25) 9422(2) 1721(3) 4015(1) 19(1) C(26) 10166(3) 959(4) 4602(1) 32(1) C(27) 8304(3) 2979(3) 4089(1) 25(1)

9.Appendix

261

C(28) 6577(2) -355(3) 3376(1) 18(1) N(2) 5448(2) 662(2) 3156(1) 22(1) C(29) 4475(2) 925(3) 3619(1) 28(1) C(30) 4588(3) 222(3) 4304(1) 28(1) C(31) 5740(3) -830(3) 4526(1) 27(1) C(32) 6740(2) -1135(3) 4052(1) 20(1) O(1) 4703(2) 3060(2) 2043(1) 20(1) O(2) 4678(2) 4780(2) 3600(1) 26(1) O(3) 8666(2) -1852(2) 3064(1) 19(1) O(4) 8250(2) -190(2) 1431(1) 31(1) Table 3. Bond lengths [Å] and angles [°] for s3868ma. C(1)-O(1) 1.417(3) C(1)-C(12) 1.525(3) C(1)-C(2) 1.540(3) C(1)-H(1) 1.0000 C(2)-C(3) 1.534(3) C(2)-C(7) 1.547(3) C(2)-H(2) 1.0000 C(3)-O(2) 1.216(3) C(3)-C(4) 1.478(3) C(4)-C(5) 1.331(3) C(4)-C(8) 1.505(3) C(5)-C(6) 1.494(3) C(5)-H(5) 0.9500 C(6)-C(7) 1.536(3) C(6)-H(6A) 0.9900 C(6)-H(6B) 0.9900 C(7)-C(9) 1.518(3) C(7)-H(7) 1.0000 C(8)-H(8A) 0.9800 C(8)-H(8B) 0.9800 C(8)-H(8C) 0.9800 C(9)-C(10) 1.343(3) C(9)-C(11) 1.492(3) C(10)-H(10A) 0.9500 C(10)-H(10B) 0.9500 C(11)-H(11A) 0.9800 C(11)-H(11B) 0.9800

9.Appendix

262

C(11)-H(11C) 0.9800 C(12)-N(1) 1.345(3) C(12)-C(16) 1.387(3) N(1)-C(13) 1.346(3) C(13)-C(14) 1.381(3) C(13)-H(13) 0.9500 C(14)-C(15) 1.376(3) C(14)-H(14) 0.9500 C(15)-C(16) 1.393(3) C(15)-H(15) 0.9500 C(16)-H(16) 0.9500 C(17)-O(3) 1.424(2) C(17)-C(28) 1.514(3) C(17)-C(18) 1.544(3) C(17)-H(17) 1.0000 C(18)-C(19) 1.540(3) C(18)-C(23) 1.543(3) C(18)-H(18) 1.0000 C(19)-O(4) 1.215(3) C(19)-C(20) 1.481(3) C(20)-C(21) 1.333(3) C(20)-C(24) 1.509(3) C(21)-C(22) 1.492(3) C(21)-H(21) 0.9500 C(22)-C(23) 1.540(3) C(22)-H(22A) 0.9900 C(22)-H(22B) 0.9900 C(23)-C(25) 1.517(3) C(23)-H(23) 1.0000 C(24)-H(24A) 0.9800 C(24)-H(24B) 0.9800 C(24)-H(24C) 0.9800 C(25)-C(26) 1.337(3) C(25)-C(27) 1.490(3) C(26)-H(26A) 0.9500 C(26)-H(26B) 0.9500 C(27)-H(27A) 0.9800 C(27)-H(27B) 0.9800 C(27)-H(27C) 0.9800

9.Appendix

263

C(28)-N(2) 1.347(3) C(28)-C(32) 1.388(3) N(2)-C(29) 1.345(3) C(29)-C(30) 1.376(4) C(29)-H(29) 0.9500 C(30)-C(31) 1.381(3) C(30)-H(30) 0.9500 C(31)-C(32) 1.384(3) C(31)-H(31) 0.9500 C(32)-H(32) 0.9500 O(1)-H(1A) 0.8400 O(3)-H(3) 0.8400 O(1)-C(1)-C(12) 111.91(17) O(1)-C(1)-C(2) 109.76(15) C(12)-C(1)-C(2) 112.52(18) O(1)-C(1)-H(1) 107.5 C(12)-C(1)-H(1) 107.5 C(2)-C(1)-H(1) 107.5 C(3)-C(2)-C(1) 109.11(17) C(3)-C(2)-C(7) 110.02(16) C(1)-C(2)-C(7) 115.70(17) C(3)-C(2)-H(2) 107.2 C(1)-C(2)-H(2) 107.2 C(7)-C(2)-H(2) 107.2 O(2)-C(3)-C(4) 120.51(19) O(2)-C(3)-C(2) 121.32(19) C(4)-C(3)-C(2) 118.16(18) C(5)-C(4)-C(3) 119.9(2) C(5)-C(4)-C(8) 122.9(2) C(3)-C(4)-C(8) 117.14(19) C(4)-C(5)-C(6) 124.1(2) C(4)-C(5)-H(5) 118.0 C(6)-C(5)-H(5) 118.0 C(5)-C(6)-C(7) 111.51(18) C(5)-C(6)-H(6A) 109.3 C(7)-C(6)-H(6A) 109.3 C(5)-C(6)-H(6B) 109.3 C(7)-C(6)-H(6B) 109.3

9.Appendix

264

H(6A)-C(6)-H(6B) 108.0 C(9)-C(7)-C(6) 110.57(18) C(9)-C(7)-C(2) 116.41(16) C(6)-C(7)-C(2) 107.88(17) C(9)-C(7)-H(7) 107.2 C(6)-C(7)-H(7) 107.2 C(2)-C(7)-H(7) 107.2 C(4)-C(8)-H(8A) 109.5 C(4)-C(8)-H(8B) 109.5 H(8A)-C(8)-H(8B) 109.5 C(4)-C(8)-H(8C) 109.5 H(8A)-C(8)-H(8C) 109.5 H(8B)-C(8)-H(8C) 109.5 C(10)-C(9)-C(11) 121.7(2) C(10)-C(9)-C(7) 119.6(2) C(11)-C(9)-C(7) 118.64(19) C(9)-C(10)-H(10A) 120.0 C(9)-C(10)-H(10B) 120.0 H(10A)-C(10)-H(10B) 120.0 C(9)-C(11)-H(11A) 109.5 C(9)-C(11)-H(11B) 109.5 H(11A)-C(11)-H(11B) 109.5 C(9)-C(11)-H(11C) 109.5 H(11A)-C(11)-H(11C) 109.5 H(11B)-C(11)-H(11C) 109.5 N(1)-C(12)-C(16) 122.37(19) N(1)-C(12)-C(1) 115.38(18) C(16)-C(12)-C(1) 122.25(19) C(12)-N(1)-C(13) 117.72(18) N(1)-C(13)-C(14) 123.4(2) N(1)-C(13)-H(13) 118.3 C(14)-C(13)-H(13) 118.3 C(15)-C(14)-C(13) 118.5(2) C(15)-C(14)-H(14) 120.7 C(13)-C(14)-H(14) 120.7 C(14)-C(15)-C(16) 119.2(2) C(14)-C(15)-H(15) 120.4 C(16)-C(15)-H(15) 120.4 C(12)-C(16)-C(15) 118.8(2)

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265

C(12)-C(16)-H(16) 120.6 C(15)-C(16)-H(16) 120.6 O(3)-C(17)-C(28) 111.52(17) O(3)-C(17)-C(18) 109.24(16) C(28)-C(17)-C(18) 114.06(17) O(3)-C(17)-H(17) 107.2 C(28)-C(17)-H(17) 107.2 C(18)-C(17)-H(17) 107.2 C(19)-C(18)-C(23) 111.28(16) C(19)-C(18)-C(17) 108.00(17) C(23)-C(18)-C(17) 114.96(17) C(19)-C(18)-H(18) 107.4 C(23)-C(18)-H(18) 107.4 C(17)-C(18)-H(18) 107.4 O(4)-C(19)-C(20) 120.3(2) O(4)-C(19)-C(18) 120.6(2) C(20)-C(19)-C(18) 119.08(18) C(21)-C(20)-C(19) 119.9(2) C(21)-C(20)-C(24) 123.0(2) C(19)-C(20)-C(24) 117.0(2) C(20)-C(21)-C(22) 123.7(2) C(20)-C(21)-H(21) 118.1 C(22)-C(21)-H(21) 118.1 C(21)-C(22)-C(23) 111.68(19) C(21)-C(22)-H(22A) 109.3 C(23)-C(22)-H(22A) 109.3 C(21)-C(22)-H(22B) 109.3 C(23)-C(22)-H(22B) 109.3 H(22A)-C(22)-H(22B) 107.9 C(25)-C(23)-C(22) 109.57(17) C(25)-C(23)-C(18) 116.04(16) C(22)-C(23)-C(18) 109.05(17) C(25)-C(23)-H(23) 107.3 C(22)-C(23)-H(23) 107.3 C(18)-C(23)-H(23) 107.3 C(20)-C(24)-H(24A) 109.5 C(20)-C(24)-H(24B) 109.5 H(24A)-C(24)-H(24B) 109.5 C(20)-C(24)-H(24C) 109.5

9.Appendix

266

H(24A)-C(24)-H(24C) 109.5 H(24B)-C(24)-H(24C) 109.5 C(26)-C(25)-C(27) 121.9(2) C(26)-C(25)-C(23) 118.9(2) C(27)-C(25)-C(23) 119.06(18) C(25)-C(26)-H(26A) 120.0 C(25)-C(26)-H(26B) 120.0 H(26A)-C(26)-H(26B) 120.0 C(25)-C(27)-H(27A) 109.5 C(25)-C(27)-H(27B) 109.5 H(27A)-C(27)-H(27B) 109.5 C(25)-C(27)-H(27C) 109.5 H(27A)-C(27)-H(27C) 109.5 H(27B)-C(27)-H(27C) 109.5 N(2)-C(28)-C(32) 122.0(2) N(2)-C(28)-C(17) 115.35(18) C(32)-C(28)-C(17) 122.66(19) C(29)-N(2)-C(28) 117.8(2) N(2)-C(29)-C(30) 123.3(2) N(2)-C(29)-H(29) 118.3 C(30)-C(29)-H(29) 118.3 C(29)-C(30)-C(31) 118.7(2) C(29)-C(30)-H(30) 120.7 C(31)-C(30)-H(30) 120.7 C(30)-C(31)-C(32) 118.8(2) C(30)-C(31)-H(31) 120.6 C(32)-C(31)-H(31) 120.6 C(31)-C(32)-C(28) 119.3(2) C(31)-C(32)-H(32) 120.3 C(28)-C(32)-H(32) 120.3 C(1)-O(1)-H(1A) 109.5 C(17)-O(3)-H(3) 109.5

9.Appendix

267

Symmetry transformations used to generate equivalent atoms: Table 4. Anisotropic displacement parameters (Å2x 103) for s3868ma. The anisotropic displacement factor exponent takes the form: -2p2[ h2 a*2U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 U23 U13 U12 C(1) 14(1) 14(1) 21(1) -1(1) 1(1) 1(1) C(2) 14(1) 16(1) 18(1) 0(1) 4(1) 0(1) C(3) 17(1) 18(1) 20(1) -3(1) 4(1) 0(1) C(4) 18(1) 19(1) 23(1) -4(1) 4(1) 0(1) C(5) 18(1) 21(1) 30(1) -4(1) 7(1) 4(1) C(6) 18(1) 17(1) 27(1) 3(1) 2(1) 4(1) C(7) 14(1) 16(1) 20(1) 1(1) 3(1) 1(1) C(8) 25(1) 26(1) 26(1) -6(1) 9(1) 2(1) C(9) 16(1) 19(1) 19(1) 4(1) 3(1) 6(1) C(10) 24(1) 30(1) 20(1) 3(1) 4(1) 4(1) C(11) 24(1) 28(1) 21(1) 7(1) 4(1) 0(1) C(12) 14(1) 16(1) 20(1) 2(1) 1(1) 4(1) N(1) 13(1) 18(1) 24(1) -2(1) 4(1) 1(1) C(13) 16(1) 19(1) 32(1) -1(1) 6(1) -2(1) C(14) 19(1) 28(1) 24(1) 3(1) 8(1) 4(1) C(15) 22(1) 28(1) 20(1) -1(1) 4(1) 4(1) C(16) 15(1) 20(1) 22(1) -1(1) 0(1) 1(1) C(17) 16(1) 14(1) 20(1) 0(1) 1(1) 1(1) C(18) 15(1) 15(1) 19(1) 2(1) 4(1) 0(1) C(19) 24(1) 16(1) 18(1) 2(1) 3(1) 1(1) C(20) 23(1) 24(1) 24(1) 8(1) 9(1) 8(1) C(21) 18(1) 24(1) 34(1) 10(1) 8(1) 0(1) C(22) 17(1) 16(1) 30(1) 3(1) 3(1) -2(1) C(23) 14(1) 15(1) 20(1) -2(1) 0(1) 1(1) C(24) 39(1) 34(2) 33(1) 8(1) 20(1) 14(1) C(25) 16(1) 19(1) 23(1) -4(1) 2(1) -4(1) C(26) 28(1) 45(2) 23(1) -1(1) 4(1) 7(1) C(27) 27(1) 23(1) 25(1) -6(1) 3(1) 4(1) C(28) 14(1) 14(1) 24(1) -1(1) 1(1) -4(1) N(2) 16(1) 19(1) 31(1) 1(1) 8(1) -1(1) C(29) 20(1) 22(1) 46(2) 1(1) 12(1) 2(1) C(30) 24(1) 24(1) 41(1) -7(1) 19(1) -6(1) C(31) 33(1) 24(1) 26(1) -1(1) 13(1) -10(1) C(32) 21(1) 18(1) 22(1) 2(1) 5(1) 0(1)

9.Appendix

268

O(1) 18(1) 16(1) 27(1) 2(1) 4(1) -2(1) O(2) 30(1) 28(1) 22(1) 7(1) 9(1) 9(1) O(3) 17(1) 15(1) 26(1) -2(1) 2(1) 2(1) O(4) 42(1) 29(1) 22(1) -3(1) 8(1) -8(1) Table 5. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 10 3) for s3868ma. x y z U(eq) H(1) 6246 4038 2784 20 H(2) 5478 6756 2452 19 H(5) 1486 8373 2756 27 H(6A) 3500 8688 1947 25 H(6B) 1912 8044 1549 25 H(7) 2741 5392 1721 20 H(8A) 1472 7335 3932 38 H(8B) 3123 6919 4337 38 H(8C) 2005 5521 4018 38 H(10A) 3516 5500 -46 30 H(10B) 2622 4592 543 30 H(11A) 5256 7493 347 37 H(11B) 5925 7541 1211 37 H(11C) 4560 8672 883 37 H(13) 9637 6764 1735 26 H(14) 9630 5500 612 27 H(15) 7798 3589 216 28 H(16) 6015 3005 971 23 H(17) 7004 -976 2354 20 H(18) 7744 1792 2601 20 H(21) 11889 3014 2132 30 H(22A) 9931 3736 2941 26 H(22B) 11496 3047 3347 26 H(23) 10509 451 3314 20 H(24A) 10013 1771 619 51 H(24B) 10738 92 888 51 H(24C) 11730 1672 991 51 H(26A) 9989 1215 5084 39 H(26B) 10873 162 4538 39 H(27A) 8205 3092 4610 38 H(27B) 7339 2680 3799 38

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269

H(27C) 8631 3992 3905 38 H(29) 3668 1632 3466 34 H(30) 3886 456 4618 34 H(31) 5845 -1336 4995 32 H(32) 7531 -1871 4189 24 H(1A) 4938 2262 2313 31 H(3) 8417 -2680 2818 29 Table 6. Torsion angles [°] for s3868ma. O(1)-C(1)-C(2)-C(3) 76.6(2) C(12)-C(1)-C(2)-C(3) -158.06(16) O(1)-C(1)-C(2)-C(7) -48.0(2) C(12)-C(1)-C(2)-C(7) 77.3(2) C(1)-C(2)-C(3)-O(2) 20.6(3) C(7)-C(2)-C(3)-O(2) 148.5(2) C(1)-C(2)-C(3)-C(4) -160.81(18) C(7)-C(2)-C(3)-C(4) -32.9(3) O(2)-C(3)-C(4)-C(5) -179.6(2) C(2)-C(3)-C(4)-C(5) 1.7(3) O(2)-C(3)-C(4)-C(8) 2.1(3) C(2)-C(3)-C(4)-C(8) -176.60(19) C(3)-C(4)-C(5)-C(6) 2.7(3) C(8)-C(4)-C(5)-C(6) -179.1(2) C(4)-C(5)-C(6)-C(7) 24.9(3) C(5)-C(6)-C(7)-C(9) 177.39(17) C(5)-C(6)-C(7)-C(2) -54.3(2) C(3)-C(2)-C(7)-C(9) -177.18(18) C(1)-C(2)-C(7)-C(9) -53.0(3) C(3)-C(2)-C(7)-C(6) 57.9(2) C(1)-C(2)-C(7)-C(6) -177.90(17) C(6)-C(7)-C(9)-C(10) -116.3(2) C(2)-C(7)-C(9)-C(10) 120.2(2) C(6)-C(7)-C(9)-C(11) 59.5(2) C(2)-C(7)-C(9)-C(11) -64.0(3) O(1)-C(1)-C(12)-N(1) -173.53(17) C(2)-C(1)-C(12)-N(1) 62.3(2) O(1)-C(1)-C(12)-C(16) 5.5(3) C(2)-C(1)-C(12)-C(16) -118.6(2) C(16)-C(12)-N(1)-C(13) 1.5(3)

9.Appendix

270

C(1)-C(12)-N(1)-C(13) -179.45(19) C(12)-N(1)-C(13)-C(14) -0.2(3) N(1)-C(13)-C(14)-C(15) -0.5(3) C(13)-C(14)-C(15)-C(16) 0.1(3) N(1)-C(12)-C(16)-C(15) -1.9(3) C(1)-C(12)-C(16)-C(15) 179.09(19) C(14)-C(15)-C(16)-C(12) 1.1(3) O(3)-C(17)-C(18)-C(19) 76.7(2) C(28)-C(17)-C(18)-C(19) -157.72(16) O(3)-C(17)-C(18)-C(23) -48.2(2) C(28)-C(17)-C(18)-C(23) 77.4(2) C(23)-C(18)-C(19)-O(4) 156.1(2) C(17)-C(18)-C(19)-O(4) 29.0(3) C(23)-C(18)-C(19)-C(20) -25.5(3) C(17)-C(18)-C(19)-C(20) -152.54(18) O(4)-C(19)-C(20)-C(21) 176.1(2) C(18)-C(19)-C(20)-C(21) -2.4(3) O(4)-C(19)-C(20)-C(24) -0.6(3) C(18)-C(19)-C(20)-C(24) -179.0(2) C(19)-C(20)-C(21)-C(22) 0.9(3) C(24)-C(20)-C(21)-C(22) 177.3(2) C(20)-C(21)-C(22)-C(23) 28.5(3) C(21)-C(22)-C(23)-C(25) 177.77(17) C(21)-C(22)-C(23)-C(18) -54.2(2) C(19)-C(18)-C(23)-C(25) 176.71(18) C(17)-C(18)-C(23)-C(25) -60.1(2) C(19)-C(18)-C(23)-C(22) 52.4(2) C(17)-C(18)-C(23)-C(22) 175.61(17) C(22)-C(23)-C(25)-C(26) -109.3(2) C(18)-C(23)-C(25)-C(26) 126.7(2) C(22)-C(23)-C(25)-C(27) 67.2(2) C(18)-C(23)-C(25)-C(27) -56.8(3) O(3)-C(17)-C(28)-N(2) -172.69(17) C(18)-C(17)-C(28)-N(2) 63.0(2) O(3)-C(17)-C(28)-C(32) 5.7(3) C(18)-C(17)-C(28)-C(32) -118.7(2) C(32)-C(28)-N(2)-C(29) 0.9(3) C(17)-C(28)-N(2)-C(29) 179.22(19) C(28)-N(2)-C(29)-C(30) 0.7(3)

9.Appendix

271

N(2)-C(29)-C(30)-C(31) -1.2(4) C(29)-C(30)-C(31)-C(32) 0.2(4) C(30)-C(31)-C(32)-C(28) 1.3(3) N(2)-C(28)-C(32)-C(31) -1.8(3) C(17)-C(28)-C(32)-C(31) 179.9(2) ________________________________________________________________ Symmetry transformations used to generate equivalent atoms: Table 7. Hydrogen bonds for s3868ma [Å and °]. D-H...A d(D-H) d(H...A) d(D...A) <(DHA) O(3)-H(3)...N(1)#1 0.84 2.01 2.834(2) 165.4 O(1)-H(1A)...N(2) 0.84 2.04 2.869(2) 167.2 ____________________________________________________________________________ Symmetry transformations used to generate equivalent atoms: #1 x, y-1,z

9.Appendix

272


Table 1. Crystal data and structure refinement for s3965qa. Identification code s3965qa Empirical formula C21 H22 O2 Formula weight 306.39 Temperature 100(2) K Wavelength 1.5418 Å Crystal system Triclinic Space group P 1 Unit cell dimensions a = 11.2771(4) Å a= 89.977(3)°. b = 12.6946(4) Å b= 90.111(3)°. c = 11.5699(4) Å g = 90.071(3)°. Volume 1656.33(10) Å3 Z 4 Density (calculated) 1.229 Mg/m3 Absorption coefficient 0.606 mm-1 F(000) 656 Crystal size 0.29 x 0.15 x 0.11 mm3 Theta range for data collection 3.4787 to 74.4664°. Index ranges -13<=h<=13, -12<=k<=14, -15<=l<=15 Reflections collected 13244 Independent reflections 7419 [R(int) = 0.0433] Completeness to theta = 67.00° 92.3 %

O

HO HH

H

9.Appendix

273

Absorption correction Semi-empirical from equivalents Max. and min. transmission 1.00000 and 0.88985 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 7419 / 99 / 746 Goodness-of-fit on F2 1.888 Final R indices [I>2sigma(I)] R1 = 0.1354, wR2 = 0.3681 R indices (all data) R1 = 0.1406, wR2 = 0.3838 Absolute structure parameter -0.5(6) Largest diff. peak and hole 1.307 and -0.643 e.Å-3 Table 2. Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103) for s3965qa. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x y z U(eq) C(1) 10020(5) 9641(5) 7323(5) 153(17) C(2) 11181(5) 9835(5) 6979(5) 34(2) C(3) 11868(4) 10565(5) 7575(6) 52(3) C(4) 11396(5) 11101(5) 8514(5) 49(3) C(5) 10235(4) 10907(4) 8858(4) 43(2) C(6) 9548(4) 10177(4) 8263(4) 46(2) C(7) 8387(4) 9983(5) 8606(5) 43(2) C(8) 7915(5) 10519(6) 9546(5) 54(3) C(9) 8603(6) 11249(6) 10141(5) 44(3) C(10) 9763(6) 11443(5) 9798(5) 81(6) C(11) 9231(12) 8862(10) 6534(11) 45(3) C(12) 8682(10) 9411(8) 5449(9) 35(2) C(13) 7838(10) 10275(9) 5824(9) 35(2) C(14) 6606(10) 9998(10) 6230(10) 42(2) C(15) 6285(11) 9017(9) 5965(12) 45(3) C(16) 6873(15) 8168(10) 5250(12) 54(3) C(17) 8083(14) 8596(11) 4672(9) 49(3) C(18) 5959(15) 10787(12) 6876(14) 57(3) C(19) 7794(16) 9156(10) 3486(12) 58(4) C(20) 7152(14) 9975(14) 3271(13) 59(3) C(21) 8511(15) 8790(30) 2661(14) 131(15) C(22) 4966(7) 8537(7) -248(6) 52(3) C(23) 6120(7) 8319(9) 102(7) 70(5) C(24) 6794(7) 7588(11) -503(9) 132(14) C(25) 6315(7) 7076(9) -1457(9) 136(11)

