Building blocks for multinulear near-IR luminescent ... luminescent lanthanide complexes Caroline Bischof Ruhr-Universität Bochum Faculty of Chemistry and Biochemistry Building blocks

Dissertatio

n

Building blocks for multinuclear

near-ir luminescent lanthanide complexes

Caroline Bischof

Ruhr-Universität Bochum

Faculty of Chemistry and Biochemistry

Building blocks for multinuclearnear-IR luminescent lanthanide complexes

by

Caroline Bischof

A dissertation submitted in partial satisfaction

of the requirements for the degree of a

Doctor of Philosophy (Dr. rer. nat.) in Chemistry

at the

Faculty of Chemistry and Biochemistry

Ruhr-Universität Bochum

March 2013

Committee in charge:

Dr. Michael Seitz

Prof. Dr. Martin Feigel

Thesis defense date:

April 12th 2013

The present work was performed in between October 2009 until March 2013

at the Chair of Inorganic Chemistry I at the Ruhr-University Bochum

(Germany) under the supervision of Dr. Michael Seitz.

I hereby declare, on oath, that I have written the present dissertation by my own

and have not used other than the acknowledged resources and aids.

Acknowledgement

Although only my name appears on the title page, there have been many people who

contributed to this work and to the continuous learning process I went through dur-

ing the past 3.5 years. I am very grateful for their help and I would like to thank

particularly the following people:

Dr. Michael Seitz for the supervision of this work, the given scientific freedom in pur-

suing my ideas and valuable lessons on organic chemistry. I would further like to

thank him for his great support and the chance to participate in several international

conferences.

Prof. Feigel for kindly acting as second referee of my doctoral thesis.

Prof. Nils Metzler-Nolte for his continuous support over many years.

My former Bachelor student Felix Stog and my former Master students Julia Scholten

and Simon Trosien, for working with me on the deuterated cryptates.

Martin Gartmann, Gregor Barchan and Hans Jochen Hauswald for measuring several

NMR spectra for me and, more importantly, for helping out when the spectromet-

ers and me had a different opinions of what to do with my sample.

Bauke Albada, Nicola Alzakhem, Christine Doffek, Sven Hennig and Hendrik Pfeiffer for

proof-reading this thesis.

Dr. Klaus Merz and Vera Vasylyeva for their help with X-ray crystals determinations.

I would also like to thank Herr Klaus for letting me supervise students in the Ad-

vanced Inorganic Chemical Practical, the introduction to numerous good reads and

lots of cheerful laughter.

Gundula Talbot for her lending me her ear, for helpful advices and for smiling.

My climbing friends for experimenting with me in the areas of gravity and vertical

limits and producing visible results.

My bio-connection: Sabrina Beck, Rebecca Gentek, Jan Gleichhagen, Sven Hennig, Alex-

ander Neuhaus, Steffen van der Wal and Bauke Albada for their input on biochemical

matters and for sharing their different perspectives with me. I particularly thank Jan

for providing litres of different buffers. Also, thank you Sabrina and Becci for being

“my girls”!

My colleagues from the ACI, especially Jan Dittrich, Tuba Güden, Nina Hüsken, Qian

Kun, Mingyan Ma, Julia Norman, Hendrik Pfeiffer, Lukasz Raszeja and Thomas Sowik for

making the past years a colourful and memorable time, and also for sharing lots of

delicious food. I particularly thank Hendrik and Thomas for their mental support.

My partners in crime: David Bulfield, David Köster, Malay Patra, Anna Sosniak, Felix

Stog and Jessica Wahsner for being such wonderful labmates and supportive friends.

Whenever I faced a problem or just wanted to share the latest climbing story, they

were at my side listening, discussing, helping out, cheering up and laughing.

My faithful companion Nicola Alzakhem, who is an outstandingly talented chemist and

has been a patient teacher for me. With him, I celebrated clean NMR spectra, waltzed

through the lab, discussed organic chemistry from A to Z, and went for numerous

“walkabouts” to lectures, glass containers and countries. I consider myself very lucky

to have him in my life.

Ein besonderer Dank gilt meiner Familie, vor allem meinen Brüdern und meinen El-

tern. Sie haben mich stets ermuntert frohen Mutes meinen Weg zu gehen und dafür

Sorge getragen, dass ich im Gedankennebel meiner Promotion nicht die wesentlichen

Dinge im Leben aus den Augen verliere.

Last but not least I am grateful for the financial support from the International Max

Planck Research School in Chemical Biology and from the Ruhr University Research

School.

for Jürgen and Claus Bischof,

who were once joking about “Acetan Hydrid”

“ The only true voyage of discovery would be not to visit new landscapes

but to possess other eyes, to behold the universe through the eyes of another...”Marcel Proust, La Prisonnière

Contents

1 Introduction 1

1.1 Imaging in biological systems . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1.1 Imaging in medicine and biology . . . . . . . . . . . . . . . . . . 1

1.1.2 Optical imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.2 Fluorescent probes for optical imaging . . . . . . . . . . . . . . . . . . . 4

1.2.1 Luminescence, fluorescence and phosphorescence . . . . . . . . . 4

1.2.2 Properties of fluorescent probes . . . . . . . . . . . . . . . . . . . 5

1.2.3 Classification of fluorophores . . . . . . . . . . . . . . . . . . . . . 7

1.3 Near-IR optical imaging with lanthanides . . . . . . . . . . . . . . . . . . 10

1.3.1 Upconversion processes . . . . . . . . . . . . . . . . . . . . . . . . 11

1.3.2 Activators and sensitisers . . . . . . . . . . . . . . . . . . . . . . . 11

1.3.3 Upconversion nanophosphors . . . . . . . . . . . . . . . . . . . . 13

1.3.4 Upconversion in molecular lanthanide complexes . . . . . . . . . 13

2 Concept of the project 16

3 Development of binuclear cryptates 18

3.1 Deuterated cryptates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3.1.1 Retrosynthetic analysis . . . . . . . . . . . . . . . . . . . . . . . . 21

3.1.2 Synthesis of building blocks and cryptates . . . . . . . . . . . . . 22

3.1.3 Photophysical properties . . . . . . . . . . . . . . . . . . . . . . . 27

3.2 Functionalised cryptates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3.2.1 Retrosynthetic analysis of functionalised cryptates . . . . . . . . 34

3.2.2 Synthesis of carboxy functionalised cryptates . . . . . . . . . . . 35

3.2.3 Synthesis of amino functionalised cryptates . . . . . . . . . . . . 39

3.3 Binuclear cryptates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

3.3.1 Strategy for the coupling of cryptates . . . . . . . . . . . . . . . . 45

3.3.2 Functionalised cryptates for coupling reactions . . . . . . . . . . 45

3.3.3 First approaches towards the coupling of cryptates . . . . . . . . 47

3.3.4 Preparation of the first binuclear TBP cryptate . . . . . . . . . . . 48

4 Chemical unclicking of amide bonds 51

4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

4.2 Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

4.3 Preliminary work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

4.4 Retrosynthetic analysis of Unclick serine . . . . . . . . . . . . . . . . . . 56

4.5 Investigation of different Unclick systems . . . . . . . . . . . . . . . . . . 57

4.5.1 Fmoc-Unclick serine . . . . . . . . . . . . . . . . . . . . . . . . . . 57

4.5.2 Acetyl-Unclick-serine . . . . . . . . . . . . . . . . . . . . . . . . . 60

4.5.3 Acetyl-Unclick-serine via Boc-cycloserine . . . . . . . . . . . . . . 62

4.5.4 Fmoc-Gly-Unclick-serine . . . . . . . . . . . . . . . . . . . . . . . 66

5 Summary 70

6 Experimental Section 74

6.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

6.2 Luminescence measurements . . . . . . . . . . . . . . . . . . . . . . . . . 75

6.3 Simple bipyridine building blocks . . . . . . . . . . . . . . . . . . . . . . 76

6.4 Bipyridine ester building blocks . . . . . . . . . . . . . . . . . . . . . . . 87

6.5 Nitro and amino bipyridine building blocks . . . . . . . . . . . . . . . . 93

6.6 Simple macrocycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

6.7 Nitro and amino functionalised macrocycles . . . . . . . . . . . . . . . . 99

6.8 Simple deuterated cryptates . . . . . . . . . . . . . . . . . . . . . . . . . . 101

6.9 Carboxylic acid ester functionalised crypates . . . . . . . . . . . . . . . . 104

6.10 Amino functionalised cryptates . . . . . . . . . . . . . . . . . . . . . . . . 106

6.11 Derivatives of functionalised cryptates . . . . . . . . . . . . . . . . . . . . 108

6.12 Lanthanide complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

6.13 Fmoc-protected serines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

6.14 Acetyl-protected serines . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

6.15 Boc-protected serines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

6.16 Other serines and related molecules . . . . . . . . . . . . . . . . . . . . . 125

7 Bibliography 129

Appendix i

A Data for crystal structures . . . . . . . . . . . . . . . . . . . . . . . . . . . i

B Publication list . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

List of abbreviations

AA any amino acid

Boc tert-butyloxycarbonyl

CT computed tomography

CFP cyan fluorescent protein

DELFIA dissociation enhanced lanthanide fluorescence immunoassay

DIPEA N,N’-diisopropylethylenediamine

DMF dimethylformamide

DMSO dimethylsulfoxide

DOTA 1,4,7,10-tetraazacyclododecane-

1,4,7,10-tetraacetic acid

EDC 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide

EI electron impact mass spectrometry

ESI electro-spray ionisation mass spectrometry

ETU energy transfer upconversion

FAB fast atom bombardment mass spectrometry

FRET fluorescence resonance energy transfer

Fmoc 9-fluorenylmethyloxycarbonyl

GFP green fluorescent protein

HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-

triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate

MRI magnetic resonance imaging

PET positron emission tomography

PBS phosphate-buffered saline

PDB protein data bank

QD quantum dots

RFP red fluorescent protein

rt room temperature

SPECT single-photon emission computed tomography

SPPS solid phase peptide synthesis

TBP tris(bipyridine)

TFA trifluoroacetic acid

THF tetrahydrofurane

TLC thin layer chromatography

TMS trimethylsilyl

TRF time-resolved detection

UC upconversion

YFP yellow fluorescent protein

1 Introduction

1.1 Imaging in biological systems

1.1.1 Imaging in medicine and biology

Bioimaging is a collective term for a wide range of visualisation techniques in medi-

cine and modern biology. These techniques are used for the non-invasive visualisa-

tion of morphological and physiological details of living systems. Hence, bioimaging

methods are powerful tools for the diagnosis of diseases and also for the understand-

ing of biological processes. Bioimaging comprises

• anatomical imaging:

the creation of images of anatomic structures (organs or tissues),

• functional imaging:

the studies of organ functions during physiological stimulation,

• molecular imaging:

the in vivo probing of biological processes on a molecular level.

Anatomical and functional imaging together form the field of classical diagnostic med-

ical imaging. The commonly applied methods are based on ultrasound, X-ray, radi-

onuclides or nuclear magnetic resonance. The scope of information gathered by em-

ploying these techniques can even be extended by the use of contrast agents. Import-

ant advantages and potential risks of these methods are summarised in Table 1.1. [1–4]

The desire to investigate biological processes in detail led to the development of

imaging techniques, that allow to probe details on a molecular level. Molecular ima-

ging plays a vital role in drug discovery, in fundamental biological research and for the

characterisation of disease-related molecular events. Applications range from simple

cell localisation to the visualisation of complex events. For example, protein-protein

interactions can be monitored with positron emission tomography (PET), and receptor

assays or enzymatic pathways can be probed with radiotracers. Optical imaging

provides an alternative to these methods and is often used for the visualisation of

structures and pathways in biological systems.

1

1.1 Imaging in biological systems

Table 1.1: Benefits and limitations of methods in diagnostic medical imaging [4,5]

Imaging method Benefits Limitations

Ultrasound low patient risk; restricted to superficial structuresquick examination poor spatial resolution (0.1-1mm) [6]

X-ray and X-ray versatile diagnosis of bones exposition to ionising radiation hascomputed tomography (CT) or organs to be kept low

Radionuclide imaging high sensitivity; incorporation exposition to radiation released by(PET, SPECT) or attachment to substances for radionuclides has to be kept low;

specific accumulation possible; poor spatial and temporal resolutionallows whole body scanning

Magnetic resonance imaging does not require cannot be used for patients with(MRI) ionising radiation metal-containing implants due to

large magnetic field strengths

1.1.2 Optical imaging

Optical technologies rely on the use of light and provide essential tools for non-

invasive, high-resolution imaging . [7,8] The detected light can either originate from

the target structure itself or from reporter molecules, so-called optical probes. In the

first case, the observed reflection of light or change of polarisation is translated into an

image. However, the commonly low intrinsic contrast of the investigated structures

often limits the amount of gained information. A much better signal-to-background

ratio and, thus, a higher degree of information can be achieved by the utilisation of

optical probes. The most frequently used reporter molecules are fluorescence probes,

which are molecules that emit light from an electronically excited state upon relaxa-

tion to a ground state. Advanced fluorescent probes for medical diagnostics possess

three functional parts: [3]

• a signalling component,

• a carrier with suitable pharmacokinetics,

• and a targeting moiety for the delivery to a specific structure.

Such probes are incorporated into the system of interest and are excited by an external

excitation source. Usually, the excitation and emission wavelengths of these probes

are in the visible region of the electromagnetic spectrum. The performance of optical

probes is often optimised by maximising or differentiating the target signal e.g. by

the use of activatable imaging probes.

According to the underlying chemical mechanisms, activatable imaging probes are di-

vided into three classes. [3] First, there are small molecules with an intrinsic activation

2

1 Introduction

mechanism (Figure 1.1a). They are highly specific and react with an analyte or un-

dergo an intramolecular alteration. This results in a modification in the absorption

or emission characteristics (e.g. intensity, wavelength). The second class comprises

large molecules, whose luminescence is quenched due to deaggregation (Figure 1.1b).

This process might be a result of changed environmental conditions (e.g. pH) or the

interaction with an enzyme. Such macromolecules can be selectively accumulated

in target structures, which results in high resolutions. However, due to their size,

they are slowly metabolised and slowly excreted from the body. The last class are

assemblies of macromolecular targeting moieties and small molecule signalling moi-

eties (Figure 1.1c). They combine the advantages of the two former classes for they

provide high resolutions after selective activation. Typical activatable imaging probes

are short peptides or nucleic acids strands. [9–11]

++

a)

b)

c)

Figure 1.1: Activatable optical imaging probes. Blue and red structures are intact and altered optical

probes, respectively. Grey spheres stand for non-luminescent molecules and white struc-

tures for analytes. a) Small molecules with an intrinsic activation mechanism. b) Large

molecules, whose luminescence is quenched due to deaggregation. c) Molecular assemblies

of macromolecules with small signalling moieties.

Optical imaging with suitable probes provides several advantages, e.g. high sens-

itivity, low toxicity, low cost, the possibility to target designated structures and the

option of selective signal activation or quenching. [3,12] Hence, this technique repres-

ents an interesting alternative to the use of radioisotopes, which are often employed

for the detection and quantification of molecules in a biological environment. [13–17]

Applications of fluorescence probes offer a broad selection of emission penetration

depths and spatial resolutions, ranging from micrometers to centimetres. Thus, ver-

satile structures from small viruses to eukaryotic cells and large tissues can be mon-

itored. Important optical in vivo imaging techniques are listed in Table 1.2.

3

1.2 Fluorescent probes for optical imaging

Table 1.2: Optical imaging techniques [8]

Technique Contrasta Depth Common Clinical

wavelength potential

Microscopic resolutionEpi A, Fl 20 µm visible experimentalConfocal Fl 500 µm visible experimentalTwo-photon Fl 800 µm visible yes

Mesoscopic resolutionOptical projection tomography A, Fl 15 µmb visible noOptical coherence tomography S 2 mm vis. + nIR yesLaser speckle imaging S 1 mm vis. + nIR yes

Macroscopic resolution intrinsicHyperspectral imaging A, S, Fl <5 mm visible yesEndoscopy A, S, Fl <5 mm visible yesPolarization imaging A, S <1.5 cm vis. + nIR yesFl. reflectance imaging (FRI) A, Fl <7 mm nIR yesDiffuse optical tomography (DOT) A, Fl <20cm nIR yes

Macroscopic resolution molecularFl. resonance imaging (FRI) A, Fl <7 mm nIR yesFl. molecular tomography (FMT) Fl <20 cm nIR yesBioluminescence imaging (BLI) E <3 cm 500-600 nm no

a A=absorption, E=emission, S=scattering, Fl=fluorescence. b In cleared specimen.

The development of clinical optical imaging techniques means the extension of ex-

isting assays and the attempt to translate in vitro or 2D applications into in vivo or

optical tomography (3D) techniques. The underlying methods are already limited

due to photobleaching of the probes as well as strong absorption, light scattering and

autofluorescence in biological specimens. The novel techniques also have to overcome

weak tissue penetration and master the challenge to obtain high-resolution images of

cells located within deep tissues. [12] Hence, optical imaging is still regarded a highly

experimental technique. [7] One option to solve the mentioned problems and to expand

applications from through-skin-visualisation of superficial tissues (e.g., breast, [18,19]

lymph nodes [20,21]) is the use of endoscopy [20,21] or surgery [22,23].


1.2.1 Luminescence, fluorescence and phosphorescence

Luminescence means the emission of light by a substance, when an electron returns

from an electronically excited state to the ground state. Depending on the spin before

and after this transitions, two categories are distinguished (Table 1.3).

4

1 Introduction

During fluorescence, transitions are spin-allowed (∆S=0) and, consequently, the emis-

sion rates are fast. In contrast, in the case of phosphorescence, the spin changes during

the transition from the triplet excited state (T1) to the ground state (S0). This process

is spin-forbidden (∆S>0) and, hence, emission rates are slow. Since the time-scale

of this process is significantly longer than the autofluorescence in biological systems,

time-resolved detection (TRD) procedures can be employed. During these experi-

ments, the background fluorescence is allow to decay to negligible levels before the

phosphorescence signal is measured. [24,25] A general problem of phosphorescence is

that the triplet state might be subject to photochemical reactions, which destroy the

luminophore (photobleaching).

Table 1.3: Comparison of different types of luminescence [24]

Fluorescence Phosphorescence

Spin selection rule∆S=0 ∆S>0

(allowed) (forbidden)

Transition path S0 → S1 → S1 S0 → S1ISC→ T1 → S0

Emission ratesfast slow

107 − 109 s−1 1− 103 s−1

Lifetimesshort long

0.1 µs − 1 ns 1 s − 1 ms

1.2.2 Properties of fluorescent probes

Most commonly, fluorescence probes are excited with photons from an external light

source and relax back to the ground state under the emission of usually less energetic

photons. [7,8] For this processes to be efficient, certain factors have to be taken into

account.

The wavelengths of absorption and emission are the first properties to be considered.

High energy radiation (blue, green), which in general can cause tissue damage, has

a short tissue penetration depth and is therefore only suitable for the imaging of

superficial structures. [26] Lower energy excitation (yellow, red) provides an increased

penetration depth, but also goes along with strong autofluorescence, since biological

specimens mainly absorb in this spectral region (Figure 1.2). [27] An advantageous

combination of deep tissue penetration and low autofluorescence can be achieve by

the use of near-IR radiation. However, it was also reported that its use can cause tissue

heating. [28] Obviously, the use of every sort of radiation possesses advantages and

5


drawbacks and, therefore, wavelengths always have to be adjusted to the respective

specimen. Still, in every case the absorption and emission spectra should be separated

by large Stoke’s shifts to allow for an efficient optical separation of excitation and

fluorescence. [3]

Figure 1.2: Wavelength-dependent absorption coefficient of normally oxygenated tissue (saturation of

70%) with 50% water, 15% lipids and 50 mM hemoglobin concentration of 50 mM. [8]

The intensities of absorption and emission are also of importance. The absorption

intensity is a measure for the excitation probability. When it increases, less light is

required for the excitation of the probe, which simultaneously reduces the odds of

tissue damage. The fluorescence intensity, which is determined by the product of

quantum yield and extinction coefficient, reflects the brightness of a probe. With

higher fluorescence intensity, deeper penetration depths and higher signal-to-noise

ratios can be achieved. However, the latter is often only attained using larger emitters

(e.g. green fluorescent protein, quantum dots). [3] In addition to these photophysical

requirements, one also has to consider certain chemical properties of the probes.

Regarding the stability, the main problems are photobleaching and metabolisation.

Photobleaching can be reduced by decreasing the light intensity (e.g. by reducing

the number of scans), and simultaneous utilisation of highly sensitive video cameras

or photographic films. Another approach is the design photostable probes or repeat

probe injection. In vivo stability is much more difficult to achieve, because fluorescence

probes are often decomposed in the presences of lysosomes and enzymes and lose

their fluorescence properties. Still, for some compounds, degradation can be desired,

so they can be metabolised and excreted. [3,29]

6

1 Introduction

One last factor that should be considered is that fluorescent probes can induce

an alteration of pharmacokinetics. When several fluorophores or large molecules (e.g.

nanoparticles or quantum dots) are attached to the target structure, its bioavailabilty,

distribution and clearance can be significantly altered. As a consequence, size and

form have to be considered carefully, keeping in mind the target structure. [3]

1.2.3 Classification of fluorophores

Fluorescence probes can be divided into three major classes: genetically encoded

fluorophores, inorganic materials and small molecule fluorophores. [3]

The class of genetically encoded fluorophores [3,30,31] comprises fluorescent molecules

that naturally occur in eukaryotic cells. The most prominent examples is the green

fluorescent protein (GFP) (Figure 1.3a). The fluorophore of this protein, an imidazoline

moiety (λex 488 nm, λem 509 nm), is derived from sequential serine-tyrosine-glycine

residues, [32–34] and is embedded into a beta-barrel. Mutations or circular permuta-

tions of these or other amino acids result in new spectral variants e.g. blue fluores-

cence (Cyan Fluorescent Protein, CFP). [32,35] Other representatives of this group are

the endogenous yellow (YFP) and red fluorescent proteins (RFP), artificial endogen-

ous proteins with “unnatural wavelengths” (e.g. in the near-IR), and certain fusion

proteins. All these molecules are relatively large (30-50 kDa), which limits their tar-

geted delivery and potentially affects normal protein functions. Fluorescent proteins

have usually broad excitation and emission spectra as well as low quantum yields.

Still, they can be obtained without cumbersome synthetic procedures and possess ex-

cellent photostability. These important advantages mostly explain their routine use

in functional and molecular imaging e.g. for site-specific labelling of proteins or for

monitoring gene expression. However, translation of these applications for clinical

use is highly impracticable, since the DNA code for the respective protein needs to be

transferred into the host cells.

The most commonly used inorganic materials for bioimaging are quantum dots (QD)

(Figure 1.3b). [3,12,30,31] These are semiconductor nanocrystals, which have sizes ran-

ging from 2 to 50 nm, typically consist of CdSe or CdTe cores and a ZnS coating. [36]

Their emission wavelength can be tuned by changing the particle size, where an in-

crease in size results in a red-shift of emission. QD are commercially available in

various sizes with emissions across the visible spectrum. They possess superb photo-

physical properties, e.g. high photostability, high fluorescence quantum yields (>80%

in organic solvents), broad excitation spectra and narrow emission bandwidths. All

these factors contribute to excpetionally high signal-to-noise ratios. In addition, their

7


functionalisation is also feasible e.g. to enhance water solubility or to attach bio-

molecules to them. [37] Therefore, QD have become regularly used fluorophores for

microscopy [38] and for the monitoring of analytes in living cells. [39] There are two

drawbacks which limit the use of quantum dots. On the one hand, they contain

heavy metals (Se, Cd), which are potentially cytotoxic. On the other hand, their size

often exceeds the renal excreation limit (~6 nm). Instead, they are mostly excreted

through liver and bile without metabolism which implies a prolonged residence time

in the blood. If they are attachted to targetting moieties, the clearance from the body

is even more delayed. In addition to quantum dots, carbon nanotubes and gold nan-

oparticles are also promising inorganic materials for in vivo imaging. However, they

also raise concerns regarding biodegradability and toxicity, which still impedes their

clinical use. [40] Other important inorganic materials are lanthanide-based luminescent

nanoparticles, which will be discussed in section 1.3.3.

10 Å

CdSe coreZnS shell

β-barrel

imidazoline

10 Å

a) b) c)

OHN NH

O

O

Cl

ca. 20-500 Å

Figure 1.3: Typical representatives for different classes of fluorophores. (a) The green fluorescent

protein [41] (PDB Code 1GFL, genetically encoded fluorophore), (b) a simple quantum dot [42]

(inorganic materials) and (c) Rhodamine 6G (small-molecule fluorophore).

The class of small-molecule fluorophores [3,31,43] comprises a large number of organic and

inorganic compounds with low molecular weights (300-2000 g/mol). The majority

of them are commercially available organic dyes with core structures of cyanines [44],

boradiazaindacenes [45], coumarins [46], and xanthenes [47,48]. Well-known representat-

ives of organic fluorophores are fluoresceins and rhodamines (Figure 1.3c), which are

both derived from a xanthene motif. Small molecule fluorophores can have emis-

sion wavelengths from blue to near-infrared, and thus cover the greater part of the

electromagnetic spectrum. Depending on the molecule, the emission wavelength or

intensity can also be influenced by pH, solvent or the presence of metal ions. This

sensitive emission as well as their small size and monodispersity make them versatile

8

1 Introduction

tools for biomolecular labelling, cellular staining or the indication of environmental

factors. However, limitations may arise from their susceptibility to photobleaching,

lacking stability under physiological conditions, strong background fluorescence in

the visible spectrum and broad emission bands. [49]

Prominent examples for inorganic small-molecule fluorophores are transition metal

complexes such as [Ru(bpy)3]Cl2, and lanthanide complexes with macrocyclic lig-

ands, such as cryptates [50–53] (Figure 1.4a) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-

tetraacetic acid (DOTA) [54,55] (Figure 1.4b). Lanthanides exhibit elementspecific lu-

minescence, which covers the whole spectral range from UV to near-IR. However,

they possess low absorption coefficients (<10 M−1 cm−1) due to Laporte-forbidden

transitions within the 4f orbitals. [56] As a result, excited states are not populated read-

ily, leading to weak emission intensities and limiting the detection level. Hence, the

surrounding of the lanthanide has to be equipped with a strongly absorbing chromo-

phore. This can be the ligand itself or a moiety attached to it. [57]

N

N

N

NN

N

N

N

Ln

3+ -

N N

NN

O

O

O

O

O

OO

O Ln

a) b)

Figure 1.4: Lanthanide-based small molecule fluorophores. Structures of lanthanide complexes with

a trisbipyridine (TBP) cryptand (a) and a DOTA ligand (b).

Irradiation of this "antenna" at a suitable wavelength leads to the excitation of a

singlet excited state (S0), followed by intersystem crossing to a triplet excited state (T1)

(Figure 1.5). Subsequent energy transfer to matching lanthanide emissive states res-

ults in efficient sensitation of luminescence (Figure 1.5c). [58] The observed lanthanide

emission bands are narrow (full width half maximum <12 nm), [59] since the electronic

states are not subject to coupling with vibrational modes. Moreover, the f-electrons are

shielded by outlying 5s and 5p electrons, so that ligand field effects marginally influ-

ence the spectroscopic properties. Consequently, lanthanides are mostly insensitive to

changes in their coordination sphere. Concentration dependent self-absorption does

also not occur to a great extend, since absorption and emission bands are separated

by large Stoke’s shifts (~200 nm). [24,25] Overall, their unique photophysical properties

make molecular lanthanide complexes excellent candidates for optical probes, which

can even be used for time-resolved measurements.

9

1.3 Near-IR optical imaging with lanthanides

Ligand Ln3+

S1

T1

S0

4f*

emis

sion

abso

rptio

nEne

rgy

Figure 1.5: Jablonski diagram that illustrates the antenna effect. The dashed-dotted, dashed, dotted,

and full arrows represent photon excitation, energy transfer, multiphonon relaxation, and

emission processes, respectively.

Thus, they form a low-cost alternative to inorganic materials. Typical applications

of luminescent lanthanide complexes are DELFIA (dissociation enhanced lanthanide

fluorescence immunoassay) and time-resolved FRET (fluorescence resonance energy

transfer) assays. [60–62] The biggest challenge in employing lanthanide complexes is to

find suitable antenna ligands and protect the lanthanide efficiently from quenching

molecules in its proximity.


Optical imaging techniques that rely on lanthanide luminescence often employ probes

that emit in the visible region e.g. Eu3+ or Tb3+ complexes. [15–17,63,64] However, there is

a growing interest in the combination of the excellent photophysical properties of the

lanthanides with the advantages of near-IR radiation. [65,66] As mentioned previously,

the usefulness or near-IR light in bioimaging arises from its deep penetration depth

and the minor autofluorescence in this spectral window (650-900 nm). Furthermore,

the light scattering decreases with increasing wavelengths, which can also contribute

to a higher sensitivity of the probe.

There are two different options to combine the merits of lanthanide luminescence and

near-IR radiation. On the one hand, near-IR emission e.g. from Yb3+, Nd3+ and Er3+

complexes can be sensitised, using biologically compatible, visible light. [67–70] On the

other hand, lanthanides can convert near-IR long-wavelength excitation into shorter-

wavelength emission through upconversion processes. In contrast to the former case,

such systems (usually inorganic materials) are preferably used for bioimaging, since

their emission is less prone to non-radiative deactivation and, simultaneously, they

provide the chance to excite and detect in the near-IR region. [49,71]

10

1 Introduction

1.3.1 Upconversion processes

During simple linear optical processes (Figure 1.6 left), an activator in its ground state

absorbs a photon, which leads to the population of an excited state. Radiative de-

activation then results in the emission of light. It is also possible, that the activator

interacts with a nearby sensitiser to get promoted to an excited state, while the sens-

itiser relaxes to its ground state.