9.Appendix

274

C(26) 5162(7) 7294(7) -1807(6) 75(5) C(27) 4487(6) 8024(6) -1203(5) 101(7) C(28) 3333(7) 8242(10) -1553(8) 54(3) C(29) 2854(8) 7729(12) -2507(8) 800(200) C(30) 3529(10) 6999(12) -3112(7) 490(90) C(31) 4683(9) 6781(9) -2762(7) 151(17) C(32) 4009(17) 9350(9) 326(17) 76(6) C(33) 3544(10) 8796(8) 1395(11) 39(2) C(34) 2713(9) 7883(8) 1121(8) 32(2) C(35) 1536(11) 8176(10) 771(10) 41(2) C(36) 1099(9) 9161(8) 989(8) 32(2) C(37) 1799(8) 9915(8) 1671(8) 29(2) C(38) 2870(8) 9550(7) 2246(10) 30(2) C(39) 817(9) 7349(10) 126(11) 43(3) C(40) 2696(10) 9102(10) 3403(9) 41(2) C(41) 1591(12) 8632(10) 3737(9) 46(2) C(42) 3703(17) 8965(13) 4167(18) 71(5) C(43) 1843(5) 3509(4) 2286(4) 40(2) C(44) 685(5) 3309(5) 1936(5) 43(3) C(45) -4(4) 2585(6) 2537(6) 68(5) C(46) 465(5) 2061(5) 3488(6) 49(3) C(47) 1623(5) 2261(4) 3839(4) 56(4) C(48) 2312(4) 2985(4) 3238(4) 32(2) C(49) 3470(4) 3185(6) 3588(5) 43(3) C(50) 3939(5) 2661(7) 4540(6) 65(4) C(51) 3250(7) 1937(7) 5141(5) 65(4) C(52) 2092(7) 1737(6) 4790(5) 62(4) C(53) 2627(13) 4323(11) 1590(13) 54(3) C(54) 3164(11) 3792(10) 479(10) 42(2) C(55) 4003(10) 2840(8) 865(10) 37(2) C(56) 5201(12) 3143(10) 1207(11) 47(3) C(57) 5558(10) 4172(9) 948(11) 41(2) C(58) 4926(11) 4957(9) 252(12) 46(3) C(59) 3823(10) 4536(9) -336(9) 38(2) C(60) 5902(10) 2376(9) 1874(12) 44(3) C(61) 4044(14) 4043(12) -1531(10) 52(3) C(62) 3361(14) 4290(20) -2354(14) 83(7) C(63) 5270(20) 3692(18) -1810(20) 84(5) C(64) 6704(9) 4667(8) 4733(7) 240(30)

9.Appendix

275

C(65) 5545(9) 4848(8) 5087(8) 66(4) C(66) 4836(7) 5554(7) 4483(8) 98(9) C(67) 5286(7) 6080(7) 3524(8) 127(13) C(68) 6445(7) 5899(5) 3171(6) 42(2) C(69) 7154(7) 5193(6) 3775(5) 310(50) C(70) 8313(7) 5012(8) 3421(7) 130(14) C(71) 8763(7) 5537(9) 2463(8) 97(9) C(72) 8054(9) 6244(9) 1859(8) 390(70) C(73) 6895(9) 6425(8) 2212(8) 139(16) C(74) 7770(20) 3809(10) 5330(13) 72(5) C(75) 8238(10) 4389(8) 6405(12) 41(3) C(76) 9054(10) 5242(10) 6133(9) 38(2) C(77) 10249(11) 4960(8) 5751(9) 39(2) C(78) 10712(13) 4034(10) 5974(10) 48(3) C(79) 10036(12) 3217(8) 6665(9) 41(2) C(80) 8846(11) 3586(9) 7216(10) 40(2) C(81) 10942(14) 5847(14) 5101(15) 62(4) C(82) 9040(11) 4073(9) 8392(9) 39(2) C(83) 9980(20) 4694(18) 8673(15) 79(5) O(1) 10037(10) 8016(9) 6211(9) 60(3) O(2) 8185(9) 11216(7) 5845(8) 49(2) O(3) 4921(9) 10083(7) 653(12) 61(3) O(4) 3079(7) 6973(5) 1159(8) 40(2) O(5) 1789(7) 5103(6) 1223(7) 40(2) O(6) 3617(9) 1943(7) 810(9) 51(2) O(7) 6915(11) 3080(7) 5608(15) 78(4) O(8) 8721(8) 6199(6) 6166(8) 41(2) C(84) 7986(16) 4150(20) 9160(20) 90(6)

Table 3. Bond lengths [Å] and angles [°] for s3965qa. C(1)-C(2) 1.3900 C(1)-C(6) 1.3900 C(1)-C(11) 1.612(13) C(2)-C(3) 1.3900 C(2)-H(2) 0.9500 C(3)-C(4) 1.3900 C(3)-H(3) 0.9500 C(4)-C(5) 1.3900

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C(4)-H(4) 0.9500 C(5)-C(6) 1.3900 C(5)-C(10) 1.3900 C(6)-C(7) 1.3900 C(7)-C(8) 1.3900 C(7)-H(7) 0.9500 C(8)-C(9) 1.3900 C(8)-H(8) 0.9500 C(9)-C(10) 1.3900 C(9)-H(9) 0.9500 C(10)-H(10) 0.9500 C(11)-O(1) 1.457(18) C(11)-C(12) 1.563(16) C(11)-H(11) 1.0000 C(12)-C(13) 1.517(15) C(12)-C(17) 1.528(14) C(12)-H(12) 1.0000 C(13)-O(2) 1.256(14) C(13)-C(14) 1.509(16) C(14)-C(15) 1.332(19) C(14)-C(18) 1.45(2) C(15)-C(16) 1.512(18) C(15)-H(15) 0.9500 C(16)-C(17) 1.61(2) C(16)-H(16A) 0.9900 C(16)-H(16B) 0.9900 C(17)-C(19) 1.578(19) C(17)-H(17) 1.0000 C(18)-H(18A) 0.9800 C(18)-H(18B) 0.9800 C(18)-H(18C) 0.9800 C(19)-C(20) 1.29(2) C(19)-C(21) 1.34(3) C(20)-H(20A) 0.9500 C(20)-H(20B) 0.9500 C(21)-H(21A) 0.9800 C(21)-H(21B) 0.9800 C(21)-H(21C) 0.9800 C(22)-C(23) 1.3900

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C(22)-C(27) 1.3900 C(22)-C(32) 1.636(14) C(23)-C(24) 1.3900 C(23)-H(23) 0.9500 C(24)-C(25) 1.3900 C(24)-H(24) 0.9500 C(25)-C(26) 1.3900 C(25)-H(25) 0.9500 C(26)-C(27) 1.3900 C(26)-C(31) 1.3900 C(27)-C(28) 1.3900 C(28)-C(29) 1.3900 C(28)-H(28) 0.9500 C(29)-C(30) 1.3900 C(29)-H(29) 0.9500 C(30)-C(31) 1.3900 C(30)-H(30) 0.9500 C(31)-H(31) 0.9500 C(32)-O(3) 1.44(2) C(32)-C(33) 1.517(17) C(32)-H(32) 1.0000 C(33)-C(34) 1.523(14) C(33)-C(38) 1.571(13) C(33)-H(33) 1.0000 C(34)-O(4) 1.229(14) C(34)-C(35) 1.437(16) C(35)-C(36) 1.368(16) C(35)-C(39) 1.521(14) C(36)-C(37) 1.468(14) C(36)-H(36) 0.9500 C(37)-C(38) 1.454(13) C(37)-H(37A) 0.9900 C(37)-H(37B) 0.9900 C(38)-C(40) 1.468(16) C(38)-H(38) 1.0000 C(39)-H(39A) 0.9800 C(39)-H(39B) 0.9800 C(39)-H(39C) 0.9800 C(40)-C(41) 1.435(17)

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C(40)-C(42) 1.448(17) C(41)-H(41A) 0.9800 C(41)-H(41B) 0.9800 C(41)-H(41C) 0.9800 C(42)-H(42A) 0.9500 C(42)-H(42B) 0.9500 C(43)-C(44) 1.3900 C(43)-C(48) 1.3900 C(43)-C(53) 1.581(13) C(44)-C(45) 1.3900 C(44)-H(44) 0.9500 C(45)-C(46) 1.3900 C(45)-H(45) 0.9500 C(46)-C(47) 1.3900 C(46)-H(46) 0.9500 C(47)-C(48) 1.3900 C(47)-C(52) 1.3900 C(48)-C(49) 1.3900 C(49)-C(50) 1.3900 C(49)-H(49) 0.9500 C(50)-C(51) 1.3900 C(50)-H(50) 0.9500 C(51)-C(52) 1.3900 C(51)-H(51) 0.9500 C(52)-H(52) 0.9500 C(53)-O(5) 1.432(18) C(53)-C(54) 1.574(16) C(53)-H(53) 1.0000 C(54)-C(59) 1.527(16) C(54)-C(55) 1.599(17) C(54)-H(54) 1.0000 C(55)-O(6) 1.220(14) C(55)-C(56) 1.459(17) C(56)-C(57) 1.399(17) C(56)-C(60) 1.47(2) C(57)-C(58) 1.47(2) C(57)-H(57) 0.9500 C(58)-C(59) 1.513(15) C(58)-H(58A) 0.9900

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C(58)-H(58B) 0.9900 C(59)-C(61) 1.539(14) C(59)-H(59) 1.0000 C(60)-H(60A) 0.9800 C(60)-H(60B) 0.9800 C(60)-H(60C) 0.9800 C(61)-C(62) 1.26(2) C(61)-C(63) 1.49(3) C(62)-H(62A) 0.9800 C(62)-H(62B) 0.9800 C(62)-H(62C) 0.9800 C(63)-H(63A) 0.9500 C(63)-H(63B) 0.9500 C(64)-C(65) 1.3900 C(64)-C(69) 1.3900 C(64)-C(74) 1.76(2) C(65)-C(66) 1.3900 C(65)-H(65) 0.9500 C(66)-C(67) 1.3900 C(66)-H(66) 0.9500 C(67)-C(68) 1.3900 C(67)-H(67) 0.9500 C(68)-C(69) 1.3900 C(68)-C(73) 1.3900 C(69)-C(70) 1.3900 C(70)-C(71) 1.3900 C(70)-H(70) 0.9500 C(71)-C(72) 1.3900 C(71)-H(71) 0.9500 C(72)-C(73) 1.3900 C(72)-H(72) 0.9500 C(73)-H(73) 0.9500 C(74)-O(7) 1.38(2) C(74)-C(75) 1.537(14) C(74)-H(74) 1.0000 C(75)-C(76) 1.455(16) C(75)-C(80) 1.546(16) C(75)-H(75) 1.0000 C(76)-O(8) 1.273(15)

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280

C(76)-C(77) 1.463(16) C(77)-C(78) 1.31(2) C(77)-C(81) 1.563(18) C(78)-C(79) 1.515(16) C(78)-H(78) 0.9500 C(79)-C(80) 1.560(17) C(79)-H(79A) 0.9900 C(79)-H(79B) 0.9900 C(80)-C(82) 1.510(15) C(80)-H(80) 1.0000 C(81)-H(81A) 0.9800 C(81)-H(81B) 0.9800 C(81)-H(81C) 0.9800 C(82)-C(83) 1.36(2) C(82)-C(84) 1.49(2) C(83)-H(83A) 0.9500 C(83)-H(83B) 0.9500 O(1)-H(1) 0.8400 O(3)-H(3A) 0.8400 O(5)-H(5) 0.8400 O(7)-H(7A) 0.8400 C(84)-H(84A) 0.9800 C(84)-H(84B) 0.9800 C(84)-H(84C) 0.9800 C(2)-C(1)-C(6) 120.0 C(2)-C(1)-C(11) 117.7(5) C(6)-C(1)-C(11) 122.1(5) C(1)-C(2)-C(3) 120.0 C(1)-C(2)-H(2) 120.0 C(3)-C(2)-H(2) 120.0 C(4)-C(3)-C(2) 120.0 C(4)-C(3)-H(3) 120.0 C(2)-C(3)-H(3) 120.0 C(5)-C(4)-C(3) 120.0 C(5)-C(4)-H(4) 120.0 C(3)-C(4)-H(4) 120.0 C(4)-C(5)-C(6) 120.0 C(4)-C(5)-C(10) 120.0

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C(6)-C(5)-C(10) 120.0 C(7)-C(6)-C(5) 120.0 C(7)-C(6)-C(1) 120.0 C(5)-C(6)-C(1) 120.0 C(6)-C(7)-C(8) 120.0 C(6)-C(7)-H(7) 120.0 C(8)-C(7)-H(7) 120.0 C(9)-C(8)-C(7) 120.0 C(9)-C(8)-H(8) 120.0 C(7)-C(8)-H(8) 120.0 C(8)-C(9)-C(10) 120.0 C(8)-C(9)-H(9) 120.0 C(10)-C(9)-H(9) 120.0 C(9)-C(10)-C(5) 120.0 C(9)-C(10)-H(10) 120.0 C(5)-C(10)-H(10) 120.0 O(1)-C(11)-C(12) 111.7(10) O(1)-C(11)-C(1) 104.7(9) C(12)-C(11)-C(1) 113.5(9) O(1)-C(11)-H(11) 108.9 C(12)-C(11)-H(11) 108.9 C(1)-C(11)-H(11) 108.9 C(13)-C(12)-C(17) 112.4(10) C(13)-C(12)-C(11) 110.0(9) C(17)-C(12)-C(11) 110.2(9) C(13)-C(12)-H(12) 108.0 C(17)-C(12)-H(12) 108.1 C(11)-C(12)-H(12) 108.1 O(2)-C(13)-C(14) 120.1(11) O(2)-C(13)-C(12) 119.9(10) C(14)-C(13)-C(12) 120.0(10) C(15)-C(14)-C(18) 129.0(13) C(15)-C(14)-C(13) 113.3(11) C(18)-C(14)-C(13) 117.7(12) C(14)-C(15)-C(16) 132.3(12) C(14)-C(15)-H(15) 113.8 C(16)-C(15)-H(15) 113.8 C(15)-C(16)-C(17) 111.0(10) C(15)-C(16)-H(16A) 109.4

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C(17)-C(16)-H(16A) 109.4 C(15)-C(16)-H(16B) 109.4 C(17)-C(16)-H(16B) 109.4 H(16A)-C(16)-H(16B) 108.0 C(12)-C(17)-C(19) 107.3(10) C(12)-C(17)-C(16) 110.9(10) C(19)-C(17)-C(16) 109.8(13) C(12)-C(17)-H(17) 109.6 C(19)-C(17)-H(17) 109.6 C(16)-C(17)-H(17) 109.6 C(14)-C(18)-H(18A) 109.4 C(14)-C(18)-H(18B) 109.5 H(18A)-C(18)-H(18B) 109.5 C(14)-C(18)-H(18C) 109.5 H(18A)-C(18)-H(18C) 109.5 H(18B)-C(18)-H(18C) 109.5 C(20)-C(19)-C(21) 118.9(18) C(20)-C(19)-C(17) 130.1(14) C(21)-C(19)-C(17) 109.9(16) C(19)-C(20)-H(20A) 120.0 C(19)-C(20)-H(20B) 120.0 H(20A)-C(20)-H(20B) 120.0 C(19)-C(21)-H(21A) 109.5 C(19)-C(21)-H(21B) 109.5 H(21A)-C(21)-H(21B) 109.5 C(19)-C(21)-H(21C) 109.4 H(21A)-C(21)-H(21C) 109.5 H(21B)-C(21)-H(21C) 109.5 C(23)-C(22)-C(27) 120.0 C(23)-C(22)-C(32) 128.7(10) C(27)-C(22)-C(32) 111.3(10) C(24)-C(23)-C(22) 120.0 C(24)-C(23)-H(23) 120.0 C(22)-C(23)-H(23) 120.0 C(23)-C(24)-C(25) 120.0 C(23)-C(24)-H(24) 120.0 C(25)-C(24)-H(24) 120.0 C(26)-C(25)-C(24) 120.0 C(26)-C(25)-H(25) 120.0

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C(24)-C(25)-H(25) 120.0 C(25)-C(26)-C(27) 120.0 C(25)-C(26)-C(31) 120.0 C(27)-C(26)-C(31) 120.0 C(26)-C(27)-C(28) 120.0 C(26)-C(27)-C(22) 120.0 C(28)-C(27)-C(22) 120.0 C(29)-C(28)-C(27) 120.0 C(29)-C(28)-H(28) 120.0 C(27)-C(28)-H(28) 120.0 C(28)-C(29)-C(30) 120.0 C(28)-C(29)-H(29) 120.0 C(30)-C(29)-H(29) 120.0 C(31)-C(30)-C(29) 120.0 C(31)-C(30)-H(30) 120.0 C(29)-C(30)-H(30) 120.0 C(30)-C(31)-C(26) 120.0 C(30)-C(31)-H(31) 120.0 C(26)-C(31)-H(31) 120.0 O(3)-C(32)-C(33) 109.4(16) O(3)-C(32)-C(22) 92.5(11) C(33)-C(32)-C(22) 105.5(9) O(3)-C(32)-H(32) 115.6 C(33)-C(32)-H(32) 115.6 C(22)-C(32)-H(32) 115.6 C(32)-C(33)-C(34) 113.3(12) C(32)-C(33)-C(38) 113.4(9) C(34)-C(33)-C(38) 107.2(9) C(32)-C(33)-H(33) 107.5 C(34)-C(33)-H(33) 107.5 C(38)-C(33)-H(33) 107.5 O(4)-C(34)-C(35) 124.4(9) O(4)-C(34)-C(33) 120.1(10) C(35)-C(34)-C(33) 115.4(10) C(36)-C(35)-C(34) 121.3(10) C(36)-C(35)-C(39) 122.0(11) C(34)-C(35)-C(39) 116.7(10) C(35)-C(36)-C(37) 120.1(10) C(35)-C(36)-H(36) 120.0

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C(37)-C(36)-H(36) 120.0 C(38)-C(37)-C(36) 118.9(9) C(38)-C(37)-H(37A) 107.6 C(36)-C(37)-H(37A) 107.6 C(38)-C(37)-H(37B) 107.6 C(36)-C(37)-H(37B) 107.6 H(37A)-C(37)-H(37B) 107.0 C(37)-C(38)-C(40) 115.4(8) C(37)-C(38)-C(33) 108.1(8) C(40)-C(38)-C(33) 113.6(9) C(37)-C(38)-H(38) 106.4 C(40)-C(38)-H(38) 106.4 C(33)-C(38)-H(38) 106.4 C(35)-C(39)-H(39A) 109.5 C(35)-C(39)-H(39B) 109.5 H(39A)-C(39)-H(39B) 109.5 C(35)-C(39)-H(39C) 109.5 H(39A)-C(39)-H(39C) 109.5 H(39B)-C(39)-H(39C) 109.5 C(41)-C(40)-C(42) 117.8(13) C(41)-C(40)-C(38) 121.6(9) C(42)-C(40)-C(38) 119.8(13) C(40)-C(41)-H(41A) 109.5 C(40)-C(41)-H(41B) 109.5 H(41A)-C(41)-H(41B) 109.5 C(40)-C(41)-H(41C) 109.5 H(41A)-C(41)-H(41C) 109.5 H(41B)-C(41)-H(41C) 109.5 C(40)-C(42)-H(42A) 120.0 C(40)-C(42)-H(42B) 120.0 H(42A)-C(42)-H(42B) 120.0 C(44)-C(43)-C(48) 120.0 C(44)-C(43)-C(53) 119.8(7) C(48)-C(43)-C(53) 120.2(7) C(45)-C(44)-C(43) 120.0 C(45)-C(44)-H(44) 120.0 C(43)-C(44)-H(44) 120.0 C(46)-C(45)-C(44) 120.0 C(46)-C(45)-H(45) 120.0

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C(44)-C(45)-H(45) 120.0 C(47)-C(46)-C(45) 120.0 C(47)-C(46)-H(46) 120.0 C(45)-C(46)-H(46) 120.0 C(46)-C(47)-C(48) 120.0 C(46)-C(47)-C(52) 120.0 C(48)-C(47)-C(52) 120.0 C(49)-C(48)-C(47) 120.0 C(49)-C(48)-C(43) 120.0 C(47)-C(48)-C(43) 120.0 C(50)-C(49)-C(48) 120.0 C(50)-C(49)-H(49) 120.0 C(48)-C(49)-H(49) 120.0 C(49)-C(50)-C(51) 120.0 C(49)-C(50)-H(50) 120.0 C(51)-C(50)-H(50) 120.0 C(50)-C(51)-C(52) 120.0 C(50)-C(51)-H(51) 120.0 C(52)-C(51)-H(51) 120.0 C(51)-C(52)-C(47) 120.0 C(51)-C(52)-H(52) 120.0 C(47)-C(52)-H(52) 120.0 O(5)-C(53)-C(54) 108.0(11) O(5)-C(53)-C(43) 103.5(9) C(54)-C(53)-C(43) 110.6(10) O(5)-C(53)-H(53) 111.5 C(54)-C(53)-H(53) 111.5 C(43)-C(53)-H(53) 111.5 C(59)-C(54)-C(53) 115.3(10) C(59)-C(54)-C(55) 110.5(9) C(53)-C(54)-C(55) 109.0(10) C(59)-C(54)-H(54) 107.2 C(53)-C(54)-H(54) 107.2 C(55)-C(54)-H(54) 107.2 O(6)-C(55)-C(56) 126.1(12) O(6)-C(55)-C(54) 118.7(11) C(56)-C(55)-C(54) 115.1(10) C(57)-C(56)-C(55) 117.0(13) C(57)-C(56)-C(60) 125.1(11)

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286

C(55)-C(56)-C(60) 117.7(10) C(56)-C(57)-C(58) 127.8(11) C(56)-C(57)-H(57) 116.1 C(58)-C(57)-H(57) 116.1 C(57)-C(58)-C(59) 114.0(9) C(57)-C(58)-H(58A) 108.8 C(59)-C(58)-H(58A) 108.8 C(57)-C(58)-H(58B) 108.8 C(59)-C(58)-H(58B) 108.8 H(58A)-C(58)-H(58B) 107.6 C(58)-C(59)-C(61) 114.4(10) C(58)-C(59)-C(54) 109.9(10) C(61)-C(59)-C(54) 112.6(10) C(58)-C(59)-H(59) 106.5 C(61)-C(59)-H(59) 106.5 C(54)-C(59)-H(59) 106.5 C(56)-C(60)-H(60A) 109.5 C(56)-C(60)-H(60B) 109.5 H(60A)-C(60)-H(60B) 109.5 C(56)-C(60)-H(60C) 109.5 H(60A)-C(60)-H(60C) 109.5 H(60B)-C(60)-H(60C) 109.5 C(62)-C(61)-C(63) 118.4(14) C(62)-C(61)-C(59) 118.3(13) C(63)-C(61)-C(59) 118.1(14) C(61)-C(62)-H(62A) 109.5 C(61)-C(62)-H(62B) 109.5 H(62A)-C(62)-H(62B) 109.5 C(61)-C(62)-H(62C) 109.4 H(62A)-C(62)-H(62C) 109.5 H(62B)-C(62)-H(62C) 109.5 C(61)-C(63)-H(63A) 120.0 C(61)-C(63)-H(63B) 120.0 H(63A)-C(63)-H(63B) 120.0 C(65)-C(64)-C(69) 120.0 C(65)-C(64)-C(74) 129.0(8) C(69)-C(64)-C(74) 111.0(8) C(66)-C(65)-C(64) 120.0 C(66)-C(65)-H(65) 120.0

9.Appendix

287

C(64)-C(65)-H(65) 120.0 C(65)-C(66)-C(67) 120.0 C(65)-C(66)-H(66) 120.0 C(67)-C(66)-H(66) 120.0 C(68)-C(67)-C(66) 120.0 C(68)-C(67)-H(67) 120.0 C(66)-C(67)-H(67) 120.0 C(67)-C(68)-C(69) 120.0 C(67)-C(68)-C(73) 120.0 C(69)-C(68)-C(73) 120.0 C(70)-C(69)-C(68) 120.0 C(70)-C(69)-C(64) 120.0 C(68)-C(69)-C(64) 120.0 C(71)-C(70)-C(69) 120.0 C(71)-C(70)-H(70) 120.0 C(69)-C(70)-H(70) 120.0 C(72)-C(71)-C(70) 120.0 C(72)-C(71)-H(71) 120.0 C(70)-C(71)-H(71) 120.0 C(71)-C(72)-C(73) 120.0 C(71)-C(72)-H(72) 120.0 C(73)-C(72)-H(72) 120.0 C(72)-C(73)-C(68) 120.0 C(72)-C(73)-H(73) 120.0 C(68)-C(73)-H(73) 120.0 O(7)-C(74)-C(75) 111.8(14) O(7)-C(74)-C(64) 91.6(13) C(75)-C(74)-C(64) 104.6(10) O(7)-C(74)-H(74) 115.4 C(75)-C(74)-H(74) 115.4 C(64)-C(74)-H(74) 115.4 C(76)-C(75)-C(74) 113.3(12) C(76)-C(75)-C(80) 110.0(9) C(74)-C(75)-C(80) 109.0(9) C(76)-C(75)-H(75) 108.1 C(74)-C(75)-H(75) 108.1 C(80)-C(75)-H(75) 108.1 O(8)-C(76)-C(75) 121.1(10) O(8)-C(76)-C(77) 121.0(10)

9.Appendix

288

C(75)-C(76)-C(77) 117.8(10) C(78)-C(77)-C(76) 121.8(11) C(78)-C(77)-C(81) 122.7(12) C(76)-C(77)-C(81) 115.5(11) C(77)-C(78)-C(79) 121.1(12) C(77)-C(78)-H(78) 119.4 C(79)-C(78)-H(78) 119.4 C(78)-C(79)-C(80) 116.3(10) C(78)-C(79)-H(79A) 108.2 C(80)-C(79)-H(79A) 108.2 C(78)-C(79)-H(79B) 108.2 C(80)-C(79)-H(79B) 108.2 H(79A)-C(79)-H(79B) 107.4 C(82)-C(80)-C(75) 109.9(9) C(82)-C(80)-C(79) 111.6(10) C(75)-C(80)-C(79) 109.4(9) C(82)-C(80)-H(80) 108.6 C(75)-C(80)-H(80) 108.6 C(79)-C(80)-H(80) 108.6 C(77)-C(81)-H(81A) 109.4 C(77)-C(81)-H(81B) 109.5 H(81A)-C(81)-H(81B) 109.5 C(77)-C(81)-H(81C) 109.5 H(81A)-C(81)-H(81C) 109.5 H(81B)-C(81)-H(81C) 109.5 C(83)-C(82)-C(84) 116.0(15) C(83)-C(82)-C(80) 124.4(13) C(84)-C(82)-C(80) 116.9(14) C(82)-C(83)-H(83A) 120.0 C(82)-C(83)-H(83B) 120.0 H(83A)-C(83)-H(83B) 120.0 C(11)-O(1)-H(1) 109.5 C(32)-O(3)-H(3A) 109.5 C(53)-O(5)-H(5) 109.5 C(74)-O(7)-H(7A) 109.5 C(82)-C(84)-H(84A) 109.4 C(82)-C(84)-H(84B) 109.5 H(84A)-C(84)-H(84B) 109.5 C(82)-C(84)-H(84C) 109.5

9.Appendix

289

H(84A)-C(84)-H(84C) 109.5 H(84B)-C(84)-H(84C) 109.5 Symmetry transformations used to generate equivalent atoms: Table 4. Anisotropic displacement parameters (Å2x 103) for s3965qa. The anisotropic displacement factor exponent takes the form: -2p2[ h2 a*2U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 U23 U13 U12 C(1) 55(10) 250(30) 160(20) 150(30) 77(14) 90(17) C(2) 33(5) 31(5) 38(4) -6(4) -4(4) -14(4) C(3) 46(6) 29(5) 80(9) 1(5) -31(6) -11(5) C(4) 46(4) 45(4) 57(4) 15(3) 7(3) -5(3) C(5) 39(5) 38(6) 51(6) 18(5) -9(4) -1(4) C(6) 53(4) 41(4) 44(4) 0(3) -8(3) -3(3) C(7) 33(5) 44(6) 54(6) 6(5) -7(4) -5(4) C(8) 85(9) 29(6) 48(6) -7(5) 2(6) -8(6) C(9) 56(6) 34(5) 43(5) -15(4) 17(5) -16(5) C(10) 106(14) 88(13) 49(7) -2(7) 12(8) -61(12) C(11) 51(6) 39(6) 44(5) 0(5) -4(5) -1(5) C(12) 46(6) 24(5) 37(5) 7(4) -7(4) -3(4) C(13) 42(3) 24(3) 40(3) 5(3) 1(3) -4(3) C(14) 38(5) 46(7) 42(5) 7(4) -13(4) 12(4) C(15) 40(6) 29(6) 68(7) 9(5) 6(5) -7(4) C(16) 78(9) 29(6) 53(7) 5(5) 1(6) -1(6) C(17) 76(8) 43(7) 28(5) 5(4) -3(5) -7(6) C(18) 65(8) 42(7) 65(8) 17(6) -4(6) 3(6) C(19) 89(10) 29(6) 56(7) -10(5) -15(7) -18(6) C(20) 56(6) 67(6) 54(5) 7(5) -17(5) 5(5) C(21) 39(7) 310(40) 42(7) 49(15) 4(6) 43(14) C(22) 56(4) 46(4) 53(4) -12(3) 6(3) 8(3) C(23) 32(6) 112(14) 67(8) 21(8) -14(5) -36(7) C(24) 190(40) 110(20) 95(18) -15(15) 70(20) 20(20) C(25) 136(12) 134(12) 137(12) 0(4) -2(4) -3(4) C(26) 147(17) 54(8) 24(5) 1(5) -15(7) -13(9) C(27) 102(8) 100(8) 101(8) 4(4) 2(4) 0(4) C(28) 50(4) 60(4) 52(4) 4(3) -5(3) 2(3) C(29) 2000(700) 270(70) 22(10) -5(19) -60(60) -400(190) C(30) 100(20) 150(40) 1200(300) -180(80) 290(70) -80(30) C(31) 115(17) 300(40) 37(7) 37(13) -25(9) -160(30)

9.Appendix

290

C(32) 113(13) 8(4) 106(12) 26(6) 85(11) 16(6) C(33) 36(5) 24(5) 57(6) -4(5) -3(4) 10(4) C(34) 34(5) 27(5) 33(4) 4(3) 11(3) -10(4) C(35) 48(4) 33(4) 43(4) 6(3) -5(3) 4(3) C(36) 38(5) 31(5) 28(4) 19(3) 6(3) 1(4) C(37) 23(4) 28(5) 36(4) 1(4) 14(3) 5(3) C(38) 21(4) 20(4) 50(5) 10(4) 5(4) -1(3) C(39) 29(5) 39(6) 61(7) -21(5) 2(4) -11(4) C(40) 44(6) 46(7) 33(5) 10(4) -8(4) 4(5) C(41) 57(5) 44(5) 37(4) 11(4) -1(4) -5(4) C(42) 68(9) 54(8) 90(11) 2(7) -41(9) 1(7) C(43) 39(4) 41(4) 41(3) 4(3) 3(3) -7(3) C(44) 25(4) 49(6) 54(6) -10(5) -27(4) 10(4) C(45) 26(5) 100(13) 77(9) -18(9) 15(5) -13(6) C(46) 68(8) 19(5) 59(7) 0(5) -9(6) -2(5) C(47) 91(10) 25(6) 52(7) -4(5) 19(7) -19(6) C(48) 25(4) 29(5) 43(5) 14(4) -3(3) -6(3) C(49) 17(4) 64(7) 49(6) -22(5) -12(4) -9(4) C(50) 63(5) 65(5) 67(5) 0(4) 0(4) 1(4) C(51) 71(9) 77(11) 48(7) 11(6) -19(6) 12(8) C(52) 49(7) 82(10) 55(7) -32(7) 4(5) -7(6) C(53) 58(7) 39(7) 66(8) 13(6) 29(6) -6(5) C(54) 50(6) 38(6) 38(5) 0(4) 10(4) 6(5) C(55) 41(5) 17(4) 52(5) -8(4) 19(4) -11(4) C(56) 50(7) 43(7) 48(6) -7(5) 19(5) -18(5) C(57) 29(5) 35(5) 59(6) -14(5) 5(4) -10(4) C(58) 45(6) 29(5) 64(7) -8(5) 18(5) -13(4) C(59) 39(5) 35(6) 41(5) 4(4) -4(4) -13(4) C(60) 33(5) 26(5) 74(7) 2(5) 12(5) 6(4) C(61) 64(8) 57(8) 34(5) -2(5) 8(5) 6(6) C(62) 41(7) 150(20) 55(7) -3(10) 14(6) 34(10) C(63) 86(8) 85(8) 80(7) -5(6) 9(6) 20(7) C(64) 240(30) 240(30) 240(30) -2(4) 0(4) -1(4) C(65) 82(11) 45(8) 71(9) -5(7) -10(8) 26(7) C(66) 95(14) 28(7) 170(20) 18(10) -82(16) -14(7) C(67) 30(6) 110(16) 240(30) -130(20) 33(11) -22(8) C(68) 53(6) 40(6) 34(5) 1(4) 3(4) 5(5) C(69) 340(80) 360(70) 210(40) -230(50) 150(50) -260(70) C(70) 200(30) 150(20) 43(8) 22(11) -46(13) -100(20)

9.Appendix

291

C(71) 210(30) 38(7) 49(7) 2(6) -54(12) -15(11) C(72) 490(110) 180(40) 500(100) -270(60) -400(100) 240(60) C(73) 180(30) 130(20) 107(19) -33(17) 80(20) -80(20) C(74) 131(15) 28(6) 58(7) -9(6) -33(9) -30(8) C(75) 38(5) 13(4) 72(7) -7(5) -15(5) 8(4) C(76) 41(4) 37(4) 36(3) 1(3) -3(3) -2(3) C(77) 53(6) 24(5) 40(5) -9(4) 7(4) -5(4) C(78) 72(8) 33(6) 38(5) 1(5) 8(5) -22(5) C(79) 63(7) 18(4) 41(5) 6(4) 7(5) 16(4) C(80) 54(6) 27(5) 38(5) 0(4) -10(5) -7(4) C(81) 54(7) 54(8) 78(9) -8(8) 16(7) -20(6) C(82) 50(6) 29(6) 39(5) 14(4) -6(4) 16(4) C(83) 82(7) 93(8) 61(6) -13(6) 0(6) -19(6) O(1) 69(6) 49(5) 61(5) 5(4) -21(5) -26(5) O(2) 63(5) 32(5) 53(4) -8(4) 6(4) 1(4) O(3) 50(5) 21(4) 112(8) 5(5) 24(5) -5(3) O(4) 49(4) 12(3) 60(4) 10(3) 0(3) 6(3) O(5) 44(4) 17(3) 59(4) 11(3) 4(3) 2(3) O(6) 60(6) 33(5) 59(5) 5(4) 5(4) 0(4) O(7) 61(6) 22(4) 149(12) -12(5) -55(7) 11(4) O(8) 47(4) 19(4) 56(4) 3(3) -5(3) 5(3) C(84) 50(9) 123(18) 96(13) 1(12) 24(9) 24(10) Table 5. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 10 3) for s3965qa. x y z U(eq) H(2) 11503 9469 6337 41 H(3) 12661 10698 7340 62 H(4) 11866 11600 8922 59 H(7) 7917 9484 8199 52 H(8) 7122 10387 9780 65 H(9) 8280 11616 10783 53 H(10) 10233 11942 10205 97 H(11) 8577 8562 7015 54 H(12) 9341 9745 5002 43 H(15) 5546 8810 6287 55 H(16A) 6321 7933 4635 64 H(16B) 7053 7554 5749 64

9.Appendix

292

H(17) 8635 7994 4536 59 H(18A) 5328 10446 7322 86 H(18B) 5608 11297 6339 86 H(18C) 6502 11152 7403 86 H(20A) 7194 10307 2536 71 H(20B) 6638 10246 3848 71 H(21A) 9109 9324 2475 197 H(21B) 8044 8628 1968 197 H(21C) 8904 8148 2935 197 H(23) 6447 8669 754 84 H(24) 7583 7440 -263 158 H(25) 6777 6577 -1870 163 H(28) 2872 8741 -1140 65 H(29) 2066 7878 -2747 917 H(30) 3202 6649 -3764 593 H(31) 5144 6282 -3175 181 H(32) 3391 9630 -213 91 H(33) 4242 8502 1820 47 H(36) 342 9358 699 38 H(37A) 2025 10501 1151 35 H(37B) 1271 10213 2270 35 H(38) 3387 10184 2348 36 H(39A) 140 7686 -256 64 H(39B) 530 6818 674 64 H(39C) 1319 7010 -456 64 H(41A) 1342 8123 3147 69 H(41B) 987 9181 3813 69 H(41C) 1690 8269 4479 69 H(42A) 3618 8575 4863 85 H(42B) 4448 9266 3972 85 H(44) 364 3667 1285 51 H(45) -796 2448 2297 81 H(46) -6 1566 3899 58 H(49) 3941 3680 3177 52 H(50) 4731 2798 4779 78 H(51) 3571 1579 5791 78 H(52) 1621 1242 5201 74 H(53) 3261 4637 2089 65 H(54) 2488 3482 34 50

9.Appendix

293

H(57) 6299 4388 1261 49 H(58A) 5471 5233 -345 55 H(58B) 4699 5552 757 55 H(59) 3290 5154 -465 46 H(60A) 6510 2748 2322 66 H(60B) 6283 1880 1343 66 H(60C) 5379 1990 2401 66 H(62A) 2726 4748 -2061 125 H(62B) 3015 3656 -2692 125 H(62C) 3812 4674 -2945 125 H(63A) 5520 3660 -2595 100 H(63B) 5798 3499 -1210 100 H(65) 5238 4488 5742 79 H(66) 4044 5678 4724 117 H(67) 4801 6563 3112 153 H(70) 8798 4529 3834 155 H(71) 9555 5414 2221 117 H(72) 8361 6603 1204 467 H(73) 6410 6907 1799 167 H(74) 8394 3558 4781 87 H(75) 7543 4695 6822 49 H(78) 11483 3873 5698 57 H(79A) 10560 2960 7293 49 H(79B) 9865 2611 6153 49 H(80) 8313 2961 7304 48 H(81A) 11558 5529 4618 93 H(81B) 11310 6321 5666 93 H(81C) 10392 6246 4613 93 H(83A) 9935 5144 9328 95 H(83B) 10675 4680 8215 95 H(1) 9646 7467 6071 90 H(3A) 5588 9828 495 92 H(5) 1579 5465 1794 60 H(7A) 7230 2568 5952 116 H(84A) 7394 3626 8930 135 H(84B) 7642 4858 9098 135 H(84C) 8227 4023 9963 135

9.Appendix

294

Table 6. Torsion angles [°] for s3965qa. C(6)-C(1)-C(2)-C(3) 0.0 C(11)-C(1)-C(2)-C(3) 174.9(8) C(1)-C(2)-C(3)-C(4) 0.0 C(2)-C(3)-C(4)-C(5) 0.0 C(3)-C(4)-C(5)-C(6) 0.0 C(3)-C(4)-C(5)-C(10) 180.0 C(4)-C(5)-C(6)-C(7) 180.0 C(10)-C(5)-C(6)-C(7) 0.0 C(4)-C(5)-C(6)-C(1) 0.0 C(10)-C(5)-C(6)-C(1) 180.0 C(2)-C(1)-C(6)-C(7) 180.0 C(11)-C(1)-C(6)-C(7) 5.4(8) C(2)-C(1)-C(6)-C(5) 0.0 C(11)-C(1)-C(6)-C(5) -174.6(8) C(5)-C(6)-C(7)-C(8) 0.0 C(1)-C(6)-C(7)-C(8) 180.0 C(6)-C(7)-C(8)-C(9) 0.0 C(7)-C(8)-C(9)-C(10) 0.0 C(8)-C(9)-C(10)-C(5) 0.0 C(4)-C(5)-C(10)-C(9) 180.0 C(6)-C(5)-C(10)-C(9) 0.0 C(2)-C(1)-C(11)-O(1) 40.6(11) C(6)-C(1)-C(11)-O(1) -144.6(7) C(2)-C(1)-C(11)-C(12) -81.4(10) C(6)-C(1)-C(11)-C(12) 93.4(9) O(1)-C(11)-C(12)-C(13) 179.3(9) C(1)-C(11)-C(12)-C(13) -62.7(12) O(1)-C(11)-C(12)-C(17) 54.8(14) C(1)-C(11)-C(12)-C(17) 172.9(10) C(17)-C(12)-C(13)-O(2) -138.7(11) C(11)-C(12)-C(13)-O(2) 98.2(12) C(17)-C(12)-C(13)-C(14) 44.5(13) C(11)-C(12)-C(13)-C(14) −78.7(12) O(2)-C(13)-C(14)-C(15) 168.5(12) C(12)-C(13)-C(14)-C(15) -14.7(15) O(2)-C(13)-C(14)-C(18) -13.9(17) C(12)-C(13)-C(14)-C(18) 162.9(11)

9.Appendix

295

C(18)-C(14)-C(15)-C(16) 176.4(14) C(13)-C(14)-C(15)-C(16) -6(2) C(14)-C(15)-C(16)-C(17) -3(2) C(13)-C(12)-C(17)-C(19) 68.8(14) C(11)-C(12)-C(17)-C(19) -168.2(11) C(13)-C(12)-C(17)-C(16) -51.1(13) C(11)-C(12)-C(17)-C(16) 72.0(13) C(15)-C(16)-C(17)-C(12) 32.1(16) C(15)-C(16)-C(17)-C(19) -86.3(13) C(12)-C(17)-C(19)-C(20) -59(2) C(16)-C(17)-C(19)-C(20) 61.6(19) C(12)-C(17)-C(19)-C(21) 108.8(19) C(16)-C(17)-C(19)-C(21) -130.7(19) C(27)-C(22)-C(23)-C(24) 0.0 C(32)-C(22)-C(23)-C(24) 179.5(9) C(22)-C(23)-C(24)-C(25) 0.0 C(23)-C(24)-C(25)-C(26) 0.0 C(24)-C(25)-C(26)-C(27) 0.0 C(24)-C(25)-C(26)-C(31) 180.0 C(25)-C(26)-C(27)-C(28) 180.0 C(31)-C(26)-C(27)-C(28) 0.0 C(25)-C(26)-C(27)-C(22) 0.0 C(31)-C(26)-C(27)-C(22) 180.0 C(23)-C(22)-C(27)-C(26) 0.0 C(32)-C(22)-C(27)-C(26) -179.6(8) C(23)-C(22)-C(27)-C(28) 180.0 C(32)-C(22)-C(27)-C(28) 0.4(8) C(26)-C(27)-C(28)-C(29) 0.0 C(22)-C(27)-C(28)-C(29) 180.0 C(27)-C(28)-C(29)-C(30) 0.0 C(28)-C(29)-C(30)-C(31) 0.0 C(29)-C(30)-C(31)-C(26) 0.0 C(25)-C(26)-C(31)-C(30) 180.0 C(27)-C(26)-C(31)-C(30) 0.0 C(23)-C(22)-C(32)-O(3) 34.3(13) C(27)-C(22)-C(32)-O(3) -146.2(9) C(23)-C(22)-C(32)-C(33) -76.7(14) C(27)-C(22)-C(32)-C(33) 102.9(13) O(3)-C(32)-C(33)-C(34) -171.0(9)

9.Appendix

296

C(22)-C(32)-C(33)-C(34) -72.6(16) O(3)-C(32)-C(33)-C(38) 66.5(14) C(22)-C(32)-C(33)-C(38) 164.9(10) C(32)-C(33)-C(34)-O(4) 100.8(12) C(38)-C(33)-C(34)-O(4) -133.3(10) C(32)-C(33)-C(34)-C(35) -76.1(12) C(38)-C(33)-C(34)-C(35) 49.8(12) O(4)-C(34)-C(35)-C(36) 165.3(11) C(33)-C(34)-C(35)-C(36) -18.0(15) O(4)-C(34)-C(35)-C(39) -16.1(16) C(33)-C(34)-C(35)-C(39) 160.6(11) C(34)-C(35)-C(36)-C(37) -4.8(15) C(39)-C(35)-C(36)-C(37) 176.6(10) C(35)-C(36)-C(37)-C(38) -8.9(13) C(36)-C(37)-C(38)-C(40) -87.0(11) C(36)-C(37)-C(38)-C(33) 41.5(11) C(32)-C(33)-C(38)-C(37) 65.9(14) C(34)-C(33)-C(38)-C(37) -59.9(11) C(32)-C(33)-C(38)-C(40) -164.7(13) C(34)-C(33)-C(38)-C(40) 69.5(11) C(37)-C(38)-C(40)-C(41) 26.2(16) C(33)-C(38)-C(40)-C(41) -99.4(13) C(37)-C(38)-C(40)-C(42) -164.2(12) C(33)-C(38)-C(40)-C(42) 70.2(15) C(48)-C(43)-C(44)-C(45) 0.0 C(53)-C(43)-C(44)-C(45) 179.3(8) C(43)-C(44)-C(45)-C(46) 0.0 C(44)-C(45)-C(46)-C(47) 0.0 C(45)-C(46)-C(47)-C(48) 0.0 C(45)-C(46)-C(47)-C(52) 180.0 C(46)-C(47)-C(48)-C(49) 180.0 C(52)-C(47)-C(48)-C(49) 0.0 C(46)-C(47)-C(48)-C(43) 0.0 C(52)-C(47)-C(48)-C(43) 180.0 C(44)-C(43)-C(48)-C(49) 180.0 C(53)-C(43)-C(48)-C(49) 0.7(8) C(44)-C(43)-C(48)-C(47) 0.0 C(53)-C(43)-C(48)-C(47) -179.3(8) C(47)-C(48)-C(49)-C(50) 0.0

9.Appendix

297

C(43)-C(48)-C(49)-C(50) 180.0 C(48)-C(49)-C(50)-C(51) 0.0 C(49)-C(50)-C(51)-C(52) 0.0 C(50)-C(51)-C(52)-C(47) 0.0 C(46)-C(47)-C(52)-C(51) 180.0 C(48)-C(47)-C(52)-C(51) 0.0 C(44)-C(43)-C(53)-O(5) 34.5(11) C(48)-C(43)-C(53)-O(5) -146.2(7) C(44)-C(43)-C(53)-C(54) -80.9(11) C(48)-C(43)-C(53)-C(54) 98.4(11) O(5)-C(53)-C(54)-C(59) 60.8(14) C(43)-C(53)-C(54)-C(59) 173.3(10) O(5)-C(53)-C(54)-C(55) -174.3(9) C(43)-C(53)-C(54)-C(55) -61.7(13) C(59)-C(54)-C(55)-O(6) -131.9(11) C(53)-C(54)-C(55)-O(6) 100.4(12) C(59)-C(54)-C(55)-C(56) 45.4(12) C(53)-C(54)-C(55)-C(56) -82.3(12) O(6)-C(55)-C(56)-C(57) 164.8(12) C(54)-C(55)-C(56)-C(57) -12.3(14) O(6)-C(55)-C(56)-C(60) -19.9(16) C(54)-C(55)-C(56)-C(60) 163.0(10) C(55)-C(56)-C(57)-C(58) -7.7(17) C(60)-C(56)-C(57)-C(58) 177.3(11) C(56)-C(57)-C(58)-C(59) -6.6(17) C(57)-C(58)-C(59)-C(61) -87.9(13) C(57)-C(58)-C(59)-C(54) 39.8(13) C(53)-C(54)-C(59)-C(58) 65.9(14) C(55)-C(54)-C(59)-C(58) -58.2(12) C(53)-C(54)-C(59)-C(61) -165.4(12) C(55)-C(54)-C(59)-C(61) 70.5(12) C(58)-C(59)-C(61)-C(62) -133.6(18) C(54)-C(59)-C(61)-C(62) 100.1(19) C(58)-C(59)-C(61)-C(63) 20.6(19) C(54)-C(59)-C(61)-C(63) -105.8(17) C(69)-C(64)-C(65)-C(66) 0.0 C(74)-C(64)-C(65)-C(66) -179.2(10) C(64)-C(65)-C(66)-C(67) 0.0 C(65)-C(66)-C(67)-C(68) 0.0