In a non-linear optical process (Figure 1.6 right), the activator is first promoted to

a metastable excited level by absorption of a pump photon. Non-radiative energy

transfer to this system by a nearby sensitiser leads to the promotion of the activator

to a higher excited state. [72–75] The sequential absorption of two or more photons of

lower energy (e.g. near-IR) thus produces emission of higher energy (e.g. visible

light). This process is referred to as upconversion (UC) luminescence. It is considered

a non-linear process, since the luminescence intensity is proportional to the square of

the laser power (I ∝ P2).

G

E1

G

E1

G

E1

E2

Ene

rgy

Upconversion luminescenceLuminescence

AS AS

Figure 1.6: Energy schemes illustrating the difference between linear and non-linear optical pro-

cesses. A and S stand for activator and sensitiser, respecivley. Ground and excited states

are marked with G and E1 or E2, respectively. In simple systems, activators are excited dir-

ectly (left) or by energy transfer from a sensitiser (middle). Upconversion can be achieved

by energy transfer upconversion (ETU, right). The dashed-dotted, dashed, dotted, and full

arrows represent photon excitation, energy transfer, multiphonon relaxation, and emission

processes, respectively.

1.3.2 Activators and sensitisers

In general, all lanthanides with multiple long-lived metastable excited levels can act

as activators. However, the energetic distances between ground state, metastable in-

termediate level and excited level have to be approximately the same for an efficient

energy transfer (Figure 1.7). For example, in an Er3+ ion the energy gaps between

11


4 I15/2 and 4 I11/2 (10 350 cm−1) and between the excited states 4 I11/2 and 4F7/2 (10 370

cm−1) are very similar. Hence, 4F7/2 can be populated readily by non-linear energy

transfer from a sensitiser. Subsequent non-radiative deactivation leads to the popu-

lation of lower-lying metastable levels (e.g. 2H11/2, 4S3/2, 4F9/2), which relax to the

ground state under UC emission. In addition to Er3+, the lanthanides Tm3+ and Ho3+

are also frequently used activators. [49]

The sensitiser, which can be either a lanthanide, a transition metal or an organic

chromophore has to be located in the proximity of the activator. The most often used

sensitiser is Yb3+, because it has just one excited level and a higher absorption cross

section than most other lanthanides. Yb3+ absorbs low-energy photons at around

980 nm due to the 2F7/2 → 2F5/2 transition, which is resonant with f-f-transitions of

Er3+, Tm3+ and Ho3+.

Particularly interesting combinations for bioimaging are Yb/Er (λexc 980 nm, λem

655 nm) and Yb/Tm (λexc 980 nm, λem 800 nm), which have both, excitation as well

as emission wavelengths, in the near-IR. [49,59,76]

4F7/2

2H11/24S3/2

4F9/2

4I9/2

4I11/2

4I13/2

4I15/22F7/2

2F5/2

0

5

10

15

20

E/(

10

3cm

-1)

Yb3+ Er3+

980

nm

655

nm

540

nm

520

nm

Figure 1.7: Energy transfer mechanisms for UC processes involving an Yb3+ sensitiser and an Er3+

activator using 980 nm excitation. The dashed-dotted, dashed, dotted, and full arrows

represent photon excitation, energy transfer, multiphonon relaxation, and near-IR emission

processes, respectively.

Regardless of the specific lanthanide combination, it is important that the sensitiser

content is much lower than the activator content to avoid an energy loss during cross-

relaxation. In addition, non-radiative deactivation by other molecules (e.g. solvents)

has to be avoided. In practice, this is achieved by the controlled doping of inorganic

matrices with lanthanides. [49,55]

12

1 Introduction

1.3.3 Upconversion nanophosphors

Upconversion nanophosphors are lanthanide-doped crystalline materials, [38] that can

have different shapes (e.g., nanospheres, nanorods, nanocubes or nanoplates) and

sizes (sub-nm to sub-µm range). Under continuous wave excitation at 980 nm, they

generate violet to near-IR emission. Their UC efficiency and emission colour can be

adjusted by the choice of host lattice (material, structure) and dopant ions (concentra-

tion, combination). [59,76,77]

The host lattice has to meet several requirements, such as consistent defined shape

and nanoscale size, low cytotoxicity, water stability and low lattice phonon ener-

gies to prevent non-radiative deactivation processes. Commonly used host matrices

are transparent crystalline materials based on fluorides, oxides, chlorides, bromides,

oxysulfides, phosphates or vanadates. These materials also contain alkali metals (e.g.

Na+) or rare earth elements (e.g. Y3+, La3+, Gd3+) as counter ions. Among the re-

ported lattices, hexagonal 100 nm [NaYF4:Yb,Er] nanocrystals proved to be the most

efficient matrix with absolute UC luminescence efficiencies of up to 0.3%. The dopants

are embedded into the host lattice in relatively low concentrations (usually <2 mol%)

to create sufficient distances between neighbouring ions. [49,60,76]

The most important benefits of nanophosphors are their photostable luminescence

and minimised background autofluorescence. Additionally, they can be excited with

an inexpensive continuous wave near-IR diode laser and in comparison with quan-

tum dots, they possess low toxicity. Drawbacks, which limit their standardised use are

the lack of generalised protocols for synthesis and modification, and also the low UC

luminescence efficiency due to vibrational deactivation and surface defects. [59]

Nevertheless, upconversion nanophosphors are already in use in several applications

as alternatives to quantum dots or organic fluorophores. They are used in vitro for

immunochromatographic assays, bioaffinity assays, and homogeneous bioanalytical

assays based on FRET. [49,76,77] Since their emission has a deeper penetration depth

than quantum dots, upconversion nanophosphors were successfully applied in vivo

for small-animal imaging e.g. to visualise tumours, the lymphatic and vascular system

or to track cells. [59]

1.3.4 Upconversion in molecular lanthanide complexes

There are only few precedences for systems, which combine the advantages of small

molecule fluorophores with the benefits of upconverted near-IR emission. In com-

parison with UC nanophosphors, the reported compounds also contain a lanthanide

13


activator, but the sensitisers are either organic chromophores or transition metals.

When organic chromophores are employed as sensitisers, multiphoton absorption of

the organic moiety causes the population of its singlet excited state. By definition, this

intersystem crossing and energy transfer to matching lanthanide emissive states (e.g.

of Eu, Tb, Nd, Er, Tm) then results in sensitised emission (Figure 1.8a). This process

displays a special variant of the antenna effect, for which low energy radiation can

be employed. It was observed for homonuclear lanthanide complexes and also in

lanthanide coordination polymers. [78–82]

In the recently reported heteronuclear complex developed by Piguet et al., a trans-

ition metal served as the sensitiser. Its development was the result of a sophisticated

molecular design, which took into account three requirements for efficient upconver-

sion processes. First, the activator has to be surrounded by at least two equidistant

sensitisers. Second, the activator has to be protected from high-frequency oscillators

to reduce energy loss due to non-radiative relaxation of 4f excited levels.

sensitiser activator

Si S1 T1

S0

sensitiser sensitiser

a) b)

4f*

emis

sion

activator

NN

N N

N

NN NN

NNCr

Er

Cr

540 nm

750 nm 750 nm

Ligand Ln3+

Figure 1.8: Upconversion luminescence in molecular systems. (a) Jablonski diagram that illustrates

molecular upconversion complexes with organic sensitisers. The dashed-dotted, dashed,

dotted, and full arrows represent photon excitation, energy transfer, multiphonon relax-

ation, and emission processes, respectively. (b) Heteronuclear upconversion complex de-

veloped by Piguet et al., in which Cr3+ served as the sensitiser. Two of the three ligands are

omitted for clarity. [83]

Third, the excited state lifetimes of the sensitiser have to be long enough for the in-

tramolecular energy transfer to occur. In the reported complex, a central Er3+ activator

is embedded between two peripherical chromium(III) sensitisers (Figure 1.8b). The

latter possess a sufficiently long excited state lifetimes to enable the energy transfer to

the activator. Hence, when the transition metal is irradiated with near-IR light, the in-

termetallic non-linear energy transfer results in lanthanide-centered near-IR emission.

14

1 Introduction

This compound was the first molecular system for which two-photon upconverted Er-

centered emission could be observed. [83]

However, to the present date there are no reports on lanthanides complexes that

enable UC luminescence in solution, because under these conditions long-lived lanthan-

ide luminescence is prone to several non-radiative deactivation pathways. The latter

is mainly caused by vibrational energy transfer from the metal emissive states to O-H

oscillators, especially those of water. [84–86]

There is also clear evidence, that high-energy vibrations of N-H (3300− 3500 cm−1)

and C-H (2900− 3100 cm−1) oscillators significantly contribute to the quenching of lu-

minescence by energy transfer into their higher harmonics. [87] Still, since their stretch-

ing vibrations are lower than these of O-H (3300− 3500 cm−1), the quenching effect is

expected to be lower. [85,88]

In general, a reduction of quenching can be facilitated by the use of a rigid ligand, that

shields the lanthanide efficiently from coordinating or closely diffusing water, and by

excluding high-energy vibrators from the ligand sphere. One strategy to achieve the

latter is the selective X-D/X-H (X = O,N,C) replacement.

Ene

rgy

[cm

-1]

≈10

250

cm

-1

Yb3+

2F7/2

2F5/2

0

5 000

10 000

15 000

4

3

2

1

0νC-H

4

3

2

1

0

5

νC-D

Figure 1.9: Non-radiative deactivation pathways of excited Yb3+ by C-H and C-D moieties. In com-

parison to C-H oscillators, C-D oscillators have to be excited into a higher vibrational level

in order to bridge the same energy gap. The dashed-dotted, dashed and dotted arrows

represent photon excitation, energy transfer and multiphonon relaxation, respectively.

X-D stretching vibrations are of lower energy than those of X-H, so that excitation

into a higher vibrational level is required to bridge the same energy gap (∆E) between

emissive and next lower state (Figure 1.9). [84–86] As the Franck-Condon overlap of the

wavefunctions of ground (ν”) and excited state vibrational levels (ν’) decreases with

higher ν’, the probability to excite an oscillator (e.g. X-D) to a higher vibrational level

also decreases. In other words, the possibility for non-radiative deactivation can be

reduced by replacing X-H by X-D groups

15

2 Concept of the project

The long-term goal of this project is the development of a monodispers, molecular

upconversion system in form of a binuclear lanthanide complex (Figure 2.1). The

covalent linkage of the two complexes brings the activator and the sensitiser of an

upconversion system in a close and defined distance from each other, independent

from the concentration of the binuclear compound in solution. The envisaged system

should be excited with near-IR radiation, followed by emission of light of shorter

wavelengths.

655 nm980 nm

Yb Er

D

D D

D

Figure 2.1: Model for a molecular upconversion complex.

The molecular upconversion complex has to meet two important requirements. On

the one hand, non-radiative deactivation by X-H oscillators in the proximity of the

lanthanide has to be minimised. On the other hand, sufficient stability of the lanthan-

ide complexes in a biological environment has to be ensured. Both challenges can be

addressed by employing tris(bipyridine) (TBP) cryptands (Figure 2.2).

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Figure 2.2: Molecular structure of a dinuclear TBP cryptand

16

2 Concept of the project

These ligands reportedly form kinetically stable complexes with different lanthan-

ides and sensitise their emission. [50,89,90] Furthermore, TBP cryptands efficiently pro-

tect the coordinated metal from interactions with surrounding solvent molecules,

which potentially cause luminescence quenching. [52] These ligands are also devoid

of N-H and O-H oscillators and convenient protocols for bipyridine perdeuteration

can be employed for the replacement of C-H groups. [91]

As part of the development of such molecular upconversion systems, the achievement

of three specific goals was aimed in this work.

• First, the evaluation of the influence of C-H oscillators on near-IR emissive

lanthanide TBP cryptates. This implies the synthesis and photophysical invest-

igation of different deuterated lanthanide TBP cryptates.

• Second, the introduction of functional groups to TBP cryptates.

• Third, the selective coupling of suitably functionalised TBP cryptates.

17

3 Development of binuclear cryptates

The envisaged binuclear system should be accessible from functionalised, deuterated

tris(bipyridine) cryptates. As mentioned before, these cryptates possess a high kinetic

stability and efficiently sensitise lanthanide luminescence. [50,89] These properties can

both be further improved by the introduction of N-oxides (Figure 3.1). [92,93] While

initial studies primarily employed the simple compound, later experiments were con-

ducted with the rigid, oxidised analogue. It is worth mentioning, that the introduction

of an axially chiral bipyridine-N,N’-dioxide moiety to the helical structure of such a

cryptate results in the formation of two different enantiomers. Still, within this work

only the racemic mixtures thereof were synthesised.

N

N

N

N

N

N

N

NOO

N

N

N

N

N

N

N

N

Figure 3.1: Structures of tris(bipyridine) (TBP) cryptates. Left: (bpy.bpy.bpy). Right: (bpy.bpy.bpy)O2 .

The strategy for the development of binuclear cryptates comprised the success-

ive preparation and characterisation of functionalised and deuterated cryptates (Fig-

ure 3.2). At a later stage, coupling experiments to the binuclear compound had to be

conducted.

a) b) c)

D

D

D

D

Figure 3.2: Milestones for the development of binuclear cryptates The blue and grey structures stand

for TBP cryptands, the yellow spheres stand for a coordinated metal. a) Deuterated cryptate.

b) Cryptate with a functional group. c) Deuterated cryptate with a functional group.

18


First, a deuterated cryptand (Figure 3.2a) had to be prepared. The apparent ap-

proach was to apply literature known protocols for perdeuteration, [91] and to find

novel procedures for the partial deuteration of bipyridines. These deuterated build-

ing blocks were assembled to the desired cryptands, whose effect on the luminescence

performance of different near-IR emissive lanthanides was evaluated.

Second, suitable functional groups that allow for selective coupling reactions were

introduced to the cryptand (Figure 3.2b). Since reportedly carboxyl functionalised

cryptates can be successfully coupled to other molecules (e.g. antibodies, DNA, pro-

teins), [94] a carboxylic acid ester was regarded as most promising functional group

for this work. Amino groups can also enable coupling to amino acids or to carboxyl

functionalised cryptates via an amide bond and were therefore chosen as alternative

functional group. While the synthesis of carboxylic acid ester derivatives is already

known in the literature, [95,96] novel synthetic pathways for the preparation of amino

functionalised cryptands had to be developed.

For the preparation of the rigid, functionalised cryptates, again the chirality had

be taken into account. There are two possibilities to incorporate an N,N’-dioxide and

a functional group into one cryptate (Figure 3.3). In the first case, one bipyridine

features both structural elements, so that the axially chiral bipyridine represents the

only stereogenic element within the cryptate. Consequently, racemic mixtures are

obtained. In the second case, the N,N’-dioxide and the functional group are attached

to different bipyridines. Hence, all three bipyridine moieties are inequivalent and

one of them is unsymmetrical, which creates an unusual form of chirality. The latter

adds to the axial chirality of the N,N’-dioxide, so that mixtures of diastereomers are

expected which were, however, not aimed to be separated within this work.

N

N

N

N

N

N

N

N

X

OO

N

N

N

N

N

N

N

NOO

X

Figure 3.3: Possible structures of functionalised, rigid TBP cryptates. When N,N’-dioxide and a func-

tional group X are locate at the same bipyridine, racemic mixtures are obtained (left). When

they are located at different bipyridines different diastereomers result (right).

Third, a functionalised, deuterated cryptand had to be synthesised (Figure 3.2c).

Also, the consistency of the photophysical properties of the corresponding lanthanide

complexes, compared to the unfunctionalised counterpart, had to be verified.

19

Last, protocols for the selective coupling of two functionalised cryptands had to be

developed (Figure 3.4). In case of the cryptates that carry an N,N-dioxide moiety, the

coupling to a binuclear compound is anticipated to further increases the stereochem-

ical complexity.

+

Figure 3.4: Selective coupling of two cryptates. The grey structures stand for TBP cryptands, the

yellow spheres represent any coordinated metal.

20


3.1 Deuterated cryptates

3.1.1 Retrosynthetic analysis

Deuterated TBP cryptands can be derived from deuterated building blocks, which can

be assembled in two different ways. On the one hand, it is possible to react a bipyrid-

ine moiety with an azamacrocycle, where the latter is obtained from two identical

bipyridine building blocks (Scheme 3.1 top). On the other hand, three bipyridines

with aminomethyl and bromomethyl groups can be connected directly to the target

compound in one step (Scheme 3.1 bottom).

N

NH2N

H2N

N

NBr

Br

2 +

N

N

N

N

N

N

N

N

2N

N

N

N

N

N

H

H

N

NBr

Br

+N

NBr

Br

OR

[Dx] [Dy] [Dy] [Dy]

[Dx] [Dy]

[Dz]

Scheme 3.1: Retrosynthetic analysis of a deuterated TBP cryptand. Dx, Dy and Dz stand for any

deuteration level

Both strategies have to be employed to combine different deuterated and undeuter-

ated bipyridines and, thus, preparing a series of isotopologic cryptates. As candid-

ates for deuterated building blocks, perdeuterated bipyridines and those deuterated

in benzylic positions were considered as the most promising (Figure 3.5).

N

NZ

Z

D D

D D

N

N

DD

DD

D

D

Z

Z

D D

D D

N

NZ

Z

OR

[Dx/y]

Figure 3.5: Required deuterated bipyridine building blocks. Dx,y stands for the deuteration level (D4

or D10) and Z for Br or NH2.

21


3.1.2 Synthesis of building blocks and cryptates

Undeuterated bipyridines

The simple, undeuterated building blocks were prepared from commercially available

2-amino-6-picoline (1) (Scheme 3.2), starting with a Sandmeyer reaction. In accord-

ance to the literature procedure, 1 was diazotised in the presence of NaNO2 and a

mineral acid (HBr) to generate a labile diazonium salt. The diazonium salt directly

underwent a SNAr reaction with Br− to give 2 in 58% yield after distillation. [97]

N

N

N

N

N

N

Br

Br

N

Z

a

b c d

Z=NH2

Z=Br

OO

N

N

NH

HN

N

N

e

1

2

3 4 5 6

Scheme 3.2: Synthesis of undeuterated building blocks. (a) i. HBr, Br2, rt→-20 °C; ii. NaNO2, NaOH,

-20 °C→rt (58%). (b) i. dry toluene, Raney-Ni, reflux; ii. H2O, 40 °C (17%). (c) mCPBA,

CHCl3, 0 °C→rt (80%). (d) i. TFA anhydride, dry CH2Cl2, reflux; ii. dry DMF/THF, LiBr,

rt (48%). (e) i. EtOH, tosylamide monosodium salt, reflux; ii. conc. H2SO4, 110 °C (57%).

In the next step, 2 was subjected to a Nickel catalysed reductive coupling to give

3. This reaction proceeded in two steps (Scheme 3.3). First, The two-fold oxidative

addition of 2-bromo-6-picoline (2) to Ni0 and coupling of the pyridine rings gave a

NiI I complex, which was hydrolysed to release of 6,6’-dimethyl-2,2’-bipyridine (3). [98]

N Br N N N NNi

Br Br

Raney-Ni H2O

Ni(H2O)2Br2

2 3

Scheme 3.3: Nickel-catalysed reductive coupling of 2-bromo-6-picoline (2) to 6,6’-dimethyl-2,2’-

bipyridine (3).

3 was then treated with meta-chloroperbenzoic acid (mCPBA) to form the corres-

ponding N,N’-dioxide 4. Traces of the reactant and the mono-oxidised side product

were removed by column chromatography to afford the pure product in an excellent

yield of 80%. [99]

22


The obtained N,N’-dioxide 4 was treated with trifluoroacetic acid (TFA) anhydride

to generate a trifluoroacetate species via a Boekelheide rearrangement. The resulting

reactive intermediate had to be handled under the strict exclusion of moisture to

avoid the hydrolysis to the corresponding, non-reactive alcohols. Following addition

of LiBr at room temperature yielded 5 in 48% yield after column chromatography. [100]

This nucleophilic substitution requires the strict exclusion of water and the use of an

aprotic solvent like DMF to solvate the lithium ions and generate “naked”, highly

nucleophilic bromide ions.

Finally, two equivalents of 5 were assembled to an azamacrocycle in a two-step

procedure. [101] For this purpose, an ethanolic solution of the bipyridine building block

(5) was first heated at reflux in the presence of tosylamide monosodium salt. [101] After

isolation of the tosylated macrocycle, the amine was then deprotected under acidic

conditions (H2SO4) to give the desired macrocycle 6 in 57% yield.

Partially deuterated bipyridines

The partially deuterated bipyridine units were derived from previously prepared 6,6’-

dimethyl-2,2’-bipyridine (3) (Scheme 3.4). At the beginning, 3 was converted into the

corresponding dicarboxylic acid 7 via a CrO3-mediated oxidation in the presence of

H2SO4. [102]

N

N

N

N

Z

Z

a

b Z=OH

Z=OEt

N

N

Z

Z

c

d Z=OH

Z=Br

O

O

DD

DD

eZ=NH2 3HBr H2O

3 7

8

9

10

11

Scheme 3.4: Synthesis of building blocks deuterated in benzylic position. (a) conc. H2SO4, 65 °C,

CrO3, 70 °C. (b) EtOH, conc. H2SO4, reflux (73% over two steps). (c) NaBD4, CD3OD,

rt (65%, >99%D). (d) PBr3, dry DMF, rt (92%, 98%D). (e) i. CHCl3, urotropine, re-

flux; ii. H2O/EtOH/HBr), 75 °C→rt (61%, 97%D).

Subsequent treatment with EtOH under acidic conditions gave the corresponding

ethyl ester 8 [103] in 73% over two steps. In the following key step, the ester was

reduced with NaBD4 in CD3OD to generate a bipyridine with selectively deuterated

23


benzylic positions. The pure product (9) was obtained in 65% yield after column

chromatography. The overall deuteration level of >99% was confirmed using ESI

mass spectrometry.

Nucleophilic substitution of the hydroxyl groups by bromine was achieved by treat-

ment of 9 with PBr3 in DMF and gave 10 in an excellent yield of 92% (98%[D]). Fi-

nally, a Delépine reaction was used for the synthesis of 11. [104] This reaction pro-

ceeded via an SN2 reaction of 10 with urotropine, followed by acidic hydrolysis

(HBr/EtOH/H2O) of the urotropine salt and gave the desired primary amine (61%

yield, 97% [D]).

Remarkably, the deuteration levels of the cryptate building blocks 10 and 11 are just

a little lower than for 9, although no deuterated solvents or reagents were used after

the reaction with NaBD4.

Perdeuterated bipyrdines

All perdeuterated compounds were prepared employing procedures developed by

J. Wahsner (Scheme 3.5). [105] Their synthesis started with the base-catalysed deu-

terium exchange of previously synthesised N,N’-dioxide 4. In analogy to the pro-

cedure for 2,2’-bipyridine, this reaction was performed in an autoclave with D2O as

deuterium source and NaOD as base. [91]

c Z=OAc

Z=OHd

Z=Br

N

N

N

N

Z

Z

a bN

N

e

[D10][D12]

OO

OO

Z=NH2 3HBr H2O

4 12 13

14

15

16

Scheme 3.5: Synthesis of benzylic deuterated building blocks. (a) NaOD (40 wt% in D2O), D2O,

autoclave, 150 °C (78%,>99%D). (b) Ac2O, reflux (57%, 98%D). (c) K2CO3, dry EtOH, rt

(57%, 98%D). (d) PBr3, dry DMF (8 ml), rt (53%, 99%D). (e) i. CHCl3, urotropine, re-

flux; ii. H2O/EtOH/HBr), 75 °C→rt (73%, 98%D).

After a total reaction time of four days, the crude product was purified with column

chromatography to give the desired perdeuterated compound 12 in 78% yield. The

excellent overall-deuteration level of over 99% was confirmed by ESI mass spectro-

24


metry (Figure 3.6). Despite no deuterated reagents or solvents were used after the

H/D exchange, deuteration levels did not decrease significantly during the following

four synthetic steps.

1 5 3 1 7 0 1 8 7 2 0 4 2 2 1 2 3 8 2 5 5 2 7 2 2 8 9

2 2 8 2 2 9 2 3 0 2 3 1

O ON N

D 1 2

E x a c t m a s s : 2 2 8 . 1 7

m / z

2 2 8 . 9 8[ M + H ] +

Figure 3.6: ESI+ mass spectrum of 12 after deuteration in an autoclave with D2O/NaOD, which con-

firms an overall deuteration level >99%.

In analogy to the procedures for the undeuterated compound, [99] 12 was reacted

in a standard Boekelheide rearrangement with acetic anhydride to give 13, followed

by saponification with K2CO3 in EtOH provided the alcohol 14 in 56% yield after

column chromatography. [105] Nucleophilic substitution (SN2) of the hydroxyl groups

by treatment with PBr3 gave the brominated analogue 15 in 53% yield (99% [D]) after

purification. [105] Last, 15 was subjected to a Delépine reaction, which provided the

bis(methylamine) 16 in 73% yield with an overall deuteration level of 98%. [105] Slow

cooling of the reaction mixture also afforded crystals, which were suitable for a X-ray

single crystal structure determination (Figure 3.7).

Figure 3.7: X-ray crystal structure of [D10]-6,6’-bis(aminomethyl)-2,2’-bipyridine trihydrobromide hy-

drate (16, monoclinic, P21/n). Ellipsoids are drawn at 50% probability level.

25


Synthesis of isotopologic cryptates

With the necessary building blocks in hand, a series of isotopologic sodium cryptates

was prepared (Scheme 3.6). As depicted, the [D4]-cryptate 17 was obtained by the

reaction of bipyridine moiety 10 with macrocyle 6.

N

N

N

N

N

N

N

NNa Br

+N

N

NH

HN

N

N

+N

N

Br

Br[Dx]

H2N

H2N

N

N

[Dy]

x=4

x=10

x=4

x=10

2

[Dy][Dx]

[Dx]

x=4, y=0

x=y=4

x=y=10

x=4 x=0

a

a

N

N

Br

Br[D4]

N

N

N

N

N

N

N

NNa Br

[D4]

10

10

10 6

15

18

11

16

17

19

20

21

Scheme 3.6: Synthesis of the isotopologic cryptates. Top: [D4]-(bpy.bpy.bpy). Bottom: [D8]-, [D12]-,

and [D30]-(bpy.bpy.bpy). (a) Na2CO3, CH3CN, reflux (40-58%,>97%[D]).

On the contrary, the [D8]-, [D12]-, and [D30]-analogues 19, 20, and 21 were obtained

from 6,6’-bis(bromomethyl)- and 6,6’-bis(aminomethyl) bipyridines as illustrated. In

all cases, suspensions of the respective building blocks with Na2CO3 in CH3CN were

heated at reflux and, after purification by column chromatography, products were

isolated in yields between 40% and 58% with deuteration levels >97%. In comparison,

previously reported deuterated DOTA ligands did not exceed a deuteration degree of

93%. [88] One exemplary ESI mass spectrum for 21 is shown in Figure 3.8.

Finally, the sodium cryptates were converted into the corresponding lanthanide

complexes by refluxing them in CH3CN in the presence of YbCl3·6H2O or NdCl3·6H2O,

respectively. The obtained complexes were characterised by analytical HPLC as well

as positive ESI mass spectrometry.

26


2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0

6 2 4 6 2 6 6 2 8 6 3 0

N

N

NN

N

N

N

N N a

[ D 3 0 ]E x a c t m a s s : 6 2 7 . 4 4

m / z

6 2 7 . 2 5[ M ] +

6 2 7 . 2

6 2 6 . 3

6 2 4 . 3

6 2 8 . 2

6 2 9 . 3

6 2 5 . 3

Figure 3.8: ESI+ mass spectrum of 21, which confirms an overall deuteration level of 98%

In this way, two sets of isotopologic [Dx]-TBP lanthanide cryptates (Ln = Yb, Nd)

were obtained. For the completion of the series (Figure 3.9), the [D0]-, [D10]- and

[D20]-TBP cryptates were also prepared within the Seitz group. [105]

3+

Ln = Nd, Yb

[Dx] = [D0] [D4], [D8], [D12] [D10], [D20], [D30]

N

N

N

N

N

N

N

NLn

[Dx]

Figure 3.9: Series of isotopologic lanthanide cryptates. The [D4]-, [D8]-, and [D12]-TBP cryptates

possess either one, two or three benzylically deuterated bipyridines. [D10]-, [D20]-, and

[D30]-TBP cryptates contain either one, two or three perdeuterated bipyridines.

3.1.3 Photophysical properties

For the investigation of the luminescence behaviour of the selectively deuterated

cryptates, [D6]-DMSO for [Dx]-Nd and D2O for [Dx]-Yb were chosen as solvents in or-

der to minimize quenching effects in solution. Thus, the most intensive luminescence

signals with monoexponential decay profiles could be measured. First, emission spec-

tra of the lanthanide cryptates were recorded, which showed the expected bands in

the near-IR region (Figure 3.10).

27


Figure 3.10: Normalized emission spectra (298 K, ca. 100 µM) of [D30]-Yb (red) (D2O, λexc 306

nm, 4.0 nm bandwidth) and [D30]-Nd (black) ([D6]-DMSO, λexc 306 nm, 6.0 nm band-

width). [105]

Then, the decrease of the luminescence intensity over time was studied and lu-

minescence lifetimes (τobs) as well as decay rates (kobs) were determined (Table 3.1).

The latter represents the sum of rate constants for radiative (k0) and non-radiative

deactivation processes (knr) (equation 1). [25]

kobs = k0 + ∑ kinr =

1τobs

(1)

The observed luminescence decays were monoexponential in all cases. The de-

termined luminescence lifetimes, which are listed in Table 3.1, increase with higher

deuteration level. This means that with fewer C-H oscillators present in the molecule,

less luminescence quenching is observed. The longest lifetimes were reached for the

perdeuterated compounds with 10.8 µs for [D30]-Yb and 9.09 µs for [D30]-Nd. In com-

parison with the undeuterated analogues, this means a prolongation by the factor of

1.6 for Yb and 6.9 for Nd. Indeed, the latter represents one of the longest lifetimes for

neodymium complexes in solution reported so far.

In order to specify the contribution of benzylic and aromatic oscillators to the overall

quenching rate, the kobs values from Table 3.1 were plotted against the number of

deuterated methylene groups y or pyridine rings z, respectively.