9.Appendix

298

C(66)-C(67)-C(68)-C(69) 0.0 C(66)-C(67)-C(68)-C(73) 180.0 C(67)-C(68)-C(69)-C(70) 180.0 C(73)-C(68)-C(69)-C(70) 0.0 C(67)-C(68)-C(69)-C(64) 0.0 C(73)-C(68)-C(69)-C(64) 180.0 C(65)-C(64)-C(69)-C(70) 180.0 C(74)-C(64)-C(69)-C(70) -0.7(9) C(65)-C(64)-C(69)-C(68) 0.0 C(74)-C(64)-C(69)-C(68) 179.3(9) C(68)-C(69)-C(70)-C(71) 0.0 C(64)-C(69)-C(70)-C(71) 180.0 C(69)-C(70)-C(71)-C(72) 0.0 C(70)-C(71)-C(72)-C(73) 0.0 C(71)-C(72)-C(73)-C(68) 0.0 C(67)-C(68)-C(73)-C(72) 180.0 C(69)-C(68)-C(73)-C(72) 0.0 C(65)-C(64)-C(74)-O(7) 35.1(12) C(69)-C(64)-C(74)-O(7) -144.2(10) C(65)-C(64)-C(74)-C(75) −78.0(13) C(69)-C(64)-C(74)-C(75) 102.8(12) O(7)-C(74)-C(75)-C(76) -173.9(12) C(64)-C(74)-C(75)-C(76) -76.2(14) O(7)-C(74)-C(75)-C(80) 63.3(18) C(64)-C(74)-C(75)-C(80) 161.0(10) C(74)-C(75)-C(76)-O(8) 103.1(14) C(80)-C(75)-C(76)-O(8) -134.6(11) C(74)-C(75)-C(76)-C(77) -73.3(14) C(80)-C(75)-C(76)-C(77) 49.0(13) O(8)-C(76)-C(77)-C(78) 162.8(11) C(75)-C(76)-C(77)-C(78) -20.9(16) O(8)-C(76)-C(77)-C(81) -15.3(15) C(75)-C(76)-C(77)-C(81) 161.1(11) C(76)-C(77)-C(78)-C(79) -1.4(17) C(81)-C(77)-C(78)-C(79) 176.5(11) C(77)-C(78)-C(79)-C(80) -7.3(17) C(76)-C(75)-C(80)-C(82) 69.0(13) C(74)-C(75)-C(80)-C(82) -166.1(13) C(76)-C(75)-C(80)-C(79) -53.9(12)

9.Appendix

299

C(74)-C(75)-C(80)-C(79) 71.0(14) C(78)-C(79)-C(80)-C(82) -87.3(12) C(78)-C(79)-C(80)-C(75) 34.6(13) C(75)-C(80)-C(82)-C(83) -83.7(18) C(79)-C(80)-C(82)-C(83) 37.9(18) C(75)-C(80)-C(82)-C(84) 76.9(17) C(79)-C(80)-C(82)-C(84) -161.5(14) ________________________________________________________________ Symmetry transformations used to generate equivalent atoms: Table 7. Hydrogen bonds for s3965qa [Å and °]. D-H...A d(D-H) d(H...A) d(D...A) <(DHA) O(1)-H(1)...O(8) 0.84 1.92 2.742(12) 165.6 O(5)-H(5)...O(4) 0.84 2.66 2.783(11) 89.7 O(7)-H(7A)...O(2)#1 0.84 2.03 2.781(13) 148.1 Symmetry transformations used to generate equivalent atoms: #1 x,y-1,z

9.Appendix

300


Table 1. Crystal data and structure refinement for s3921ma. Identification code s3921ma Empirical formula C17H17NO3 Formula weight 283.32 Temperature 180(2) K Wavelength 1.54178 A Crystal system, space group Monoclinic, P2(1) Unit cell dimensions a = 13.7951(2) A alpha = 90 deg. b = 6.60970(10) A beta=90.6450(10) deg. c = 15.9700(2) A gamma = 90 deg. Volume 1456.08(4) A^3 Z, Calculated density 4, 1.292 Mg/m^3 Absorption coefficient 0.721 mm^-1 F(000) 600 Crystal size 0.29 x 0.28 x 0.06 mm Theta range for data collection 2.77 to 69.63 deg. Limiting indices -15<=h<=16, -7<=k<=8, -13<=l<=19 Reflections collected / unique 6846 /4547 [R(int) =0.0186] Completeness to theta = 66.60 98.2 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.9580 and 0.821397

(S)(S)

(Z) O

(E)

NO2

9.Appendix

301

Refinement method Full-matrix least-squares on F^2 Data / restraints / parameters 4547 / 1 / 383 Goodness-of-fit on F^2 1.022 Final R indices [I>2sigma(I)] R1 = 0.0296, wR2 = 0.0768 R indices (all data) R1 = 0.0309, wR2 = 0.0779 Absolute structure parameter 0.10(15) Largest diff. peak and hole 0.157 and -0.165 e.A^-3 Table 2. Atomic coordinates ( x 10^4) and equivalent isotropic displacement parameters (A^2 x 10^3) for s3921ma. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x y z U(eq) C(1) 7630(1) 8993(2) 9877(1) 23(1) C(2) 7179(1) 9534(2) 10697(1) 25(1) C(3) 6740(1) 7862(3) 11172(1) 28(1) C(4) 6778(1) 5982(3) 10866(1) 30(1) C(5) 7132(1) 5454(2) 10006(1) 29(1) C(6) 7128(1) 7288(2) 9415(1) 24(1) C(7) 8474(1) 9873(2) 9702(1) 26(1) C(8) 9119(1) 9466(2) 8992(1) 25(1) C(9) 8786(1) 9233(3) 8168(1) 27(1) C(10) 9426(1) 8879(2) 7524(1) 27(1) C(11)10401(1) 8753(2) 7711(1) 26(1) C(12) 10766(1) 8972(3) 8517(1) 28(1) C(13) 10118(1) 9352(3) 9151(1) 28(1) C(14) 6315(1) 8389(3) 12010(1) 38(1) C(15) 6117(1) 7928(3) 9106(1) 27(1) C(16) 5312(1) 6950(3) 9300(1) 39(1) C(17) 6090(1) 9733(3) 8540(1) 42(1) C(18) 2351(1) 1773(2) 4821(1) 24(1) C(19) 2769(1) 1760(3) 3955(1) 27(1) C(20) 3209(1) -146(3) 3662(1) 28(1) C(21) 3156(1) -1804(3) 4138(1) 32(1) C(22) 2744(1) -1837(3) 5002(1) 31(1) C(23) 2783(1) 260(2) 5422(1) 26(1) C(24) 1603(1) 3008(2) 4967(1) 27(1) C(25) 1077(1) 3263(2) 5759(1) 27(1) C(26) 69(1) 3468(3) 5732(1) 32(1) C(27) -455(1) 3695(3) 6462(1) 32(1) C(28) 41(1) 3779(2) 7218(1) 28(1) C(29) 1040(1) 3626(3) 7268(1) 30(1) C(30) 1553(1) 3371(3) 6533(1) 30(1)

9.Appendix

302

C(31) 3653(1) -128(3) 2807(1) 40(1) C(32) 3796(1) 855(3) 5729(1) 29(1) C(33) 4171(1) 2647(3) 5583(1) 40(1) C(34) 4322(1) -688(4) 6256(1) 45(1) N(1) 11084(1) 8419(2) 7028(1) 30(1) N(2) -512(1) 4037(2) 7993(1) 33(1) O(1) 11948(1) 8285(2) 7203(1) 42(1) O(2) 10759(1) 8289(2) 6312(1) 40(1) O(3) 7229(1) 11252(2) 10982(1) 34(1) O(4) -58(1) 4179(2) 8654(1) 44(1) O(5) -1396(1) 4100(2) 7941(1) 46(1) O(6) 2707(1) 3246(2) 3499(1) 37(1) Table 3. Bond lengths [A] and angles [deg] for s3921ma. C(1)-C(7) 1.333(2) C(1)-C(2) 1.4998(19) C(1)-C(6) 1.511(2) C(2)-O(3) 1.224(2) C(2)-C(3) 1.474(2) C(3)-C(4) 1.336(3) C(3)-C(14) 1.508(2) C(4)-C(5) 1.504(2) C(4)-H(4) 0.9500 C(5)-C(6) 1.537(2) C(5)-H(5A) 0.9900 C(5)-H(5B) 0.9900 C(6)-C(15) 1.5337(19) C(6)-H(6) 1.0000 C(7)-C(8) 1.474(2) C(7)-H(7) 0.9500 C(8)-C(9) 1.397(2) C(8)-C(13) 1.401(2) C(9)-C(10) 1.383(2) C(9)-H(9) 0.9500 C(10)-C(11) 1.378(2) C(10)-H(10) 0.9500 C(11)-C(12) 1.385(2) C(11)-N(1) 1.4648(18) C(12)-C(13) 1.381(2) C(12)-H(12) 0.9500 C(13)-H(13) 0.9500 C(14)-H(14A) 0.9800 C(14)-H(14B) 0.9800 C(14)-H(14C) 0.9800 C(15)-C(16) 1.326(2) C(15)-C(17) 1.497(2) C(16)-H(16A) 0.9500 C(16)-H(16B) 0.9500 C(17)-H(17A) 0.9800 C(17)-H(17B) 0.9800

9.Appendix

303

C(17)-H(17C) 0.9800 C(18)-C(24) 1.338(2) C(18)-C(19) 1.504(2) C(18)-C(23) 1.506(2) C(19)-O(6) 1.226(2) C(19)-C(20) 1.477(2) C(20)-C(21) 1.337(2) C(20)-C(31) 1.502(2) C(21)-C(22) 1.499(2) C(21)-H(21) 0.9500 C(22)-C(23) 1.541(2) C(22)-H(22A) 0.9900 C(22)-H(22B) 0.9900 C(23)-C(32) 1.527(2) C(23)-H(23) 1.0000 C(24)-C(25) 1.474(2) C(24)-H(24) 0.9500 C(25)-C(30) 1.395(2) C(25)-C(26) 1.397(2) C(26)-C(27) 1.387(2) C(26)-H(26) 0.9500 C(27)-C(28) 1.382(2) C(27)-H(27) 0.9500 C(28)-C(29) 1.383(2) C(28)-N(2) 1.4709(19) C(29)-C(30) 1.388(2) C(29)-H(29) 0.9500 C(30)-H(30) 0.9500 C(31)-H(31A) 0.9800 C(31)-H(31B) 0.9800 C(31)-H(31C) 0.9800 C(32)-C(33) 1.314(3) C(32)-C(34) 1.504(2) C(33)-H(33A) 0.9500 C(33)-H(33B) 0.9500 C(34)-H(34A) 0.9800 C(34)-H(34B) 0.9800 C(34)-H(34C) 0.9800 N(1)-O(1) 1.2248(17) N(1)-O(2) 1.2277(17) N(2)-O(5) 1.2221(19) N(2)-O(4) 1.2249(18) C(7)-C(1)-C(2) 116.86(13) C(7)-C(1)-C(6) 128.21(13) C(2)-C(1)-C(6) 114.34(12) O(3)-C(2)-C(3) 121.73(13) O(3)-C(2)-C(1) 121.55(14) C(3)-C(2)-C(1) 116.55(14) C(4)-C(3)-C(2) 119.42(14) C(4)-C(3)-C(14) 123.82(16)

9.Appendix

304

C(2)-C(3)-C(14) 116.68(15) C(3)-C(4)-C(5) 124.36(14) C(3)-C(4)-H(4) 117.8 C(5)-C(4)-H(4) 117.8 C(4)-C(5)-C(6) 112.28(13) C(4)-C(5)-H(5A) 109.1 C(6)-C(5)-H(5A) 109.1 C(4)-C(5)-H(5B) 109.1 C(6)-C(5)-H(5B) 109.1 H(5A)-C(5)-H(5B) 107.9 C(1)-C(6)-C(15) 111.21(12) C(1)-C(6)-C(5) 106.84(11) C(15)-C(6)-C(5) 114.32(13) C(1)-C(6)-H(6) 108.1 C(15)-C(6)-H(6) 108.1 C(5)-C(6)-H(6) 108.1 C(1)-C(7)-C(8) 128.20(13) C(1)-C(7)-H(7) 115.9 C(8)-C(7)-H(7) 115.9 C(9)-C(8)-C(13) 118.54(13) C(9)-C(8)-C(7) 123.29(13) C(13)-C(8)-C(7) 118.15(13) C(10)-C(9)-C(8) 120.83(13) C(10)-C(9)-H(9) 119.6 C(8)-C(9)-H(9) 119.6 C(11)-C(10)-C(9) 118.69(13) C(11)-C(10)-H(10) 120.7 C(9)-C(10)-H(10) 120.7 C(10)-C(11)-C(12) 122.55(13) C(10)-C(11)-N(1) 118.90(13) C(12)-C(11)-N(1) 118.54(13) C(13)-C(12)-C(11) 118.01(13) C(13)-C(12)-H(12) 121.0 C(11)-C(12)-H(12) 121.0 C(12)-C(13)-C(8) 121.36(13) C(12)-C(13)-H(13) 119.3 C(8)-C(13)-H(13) 119.3 C(3)-C(14)-H(14A) 109.5 C(3)-C(14)-H(14B) 109.5 H(14A)-C(14)-H(14B) 109.5 C(3)-C(14)-H(14C) 109.5 H(14A)-C(14)-H(14C) 109.5 H(14B)-C(14)-H(14C) 109.5 C(16)-C(15)-C(17) 120.99(15) C(16)-C(15)-C(6) 123.46(15) C(17)-C(15)-C(6) 115.53(13) C(15)-C(16)-H(16A) 120.0 C(15)-C(16)-H(16B) 120.0 H(16A)-C(16)-H(16B) 120.0 C(15)-C(17)-H(17A) 109.5 C(15)-C(17)-H(17B) 109.5

9.Appendix

305

H(17A)-C(17)-H(17B) 109.5 C(15)-C(17)-H(17C) 109.5 H(17A)-C(17)-H(17C) 109.5 H(17B)-C(17)-H(17C) 109.5 C(24)-C(18)-C(19) 117.99(13) C(24)-C(18)-C(23) 126.43(13) C(19)-C(18)-C(23) 115.42(12) O(6)-C(19)-C(20) 121.39(13) O(6)-C(19)-C(18) 121.10(14) C(20)-C(19)-C(18) 117.42(13) C(21)-C(20)-C(19) 119.51(13) C(21)-C(20)-C(31) 123.32(15) C(19)-C(20)-C(31) 117.09(14) C(20)-C(21)-C(22) 124.08(15) C(20)-C(21)-H(21) 118.0 C(22)-C(21)-H(21) 118.0 C(21)-C(22)-C(23) 112.08(13) C(21)-C(22)-H(22A) 109.2 C(23)-C(22)-H(22A) 109.2 C(21)-C(22)-H(22B) 109.2 C(23)-C(22)-H(22B) 109.2 H(22A)-C(22)-H(22B) 107.9 C(18)-C(23)-C(32) 112.82(13) C(18)-C(23)-C(22) 107.93(12) C(32)-C(23)-C(22) 113.53(13) C(18)-C(23)-H(23) 107.4 C(32)-C(23)-H(23) 107.4 C(22)-C(23)-H(23) 107.4 C(18)-C(24)-C(25) 127.42(14) C(18)-C(24)-H(24) 116.3 C(25)-C(24)-H(24) 116.3 C(30)-C(25)-C(26) 118.74(13) C(30)-C(25)-C(24) 122.33(13) C(26)-C(25)-C(24) 118.90(13) C(27)-C(26)-C(25) 120.86(14) C(27)-C(26)-H(26) 119.6 C(25)-C(26)-H(26) 119.6 C(28)-C(27)-C(26) 118.70(14) C(28)-C(27)-H(27) 120.7 C(26)-C(27)-H(27) 120.7 C(27)-C(28)-C(29) 122.12(14) C(27)-C(28)-N(2) 118.87(14) C(29)-C(28)-N(2) 119.01(13) C(28)-C(29)-C(30) 118.47(13) C(28)-C(29)-H(29) 120.8 C(30)-C(29)-H(29) 120.8 C(29)-C(30)-C(25) 121.07(14) C(29)-C(30)-H(30) 119.5 C(25)-C(30)-H(30) 119.5 C(20)-C(31)-H(31A) 109.5 C(20)-C(31)-H(31B) 109.5

9.Appendix

306

H(31A)-C(31)-H(31B) 109.5 C(20)-C(31)-H(31C) 109.5 H(31A)-C(31)-H(31C) 109.5 H(31B)-C(31)-H(31C) 109.5 C(33)-C(32)-C(34) 121.50(16) C(33)-C(32)-C(23) 122.41(15) C(34)-C(32)-C(23) 115.99(15) C(32)-C(33)-H(33A) 120.0 C(32)-C(33)-H(33B) 120.0 H(33A)-C(33)-H(33B) 120.0 C(32)-C(34)-H(34A) 109.5 C(32)-C(34)-H(34B) 109.5 H(34A)-C(34)-H(34B) 109.5 C(32)-C(34)-H(34C) 109.5 H(34A)-C(34)-H(34C) 109.5 H(34B)-C(34)-H(34C) 109.5 O(1)-N(1)-O(2) 123.48(12) O(1)-N(1)-C(11) 118.31(12) O(2)-N(1)-C(11) 118.21(13) O(5)-N(2)-O(4) 123.72(14) O(5)-N(2)-C(28) 118.30(13) O(4)-N(2)-C(28) 117.99(14) _____________________________________________________________ Symmetry transformations used to generate equivalent atoms: Table 4. Anisotropic displacement parameters (A^2 x 10^3) for s3921ma. The anisotropic displacement factor exponent takes the form: -2 pi^2 [ h^2 a*^2 U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 U23 U13 U12 C(1) 22(1) 24(1) 23(1) 1(1) 0(1) 2(1) C(2) 18(1) 30(1) 25(1) -1(1) -2(1) 1(1) C(3) 20(1) 39(1) 25(1) 5(1) -1(1) 0(1) C(4) 26(1) 34(1) 32(1) 10(1) 0(1) 1(1) C(5) 27(1) 24(1) 36(1) 2(1) 0(1) 2(1) C(6) 21(1) 28(1) 23(1) -1(1) 1(1) 0(1) C(7) 27(1) 27(1) 24(1) -2(1) 0(1 -1(1) C(8) 25(1) 23(1) 27(1) 2(1) 3(1) -4(1) C(9) 20(1) 32(1) 29(1) 3(1) -2(1) -3(1) C(10) 30(1) 30(1) 22(1) 4(1) -1(1) -4(1) C(11) 28(1) 26(1) 24(1) 4(1) 5(1) -3(1) C(12) 22(1) 34(1) 27(1) 4(1) 0(1) -2(1) C(13) 26(1) 35(1) 22(1) 0(1) -1(1) -3(1) C(14) 33(1) 54(1) 26(1) 3(1) 5(1) -3(1) C(15) 24(1) 35(1) 23(1) -6(1) -1(1) 3(1) C(16) 24(1) 50(1) 44(1) 3(1) -4(1) -2(1) C(17) 33(1) 44(1) 48(1) 11(1) -6(1) 4(1) C(18) 20(1) 26(1) 24(1) -2(1) -1(1) -2(1) C(19) 21(1) 31(1) 28(1) 2(1) -1(1) -1(1)

9.Appendix

307

C(20) 20(1) 37(1) 29(1) -4(1) 1(1) 0(1) C(21) 25(1) 31(1) 40(1) -8(1) 2(1) 3(1) C(22) 28(1) 28(1) 38(1) 4(1) 4(1) 2(1) C(23) 21(1) 31(1) 25(1) 2(1) 6(1) 3(1) C(24) 25(1) 29(1) 26(1) 0(1) -5(1) 1(1) C(25) 26(1) 25(1) 29(1) -2(1) -1(1) 5(1) C(26) 28(1) 38(1) 29(1) -3(1) -5(1) 7(1) C(27) 24(1) 36(1) 36(1) -2(1) 0(1) 7(1) C(28) 32(1) 23(1) 30(1) 0(1) 4(1) 3(1) C(29) 32(1) 33(1) 26(1) -3(1) -4(1) 3(1) C(30) 24(1) 33(1) 32(1) -4(1) -2(1) 4(1) C(31) 37(1) 52(1) 32(1) -5(1) 9(1) 6(1) C(32) 22(1) 44(1) 22(1) -2(1) 2(1) 6(1) C(33) 27(1) 50(1) 44(1) -3(1) -7(1) -5(1) C(34) 35(1) 62(1) 38(1) 4(1) -6(1) 13(1) N(1) 32(1) 33(1) 25(1) 5(1) 6(1) -4(1) N(2) 41(1) 26(1) 33(1) 0(1) 7(1) 2(1) O(1) 28(1) 64(1) 35(1) 9(1) 10(1) 4(1) O(2) 42(1) 54(1) 23(1) 0(1) 7(1) -6(1) O(3) 38(1) 32(1) 33(1) -7(1) 8(1) -2(1) O(4) 56(1) 50(1) 28(1) -2(1) 5(1) -2(1) O(5) 38(1) 54(1) 47(1) -3(1) 14(1) 9(1) O(6) 44(1) 38(1) 30(1) 6(1) 6(1) 4(1) Table 5. Hydrogen coordinates ( x 10^4) and isotropic displacement parameters (A^2 x 10^3) for s3921ma. x y z U(eq) H(4) 6567 4908 11215 37 H(5A) 6713 4381 9764 35 H(5B) 7799 4910 10052 35 H(6) 7525 6939 8915 29 H(7) 8685 10890 10082 31 H(9) 8110 9318 8049 33 H(10) 9197 8726 6964 33 H(12) 11441 8864 8631 33 H(13) 10355 9541 9706 33 H(14A) 6239 7155 12342 56 H(14B) 5680 9029 11925 56 H(14C) 6748 9328 12306 56 H(16A) 4705 7392 9080 47 H(16B) 5341 5807 9661 47 H(17A) 5415 10118 8429 63 H(17B) 6405 9399 8010 63 H(17C) 6432 10862 8809 63 H(21) 3393 -3038 3915 38 H(22A) 3112 -2817 5350 38 H(22B) 2062 -2300 4972 38 H(23) 2355 209 5923 31 H(24) 1386 3811 4509 32 H(26) -261 3451 5207 38

9.Appendix

308

H(27) -1142 3792 6443 39 H(29) 1367 3695 7794 37 H(30) 2240 3267 6558 36 H(31A) 3885 -1488 2670 61 H(31B) 4198 822 2802 61 H(31C) 3165 293 2392 61 H(33A) 4787 2992 5812 48 H(33B) 3826 3598 5247 48 H(34A) 4883 -53 6530 67 H(34B) 4541 -1801 5900 67 H(34C) 3883 -1215 6683 67 Table 6. Torsion angles [deg] for s3921ma. C(7)-C(1)-C(2)-O(3) 36.9(2) C(6)-C(1)-C(2)-O(3) -151.25(14) C(7)-C(1)-C(2)-C(3) -138.52(14) C(6)-C(1)-C(2)-C(3) 33.37(17) O(3)-C(2)-C(3)-C(4) -174.23(15) C(1)-C(2)-C(3)-C(4) 1.1(2) O(3)-C(2)-C(3)-C(14) 2.7(2) C(1)-C(2)-C(3)-C(14) 178.03(13) C(2)-C(3)-C(4)-C(5) -8.1(2) C(14)-C(3)-C(4)-C(5) 175.25(14) C(3)-C(4)-C(5)-C(6) -18.9(2) C(7)-C(1)-C(6)-C(15) -121.29(16) C(2)-C(1)-C(6)-C(15) 67.93(15) C(7)-C(1)-C(6)-C(5) 113.34(17) C(2)-C(1)-C(6)-C(5) -57.44(16) C(4)-C(5)-C(6)-C(1) 49.27(17) C(4)-C(5)-C(6)-C(15) -74.20(16) C(2)-C(1)-C(7)-C(8) 173.14(15) C(6)-C(1)-C(7)-C(8) 2.6(3) C(1)-C(7)-C(8)-C(9) 44.5(2) C(1)-C(7)-C(8)-C(13) -136.86(17) C(13)-C(8)-C(9)-C(10) 0.5(2) C(7)-C(8)-C(9)-C(10) 179.16(15) C(8)-C(9)-C(10)-C(11) 0.3(2) C(9)-C(10)-C(11)-C(12) -0.2(2) C(9)-C(10)-C(11)-N(1) -178.78(15) C(10)-C(11)-C(12)-C(13) -0.7(2) N(1)-C(11)-C(12)-C(13) 177.89(15) C(11)-C(12)-C(13)-C(8) 1.5(2) C(9)-C(8)-C(13)-C(12) -1.5(2) C(7)-C(8)-C(13)-C(12) 179.84(16) C(1)-C(6)-C(15)-C(16) 123.57(17) C(5)-C(6)-C(15)-C(16) -2.5(2) C(1)-C(6)-C(15)-C(17) 57.95(17) C(5)-C(6)-C(15)-C(17) 179.04(14) C(24)-C(18)-C(19)-O(6) 26.5(2)

9.Appendix

309

C(23)-C(18)-C(19)-O(6) -157.62(14) C(24)-C(18)-C(19)-C(20) -150.16(15) C(23)-C(18)-C(19)-C(20) 25.69(19) O(6)-C(19)-C(20)-C(21) -171.64(15) C(18)-C(19)-C(20)-C(21) 5.0(2) O(6)-C(19)-C(20)-C(31) 5.2(2) C(18)-C(19)-C(20)-C(31) -178.16(13) C(19)-C(20)-C(21)-C(22) -5.4(2) C(31)-C(20)-C(21)-C(22) 178.00(15) C(20)-C(21)-C(22)-C(23) -23.8(2) C(24)-C(18)-C(23)-C(32) -110.62(17) C(19)-C(18)-C(23)-C(32) 73.94(16) C(24)-C(18)-C(23)-C(22) 123.13(16) C(19)-C(18)-C(23)-C(22) -52.30(16) C(21)-C(22)-C(23)-C(18) 50.41(17) C(21)-C(22)-C(23)-C(32) -75.42(16) C(19)-C(18)-C(24)-C(25) 179.63(14) C(23)-C(18)-C(24)-C(25) 4.3(3) C(18)-C(24)-C(25)-C(30) 42.5(2) C(18)-C(24)-C(25)-C(26) -139.53(18) C(30)-C(25)-C(26)-C(27) -2.5(3) C(24)-C(25)-C(26)-C(27) 179.48(17) C(25)-C(26)-C(27)-C(28) 2.0(3) C(26)-C(27)-C(28)-C(29) -0.5(3) C(26)-C(27)-C(28)-N(2) 179.52(15) C(27)-C(28)-C(29)-C(30) -0.3(2) N(2)-C(28)-C(29)-C(30) 179.60(15) C(28)-C(29)-C(30)-C(25) -0.2(3) C(26)-C(25)-C(30)-C(29) 1.5(2) C(24)-C(25)-C(30)-C(29) 179.54(16) C(18)-C(23)-C(32)-C(33) 10.2(2) C(22)-C(23)-C(32)-C(33) 133.39(16) C(18)-C(23)-C(32)-C(34) -173.47(13) C(22)-C(23)-C(32)-C(34) 50.29(17) C(10)-C(11)-N(1)-O(1) 179.14(15) C(12)-C(11)-N(1)-O(1) 2.2(2) C(10)-C(11)-N(1)-O(2) 0.9(2) C(12)-C(11)-N(1)-O(2) 177.79(16) C(27)-C(28)-N(2)-O(5) 2.4(2) C(29)-C(28)-N(2)-O(5) 177.52(16) C(27)-C(28)-N(2)-O(4) 177.48(16) C(29)-C(28)-N(2)-O(4) 2.6(2) Symmetry transformations used to generate equivalent atoms: Table 7. Hydrogen bonds for s3921ma [A and deg.]. D-H...A d(D-H) d(H...A) d(D...A) <(DHA)

9.Appendix

310


Table 1. Crystal data and structure refinement for s4255ma. Identification code s4255ma Empirical formula C16H21NO2 Formula weight 259.34 Temperature 100(2) K Wavelength 1.54178 Å Crystal system Monoclinic Space group P2(1) Unit cell dimensions a = 7.3315(2) Å a= 90°. b = 9.4721(3) Å b= 96.634(2)°. c = 10.0951(3) Å g = 90°. Volume 696.36(4) Å3 Z 2 Density (calculated) 1.237 Mg/m3 Absorption coefficient 0.641 mm-1 F(000) 280 Crystal size 0.19 x 0.16 x 0.09 mm3 Theta range for data collection 4.41 to 72.06°. Index ranges -9<=h<=8, -11<=k<=11, -11<=l<=12 Reflections collected 7189

O

N

OH

HH

9.Appendix

311

Independent reflections 2587 [R(int) = 0.0196] Completeness to theta = 67.00° 97.7 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.9445 and 0.869291 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 2587 / 1 / 175 Goodness-of-fit on F2 1.056 Final R indices [I>2sigma(I)] R1 = 0.0253, wR2 = 0.0669 R indices (all data) R1 = 0.0260, wR2 = 0.0676 Absolute structure parameter -0.05(16) Largest diff. peak and hole 0.220 and -0.154 e.Å-3 Table 2. Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103) for s4255ma. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x y z U(eq) C(1) 10526(2) 2555(1) 1821(1) 16(1) C(2) 10502(2) 781(1) 3371(1) 22(1) C(3) 11076(2) 1685(2) 4419(1) 24(1) C(4) 11417(2) 3077(1) 4122(1) 24(1) C(5) 11138(2) 3525(1) 2810(1) 20(1) C(6) 10210(2) 2968(1) 359(1) 14(1) C(7) 8261(1) 2632(1) -314(1) 14(1) C(8) 8298(2) 2509(1) -1819(1) 15(1) C(9) 6466(2) 2229(1) -2639(1) 18(1) C(10) 5085(2) 3356(2) -2296(1) 22(1) C(11) 4911(2) 3358(1) -811(1) 19(1) C(12) 6746(2) 3677(1) 36(1) 15(1) C(13) 6608(2) 2137(2) -4125(1) 25(1) C(14) 6511(2) 3638(1) 1510(1) 17(1) C(15) 6944(2) 4742(1) 2290(1) 23(1) C(16) 5740(2) 2309(2) 2046(1) 22(1) N(1) 10240(1) 1190(1) 2088(1) 18(1) O(1) 10659(1) 4417(1) 257(1) 18(1) O(2) 9717(1) 2604(1) -2330(1) 19(1) Table 3. Bond lengths [Å] and angles [°] for s4255ma. C(1)-N(1) 1.3425(17) C(1)-C(5) 1.3922(17)

9.Appendix

312

C(1)-C(6) 1.5181(15) C(2)-N(1) 1.3445(17) C(2)-C(3) 1.387(2) C(2)-H(2) 0.9500 C(3)-C(4) 1.382(2) C(3)-H(3) 0.9500 C(4)-C(5) 1.3834(18) C(4)-H(4) 0.9500 C(5)-H(5) 0.9500 C(6)-O(1) 1.4178(14) C(6)-C(7) 1.5430(15) C(6)-H(6) 1.0000 C(7)-C(8) 1.5262(15) C(7)-C(12) 1.5591(15) C(7)-H(7) 1.0000 C(8)-O(2) 1.2165(15) C(8)-C(9) 1.5180(15) C(9)-C(13) 1.5189(16) C(9)-C(10) 1.5376(18) C(9)-H(9) 1.0000 C(10)-C(11) 1.5192(17) C(10)-H(10A) 0.9900 C(10)-H(10B) 0.9900 C(11)-C(12) 1.5388(15) C(11)-H(11A) 0.9900 C(11)-H(11B) 0.9900 C(12)-C(14) 1.5173(16) C(12)-H(12) 1.0000 C(13)-H(13A) 0.9800 C(13)-H(13B) 0.9800 C(13)-H(13C) 0.9800 C(14)-C(15) 1.3268(18) C(14)-C(16) 1.5054(18) C(15)-H(15A) 0.9500 C(15)-H(15B) 0.9500 C(16)-H(16A) 0.9800 C(16)-H(16B) 0.9800 C(16)-H(16C) 0.9800 O(1)-H(1) 0.8400

9.Appendix

313

N(1)-C(1)-C(5) 122.47(11) N(1)-C(1)-C(6) 115.69(10) C(5)-C(1)-C(6) 121.81(11) N(1)-C(2)-C(3) 123.64(13) N(1)-C(2)-H(2) 118.2 C(3)-C(2)-H(2) 118.2 C(4)-C(3)-C(2) 118.01(12) C(4)-C(3)-H(3) 121.0 C(2)-C(3)-H(3) 121.0 C(3)-C(4)-C(5) 119.35(12) C(3)-C(4)-H(4) 120.3 C(5)-C(4)-H(4) 120.3 C(4)-C(5)-C(1) 118.93(12) C(4)-C(5)-H(5) 120.5 C(1)-C(5)-H(5) 120.5 O(1)-C(6)-C(1) 108.03(9) O(1)-C(6)-C(7) 112.26(9) C(1)-C(6)-C(7) 113.90(9) O(1)-C(6)-H(6) 107.5 C(1)-C(6)-H(6) 107.5 C(7)-C(6)-H(6) 107.5 C(8)-C(7)-C(6) 109.40(9) C(8)-C(7)-C(12) 111.69(9) C(6)-C(7)-C(12) 114.61(9) C(8)-C(7)-H(7) 106.9 C(6)-C(7)-H(7) 106.9 C(12)-C(7)-H(7) 106.9 O(2)-C(8)-C(9) 121.88(10) O(2)-C(8)-C(7) 122.05(10) C(9)-C(8)-C(7) 116.06(9) C(8)-C(9)-C(13) 113.02(10) C(8)-C(9)-C(10) 108.68(10) C(13)-C(9)-C(10) 112.64(10) C(8)-C(9)-H(9) 107.4 C(13)-C(9)-H(9) 107.4 C(10)-C(9)-H(9) 107.4 C(11)-C(10)-C(9) 110.81(10) C(11)-C(10)-H(10A) 109.5

9.Appendix

314

C(9)-C(10)-H(10A) 109.5 C(11)-C(10)-H(10B) 109.5 C(9)-C(10)-H(10B) 109.5 H(10A)-C(10)-H(10B) 108.1 C(10)-C(11)-C(12) 112.38(9) C(10)-C(11)-H(11A) 109.1 C(12)-C(11)-H(11A) 109.1 C(10)-C(11)-H(11B) 109.1 C(12)-C(11)-H(11B) 109.1 H(11A)-C(11)-H(11B) 107.9 C(14)-C(12)-C(11) 110.41(9) C(14)-C(12)-C(7) 111.81(9) C(11)-C(12)-C(7) 110.42(9) C(14)-C(12)-H(12) 108.0 C(11)-C(12)-H(12) 108.0 C(7)-C(12)-H(12) 108.0 C(9)-C(13)-H(13A) 109.5 C(9)-C(13)-H(13B) 109.5 H(13A)-C(13)-H(13B) 109.5 C(9)-C(13)-H(13C) 109.5 H(13A)-C(13)-H(13C) 109.5 H(13B)-C(13)-H(13C) 109.5 C(15)-C(14)-C(16) 121.30(11) C(15)-C(14)-C(12) 121.08(12) C(16)-C(14)-C(12) 117.61(10) C(14)-C(15)-H(15A) 120.0 C(14)-C(15)-H(15B) 120.0 H(15A)-C(15)-H(15B) 120.0 C(14)-C(16)-H(16A) 109.5 C(14)-C(16)-H(16B) 109.5 H(16A)-C(16)-H(16B) 109.5 C(14)-C(16)-H(16C) 109.5 H(16A)-C(16)-H(16C) 109.5 H(16B)-C(16)-H(16C) 109.5 C(1)-N(1)-C(2) 117.55(11) C(6)-O(1)-H(1) 109.5 Symmetry transformations used to generate equivalent atoms:

9.Appendix

315

Table 4. Anisotropic displacement parameters (Å2x 103) for s4255ma. The anisotropic displacement factor exponent takes the form: -2p2[ h2 a*2U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 U23 U13 U12 C(1) 10(1) 20(1) 17(1) 0(1) 2(1) 3(1) C(2) 24(1) 22(1) 19(1) 4(1) 4(1) 5(1) C(3) 25(1) 32(1) 15(1) 3(1) 1(1) 9(1) C(4) 23(1) 30(1) 17(1) -5(1) -3(1) 6(1) C(5) 19(1) 20(1) 20(1) -2(1) 0(1) 2(1) C(6) 13(1) 14(1) 16(1) -1(1) 2(1) -1(1) C(7) 12(1) 14(1) 15(1) 1(1) 2(1) 0(1) C(8) 16(1) 12(1) 18(1) 0(1) 2(1) 1(1) C(9) 18(1) 22(1) 15(1) 0(1) 1(1) -1(1) C(10) 16(1) 30(1) 18(1) 2(1) -2(1) 3(1) C(11) 14(1) 26(1) 17(1) -1(1) 2(1) 3(1) C(12) 14(1) 15(1) 17(1) 0(1) 3(1) 2(1) C(13) 23(1) 36(1) 15(1) -1(1) 1(1) -2(1) C(14) 12(1) 22(1) 17(1) -1(1) 2(1) 5(1) C(15) 22(1) 26(1) 19(1) -4(1) 1(1) 5(1) C(16) 22(1) 28(1) 18(1) 1(1) 7(1) -1(1) N(1) 18(1) 20(1) 17(1) 1(1) 2(1) 2(1) O(1) 19(1) 16(1) 17(1) 1(1) 0(1) -4(1) O(2) 17(1) 24(1) 18(1) -1(1) 5(1) -1(1 Table 5. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 10 3) for s4255ma. x y z U(eq) H(2) 10283 -180 3572 26 H(3) 11229 1356 5314 29 H(4) 11840 3721 4812 28 H(5) 11360 4480 2587 24 H(6) 11096 2418 -119 17 H(7) 7927 1679 10 16 H(9) 6012 1295 -2351 22 H(10A) 5498 4298 -2565 26 H(10B) 3870 3164 -2799 26 H(11A) 4459 2424 -553 23 H(11B) 3993 4075 -621 23 H(12) 7132 4654 -185 18 H(13A) 6949 3062 -4455 37 H(13B) 5421 1847 -4593 37

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316

H(13C) 7547 1441 -4287 37 H(15A) 6770 4705 3208 27 H(15B) 7429 5571 1932 27 H(16A) 5785 2379 3018 33 H(16B) 6469 1497 1817 33 H(16C) 4464 2188 1654 33 H(1) 10371 4691 -531 26 Table 6. Torsion angles [°] for s4255ma. N(1)-C(2)-C(3)-C(4) -0.85(19) C(2)-C(3)-C(4)-C(5) 1.50(19) C(3)-C(4)-C(5)-C(1) -0.30(18) N(1)-C(1)-C(5)-C(4) -1.72(18) C(6)-C(1)-C(5)-C(4) -179.35(11) N(1)-C(1)-C(6)-O(1) -177.17(9) C(5)-C(1)-C(6)-O(1) 0.61(14) N(1)-C(1)-C(6)-C(7) 57.38(13) C(5)-C(1)-C(6)-C(7) -124.84(12) O(1)-C(6)-C(7)-C(8) 79.72(12) C(1)-C(6)-C(7)-C(8) -157.10(9) O(1)-C(6)-C(7)-C(12) -46.59(13) C(1)-C(6)-C(7)-C(12) 76.58(12) C(6)-C(7)-C(8)-O(2) 3.32(15) C(12)-C(7)-C(8)-O(2) 131.29(11) C(6)-C(7)-C(8)-C(9) -177.96(9) C(12)-C(7)-C(8)-C(9) -50.00(13) O(2)-C(8)-C(9)-C(13) -1.91(17) C(7)-C(8)-C(9)-C(13) 179.38(10) O(2)-C(8)-C(9)-C(10) -127.72(12) C(7)-C(8)-C(9)-C(10) 53.57(13) C(8)-C(9)-C(10)-C(11) -57.02(13) C(13)-C(9)-C(10)-C(11) 176.94(10) C(9)-C(10)-C(11)-C(12) 60.20(14) C(10)-C(11)-C(12)-C(14) -178.85(10) C(10)-C(11)-C(12)-C(7) -54.69(13) C(8)-C(7)-C(12)-C(14) 171.43(9) C(6)-C(7)-C(12)-C(14) -63.45(13) C(8)-C(7)-C(12)-C(11) 48.08(12)

9.Appendix

317

C(6)-C(7)-C(12)-C(11) 173.20(9) C(11)-C(12)-C(14)-C(15) -123.22(12) C(7)-C(12)-C(14)-C(15) 113.42(12) C(11)-C(12)-C(14)-C(16) 55.82(13) C(7)-C(12)-C(14)-C(16) -67.54(13) C(5)-C(1)-N(1)-C(2) 2.37(17) C(6)-C(1)-N(1)-C(2) -179.87(10) C(3)-C(2)-N(1)-C(1) -1.07(18) Symmetry transformations used to generate equivalent atoms: Table 7. Hydrogen bonds for s4255ma [Å and °]. D-H...A d(D-H) d(H...A) d(D...A) <(DHA) O(1)-H(1)...N(1)#1 0.84 2.13 2.9149(13) 156.1 Symmetry transformations used to generate equivalent atoms: #1 -x+2,y+1/2,-z

9.Appendix

318


Table 1. Crystal data and structure refinement for s3974pat5. Identification code s3974pat5 Empirical formula C16H23NO2 Formula weight 261.35 Temperature 100(2) K Wavelength 1.5418 Å Crystal system Triclinic Space group P 1 Unit cell dimensions a = 8.5624(5) Å a= 89.581(4)°. b = 9.5310(5) Å b= 73.708(5)°. c = 9.5638(5) Å g = 74.791(5)°. Volume 721.06(7) Å3 Z 2 Density (calculated) 1.204 Mg/m3 Absorption coefficient 0.620 mm-1 F(000) 284 Crystal size 0.20 x 0.16 x 0.09 mm3 Theta range for data collection 4.8258 to 74.1558°. Index ranges -10<=h<=10, -11<=k<=9, -11<=l<=11 Reflections collected 7159

O

HH

HO HN

9.Appendix

319

Independent reflections 3677 [R(int) = 0.0837] Completeness to theta = 67.00° 97.2 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 1.00000 and 0.51089 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 3677 / 3 / 352 Goodness-of-fit on F2 1.046 Final R indices [I>2sigma(I)] R1 = 0.0731, wR2 = 0.1797 R indices (all data) R1 = 0.0821, wR2 = 0.1905 Absolute structure parameter 0.0(4) Largest diff. peak and hole 0.450 and -0.456 e.Å-3 Table 2. Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103) for s3974pat5. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x y z U(eq) C(1) 2210(5) 2979(4) 5675(4) 18(1) C(2) -197(5) 4601(5) 5392(5) 21(1) C(3) -1222(5) 4200(5) 6650(5) 24(1) C(4) -470(5) 3166(5) 7449(5) 24(1) C(5) 1263(5) 2530(5) 6952(4) 22(1) C(6) 4107(5) 2363(4) 5140(4) 18(1) C(7) 4845(5) 2422(4) 3492(4) 15(1) C(8) 4120(5) 1549(4) 2624(4) 18(1) C(9) 4769(5) 1496(4) 969(4) 19(1) C(10) 6718(5) 980(5) 551(4) 24(1) C(11) 7455(5) 1922(5) 1322(4) 22(1) C(12) 6810(5) 1915(4) 2982(4) 18(1) C(13) 4071(5) 2943(4) 358(4) 20(1) C(14) 2119(6) 3392(5) 852(5) 30(1) C(15) 4722(6) 2834(5) -1321(5) 28(1) C(16) 7562(5) 2858(5) 3739(5) 25(1) C(17) 4883(5) 7306(4) 3152(4) 18(1) C(18) 7245(5) 7779(4) 3485(4) 21(1) C(19) 8317(5) 6830(5) 2311(5) 22(1) C(20) 7589(6) 6129(5) 1504(5) 26(1) C(21) 5846(5) 6360(5) 1928(4) 22(1) C(22) 2955(5) 7613(4) 3662(4) 17(1) C(23) 2205(5) 8005(4) 5309(4) 16(1) C(24) 2931(5) 6805(4) 6198(4) 18(1)

9.Appendix

320

C(25) 2248(5) 7070(4) 7851(4) 22(1) C(26) 315(6) 7359(6) 8220(5) 30(1) C(27) -456(5) 8649(5) 7449(5) 27(1) C(28) 247(5) 8428(4) 5786(4) 20(1) C(29) 2844(5) 8291(5) 8441(4) 21(1) C(30) 4776(6) 7892(6) 8032(5) 32(1) C(31) 2073(6) 8551(5) 10105(5) 28(1) C(32) -481(6) 9788(5) 5082(6) 28(1) N(1) 1484(4) 4023(4) 4923(4) 19(1) N(2) 5552(4) 8037(4) 3899(4) 20(1) O(1) 4654(3) 953(3) 5633(3) 20(1) O(2) 3104(4) 894(3) 3225(3) 24(1) O(3) 2400(4) 6476(3) 3150(3) 19(1) O(4) 3964(4) 5672(3) 5623(3) 22(1) Table 3. Bond lengths [Å] and angles [°] for s3974pat5. C(1)-N(1) 1.345(5) C(1)-C(5) 1.396(6) C(1)-C(6) 1.511(5) C(2)-N(1) 1.343(5) C(2)-C(3) 1.393(6) C(2)-H(2) 0.9500 C(3)-C(4) 1.379(7) C(3)-H(3) 0.9500 C(4)-C(5) 1.390(6) C(4)-H(4) 0.9500 C(5)-H(5) 0.9500 C(6)-O(1) 1.426(5) C(6)-C(7) 1.531(5) C(6)-H(6) 1.0000 C(7)-C(8) 1.528(5) C(7)-C(12) 1.555(5) C(7)-H(7) 1.0000 C(8)-O(2) 1.214(5) C(8)-C(9) 1.520(5) C(9)-C(13) 1.534(6) C(9)-C(10) 1.544(5) C(9)-H(9) 1.0000 C(10)-C(11) 1.522(6)

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C(10)-H(10A) 0.9900 C(10)-H(10B) 0.9900 C(11)-C(12) 1.529(5) C(11)-H(11A) 0.9900 C(11)-H(11B) 0.9900 C(12)-C(16) 1.523(6) C(12)-H(12) 1.0000 C(13)-C(15) 1.539(5) C(13)-C(14) 1.545(6) C(13)-H(13) 1.0000 C(14)-H(14A) 0.9800 C(14)-H(14B) 0.9800 C(14)-H(14C) 0.9800 C(15)-H(15A) 0.9800 C(15)-H(15B) 0.9800 C(15)-H(15C) 0.9800 C(16)-H(16A) 0.9800 C(16)-H(16B) 0.9800 C(16)-H(16C) 0.9800 C(17)-N(2) 1.330(5) C(17)-C(21) 1.396(6) C(17)-C(22) 1.532(5) C(18)-N(2) 1.346(5) C(18)-C(19) 1.387(6) C(18)-H(18) 0.9500 C(19)-C(20) 1.388(7) C(19)-H(19) 0.9500 C(20)-C(21) 1.389(6) C(20)-H(20) 0.9500 C(21)-H(21) 0.9500 C(22)-O(3) 1.431(5) C(22)-C(23) 1.533(5) C(22)-H(22) 1.0000 C(23)-C(24) 1.525(5) C(23)-C(28) 1.549(5) C(23)-H(23) 1.0000 C(24)-O(4) 1.219(5) C(24)-C(25) 1.521(5) C(25)-C(26) 1.541(6)

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C(25)-C(29) 1.553(6) C(25)-H(25) 1.0000 C(26)-C(27) 1.524(7) C(26)-H(26A) 0.9900 C(26)-H(26B) 0.9900 C(27)-C(28) 1.529(6) C(27)-H(27A) 0.9900 C(27)-H(27B) 0.9900 C(28)-C(32) 1.524(6) C(28)-H(28) 1.0000 C(29)-C(30) 1.531(6) C(29)-C(31) 1.537(5) C(29)-H(29) 1.0000 C(30)-H(30A) 0.9800 C(30)-H(30B) 0.9800 C(30)-H(30C) 0.9800 C(31)-H(31A) 0.9800 C(31)-H(31B) 0.9800 C(31)-H(31C) 0.9800 C(32)-H(32A) 0.9800 C(32)-H(32B) 0.9800 C(32)-H(32C) 0.9800 O(1)-H(1) 0.8400 O(3)-H(3A) 0.8400 N(1)-C(1)-C(5) 121.5(4) N(1)-C(1)-C(6) 117.7(3) C(5)-C(1)-C(6) 120.8(4) N(1)-C(2)-C(3) 123.0(4) N(1)-C(2)-H(2) 118.5 C(3)-C(2)-H(2) 118.5 C(4)-C(3)-C(2) 118.3(4) C(4)-C(3)-H(3) 120.8 C(2)-C(3)-H(3) 120.8 C(3)-C(4)-C(5) 119.2(4) C(3)-C(4)-H(4) 120.4 C(5)-C(4)-H(4) 120.4 C(4)-C(5)-C(1) 119.3(4) C(4)-C(5)-H(5) 120.4

9.Appendix

323

C(1)-C(5)-H(5) 120.4 O(1)-C(6)-C(1) 112.2(3) O(1)-C(6)-C(7) 112.8(3) C(1)-C(6)-C(7) 113.6(3) O(1)-C(6)-H(6) 105.8 C(1)-C(6)-H(6) 105.8 C(7)-C(6)-H(6) 105.8 C(8)-C(7)-C(6) 111.5(3) C(8)-C(7)-C(12) 110.4(3) C(6)-C(7)-C(12) 112.9(3) C(8)-C(7)-H(7) 107.3 C(6)-C(7)-H(7) 107.3 C(12)-C(7)-H(7) 107.3 O(2)-C(8)-C(9) 121.2(4) O(2)-C(8)-C(7) 121.7(3) C(9)-C(8)-C(7) 117.0(3) C(8)-C(9)-C(13) 112.5(3) C(8)-C(9)-C(10) 107.2(3) C(13)-C(9)-C(10) 114.2(3) C(8)-C(9)-H(9) 107.6 C(13)-C(9)-H(9) 107.6 C(10)-C(9)-H(9) 107.6 C(11)-C(10)-C(9) 112.5(3) C(11)-C(10)-H(10A) 109.1 C(9)-C(10)-H(10A) 109.1 C(11)-C(10)-H(10B) 109.1 C(9)-C(10)-H(10B) 109.1 H(10A)-C(10)-H(10B) 107.8 C(10)-C(11)-C(12) 111.8(3) C(10)-C(11)-H(11A) 109.3 C(12)-C(11)-H(11A) 109.3 C(10)-C(11)-H(11B) 109.3 C(12)-C(11)-H(11B) 109.3 H(11A)-C(11)-H(11B) 107.9 C(16)-C(12)-C(11) 111.2(3) C(16)-C(12)-C(7) 111.3(3) C(11)-C(12)-C(7) 110.0(3) C(16)-C(12)-H(12) 108.1 C(11)-C(12)-H(12) 108.1