28


Table 3.1: Luminescence lifetimes τobs and excited state deactivation rates kobs of

Yb (D2O, λexc 335 nm, λem 980 nm) and Nd complexes ([D6]-DMSO,

λexc 306 nm, λem 1064 nm)

y=no. z=no. of deuter- τobs kobs calcd. kobsComplex of CD2 ated pyridine rings µsa [102 ms−1] [102 ms−1]b

[D12]-Yb 6 0 9.6 1.0 1.0[D8]-Yb 4 0 8.6 1.2 1.2[D4]-Yb 2 0 7.5 1.3 1.3[D0]-Yb 0 0 6.7 1.5 1.5[D10]-Yb 2 2 7.7 1.3 1.3[D20]-Yb 4 4 9.2 1.1 1.1[D30]-Yb 6 6 11.0c 0.93c -

[D12]-Nd 6 0 5.4 1.9 2.0[D8]-Nd 4 0 2.5 4.0 3.9[D4]-Nd 2 0 1.7 5.8 5.7[D0]-Nd 0 0 1.3 7.6 7.6[D10]-Nd 2 2 1.8 5.5 5.6[D20]-Nd 4 4 2.8 3.5 3.5[D30]-Nd 6 6 9.1c 1.1c -a Estimated error: ±15% for τobs > 3 µs and ±20% for τobs < 3 µsb Calculated using the parameters obtained from the global fitting procedurec value not used for the global fit

The 2D-projections reveal for both series of lanthanide complexes a linear correla-

tion as depicted exemplarily in Figure 5.2. In contrast to the commonly employed,

error-prone two-point calibrations (perdeuterated vs. undeuterated), this linearity is

established using multiple data points. The plotted values were modeled globally

with a three-dimensional planar fit function (equation 2).

kobs = k0 − y∆kbenzyl − z∆kpy (2)

k0 kobs of [D0]-Ln∆kbenzyl , ∆kpy quenching rate difference for the two/three C-H

oscillators of one benzylic methylene group or one pyridine ring

It should be noted, that the [D30]-Ln complexes were not included in the fitting

procedures, because at the time the obtained data (e.g, triplet level of [D30]-Gd or the

emission band shape of [D30]-Yb), suggested a different photophysical behaviour of

the perdeuterated cryptates.

29


Figure 3.11: Deactivation rates kobs of Nd complexes for the two series [D0]-[D4]-[D8]-[D12] (∆) and

[D0]-[D10]-[D20] (∇) with the corresponding 2D-projections (solid lines) of the 3D-global

fit planes. Yellow spheres stand for Nd, grey and blue structures represent undeuterated

and deuterated bipyridine moities, respectively. [105]

The rates (∆k) of the different oscillator groups (benzylic C-H, aromatic C-H) ob-

tained by the fitting procedures are shown in Table 3.2. Independent of the lanthanide,

benzylic C-H oscillators have a stronger impact on luminescence quenching than aro-

matic C-H moieties. Moreover, the deactivation rates are always greater for Nd com-

pared to Yb because of the smaller energy gap between emissive state and highest

receiving state (∆ENd ~5400 cm−1 vs. ∆EYb ~10 250 cm−1).

Table 3.2: Quenching rate differences ∆k for benzylic

and aromatic C-(H/D) oscillator groups in

[Dx]-Yb and [Dx]-Nda

k0 ∆kbenzyl ∆kpy kobsComplex [102ms-1]a [ms-1]a [ms-1]a R2

[Dx]-Yb 1.5 7.7 2.2 0.997[Dx]-Nd 7.6 94.1 9.4 0.997

a Estimated error: ±25%

30


In comparison to literature data, the determined deactivation rate ∆kbenzyl for Yb

luminescence of 7.7 ms−1 lies within the range of previously reported values (3-13

ms−1) for methylene groups close to Yb (~3.5-4.5 Å). [88,106]. All other values are

unprecedented.

To further deconvolute the contributions of individual C-H oscillators, two factors

were considered: the Franck-Condon overlaps with lanthanide excited states, and the

distance of an oscillator from the metal centre (rLn−H). The overlap factors were re-

garded to be similar within a particular group of C-H oscillators (either benzylic or

aromatic). This assumption simplifies the calculations, because then the quenching

rate of an oscillator only depends on its distance from the metal centre. Typical val-

ues for the distances rLn−H were obtained from the Cambridge Structural Database7

(CSD, version 5.31, February 2010) of structurally similar compounds as indicated in

Figure 3.12. The results of this search as well as overall averages of the respective

distances are listed in Table 3.3 to Table 3.6.

A B

N N

H3H4

H5

LnN

Heq

N

Hax

Ln**

*

*

H4

H5

H3

*

*

*

*

*

(Ln = Nd, Yb)

Figure 3.12: Structural motifs used for the Cambridge Structural Database search to obtain typical

distances for rLn−H . Positions allowed for substitutions are marked with an asterisc.

Table 3.3: Average distances rNd−H in

crystal structures with motif A

CSD Code Nd-H3 Nd-H4 Nd-H3

[Å] [Å] [Å]

XISFOV 5.573 6.346 5.525XIFMAA 5.542 6.338 5.479XAQLIL 5.539 6.270 5.471XAKWAH 5.440 6.291 5.578QIRDOK 5.571 6.332 5.486NAZHOM 5.546 6.310 5.483LEWNUX 5.580 6.339 5.531IDUQAA 5.555 6.312 5.487GUHJAU 5.492 6.270 5.466DOJHIU 5.577 6.337 5.512DICNUZ 5.560 6.330 5.501AKIPIT 5.542 6.307 5.491

Overall avg. 5.543 6.315 5.501

Table 3.4: Average distances rYb−H in

crystal structures with motif A

CSD Code Yb-H3 Yb-H4 Yb-H3

[Å] [Å] [Å]

XEWVUQ 5.390 6.172 5.396XEPLIN 5.360 6.155 5.386XEFBAM 5.440 6.227 5.442XAWSAQ 5.398 6.240 5.373RENXIR 5.300 6.095 5.344PAFBII 5.402 6.180 5.394LEWNIL 5.399 6.175 5.432LEGDAD 5.377 6.144 5.345JAPQOG 5.369 6.140 5.357DEFTEO 5.416 6.190 5.390

Overall avg. 5.385 6.172 5.386

31


Table 3.5: Average distances rNd−H

in crystal structures with

motif B

CSD Code Nd-Heq Nd-Hax

[Å] [Å]

AYEJAP 3.569 4.372BEJTIU 3.622 4.443HIZHIH 3.617 4.374LARGOB 3.512 4.347

Overall avg. 3.580 4.384

Table 3.6: Average distances rYb−H

in crystal structures with

motif B

CSD Code Yb-Heq Yb-Hax

[Å] [Å]

QAXYAP 3.356 4.202REPCAR 3.572 4.275YIPJIR 3.639 4.277YIPJOX 3.616 4.184

Overall avg. 3.546 4.235

For the theoretical deconvolution, one has to bear in mind that vibrational energy

transfer from the lanthanide ion to an oscillator is inversely proportional to the sixth

power of the distance. This can be derived from the general energy transfer formula

of Förster. [107,108]

∆ki ∝ (rLn−H)−6 (3)

Additionally, it was assumed that individual contributions to the overall quenching

rate are additive (equation 4).

∆ktot = ∑i

∆ki (4)

With these considerations and the determined average distances, the quenching

rates of individual C-H oscillators (∆ki) were calculated according to equation 5.

∆ki =∆ktot · (rLn−H)

−6

∑i(rLn−H)−6 (5)

The used parameters and results are summarised in Table 3.7 for Nd and Table 3.8

for Yb. The values for ∆ki are estimates for quenching rate differences, meaning

the rate by which single C-H groups quench lminescence stronger than single C-D

groups. Figure 3.13 also gives a graphical representation of the calculated estimates.

32


Table 3.7: Nd deconvolution of individual C-H deactivation rates ∆ki

based on typical crystal structure values for rLn−H .

benzylic H aromatic HHeq Hax H3 H4 H5

rNd−H [Å] 3.580a 4.384a 5.543b 6.315b 5.501b

(rNd−H)−6 10−5[Å]−6 47.5 14.1 3.45 1.58 3.61

∆ktot [ms−1] ��94.1c�� 9.4c��∆ki [ms−1] 72.6 21.5 3.8 1.7 3.9a, b, c values taken from Table 3.5, Table 3.3,Table 3.2, respectively

Table 3.8: Yb deconvolution of individual C-H deactivation rates ∆ki

based on typical crystal structure values for rLn−H .

benzylic H aromatic HHeq Hax H3 H4 H5

rYb−H [Å] 3.567a 4.235a 5.393b 6.183b 5.389b

(rYb−H)−6 10−5[Å]−6 50.3 17.3 4.10 1.81 4.10

∆ktot [ms−1] ��7.7c�� 2.2c��∆ki [ms−1] 5.7 2.0 0.9 0.4 0.9a, b, c values taken from Table 3.6, Table 3.4,Table 3.2, respectively

For both lanthanides, similar trends are observed. Among the aromatic oscillators,

the C-H groups in 3 and 5 position quench about twice as much as those in 4 posi-

tion. Among the benzylic oscillators, deactivation rates of closer lying equatorial C-H

groups are about the factor three higher compared to further away axial C-H groups.

Figure 3.13: Estimates for quenching rate differences in (∆ki in ms−1) for individual C-(H/D) oscillat-

ors in the Nd (upper value) and Yb complexes (lower value). [105]

33

3.2 Functionalised cryptates


3.2.1 Retrosynthetic analysis of functionalised cryptates

Monofunctionalised cryptates are usually obtained from a bipyridine and a macro-

cycle. Since either of the two reactants can carry the functional group, two possible

combinations result (Scheme 3.7). The first was used for the synthesis of the majority

of the functionlised cryptates, while the second was only applied for a cryptate that

possesses a functional group and an N,N’-dioxide moiety at different bipyridine arms.

N

N

N

N

N

N

N

N

N

N

N

N

N

N

H

H

N

NBr

Br

+

OR

Z

N

N

N

N

N

N

H

H

N

NBr

Br

+

Z

Z

Scheme 3.7: Retrosynthetic analysis of a TBP cryptand with a functional group X.

The functionalised macrocycle, which is required for the second outlined syn-

thesis, can be prepared from a simple and a functionalised bipyridine as depicted

in Scheme 3.8.

N

N

N

N

N

N

H

H

N

NNH2

NH2Z

N

NBr

Br

+

Z

Scheme 3.8: Retrosynthetic analysis of an azamacrocycle with a functional group X.

34


3.2.2 Synthesis of carboxy functionalised cryptates

Undeuterated building blocks and cryptates

The synthesis of carboxy functionalised cryptates started with the preparation of func-

tionalised bipyridines (Scheme 3.9). 2-(Tributylstannyl)-6-methylpyridine (22) was de-

rived from previously prepared 2. [109] In this conversion, a halogen-metal replacement

using n-BuLi was followed by a metal exchange with Bu3SnCl. Instead of purification

by column chromatography as reported in the literature, a fractional vacuum distilla-

tion was performed. Although this procedure is tedious, it reduces the contact with

toxic tin compounds and provided 22 in 60% yield and excellent purity (Figure 3.14).

Following Stille coupling with an isonicotinic acid methyl ester triflate 23 provided

bipyridine 24 in 59% after two-fold purification by column chromatography. [109] Com-

pound 24 was treated with mCPBA to provide the corresponding N,N’-dioxide 25 in

71% yield after column chromatography. The N,N’-dioxide 25 was subjected to a

Boekelheide reaction with TFA, followed by treatment with anhydrous LiBr. The res-

ulting methyl ester functionalised bipyridine building block 26 was obtained in 53%

yield. Compound 27 was prepared from 26 by treatment with peroxytrifluoroacetic

acid. This strong oxidising agent was generated in situ from urea hydrogen peroxide,

which represents a solid source of anhydrous H2O2, and TFA anhydride. [110,111]

N

OTf

MeOOCN

NN

Z

a

c

Z=Br

Z=SnBu3

MeOOC

b+

N

N

Br

Br

MeOOC

N

N

MeOOC

OO

Br

Br

N

N

MeOOC

OO

e

d

2

22

23 24 25

2627

Scheme 3.9: Synthesis of bipyridine building blocks that carry a methyl ester. (a) i. n-BuLi, dry THF,

−78 °C; ii. Bu3SnCl, −78 °C→rt (60%). (b) PdCl2(PPh3)2, PPh3, dry xylene, reflux (59%).

(c) mCPBA, CH2Cl2, 0 °C→rt (71%). (d) i. TFA anhydride, dry CH2Cl2, reflux; ii. dry

DMF/THF, LiBr, rt (53%). (e) urea·H2O2, CH2Cl2, TFA anhydride, 0 °C→rt (31%).

35


0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5f1 (ppm)

1

2

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5f1 (ppm)

36.4

66.

9836

.56

17.9

7

3.28

1.05

0.98

1.00

9.00

6.15

6.64

5.82

2.90

0.94

0.88

0.92

0.50.70.91.11.31.51.71.9f1 (ppm)

Figure 3.14: 1H NMR spectra (200 MHz, CDCl3) of 22 (below) and the major by-product (above) of the

halogen-metal exchange reaction.

Following the literature procedures, the reactions (Scheme 3.10) of the bipyridines

28 and 29 with macrocycle 6 finally provided the cryptates 28 [95] and 29 [112] in 36%

and 39% yield after column chromatography.

N

N

N

N

N

N

N

NNa

N

N

COOMe

OO

Br

Br

N

N

N

N

N

N

N

NNa O

O

Br

Br

COOMe

COOMe

+N

N

NH

HN

N

N

aN

N

Br

Br

COOMe

N

N

NH

HN

N

N

+ b

6 26 28

6 27 29

Scheme 3.10: Synthesis of cryptates with a carboxylic acid methyl ester. (a) Na2CO3, CH3CN, reflux

(36%). (b) Na2CO3, CH3CN, reflux (39%).

36


Deuterated building blocks and cryptate

In the next stage of development, it was investigated if the deuteration protocols can

also be applied to the carboxy functionalised cryptates. Cryptate 28 was chosen as

the model system for this endeavour. The synthesis of its perdeuterated analogue

30 was envisaged by the coupling of two perdeuterated precursors, with one being

the previously synthesised [D20]-macrocycle 31 and the other being a perdeuterated

functionalised bipydine 32.

aN

N

MeOOC

OO

[D11]

N

N

MeOOC

OO

b N

N

MeOOC

d

[D9]

OAc

OAc

N

N

MeOOC

c

[D9]

Br

Br

N

N

MeOOC

OO

[D9]

Br

Br

25 33 34 32

32

Scheme 3.11: Synthesis of a deuterated bipyridine building block that carries a methyl ester. (a)

i. NaOD (40 wt% in D2O), D2O, autoclave, 150 °C. ii. conc. H2SO4, MeOH, reflux

(62%, 98%D). (b) Ac2O, reflux (67%, 98%D). (c) HBr (47%) in HOAc, reflux. (d) i. TFA

anhydride, dry CH2Cl2, reflux; ii. dry DMF/THF, LiBr, rt (45%, >99%).

For the synthesis of a perdeuterated, functionalised bipyridine (Scheme 3.11), the

same strategy as for the simple bipyridines (see section 3.1.2) was applied. [105] Perdeu-

teration was performed in an autoclave with D2O as deuterium source and NaOD as

base. This reaction was repeated twice to ensure a sufficiently high deuteration level

of the crude product. Due to the basic conditions employed in this step, the methyl

ester was saponified and had to be restored using a Fisher esterificaton. Thus, the

desired product 33 was obtained in 62% yield with a deuteration level of 98%D. Sub-

sequent Boekelheide rearrangment with acetic anhydride gave 34 in 67% yield (98%D)

after two-fold column chromatography. However, the nucleophilic substitution of the

acetoxy group using HBr/HOAc as bromine source did not work well. [95]. In repeated

attempts this reaction only yielded small amounts of 32 beside the mono-brominated

analogue and the corresponding dialcohol, which is the hydrolysis product of 34.

37


Hence, the alternative reaction of 33 with TFA anhydride, followed by a treatment

with LiBr was tried. This approach was successful and 32 could be isolated in 45%

yield with a deuteration level >99%, as determined from its ESI mass spectrum.

The prepared carboxy-functionalised, perdeuterated building block 32 was reacted

with the previously synthesised perdeuterated macrocyle 31 using standard condi-

tions for the synthesis of TBP cryptates. As a result, the sodium complex was obtained

in 26% (98%D) yield after column chromatography.

+N

N

NH

HN

N

N

aN

N

Br

Br

N

N

N

N

N

N

N

NM

COOMeCOOMe

bM=Yb3+, n=3

[D9][D20] [D29]

M=Na+, n=1

nXX=Cl or Br

31 32 35

36

Scheme 3.12: Synthesis of deuterated sodium and ytterbium cryptates with a methyl ester function-

ality. (a) Na2CO3, CH3CN, reflux (26%, 98%D). (b) YbCl3·6H2O, CH3CN, reflux.

Subsequent reaction with YbCl3·6H2O afforded the corresponding ytterbium crypt-

ate 36, which was purified by precipitation from its methanolic solution. For this

compound, the ESI mass spectrum only showed the signal for the reactant and not

for the triple charged product. However, the recorded steady state emission spectrum

(Figure 3.15) confirmed the expected near-IR emission of ytterbium. Consequently, the

synthesis of the first perdeuterated functionalised ytterbium cryptate was successful.

8 5 0 9 0 0 9 5 0 1 0 0 0 1 0 5 0 1 1 0 0 1 1 5 0

0 . 2

0 . 4

0 . 6

0 . 8

1 . 0 2 F 5 / 2 2 F 7 / 2

relati

ve em

ission

inten

sity

w a v e l e n g t h i n n m

9 7 5 . 5 n m

9 9 8 . 5 n m1 0 1 0 . 5 n m

Figure 3.15: Steady state emission spectrum of 36. The spectrum was recorded at room temperature

with an excitation wavelength of 312 nm in D2O.

38


3.2.3 Synthesis of amino functionalised cryptates

Simple building blocks and cryptate

For the preparation of a amino functionalised TBP cryptate, a macrocycle and a

bipyridine with an amino group were required.

N

N

Z

N

N

N

N

N

N

a b

O2N

N

N

c

Z

d Z=NH2

Z=NHAc

Z=NH2, NHAc

e

OO

O O

3 37 38 39

40

Scheme 3.13: Synthetic approach towards an amino functionalised bipyridine building block. (a)

mCPBA, CHCl3, 0 °C→rt (81%). (b) i. H2SO4/HNO3, 100 °C; ii. aq. NaOH, rt (61%).

(c) NaBH4, Pd/C, MeOH, 0 °C→rt (68%). (d) AcCl, py, rt (37%). (e) mCPBA, CH2Cl2,

0 °C→rt.

The multistep synthesis of the latter (Scheme 3.13) started with the reaction of 6,6’-

dimethyl-2,2’-bipyridine (3) with one equivalent of mCPBA to give mono-N-oxide 37

in 81% yield after column chromatography. [99] The activated pyridine ring was select-

ively nitrated by treatment with a mixture of nitric and sulfuric acid at elevated tem-

peratures (100 °C). [113] Aqueous workup of the reaction mixture led to precipitation

of the nitro functionalised bipyridine (38), which was isolated in 61% yield. The nitro

group was subsequently reduced to the corresponding amine with Pd/NaBH4. [114]

The clean product 39 (Figure 3.16 bottom) was obtained in 68% yield after aqueous

workup and was used without further purification.

Compound 39 was susbsequently treated with two equivalents of mCPBA. How-

ever, TLC and 1H NMR spectrum of the isolated residue indicated that the presumed

N,N’-dioxide was not formed. Instead the applied conditions only caused reoxidition

of the amino to a nitro group. Indeed, there is already literature precedence for the

closely related hydrogen peroxide induced oxidation of amino groups attached to a

pyridine. [115]

Accordingly, a modification of the synthetic strategy was necessary and an addi-

tional step was introduced to protect the amino function with an acetyl group. While

the acetylation with acetic anhydride failed, [116] the alternative reaction of 39 with

acetyl chloride in dry pyridine was more successful, [117] and gave 40 in 37% yield.

However, despite purification by column chromatography, the 1H NMR of the isol-

ated material confirmed the presence of acetyl containing impurities (Figure 3.16 top).

39


1.82.02.22.42.62.83.03.23.43.63.84.04.24.46.26.46.66.87.07.27.47.67.88.08.28.48.68.8f1 (ppm)

1

2

3.0

1.0

1.0

0.9

0.8

1.0

1.0

2.9

3.3

1.7

1.0

1.0

1.0

1.0

1.0

5.9

CDCl3

CDCl3

EtOH

Figure 3.16: 1H NMR spectra of 39 (400 MHz, blue trace) and 40 (250 MHz, red trace) in CDCl3.

Nevertheless, oxidation of 40 was attempted by treatment with mCPBA, but inde-

pendent of increasing amounts of oxidising agent or prolonged reaction times, 1H

NMR and ESI mass spectra only proved the formation of the mono-N-oxide.

Consequently, the strategy was changed again and the conversion of the nitro to

an amino group was shifted from an early stage of the synthesis pathway to the

end. This means, that the novel primary synthetic target was the formation of a nitro

functionalised cryptate, which should be reduced to the amino functionalised cryptate

in the last step. Hence, a nitro functionalised bipyridine building block was required

for the cryptate synthesis (Scheme 3.14).

N

N

O2N

bN

N

Br

Br

O2N

N

N

O

O2N

a OO

38 41 42

Scheme 3.14: Synthesis of a nitro functionalised bipyridine building block. (a) mCPBA, CHCl3,

0 °C→rt (82%). (b) i. TFA anhydride, dry CH2Cl2, reflux; ii. dry DMF/THF, LiBr, rt

(56%).

40


Therefore, previously prepared 38 was again oxidised with mCPBA to give 41 in

82% yield after column chromatography. The Boekelheide rearrangement of 41 with

TFA anhydride, followed by treatment with LiBr afforded the nitro functionalised

bipyridine building block 42 in 56% yield after purification. The latter was reacted

with 6 using standard conditions for the assembly of TBP cryptands, which provided

the desired nitro functionalised cryptate 43 in 33% yield.

+N

NBr

N

N

NH

HN

N

N

a

N

N

N

N

N

N

N

NNa Br

O2NBr Z

b Z=NO2

Z=NH2

42 6 43

44

Scheme 3.15: Synthesis of an amino functionalised cryptate. (a) Na2CO3, CH3CN, reflux (33%). (b)

N2H4 · H2O, Pd/C, EtOH, reflux (68%).

Final reduction of the nitro group with hydrazine hydrate in the presence of palla-

dium gave the pure target compound 44 (Figure 3.17) in an excellent yield of 68% after

column chromatography. The successful synthesis of both cryptates was confirmed

by 1H and 13C NMR, as well as ESI mass spectrometry and elemental analysis.

3.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)

1.9

10.0

0.9

0.8

0.8

4.1

6.4

4.1

A (d)8.01

B (m)7.85

C (d)7.35

D (d)7.32

E (d)7.23

F (d)6.58

G (m)3.81

H (m)3.64

3.62

3.65

3.80

3.80

3.82

3.83

6.58

6.58

7.22

7.23

7.31

7.33

7.34

7.36

7.80

7.82

7.84

7.84

7.84

7.86

7.86

7.88

7.88

7.89

7.90

8.00

8.02

CD2Cl2

water

CD3OD

Figure 3.17: 1H NMR (400 MHz, CD2Cl2/CD3OD 1:1) of 44

41


A cryptate with a rigid N,N’-dioxide moiety

For the development of an analogous rigid, amino functionalised cryptate, one has

to keep in mind that the N,N’-dioxides and the amino group cannot be present at

the same bipyridine (see section 3.2.3). During the synthesis, oxidising or reducing

agents affect the pyridine and amino nitrogens similarly and, on that account, these

structural features have to be introduced with different building blocks (Figure 3.18).

N

N

N

N

N

N

N

NNa Br

N

N

N

N

N

N

N

NNa Br

H2N

D30

OO

OO

45 46

Figure 3.18: Rigid TBP cryptates. Left: deuterated parent compound developed by Doffek et al. [93]

Right: envisaged functionalised analogue.

Since it was difficult to prepare an N,N’-dioxidised macrocycle, the second retrosyn-

thetic pathway (see Scheme 3.7) was applied. Accordingly, a functionalised building

block first has to be coupled to a simple bipyridine to form a macrocycle. The latter

can subsequently react with an N,N’-dioxide moiety to build the desired cryptate.

While the nitro functionalised bipyridine 42 was already at hand, its counterpart for

the macrocycle synthesis was prepared from previously synthesised 5 (Scheme 3.16

left).

N

N

N

N

a

bZ=NHTs

Z

Z

Br

Br

N

N

c

Br

Br

OO

Z=NH2 3HBr H2O 518

47

48

Scheme 3.16: Synthesis of additional required bipyridine building blocks for a functionalised rigid

cryptate. (a) i. CHCl3, urotropine, reflux; ii. H2O/EtOH/HBr, 75 °C→rt (50%). (b) aq.

NaOH, TosCl, Et2O, 0 °C→rt (49%). (c) urea · H2O2, CH2Cl2, TFA anhydride, 0 °C→rt

(84%).

A Delépine reaction was used for the conversion to amine 18, followed by pro-

tection of the primary amine with tosyl chloride to give 47 in 49% yield. [112] The

42


bipyridine-N,N’-dioxide building block was prepared from the same precursor by

treatment with peroxytrifluoroacetic acid (Scheme 3.16 right). [118] In comparison to

the literature known oxidation of 5 with mCPBA, [92] this reaction is less tedious and

gave 48 in relatively high yield (84%) after column chromatography.

After their successful preparation, the three building blocks were assembled step-

wise, starting with the reaction of 47 and 42 under slightly basic conditions. [112]

Thereby, the tosyl protecting groups prevented multiple substitution at the amines

of 47, so that the macrocycle and not the cryptate was obtained. Acid-mediated de-

protection of the amines gave the poorly soluble nitrated macrocycle 49 in 52% yield.

N

N

NH

HN

N

N

a

NO2

+N

NNHTs

NHTsN

NBr

Br

N

N

NH

HN

N

N

NH2

NO2

N

N

N

N

N

N

N

NNa Br

H2N

c

b

OO

47 42 49

50

48

46

Scheme 3.17: Synthesis of a rigid, amino functionalised TBP cryptand. (a) i. DMF, K2CO3, 50 °C→rt;

ii. H2SO4, 110 °C (52%). (b) N2H4 · H2O, Pd/C, EtOH, reflux (85%) (c) Na2CO3, CH3CN,

reflux (8%).

In analogy to the procedure for amino cryptate 44, compound 49 was reduced with

N2H4 · H2O to the corresponding amino macrocycle. The isolated crude product 50

was used without further purification for the final step, during which it was coupled

to the third building block (48). As a result, the desired cryptate 46 was formed, which

was isolated in 8% yield after purification. As for the other amino functionalised

cryptate, the identity of the target compound was confirmed by 1H and 13C NMR,

ESI mass spectrometry as well as elemental analysis. It is worth mentioning, that the1H spectrum of 46 (Figure 3.19) shows numerous signals and is far more complicated

than that of 44. This can be explained by the existence of diastereomers.

43


As mentioned previously (see Figure 3.3), the cryptate is devoid of symmetry ele-

ments, because all three bipyridine moieties are inequivalent and one of them is un-

symmetrical. Hence, 46 possesses a special form of chirality. The latter adds to the

axial chirality of bpyridine-N,N’-dioxide and, as a consequence, different diastereo-

mers result. Because of their great similarity it was not possible to unambiguously

distinguish these species spectroscopically.

3.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.67.88.0f1 (ppm)

2.0

2.9

4.3

2.0

0.7

0.8

0.9

3.3

1.7

10.0

A (d)4.45

B (m)7.74

C (m)7.50

D (m)7.39

E (m)7.01

F (s)6.62

G (m)4.30

H (m)3.85

I (m)3.45

J (m)3.14

3.28

3.30

3.33

3.37

3.39

3.46

3.49

3.51

3.52

3.75

3.80

3.82

3.84

3.87

3.88

3.93

4.28

4.30

4.32

4.43

4.48

6.62

6.98

6.99

7.02

7.03

7.32

7.35

7.36

7.38

7.38

7.38

7.39

7.41

7.42

7.45

7.48

7.49

7.49

7.51

7.52

7.52

7.59

7.60

7.61

7.62

7.63

7.64

7.64

7.66

7.67

7.67

7.70

7.70

7.73

7.74

7.76

7.76

7.77

7.79

7.80

7.81

7.82

7.82

7.83

7.83

7.83

7.84

7.85

7.86

7.88

CD2Cl2

CD3ODwater

Figure 3.19: 1H NMR (250 MHz, CD2Cl2/CD3OD 1:1) of 46

44


3.3 Binuclear cryptates

3.3.1 Strategy for the coupling of cryptates

There are different possibilities to connect two cryptates (Figure 3.20). The shortest

linkage is an amide bond, for which two cryptates with different functional groups

are required. However, from a synthetic point of view, this approach is rather cum-

bersome, since both coupling partners have to be synthesised in sufficient amounts.

Furthermore, an amide bond formation with an aromatic amino group is often prob-

lematic due to its low nucleophilicity.

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NNa

O

NH

O

NHNH

OO

NHNH

O

NHNH

O

Na

Figure 3.20: Molecular architecture of binuclear TBP cryptates. The dotted line stands for the possible

linkages, which are shown below.

In comparison, the connection via one or two ethylendiamine bridges only involves

aliphatic amino groups and one functionalised cryptate. Consequently, this type of

linkage was favoured for the first coupling reactions of two TBP cryptates. It is also

worth noting, that only undeuterated, less precious cryptates were used for the de-

velopment of these reactions.

3.3.2 Functionalised cryptates for coupling reactions

The synthesis of cryptates suitable for coupling reactions started with the preparation

of the two required coupling partners from the methyl ester functionalised cryptate

28 (Scheme 3.18). On the one hand, the methyl ester 28 was saponified with NaOH

to give the corresponding carboxyl functionalised cryptate 51 (80% yield). [119] On

45


the other hand, the reaction of 28 with ethylenediamine under anhydrous conditions

resulted in the formation of amine 52.

N

N

N

N

N

N

N

NNa

N

N

N

N

N

N

N

NNa

O

NH

NH2

O

OMe

a

Br

N

N

N

N

N

N

N

NNa

O

Br

O

b

2851 52

Scheme 3.18: Synthesis of the coupling partners from a common precursor. (a) aq. NaOH, MeOH,

40 °C (80%). (b) ethylendiamine, dry MeOH, 0 °C→rt (87%).

The crude product of this reaction was purified with reversed-phase column chro-

matography using silanised silica (RP-2) as solid phase and CH2Cl2/MeOH (15:1 v/v)

as eluent. For thin layer chromatography, the employed RP-18 plates were developed

first with iodine to confirm the presence of the cryptate and, subsequently, with nin-

hydrin to detect the primary amine. This purification could be performed on a 50 mg

scale and enabled the isolation of the pure 52 in 87% yield.

3.03.23.43.63.84.04.26.87.07.27.47.67.88.08.28.48.68.89.0f1 (ppm)

1

2

3

7.3

1.7

2.7

4.9

10.9

1.2

11.7

5.2

1.1

5.4

4.3

1.3

1.1

2.2

2.2

11.7

4.9

6.0

4.2

1.0

1.0

CDCl3

MeOH CD3OD

CD3OD

Figure 3.21: 1H NMR spectra of 28 (200 MHz, CDCl3), 51 (250 MHz, CD2Cl2/CD3OD 1:1) and 52 (250

MHz, CD2Cl2/CD3OD 1:1).

46


The direct comparison of the 1H NMR spectra of 28, 51 and 52 (Scheme 3.18) re-

veals, that both derivatisation reactions go along with the loss of the methyl ester

moiety (peak at 4.00 ppm). While no new signal are observed in the spectrum of the

carboxylic acid 51, the 1H NMR spectrum of amine 52 displays two novel triplets,

which could be assigned to the two methylene groups of the ethylenediamine.

3.3.3 First approaches towards the coupling of cryptates

In the first attempt to form a binuclear TBP cryptate, it was tried to couple two methyl

ester functionalised cryptates (28) with one equivalent of ethylenediamine. Still, this

reaction only gave the monocoupled product 52. Therefore, the selective coupling of

the latter with a carboxyl functionalised cryptate 51 was tried.

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NNa

O

NH

HN

O

Na

Br

Br

53

Figure 3.22: Binuclear TBP cryptate, which was observed in the ESI mass spectrum.

The bottle-neck for all amide bond formations is the choice of an appropriate ac-

tivating agent, which enhances the electrophilicity of the carboxylate group. On that

account, several commonly used compounds were tested for this coupling reaction.

When employing oxalyl chloride, DCC/HOBt or EDCI/HOBt , no product formation

was observed. In contrast, after activation of acid 51 with HATU in the presence of

DIPEA or NEt3, ESI mass spectra indicated the the formation of the binuclear com-

pound 53. Nevertheless, it was not possible to gain sufficient amounts of this species

for a complete characterisation. It was concluded that major obstacles for this syn-

thetic pathway are the inefficient activation of the carboxylic acid, and the loss of

material during the purification procedure, which might be related to stability issues.

47


3.3.4 Preparation of the first binuclear TBP cryptate

To overcome the described problems, the synthetic strategy for the preparation of the

binuclear cryptates was modified regarding two important points. First, instead of

coupling to a carboxyl functionalised cryptate, it was aimed to connect to ethylene-

diamine linked cryptates via a carbamide. This way, one circumvents the difficult

activation of the carboxylic acid of a cryptate and, in addition, the synthetic effort

is reduced, since only one sort of functionalised cryptate needs to be prepared. As

second alteration, the cryptates were equipped with an N,N’-dioxide moiety which is

known to increase their stability. [93] The resulting novel target structure is in shown

in Figure 3.23.

N

N

N

N

N

N

N

NNa

Br

O

NH

HN

N

N

N

N

N

N

N

NNa

Br

O

NH

HN

OOO

OO

54

Figure 3.23: Revised target structure of a binuclear TBP cryptate.

The ethylenediamine linked cryptate 55 was prepared by treatment of 29 with neat

ethylenediamine. Purification with reversed-phase column chromatography using the

above discussed conditions afforded the pure compound in an excellent yield of 76%.

a

N

N

N

N

N

N

N

NNa O

OBr

N

N

N

N

N

N

N

NNa O

OBr

O

O

O

NH

NH2

29 55

Scheme 3.19: Preparation of a rigid, ethylenediamine linked cryptate. (a) ethylendiamine, neat,

0 °C→rt (76%).

For the coupling to the binuclear compound (Scheme 3.20), six equivalents of 55

were treated with one equivalent of the urea synthon triphosgene, which was chosen

as a safer and more practical alternative to phosgene. The reaction was performed un-

der anhydrous conditions in the presence of DIPEA and yielded the target compound

besides an isocyanate by-product. The latter was removed with two-fold column

48


chromatography, so that target molecule 54 could be isolated in 50% yield. Successful

separation was confirmed by 1H NMR spectroscopy, whereas the spectra could be

assigned to the respective species with the aid of their ESI mass spectra (Figure 3.24).

O

O

O

Cl

ClCl

Cl

ClCl+ a6

N

N

N

N

N

N

N

NNa O

OBr

O

NH

NH2

55

54

Scheme 3.20: Coupling of two ethylenediamine linked cryptates to a binculear TBP cryptate. (a)

DIPEA, dry CH2Cl2, 0 °C→rt (50%).

The 1H NMR specta of reactant 55 and product 54 are shown in Figure 3.25. As

expected, the spectra do not differ in terms of integral ratios, since no new protons

were introduced during the reaction. However, the shape and the complexity of the

whole spectrum is altered significantly after the coupling. The product shows a larger

number of overlapping peaks and it is also noticable, that the signals in the aromatic

region are more condensed. In summary, the synthesis and purification of the first

binuclear TBP cryptate was successful.

6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0 1 6 0 0

E x a c t M a s s o f 5 4 : 1 6 1 4 . 3 9

1 4 9 1 . 8 9 [ M + H - 2 N a - B r ] +

7 4 1 . 0 1 i s o c y a n a t e b y p r o d u c t

7 2 8 . 5 5 [ M - 2 B r ] 2 +

m / zFigure 3.24: ESI+ MS spectrum of 54

49


3.03.23.43.63.84.04.24.46.87.07.27.47.67.88.08.28.48.6f1 (ppm)

1.9

4.5

3.9

4.1

2.1

4.0

1.1

1.0

9.2

0.9

0.9

19.0

8.1

0.8

4.2

0.8

1.9

6.9

4.2

17.0

1.9

0.8

CD3OD

CD3OD

Figure 3.25: 1H NMR spectra (250 MHz, CD2Cl2/CD3OD 1:1) of 55 (top) and 54 (bottom).

50

4 Chemical unclicking of amide bonds

4.1 Introduction

The first part of this work focussed on the development of binuclear cryptates to

prepare a promising molecular environment for an upconversion system. This part

deals with the design of a synthetic concept, that allows to selectively disconnect

binuclear lanthanide cryptates and thus switch off potential upconversion emission.

As it was outlined in the previous section, cryptates can be directly connected via

an amide bond or a short linker. Another approach would be the integration of two

cryptates into a tripeptide. In this case, the cleavage of a peptide bond would also

result in a separation of two cryptates as depicted in Figure 4.1.

Ln1

AA1 AA2 AA3

Ln2 Ln1

AA1 AA2 AA3

Ln2

+

Figure 4.1: Envisaged selective cleavage of a tripeptide with two cryptates. Blue structures stand for

cryptands, yellow spheres for lanthanides, and grey spheres for amino acids.

With the potential application as optical probe in biological systems in mind, two

restrictions were made towards the synthetic concept. First, the trigger of the cleav-

age had to be bioorthogonal, meaning that it is not associated with and chemically

inert within a biological system. [120] Second, the reaction itself should be a straight-

forward, simple chemical transformation, which proceeds under mild conditions (e.g.

temperatures between 25 °C and 40 °C, aqueous solutions, neutral pH). With these

requirements fulfilled, this idea of selective bond cleavage (“Unclick chemistry”) rep-

resents the counterpart of the well-established “Click chemistry”. [121]

51

4.2 Concept

4.2 Concept

Specific peptide bonds are routinely cleaved with proteolytic enzymes. [122] As an al-

ternative, non-enzymatic, chemical methods were developed, which take advantage

of the nature of certain amino acid residues. [123] In contrast to proteases, these mo-

lecules represent the smallest possible entity to cleave an amide bond and, therefore,

are expected to marginally disturb other molecules within a biological environment.

In order to keep Unclick chemistry as simple as possible, an artificial amino acid, that

possesses a specially protected side chain, was chosen as the key molecule.

R1

R1

+

Figure 4.2: The principle of Unclick chemistry.

The unprotected side chain is thought to contain a nucleophilic moiety, which at-

tacks the amide bond in an intramolecular cyclisation reaction. As a result, the amide

is cleaved and the cyclic form of the key molecule, as well as an amino acid or pep-

tide with a free N-terminus is obtained. The designated side-chain protecting group

masks the nucleophilic moiety, until it is removed by applying a bioorthogonal chem-

ical trigger (Figure 4.2, key and lock). Therewith, it provides a precise spatial and

temporal control over the reaction.

The molecular design of the Unclick amino acid was inspired by L-asparaginyl-

residues. Peptides, which possess the latter moiety, undergo an intramolecular re-

action at basic pH (7.4-13.8), during which the side chain amide nitrogen nucleo-

philically attacks the amide carbon. As a result, the peptide bond is cleaved and a

C-terminal succinimidyl-residue as well as a free N-terminus are formed. [124]

52


HN

PG NH

O

O

NH2

R

ONH

OHN

H2N R+5-exo-trig PG

Figure 4.3: Core structure of the Unclick amino acid and envisaged peptide bond cleavage. PG =

protecting group.

The envisaged nucleophilic moiety of the target molecule was also thought to pos-

sess an amino group, which is however deprotonated under physiological conditions

(pH 7.4). Organic hydroxyl amines display pKa values beyond 7.4 (e.g. N-methyl hy-

droxylamine 5.96, N,O-dimethyl hydroxylamine 4.75), [125] and, hence, an aminoserine

was chosen as the lead structure for the Unclick amino acid. The hydroxylamine ni-

trogen of this molecule is expected to react intramolecularly with the amide bond

to form an O-alkyl hydroxamic acid (Figure 4.3). This 5-exo-trig reaction is kinetically

and entropically favoured and, moreover, it is driven by the protonation of the formed

amine in a physiological environment.

HN

PG NH

O

O

NH

R

HN

PG NH

O

O

N O

O

N3

HN

PG NH

O

O

N O

O

NH2

R R

PPh3

Staudinger reaction

Figure 4.4: Structure of “Unclick serine” and deprotection of the Azoc side chain protecting group.

PG = protecting group.

The hydroxylamine was thought to be protected with a bioorthogonal azide-based

carbamate group (Azoc), [126] which is reportedly stable under solid phase peptide

synthesis (SPPS) conditions and orthogonal to Fmoc and Boc. Furthermore, it is se-

lectivley and rapidly deprotected by treatment with bioorthogonal PPh3. For the

introduction of Azoc to the aminoserine, the N- and C-terminus also have to be pro-

tected and the hydroxylamine nitrogen has to be monoalkylated in order to prevent

side reactions.

53

4.3 Preliminary work

4.3 Preliminary work

Before the preparation of Unclick serine was initiated, it was elucidated, if basic re-

quirements for the selective cleavage of cryptate carrying peptides are fulfilled. On

the one hand, it had to be verified that cryptates can be efficiently conjugated to amino

acids. On the other hand, it had to be validated that a 5-exo-trig reaction is general

possible within such a system.

Preparation of an amino acid cryptate conjugate

The conjugation of cryptates to amino acids was already pursued by N. Alzakhem,

who coupled L-lysine to a rigid, carboxyl functionalised TBP cryptand. [112] During

this work, he was faced problems with the activation of the carboxylic acid attached

to the cryptate, which considerably limited the yield of the coupling reaction.

On the account of his findings, a different approach was chosen, which involved

the side chain coupling of L-glutamic acid to the ethylenediamine linked, rigid TBP

cryptate 55. As depicted in Scheme 4.1, the employed L-glutamic acid beared an N-

terminal Fmoc protecting group, which is usually required for the standard coupling

of amino acids using solid phase peptide synthesis (SPPS). Moreover, this moiety as

well as the C-terminal tert-butyl ester are orthogonal to the Azoc group of Unclick

serine.

N

N

N

N

N

N

N

NNa

Br

O

NH

HN O

FmocHNO

OtBu

N

N

N

N

N

N

N

NNa

Br

O

NH

NH2

O

FmocHNO

OtBu

HO

+a O

OOO

55 56

Scheme 4.1: Coupling of a rigid, functionalised TBP cryptate to L-Glu. (a) HATU, DIPEA, DMF,

0 °C→rt (52%).

The reaction of 55 with Fmoc-Glu-OtBu was facilitated by HATU in the presence of

DIPEA and resulted in formation of the amino acid conjugate 56, which was isolated

in 52% yield after two-fold column chromatography. Identity and excellent purity of

the product could be confirmed by ESI mass spectrometry, and by 1H NMR spetro-

scopy (Figure 4.5). Both spectra only displayed the expected signals for pure 56.

54


1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)

9.5

2.4

2.1

10.5

4.3

6.4

1.0

1.1

8.3

1.3

15.2

1.0

1.0

CH2Cl2

CHCl3

N-H N-H

enCH2

benz.CH2

GluCH2

GluCH2

Fmoc& Glu & cryp

tBu

N-Harom.CH2

arom.CH2

Fmoc

Figure 4.5: 1H NMR (250 MHz, CDCl3) of 56.

5-exo-trig test reaction

To evaluate the chances for a 5-exo-trig reaction to occur in Unclick serine, the struc-

turally related, commercially available 1-phenyl-3-pyrazolidinone (57) was chosen as

a suitable model system for test reactions. In comparison to Unclick serine, this com-

pound possesses a hydrazine instead of a hydroxylamine moiety, and no N-terminus.

At first, the starting material 57 was reacted with HCl/MeOH to form methyl 3-(1-

phenylhydrazinyl)propanoate hydrochloride (58). Then, the latter was treated with

aqueous NaHCO3 to remove the hydrochloride and, therewith, enhance the nucleo-

philicity of the terminal nitrogen.

NNH

O

Ph

O

O

NPh

NH2 HCl

a

b

57 58

Scheme 4.2: Reversible ring opening of phenylpyrazolidinone. (a) HCl/MeOH, 0°C→rt. (b)

NaHCO3, CHCl3, D2O.

55

4.4 Retrosynthetic analysis of Unclick serine

The behaviour of the free hydrazine in CDCl3, [D6]-DMSO or D2O was followed

with 1H NMR spectroscopy. While no spectral changes were observed in organic

solution, the spectrum of the aqueous mixture after two hours at room temperature

proved the recovery of 57. This result encouraged the working hypothesis regarding

the 5-exo-trig reaction.

4.4 Retrosynthetic analysis of Unclick serine

An N-terminal protected Unclick serine (59, Scheme 4.3) can be derived from an Azoc-

free, N-terminal protected aminoserine (60). This precursor is accessible by ring open-

ing of 61. The required N-methylated, protected cyclic amino acid may arise from the

selective protection and methylation of D-cycloserine (62).

ONH

OH2N

ON

OHN

PG

HN

PG OMe

O

O

HN

HN

PG OH

O

O

N O

O

N3

methylationprotectionAzoc

protectionringopening

59 60 61 62

Scheme 4.3: Retrosynthetic analysis of Unclick serine.

56


4.5 Investigation of different Unclick systems

4.5.1 Fmoc-Unclick serine

Fmoc-protected Unclick serine (63) was prepared from commercially available D-

cycloserine in six steps, during which the Fmoc and the Azoc protecting group were

successively introduced to the molecule.

ONH

OHN

Z

ON

OHN

Fmoc

HN

Fmoc OMe

O

O

HN

HN

Fmoc OMe

O

O

N O

O

Z

HCl

b c d

a Z=H

Z=Fmoce Z=Cl

Z=N3

62

6465 66

67

63

Scheme 4.4: Synthesis of Fmoc-Unclick-serine. (a) 1. BSA, dry CH2Cl2, rt; 2. pyridine, Fmoc-OSu, dry

CH2Cl2, 0 °C→rt (84%). (b) MeI, K2CO3, acetone, reflux (70%). (c) HCl/MeOH, 0 °C→rt

(97%). (d) chloromethyl chloroformate, NEt3, dry CH2Cl2, -20 °C (15%). (e) NaN3, DMF,

rt (44%).

Fmoc protection of D-cycloserine (62) was performed according to the literature

procedure of Gordeev et al. [127] Accordingly, the reactant was treated with N-(9-

fluorenyl-methoxycarbonyloxy) succinimide (Fmoc-OSu) in the presence of pyridine,

which functioned as a proton acceptor, and N,O-bis(trimethylsilyl) acetamide (BSA)

to mask the lactam nitrogen. Due to the poor solubility of the obtained crude product,

workup and purification were cumbersome and Fmoc-cycloserine (64) could only be

isolated in low yield (15%). However, a minor modification of the procedure signific-

antly improved the step. When the reaction was not carried out at room temperature

with solid BSA, but at 0 °C with BSA dissolved in CH2Cl2, the product could be

obtained in high yield (84%) and purity, after a short and simple work up.

In the next step, the lactame nitrogen of 62 was alkylated with MeI in the presence

of K2CO3. By carefully adjusting the reaction time, it was possible to facilitate methyl-

ation and, simultaneously, prevent Fmoc deprotection, so that 65 could be isolated in

70% yield after column chromatography. The regioselectivity of this reaction was in-

directly confirmed by the analytic data of the follow up product. The methanolysis

of the lactam with HCl/MeOH gave the acyclic aminoserine hydrochloride 66 in 97%

yield after coevaporation with MeOH.

57


The introduction of the Azoc protection group was realised using the two-step liter-

ature procedure of Pothukanuri et al. [126] First, 66 was reacted with chloromethyl chlo-

roformate at -20 °C to give 67 in 15% after column chromatography. In the second

step, 67 was treated with NaN3 to substitute the chloro moiety. In order to reveal

the best solvent for this exchange, test runs were performed in deuterated CH3CN,

DMSO as well as DMF, and monitored with 1H NMR spectroscopy. It was found that

formation of product 63 proceeded very slowly in deuterated CH3CN and DMSO. In

contrast, when DMF was used, the conversion was completed after two hours, how-

ever, significant amounts of a side product were observed after three hours. Hence,

the nucleophilic substitution was performed on larger scale in DMF with a reaction

time of two hours to obtain 63 in 44% yield.

ON

OHN

Fmoc

HN

Fmoc OMe

O

O

HN

HN

Fmoc OMe

O

O

N O

O

N3

a

HN

Fmoc OMe

O

O

HN HCl

b

63

68 65

66

Scheme 4.5: Attempted Unclick reaction. (a) PPh3, [D8]-THF/H2O (9:1). (b) aq. NaHCO3, CHCl2(83%).

To investigate the proposed Unclick mechanism, a solution of Fmoc-Unclick serine

(63) in [D8]-THF/H2O (9:1) was treated with PPh3 (Scheme 4.5 (a)), and the reac-

tion was followed with 1H NMR spectroscopy. It was expected, that the induced

Staudinger reaction triggers Azoc deprotection, and that the resulting free hydroxyl

amine 68 cyclises to 65. Indeed, the spectra indicated a rapid loss of Azoc, but also

the retention of the methyl ester. Thereupon, it was explored, if the cyclisation is pH

dependent by repeating the reaction in mixtures of [D8]-THF/PBS (9:1) at pH 5.2, 7.0

and 8.8. However, the resulting 1H NMR spectra strongly resembled the former one.

58


In order to prove the assumption, that the common product of the reactions was 68,

compound 66 treated with aqueous NaHCO3 in CH2Cl2 to remove the hydrochloride

(Scheme 4.5 (b)). The 1H NMR spectra before and after base treatment as well as the

desired cyclic structure are displayed in Figure 4.6.

3.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)

1

2

3

2.6

3.2

1.7

1.3

2.1

0.9

0.7

4.4

2.1

2.2

Fmoc

Fmoc

Fmoc

0.9

1.1

1.8

1.1

0.8

0.6

4.0

2.0

2.0

2.75

3.33

1.45

2.40

1.31

1.60

0.70

4.29

2.37

2.32

CH2Cl2 OMe N-Me

N-Me

N-Me

+ NaHCO3

NH

NH

NH

CH2Cl2

CHCl3

CHCl3

CHCl3

OMe

Figure 4.6: 1H NMR of 65 (top, 250 MHz, CDCl3), 66 (middle, 200 MHz, CDCl3) and 68 (bottom, 250

MHz, CDCl3).

The spectrum, which is obtained after the reaction with NaHCO3, is in principle

identical with the spectra obtained from the test reactions. It displays the signal for

the methyl ester and, in comparison to its precursor with the hydrochloride (66), an

upfield shifted N-methyl signal. It was concluded, that Fmoc-Unclick serine (68) fails

to undergo the 5-exo-trig reaction, which might be due to the steric hinderance of the

bulky Fmoc group or the electronic structure of the carbamate moiety.

59


4.5.2 Acetyl-Unclick-serine

The comparably small acetyl group was chosen as alternative to the Fmoc protecting

group. In comparison to the carbamate, the amide causes a strong electron withdraw-

ing effect, which is not diminished by the donation of a lone pair from an alkoxy

substituent. As a result, the C-terminus of the acetyl-protected amino acid is more

electrophilic, which was presumed to facilitate the ring closure.

ONH

OHN

Z

ON

OHN

Ac

HN

Ac OMe

O

O

HNHCl

b c

a Z=H

Z=Ac

62

6970 71

Scheme 4.6: Synthesis of Acetyl-aminoserine hydrochloride. (a) Ac2O, MeOH, rt (58%). (b) MeI,

K2CO3, acetone, reflux (27%). (c) HCl/MeOH, 0 °C→rt.

The synthesis of Acetyl-Unclick serine started with the N-terminal protection of

D-cycloserine (62) (Scheme 4.6). Therefore, 62 was treated with Ac2O in refluxing

methanol. [128] Purification of the crude product by recrystallisation in EtOH afforded

69 in the form of crystals, which were suitable for X-ray crystal structure analysis

(Figure 4.7).

Figure 4.7: X-ray crystal strcture of D-acetylcycloserine (69, orthorhombic, P212121). Ellipsoids are

drawn at 50% probability level.

Subsequently, compound 69 was treated with MeI to selectively alkylate the ring

nitrogen. Due to the poor solubility of the reactant and, despite some effort to op-

timise the reaction and the following work up procedure, the methylated product 70

could only be isolated in low yields (max. 27%). Nevertheless, it was possible to

obtain crystals suitable for X-ray crystal structure determination by layering an eth-

60


anolic solution of 69 with Et2O. According to the 1H NMR spectrum (Figure 4.9 top),

these crystals contained only one species, so that together with the resulting struc-

ture solution (Figure 4.8) the exclusive methylation of the lactam nitrogen could be

confirmed.

Figure 4.8: X-ray crystal strcture of D-N(Me)-acetylcycloserine (70, orthorhombic, P212121). Ellipsoids

are drawn at 50% probability level.

For the preparation of acyclic 71, N-methylated acetylcycloserine 70 was treated

with HCl/MeOH. Then, a test reaction was performed to study the potential of the

envisaged Ac-Unlick serine to undergo a 5-exo-trig reaction. Hence, the crude material

of the this reaction was treated with aqueous NaHCO3 to remove the hydrochloride.

1.52.02.53.03.54.04.55.05.56.06.5f1 (ppm)

1

2

3

3.0

2.9

1.1

1.0

1.0

0.7

NH

aa -CHbb -CH

bb -CH

N-Me Ac

3.0

2.8

3.1

2.2

2.3

aa -CH NHbb -CH2

N-Me

AcOMe

aa -CH

bb -CHbb -CH

Ac

3.0

3.0

1.0

1.0

1.0

H2O DMSO- D6

+ NaHCO3

N-Me

Figure 4.9: 1H NMR of 70 (top, 200 MHz, DMSO-D6) and 71 before (middle, 250 MHz, DMSO-D6) and

after (bottom, 200 MHz, CDCl3) treatment with aq. NaHCO3 in CH2Cl2.

61


The comparison of the 1H NMR spectra (Figure 4.9) of 71 before and after base

treatment is difficult, since different solvents were used. Still, it reveals that the latter

reaction causes a loss of the methyl ester signal. Also, the sharp signals of the product,

which resemble those of the 1H NMR spectrum of 70, suggest the recovery of the cyclic

structure.

Since these results were very promising, it was further intended to prepare a larger

amount of 71. However, multiple repetition of the methanolysis of 70 applying the

initial as well as different reaction conditions did not yield the pure product as ex-

pected, but mixtures with unpredictable amounts of an unidentified by-product. Due

to the poor solubility of this mixtures in organic solvents, neither purification nor the

subsequent reaction with chloromethyl chloroformate were successful.

4.5.3 Acetyl-Unclick-serine via Boc-cycloserine

Because of the synthetic unavailability of acetyl-protected Unclick serine 72, the mo-

lecular structure of the target molecule and the synthetic approach were reconsidered.

The resulting novel synthesis sequence does however not allow N-methylation of

the hydroxylamine, which also implies a slightly different target structure (73) (Fig-

ure 4.10).

HN

Ac OMe

O

O

HN O

O

N3

HN

Ac OMe

O

O

N O

O

N3

72 73

Figure 4.10: Structures of the previous (left) and the new synthetic target (right).

Therefore, it was planned to successively protect D-cycloserine with Boc and also

with chloromethyl carbamate (chloroc) prior to the ring opening (Figure 4.10). Taking

this new synthetic pathway (Scheme 4.7), D-cycloserine was first reacted with Boc2O

to give the N-terminal protected amino acid (74) in 54% yield after column chromato-

graphy. [129,130] The subsequent reaction with chloromethyl chloroformate afforded the

diprotected D-cycloserine (75) in 56% after purification. Treatment of the latter with

a HCl/ MeOH not only caused ring opening of the reactant, but also the removal of

Boc to quantitatively yield 76. Thus, the new reaction sequence provides the chance

to modify the N-terminal protecting group at a late stage of the synthetic pathway.

62


ONH

OH2N

ONH

OHN

BocO

N

OHN

Boc

H2NOMe

O

O

HN O

O

Cl

O

O

Cl

HN

Ac OMe

O

O

HN O

O

Z

e

a

Z=Cl

Z=N3

b c d

62 74 75 76 77

73

Scheme 4.7: Synthesis of Ac-Unclick serine without the N-methyl group. (a) Boc2O, NEt3, THF/H2O,

0 °C→rt (54%) (b) chloromethyl chloroformate, NEt3, dry CH2Cl2, -20 °C (56%). (c)

HCl/MeOH, 0 °C→rt (quantitative). (d) Ac2O/DIPEA/DMF, rt (47%). (e) NaN3, DMF, rt.

Unexpectedly, the acetylation of 76 appeared to be very difficult and several differ-

ent reaction conditions were tested to optimise this reaction (Table 4.1).

Table 4.1: Overview over tested reaction conditions for the acetylation of 76

reagent base solvent temperature time remark

Ac2O - MeOH rt 2 h incomplete acetylationloss of chloroc during purification

AcCl NEt3 CHCl3 0°C→rt 2 h no acetylationloss of chloroc

AcCl NEt3 CH2Cl2 0°C→rt 1 h three acetyl signals in 1H NMRno separation possible

Ac2O DIPEA DMF rt 15 min simultaneous additionmoderate yield

Ac2O DIPEA DMF rt 15 min successive additionvery low yield

Ac2O NEt3 CH2Cl2 rt 3 h several acetyl signals in 1H NMRpartial loss of chloroc

Ac2O NEt3 CH2Cl2 -50°C, 3 h, then three acetyl signals in 1H NMRthen rt overnight partial loss of chloroc

When Ac2O was used, the resulting 1H NMR spectrum of the crude product indic-

ated incomplete acetylation. Purification with column chromatography to remove the

reactant and traces of Ac2O was not successful, but only led to the loss of chloroc.

Alternatively, the use of AcCl in the presence of a non-nucleophilic base was tested,

employing conditions reported by Griesbeck et al. for the synthesis of N-acetylamino

acid methyl esters. [131] Accordingly, 76 was treated with two equivalents of NEt3 to

remove the hydrochloride and to deprotonate the N-terminal nitrogen. Then, AcCl

was added at low temperature to prevent multiple acetylation of the same nitrogen.

63


However, this reaction completely failed to produce 77. In contrast, when CHCl3was used as reaction medium, the isolated crude material showed several acetyl and

methylene groups in the 1H NMR spectrum. Still, further purification with column

chromatography was not successful.

Since the reactions with AcCl were rather discouraging, attention was turned back

to the use of Ac2O, while keeping the idea of employing a non-nucleophilic base. A

simple method that combines both is the utilisation of a capping agent, which is a

mixture of Ac2O and DIPEA in DMF that is normally used in solid phase peptide

synthesis to cap unreacted amino acids. Indeed, treatment of 76 with this mixture,

followed by repeated column chromatography of the crude product provided 77 in

47% yield. In contrast, the successive addition of DIPEA and Ac2O provided the

acetylated product in only 21% yield after column chromatography.

The reaction could not be further improved by replacement of DIPEA with NEt3 and

simultaneous extension of the reaction time. Addition of Ac2O at low temperature

did also not aid product formation. Instead, a partial loss of the chloroc group and

multiple acetylation was observed.