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C(7)-C(12)-H(12) 108.1 C(9)-C(13)-C(15) 110.9(3) C(9)-C(13)-C(14) 110.7(3) C(15)-C(13)-C(14) 109.8(4) C(9)-C(13)-H(13) 108.5 C(15)-C(13)-H(13) 108.5 C(14)-C(13)-H(13) 108.5 C(13)-C(14)-H(14A) 109.5 C(13)-C(14)-H(14B) 109.5 H(14A)-C(14)-H(14B) 109.5 C(13)-C(14)-H(14C) 109.5 H(14A)-C(14)-H(14C) 109.5 H(14B)-C(14)-H(14C) 109.5 C(13)-C(15)-H(15A) 109.5 C(13)-C(15)-H(15B) 109.5 H(15A)-C(15)-H(15B) 109.5 C(13)-C(15)-H(15C) 109.5 H(15A)-C(15)-H(15C) 109.5 H(15B)-C(15)-H(15C) 109.5 C(12)-C(16)-H(16A) 109.5 C(12)-C(16)-H(16B) 109.5 H(16A)-C(16)-H(16B) 109.5 C(12)-C(16)-H(16C) 109.5 H(16A)-C(16)-H(16C) 109.5 H(16B)-C(16)-H(16C) 109.5 N(2)-C(17)-C(21) 123.0(4) N(2)-C(17)-C(22) 116.2(3) C(21)-C(17)-C(22) 120.7(4) N(2)-C(18)-C(19) 123.9(4) N(2)-C(18)-H(18) 118.0 C(19)-C(18)-H(18) 118.0 C(18)-C(19)-C(20) 117.6(4) C(18)-C(19)-H(19) 121.2 C(20)-C(19)-H(19) 121.2 C(19)-C(20)-C(21) 119.4(4) C(19)-C(20)-H(20) 120.3 C(21)-C(20)-H(20) 120.3 C(20)-C(21)-C(17) 118.5(4) C(20)-C(21)-H(21) 120.8

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C(17)-C(21)-H(21) 120.8 O(3)-C(22)-C(17) 111.9(3) O(3)-C(22)-C(23) 113.3(3) C(17)-C(22)-C(23) 112.9(3) O(3)-C(22)-H(22) 106.0 C(17)-C(22)-H(22) 106.0 C(23)-C(22)-H(22) 106.0 C(24)-C(23)-C(22) 112.1(3) C(24)-C(23)-C(28) 111.6(3) C(22)-C(23)-C(28) 112.2(3) C(24)-C(23)-H(23) 106.9 C(22)-C(23)-H(23) 106.9 C(28)-C(23)-H(23) 106.9 O(4)-C(24)-C(25) 121.4(4) O(4)-C(24)-C(23) 122.1(3) C(25)-C(24)-C(23) 116.4(3) C(24)-C(25)-C(26) 106.2(3) C(24)-C(25)-C(29) 112.2(3) C(26)-C(25)-C(29) 114.7(3) C(24)-C(25)-H(25) 107.8 C(26)-C(25)-H(25) 107.8 C(29)-C(25)-H(25) 107.8 C(27)-C(26)-C(25) 111.7(3) C(27)-C(26)-H(26A) 109.3 C(25)-C(26)-H(26A) 109.3 C(27)-C(26)-H(26B) 109.3 C(25)-C(26)-H(26B) 109.3 H(26A)-C(26)-H(26B) 107.9 C(26)-C(27)-C(28) 112.8(3) C(26)-C(27)-H(27A) 109.0 C(28)-C(27)-H(27A) 109.0 C(26)-C(27)-H(27B) 109.0 C(28)-C(27)-H(27B) 109.0 H(27A)-C(27)-H(27B) 107.8 C(32)-C(28)-C(27) 110.4(3) C(32)-C(28)-C(23) 111.5(3) C(27)-C(28)-C(23) 110.9(3) C(32)-C(28)-H(28) 108.0 C(27)-C(28)-H(28) 108.0

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326

C(23)-C(28)-H(28) 108.0 C(30)-C(29)-C(31) 110.6(3) C(30)-C(29)-C(25) 110.6(3) C(31)-C(29)-C(25) 110.1(3) C(30)-C(29)-H(29) 108.5 C(31)-C(29)-H(29) 108.5 C(25)-C(29)-H(29) 108.5 C(29)-C(30)-H(30A) 109.5 C(29)-C(30)-H(30B) 109.5 H(30A)-C(30)-H(30B) 109.5 C(29)-C(30)-H(30C) 109.5 H(30A)-C(30)-H(30C) 109.5 H(30B)-C(30)-H(30C) 109.5 C(29)-C(31)-H(31A) 109.5 C(29)-C(31)-H(31B) 109.5 H(31A)-C(31)-H(31B) 109.5 C(29)-C(31)-H(31C) 109.5 H(31A)-C(31)-H(31C) 109.5 H(31B)-C(31)-H(31C) 109.5 C(28)-C(32)-H(32A) 109.5 C(28)-C(32)-H(32B) 109.5 H(32A)-C(32)-H(32B) 109.5 C(28)-C(32)-H(32C) 109.5 H(32A)-C(32)-H(32C) 109.5 H(32B)-C(32)-H(32C) 109.5 C(2)-N(1)-C(1) 118.7(4) C(17)-N(2)-C(18) 117.5(3) C(6)-O(1)-H(1) 109.5 C(22)-O(3)-H(3A) 109.5 Symmetry transformations used to generate equivalent atoms: Table 4. Anisotropic displacement parameters (Å2x 103) for s3974pat5. The anisotropic displacement factor exponent takes the form: -2p2[ h2 a*2U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 U23 U13 U12 C(1) 22(2) 14(2) 19(2) -4(1) -5(2) -8(2) C(2) 18(2) 20(2) 26(2) -2(2) -7(2) -6(2) C(3) 20(2) 22(2) 30(2) -3(2) -3(2) -7(2)

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327

C(4) 26(2) 27(2) 20(2) -1(2) -2(2) -13(2) C(5) 27(2) 18(2) 23(2) 1(2) -6(2) -10(2) C(6) 21(2) 14(2) 21(2) 0(1) -5(1) -5(2) C(7) 18(2) 11(2) 17(2) 1(1) -4(1) -6(1) C(8) 20(2) 14(2) 19(2) 3(1) -6(1) -2(2) C(9) 21(2) 18(2) 19(2) -2(1) -5(2) -7(2) C(10) 26(2) 21(2) 20(2) -2(2) -2(2) -2(2) C(11) 18(2) 25(2) 22(2) 2(2) -5(2) -3(2) C(12) 16(2) 16(2) 22(2) 4(1) -4(1) -3(2) C(13) 22(2) 18(2) 20(2) -2(1) -4(1) -6(2) C(14) 24(2) 31(2) 30(2) 3(2) -5(2) -1(2) C(15) 30(2) 31(2) 20(2) 5(2) -7(2) -8(2) C(16) 24(2) 22(2) 31(2) 1(2) -10(2) -8(2) C(17) 20(2) 17(2) 18(2) 3(2) -2(2) -9(2) C(18) 21(2) 19(2) 26(2) 5(2) -6(2) -11(2) C(19) 19(2) 19(2) 28(2) 7(2) -2(2) -7(2) C(20) 29(2) 17(2) 26(2) -2(2) 4(2) -7(2) C(21) 28(2) 19(2) 20(2) 0(2) -5(2) -10(2) C(22) 19(2) 16(2) 20(2) 2(1) -8(1) -7(2) C(23) 15(2) 12(2) 22(2) -1(1) -5(1) -4(1) C(24) 24(2) 13(2) 20(2) -2(1) -4(1) -13(2) C(25) 33(2) 17(2) 16(2) 3(1) -5(2) -11(2) C(26) 36(2) 38(3) 18(2) -4(2) 4(2) -26(2) C(27) 18(2) 30(2) 31(2) -10(2) 1(2) -10(2) C(28) 16(2) 18(2) 26(2) -5(2) -6(1) -6(2) C(29) 27(2) 18(2) 19(2) 1(1) -6(2) -8(2) C(30) 28(2) 45(3) 23(2) -6(2) -6(2) -12(2) C(31) 35(2) 31(3) 21(2) -1(2) -9(2) -15(2) C(32) 23(2) 19(2) 44(3) -4(2) -16(2) -2(2) N(1) 20(2) 17(2) 21(2) 0(1) -4(1) -8(1) N(2) 23(2) 18(2) 21(2) 0(1) -4(1) -9(1) O(1) 25(1) 14(1) 20(1) 3(1) -9(1) -5(1) O(2) 27(1) 22(2) 28(2) 1(1) -8(1) -14(1) O(3) 26(1) 17(1) 20(1) 3(1) -9(1) -12(1) O(4) 30(2) 14(1) 22(1) 2(1) -7(1) -6(1)

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Table 5. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 10 3)for s3974pat5. x y z U(eq) H(2) -711 5315 4842 25 H(3) -2409 4628 6950 29 H(4) -1129 2891 8328 29 H(5) 1798 1799 7476 26 H(6) 4584 3016 5612 22 H(7) 4498 3463 3263 18 H(9) 4374 733 563 23 H(10A) 7069 -41 806 29 H(10B) 7183 1001 -518 29 H(11A) 7148 2935 1035 27 H(11B) 8704 1556 1007 27 H(12) 7175 891 3254 22 H(13) 4469 3715 745 24 H(14A) 1698 4314 445 45 H(14B) 1710 3514 1920 45 H(14C) 1709 2633 503 45 H(15A) 5927 2791 -1625 41 H(15B) 4092 3691 -1696 41 H(15C) 4561 1951 -1709 41 H(16A) 8793 2587 3318 38 H(16B) 7282 2707 4785 38 H(16C) 7096 3886 3600 38 H(18) 7734 8276 4031 26 H(19) 9508 6666 2067 27 H(20) 8275 5498 670 32 H(21) 5322 5885 1397 26 H(22) 2508 8503 3180 21 H(23) 2545 8886 5528 20 H(25) 2692 6148 8287 26 H(26A) 46 6478 7924 36 H(26B) -192 7558 9289 36 H(27A) -234 9537 7786 33 H(27B) -1696 8804 7726 33 H(28) -116 7606 5453 23 H(29) 2440 9211 7984 25 H(30A) 5129 8675 8406 48

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H(30B) 5244 7761 6967 48 H(30C) 5192 6983 8462 48 H(31A) 2262 7617 10553 41 H(31B) 856 9014 10335 41 H(31C) 2610 9189 10489 41 H(32A) -1718 10030 5409 42 H(32B) -83 9606 4017 42 H(32C) -109 10603 5367 42 H(1) 4563 320 5076 29 H(3A) 2566 5756 3654 29 Table 6. Torsion angles [°] for s3974pat5. N(1)-C(2)-C(3)-C(4) 0.2(6) C(2)-C(3)-C(4)-C(5) -2.0(6) C(3)-C(4)-C(5)-C(1) 1.6(6) N(1)-C(1)-C(5)-C(4) 0.7(6) C(6)-C(1)-C(5)-C(4) 177.2(4) N(1)-C(1)-C(6)-O(1) -157.8(3) C(5)-C(1)-C(6)-O(1) 25.6(5) N(1)-C(1)-C(6)-C(7) -28.4(5) C(5)-C(1)-C(6)-C(7) 155.0(4) O(1)-C(6)-C(7)-C(8) 68.8(4) C(1)-C(6)-C(7)-C(8) -60.3(4) O(1)-C(6)-C(7)-C(12) -56.2(4) C(1)-C(6)-C(7)-C(12) 174.8(3) C(6)-C(7)-C(8)-O(2) -0.9(5) C(12)-C(7)-C(8)-O(2) 125.4(4) C(6)-C(7)-C(8)-C(9) -179.1(3) C(12)-C(7)-C(8)-C(9) -52.8(4) O(2)-C(8)-C(9)-C(13) 108.6(4) C(7)-C(8)-C(9)-C(13) -73.2(4) O(2)-C(8)-C(9)-C(10) -125.2(4) C(7)-C(8)-C(9)-C(10) 53.0(4) C(8)-C(9)-C(10)-C(11) -54.8(4) C(13)-C(9)-C(10)-C(11) 70.4(4) C(9)-C(10)-C(11)-C(12) 59.8(4) C(10)-C(11)-C(12)-C(16) 179.8(3) C(10)-C(11)-C(12)-C(7) -56.4(4) C(8)-C(7)-C(12)-C(16) 175.1(3)

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330

C(6)-C(7)-C(12)-C(16) -59.4(4) C(8)-C(7)-C(12)-C(11) 51.4(4) C(6)-C(7)-C(12)-C(11) 176.9(3) C(8)-C(9)-C(13)-C(15) -178.6(3) C(10)-C(9)-C(13)-C(15) 59.0(4) C(8)-C(9)-C(13)-C(14) -56.5(4) C(10)-C(9)-C(13)-C(14) -178.9(3) N(2)-C(18)-C(19)-C(20) 1.2(6) C(18)-C(19)-C(20)-C(21) -2.3(6) C(19)-C(20)-C(21)-C(17) 0.6(6) N(2)-C(17)-C(21)-C(20) 2.5(6) C(22)-C(17)-C(21)-C(20) 179.4(4) N(2)-C(17)-C(22)-O(3) -160.5(3) C(21)-C(17)-C(22)-O(3) 22.4(5) N(2)-C(17)-C(22)-C(23) -31.2(5) C(21)-C(17)-C(22)-C(23) 151.7(4) O(3)-C(22)-C(23)-C(24) 70.6(4) C(17)-C(22)-C(23)-C(24) -58.0(4) O(3)-C(22)-C(23)-C(28) -55.9(4) C(17)-C(22)-C(23)-C(28) 175.6(3) C(22)-C(23)-C(24)-O(4) -1.2(5) C(28)-C(23)-C(24)-O(4) 125.6(4) C(22)-C(23)-C(24)-C(25) -178.9(3) C(28)-C(23)-C(24)-C(25) -52.2(4) O(4)-C(24)-C(25)-C(26) -121.7(4) C(23)-C(24)-C(25)-C(26) 56.0(4) O(4)-C(24)-C(25)-C(29) 112.2(4) C(23)-C(24)-C(25)-C(29) -70.0(4) C(24)-C(25)-C(26)-C(27) -57.9(4) C(29)-C(25)-C(26)-C(27) 66.6(4) C(25)-C(26)-C(27)-C(28) 59.5(4) C(26)-C(27)-C(28)-C(32) -176.2(3) C(26)-C(27)-C(28)-C(23) -52.2(4) C(24)-C(23)-C(28)-C(32) 170.5(3) C(22)-C(23)-C(28)-C(32) -62.8(4) C(24)-C(23)-C(28)-C(27) 47.1(4) C(22)-C(23)-C(28)-C(27) 173.8(3) C(24)-C(25)-C(29)-C(30) -59.4(5) C(26)-C(25)-C(29)-C(30) 179.3(4)

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331

C(24)-C(25)-C(29)-C(31) 178.1(4) C(26)-C(25)-C(29)-C(31) 56.8(5) C(3)-C(2)-N(1)-C(1) 2.0(6) C(5)-C(1)-N(1)-C(2) -2.5(6) C(6)-C(1)-N(1)-C(2) -179.1(3) C(21)-C(17)-N(2)-C(18) -3.6(6) C(22)-C(17)-N(2)-C(18) 179.4(4) C(19)-C(18)-N(2)-C(17) 1.7(6) ________________________________________________________________ Symmetry transformations used to generate equivalent atoms: Table 7. Hydrogen bonds for s3974pat5 [Å and °]. D-H...A d(D-H) d(H...A) d(D...A) <(DHA) O(1)-H(1)...N(2)#1 0.84 2.29 3.049(4) 150.0 O(1)-H(1)...O(2) 0.84 2.42 2.970(4) 123.7 O(3)-H(3A)...N(1) 0.84 2.28 3.019(5) 146.8 O(3)-H(3A)...O(4) 0.84 2.49 3.031(4) 123.1 ____________________________________________________________________________ Symmetry transformations used to generate equivalent atoms: #1 x,y-1,z

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Table 1. Crystal data and structure refinement for s3947oa. Identification code s3947oa Empirical formula C15H28O2 Formula weight 240.37 Temperature 150.04(18) K Wavelength 0.7107 Å Crystal system Monoclinic Space group P 21 Unit cell dimensions a = 5.7198(5) Å a= 90°. b = 19.7983(19) Å b= 90.038(7)°. c = 13.0031(10) Å g = 90°. Volume 1472.5(2) Å3 Z 4 Density (calculated) 1.084 Mg/m3 Absorption coefficient 0.069 mm-1 F(000) 536 Crystal size 0.29 x 0.27 x 0.23 mm3 Theta range for data collection 3.09 to 28.95°. Index ranges -7<=h<=7, -26<=k<=22, -16<=l<=16 Reflections collected 11259 Independent reflections 5803 [R(int) = 0.0580] Completeness to theta = 28.95° 89.2 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 1.00000 and 0.65395 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 5803 / 1 / 320 Goodness-of-fit on F2 1.667

O

HH

HO H

9.Appendix

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Final R indices [I>2sigma(I)] R1 = 0.1597, wR2 = 0.4070 R indices (all data) R1 = 0.1693, wR2 = 0.4254 Absolute structure parameter -2(5) Largest diff. peak and hole 0.570 and -0.566 e.Å-3 Table 2. Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103) for s3947oa. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x y z U(eq) C(1) 6421(14) 4597(4) 4074(6) 27(2) C(2) 5995(16) 4692(4) 5244(7) 28(2) C(3) 8049(16) 4909(5) 5854(7) 32(2) C(4) 9295(17) 5526(4) 5357(8) 34(2) C(5) 9767(19) 5412(5) 4250(8) 37(2) C(6) 7627(17) 5220(5) 3601(6) 31(2) C(7) 7721(15) 3931(5) 3873(6) 28(2) C(8) 6519(18) 3334(4) 4385(7) 34(2) C(9) 7306(17) 2653(4) 4007(7) 30(2) C(10) 6150(20) 2095(5) 4598(9) 46(3) C(11) 9980(20) 2554(6) 4079(8) 44(2) C(12) 7570(19) 5008(6) 7001(7) 40(2) C(13) 9860(20) 5011(7) 7658(8) 45(2) C(14) 6090(20) 5646(6) 7241(7) 44(2) C(15) 5930(20) 5824(6) 3495(8) 46(3) C(16) 813(16) 3097(4) 10822(6) 25(2) C(17) -26(16) 3144(5) 9699(7) 28(2) C(18) 1755(18) 2924(5) 8880(8) 39(2) C(19) 2713(18) 2236(6) 9148(7) 37(2) C(20) 3636(18) 2187(5) 10247(9) 40(2) C(21) 1827(18) 2388(5) 11032(7) 34(2) C(22) 2613(17) 3674(5) 11068(6) 28(2) C(23) 1630(20) 4355(6) 10740(8) 43(2) C(24) 3330(20) 4929(6) 10975(9) 48(3) C(25) 2100(30) 5605(6) 10932(12) 64(4) C(26) 5350(30) 4938(9) 10228(17) 86(6) C(27) 820(20) 3025(6) 7790(8) 43(2) C(28) -810(30) 2452(7) 7472(10) 55(3) C(29) 2770(30) 3090(7) 7043(8) 56(3) C(30) -90(20) 1864(6) 11128(9) 46(2)

9.Appendix

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O(1) 4146(12) 4603(4) 5622(5) 42(2) O(2) 7878(15) 3842(4) 2788(5) 43(2) O(3) -1973(13) 3294(4) 9496(5) 43(2) O(4) 3106(16) 3649(4) 12143(5) 42(2) Table 3. Bond lengths [Å] and angles [°] for s3947oa. C(1)-C(7) 1.537(12) C(1)-C(6) 1.541(12) C(1)-C(2) 1.552(11) C(1)-H(1) 1.0000 C(2)-O(1) 1.180(11) C(2)-C(3) 1.481(13) C(3)-C(12) 1.528(12) C(3)-C(4) 1.556(14) C(3)-H(3) 1.0000 C(4)-C(5) 1.482(15) C(4)-H(4A) 0.9900 C(4)-H(4B) 0.9900 C(5)-C(6) 1.535(14) C(5)-H(5A) 0.9900 C(5)-H(5B) 0.9900 C(6)-C(15) 1.545(16) C(6)-H(6) 1.0000 C(7)-O(2) 1.425(9) C(7)-C(8) 1.521(12) C(7)-H(7) 1.0000 C(8)-C(9) 1.505(12) C(8)-H(8A) 0.9900 C(8)-H(8B) 0.9900 C(9)-C(10) 1.500(14) C(9)-C(11) 1.542(15) C(9)-H(9) 1.0000 C(10)-H(10A) 0.9800 C(10)-H(10B) 0.9800 C(10)-H(10C) 0.9800 C(11)-H(11A) 0.9800 C(11)-H(11B) 0.9800 C(11)-H(11C) 0.9800 C(12)-C(14) 1.554(16)

9.Appendix

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C(12)-C(13) 1.562(15) C(12)-H(12) 1.0000 C(13)-H(13A) 0.9800 C(13)-H(13B) 0.9800 C(13)-H(13C) 0.9800 C(14)-H(14A) 0.9800 C(14)-H(14B) 0.9800 C(14)-H(14C) 0.9800 C(15)-H(15A) 0.9800 C(15)-H(15B) 0.9800 C(15)-H(15C) 0.9800 C(16)-C(17) 1.539(11) C(16)-C(21) 1.544(13) C(16)-C(22) 1.570(13) C(16)-H(16) 1.0000 C(17)-O(3) 1.182(13) C(17)-C(18) 1.537(12) C(18)-C(19) 1.509(15) C(18)-C(27) 1.527(16) C(18)-H(18) 1.0000 C(19)-C(20) 1.525(15) C(19)-H(19A) 0.9900 C(19)-H(19B) 0.9900 C(20)-C(21) 1.508(14) C(20)-H(20A) 0.9900 C(20)-H(20B) 0.9900 C(21)-C(30) 1.515(15) C(21)-H(21) 1.0000 C(22)-O(4) 1.427(9) C(22)-C(23) 1.523(12) C(22)-H(22) 1.0000 C(23)-C(24) 1.528(17) C(23)-H(23A) 0.9900 C(23)-H(23B) 0.9900 C(24)-C(26) 1.509(19) C(24)-C(25) 1.512(19) C(24)-H(24) 1.0000 C(25)-H(25A) 0.9800 C(25)-H(25B) 0.9800

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C(25)-H(25C) 0.9800 C(26)-H(26A) 0.9800 C(26)-H(26B) 0.9800 C(26)-H(26C) 0.9800 C(27)-C(29) 1.485(16) C(27)-C(28) 1.528(18) C(27)-H(27) 1.0000 C(28)-H(28A) 0.9800 C(28)-H(28B) 0.9800 C(28)-H(28C) 0.9800 C(29)-H(29A) 0.9800 C(29)-H(29B) 0.9800 C(29)-H(29C) 0.9800 C(30)-H(30A) 0.9800 C(30)-H(30B) 0.9800 C(30)-H(30C) 0.9800 O(2)-H(2) 0.8400 O(4)-H(4) 0.8400 C(7)-C(1)-C(6) 113.7(6) C(7)-C(1)-C(2) 110.3(7) C(6)-C(1)-C(2) 111.5(7) C(7)-C(1)-H(1) 107.0 C(6)-C(1)-H(1) 107.0 C(2)-C(1)-H(1) 107.0 O(1)-C(2)-C(3) 122.1(8) O(1)-C(2)-C(1) 122.1(9) C(3)-C(2)-C(1) 115.8(7) C(2)-C(3)-C(12) 114.7(8) C(2)-C(3)-C(4) 111.7(7) C(12)-C(3)-C(4) 112.8(9) C(2)-C(3)-H(3) 105.6 C(12)-C(3)-H(3) 105.6 C(4)-C(3)-H(3) 105.6 C(5)-C(4)-C(3) 111.5(8) C(5)-C(4)-H(4A) 109.3 C(3)-C(4)-H(4A) 109.3 C(5)-C(4)-H(4B) 109.3 C(3)-C(4)-H(4B) 109.3