1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)

1

2

2.8

3.0

2.1

1.3

1.6

3.0

3.0

1.8

1.0

1.7

0.8

0.8

CDCl3

NH

-CH2-Cl

aa -CH

bb -CH2

OMe Ac

NH

-CH2-N3

aa -CH

bb -CH2

OMe AcCDCl3

Figure 4.11: 1H NMR of 77 (top, 250 MHz, CD3OD) and of 73 (bottom, 200 MHz, CDCl3).

64


After optimisation of the acetylation, 77 was finally converted into Ac-Unclick ser-

ine (73) by treatment with NaN3. As the 1H NMR spectra show (Figure 4.11), the

substitution of the chloro moiety by an azide group causes an upfield shift of the

methylene protons, while the remaining signals show the same chemical shifts and

integrals as before. However, the result of this test reaction could not be reproduced

when this reaction was repeated on larger scale. The analytical data of the obtained

products always indicated a loss or decomposition of the chloroc moiety.

65


4.5.4 Fmoc-Gly-Unclick-serine

For the repeated reconsideration of the Unclick concept, all previous findings were

taken into account. To this point, the following was known:

• Fmoc-protected (cyclo)serine derivatives provide good solubility in organic media, can be purifiedwith reasonable effort and are obtained in satisfying yields.

• Fmoc(N-Me) Unclick serine does not undergo the attempted ring closure after Azoc removal,presumably due to the presence of a bulky carbamate.

• Ac-protected (cyclo)serine derivatives provide very limited solubility in organic media, are usuallypurified by crystallisation and are isolated in low yields.

• Although it was shown, that ring closure in Ac(N-Me) aminoserine occurs readily, its base labilityalso prevents the introduction of Azoc.

• Boc-protected cycloserine derivatives are comparable to Fmoc analogues in solubility, purificationeffort and obtained yield.

• Boc-cycloserine-Chloroc is deprotected during the acidic ring opening. Subsequent acetylation ischallenging and the Cl/N3 replacement is not reproducible. Especially the lack of N-methylationseems to favour side reactions.

From the gathered knowledge, the requirements for an improved Unclick system

were derived. First, an Fmoc or Boc protecting group is necessary to provide sufficient

solution in organic media. This is equally important for the reactions as well as puri-

fication procedures and has, in general, a positive influence on the yield. Second, the

N-terminus has to be equipped with an amide group which causes a stronger electron

withdrawing effect than a carbamate and, consequently, enhances the electrophilicity

of the C-terminus. Third, the hydroxyl carbamate nitrogen has to be methylated to

prevent side reactions. A lead structure, which fulfils all criteria, is shown in Fig-

ure 4.12. It can be obtained by coupling of D-cycloserine to L-glycine, followed by

methylation, ring opening, and Azoc introduction.

NH

O

OR

O

HN

O

OH

O

N O

O

N3

Figure 4.12: The newly developed Unclick system possesses three important features, which are expec-

ted to contribute to a high solubility in organic media (carbamate), a readily occuring ring

closure (N-terminal amide) and a reduced chance of side reactions (N-methylation).

66


The synthesis of this dipeptide was attempted via the trimethylsilyl/acyl chloride

method, which was adapted from a procedure of De et al. [132] This rather uncommon

method for peptide coupling in solution was chosen, since silylation of both nitrogens

of 62 significantly increases the solubility of the starting material in organic media. [133]

In addition, when treated with a carboxylic acid chloride, the very good leaving group

trimethylsilylchloride is formed as a byproduct during peptide coupling. [134]

The peptide coupling was prepared by the reaction of Fmoc-Gly-OH (78) with

oxalyl chloride to form acyl chloride 79. At the same time, D-cycloserine (62) was

silylated with BSA to 80 in a solvent mixture of N-methyl-2-pyrrolidone (NMP) and

CH2Cl2. The isolated acid chloride and an excess of pyridine were added to the latter

reaction mixture at low temperature to form dipeptide (81), which was isolated in

42% yield after column chromatography.

+Cl

OCl

+O

Si

NSi

NH

Fmoc

O

OH

NH

Fmoc

O

HN

ONH

O

O

NH

Fmoc

O

Cl

ONH

H2NO

ON

HN

O

TMSTMS

a

b

c78 79

62 80

81

Scheme 4.8: Synthesis of Fmoc-glycine-cycloserine via the trimethylsilyl/acyl chloride method. (a)

neat, 0 °C. (b) BSA, NMP, dry CH2Cl2, rt. (c) pyridine, 0 °C→rt (42%).

The subsequent reactions (Scheme 4.9) were performed in analogy to the synthesis

pathway of Fmoc-Unclick serine. At first, the ring nitrogen was alkylated with MeI in

acetone under slightly basic conditions to yield the compound 82 in 67% yield after

column chromatography.

Following treatment with HCl/MeOH resulted in the formation of two products,

which were separated by column chromatography and identified as 83 (50% yield)

and Fmoc-Gly-OMe (16% yield). This observation implies a partial decomposition

of the dipeptide, which is accompanied by acidic esterification of Fmoc-glycine-OH.

Indeed, it is well-known, that the applied conditions can be used for the C-terminal

esterification unprotected amino acid, [135,136] but are also strong enough to cause the

partial cleave of peptide bonds. [137,138]

67


NH

Fmoc

O

HN

ON

O

NH

Fmoc

O

HN

ONH

O

NH

Fmoc

O

HN

O

O

NH

ONH

Fmoc

O

HN

O

O

N

O

O

O

Z

a

Z=Cl

Z=N3

b

d

c

81 82

8384

85

Scheme 4.9: Synthetic pathway for the synthesis of Fmoc-Gly-Unclick serine. (a) MeI, K2CO3, acetone,

reflux (67%). (b) 1.25M HCl/MeOH, 0°C → RT (50%). (c) chloromethyl chloroformate,

NEt3, CH2Cl2, -20°C (71%). (d) NaN3, DMF, rt (72%).

Retrospectively, this acidic amide hydrolysis might also explain the previous ob-

servation of several products after treatment of 70 with HCl/MeOH. It is also worth

mentioning that aminoserine 83 is highly unpolar, which allowed its purification with

column chromatography and, therewith, suggests the absence of a hydrogen chloride

is formed. This hypothesis was strengthened by a test reaction during which 83 was

dissolved in CH2Cl2 and treated with an excess of aqueous NaHCO3. Even after four

days, no changes in the 1H NMR spectrum occurred, which would go along with the

removal of the hydrogen chloride (observed for Fmoc-protected aminoserine) or ring

closure (observed for Ac-protected aminoserine).

Regardless, Azoc introduction was attempted as usual by a two-step procedure.

First, 83 was reacted with chloromethyl chloroformate in CH2Cl2 at -20°C to give 84,

which was obtained in 71% yield after column chromatography. Subsequent reaction

with NaN3 provided 85, which was isolated in 72% yield after purification.

Finally, with the target compound at hand, it was studied, if a 5-exo-trig reaction can

be induced. Therefore, Fmoc-Gly-Unclick serine was treated with PPh3 in aqueous

THF and the reaction was monitored with 1H NMR spectroscopy. Unfortunately, the

resulting 1H NMR spectrum revealed (Figure 4.13), that the product of this reaction

was the unprotected aminoserine 83.

68


1.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)

1

2

3

2.9

3.0

1.9

5.2

1.0

1.6

4.1

1.9

2.0

2.7

3.1

2.0

4.3

1.0

2.6

2.8

4.0

1.2

2.2

1.2

1.1

4.8

2.0

2.0

+ PPh3+ H2O

Fmoc

Fmoc

Fmoc + PPh3 + O=PPh3

NH

OMe

OMe

OMe N-Me

N-Me

N-Me

Figure 4.13: 1H NMR spectra of 85 before (top, THF-D8/D20, 250 MHz) and after (middle, THF-

D8/D20, 250 MHz) reaction with PPh3. For comparison, the spectrum of the desired

product of this reaction (83) (bottom, CDCl3, 200 MHz) is also displayed.

In other words, the applied conditions lead to the expected removal of Azoc, but

not to the intended ring-closure. This result is in accordance with the stability of 83

under basic conditions. It was concluded, that the N-terminal protecting group has

a small influence on the ring closure and that the pKa value of the hydroxyl amine

is of greater importance. In a final approach (Scheme 4.10) it was tried to synthesise

the related Fmoc-Gly-Unclick serine and Fmoc-Unclick serine, without the N-methyl

group. Unfortunately, both attempts failed due to the poor stability of the chloroc

moiety, which prevented the isolation and purification of 86 and 87, respectively.

ONH

OHN

ZO

N

OHN

Z

O

O

Cl

Z=Fmoc

Z=Fmoc-Gly

Z=Fmoc

Z=Fmoc-Gly

64

81

86

87

Scheme 4.10: Attempted synthesis of chloroc protected cycloserine derivatives.

69

5 Summary

The first part of this work dealt with the development of a binuclear ligand for a

lanthanide-based, near-IR emissive upconversion complex. The structure of this sys-

tem is based on tris(bipyridine) (TBP) cryptands. These ligands form stable, lumin-

escent lanthanide complexes, are devoid of N-H and O-H oscillators and protect the

coordinated metal efficiently from other oscillators e.g. in the solvent. Within this

work, it was aimed to evaluate, if exclusion of C-H groups from the TBP ligand re-

duces the quenching of lanthanide-centered near-IR luminescence. In addition, it was

intended to introduce functional groups to TBP cryptates and to selectively couple

them to a binuclear ligand.

To address the first matter, efficient protocols for the synthesis of selectively deuter-

ated TBP cryptates were developed. In comparison to the undeuterated analogue, the

preparation of the isotopologues required just one additional synthetic step during

which the H/D exchange was performed using a readily available, cheap deuterium

sources (D2O or NaBD4). Thus, it was possible to prepare a series of isotopologic

cryptates (Figure 5.1) with excellent overall deuteration levels (>97%).

N

N

N

N

N

N

N

N

DD

DD

DD D D

D D DD

DD

DDDD

D D

Na Br

N

N

N

N

N

N

N

N

D D DD

DD

DDDD

Na Br

N

N

N

N

N

N

N

N

D D

DDD D

DDDD

D D

Na Br

Figure 5.1: Examples of synthesised isotopologic cryptates

Photophysical studies of the corresponding Nd3+ and Yb3+ complexes revealed,

that a decreasing number of C-H groups goes along with prolonged luminescence

lifetimes and, hence, with reduced quenching (Figure 5.2). Accordingly, the smallest

quenching rates were observed for the perdeuterated complexes. Compared to the

undeuterated counterparts, the determined luminescence lifetimes were increased by

70

5 Summary

the factor 1.6 for Yb3+ and 6.9 for Nd3+, the latter being among the longest lifetimes

for Nd3+ complexes in solution reported so far. Moreover, the deactivation rate contri-

butions of individual C-H groups were accurately quantified. This study represented

the first systematic investigation of C-H induced luminescence quenching in near-IR

emissive lanthanide cryptates.

Figure 5.2: Decrease of luminescence quenching with increasing degree of deuteration

For the second aim, the selective coupling of functionalised TBP cryptates, carboxy-

lic acid esters and amino groups were regarded the most attractive functional groups.

While the carboxy functionalised cryptates were prepared employing reported pro-

cedures, two novel synthetic pathways were developed, which enabled the prepara-

tion of previously unknown amino functionalised cryptates. Furthermore, the above

mentioned deuteration protocols could be successfully adapted for the synthesis of

the first deuterated carboxy functionalised cryptate (98%D) (Figure 5.3).

N

N

N

N

N

N

N

N

DD

DD

DD D D

DDDD D

DD

DDDD

DD

DD

D

D

DD

D D

Na

COOMe

Br

Figure 5.3: Synthesised perdeuterated functionalised cryptate

71

The synthesis of a binuclear cryptate was initially attempted by the successive coup-

ling of two carboxy functionalised cryptates to one ethylenediamine linker. However,

this approach only provided minimal yields, so that an alternative pathway was pur-

sued. Therefore, two carboxy functionalised cryptates were linked to one ethylene-

diamine each and then coupled via a carbamide. Using this procedure, it was possible

for the first time to successfully prepare, isolate and characterise a dinuclear TBP

crpytate (Figure 5.4).

N

N

N

N

N

N

N

NNa

Br

O

NH

HN

N

N

N

N

N

N

N

NNa

Br

O

NH

HN

OOO

OO

Figure 5.4: Synthesised binuclear cryptate

The second part of this work dealt with the development of a molecular system, that

allows the straightforward selective cleavage of peptide bonds by applying a bioortho-

gonal, chemical trigger. This simple chemical transformation, which proceeds under

mild conditions, represents the counterpart of the well-established “Click chemistry”

and was therefore termed “Unclick chemistry”.

The key molecule of this system was a D-aminoserine with a bioorthogonal azide

carbamate (Azoc) protecting group in its side chain. Within this work, it was aimed

to synthesise such an “Unclick”-serine and to prove the PPh3-mediated Azoc depro-

tection, as well as 5-exo-trig reactions of this molecule. Hence, several D-aminoserines

with different N-terminal protecting groups (Fmoc, acetyl, Boc) or residues (Fmoc-

Glu) were prepared. While it was not possible to prove Azoc deprotection and 5-exo-

trig reaction with the same molecule, still two important goals were achieved.

HN

Fmoc OMe

O

O

HN

HN

Fmoc OMe

O

O

N O

O

N3

PPh3

H2O

Figure 5.5: Selective Azoc-deprotection of Fmoc-protected Unclick serine

72

5 Summary

Using the Fmoc protected D-aminoserine it was shown, that Azoc can be easily

introduced to this amino acid and that deprotection with PPh3 readily occurs under

physiological conditions (Figure 5.5). In addition, it was found that the 5-exo-trig

reaction indeed proceeds within acetyl protected D-aminoserine (Figure 5.6).

HN

Ac O

O

O

NHO

NH

OHN5-exo-trig Ac

Figure 5.6: Ringclosure of an acetyl-protected aminoserine

73

6 Experimental Section

6.1 General

Chemicals were purchased from commercial suppliers and used as received unless

stated otherwise. Deuterated solvents/reagents had deuterium contents > 99.8% with

the exception of NaBD4 (> 98%D). CH3CN for the synthesis of the cryptates was

HPLC grade and was stored under nitrogen. DMF for the synthesis of cryptate amino

acid derivatives was SPPS grade. Dry DMF for other reactions was used as pur-

chased. THF was dried using an MBraun Solvent Purification System. Other solvents

were dried by standard procedures (EtOH (Mg)), CH2Cl2 (CaH2), MeOH (Mg), ethyl-

enediamine (KOH), pyridine (KOH)). Air-sensitive reactions were carried out under

a dry, dioxygen-free atmosphere of N2 using Schlenk technique. Column chroma-

tography was performed with silica gel 60 (Merck, 0.063-0.200 mm) or silica gel 60

silanised (Merck, 0.063-0.200 mm). Analytical thin layer chromatography (TLC) was

done on silica gel 60 F254 plates (Merck, coated on aluminium sheets) or silica gel C18

plates (Machery-Nagel, pre-coated on aluminium sheets, Alugram RP-18 W/UV254)

Product spots were visualized by UV light at 254 nm, and subsequently developed

using I2 vapour or ninhydrin solution as appropriate. Overall deuteration levels were

established by deconvolution of the corresponding mass spectra. ESI mass spectro-

metry was done using Bruker Daltonics Esquire6000. NMR spectra were measured

at room temperature on either a Bruker DPX-200, Bruker DPX-250, or a DRX-400. 1H

NMR spectra were recorded at 200 MHz, 250 MHz, or 400 MHz and 13C NMR at

50.3 MHz, 62.9 MHz, or 100.6 MHz, respectively. The chemical shifts δ are repor-

ted in parts per million (ppm) downfield of tetramethylsilane (TMS), using residual

protonated solvent as internal standard. For NMR spectra recorded in a mixture of

CD2Cl2/CD3OD (1:1), the pure solvent mixture was referenced to tetramethylsilane

and the CD3OD peak (1H NMR: 3.33 ppm, 13C NMR: 49.07 ppm) used as secondary

reference within the sample. Coupling constants J are quoted to the nearest 0.1 Hz.

The abbreviations used in the description of resonances are: s (singlet), d (doublet), t

(triplet), q, (quartet), br (broad), m (multiplet).

74


IR spectra were aquired on a Bruker Tensor 27 FT-IR spectrometer equipped with a

diamond ATR setup. Analytical reversed-phase HPLC was performed on Lichrospher

RP-18e (Merck, 125 × 4mm-5µm, flow rate: 1 ml min−1, UV detection: 300 nm).

X-ray crystal structure determinations were performed by placing the respective

crytal on a glass fiber using perfluoropolyether oil. The measurement was carried

out on an Oxford Xcalibur 2 diffractometer using monochromated Mo Kα radiation.

Frames corresponding to an arbitrary hemisphere of data were collected using ω

scans. The structure was solved within the Wingx [139] package using direct meth-

ods (SIR92 [140]) and expanded using Fourier techniques (SHELXL-97 [141]). Hydrogen

atoms were included but not refined.

6.2 Luminescence measurements

Corrected steady state emission spectra were acquired on a PTI Quantamaster QM4

spectrofluorimeter using 1.0 cm quartz cuvettes with ca. 100 µM solutions at 298 K.

D2O and [D6]-DMSO (NMR grade, 99.9%D) were purchased from commercial sup-

pliers and used as received. The excitation light source was a 75 W continuous xenon

short arc lamp. Emission spectra were collected at 90° to the excitation beam using a

PTI R928 photomultiplier tube (operated at -1000 V) as the detector. Spectral selection

was achieved by single grating monochromators (excitation:1200 grooves/mm, blazed

at 300 nm; visible emission: 1200 grooves/mm, blazed at 400 nm; near-IR emission:

600 grooves/mm, blazed at 1200 nm).

Luminescence lifetimes were measured with the same spectrofluorimeter equipped

with an additional Q4 phosphorescence module. The light source for these measure-

ments was a xenon flash lamp (Hamamatsu L4633: 10 Hz repetition rate, pulse width

ca. 1.5 µs FWHM). Emission was measured at 90° using a PTI P1.7R detector mod-

ule (Hamamatsu PMT R5509-72 with a Hamamatsu C9525 power supply operated

at -1500 V and a Hamamatsu liquid N2 cooling unit C9940 set to -80 °C). Lifetime

data analysis (deconvolution, statistical parameters, etc.) was performed using the

software package FeliX32 from PTI. The instrument response function (IRF) for the

spectral deconvolution procedure was determined using a dilute aqueous dispersion

of colloidal silica (Ludox® AM-30). The given values are averages of three independ-

ent measurements. The global fitting of the lifetime data was performed with Origin

Pro 8G using the “nonlinear surface fit” function constrained to a first order polyno-

mial in y and z: kobs(y,z) = k0 - y ∆kbenzyl - z ∆kpy.

75

6.3 Simple bipyridine building blocks


Undeuterated compounds

2-Bromo-6-picoline (2)

N Br2

2 was prepared according to the literature procedure of Maheswari Palanisamy et al. [97]

In a 3-necked round bottom flask equipped with a dropping funnel and mechanical

stirrer, 2-aminopicoline (1) (50.0 g, 0.462 mol, 1.0 equiv) was added in small portions

to gently stirred HBr (47%, 300 ml) at room temperature. The obtained clear yellow

solution was cooled to -20 °C and cooled Br2 (207.0 g, 66.3 ml, 1.295 mol, 2.8 equiv),

was added dropwise over a period of 40 min while the temperature was maintained.

During addition, the solution turned from orange to red and finally a bright yellow

suspension was obtained. After stirring for 90 min at -20 °C, aqueous NaNO2 (86.0 g

in 125 ml water, 1.247 mol, 2.7 equiv) was added dropwise via a dropping funnel (gas

formation!). The brown suspension was allowed to warm to 15 °C and then stirred

for 45 min. Then, it was cooled to -20 °C again and aqueous NaOH (335 g in 500 ml

water) was added dropwise while the temperature was kept between −10 °C and

−20 °C. Subsequently, the mixture was allowed to warm to room temperature and

then stirred for another hour. After extraction with CHCl3, the organic layer was

dried over MgSO4 and the solvent removed in vacuo to leave a brown oil behind. The

crude product was destilled in vacuo (7.1 mbar, 70-72 °C, bath 120 °C) to yield 2 as

colourless liquid.

Yield: 45.7 g (0.266 mol, 58%). 1H NMR (250 MHz, CDCl3): δ 7.42 (t, 3J = 7.7 Hz, 1H),

7.29 (d, 3J = 7.8 Hz, 1H), 7.10 (d, 3J = 7.5 Hz, 2.53 (s, 3H) ppm.

6,6’-Dimethyl-2,2’-bipyridine (3)

N N3

3 was prepared according to the literature procedure of Rode et al. [98] Under nitrogen

and with vigorous stirring, a solution of 2-bromo-6-picoline (2) (21.9 g, 127.1 mmol,

76


2.0 equiv) in dry toluene (100 ml) was added dropwise to Raney-Nickel (3.7 g, 63.6

mmol, 1.0 equiv) over the course of 10 min. The resulting reaction mixture was heated

at reflux for 18 h (bath: 125 °C) to obtain a dark violet suspension, which was filtered

over a Schlenk frit. The violet solid was washed with dry toluene (2 × 40 ml) and then

dried in vacuo. The blue filtrate was treated with HCl/H2O to neutralise remaining

Ni prior to disposal. To hydrolyse the obtained nickel complex, the violet solid was

added to 40 °C warm water (250 ml) in small portions and stirred for 2 h, whereas

the colour turned from violet to green. The suspension was filtered until the filter

was plugged and the aqueous layer was extracted with CHCl3 (2 × 200 ml). The dark

sticky residue was washed with CHCl3 (5 × 100 ml) and filtered again. The combined

organic extracts were dried over MgSO4 and the solvent removed in vacuo to leave

the brown crude product behind (3.58 g). Recrystallisation from boiling petrolether

yielded 3 as colourless crystals.

Yield: 2.0 g (11.1 mmol, 17%). 1H NMR (250 MHz, CDCl3): δ 8.19 (t, 3J = 7.8 Hz, 2H),

7.68 (d, 3J = 7.7 Hz, 2H), 7.15 (d, 3J = 7.6 Hz, 2.63 (s, 6H) ppm.

6,6’-Dimethyl-2,2’-bipyridine-N,N’-dioxide (4)

N NO O

4

4 was prepared according to the literature procedure of Newkome et al. [99] With cool-

ing in an ice bath, a solution of m-chloroperoxybenzoic acid (18.0 g of 77 wt%, 13.9 g

pure, 80.3 mmol, 2.5 equiv) in CHCl3 (450 ml) was added dropwise to a solution of 3

(6.0 g, 32.6 mmol, 1.0 equiv) in CHCl3 (450 ml) over the course of 3 h. The reaction

mixture was allowed to slowly warm to room temperature and stirred overnight.

Then, the organic phase was extracted with saturated NaHCO3 (10 ml), aqueous

Na2S2O3 (10 ml), dried over MgSO4 and evaporated to dryness. The crude product

was purified by column chromatography (SiO2, CH2Cl2/MeOH 24:1→ 9:1) to give 4

as an off-white solid.

Yield: 5.7 g (26.2 mmol, 80%). Rf = 0.38 (SiO2, CH2Cl2/MeOH 9:1, detection UV).

MS (ESI+): m/z (%) 217.04 (7, [M+H]+), 238.98 (100, [M+Na]+), 254.92 (6, [M+K]+).1H NMR (200 MHz, CHCl3): δ 7.39–7.31 (m, 4H), 7.27–7.16 (m, 2H), 2.58 (s, 6H) ppm.13C NMR (50.3 MHz, CHCl3): δ 149.7, 143.6, 126.8, 125.4, 124.5, 17.9 ppm.IR (FT-ATR):

ν̃ 3074 (w, C-Harom), 3046 (w, C-Harom), 2953 (w, CH3), 2910 (w, CH3), 1701 (w), 1608 (w,

77


C=Carom), 1560 (w, C=Carom), 1475 (m), 1443 (m), 1360 (m), 1263 (s, N-oxide), 1246 (s),

848 (s), 743 (s) cm-1.

6,6’-Bis(bromomethyl)-2,2’-bipyridine (5)

NN

BrBr5

5 was prepared in analogy to a procedure of Psychogios et al. [100] Under nitrogen,

TFA anhydride (55 ml) was added quickly via syringe to a yellow solution of 4 (2.7 g,

12.6 mmol, 1.0 equiv) in dry CH2Cl2 (55 ml). The orange reaction mixture was heated

at reflux for 1.5 h before the solvents were removed in vacuo. The orange residue

was dissolved in a mixture of dry DMF/THF (2:1, 60 ml) and the obtained solution

was added to solid LiBr (11.9 g, 137.4 mmol, 11.0 equiv), which was dried before-

hand in vacuo at 180 °C for 5 h. The reaction mixture was stirred at room temperat-

ure overnight before the solvents were removed in vacuo at 60 °C. The bright orange

residue was dissolved in CH2Cl2 (200 ml) and washed with water (3 × 200 ml). The

combined organic layers were dried over MgSO4 and evaporated to dryness. The

crude product was dry-loaded onto silica and purified by column chromatography

(SiO2, CH2Cl2/MeOH 100:1) to give 5 as a bright yellow solid.


MS (ESI+): m/z (%) 362.85 (60, [M+Na]+), 338.81 (26, [M+H+CF3CO]+). 1H NMR

(250 MHz, CDCl3): δ 8.39 (dd, 3J = 8.0 Hz, 4J = 1.1 Hz, 1H), 7.82 (t, 3J = 7.8 Hz, 1H),

7.47 (dd, 3J = 7.7 Hz, 4J = 1.1 Hz, 1H), 4.63 (s, 2H) ppm. 13C NMR (50.3 MHz, CDCl3):

δ 156.4, 155.7, 138.1, 123.7, 120.7, 34.2 ppm. IR (FT-ATR): ν̃ 3039 (w, C-Harom), 2974 (w,

CH2), 1579 (m, C=Carom), 1567 (m, C=Carom), 1438 (m), 1260 (s), 1080 (m, C-Br), 993 (m),

806 (s), 749 (s), 632 (s, C-Br) cm-1.

6,6’-Bis(bromomethyl)-2,2’-bipyridine-N,N’-dioxide (48)

NNOO

BrBr48

78


48 was prepared by a modified procedure of Caron et al. [118] Under nitrogen and with

cooling in an ice bath, urea-hydrogen peroxide (415.3 mg, 4.414 mmol, 3 equiv) was

added as a solid to a solution of 5 (502.0 mg, 1.468 mmol, 1 equiv) in dry CH2Cl2(30 ml) followed by dropwise addition of TFA anhydride (1.511 g, 1 ml, 7.194 mmol,

4.9 equiv) over the course of 15 min. The reaction mixture was slowly warmed to

room temperature and stirred for 2 d. The obtained clear yellow solution was diluted

with CH2Cl2 (50 ml) before sat. Na2S2O3 (15 ml) was added. After stirring for 1 h,

the phases were separated and the aqueous layer extracted with CH2Cl2 (3 × 50 ml).

The combined organic extracts were dried over MgSO4 and the solvent removed in

vacuo. The yellow crude product was dry-loaded onto silica and purified by column

chromatography (SiO2, CH2Cl2/MeOH 50:1→ 24:1) to give 48 as a white solid.

Yield: 462.8 mg (1.237 mmol, 84%). Rf = 0.17 (SiO2, CH2Cl2/MeOH 24:1, detec-

tion UV). MS (ESI+): m/z (%) 372.96 (5, [M+H]+), 394.86 (49, [M+Na]+). 1H NMR

(200 MHz, CDCl3): δ 7.68–7.64 (m, 1H), 7.64–7.61 (m, 1H), 7.61–7.58 (m, 1H), 7.57–7.54

(m, 1H), 7.35 (s, 1H), 7.31 (s, 1H), 4.73 (s, 4H) ppm. IR (FT-ATR): ν̃ 3067 (w, C-Harom),

3048 (w, C-Harom), 3023 (w, C-Harom), 2980 (w, CH2, 1477 (w), 1410 (m), 1384 (s), 1250

(s, N-oxide), 1205 (s), 887 (m), 839 (s), 790 (s), 646 (m, C-Br) cm-1.

6,6’-Bis(aminomethyl)-2,2’-bipyridine trihydrobromide hydrate (18)

NN

H2N

H2O

3 HBr

NH218

18 was prepared according to the literature procedure. [104] 5 (1.5 g, 4.4 mmol, 1 equiv)

in CHCl3 (60 ml) was heated at reflux until dissolution. Then, a solution of hexa-

methylenetetramine (1.3 g, 9.4 mmol, 2.15 equiv) in CHCl3 (40 ml) was added drop-

wise. After refluxing for 3 h, the mixture was cooled to room temperature and stored

at 8 °C overnight. The precipitate was collected on a Buchner funnel, washed with

a small amount of CHCl3 and dried in vacuo. The white solid was suspended in

H2O/EtOH/47% HBr (16 ml, 62 ml, 10 ml) and stirred at 75 °C until dissolution.

Upon cooling in an ice bath, 18 crystallised as slightly yellow needles, which were

filtered off, washed with a small amount of cold EtOH and dried in vacuo.

Yield: 1.1 g (2.2 mmol, 50%). MS (ESI+): m/z (%) 215.01 (100, [M+H2O-3HBr]+).1H NMR (200 MHz, D2O): δ 8.35 (d, 3J = 8.0 Hz, 2H), 8.04 (dd, 3J = 8.3 Hz, 3J = 7.3

Hz, 2H), 7.53 (d, 3J = 7.8 Hz, 2H), 4.43 (s, 4H) ppm. 13C NMR (50.3 MHz, D2O): δ

79


152.3, 151.2, 141.0, 124.3, 122.3, 42.4 ppm. IR (FT-ATR): ν̃ 3306 (w, NH+3 ), 3030 (m,

CH3), 2928 (m, CH3), 1612 (m, C=Carom), 1592 (m, C=Carom), 1561 (m, NH+3 ), 1523 (m,

C=Carom), 1508 (m, NH+3 ), 1420 (m), 1265 (m), 1183 (m), 995 (m), 884 (s), 795 (s) cm-1.

6,6’-Bis((N-tosyl)aminomethyl)-2,2’-bipyridine (47)

NN

TsHNNHTs47

47 was prepared according to the literature procedure. [142] 18 (950 mg, 2.0 mmol,

1 equiv) was added to a solution of NaOH (672 mg, 16.8 mmol, 8.5 equiv) in H2O

(6.5 ml). With vigorous stirring, a solution of tosyl chloride (827 mg, 4.3 mmol,

2.2 equiv) in Et2O (3 ml) was added at 0 °C before the yellow suspension was al-

lowed to warm to room temperature. After stirring overnight, the precipitate was

collected on a Buchner funnel and dried in vacuo at 60◦C for 1 d to give 47 as a white

solid.

Yield: 512.4 mg (0.98 mmol, 49%). MS (ESI+): m/z (%) 523.09 (78, [M+H]+), 545.04

(100, [M+Na]+) . 1H NMR (200 MHz, CDCl3): δ 8.17 (d, 3J = 7.8 Hz, 2H), 7.80–7.69

(m, 6H), 7.24–7.13 (m, 6H), 5.81 (t, 3J = 4.8 Hz, 2H), 4.34 (d, 3J = 5.2 Hz, 4H), 2.35 (s,

6H) ppm. 13C NMR (50.3 MHz, CDCl3): δ 154.8, 154.3, 143.6, 137.9, 136.8, 129.8, 127.3,

122.2, 120.0, 47.5, 21.6 ppm. IR (FT-ATR): ν̃ 3261 (w, C-Harom), 1575 (w, C=Carom), 1437

(w), 1337 (m, R-SO2-N), 1160 (s, R-SO2-N), 1074 (m), 898 (w), 812 (m), 798 (m), 667 (s)

cm-1.

2,2’-bipyridine-6,6’dicarboxylic acid (7)

NN

HOOHOO

7

7 was prepared in analogy to the procedure of Alyapyshev et al. [102] 3 (12.1 g, 65.7 mmol,

1.0 equiv) was added in portions to conc. H2SO4 (140 ml) and the mixture was heated

to 65 °C (bath temperature). CrO3 (29.5 g, 295.0 mmol, 4.5 equiv) was added at a

rate that the internal temperature did not rise above 70 °C. After complete addition,

the dark solution was heated to 70 °C (bath temperature) for additional 60 min. The

80


mixture was poured onto crushed ice (ca. 350 g), the precipitate was collected on a

filter, washed with cold water, and dried in vacuo at 100 °C for several hours. The

crude product 7 was obtained as a light-yellow solid. The isolated material was used

for the following reaction without further purification.

Yield: 14.5 g. 1H NMR (200 MHz, DMSO−D6): δ 8.76 (dd, 3J = 7.6 Hz, 4J = 1.1 Hz, 2

H), 8.21 (t, 3J = 7.6 Hz, 2 H), 8.15 (dd, 3J = 7.6 Hz, 3J = 1.1 Hz, 2 H) ppm.

2,2’-bipyridine-6,6’dicarboxylic acid diethyl ester (8)

NN

EtOOEtOO

8

8 was prepared in analogy to the reported methyl ester of Blake et al. [103] 7 was

suspended in dry EtOH (300 mL), conc. H2SO4 (30 mL) was added cautiously, and

the mixture was refluxed for 20 h. At 0 °C, a saturated solution of Na2CO3 was added

dropwise until pH 7. The aqueous phase was extracted with CHCl3 (3 × 200 mL), the

combined organic phases were dried over MgSO4, and concentrated. The product 8

was obtained as a colorless solid.

Yield: 14.4 g (47.8 mmol, 73% over two steps). 1H NMR (200 MHz, CDCl3): δ 8.77 (d,3J = 7.8 Hz, 2 H), 8.15 (d, 3J = 7.8 Hz, 2 H), 7.98 (t, 3J = 7.8 Hz, 2H), 4.50 (q, 3J = 7.1

Hz, 4 H), 1.47 (t, 3J = 7.1 Hz, 6 H) ppm.

Deuterated compounds

[D4]-6,6’-(Bis(hydroxymethyl)-2,2’-bipyridine (9)

N N

HOOH

DD

DD

9

9 was prepared according to the literature procedure for the undeuterated compound

of Newkome et al. [99] Solid NaBD4 (357 mg, 8.52 mmol, 4.0 equiv) was added in

81


portions to a suspension of 8 (0.64 g, 2.13 mmol, 1.0 equiv) in CD3OD (5 ml). The

resulting slightly pink solution was stirred at room temperature for 20 h before D2O

(10 ml) was added and the pH adjusted to 6 through addition of 2 M DCl (in D2O).

Then, the mixture was extracted with CHCl3 (4× 25 ml), the combined organic phases

dried over MgSO4 and the solvent removed in vacuo. The remaining residue was dry-

loaded onto silica and subjected to column chromatography (SiO2, CH2Cl2/MeOH

9:1, detection UV) to yield 9 as a colorless solid.

Yield: 303 mg (1.38 mmol, 65%, overall deuteration level: 99%). Rf = 0.08 (SiO2,

CH2Cl2/MeOH 9:1, detection UV). MS (ESI+): m/z (%) 221.0 (26, [M+H]+), 242.9 (100,

[M+Na]+). 1H NMR (250 MHz, CD3OD): δ 8.22 (d, 3J = 7.8 Hz, 2H), 7.88 (t, 3J = 7.8

Hz, 2H), 7.51 (d, 3J = 7.7 Hz, 2H) ppm. 13C NMR (62.9 MHz, CD3OD): δ 161.9, 156.6,

138.9, 121.8, 120.9, 65.3 (quint, J = 22.0 Hz) ppm.

[D4]-6,6’-Bis(bromomethyl)-2,2’-bipyridine (10)

N N

BrBr

DD

DD

10

Under nitrogen and with cooling in an ice bath, PBr3 (0.60 ml, 1.72 g, 6.36 mmol,

5.0 equiv) was added dropwise via syringe to a solution of 9 (280 mg, 1.27 mmol,

1.0 equiv) in dry DMF (8 ml). The yellow-brown suspension was stirred at room

temperature for 20 h before the volatiles were removed under reduced pressure, and

H2O (30 ml) was added cautiously with ice cooling. The pH was adjusted to 6 with

sat. NaHCO3 and the aqueous phase was extracted with CHCl3 (4 × 50 ml). The

combined organic layers were dried over MgSO4 and the solvent removed in vacuo.

The light-yellow solid residue was dry-loaded onto silica and subjected to column

chromatography (SiO2, CH2Cl2/MeOH 100:1, detection UV) to yield 10 as a colorless

solid.


CH2Cl2/MeOH 100:1, detection UV) . MS (ESI+): m/z (%) 304.1 (83), 346.8 (100,

[M+H]+). 1H NMR (250 MHz, CD2Cl2): δ 8.39 (d, 3J = 7.9 Hz, 2H), 7.83 (t, 3J =

7.8 Hz, 2H), 7.47 (d, 3J = 7.7 Hz, 2H) ppm. 13C NMR (62.9 MHz, CDCl3): δ 156.3,

155.5, 138.0, 123.6, 120.3, 34.0 (quint, J = 23.2 Hz) ppm.

82


[D4]-6,6’-Bis(aminomethyl)-2,2’-bipyridine trihydrobromide hydrate (11)

NN

H2N

H2O

3 HBr

NH2

DD

DD

11


of Wang et al. [104] 10 (82.6 mg, 239 µmol, 1.0 equiv) was suspended in CHCl3 (3.1 mL)

and heated at reflux until dissolution. Then, a solution of hexamethylenetetramine

(72.4 mg, 516 µmol, 2.16 equiv) in CHCl3 (2 mL) was added dropwise. After refluxing

for 3 h, the reaction mixture was cooled to room temperature and stored at room

temperature overnight. The precipitate formed was collected on a Buchner funnel,

washed with a small amount of CHCl3 and dried in vacuo. Subsequently, the white

solid was suspended in H2O/EtOH/47% HBr) (0.7 ml, 2.9 ml, 0.50 ml) and stirred at

75 °C until dissolution. After storing the mixture at room temperature for 1 h and at

0 °C for 30 min, the precipitate was collected, washed with EtOH and dried in vacuo.

11 was obtained as a light-yellow solid.

Yield: 69.5 mg (145 µmol, 61%, overall deuteration level: 97%). MS (ESI+): m/z (%)

219.0 (48, [M+H]+), 282.1 (44), 311.0 (100). 1H NMR (250 MHz, D2O): δ 8.29 (d, 3J =

8.0 Hz, 2H), 8.00 (t, 3J = 8.0 Hz, 2H), 7.49 (d, 3J = 8.0 Hz, 2H) ppm.

[D12]-6,6’-Dimethyl-2,2’-bipyridine-N,N’-dioxide (12)

N NCD3D3C

D

DDDD

D

O O12

12 was prepared in analogy to the literature procedure of Cook et al., [91] using the

conditions of J. Wahsner. [105] A solution of NaOD (40 wt% in D2O, 3 ml, 44.4 mmol,

6.5 equiv) was added to a suspension of 4 (1.5 g, 6.8 mmol) in D2O (30 ml). The res-

ulting reaction mixture was heated in a stainless steel autoclave for 48 h (bath 150 °C)

to give a brown suspension, which was extracted with CH2Cl2 (3 × 180 ml). The

organic phase was dried over MgSO4, the solvent removed in vacuo and the obtained

yellow crude product was reacted again under the same conditions. After extraction

of the second reaction mixture with CH2Cl2 (3 × 180 ml), the white crude product

83


was purified by column chromatography (SiO2, CH2Cl2/MeOH 15:1) to afford 12 as

white solid.

Yield: 1.2 g (5.3 mmol, 78%, overall deuteration level: >99%). Rf = 0.21 (SiO2,

CH2Cl2/MeOH 9:1, detection UV). MS (ESI+): m/z (%) 228.97 (100, [M+H]+).

[D10]-6,6’-(Bis(acetoxymethyl)-2,2’-bipyridine (13)

N ND

DDDD

D

AcOOAc

DD

DD

13


of Newkome et al., [99] using the conditions of J. Wahsner. [105] Under nitrogen, 12 (1.2

g, 5.3 mmol) was dissolved in Ac2O (30 ml) and heated to 120 °C for 16 h. Then,

the solvent was removed in vacuo and the yellow crude product purified by column

chromatography (SiO2, CH2Cl2/MeOH 75:1 → 50:1) to obtain 13 as slightly yellow

powder.

Yield: 0.9 g (3.0 mmol, 57%, overall deuteration level: 98%). Rf = 0.46 (SiO2, CH2Cl2/MeOH 24:1, detection UV). MS (ESI+): m/z (%) 310.99 (100, [M+H]+), 332.96 (19,

[M+Na]+).

[D10]-6,6’-(Bis(hydroxymethyl)-2,2’-bipyridine (14)

N ND

DDDD

D

HOOH

DD

DD

14


of Newkome et al., [99] using the conditions of J. Wahsner. [105] Under nitrogen, anhyd-

rous K2CO3 (1.4 g, 138.2 mmol, 3.5 equiv) was added to a slightly beige suspension

of 13 (0.9 g, 3.0 mmol, 1.0 equiv) in dry EtOH (20 ml). The yellow reaction mixture

was stirred at room temperature for 1 h, then filtered through a paper filter and the

solid was washed with CH2Cl2/MeOH (9:1, 100 ml). After removal of the solvent,

the light-yellow crude product was dry-loaded onto silica and purified by column

chromatography (SiO2, CH2Cl2/MeOH 9:1) to afford 14 as colourless solid.

84



[M+Na]+).

[D10]-6,6’-Bis(bromomethyl)-2,2’-bipyridine (15)

N ND

DDDD

D

BrBr

DD

DD

15


of Rodriguez-Ubis et al., [89] using the conditions of J. Wahsner. [105] Under nitrogen,

PBr3 (2.3 g, 8.5 mmol, 5.0 equiv) was added carefully via syringe to a yellow solution

of 14 (0.4 g, 1.7 mmol, 1.0 equiv) in DMF (15 ml). The red reaction mixture was stirred

at room temperature for 1 d, while it turned first dark brown and then olive green.

After removal of the solvent in vacuo, H2O (50 ml) was added cautiously with cooling

in an ice bath and the pH was adjusted to 6 with aq. NaHCO3. The aqueous phase

was extracted with CHCl3 (4 × 100 ml), the organic layer dried over MgSO4 and the

solvent removed in vacuo to afford a slightly yellow crude product. Purification by

column chromatography (SiO2, CH2Cl2/MeOH 9:1) yielded the 15 as a white solid.


[M+Na]+).

[D10]-6,6’-Bis(aminomethyl)-2,2’-bipyridine trihydrobromide trihydrate (16)

NN

H2N

H2O

3 HBr

NH2

DD

D

D D

D

DD

DD

16


of Wang et al., [104] using the conditions of J. Wahsner. [105] 15 (100 mg, 0.284 mmol,

1.0 equiv) was suspended in CHCl3 (4 ml) and heated at reflux until dissolution.

Then, a solution of hexamethylenetetramine (85.6 mg, 0.611 mmol, 2.25 equiv) in

85


CHCl3 (2.5 ml) was added dropwise. After refluxing for 6 h, the reaction mixture was

cooled to room temperature and stored at 8 °C overnight. The precipitate formed was

collected on a Buchner funnel, washed with a small amount of CHCl3 and dried in

vacuo. Subsequently, the white solid was suspended in H2O/EtOH/47% HBr (1.0 ml,

4.1 ml, 0.68 ml) and stirred at 75 °C until dissolution. Upon cooling in an ice bath, 16

crystallised as slightly yellow needles, which were filtered off, washed with a small

amount of cold EtOH and dried in vacuo.

Yield: 100.1 mg (0.206 mmol, 73%, overall deuteration level: 98%). MS (ESI+): m/z

(%) 225.11 (100, [M+H]+).

86


6.4 Bipyridine ester building blocks


2-(Tributylstannyl)-6-methylpyridine (22)

NSnBu3

22

22 was prepared according to the literature procedure of Mathieu et al. [109] Under

nitrogen and at -78 °C, n-butyllithium (10.3 g, 100 ml, 160 mmol, 1.1 equiv) was ad-

ded dropwise to a slightly yellow solution of 2-bromo-picoline (2) (25.1 g, 146 mmol,

1.0 equiv) in dry THF (350 ml). The obtained red solution was stirred for another

90 min at -78 °C. Then, freshly distilled tributyltinchloride (61.5 g, 52 ml, 175 mmol,

1.2 equiv) was added dropwise and the brown reaction mixture was allowed to warm

to room temperature and stirred overnight. After addition of water (100 ml), the

phases were separated and the aqueous layer extracted with Et2O (3 × 150 ml). The

combined organic phases were dried over MgSO4 and the solvent removed in vacuo.

The reddish brown oil was subjected to vacuum destillation (p ~8·10-2 mbar) to separ-

ate the slightly yellow byproducts (bp: 70 °C and 90 °C) from 22, which was isolated

as colourless oil (bp: 120 °C).

Yield: 33.1 g (87 mmol, 60%). 1H NMR (200 MHz, CDCl3): δ 7.36 (t, 3J = 7.4 Hz, 1H),

7.19 (d, 3J = 7.4 Hz, 1H), 6.96 (dd, 3J = 7.7 Hz, 4J = 0.9 Hz, 1H), 2.56 (s, 3H), 1.72–1.46

(m, 6H), 1.44–1.25 (m, 6H), 1.23–0.97 (m, 6H), 0.90 (t, 3J = 7.2 Hz, 9H) ppm.

6,6’-Dimethyl-2,2’-bipyridine-4-carboxylic acid methyl ester (24)

N

OMe

N

O

24

24 was prepared according to the literature procedure of Mathieu et al. [109] Under ni-

trogen, PdCl2(PPh3)2 (1.2 g, 1.7 mmol, 0.05 equiv), PPh3 (0.9 g, 3.3 mmol, 0.1 equiv),

2-methyl-6-(toluene-4-sulfonyloxy)isonicotinic acid methyl ester (10.0 g, 33.3 mmol,

1.0 equiv), and 22 (12.8 g, 33.4 mmol, 1.0 equiv) were suspended in dry xylene

(250 ml). The yellow reaction mixture was degassed by four freeze-pump-thaws cycles

87


and then heated at reflux for 39 h. The obtained dark brown mixture was allowed to

warm to room temperature and poured into saturated EDTA solution (250 ml). The

aqueous layer was extracted with CH2Cl2 (3 × 500 ml), the combined organic extracts

dried over MgSO4 and the solvent removed in vacuo. The resulting deep red oil was

subjected to column chromatography (SiO2, CH2Cl2/MeOH 75:1 → 50:1) to give the

crude product as dark orange solid. Second purification by column chromatography

(SiO2, CH2Cl2/MeOH 100:1) afforded 24 as a slightly yellow solid.

Yield: 4.8 g (19.6 mmol, 59%). Rf = 0.38 (SiO2, CH2Cl2/MeOH 24:1, detection UV) 1H

NMR (200 MHz, CDCl3): δ 7.37 (d, 4J = 0.7 Hz, 1H), 8.20 (d, 3J = 7.8 Hz, 1H), 7.75–7.62

(m, 2H), 7.17 (d, 3J = 7.3 Hz, 1H), 3.97 (d, 4J = 1.3 Hz, 3H), 2.68 (s, 3H), 2.64 (s, 3H)

ppm.

6,6’-Dimethyl-2,2’-bipyridine-4-carboxylic acid methyl ester-N,N’-dioxide (25)

NN

OOMe

O O25

25 was prepared in analogy to the procedure for compound 4. With cooling in an ice

bath, a solution of m-chloroperoxybenzoic acid (1.8 g of 77 wt%, 1.4 pure, 8.1 mmol,

2.0 equiv) in CH2Cl2 (80 ml) was added dropwise to a solution of 24 (1.0 g, 4.1 mmol,

1.0 equiv) in CH2Cl2 (80 ml) over the course of 1.5 h. The pale yellow solution was

allowed to slowly warm to room temperature and stirred for 48 h. After extraction

of the reaction mixture with saturated NaHCO3 (20 ml) and aqueous Na2S2O3 (2 g in

75 ml, 3 × 20 ml), the organic layer was dried over MgSO4 and the solvent removed

in vacuo. The crude product was dry-loaded onto silica and purified by column chro-

matography (SiO2, CH2Cl2/MeOH 15:1) to afford 25 as a colourless solid.


MS (ESI+): m/z (%) 274.97 (6, [M+H]+), 296.89 (100, [M+Na]+), 312.83 (5, [M+K]+).1H NMR (200 MHz, CDCl3): δ 7.96 (d, 4J = 0.8 Hz, 2H), 7.44–7.27 (m, 3H), 3.91 (s,

3H), 2.59 (s, 3H), 2.58 (s, 3H) ppm. 13C NMR (50 MHz, CDCl3): δ 164.36, 150.2, 150.1,

143.8, 143.3, 127.3, 126.9, 125.9, 125.7, 125.1, 124.9, 52.8, 18.0 ppm. IR (FT-ATR): ν̃ 3073

(w, C-Harom), 2956 (w, CH3), 2917 (w, CH3), 1720 (s, C=O), 1629 (w, C=Carom), 1554 (w,

C=Carom), 1425 (m), 1327 (m), 1258 (s, N-oxide), 1236 (s), 936 (m), 899 (m), 848 (m), 764

(s) cm-1.

88


6,6’-bis(bromomethyl)-2,2’-bipyridine-4-carboxylic acid methyl ester (26)

N N

Br

OOMe

Br26

26 was prepared in analogy to the procedure of Psychogios et al. [100] Under nitro-

gen, TFA anhydride (3 ml) was added quickly via syringe to a yellow solution of 25

(145.4 mg, 0.530 mmol, 1.0 equiv) in dry CH2Cl2 (3 ml). The yellow reaction mixture

was heated at reflux for 1.5 h before the volatiles were removed in vacuo. The obtained

orange residue was dissolved in a mixture of dry DMF/THF (1:1, 6 ml) and, upon ice

cooling, added to solid LiBr (460.3 mg, 5.300 mmol, 10.0 equiv), which was dried

beforehand in vacuo at 180 °C for 3h. After stirring the reaction mixture at room tem-

perature overnight, the solvents were removed in vacuo. The bright orange residue

was dissolved in CH2Cl2 (5 ml), washed with water (3 × 5 ml) and the combined

organic layers were dried over MgSO4 before evaporation to dryness. The isolated

crude product was dry-loaded onto silica and purified by column chromatography

(SiO2, CH2Cl2/MeOH 100:1) to give the product 26 as a yellow solid.

Yield: 112.8 mg (0.282 mmol, 53%). Rf = 0.64 (SiO2, CH2Cl2/MeOH 100:1, detec-

tion UV). MS (ESI+): m/z (%) 258.99 (42, [reactant-O+H]+), 336.88 (46, [M-Br-O+H]+),

400.74 (100, [M+H]+), 422.69 (100, [M+K]+). 1H NMR (200 MHz, CDCl3): δ 8.92 (d, 4J

= 1.4 Hz, 1H), 8.42 (dd, 3J = 7.9 Hz, 4J = 1.0 Hz, 1H), 8.04 (d, 4J = 1.4 Hz, 1H), 7.90

(t, 3J = 7.8 Hz, 1H), 7.56 (dd, 3J = 7.7 Hz, 4J = 0.9 Hz, 1H), 4.73 (s, 2H), 4.69 (s, 2H),

4.02 (s, 4H) ppm. 13C NMR (50 MHz, CDCl3): δ 165.4, 157.6, 156.6, 156.4, 154.4, 139.9,

138.5, 124.4, 123.1, 121.0, 120.2, 53.0, 33.6, 33.4 ppm. IR (FT-ATR): ν̃ 3077 (w, C-Harom),

2950 (w, CH3), 2848 (w, CH3), 1726 (s, C=O), 1584 (w, C=Carom), 1566 (m, C=Carom),

1425 (m), 1405 (m), 1227 (s), 1202 (s), 991 (m), 912 (w), 826 (m), 771 (s), 652 (s) cm-1.

6,6’-bis(bromomethyl)-2,2’-bipyridine-4-carboxylic acid methyl ester-N,N-dioxide (27)

N N

Br

OOMe

BrO O

27

89


27 was prepared in analogy to the procedure of Sieser et al. [143] Under nitrogen and

with cooling in an ice bath, urea/H2O2 adduct (2.1 g, 22.5 mmol, 3 equiv) was added

in portions to a suspension of 26 (3.0 g, 7.5 mmol, 1.0 equiv). Then, TFA anhydride

was added dropwise over the course of 15 min, the mixture was allowed to warm to

room temperature and stirred for 2 d. After dilution with CH2Cl2 (150 ml), a solution

of saturated Na2S2O3 was added and the mixture stirred for 1 h. The phases are

separated, the aqueous layer washed with CH2Cl2 (3 × 200 ml), the combined phases

dried over MgSO4 and evaporated to dryness. The isolated crude product was then

dry-loaded onto silica and purified by column chromatography (SiO2, CH2Cl2/MeOH

50:1) to give 27 as a colourless solid.

Yield: 1.0 g (2.3 mmol, 31%). Rf = (SiO2, CH2Cl2/MeOH 50:1, detection UV+I2 va-

pour). 1H NMR (200 MHz, CDCl3): δ 8.23 (d, 4J = 2.5 Hz, 1H), 8.14 (d, 4J = 2.4 Hz,

1H), 7.67 (dd, 3J = 7.5 Hz, 4J = 1.9 Hz, 1H), 7.59–7.50 (m, 1H), 7.34 (t, J = 7.9 Hz, 1H),

4.73 (s, 2H), 4.70 (s, 2H), 3.96 (s, 3H) ppm.


[D11]-6,6’-Dimethyl-2,2’-bipyridine-4-carboxylic acid methyl ester-N,N’-dioxide (33)

N NCD3D3C

D

DDD

D

OOMe

O O33

Deuteration was performed in analogy to the procedure for compound 12 and esterifi-

cation was performed in analogy to the literature procedure for the undeuterated

dicarboxylic acid. [144] A solution of NaOD (40 wt% in D2O, 1.5 ml, 22.2 mmol, 7.5

equiv) was added to a suspension of 25 (0.8 g, 2.9 mmol, 1.0 equiv) in D2O (15 ml).

The resulting reaction mixture was heated in a stainless steel autoclave for 68 h (bath

150 °C) to give a brown suspension, which was neutralised with DCl (7.6 M in D2O)

and rotavapped to dryness to leave a beige solid behind. Reaction (48 h) and workup

were repeated to yield the carboxylic acid derivative as a beige solid. The latter was

suspended in MeOH (15 ml) and conc. H2SO4 (2 ml) and heated at reflux for 24 h.

After cooling to room temperature, the brownish yellow residue was dissolved in wa-

ter (20 ml) and neutralised cautiously with 10% NaOH. After extraction with CH2Cl2(3 × 100 ml), the organic layer was dried over MgSO4 and the crude product dry-

loaded onto silica. Purification by column chromatography afforded 33 as colourless

90


solid.

Yield: 0.5 g (1.8 mmol, 62%, overall deuteration level: 98%). MS (ESI+): m/z (%) 286.25

(36, [M+H]+), 308.11 (100, [M+Na]+).

[D9]-6,6’-(Bis(acetoxymethyl)-2,2’-bipyridine-4-carboxylic acid methyl ester (34)

N ND

DDD

D

AcOOAc

DD

OOMe

DD

34

34 was prepared in analogy to the procedure for compound 13. Under nitrogen, 33

(0.5 g, 1.8 mmol) was dissolved in Ac2O (25 ml) and heated at reflux for 16 h. Then,

the solvent was removed in vacuo and the yellow crude product was purified twice

by column chromatography (SiO2, CH2Cl2/MeOH 50:1 and CH2Cl2/MeOH 100:1) to

obtain 34 as slightly yellow solid.

Yield: 0.4 g (1.2 mmol, 67%, overall deuteration level: 98%). Rf = 0.79 (SiO2, CH2Cl2/MeOH 9:1, detection UV). MS (ESI+): m/z (%) 390.04 (100, [M+Na]+).

[D9]-6,6’-bis(bromomethyl)-2,2’-bipyridine-4-carboxylic acid methyl ester (32)

N ND

DDD

D

BrBr

DD

DD

OOMe

32

32 was prepared analogous to compound 5. Under nitrogen, TFA anhydride (10 ml)

was added quickly via syringe to a yellow solution of 33 (1.0 g, 3.5 mmol, 1.0 equiv)

in dry CH2Cl2 (10 ml). The orange reaction mixture was heated at reflux for 1.5 h

before the volatiles were removed in vacuo. The obtained residue was dissolved in

a mixture of dry DMF/THF (1:1, 10 ml) and then added upon ice cooling to solid

LiBr (3.0 g, 35.0 mmol, 10.0 equiv), which was dried beforehand in vacuo at 180 °C for

1 h. After stirring the reaction mixture at room temperature overnight, the solvents

were removed in vacuo. The bright orange residue was dissolved in CH2Cl2 (20 ml),

washed with water (5 × 20 ml) and the combined organic layers were dried over

91


MgSO4 before evaporation to dryness. The isolated crude product was dry-loaded

onto silica and purified by column chromatography (SiO2, CH2Cl2/MeOH 100:1) to

give 32 as a yellow solid.

Yield: 644 mg (1.57 mmol, 45%, overall deuteration level: >99%). MS (ESI+) m/z (%)

409.78 [M+H]+, 431.74 [M+Na]+. 1H NMR (400 MHz, CDCl3): δ 4.00 (s, 3H) ppm.13C NMR (101 MHz, CDCl3): δ 165.4, 157.3, 156.8, 156.5, 154.6, 139.6, 137.6 (t, J = 24.7

Hz), 123.7 (t, J = 24.2 Hz), 122.5 (t, J = 25.8 Hz), 120.3 (t, J = 25.8 Hz), 119.7 (t, J = 25.6

Hz), 52.9, 34.0–32.6 (m) ppm.

92


6.5 Nitro and amino bipyridine building blocks

6,6’-Dimethyl-2,2’-bipyridine-N-oxide (37)

NNO

37

37 was prepared according to the literature procedure of Newkome et al. [99] With

cooling in an ice bath, a solution of m-chloroperoxybenzoic acid (1.3 g of 77 wt%, 1.0

g pure, 5.6 mmol, 1.0 equiv) in CHCl3 (200 ml) was added dropwise to a solution of 3

(1.0 g, 5.4 mmol, 1.0 equiv) in CHCl3 (200 ml) over the course of 3 h. The reaction mix-

ture was allowed to slowly warm to room temperature and stirred overnight. Then,

the reaction mixture was washed with saturated NaHCO3 (20 ml), dried over MgSO4

and evaporated to dryness. The yellow crude product was subjected to column chro-

matography (SiO2, CH2Cl2/MeOH 100:1 → 9:1) to separate 37 as a white solid from

the reactant (Rf = 0.50) and the N,N’-dioxide byproduct (Rf = 0.24).

Yield: 0.88 g (4.39 mmol, 81%). Rf = 0.42 (SiO2, CH2Cl2/MeOH 9:1, detection UV).1H NMR (200 MHz, CDCl3): δ 8.57 (d, 3J = 7.9 Hz, 1H), 8.02–7.94 (m, 1H), 7.69 (t, 3J =

7.8 Hz, 1H), 7.31–7.27 (m, 1H), 7.25–7.15 m (m, 2H), 2.62 (s, 3H), 2.56 (s, 3H) ppm.

4-Nitro-6,6’-dimethyl-2,2’-bipyridine-N-oxide (38)

NN

NO2

O O38

38 was prepared according to the literature procedure of Mukkala et al. [113] A solution

of 37 (849 mg, 4.24 mmol) in H2SO4/HNO3 (4:3, 14 ml) was heated to 100 °C for 4 h.

After cooling to room temperature, the reaction mixture was poured into crushed ice

(~ 40 g) and the pH was adjusted to 6 with aqueous NaOH. The resulting precipitate

was collected on a Buchner funnel, washed with cold water and dried in vacuo to give

38 as white solid.