9.Appendix

337

H(4A)-C(4)-H(4B) 108.0 C(4)-C(5)-C(6) 115.3(8) C(4)-C(5)-H(5A) 108.5 C(6)-C(5)-H(5A) 108.5 C(4)-C(5)-H(5B) 108.5 C(6)-C(5)-H(5B) 108.5 H(5A)-C(5)-H(5B) 107.5 C(5)-C(6)-C(1) 109.6(7) C(5)-C(6)-C(15) 111.0(8) C(1)-C(6)-C(15) 112.0(8) C(5)-C(6)-H(6) 108.1 C(1)-C(6)-H(6) 108.1 C(15)-C(6)-H(6) 108.1 O(2)-C(7)-C(8) 111.5(8) O(2)-C(7)-C(1) 107.8(7) C(8)-C(7)-C(1) 111.9(6) O(2)-C(7)-H(7) 108.5 C(8)-C(7)-H(7) 108.5 C(1)-C(7)-H(7) 108.5 C(9)-C(8)-C(7) 114.7(7) C(9)-C(8)-H(8A) 108.6 C(7)-C(8)-H(8A) 108.6 C(9)-C(8)-H(8B) 108.6 C(7)-C(8)-H(8B) 108.6 H(8A)-C(8)-H(8B) 107.6 C(10)-C(9)-C(8) 111.1(7) C(10)-C(9)-C(11) 108.3(9) C(8)-C(9)-C(11) 112.9(8) C(10)-C(9)-H(9) 108.1 C(8)-C(9)-H(9) 108.1 C(11)-C(9)-H(9) 108.1 C(9)-C(10)-H(10A) 109.5 C(9)-C(10)-H(10B) 109.5 H(10A)-C(10)-H(10B) 109.5 C(9)-C(10)-H(10C) 109.5 H(10A)-C(10)-H(10C) 109.5 H(10B)-C(10)-H(10C) 109.5 C(9)-C(11)-H(11A) 109.5 C(9)-C(11)-H(11B) 109.5

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338

H(11A)-C(11)-H(11B) 109.5 C(9)-C(11)-H(11C) 109.5 H(11A)-C(11)-H(11C) 109.5 H(11B)-C(11)-H(11C) 109.5 C(3)-C(12)-C(14) 113.5(8) C(3)-C(12)-C(13) 112.6(8) C(14)-C(12)-C(13) 110.1(9) C(3)-C(12)-H(12) 106.8 C(14)-C(12)-H(12) 106.8 C(13)-C(12)-H(12) 106.8 C(12)-C(13)-H(13A) 109.5 C(12)-C(13)-H(13B) 109.5 H(13A)-C(13)-H(13B) 109.5 C(12)-C(13)-H(13C) 109.5 H(13A)-C(13)-H(13C) 109.5 H(13B)-C(13)-H(13C) 109.5 C(12)-C(14)-H(14A) 109.5 C(12)-C(14)-H(14B) 109.5 H(14A)-C(14)-H(14B) 109.5 C(12)-C(14)-H(14C) 109.5 H(14A)-C(14)-H(14C) 109.5 H(14B)-C(14)-H(14C) 109.5 C(6)-C(15)-H(15A) 109.5 C(6)-C(15)-H(15B) 109.5 H(15A)-C(15)-H(15B) 109.5 C(6)-C(15)-H(15C) 109.5 H(15A)-C(15)-H(15C) 109.5 H(15B)-C(15)-H(15C) 109.5 C(17)-C(16)-C(21) 109.8(7) C(17)-C(16)-C(22) 110.7(6) C(21)-C(16)-C(22) 112.2(7) C(17)-C(16)-H(16) 108.0 C(21)-C(16)-H(16) 108.0 C(22)-C(16)-H(16) 108.0 O(3)-C(17)-C(18) 122.7(9) O(3)-C(17)-C(16) 121.3(8) C(18)-C(17)-C(16) 115.7(8) C(19)-C(18)-C(27) 117.4(9) C(19)-C(18)-C(17) 109.6(8)

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C(27)-C(18)-C(17) 112.0(9) C(19)-C(18)-H(18) 105.6 C(27)-C(18)-H(18) 105.6 C(17)-C(18)-H(18) 105.6 C(18)-C(19)-C(20) 113.6(8) C(18)-C(19)-H(19A) 108.8 C(20)-C(19)-H(19A) 108.8 C(18)-C(19)-H(19B) 108.8 C(20)-C(19)-H(19B) 108.8 H(19A)-C(19)-H(19B) 107.7 C(21)-C(20)-C(19) 112.3(8) C(21)-C(20)-H(20A) 109.1 C(19)-C(20)-H(20A) 109.1 C(21)-C(20)-H(20B) 109.1 C(19)-C(20)-H(20B) 109.1 H(20A)-C(20)-H(20B) 107.9 C(20)-C(21)-C(30) 111.8(8) C(20)-C(21)-C(16) 112.2(8) C(30)-C(21)-C(16) 111.5(8) C(20)-C(21)-H(21) 107.0 C(30)-C(21)-H(21) 107.0 C(16)-C(21)-H(21) 107.0 O(4)-C(22)-C(23) 112.2(8) O(4)-C(22)-C(16) 107.7(7) C(23)-C(22)-C(16) 110.1(8) O(4)-C(22)-H(22) 108.9 C(23)-C(22)-H(22) 108.9 C(16)-C(22)-H(22) 108.9 C(22)-C(23)-C(24) 111.5(9) C(22)-C(23)-H(23A) 109.3 C(24)-C(23)-H(23A) 109.3 C(22)-C(23)-H(23B) 109.3 C(24)-C(23)-H(23B) 109.3 H(23A)-C(23)-H(23B) 108.0 C(26)-C(24)-C(25) 108.8(11) C(26)-C(24)-C(23) 111.6(11) C(25)-C(24)-C(23) 110.7(11) C(26)-C(24)-H(24) 108.6 C(25)-C(24)-H(24) 108.6

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C(23)-C(24)-H(24) 108.6 C(24)-C(25)-H(25A) 109.5 C(24)-C(25)-H(25B) 109.5 H(25A)-C(25)-H(25B) 109.5 C(24)-C(25)-H(25C) 109.5 H(25A)-C(25)-H(25C) 109.5 H(25B)-C(25)-H(25C) 109.5 C(24)-C(26)-H(26A) 109.5 C(24)-C(26)-H(26B) 109.5 H(26A)-C(26)-H(26B) 109.5 C(24)-C(26)-H(26C) 109.5 H(26A)-C(26)-H(26C) 109.5 H(26B)-C(26)-H(26C) 109.5 C(29)-C(27)-C(18) 110.9(10) C(29)-C(27)-C(28) 110.3(10) C(18)-C(27)-C(28) 111.6(9) C(29)-C(27)-H(27) 108.0 C(18)-C(27)-H(27) 108.0 C(28)-C(27)-H(27) 108.0 C(27)-C(28)-H(28A) 109.5 C(27)-C(28)-H(28B) 109.5 H(28A)-C(28)-H(28B) 109.5 C(27)-C(28)-H(28C) 109.5 H(28A)-C(28)-H(28C) 109.5 H(28B)-C(28)-H(28C) 109.5 C(27)-C(29)-H(29A) 109.5 C(27)-C(29)-H(29B) 109.5 H(29A)-C(29)-H(29B) 109.5 C(27)-C(29)-H(29C) 109.5 H(29A)-C(29)-H(29C) 109.5 H(29B)-C(29)-H(29C) 109.5 C(21)-C(30)-H(30A) 109.5 C(21)-C(30)-H(30B) 109.5 H(30A)-C(30)-H(30B) 109.5 C(21)-C(30)-H(30C) 109.5 H(30A)-C(30)-H(30C) 109.5 H(30B)-C(30)-H(30C) 109.5 C(7)-O(2)-H(2) 109.5 C(22)-O(4)-H(4) 109.5

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Symmetry transformations used to generate equivalent atoms: Table 4. Anisotropic displacement parameters (Å2x 103) for s3947oa. The anisotropic displacement factor exponent takes the form: -2p2[ h2 a*2U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 U23 U13 U12 C(1) 27(4) 27(4) 27(4) -4(3) 13(3) -3(3) C(2) 28(4) 23(4) 31(4) -2(3) 11(4) -2(3) C(3) 34(4) 38(5) 24(4) 0(3) 10(3) 13(4) C(4) 30(4) 21(4) 52(5) -6(4) -6(4) 4(3) C(5) 47(5) 23(4) 40(5) -1(4) 5(4) 0(4) C(6) 40(5) 31(4) 22(3) 2(3) 11(4) -5(4) C(7) 26(4) 44(5) 15(3) -1(3) 12(3) 0(4) C(8) 46(5) 22(4) 34(4) 2(3) 23(4) 0(4) C(9) 37(4) 25(4) 29(4) -15(3) 9(4) -3(3) C(10) 48(6) 31(5) 58(7) 3(4) 19(5) -3(4) C(11) 54(6) 45(6) 32(5) 7(4) 9(5) 7(5) C(12) 43(5) 54(6) 24(4) 3(4) 8(4) -6(5) C(13) 41(5) 60(7) 35(5) 11(5) 0(4) 6(5) C(14) 59(6) 50(6) 24(4) -17(4) 4(4) 5(5) C(15) 58(6) 43(6) 36(5) 1(4) 13(5) -11(5) C(16) 35(4) 24(4) 16(3) -3(3) 3(3) -9(3) C(17) 27(4) 34(4) 24(4) -2(3) 8(3) -7(3) C(18) 33(5) 46(6) 39(5) 4(4) 20(4) -5(4) C(19) 37(5) 45(5) 30(4) -1(4) 5(4) -4(4) C(20) 33(5) 31(5) 56(6) -5(4) 6(4) 0(4) C(21) 43(5) 34(5) 26(4) -3(3) 10(4) 2(4) C(22) 31(4) 37(5) 14(3) 1(3) 0(3) 11(3) C(23) 51(6) 40(5) 37(5) 16(4) 10(5) 22(4) C(24) 55(6) 43(6) 48(6) 2(5) -3(5) -11(5) C(25) 96(11) 31(6) 66(8) -7(5) 0(8) -20(6) C(26) 57(8) 69(10) 133(16) 25(10) 42(10) -1(7) C(27) 46(6) 46(6) 38(5) 8(5) 7(5) -1(5) C(28) 65(8) 58(7) 43(6) -7(5) -3(6) 8(6) C(29) 84(9) 60(8) 24(4) 5(4) 26(6) -7(6) C(30) 45(5) 54(7) 38(5) 5(5) 11(5) -8(5) O(1) 32(3) 59(5) 34(3) -3(3) 6(3) -5(3) O(2) 69(5) 33(4) 28(3) -3(3) 23(3) -5(4)

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O(3) 44(4) 53(4) 33(3) -6(3) 15(3) 8(3) O(4) 68(5) 33(4) 25(3) -1(3) 3(3) -2(3) Table 5. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 10

3)

for s3947oa. x y z U(eq) H(1) 4851 4557 3741 32 H(3) 9193 4528 5811 39 H(4A) 8301 5932 5437 41 H(4B) 10789 5611 5720 41 H(5A) 10945 5047 4187 44 H(5B) 10463 5828 3960 44 H(6) 8183 5095 2897 37 H(7) 9340 3970 4158 34 H(8A) 6800 3359 5135 41 H(8B) 4812 3374 4272 41 H(9) 6840 2609 3269 36 H(10A) 6740 2091 5305 69 H(10B) 6487 1661 4269 69 H(10C) 4452 2169 4606 69 H(11A) 10343 2071 4054 65 H(11B) 10548 2745 4728 65 H(11C) 10734 2784 3501 65 H(12) 6633 4609 7231 48 H(13A) 10793 4612 7492 68 H(13B) 9451 5005 8390 68 H(13C) 10759 5420 7505 68 H(14A) 7008 6052 7092 66 H(14B) 5644 5644 7969 66 H(14C) 4675 5644 6814 66 H(15A) 4414 5704 3792 69 H(15B) 5732 5935 2766 69 H(15C) 6576 6215 3859 69 H(16) -576 3161 11278 30 H(18) 3102 3243 8949 47 H(19A) 3995 2125 8666 45 H(19B) 1463 1896 9053 45 H(20A) 4141 1717 10382 48 H(20B) 5021 2483 10319 48

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343

H(21) 2640 2410 11713 41 H(22) 4089 3586 10678 33 H(23A) 136 4438 11105 51 H(23B) 1294 4347 9993 51 H(24) 3965 4863 11685 58 H(25A) 1084 5620 10326 97 H(25B) 3265 5967 10888 97 H(25C) 1159 5665 11554 97 H(26A) 6384 4555 10369 130 H(26B) 6223 5360 10307 130 H(26C) 4749 4904 9524 130 H(27) -93 3456 7778 52 H(28A) 53 2025 7464 83 H(28B) -1440 2543 6784 83 H(28C) -2107 2420 7964 83 H(29A) 3855 3441 7278 84 H(29B) 2141 3214 6368 84 H(29C) 3598 2658 6990 84 H(30A) -979 1843 10485 69 H(30B) -1139 1988 11693 69 H(30C) 608 1421 11270 69 H(2) 6533 3861 2529 65 H(4) 2164 3899 12460 63 Table 6. Torsion angles [°] for s3947oa. C(7)-C(1)-C(2)-O(1) -104.1(10) C(6)-C(1)-C(2)-O(1) 128.7(10) C(7)-C(1)-C(2)-C(3) 76.8(9) C(6)-C(1)-C(2)-C(3) -50.5(10) O(1)-C(2)-C(3)-C(12) 0.0(13) C(1)-C(2)-C(3)-C(12) 179.2(8) O(1)-C(2)-C(3)-C(4) -129.9(9) C(1)-C(2)-C(3)-C(4) 49.3(10) C(2)-C(3)-C(4)-C(5) -50.0(10) C(12)-C(3)-C(4)-C(5) 179.1(8) C(3)-C(4)-C(5)-C(6) 54.6(10) C(4)-C(5)-C(6)-C(1) -54.9(10) C(4)-C(5)-C(6)-C(15) 69.2(10)

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C(7)-C(1)-C(6)-C(5) -75.6(9) C(2)-C(1)-C(6)-C(5) 49.8(10) C(7)-C(1)-C(6)-C(15) 160.9(8) C(2)-C(1)-C(6)-C(15) -73.8(10) C(6)-C(1)-C(7)-O(2) -59.1(10) C(2)-C(1)-C(7)-O(2) 174.9(7) C(6)-C(1)-C(7)-C(8) 178.0(8) C(2)-C(1)-C(7)-C(8) 51.9(9) O(2)-C(7)-C(8)-C(9) 44.2(11) C(1)-C(7)-C(8)-C(9) 165.0(8) C(7)-C(8)-C(9)-C(10) 176.4(9) C(7)-C(8)-C(9)-C(11) 54.5(11) C(2)-C(3)-C(12)-C(14) -72.2(12) C(4)-C(3)-C(12)-C(14) 57.1(12) C(2)-C(3)-C(12)-C(13) 161.9(9) C(4)-C(3)-C(12)-C(13) -68.7(11) C(21)-C(16)-C(17)-O(3) 123.6(10) C(22)-C(16)-C(17)-O(3) -111.8(10) C(21)-C(16)-C(17)-C(18) -51.2(10) C(22)-C(16)-C(17)-C(18) 73.4(10) O(3)-C(17)-C(18)-C(19) -123.6(10) C(16)-C(17)-C(18)-C(19) 51.1(11) O(3)-C(17)-C(18)-C(27) 8.5(13) C(16)-C(17)-C(18)-C(27) -176.7(8) C(27)-C(18)-C(19)-C(20) 179.1(9) C(17)-C(18)-C(19)-C(20) -51.6(11) C(18)-C(19)-C(20)-C(21) 55.3(11) C(19)-C(20)-C(21)-C(30) 71.7(11) C(19)-C(20)-C(21)-C(16) -54.4(11) C(17)-C(16)-C(21)-C(20) 51.5(10) C(22)-C(16)-C(21)-C(20) -72.1(10) C(17)-C(16)-C(21)-C(30) -74.8(9) C(22)-C(16)-C(21)-C(30) 161.6(7) C(17)-C(16)-C(22)-O(4) 173.4(7) C(21)-C(16)-C(22)-O(4) -63.4(9) C(17)-C(16)-C(22)-C(23) 50.8(9) C(21)-C(16)-C(22)-C(23) 173.9(7) O(4)-C(22)-C(23)-C(24) 59.7(11) C(16)-C(22)-C(23)-C(24) 179.6(8)

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345

C(22)-C(23)-C(24)-C(26) 74.7(13) C(22)-C(23)-C(24)-C(25) -164.0(9) C(19)-C(18)-C(27)-C(29) -75.6(12) C(17)-C(18)-C(27)-C(29) 156.3(9) C(19)-C(18)-C(27)-C(28) 47.8(12) C(17)-C(18)-C(27)-C(28) -80.3(11) Symmetry transformations used to generate equivalent atoms: Table 7. Hydrogen bonds for s3947oa [Å and °]. D-H...A d(D-H) d(H...A) d(D...A) <(DHA) O(2)-H(2)...O(4)#1 0.84 2.07 2.880(13) 163.0 O(4)-H(4)...O(2)#2 0.84 2.49 3.130(12) 133.5 Symmetry transformations used to generate equivalent atoms: #1 x,y,z-1 #2 x-1,y,z+1

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Table 1. Crystal data and structure refinement for s4027ma. Identification code s4027ma Empirical formula C17H21NO4 Formula weight 303.35 Temperature 100(2) K Wavelength 1.54178 Å Crystal system Orthorhombic Space group P2(1)2(1)2(1) Unit cell dimensions a = 8.1285(3) Å a= 90°. b = 11.7313(4) Å b= 90°. c = 16.0877(6) Å g = 90°. Volume 1534.09(10) Å3 Z 4 Density (calculated) 1.313 Mg/m3 Absorption coefficient 0.764 mm-1 F(000) 648 Crystal size 0.30 x 0.27 x 0.17 mm3

OHO

H

H NO2

9.Appendix

347

Theta range for data collection 4.66 to 72.41°. Index ranges -9<=h<=10, -14<=k<=14, -19<=l<=17 Reflections collected 7558 Independent reflections 2965 [R(int) = 0.0217] Completeness to theta = 67.00° 99.6 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.8811 and 0.798439 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 2965 / 0 / 203 Goodness-of-fit on F2 1.071 Final R indices [I>2sigma(I)] R1 = 0.0293, wR2 = 0.0752 R indices (all data) R1 = 0.0299, wR2 = 0.0757 Absolute structure parameter 0.09(14) Largest diff. peak and hole 0.173 and -0.232 e.Å-3 Table 2. Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103) or s4027ma. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x y z U(eq) C(1) 320(1) 7023(1) 6384(1) 13(1) C(2) -1530(2) 7286(1) 6398(1) 14(1) C(3) -2100(2) 8402(1) 6798(1) 17(1) C(4) -676(2) 8992(1) 7253(1) 16(1) C(5) 450(2) 8137(1) 7740(1) 17(1) C(6) 1286(2) 7912(1) 6883(1) 16(1) C(7) -3666(2) 8252(1) 7314(1) 29(1) C(8) 757(2) 9133(1) 6629(1) 19(1) C(9) -295(2) 7125(1) 8201(1) 22(1) C(10) 1609(2) 8765(1) 8338(1) 23(1) C(11) 940(2) 6919(1) 5476(1) 14(1) C(12) 799(2) 5703(1) 5131(1) 14(1) C(13) 1996(2) 4853(1) 5226(1) 15(1) C(14) 1866(2) 3781(1) 4868(1) 19(1) C(15) 472(2) 3514(1) 4414(1) 22(1) C(16) -742(2) 4328(1) 4308(1) 22(1) C(17) -567(2) 5410(1) 4650(1) 18(1) N(1) 3494(1) 5050(1) 5724(1) 17(1) O(1) -2500(1) 6622(1) 6093(1) 17(1) O(2) 14(1) 7692(1) 4975(1) 16(1)

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348

O(3) 3418(1) 5723(1) 6307(1) 20(1) O(4) 4734(1) 4510(1) 5542(1) 28(1) Table 3. Bond lengths [Å] and angles [°] for s4027ma. C(1)-C(6) 1.5319(17) C(1)-C(2) 1.5357(17) C(1)-C(11) 1.5498(17) C(1)-H(1) 1.0000 C(2)-O(1) 1.2114(16) C(2)-C(3) 1.5301(17) C(3)-C(7) 1.5309(18) C(3)-C(4) 1.5345(18) C(3)-H(3) 1.0000 C(4)-C(8) 1.5474(18) C(4)-C(5) 1.5684(17) C(4)-H(4) 1.0000 C(5)-C(9) 1.5247(18) C(5)-C(10) 1.5340(18) C(5)-C(6) 1.5608(18) C(6)-C(8) 1.5512(17) C(6)-H(6) 1.0000 C(7)-H(7A) 0.9800 C(7)-H(7B) 0.9800 C(7)-H(7C) 0.9800 C(8)-H(8A) 0.9900 C(8)-H(8B) 0.9900 C(9)-H(9A) 0.9800 C(9)-H(9B) 0.9800 C(9)-H(9C) 0.9800 C(10)-H(10A) 0.9800 C(10)-H(10B) 0.9800 C(10)-H(10C) 0.9800 C(11)-O(2) 1.4284(14) C(11)-C(12) 1.5344(16) C(11)-H(11) 1.0000 C(12)-C(17) 1.3968(18) C(12)-C(13) 1.4018(17) C(13)-C(14) 1.3868(19) C(13)-N(1) 1.4766(16)

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349

C(14)-C(15) 1.384(2) C(14)-H(14) 0.9500 C(15)-C(16) 1.384(2) C(15)-H(15) 0.9500 C(16)-C(17) 1.3897(18) C(16)-H(16) 0.9500 C(17)-H(17) 0.9500 N(1)-O(4) 1.2255(15) N(1)-O(3) 1.2273(15) O(2)-H(2) 0.8400 C(6)-C(1)-C(2) 110.98(10) C(6)-C(1)-C(11) 112.39(10) C(2)-C(1)-C(11) 110.36(10) C(6)-C(1)-H(1) 107.6 C(2)-C(1)-H(1) 107.6 C(11)-C(1)-H(1) 107.6 O(1)-C(2)-C(3) 121.55(12) O(1)-C(2)-C(1) 120.17(11) C(3)-C(2)-C(1) 118.28(10) C(2)-C(3)-C(7) 112.45(11) C(2)-C(3)-C(4) 111.06(10) C(7)-C(3)-C(4) 114.84(11) C(2)-C(3)-H(3) 105.9 C(7)-C(3)-H(3) 105.9 C(4)-C(3)-H(3) 105.9 C(3)-C(4)-C(8) 107.83(11) C(3)-C(4)-C(5) 112.94(10) C(8)-C(4)-C(5) 87.33(9) C(3)-C(4)-H(4) 115.2 C(8)-C(4)-H(4) 115.2 C(5)-C(4)-H(4) 115.2 C(9)-C(5)-C(10) 108.30(11) C(9)-C(5)-C(6) 118.06(11) C(10)-C(5)-C(6) 111.57(11) C(9)-C(5)-C(4) 120.60(11) C(10)-C(5)-C(4) 111.36(10) C(6)-C(5)-C(4) 85.44(9) C(1)-C(6)-C(8) 110.39(10)