Yield: 629 mg (2.57 mmol, 61%). MS (ESI+): m/z (%) 246.14 (10, [M+H]+), 267.96 (100,

[M+Na]+). 1H NMR (200 MHz, CDCl3): δ 8.96 (d, 3J = 3.3 Hz, 1H), 8.59 (d, 3J = 8.0

Hz, 1H), 8.10 (d, 3J = 3.3 Hz, 1H), 7.74 (t, 3J = 7.8 Hz, 1H), 7.32–7.27 (m, 1H), 2.65 (s,

3H), 2.63 (s, 3H) ppm. 13C NMR (50 MHz, CHCl3): δ 158.9, 152.2, 151.8, 148.6, 147.8,

93


136.8, 124.9, 122.3, 120.2, 118.8, 24.7, 18.9 ppm. IR (FT-ATR): ν̃ 3043 (w, C-Harom), 1606

(w, C=Carom), 1524 (s, N=O), 1497 (w, C=Carom), 1337 (s, N=O), 1282 (s, N-oxide), 1101

(m), 848 (m), 781 (s), 738 (s) cm-1.

4-Nitro-6,6’-dimethyl-2,2’-bipyridine-N,N-dioxide (41)

NN

NO2

O O41

41 was prepared according to the procedure developed by N. Alzakhem. [112] With

cooling in an ice bath, a solution of m-chloroperoxybenzoic acid (4.4 g of 77 wt%,

3.4 g pure, 19.6 mmol, 1.5 equiv) in CHCl3 (100 ml) was added dropwise to a solution

of 38 (3.2 g, 13.1 mmol, 1.0 equiv) in CHCl3 (100 ml) over the course of 1 h. The

reaction mixture was allowed to warm to room temperature and stirred overnight.

Then, the reaction mixture was washed with saturated NaHCO3 (10 ml) and saturated

Na2S2O3 (10 ml). The combined aqueous layers were extracted with CHCl3 (3 ×20 ml). The combined organic phases were dried over MgSO4 and the solvent was

removed in vacuo. The crude product was purified twice by column chromatography

(SiO2, CH2Cl2/MeOH 24:1 → 9:1 and CH2Cl2/MeOH 24:1 → 9:1) to give pure 41 as

a yellow solid.


MS (ESI+): m/z (%) 261.96 (2, [M+H]+), 283.87 (100, [M+Na]+), 299.80 (2, [M+K]+).1H NMR (200 MHz, CDCl3): δ 8.27–8.17 (m, 2H), 7.49–7.27 (m, 3H), 2.62 (s, 3H), 2.59

(s, 3H) ppm. 13C NMR (50 MHz, CDCl3): δ 151.3, 150.3, 144.6, 142.1, 140.6, 127.8,

125.6, 124.9, 120.6, 120.2, 18.4, 18.0 ppm. IR (FT-ATR): ν̃ 3043 (w, C-Harom), 1606 (w,

C=Carom), 1524 (s, N=O), 1497 (w, C=Carom), 1337 (s, N=O), 1280 (s, N-oxide), 1101 (m),

849 (m), 781 (s), 746 (s) cm-1.

4-Nitro-6,6’-bis(bromomethyl)-2,2’-bipyridine (42)

NN

BrBr

NO2

42

94


42 was prepared in analogy to the procedure for compound 5. Under nitrogen, TFA

anhydride (17 ml) was added quickly via syringe to a yellow solution of 41 (1.0 g,

3.9 mmol, 1.0 equiv) in dry CH2Cl2 (17 ml). The orange reaction mixture was heated

at reflux for 2 h before the solvents were removed in vacuo. The orange residue was

dissolved in a mixture of dry DMF/THF (1:1, 26 ml) and the obtained solution was ad-

ded to solid LiBr (3.3 g, 38.2 mmol, 10.0 equiv), which was dried beforehand in vacuo

at 180 °C for 4 h. The reaction mixture was stirred at room temperature overnight be-

fore the solvents were removed in vacuo at 60 °C. The yellow residue was suspended

in CH2Cl2 (70 ml) and washed with water (3 × 70 ml). The combined organic layers

were dried over MgSO4 and evaporated to dryness. The crude product was dry-

loaded onto silica and purified by column chromatography (SiO2, CH2Cl2/MeOH

100:1) to give 42 as a bright yellow solid.

Yield: 840 mg (2.17 mmol, 56%). Rf = 0.48 (SiO2, CH2Cl2/MeOH 9:1, detection UV).

MS (ESI+): m/z (%) 360.17 (100), 374.11 (15), 392.09 (6), 408.05 (5, [M+Na]+). 1H NMR

(200 MHz, CDCl3): δ 9.10 (d, 4J = 2.0 Hz, 1H), 8.43 (dd, 3J = 7.9 Hz, 4J = 1.0 Hz, 1H),

8.17 (d, 4J = 2.0 Hz, 1H), 7.89 (t, 3J = 7.8 Hz, 1H), 7.57 (dd, 3J = 7.8 Hz, 4J = 1.0 Hz,

1H), 4.71 (s, 2H), 4.66 (s, 2H) ppm. 13C NMR (50 MHz, CDCl3): δ 159.3, 158.7, 157.0,

156.0, 153.2, 138.4, 125.0, 120.9, 116.0, 113.4, 33.6, 32.6 ppm. IR (FT-ATR): ν̃ 3085 (w,

C-Harom), 1607 (w, C=Carom), 1571 (m, C=Carom), 1530 (s, N=O), 1407 (m), 1353 (s, N=O),

1263 (m), 1217 (m), 1077 (m), 819 (m), 741 (s), 639 (s, C-Br) cm-1.

4-Amino-6,6’-dimethyl-2,2’-bipyridine (39)

NN

NH2

39

39 was prepared in analogy to the literature procedure for 6-amino-2,2’-bipyridine. [114]

Under nitrogen and with cooling in an ice bath, Pd/C (120 mg) was added to a solu-

tion of 38 (517 mg, 2.11 mmol, 1.0 equiv) in dry MeOH (12 ml). Sodium borohydride

(250 mg, 6.09 mmol, 3.0 equiv) was added as a solid in small portions, which resul-

ted in gas evolution and complete dissolution of the starting material. After stirring

the reaction mixture at room temperature overnight, the catalyst was removed by fil-

tration and the solvent evaporated to dryness. The remaining yellow residue was

dissolved in water (30 ml) and extracted with EtOAc (5 × 50 ml). The combined or-

ganic layers were dried over MgSO4 and the solvent was removed in vacuo to obtain

95


39 as a sticky yellow solid.

Yield: 286 mg (1.44 mmol, 68%). 1H NMR (250 MHz, CDCl3): δ 8.10 (d, 3J = 7.8 Hz,

1H), 7.62 (t, 3J = 7.7 Hz, 1H), 7.45 (d, 3J = 2.1 Hz, 1H), 7.10 (d, 3J = 7.6 Hz, 1H), 6.36

(d, 3J = 2.1 Hz, 1H), 4.22 (s, 2H), 2.59 (s, 3H), 2.45 (s, 3H) ppm.

4-Acetylamino-6,6’-dimethyl-2,2’-bipyridine (40)

NN

NHAc

40

40 was prepared analogy to the literature procedure for 6-(acetyl)amino-2,2’-bipyridine

of Hapke et al. [117] Under nitrogen, Me2bpyNH2 (39) (43.6 mg, 0.219 mmol, 1.0 equiv)

was dissolved in dry pyridine (5 ml), which was freshly distilled over KOH before-

hand. Acetyl chloride (41.2 mg, 36 µl, 0.526 mmol, 2.4 equiv) was added dropwise

and the yellow solution was stirred at room temperature overnight. The reaction was

quenched by cautious addition of water (5 ml) and the mixture was extracted with

CH2Cl2 (2 × 20 ml). The combined organic phases were dried over MgSO4 and the

solvent was removed in vacuo. The crude product was dry-loaded onto silica and

purified by column chromatography (SiO2, CH2Cl2/MeOH 30:1) to give 40 as yellow

sticky solid.

Yield: 19.4 mg (0.080 mmol, 37%). Rf = 0.46 (SiO2, CH2Cl2/MeOH 9:1, detection UV).1H NMR (400 MHz, CDCl3): δ 8.15 (d, 3J = 7.8 Hz, 1H), 7.99 (d, 4J = 1.9 Hz, 1H), 7.96

(br s, 1H), 7.80 (s, 1H), 7.67 (t, 3J = 7.7 Hz, 1H), 7.14 (d, 3J = 7.6 Hz, 1H), 2.58 (s, 3H),

2.57 (s, 3H), 2.11 (s, 3H) ppm. 13C NMR (50 MHz, CDCl3): δ 169.2, 159.8, 157.9, 156.9,

155.7, 154.1, 146.2, 137.3, 123.5, 118.5, 113.0, 108.3, 25.0, 24.8, 24.7 ppm.

96


6.6 Simple macrocycles


(bpy.bpy) (6)

NNH

N

NHN

N

6

6 was prepared according to the literature procedure of Bottino et al. [101] Under ni-

trogen, a solution of 5 (11.5 g, 33.6 mmol, 1.0 equiv) in EtOH (1.5 l) was added to a

suspension of tosylamide monosodium salt [101] (13.0 g, 67.2 mmol, 2.0 equiv). While

the reaction mixture was heated at reflux for 36 h (bath 98 °C), the tosylated aza mac-

rocycle precipitated. After cooling the mixture to 0 °C for 1 h, the white solid was

collected on a Buchner funnel and dried in vacuo (crude product: 5.8 g). The latter

was dissolved in H2SO4 (80 ml) and heated to 110 °C for 3 h. After cooling to room

temperature and additional cooling with an ice bath, water (10 ml) was added cau-

tiously. The clear brown solution was treated with 50% NaOH solution until the pH

was above 13 and a beige clowdy suspension has formed, which was extracted with

DCM (3 × 300 ml). The combined organic layers were dried over MgSO4 and the

solvent removed in vacuo to yield 6 as a white solid.

Yield: 3.8 g (9.5 mmol, 57%). 1H NMR (200 MHz, CDCl3): δ 7.70 (d, 3J = 7.7 Hz, 4H),

7.39 (t, 3J = 7.7 Hz, 4H), 6.91 (d, 3J = 7.5 Hz, 4H), 4.07 (s, 8H) ppm. 13C NMR (50 MHz,

CDCl3): δ 159.2, 156.1, 136.5, 122.4, 119.3, 56.3 ppm. MS (ESI+): m/z (%) 395.15 (53,

[M+H]+), 417.07 (100, [M+Na]+).

97

6.6 Simple macrocycles


[D20]-(bpy.bpy) (31)

N

NHN

N

N

NH

DD

DD

DD

D D

D D

D D

D D

DD

DD

DD

31

31 was prepared in analogy to the literature procedure for the undeuterated com-

pound of Bottino et al., [101] using the conditions of J. Wahsner. [119] Under nitrogen, a

solution of 15 (90 mg, 0.26 mmol, 1.0 equiv) in EtOH (5 ml) was added to a suspension

of tosylamide monosodium salt [101] (99 mg, 0.51 mmol, 2.0 equiv). While the reaction

mixture was heated at reflux for 24 h (bath 98 °C), the tosylated aza macrocycle pre-

cipitated. The white solid was collected on a Buchner funnel and dried in vacuo (crude

product: 51 mg). The latter was dissolved in D2SO4 (98 wt% in D2O, 99.5%D, 5 ml)

and heated to 110 °C for 2 h. After cooling to room temperature and additional cool-

ing with an ice bath, water (1 ml) was added cautiously and the mixture neutralised

with an aqueous solution of NaOH (excess). Then, the solution was extracted with

CH2Cl2 (3 × 30 ml), the organic layer dried over MgSO4 and the solvent removed in

vacuo to yield 31 as a white solid.

Yield: 43 mg (0.10 mmol, 77%, overall deuteration level: 98%). MS (ESI+): m/z (%)

415.14 (100, [M+H]+), 437.10 (71, [M+Na]+).

98


6.7 Nitro and amino functionalised macrocycles

(bpy.bpy)NO2 (49)

NNH

N

NHN

N

NO2

49

49 was prepared according to the procedure of N. Alzakhem. [112] Under nitrogen,

42 (118 mg, 0.31 mmol, 1.0 equiv) was dissolved in DMF (8 ml). Upon addition of

K2CO3 (635 mg, 4.59 mmol, 15.0 equiv) the yellow solution turned dark orange. After

addition of 47 (160 mg, 0.31 mmol, 1.0 equiv) as a solid, the cloudy pink mixture

was stirred at 50 °C overnight and for another 2 d at room temperature, whereas a

beige solid precipitated from a brown solution. Then, water (20 ml) was added, the

precipitate was collected on a Buchner funnel and shortly dried on the filter. Sub-

sequently, the crude product was treated with H2SO4 (4 ml) and the brown solution

heated at 110 °C. After 3 h, the reaction mixture was poured onto crushed ice (10 g)

and, upon cooling, treated with 50% NaOH until the pH was above 13. The obtained

beige clowdy suspension was extracted with CHCl3 (3 × 100 ml). The combined or-

ganic layers were dried over MgSO4 and the solvent removed in vacuo to yield 49 as

an off-white solid.

Yield: 70 mg (0.16 mmol, 52%). 1H NMR (200 MHz, CDCl3): δ 7.79–7.65 (m, 3H),

7.50–7.28 (m, 4H), 7.26–7.19 (m, 1H), 6.96–6.86 (m, 2H), 6.76 (d, 3J = 7.8 Hz, 1H), 4.18

(s, 2H), 4.10 (s, 4H), 4.06 (s, 2H) ppm. MS (ESI+): m/z (%) 395.17 (7, [M-NO2+H]+),

417.14 (22, [M-NO2+Na]+, 440.11 (68, [M+H]+), 462.04 (100, [M+Na]+).

99

6.7 Nitro and amino functionalised macrocycles

(bpy.bpy)NH2 (50)

NNH

N

NHN

N

NH2

50

Under nitrogen, Pd (10%)/C (20 mg) was added to a solution of 49 (49.7 mg, 0.113 mmol,

1.0 equiv) in dry EtOH (50 ml). Then, hydrazine hydrate (55 µl, 57 mg, 1.14 mmol,

10.0 equiv) in EtOH (10 ml) was added and the reaction mixture heated at reflux

overnight. After cooling to room temperature, the dark suspension was filtered

through a paper filter and the solvent removed in vacuo. The isolated crude product

was reacted again using the same conditions, amounts of reagents and solvent. After

cooling to room temperature, the reaction mixture was filtered again through a paper

filter and after solvent removal, 50 was isolated a yellowish brown solid.

Yield: 39.5 mg (0.096 mmol, 85%). 1H NMR (250 MHz, CD3OD): δ 7.92–7.67 (m, 6H),

7.35–7.18 (m, 3H), 7.09–7.03 (m, 1H), 6.52–6.47 (m, 1H), 4.10–3.99 (m, 6H), 3.91–3.84

(m, 2H) ppm. MS (ESI+): m/z (%) 410.04 (100, [M+H]+), 431.96 (40, [M+Na]+).

100


6.8 Simple deuterated cryptates

General procedure for the synthesis of the sodium cryptates: Under nitrogen, the

building blocks and Na2CO3 (10-14 equiv) were suspended in HPLC grade CH3CN.

The mixture was heated at reflux for 23-45 h, filtered cold, and the solvent removed

in vacuo. The residue was subjected to column chromatography to yield the desired

cryptate as a colorless solid.

[D4]-[Na(bpy.bpy.bpy)]Br (17)

N

N

N

N

N

N

N

N

DD

DD

Na Br

17

6 (45.0 mg, 114 µmol, 1.0 equiv), 10 (41.0 mg, 114 µmol, 1.0 equiv), Na2CO3 (121 mg,

1.14 mmol, 10 equiv), CH3CN (80 ml). Column chromatography (SiO2, CH2Cl2/

MeOH 15:1→ 9:1).

Yield: 31 mg (45 µmol 40%, overall deuteration level: 99%). Rf = 0.19 (SiO2, CH2Cl2/

MeOH 9:1, detection UV+I2 vapour). MS (ESI+): m/z (%) 601.1 (10, [M+H]+). 1H

NMR (250 MHz, CDCl3): δ 7.99–7.70 (m, 12H), 7.44–7.21 (m, 6H), 3.84 (s, 8H) ppm.13C NMR (62.9 MHz, CDCl3): δ 158.6, 155.3, 138.2, 124.1, 120.4, 58.8 (t, J = 20.5 Hz)

ppm.


N

N

N

N

N

N

N

N

D D

DD

DD

D D

Na Br

19

18 (52.0 mg, 109 µmol, 1.0 equiv), 10 (75.8 mg, 218 µmol, 2.0 equiv), CH3CN (250 ml),

Na2CO3 (162 mg, 1.53 mmol, 14 equiv). Column chromatography (SiO2, CH2Cl2/

101

6.8 Simple deuterated cryptates

MeOH 9:1).

Yield: 38 mg (55 µmol, 51%, overall deuteration level: > 99%). MS (ESI+): m/z (%) =

607.1 (100, [M+H]+). 1H NMR (250 MHz, CDCl3): δ 8.04–7.68 (m, 12H), 7.52–7.15 (m,

6H), 3.84 (s, 4H) ppm. 13C NMR (62.9 MHz, CDCl3): δ 158.5, 155.3, 138.2, 124.1, 120.4,

58.8 (t, J = 20.5 Hz)ppm.


N

N

N

N

N

N

N

N

D D

DDDD

DDDD

D D

Na Br

20

11 (62.0 mg, 129 µmol, 1.0 equiv), 10 (89.6 mg, 259 µmol, 2.0 equiv), CH3CN (120 ml),

Na2CO3 (191 mg, 1.81 mmol, 14 equiv). Column chromatography (SiO2, CH2Cl2/

MeOH 15:1→ 9:1).

Yield: 52 mg (75 µmol, 58%, overall deuteration level: > 99%). MS (ESI+): m/z (%) =

609.2 (100, [M]+). 1H NMR (250 MHz, CDCl3): δ 7.95–7.72 (m, 12 H), 7.39–7.25 (m, 6

H) ppm. 13C NMR (62.9 MHz, CDCl3): δ 158.5, 155.3, 138.2, 124.1, 120.4, 58.8 (t, J =

20.5 Hz) ppm.


N

N

N

N

N

N

N

N

DD

DD

DD D D

DDDD D

D

DD

DDDD

DD

DD

D

D

DD

D D

Na Br

21

21 was prepared according to the procedure of J. Wahsner. [105] 15 (108 mg, 0.29 mol,

2.0 equiv), 16 (70 mg, 0.14 mmol, 1.0 equiv), Na2CO3 (215 mg, 2.03 mmol, 14 equiv).

Column chromatography (SiO2, CH2Cl2/MeOH 24:1→ 15:1).

102



CH2Cl2/MeOH 15:1, detection UV+I2 vapour). MS (ESI+): m/z (%) 627.25 (100, [M-

Br]+).

103

6.9 Carboxylic acid ester functionalised crypates

6.9 Carboxylic acid ester functionalised crypates


[Na(bpy.bpy.bpyCOOMe)]Br (28)

N

N

N

N

N

N

N

NNa Br

COOMe

28

26 (66.0 mg, 165.0 µmol, 1.0 equiv), 6 (64.3 mg, 163.0 µmol, 1.0 equiv), NaCO3

(173.1 mg, 1.633 mmol, 10.0 equiv), CH3CN (200 ml). Column chromatography (SiO2,

CH2Cl2/ MeOH 15:1→ 9:1).

Yield: 42.7 mg (58.0 µmol, 36%). 1H NMR (200 MHz, CDCl3): δ 8.39 (s, 1H), 8.02–7.73

(m, 11H), 7.51–7.30 (m, 5H), 4.00 (s, 3H), 3.95 (s, 2H), 3.89 (s, 7H) ppm.

[Na(bpy.bpy.bpyCOOMeO2

)]Br (29)

N

N

N

N

N

N

N

NNa Br

COOMe

OO

29

27 (1.0 g, 2.3 mmol, 1.0 equiv), 6 (0.9 g, 2.3 mmol, 1.0 equiv), NaCO3 (2.5 g, 23.1 mmol,

10.0 equiv), CH3CN (3 l). The reactants and solvent were divided into four identical

batches. Column chromatography (SiO2, CH2Cl2/MeOH 15:1→ 9:1).

Yield: 688 mg (0.90 mmol, 39%). Rf = 0.45 (SiO2, CH2Cl2/MeOH 9:1, detection UV+I2

vapour). MS (ESI+): m/z (%) 687.17 (5, [M-Br]+). 1H NMR (250 MHz, CD2Cl2/CD3OD):

δ 8.32 (d, 4J = 2.5 Hz, 1H), 8.25 (d, 4J = 2.5 Hz, 1H), 7.95–7.87 (m, 8H), 7.85–7.76 (m,

2H), 7.66–7.56 (m, 1H), 7.51–7.41 (m, 4H), 4.35 (dd, 2J = 11.8 Hz, 4J = 4.8 Hz, 2H),

3.98 (s, 3H), 3.95–3.84 (m, 3H), 3.65–3.54 (m, 3H), 3.52–3.48 (m, 2H), 3.47–3.43 (m, 2H),

3.40 (s, 1H) ppm. 13C NMR (50.3 MHz, CDCl3): δ 163.6, 158.8, 158.4 157.8, 157.6,

157.1, 157.0, 156.4, 156.2, 148.6, 148.5, 145.5, 144.0, 138.32, 138.30, 130.2, 128.6, 126.9,

126.8, 125.4, 125.3, 124.74, 124.68, 122.2, 122.1, 121.3, 121.2, 60.9, 60.70, 60.66, 60.62,

104


54.6, 54.3, 53.1 ppm. 3 signals are missing, probably since signals are overlapping due to

the large number of non-equivalent carbons in this molecule. Anal. Calcd. (Found) for

C38H32BrN8NaO4 · 2 H2O (Mr = 803.64): C, 56.79 (56.61); H, 4.52 (4.28); N, 13.94

(13.73).


[D29]-[Na(bpy.bpy.bpyCOOMe)]Br (35)

N

N

N

N

N

N

N

N

DD

DD

DD D D

DDDD D

COOMe

DD

DDDD

DD

DD

D

D

DD

D D

Na Br

35

32 (25.3 mg, 62 µmol, 1.0 equiv), 31 (25.7 mg, 62 µmol, 1.0 equiv), Na2CO3 (66.1 mg,

624 µmol, 10.0 equiv), CH3CN (50 ml). Column chromatography (SiO2, CH2Cl2/

MeOH 24:1→ 15:1).

Yield: 11.9 mg (16 µmol, 26%, overall deuteration level: 98%). Rf = 0.25 (SiO2,

CH2Cl2/MeOH 15:1, detection UV+I2 vapour). MS (ESI+): m/z (%) 684.19 (100, [M-

Br]+). 13C NMR (101.6 MHz, CD2Cl2/CD3OD): δ 166.2, 160.9, 159.7, 159.4, 159.3,

157.4, 157.4, 156.2, 156.2, 155.4, 140.3, 138.9, 138.7, 138.5, 138.2, 124.6, 124.3, 124.1,

121.0, 120.8, 120.5, 59.8, 59.6, 59.6, 59.5, 59.4, 59.2, 52.4 ppm.

105

6.10 Amino functionalised cryptates

6.10 Amino functionalised cryptates

[Na(bpy.bpy.bpyNO2)]Br (43)

N

N

N

N

N

N

N

NNa Br

NO2

43

6 (200 mg, 0.51 mmol, 1.0 equiv), 42 (196 mg, 0.51 mmol, 1.0 equiv), Na2CO3 (536 mg,

5.10 mmol, 10.0 equiv), CH3CN (300 ml). Column chromatography (SiO2, CH2Cl2/

MeOH/NH3 9:1:0.05).

Yield: 129 mg (0.17 mmol, 33%). Rf = 0.47 (SiO2, CH2Cl2/MeOH/NH3 9:1:0.05, de-

tection UV+I2 vapour). MS (ESI+): m/z (%) 642.23 (100, [M-Br]+). 1H NMR (200 MHz,

CD2Cl2/CD3OD): 8.61 (d, 4J = 1.9 Hz, 1H), 8.14–7.78 (m, 11H), 7.41 (dd, 3J = 7.6 Hz, 4J

= 1.0 Hz, 1H), 7.37–7.28 (m, 4H), 4.37 (s, 2H), 4.00 (s, 2H), 3.92–3.79 (m, 8H) ppm. 13H

NMR (50.3 MHz, CD2Cl2/CD3OD): 163.0, 160.0, 159.3, 159.1, 159.0, 156.3, 156.0, 156.0,

154.3, 139.3, 139.0, 138.9, 125.7, 124.7, 124.6, 121.7, 121.1, 121.0, 116.7, 113.6, 60.1, 60.0,

59.9 ppm. Signals are missing, probably since signals are overlapping due to the large number

of non-equivalent carbons in this molecule. Anal. Calcd. (Found) for C36H29BrN9NaO2 ·1 H2O (Mr = 740.58): C, 58.38 (58.50); H, 4.22 (4.28); N, 17.02 (16.80).

[Na(bpy.bpy.bpyNH2)]Br (44)

N

N

N

N

N

N

N

NNa Br

NH2

44

Under nitrogen and with cooling in an ice bath, Pd (10%)/C (20 mg) was added to

a solution of 43 (51.1 mg, 71 µmol, 1.0 equiv) in dry EtOH (50 ml). Then, hydrazine

hydrate (172 µl, 177.4 mg, 3.543 mmol, 50 equiv) was added and the reaction mixture

heated at reflux overnight. After cooling to room temperature, the dark suspension

was filtered through a paper filter and the solvent removed in vacuo. The isolated

crude product was reacted again using the same conditions, amounts of reagents and

106


solvent. After cooling to room temperature, the reaction mixture was filtered again

through a paper filter and, subsequently, through a short pad of celite. After solvent

removal, 44 was isolated as a yellow solid.

Yield: 31.8 mg (43 µmol, 61%). MS (ESI+): m/z (%) 612.09 (100, [M-Br]+). 1H NMR

(400 MHz, CD2Cl2/CD3OD): δ 8.01 (d, 3J = 7.9 Hz, 4H), 7.92–7.79 (m, 6H), 7.35 (d, 3J =

7.7 Hz, 4H), 7.32 (d, 3J = 7.4 Hz, 1H), 7.23 (d, 4J = 2.2 Hz, 1H), 6.58 (d, 4J = 2.0 Hz, 1H),

3.90–3.74 (m, 10H), 3.67–3.58 (m, 2H) ppm. 13C NMR (101.6 MHz, CD2Cl2/CD3OD):

δ 160.2, 160.1, 160.1, 160.0, 159.5, 159.4, 156.7, 156.7, 156.6, 139.2, 139.2, 124.9, 121.4,

121.4, 120.9, 120.8, 109.9, 109.9, 106.8, 106.8, 60.8, 60.7, 60.6, 60.6, 60.6, 60.5 ppm. Anal.

Calcd. (Found) for C36H31BrN9Na · 2.5 H2O (Mr = 737.63): C, 58.62 (58.75); H, 4.92

(4.80); N, 17.09 (16.90).

[Na(bpy.bpyNH2 .bpyO2)]Br (46)

N

N

N

N

N

N

N

NNa Br

H2N

OO

46

6 (105.9 mg, 0.259 mmol, 1.0 equiv), 48 (151.4 mg, 0.405 mmol, 1.5 equiv), Na2CO3

(412.1 mg, 3.888 mmol, 15.0 equiv), CH3CN (400 ml). Column chromatography (SiO2,

CH2Cl2/MeOH/NH3 9:1:0.05).

Yield: 17.6 mg (22 µmol, 8%). Rf = 0.12 (SiO2, CH2Cl2/MeOH/NH3 9:1:0.05, detec-

tion UV+I2 vapour). MS (ESI+): m/z (%) 644.08 (100, [M-Br]+). 1H NMR (250 MHz,

CD2Cl2/CD3OD): δ 7.90–7.57 (m, 10H), 7.54–7.47 (m, 2H), 7.47–7.30 (m, 3H), 7.04–6.97

(m, 1H), 6.62 (s, 1H), 4.45 (d, 2J = 11.8 Hz, 1H), 4.33–4.26 (m, 2H), 4.08–3.59 (m, 4H),

3.54–3.33 (m, 3H), 3.25–3.12 (m, 2H) ppm. 13C NMR (101.6 MHz, CD2Cl2/CD3OD):

δ 159.6, 159.0, 158.8, 158.2, 158.1, 157.5, 149.6, 149.5, 149.4, 146.0, 145.9, 145.8, 139.0,

138.7, 138.5, 130.3, 127.9, 127.8, 127.8, 127.1, 125.1, 124.7, 122.8, 122.8, 122.1, 121.6,

110.4, 109.9, 107.9, 107.2, 62.0, 61.9, 61.8, 61.7, 61.6, 61.5 ppm. Anal. Calcd. (Found)

for C36H31BrN9NaO2 · 3.5 H2O (Mr = 787.64): C, 54.90 (54.50); H, 4.86 (4.40); N, 16.00

(16.45).

107

6.11 Derivatives of functionalised cryptates


Carboxylic acid functionalised cryptates

General procedure for the synthesis of carboxylic acid functionalised cryptates: A

solution of NaOH in H2O was added to a solution of the carboxylic acid ester func-

tionalised cryptate (1 equiv) in MeOH. While stirring the reaction mixture for 3 h

at 40 °C, the colourless solution turned yellow. After the solvent was removed in

vacuo, the crude product was dry-loaded onto silica and purified by column chroma-

tography (SiO2, CH2Cl2/MeOH 2:1). The desired cryptate was isolated as a colorless

solid.

[Na(bpy.bpy.bpyCOO)]Br (51)

N

N

N

N

N

N

N

NNa

COO

51

51 was prepared according to the literature procedure of J. Wahsner. [119] 28 (36.3 mg,

49 µmol, 1.0 equiv), MeOH (6 ml), NaOH (10.7 mg, 267 µmol, 5.5 equiv), H2O (2 ml).