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350

C(1)-C(6)-C(5) 110.82(10) C(8)-C(6)-C(5) 87.47(9) C(1)-C(6)-H(6) 115.0 C(8)-C(6)-H(6) 115.0 C(5)-C(6)-H(6) 115.0 C(3)-C(7)-H(7A) 109.5 C(3)-C(7)-H(7B) 109.5 H(7A)-C(7)-H(7B) 109.5 C(3)-C(7)-H(7C) 109.5 H(7A)-C(7)-H(7C) 109.5 H(7B)-C(7)-H(7C) 109.5 C(4)-C(8)-C(6) 86.49(9) C(4)-C(8)-H(8A) 114.2 C(6)-C(8)-H(8A) 114.2 C(4)-C(8)-H(8B) 114.2 C(6)-C(8)-H(8B) 114.2 H(8A)-C(8)-H(8B) 111.4 C(5)-C(9)-H(9A) 109.5 C(5)-C(9)-H(9B) 109.5 H(9A)-C(9)-H(9B) 109.5 C(5)-C(9)-H(9C) 109.5 H(9A)-C(9)-H(9C) 109.5 H(9B)-C(9)-H(9C) 109.5 C(5)-C(10)-H(10A) 109.5 C(5)-C(10)-H(10B) 109.5 H(10A)-C(10)-H(10B) 109.5 C(5)-C(10)-H(10C) 109.5 H(10A)-C(10)-H(10C) 109.5 H(10B)-C(10)-H(10C) 109.5 O(2)-C(11)-C(12) 110.26(9) O(2)-C(11)-C(1) 108.09(9) C(12)-C(11)-C(1) 112.99(10) O(2)-C(11)-H(11) 108.5 C(12)-C(11)-H(11) 108.5 C(1)-C(11)-H(11) 108.5 C(17)-C(12)-C(13) 115.89(11) C(17)-C(12)-C(11) 119.27(11) C(13)-C(12)-C(11) 124.77(11) C(14)-C(13)-C(12) 123.18(12)

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351

C(14)-C(13)-N(1) 115.49(11) C(12)-C(13)-N(1) 121.34(11) C(15)-C(14)-C(13) 119.14(12) C(15)-C(14)-H(14) 120.4 C(13)-C(14)-H(14) 120.4 C(14)-C(15)-C(16) 119.45(12) C(14)-C(15)-H(15) 120.3 C(16)-C(15)-H(15) 120.3 C(15)-C(16)-C(17) 120.61(13) C(15)-C(16)-H(16) 119.7 C(17)-C(16)-H(16) 119.7 C(16)-C(17)-C(12) 121.68(12) C(16)-C(17)-H(17) 119.2 C(12)-C(17)-H(17) 119.2 O(4)-N(1)-O(3) 123.86(11) O(4)-N(1)-C(13) 117.83(10) O(3)-N(1)-C(13) 118.29(10) C(11)-O(2)-H(2) 109.5 Symmetry transformations used to generate equivalent atoms: Table 4. Anisotropic displacement parameters (Å2x 103) for s4027ma. The anisotropic displacement factor exponent takes the form: -2p2[ h2 a*2U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 U23 U13 U12 C(1) 12(1) 12(1) 16(1) 0(1) 0(1) 0(1) C(2) 13(1) 15(1) 13(1) 4(1) 0(1) -1(1) C(3) 14(1) 18(1) 18(1) 0(1) 0(1) 4(1) C(4) 17(1) 13(1) 19(1) -1(1) 0(1) 2(1) C(5) 18(1) 14(1) 18(1) -2(1) -3(1) 1(1) C(6) 12(1) 15(1) 20(1) -1(1) -1(1) -1(1) C(7) 16(1) 39(1) 32(1) -13(1) 5(1) -1(1) C(8) 21(1) 13(1) 22(1) 0(1) 4(1) -3(1) C(9) 33(1) 17(1) 16(1) 1(1) -2(1) -1(1) C(10) 23(1) 22(1) 24(1) -7(1) -6(1) 2(1) C(11) 12(1) 12(1) 16(1) 3(1) 0(1) 1(1) C(12) 15(1) 15(1) 13(1) 2(1) 3(1) 0(1) C(13) 13(1) 17(1) 15(1) 2(1) 2(1) 0(1) C(14) 19(1) 17(1) 20(1) 1(1) 4(1) 5(1) C(15) 27(1) 17(1) 23(1) -6(1) 1(1) 0(1)

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352

C(16) 22(1) 23(1) 20(1) -2(1) -5(1) -1(1) C(17) 16(1) 18(1) 17(1) 0(1) -2(1) 2(1) N(1) 15(1) 13(1) 24(1) 3(1) 0(1) 1(1) O(1) 15(1) 19(1) 18(1) 1(1) 0(1) -3(1) O(2) 15(1) 14(1) 19(1) 5(1) 2(1) 3(1) O(3) 18(1) 19(1) 21(1) 0(1) -3(1) 1(1) O(4) 16(1) 25(1) 44(1) -6(1) -2(1) 8(1)

Table 5. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 10 3) for s4027ma. x y z U(eq) H(1) 485 6267 6659 16 H(3) -2404 8918 6328 20 H(4) -983 9699 7565 19 H(6) 2501 7783 6906 19 H(7A) -3403 7846 7830 44 H(7B) -4473 7811 6996 44 H(7C) -4126 9002 7448 44 H(8A) 1558 9734 6781 22 H(8B) 405 9206 6042 22 H(9A) 578 6587 8351 33 H(9B) -1097 6744 7841 33 H(9C) -845 7394 8706 33 H(10A) 988 9026 8824 34 H(10B) 2093 9424 8054 34 H(10C) 2487 8248 8516 34 H(11) 2122 7153 5459 16 H(14) 2722 3238 4934 23 H(15) 350 2777 4176 27 H(16) -1703 4147 4000 26 H(17) -1399 5963 4554 21 H(2) 623 7963 4602 24 Table 6. Torsion angles [°] for s4027ma. C(6)-C(1)-C(2)-O(1) -173.89(11) C(11)-C(1)-C(2)-O(1) 60.83(14) C(6)-C(1)-C(2)-C(3) 6.45(14) C(11)-C(1)-C(2)-C(3) -118.82(11) O(1)-C(2)-C(3)-C(7) 40.86(16)

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353

C(1)-C(2)-C(3)-C(7) -139.49(12) O(1)-C(2)-C(3)-C(4) 171.12(11) C(1)-C(2)-C(3)-C(4) -9.23(15) C(2)-C(3)-C(4)-C(8) 54.20(13) C(7)-C(3)-C(4)-C(8) -176.80(11) C(2)-C(3)-C(4)-C(5) -40.55(14) C(7)-C(3)-C(4)-C(5) 88.44(14) C(3)-C(4)-C(5)-C(9) -38.79(16) C(8)-C(4)-C(5)-C(9) -147.04(12) C(3)-C(4)-C(5)-C(10) -167.32(11) C(8)-C(4)-C(5)-C(10) 84.43(12) C(3)-C(4)-C(5)-C(6) 81.28(11) C(8)-C(4)-C(5)-C(6) -26.97(9) C(2)-C(1)-C(6)-C(8) -48.68(13) C(11)-C(1)-C(6)-C(8) 75.45(13) C(2)-C(1)-C(6)-C(5) 46.54(13) C(11)-C(1)-C(6)-C(5) 170.66(10) C(9)-C(5)-C(6)-C(1) 38.45(15) C(10)-C(5)-C(6)-C(1) 164.84(10) C(4)-C(5)-C(6)-C(1) -83.97(11) C(9)-C(5)-C(6)-C(8) 149.32(12) C(10)-C(5)-C(6)-C(8) -84.30(11) C(4)-C(5)-C(6)-C(8) 26.89(9) C(3)-C(4)-C(8)-C(6) -86.14(10) C(5)-C(4)-C(8)-C(6) 27.11(9) C(1)-C(6)-C(8)-C(4) 84.04(11) C(5)-C(6)-C(8)-C(4) -27.25(9) C(6)-C(1)-C(11)-O(2) -91.03(11) C(2)-C(1)-C(11)-O(2) 33.44(13) C(6)-C(1)-C(11)-C(12) 146.67(10) C(2)-C(1)-C(11)-C(12) -88.86(12) O(2)-C(11)-C(12)-C(17) -24.51(15) C(1)-C(11)-C(12)-C(17) 96.57(12) O(2)-C(11)-C(12)-C(13) 152.19(11) C(1)-C(11)-C(12)-C(13) -86.73(14) C(17)-C(12)-C(13)-C(14) 0.28(18) C(11)-C(12)-C(13)-C(14) -176.52(12) C(17)-C(12)-C(13)-N(1) -179.50(11) C(11)-C(12)-C(13)-N(1) 3.70(18)

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354

C(12)-C(13)-C(14)-C(15) -2.0(2) N(1)-C(13)-C(14)-C(15) 177.83(12) C(13)-C(14)-C(15)-C(16) 1.6(2) C(14)-C(15)-C(16)-C(17) 0.3(2) C(15)-C(16)-C(17)-C(12) -2.1(2) C(13)-C(12)-C(17)-C(16) 1.75(18) C(11)-C(12)-C(17)-C(16) 178.74(12) C(14)-C(13)-N(1)-O(4) 27.79(17) C(12)-C(13)-N(1)-O(4) -152.41(12) C(14)-C(13)-N(1)-O(3) -150.62(11) C(12)-C(13)-N(1)-O(3) 29.18(17) ________________________________________________________________ Symmetry transformations used to generate equivalent atoms: Table 7. Hydrogen bonds for s4027ma [Å and °].

D-H...A d(D-H) d(H...A) d(D...A) <(DHA) O(2)-H(2)...O(1)#1 0.84 1.95 2.7714(13) 164.4 Symmetry transformations used to generate equivalent atoms: #1 x+1/2,-y+3/2,-z+1

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355


Table 1. Crystal data and structure refinement for s4034ma. Identification code s4034ma Empirical formula C19H24O3 Formula weight 300.38 Temperature 100(2) K Wavelength 1.54178 Å Crystal system Orthorhombic Space group P2(1)2(1)2(1) Unit cell dimensions a = 8.0793(3) Å a= 90°.

b = 13.6945(4) Å b= 90°. c = 14.7420(4) Å g = 90°.

Volume 1631.08(9) Å3 Z 4 Density (calculated) 1.223 Mg/m3 Absorption coefficient 0.646 mm-1 F(000) 648 Crystal size 0.28 x 0.23 x 0.11 mm3 Theta range for data collection 4.41 to 72.09°. Index ranges -9<=h<=9, -16<=k<=14, -18<=l<=16

OOH

H

OAc

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356

Reflections collected 6641 Independent reflections 3071 [R(int) = 0.0217] Completeness to theta = 67.00° 99.8 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.9323 and 0.801289 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 3071 / 0 / 203 Goodness-of-fit on F2 1.053 Final R indices [I>2sigma(I)] R1 = 0.0328, wR2 = 0.0868 R indices (all data) R1 = 0.0351, wR2 = 0.0882 Absolute structure parameter 0.02(18) Largest diff. peak and hole 0.209 and -0.178 e.Å-3 Table 2. Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103) for s4034ma. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.

x y z U(eq) C(1) 907(2) 3828(1) 655(1) 18(1) C(2) -906(2) 3490(1) 646(1) 22(1) C(3) -1884(2) 3615(1) 1525(1) 26(1) C(4) -712(2) 3731(1) 2336(1) 23(1) C(5) 887(2) 3094(1) 2238(1) 20(1) C(6) 1539(2) 3944(1) 1629(1) 19(1) C(7) 1057(2) 4783(1) 104(1) 21(1) C(8) -82(2) 5050(1) -1377(1) 23(1) C(9) -189(2) 4618(1) -2310(1) 34(1) C(10) 2702(2) 5291(1) 198(1) 22(1) C(11) 4152(2) 4825(1) -78(1) 26(1) C(12) 5656(2) 5309(1) -42(1) 33(1) C(13) 5731(2) 6258(1) 287(1) 38(1) C(14) 4315(3) 6718(1) 578(1) 37(1) C(15) 2799(2) 6239(1) 531(1) 28(1) C(16) -3205(2) 2828(2) 1644(1) 40(1) C(17) 391(2) 4637(1) 2165(1) 24(1) C(18) 1836(2) 3030(1) 3131(1) 28(1) C(19) 798(2) 2074(1) 1826(1) 28(1) O(1) 862(2) 4491(1) -842(1) 26(1) O(2) -738(2) 5789(1) 1132(1) 38(1) O(3) -1531(1) 3154(1) -33(1) 33(1)

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357

Table 3. Bond lengths [Å] and angles [°] for s4034ma. C(1)-C(6) 1.5326(17) C(1)-C(2) 1.5363(19) C(1)-C(7) 1.5435(17) C(1)-H(1) 1.0000 C(2)-O(3) 1.2118(18) C(2)-C(3) 1.5268(19) C(3)-C(16) 1.527(2) C(3)-C(4) 1.534(2) C(3)-H(3) 1.0000 C(4)-C(17) 1.548(2) C(4)-C(5) 1.5658(19) C(4)-H(4) 1.0000 C(5)-C(19) 1.5237(18) C(5)-C(18) 1.5261(18) C(5)-C(6) 1.5618(18) C(6)-C(17) 1.5442(18) C(6)-H(6) 1.0000 C(7)-O(1) 1.4598(16) C(7)-C(10) 1.506(2) C(7)-H(7) 1.0000 C(8)-O(2) 1.1980(19) C(8)-O(1) 1.3369(17) C(8)-C(9) 1.500(2) C(9)-H(9A) 0.9800 C(9)-H(9B) 0.9800 C(9)-H(9C) 0.9800 C(10)-C(15) 1.390(2) C(10)-C(11) 1.395(2) C(11)-C(12) 1.386(2) C(11)-H(11) 0.9500 C(12)-C(13) 1.388(2) C(12)-H(12) 0.9500 C(13)-C(14) 1.375(3) C(13)-H(13) 0.9500 C(14)-C(15) 1.391(2) C(14)-H(14) 0.9500 C(15)-H(15) 0.9500

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358

C(16)-H(16A) 0.9800 C(16)-H(16B) 0.9800 C(16)-H(16C) 0.9800 C(17)-H(17A) 0.9900 C(17)-H(17B) 0.9900 C(18)-H(18A) 0.9800 C(18)-H(18B) 0.9800 C(18)-H(18C) 0.9800 C(19)-H(19A) 0.9800 C(19)-H(19B) 0.9800 C(19)-H(19C) 0.9800

C(6)-C(1)-C(2) 110.89(11) C(6)-C(1)-C(7) 112.26(10) C(2)-C(1)-C(7) 109.03(11) C(6)-C(1)-H(1) 108.2 C(2)-C(1)-H(1) 108.2 C(7)-C(1)-H(1) 108.2 O(3)-C(2)-C(3) 121.86(13) O(3)-C(2)-C(1) 121.22(13) C(3)-C(2)-C(1) 116.92(11) C(2)-C(3)-C(16) 112.36(13) C(2)-C(3)-C(4) 110.71(11) C(16)-C(3)-C(4) 114.48(14) C(2)-C(3)-H(3) 106.2 C(16)-C(3)-H(3) 106.2 C(4)-C(3)-H(3) 106.2 C(3)-C(4)-C(17) 108.13(12) C(3)-C(4)-C(5) 112.33(11) C(17)-C(4)-C(5) 87.50(10) C(3)-C(4)-H(4) 115.2 C(17)-C(4)-H(4) 115.2 C(5)-C(4)-H(4) 115.2 C(19)-C(5)-C(18) 108.39(12) C(19)-C(5)-C(6) 118.04(11) C(18)-C(5)-C(6) 111.66(12) C(19)-C(5)-C(4) 120.55(12) C(18)-C(5)-C(4) 111.48(11) C(6)-C(5)-C(4) 85.17(10)

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C(1)-C(6)-C(17) 110.08(11) C(1)-C(6)-C(5) 110.43(11) C(17)-C(6)-C(5) 87.79(10) C(1)-C(6)-H(6) 115.1 C(17)-C(6)-H(6) 115.1 C(5)-C(6)-H(6) 115.1 O(1)-C(7)-C(10) 108.03(11) O(1)-C(7)-C(1) 105.20(10) C(10)-C(7)-C(1) 114.34(11) O(1)-C(7)-H(7) 109.7 C(10)-C(7)-H(7) 109.7 C(1)-C(7)-H(7) 109.7 O(2)-C(8)-O(1) 123.98(14) O(2)-C(8)-C(9) 125.66(14) O(1)-C(8)-C(9) 110.36(13) C(8)-C(9)-H(9A) 109.5 C(8)-C(9)-H(9B) 109.5 H(9A)-C(9)-H(9B) 109.5 C(8)-C(9)-H(9C) 109.5 H(9A)-C(9)-H(9C) 109.5 H(9B)-C(9)-H(9C) 109.5 C(15)-C(10)-C(11) 118.87(14) C(15)-C(10)-C(7) 120.88(14) C(11)-C(10)-C(7) 120.21(12) C(12)-C(11)-C(10) 120.47(14) C(12)-C(11)-H(11) 119.8 C(10)-C(11)-H(11) 119.8 C(11)-C(12)-C(13) 119.93(17) C(11)-C(12)-H(12) 120.0 C(13)-C(12)-H(12) 120.0 C(14)-C(13)-C(12) 120.14(17) C(14)-C(13)-H(13) 119.9 C(12)-C(13)-H(13) 119.9 C(13)-C(14)-C(15) 120.05(14) C(13)-C(14)-H(14) 120.0 C(15)-C(14)-H(14) 120.0 C(10)-C(15)-C(14) 120.51(15) C(10)-C(15)-H(15) 119.7 C(14)-C(15)-H(15) 119.7

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C(3)-C(16)-H(16A) 109.5 C(3)-C(16)-H(16B) 109.5 H(16A)-C(16)-H(16B) 109.5 C(3)-C(16)-H(16C) 109.5 H(16A)-C(16)-H(16C) 109.5 H(16B)-C(16)-H(16C) 109.5 C(6)-C(17)-C(4) 86.36(10) C(6)-C(17)-H(17A) 114.3 C(4)-C(17)-H(17A) 114.3 C(6)-C(17)-H(17B) 114.3 C(4)-C(17)-H(17B) 114.3 H(17A)-C(17)-H(17B) 111.4 C(5)-C(18)-H(18A) 109.5 C(5)-C(18)-H(18B) 109.5 H(18A)-C(18)-H(18B) 109.5 C(5)-C(18)-H(18C) 109.5 H(18A)-C(18)-H(18C) 109.5 H(18B)-C(18)-H(18C) 109.5 C(5)-C(19)-H(19A) 109.5 C(5)-C(19)-H(19B) 109.5

H(19A)-C(19)-H(19B) 109.5 C(5)-C(19)-H(19C) 109.5 H(19A)-C(19)-H(19C) 109.5 H(19B)-C(19)-H(19C) 109.5 C(8)-O(1)-C(7) 117.92(11) Symmetry transformations used to generate equivalent atoms:

Table 4. Anisotropic displacement parameters (Å2x 103) for s4034ma. The anisotropic displacement factor exponent takes the form: -2p2[ h2 a*2U11 + ... + 2 h k a* b* U12 ]

U11 U22 U33 U23 U13 U12 C(1) 22(1) 15(1) 16(1) -3(1) -1(1) 3(1) C(2) 23(1) 24(1) 21(1) -4(1) -2(1) 3(1) C(3) 22(1) 34(1) 24(1) -7(1) 1(1) 2(1) C(4) 26(1) 25(1) 18(1) -5(1) 4(1) 1(1) C(5) 25(1) 20(1) 15(1) 0(1) 0(1) 0(1) C(6) 24(1) 17(1) 16(1) -1(1) -3(1) -1(1) C(7) 33(1) 19(1) 13(1) -1(1) -1(1) 6(1)

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361

C(8) 21(1) 27(1) 22(1) 8(1) -3(1) -5(1) C(9) 40(1) 42(1) 20(1) 6(1) -7(1) -6(1) C(10) 37(1) 17(1) 13(1) 3(1) 0(1) -1(1) C(11) 36(1) 22(1) 21(1) 0(1) 3(1) -1(1) C(12) 36(1) 38(1) 25(1) 7(1) 0(1) -5(1) C(13) 47(1) 40(1) 25(1) 12(1) -9(1) -19(1) C(14) 66(1) 23(1) 22(1) 4(1) -10(1) -12(1) C(15) 48(1) 18(1) 16(1) 2(1) -2(1) 1(1) C(16) 27(1) 58(1) 36(1) -14(1) 7(1) -14(1) C(17) 35(1) 19(1) 17(1) -5(1) -1(1) 1(1) C(18) 36(1) 33(1) 17(1) 4(1) -2(1) 1(1) C(19) 43(1) 17(1) 23(1) 2(1) 3(1) 1(1) O(1) 38(1) 25(1) 14(1) -1(1) -5(1) 8(1) O(2) 41(1) 38(1) 35(1) 4(1) -8(1) 16(1) O(3) 28(1) 48(1) 22(1) -10(1) -5(1) -4(1)

Table 5. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 10 3) for s4034ma.

x y z U(eq) H(1) 1590 3317 348 21 H(3) -2491 4249 1467 32 H(4) -1255 3705 2945 28 H(6) 2749 4085 1686 23 H(7) 146 5240 278 26 H(9A) -1000 4086 -2311 51 H(9B) 897 4363 -2487 51 H(9C) -534 5123 -2741 51 H(11) 4108 4171 -291 32 H(12) 6636 4992 -242 40 H(13) 6762 6590 311 45 H(14) 4372 7363 812 44 H(15) 1822 6562 727 33 H(16A) -3929 2820 1111 60 H(16B) -3863 2970 2187 60 H(16C) -2671 2190 1711 60 H(17A) -138 5150 1792 29 H(17B) 897 4914 2720 29 H(18A) 1265 2580 3542 43

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362

H(18B) 1892 3679 3410 43 H(18C) 2959 2791 3015 43 H(19A) 1920 1840 1697 41 H(19B) 159 2098 1261 41 H(19C) 258 1630 2254 41

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Table 6. Torsion angles [°] for s4034ma. C(6)-C(1)-C(2)-O(3) -163.77(13) C(7)-C(1)-C(2)-O(3) 72.13(16) C(6)-C(1)-C(2)-C(3) 17.26(16) C(7)-C(1)-C(2)-C(3) -106.84(13) O(3)-C(2)-C(3)-C(16) 32.1(2) C(1)-C(2)-C(3)-C(16) -148.89(13) O(3)-C(2)-C(3)-C(4) 161.52(14) C(1)-C(2)-C(3)-C(4) -19.52(17) C(2)-C(3)-C(4)-C(17) 59.15(15) C(16)-C(3)-C(4)-C(17) -172.61(12) C(2)-C(3)-C(4)-C(5) -35.72(17) C(16)-C(3)-C(4)-C(5) 92.51(15) C(3)-C(4)-C(5)-C(19) -38.05(17) C(17)-C(4)-C(5)-C(19) -146.64(12) C(3)-C(4)-C(5)-C(18) -166.80(12) C(17)-C(4)-C(5)-C(18) 84.62(13) C(3)-C(4)-C(5)-C(6) 81.81(13) C(17)-C(4)-C(5)-C(6) -26.78(9) C(2)-C(1)-C(6)-C(17) -54.82(14) C(7)-C(1)-C(6)-C(17) 67.42(14) C(2)-C(1)-C(6)-C(5) 40.49(14) C(7)-C(1)-C(6)-C(5) 162.72(11) C(19)-C(5)-C(6)-C(1) 38.42(17) C(18)-C(5)-C(6)-C(1) 165.00(11) C(4)-C(5)-C(6)-C(1) -83.78(11) C(19)-C(5)-C(6)-C(17) 149.05(13) C(18)-C(5)-C(6)-C(17) -84.37(12) C(4)-C(5)-C(6)-C(17) 26.85(10) C(6)-C(1)-C(7)-O(1) 164.51(11) C(2)-C(1)-C(7)-O(1) -72.20(13)

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363

C(6)-C(1)-C(7)-C(10) 46.16(15) C(2)-C(1)-C(7)-C(10) 169.44(11) O(1)-C(7)-C(10)-C(15) 122.61(13) C(1)-C(7)-C(10)-C(15) -120.65(14) O(1)-C(7)-C(10)-C(11) -55.23(15) C(1)-C(7)-C(10)-C(11) 61.50(16) C(15)-C(10)-C(11)-C(12) -1.6(2) C(7)-C(10)-C(11)-C(12) 176.24(13) C(10)-C(11)-C(12)-C(13) 1.3(2) C(11)-C(12)-C(13)-C(14) 0.1(2) C(12)-C(13)-C(14)-C(15) -1.0(2) C(11)-C(10)-C(15)-C(14) 0.7(2) C(7)-C(10)-C(15)-C(14) -177.19(13)

C(13)-C(14)-C(15)-C(10) 0.6(2) C(1)-C(6)-C(17)-C(4) 83.84(12) C(5)-C(6)-C(17)-C(4) -27.13(10) C(3)-C(4)-C(17)-C(6) -85.64(12) C(5)-C(4)-C(17)-C(6) 27.06(9) O(2)-C(8)-O(1)-C(7) 2.1(2) C(9)-C(8)-O(1)-C(7) -177.86(12) C(10)-C(7)-O(1)-C(8) -100.15(14) C(1)-C(7)-O(1)-C(8) 137.33(12) ________________________________________________________________


Table 7. Hydrogen bonds for s4034ma [Å and °].

D-H...A d(D-H) d(H...A) d(D...A) <(DHA)

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