Yield: 25.2 mg (39 µmol, 80%). Rf = 0.82 (SiO2, CH2Cl2/MeOH 2:1, detection UV+I2

vapour). 1H NMR (250 MHz, CD2Cl2/CD3OD): δ 8.45 (s, 1H), 8.04 (d, 3J = 7.7 Hz,

1H), 7.99–7.93 (m, 4H), 7.89–7.80 (m, 5H), 7.79–7.77 (m, 1H), 7.37–7.27 (m, 5H), 3.83 (s,

12H) ppm.

Ethylenediamine coupled cryptates

[Na(bpy.bpy.bpyC(O)en)]Br (52)

N

N

N

N

N

N

N

NNa

Br

O

NH

NH2

52

108


Under nitrogen and with cooling in an ice bath, 28 (51.4 mg, 70 µmol, 1.0 equiv)

was suspended in dry MeOH (6 ml) and subsequently added to freshly dried ethyl-

enediamine (768 µl, 691.2 mg, 11.501 mmol, 150 equiv). The reaction mixture was

allowed to warm to room temperature and stirred overnight. After removal of the

volatiles, the yellow crude product was dry-loaded onto silanised silica and subjected

to column chromatography (silanised SiO2, CH2Cl2/MeOH 15:1) to give pure 52 as

an off-white solid.

Yield: 46.8 mg (61 µmol, 87%). Rf = 0.31 (SiO2, CH2Cl2/MeOH 24:1, detection

UV+ninhydrin). MS (ESI+): m/z (%) 683.19 (100, [M-Br]+). 1H NMR (250 MHz,

CD2Cl2/CD3OD): δ 8.57 (s, 1H), 8.29 (d, 3J = 8.1 Hz, 1H), 8.00–7.93 (m, 4H), 7.91–7.79

(m, 6H), 7.38–7.27 (m, 5H), 3.97–3.78 (m, 12H), 3.73 (t, 3J = 5.7 Hz, 2H), 3.22 (t, 3J = 5.7

Hz, 2H) ppm.

[Na(bpy.bpy.bpyC(O)enO2

)]Br (55)

N

N

N

N

N

N

N

NNa

Br

O

NH

NH2

OO

55

Under nitrogen and with cooling in an ice bath, 29 (250.7 mg, 0.327 mmol, 1.0 equiv)

was added as a solid to freshly dried ethylenediamine (3.5 ml, 3.15 g, 52.41 mmol,

150 equiv). The slightly red suspension was allowed to warm to room temperature

and stirred for 2 d before the volatiles were removed in vacuo. The crude product was

dry-loaded onto silanised silica and subjected to column chromatography (silanised

SiO2, CH2Cl2/MeOH 24:1) to give pure 55 as an off-white solid.

Yield: 197.0 mg (0.248 mmol, 76%). Rf = 0.48 (SiO2, CH2Cl2/MeOH 4:1, detection

UV+ninhydrin). MS (ESI+): m/z (%) 357.99 (34, [M-Br+H]2+), 715.10 (100, [M-Br]+).1H NMR (250 MHz, CD2Cl2/CD3OD): δ 8.48 (d, 4J = 2.3 Hz, 1H), 8.35 (d, 4J = 2.6

Hz, 1H), 7.96–7.82 (m, 9H), 7.79 (dd, 3J = 7.8 Hz, 4J = 2.0 Hz, 1H), 7.62–7.53 (m, 1H),

7.51–7.42 (m, 4H), 4.43–4.30 (m, 2H), 4.02–3.85 (m, 4H), 3.83–3.60 (m, 4H), 3.59–3.39

(m, 4H), 3.30–3.20 (m, 2H) ppm. 13C NMR (62.9 MHz, CD2Cl2/CD3OD): δ 165.4,

159.4, 159.3, 158.5, 158.5, 157.7, 157.6, 157.1, 149.0, 145.5, 145.4, 139.0, 139.0, 138.7,

130.3, 130.1, 128.4, 128.2, 127.0, 126.3, 125.1, 122.7, 122.0, 121.9, 61.6, 61.5, 61.5, 61.4,

55.3, 55.1, 40.2, 38.2 ppm. 7 signals are missing, probably since signals are overlapping

109


due to the large number of non-equivalent carbons in this molecule. Anal. Calcd. (Found)

for C39H36BrN10NaO3 · 4 H2O (Mr = 867.72): C, 53.98 (53.85); H, 5.11 (4.92); N, 16.14

(16.00).

Other

([Na(bpy.bpy.bpyC(O)enO2

)]Br)2C=O (54)

N

N

N

N

N

N

N

NNa

BrOO

O

NH

HN

N

N

N

N

N

N

N

NNa

BrOO

O

NH

HN

O

54

Under nitrogen, 55 (49.7 mg, 63 µmol, 6.0 equiv) was suspended in dry CH2Cl2(15 ml). With cooling in an ice bath, DIPEA (10.86 µl, 8.1 mg, 63 µmol, 6.0 equiv)

was added to give a nearly clear solution. Then, a solution of triphosgene (3.2 mg,

10 µmol, 1.0 equiv) in dry CH2Cl2 was added dropwise at 0 °C before the reaction

mixture was allowed to warm to room temperature and stirred for 5 d. The volatiles

were removed in vacuo and the bright green product was purified twice by column

chromatography (SiO2, CH2Cl2/MeOH/NH3 9:1:0.05 and CH2Cl2/MeOH 15:1→ 6:1)

to give the desired dimer 54 as a colourless solid.


vapour). MS (ESI+): m/z (%) 728.55 (100, [M-2Br]2+), 741.01 (8, isocyanate byproduct),

1491.89 (6, [M+H-2Na-Br]+). 1H NMR (250 MHz, CD2Cl2/CD3OD): δ 8.12 (d, 4J =

2.6 Hz, 1H), 8.09–8.04 (m, 2H), 8.02–7.82 (m, 17H), 7.81–7.60 (m, 4H), 7.56–7.41 (m,

7H), 7.41–7.34 (m, 2H), 7.17 (t, 3J = 7.8 Hz, 1H), 4.38–4.12 (m, 4H), 4.11–3.98 (m, 1H),

3.97–3.71 (m, 8H), 3.68–3.36 (m, 19H) ppm.

110


[Na(bpy.bpy.bpyC(O)enO2

)-Glu]Br (56)

N

N

N

N

N

N

N

NNa Br

O

NH

HN O

FmocHNO

OtBu

OO

56

56 (11.1 mg, 26 µmol, 1.0 equiv) was dissolved in SPPS grade DMF (1 ml). With

cooling in an ice bath, the free acid was activated with HATU (11.9 mg, 31 µmol,

1.2 equiv) and DIPEA (11.2 µl, 8.4 mg, 65 µmol, 2.5 equiv). After stirring for 10 min,

55 (20.2 mg, 25 µmol, 1.0 equiv) in SPPS grade DMF (1 ml) was added and the reaction

mixture was allowed to slowly warm to room temperature. After stirring overnight,

the volatiles were removed in vacuo and the yellow crude product dry-loaded onto

silica. Twofold purification by column chromatography (SiO2, CH2Cl2/MeOH/NH3

9:1:0.05 and CH2Cl2/MeOH 24:1→ 9:1) affored 56 as a colourless solid.


vapour). MS (ESI+): m/z (%) 1122.64 (100, [M-Br]+). 1H NMR (250 MHz, CDCl3): δ

8.25 (s, 1H), 8.13 (s, 1H), 7.88–7.59 (m, 15H), 7.52 (br s, 1H), 7.44–7.31 (m, 8H), 6.76 (br

s, 1H), 6.02 (br s, 1H), 4.42–4.16 (m, 6H), 4.02–3.88 (m, 4H), 3.76–3.13 (m, 10H), 2.39 (s,

2H), 1.65 (s, 2H), 1.46 (s, 9H) ppm.

111

6.12 Lanthanide complexes


[Dx]-[Ln(bpy.bpy.bpy)]

N

N

N

N

N

N

N

N

DD

DD

DD D D

DDDD D

D

DD

DDDD

DD

DD

D

D

DD

D D

Ln Br

N

N

N

N

N

N

N

N

R1 R1

R2R2

R3 R3

R3R3R2R2

R1 R1

Ln Br

R1=D; R2,R3=H

R1,R2=D; R3=H

R1,R2,R3=D Ln = Nd, Yb

88

89

90

91

General procedure for the synthesis of [Dx]-Ln: The sodium cryptate (1.0 equiv) and

LnCl3·6H2O (1.05 equiv, 99.9%) were heated at reflux in dry CH3CN (ca. 0.25 ml per

µmol sodium cryptate) for 20 h. The solvent was removed in vacuo, the residue dis-

solved in a minimum amount of MeOH and the solution was layered with Et2O until

the mixture became slightly turbid. After storing the suspension at 4 °C overnight,

the colourless solid was collected on a membrane filter (nylon, 0.45 µm), and washed

with Et2O to yield the lanthanide complex as a colourless to faintly yellow powder

after drying under reduced pressure (yield: 50-70%). The purity of the compounds

was checked by analytical HPLC. In addition, the complexes were characterized by

positive ESI mass spectrometry.

HPLC: The only relevant impurity occasionally observed in the lanthanide complexes

was the corresponding sodium cryptate (starting material for the complexation reac-

tion). Analytical reversed-phase HPLC was performed in each case to rule out this

contaminant. All isotopologic complexes showed the same HPLC properties. The

complexes [Dx]-Yb are decomposing under the HPLC conditions on the timescale of

the run but [Dx]-Na is NOT present initially.

ESI+ Mass Spectrometry: Characteristic peaks for the lanthanide complexes: The iso-

topic patterns found are rather complicated due to the natural isotopic distributions

of both lanthanoids (6 isotopes for Yb and 7 for Nd) and the different deuteration

levels of the cryptands. The following table only lists the most intense peaks for the

corresponding manifolds.

112


Table 6.1: HPLC program for Nd cryptates

(A: H2O (1% TFA, v/v), B: CH3CN

(HPLC gradient grade))

min %A %B

0 85 15

5 85 15

19 45 55

25 45 55

40 85 15

50 85 15

Table 6.2: HPLC program for Yb cryptates

(A: H2O (0.1% HCOOH, v/v), B:

CH3CN (HPLC gradient grade))

min %A %B

0 50 50

5 50 50

25 75 25

35 75 25

50 50 50

60 50 50

Table 6.3: Major ESI+ MS peaks of Nd cryptates

[Dx]-Nd [M+Cl-]2+ [M+2Cl-]+

[D4]-Nd 378.5 791.8

[D8]-Nd 380.4 795.9

[D12]-Nd 382.5 799.9

[D30]-Nd 390.5 819.9

Table 6.4: Major ESI+ MS peaks of Yb cryptates

[Dx]-Yb [M+e-]2+ [M+e-+2H2O]+

[D4]-Yb 378.5 393.4

[D8]-Yb 377.0 395.4

[D12]-Yb 379.0 397.5

[D30]-Yb 387.5 404.5

[D30]-[Tm(bpy.bpy.bpy)]3+ 3X-

N

N

N

N

N

N

N

N

DD

DD

DD D D

DDDD D

D

DD

DDDD

DD

DD

D

D

DD

D D

Tm 3X3

92

21 (11.6 mg, 16 µmol, 1.0 equiv), Tm(NO3)3·5H2O (7.8 mg, 17 µmol, 1.05 equiv),

CH3CN (6 ml). Yield: 5.2 mg.

113


[D29]-[Yb(bpy.bpy.bpyCOOMe]3+ 3X-

N

N

N

N

N

N

N

N

DD

DD

DD D D

DDDD D

COOMe

DD

DDDD

DD

DD

D

D

DD

D D

Yb 3X3

36

35 (6.7 mg, 8.76 µmol, 1.0 equiv), YbCl3·6H2O (3.73 mg, 9.63 µmol, 1.0 equiv), CH3CN

(5 ml). Yield: 7.4 mg.

114


6.13 Fmoc-protected serines

Fmoc-D-cycloserine (64)

ONH

OHN

Fmoc

64

Fmoc-D-cylcoserine was prepared by a modified procedure of Gordeev et al.. [127] Un-

der nitrogen, N,O-bis(trimethylsilyl)acetamide (45 ml, 37.4 g, 0.184 mol, 2.5 equiv)

was added in one portion to a white suspension of D-cycloserine (62) (7.5 g, 0.073

mol, 1.0 equiv) in dry CH2Cl2 (70 ml). The white reaction mixture was stirred for

45 min at room temperature whereas the colour changed to slightly yellow upon

dissolution. Then, pyridine (120 ml) was added with a dropping funnel over the

course of 15 min and the mixture was stirred for another 15 min. Under nitrogen,

N-(9-fluorenylmethoxycarbonyloxy)succinimide (25.0 g, 0.074 mol, 1.0 equiv) in dry

CH2Cl2 (120 ml) was added via a dropping funnel at 0 °C over the course of 1 h.

The clear yellow reaction mixture was allowed to warm slowly to room temperature

overnight. The solvents were removed in vacuo. The yellow solid was dissolved in

EtOAc (350 ml) and stirred for 60 min. Undissolved particles were filtered off and the

bright yellow filtrate was extracted with HCl (pH 2, 3 × 50 ml) and brine (3 × 50 ml).

The combined organic extracts were dried over MgSO4 and the solvent removed in

vacuo. The slightly beige solid was suspended in EtOAc/hexanes (1:9, 200 ml), stirred

overnight and filtered to give 64 as white powder. If necessary, impurities of acetic

acid can be removed by recrystallisation from water.

Yield: 19.8 g (0.061 mol, 84%). MS (FAB+): m/z 179.1 (100, [dibenzofulvene-H]+), 325.2

(18, [M+H]+), 347.2 (11, [M+Na]+). 1H NMR (200 MHz, DMSO-D6): δ 11.43 (s, 1H),

7.98–7.81 (m, 2H), 7.71 (d, 3J = 7.1 Hz, 2H), 7.50–7.26 (m, 4H), 4.67–4.43 (m, 2H), 4.34

(d, 3J = 6.6 Hz, 2H), 4.25 (d, 3J = 6.3 Hz, 1H), 3.95 (t, 3J = 7.9 Hz, 1H) ppm. 13C NMR

(50.3 MHz, DMSO-D6): δ 170.4, 156.0, 143.8, 140.8, 127.7, 127.1, 125.2, 120.1, 71.7, 65.9,

52.8, 46.6 ppm. IR (FT-ATR): ν̃ 3312 (w, N-H), 3050 (w, C-Harom), 2963 (w, CH3), 1692

(s, C=O), 1540 (m, C=Carom), 1448 (w), 1386 (w), 1272 (m), 1181 (w), 1105 (w), 1092 (w),

1018 (w), 931 (w), 759 (m), 738 (s) cm-1.

115


Fmoc-D-cycloserine-N(Me)·5H2O (65)

ON

OHN

Fmoc

65

Methyl iodide (1.57 ml, 3.6 g, 25.2 mmol, 1.2 equiv) and K2CO3 (3.3 g, 23.7 mmol,

1.1 equiv) were added to a solution of 64 (6.8 g, 21.0 mmol, 1.0 equiv) in acetone

(200 ml). The reaction mixture was heated at reflux for 45 min and then filtered hot.

After removal of the solvent in vacuo, the crude product was dry-loaded onto silica

and subjected to column chromatography (SiO2, CH2Cl2/MeOH 100:1 → 25:1). The

second major fraction contained the product 65, which was isolated as slightly yellow

solid.

Yield: 5.2 g (14.7 mmol, 70%). Rf = 0.50 (SiO2, CH2Cl2/MeOH 25:1). MS (EI+): m/z

165.0 (35, [fluorenyl-H]+), 178.0 (100, [dibenzofulvene-H]+), 196.0 (6, [fluorenylme-

thyl+H]+), 338.0 (8, [M]+.). MS (FAB+): m/z 179.1 (100, [dibenzofulvene-H]+), 339.1

(55, [M+H]+), 361.1 (30, [M+Na]+). 1H (200 MHz, CDCl3): δ 7.76 (d, 3J = 7.3 Hz, 2H),

7.58 (d, 3J = 7.1 Hz, 2H), 7.35 (ddd, 2J = 13.5 Hz, 2J = 10.6 Hz, 3J = 6.5 Hz, 4H), 5.53

(s, br, 1H), 4.80–4.55 (m, 2H), 4.49–4.37 (m, 2H), 4.28–4.15 (m, 1H), 4.09–3.94 (m, 1H),

3.20 (s, 3H) ppm. 13C NMR (50.3 MHz, CDCl3): δ 165.7, 156.4, 143.8, 141.4, 127.8,

127.2, 125.1, 120.1, 72.9, 67.4, 53.5, 47.1, 31.9 ppm. IR (FT-ATR): ν̃ 3266 (w, C-Harom),

3056 (w, C-Harom), 1709 (s, C=O), 1683 (s, C=O), 1543 (m, C=Carom), 1449 (w), 1271 (m),

1171 (m), 1103 (w), 1020 (m), 1001 (m), 965 (m, N-O), 918 (m), 756 (m), 738 (w), cm-1.

Anal. Calcd. (Found) for C19H18N2O4·0.5 MeOH (Mr = 354.38): C, 66.09 (65.85), H,

5.69 (5.16), N, 7.90 (7.70).

Fmoc-D-serine-O-aminomethyl-OMe·HCl (66)

HN

Fmoc OMe

O

O

HN

HCl

66

With cooling in an ice bath, 65 (4.6 g, 12.9 mmol, 1.0 equiv) was suspended in ice cold

HCl/MeOH (1.25 M, 150 ml). The reaction mixture was allowed to warm to room

temperature and stirred overnight before the solvents were removed in vacuo. The

116


obtained residue was coevaporated with MeOH (2 × 50 ml) to give 66 as a colourless

solid.

Yield: 5.1 g (12.6 mmol, 97%). MS (EI+): m/z 165.0 (60, [fluorenyl-H]+), 178.0 (100,

[dibenzofulvene-H]+), 196.0 (12, [fluorenylmethanol+H]+), 354.9 (59, [M-CH2]+., 368.9

(59, [M-H]+.). 1H (200 MHz, CD3OD): δ 6.24 (d, 3J = 6.9 Hz, 2H), 6.10 (d, 3J = 6.9 Hz,

2H), 5.91–5.70 (m, 4H), 3.06 (d, 3J = 4.9 Hz, 1H), 2.86 (dd, 3J = 6.2 Hz, 3J = 3.3 Hz, 4H),

2.67 (t, 3J = 6.5 Hz, 1H), 2.21 (s, 3H), 1.75 (q, 3H) ppm. 13C NMR (50 MHz, CDCl3 ):

δ 169.2, 156.4, 143.8, 141.4, 127.9, 127.2, 125.3, 120.1, 100.1, 67.7, 53.4, 47.6, 35.6 ppm.

IR (FT-ATR): ν̃ 3259 (w, C-Harom), 2952 (w, CH3), 2368 (w), 1718 (br m, C=O), 1523 (w,

C=Carom), 1477 (w), 1448 (m), 1406 (w), 1213 (m), 1034 (m, N-O), 759 (m), 739 (s), 646

(m) cm-1. Anal. Calcd. (Found) for C20H22N2O5·HCl (Mr = 406.86): C, 59.04 (58.85),

H, 5.70 (5.86), N, 6.89 (6.61).

Fmoc-D-serine-O-aminomethyl-OMe (68)

HN

Fmoc OMe

O

O

HN68

A solution of 66 (1.0 g, 2.5 mmol, 1.0 equiv) in CH2Cl2 (100 ml) and washed with sat.

NaHCO3 (3 × 300 ml). The organic layer was dried over MgSO4, the solvent removed

in vacuo and the remaining residue dry-loaded onto silica. Purification by column

chromatography (SiO2, CH2Cl2/MeOH 75:1) afforded 68 as slightly yellow oil.

Yield: 0.77 g (2.07 mmol, 83%). Rf = 0.2 (SiO2, CH2Cl2/MeOH 50:1). 1H (250 MHz,

CDCl3): δ 7.77 (d, 3J = 7.3 Hz, 2H), 7.66–7.59 (m, 2H), 7.47–7.28 (m, 4H), 4.62–4.49 (m,

1H), 4.49–4.32 (m, 2H), 4.26 (t, 3J = 7.2 Hz, 1H), 4.14–3.93 (m, 2H). 4.63 (t, 3J = 5.0 Hz,

1H), 4.42 (dd, 3J = 6.2 Hz, 3J = 3.3 Hz, 4H), 4.24 (t, 3J = 6.5 Hz, 1H), 3.78 (s, 3H), 2.69

(s, 3H) ppm.

117


Fmoc-D-serine-O-aminomethyl-N-chloromethylcarbonyl-OMe (67 )

HN

Fmoc OMe

O

O

N O

O

Cl67

Under nitrogen, 66 (5.0 g, 12.3 mmol, 1.0 equiv) was suspended in dry CH2Cl2(100 ml) and cooled to -30 °C. Dry triethylamine (5.9 ml, 4.3 g, 41.8 mmol, 3.4 equiv)

and chloromethyl chloroformiate (1.4 ml, 2.0 g, 15.5 mmol, 1.3 equiv) were added

slowly. The stirred mixture was allowed to warm to -20 °C and kept at this temper-

ature for 45 min. The reaction was quenched by cautious addition of water (5 ml).

The mixture was acidified with aq. HCl (1 M, pH 3-4), and extracted with EtOAc

(3 × 180 ml). The combined organic extracts were washed with NaHCO3 (3 × 40 ml)

and brine (3 × 40 ml), and dried over MgSO4. The solvent was removed in vacuo,

the crude product dry-loaded onto silica gel and purified by column chromatography

(SiO2, gradient: hexanes/EtOAc 5:1→ 2:1) to afford 67 as colourless oil.

Yield: 0.9 g (2.1 mmol, 17%). Rf = 0.65 (SiO2, hexanes/EtOAc 1:1). MS (ESI+) m/z

371.07 (7, [reactant+H]+), 451.07 (2, [M+Na-Cl]+), 463.05 (2, [M+H]+), 485.02 (100,

[M+Na]+). 1H NMR (200 MHz, CDCl3) δ 7.77 (d,3J = 7.4 Hz, 2H), 7.69–7.59 (m, 2H),

7.48–7.27 (m, 4H), 5.81–5.72 (m, 2H), 4.65–4.08 (m, 6H), 3.79 (s, 3H), 3.17 (s, 3H) ppm.13C NMR (50.3 MHz, CDCl3) δ 170.1, 156.1, 154.8, 144.0, 143.8, 141.4, 127.8, 127.2,

125.2, 120.1, 74.0, 71.0, 67.3, 53.4, 52.9, 47.2, 36.4 ppm. IR (FT-ATR): ν̃ 3337 (w, N-H),

3050 (w, C-Harom), 2952 (w, CH3), 1720 (br s, C=O), 1516 (m, C=Carom), 1447 (m), 1372

(w), 1327 (m), 1294 (m), 1208 (s), 1141 (s), 1053 (s, N-O), 878 (w), 758 (s), 740 (s, C-Cl),

705 (m), 621 (m) cm-1. Anal. Calcd. (Found) for C22H23ClN2O7 (Mr = 462.88): C, 57.09

(57.53), H, 5.01 (5.32), N, 6.05 (6.28).

118


Fmoc-D-serine-O-aminomethyl-N(Azoc)-OMe (63)

HN

Fmoc OMe

O

O

N O

O

N363

Sodium azide (169.1 mg, 2.60 mmol, 1.5 equiv) was added to a solution of 67 (796.4 mg,

1.72 mmol, 1.0 equiv) in DMF (6 ml) and the mixture was stirred for 3 h at room tem-

perature. The reaction was quenched by cautious addition of water (5 ml) to give first

a clear and then a cloudy mixture. After removal of the solvents in vacuo, the residue

was dissolved in EtOAc (100 ml), washed with water (2× 30 ml) and brine (2× 30 ml).

The organic layer was dried over MgSO4 and the solvent removed in vacuo. The crude

product was dry-loaded onto silica and purified by column chromatography (SiO2,

CH2Cl2/EtOAc 5:1→ 2:1) to give 63 as a yellow oil.

Yield: 352.3 mg (0.75 mmol, 44%). Rf = 0.31 (SiO2, CH2Cl2/EtOAc 9:1). MS (ESI+) m/z

462.00 (7, [67+H]+), (491.98 (100, [M+Na]+). 1H NMR (200 MHz, CDCl3) δ 7.77 (d, 3J

= 7.3 Hz), 7.63 (m, 3J = 6.0 Hz, 2H), 7.46 - 7.28 (m, 4H), 6.11 (s, 1H), 5.17 (s, 1H), 4.62

- 4.06 (m, 6H), 3.79 (s, 3H), 3.17 (s, 3H) ppm. 13C NMR (62.5 MHz, CDCl3) δ 170.2,

156.5, 156.2, 144.00, 143.8, 141.4, 127.8, 127.2, 125.2, 120.2, 73.9, 67.3, 53.4, 52.9, 47.2,

36.4 ppm. IR (FT-ATR): ν̃ 3335 (w, N-H), 3051 (w, C-Harom), 2952 (w, CH3), 2360 (w),

2156 (w), 2109 (m, azideasym), 1715 (br s, C=O), 1609 (w), 1517 (m, C=Carom), 1449 (m),

1338 (m), 1294 (m), 1208 (s, azidesym), 1143 (s), 1055 (s, N-O), 1032 (s), 920 (m), 759 (s),

739 (s) cm-1. Anal. Calcd. (Found) for C22H23ClN5O7 (Mr = 469.45): C, 56.29 (55.83),

H, 4.94 (5.11), N, 14.92 (14.65).

119

6.14 Acetyl-protected serines


Ac-D-cycloserine (69)

ONH

OHN

Ac

69

Acetyl-cycloserine was prepared by a procedure of Howard et al.. [128] D-Cycloserine

(5.0 g, 49.0 mmol, 1.0 equiv) was suspended in MeOH (250 ml) and Ac2O (5.5 g, 5.1 ml,

53.9 mmol, 1.1 equiv) was added dropwise over the course of 10 min. The colourless

reaction mixture was stirred at room temperature for 2 h until total dissolution of

the reactant. After removal of the solvent in vacuo, the white crude product was

recrystallised from boiling EtOH (~75 ml). Upon slow cooling to room temperature,

69 was obtained as white crystals, which were suitable for X-ray crystal structure

analysis.

Yield: 4.1 g (28.2 mmol, 58%). MS (FAB+): m/z 145.1 (52, [M+H]+), 167.0 (15,

[M+Na]+). 1H NMR (200 MHz, DMSO-D6): δ 11.47 (s, 1H), 8.40 (d, 3J = 7.5 Hz,

1H), 4.81 - 4.65 (m, 1H), 4.49 (t, 3J = 8.4 Hz, 1H), 3.89 (dd, 3J = 9.6 Hz, 3J = 8.4 Hz, 1H),

1.86 (s, 3H) ppm. 13C NMR (50 MHz, DMSO-D6): δ 170.5, 169.6, 72.5, 51.1, 22.3 ppm.

IR (FT-ATR): ν̃ 3298 (m, N-H), 2960 (m, CH3), 2808 (w, C-H), 2722 (m, CH2), 1700 (s,

C=O), 1637 (s, C=O), 1556, 1368 (s, CH3bend), 1306, 1035, 777 (m, CH2bend) cm-1. Anal.

Calcd. (Found) for C5H8N2O3 (Mr = 144.13): C, 41.67 (41.85), H, 5.59 (5.75), N, 19.44

(19.46).

Ac-D-cycloserine-N(Me) (70)

ON

OHN

Ac

70

Under nitrogen, methyliodide (2.5 g, 17.3 mmol, 1.2 equiv) and K2CO3 (2.2 g, 16.1

mmol, 1.1 equiv) were added to a solution of 69 (2.0 g, 13.9 mmol, 1.0 equiv) in acetone

(300 ml). The reaction mixture was heated at reflux for 45 min (bath temperature

75 °C) and then filtered hot. After cooling to room temperature, the solvent was

removed in vacuo to leave a white sticky solid behind. The crude product was dry-

loaded onto silica and subjected to column chromatography (SiO2, CH2Cl2/MeOH

120


25:1 → 10:1) to remove unreacted 69 (0.39 g, Rf = 0.1 (SiO2, CH2Cl2/MeOH 15:1) and

obtain pure 70 as a white solid.

Yield: 0.6 g (3.8 mmol, 27%). Rf = 0.36 (SiO2, CH2Cl2/MeOH 15:1). MS (EI+): m/z

158.1 (13, [M+.]). 1H NMR (200 MHz, DMSO-D6): δ 8.44 (d, 3J = 7.5 Hz, 1H), 4.75 (dd,3J = 16.9 Hz, 3J = 8.6 Hz, 1H), 4.49 (t, 3J = 8.4 Hz, 1H), 3.89 (dd, 3J = 9.6 Hz, 3J = 8.4

Hz, 1H), 3.07 (s, 3H), 1.86 (s, 3H) ppm. 13C NMR (100.6 MHz, DMSO-D6): δ 169.5,

169.4, 70.3, 51.1, 31.9, 22.3 ppm. IR (FT-ATR): ν̃ 3280 (m, N-H), 2932 (w, CH3), 1704 (s,

C=O), 1632 (s, C=O), 1533 (s), 1362 (w), 1300 (s, CH3bend), 1094 (m), 705 (m, CH2bend)

cm-1. Anal. Calcd. (Found) for C6H10N2O3 (Mr = 158.16): C, 45.57 (45.76), H, 6.37

(6.72), N, 17.71 (17.80).

Ac-D-serine-O-aminomethyl-OMe·HCl (71)

HN

Ac OMe

O

O

HN

HCl

71

A solution of 70 (68 mg, 0.43 µmol) in ice cold HCl/MeOH (1.25 M, 70 ml) was allowed

to warm to room temperature and stirred overnight. After solvent removal in vacuo,

71 was obtained as a sticky yellow solid, which was used without further purification.

Ac-D-serine-O-amino-N-chloromethylcarbonyl-OMe (77)

HN

OMe

O

O

HN O

O

Cl

Ac

77

A mixture of 93 (100 mg, 0.38 mmol, 1.0 equiv) and capping agent (6% DIPEA & 5%

Ac2O in DMF, 1420 µl, 1.2 equiv & 2.0 equiv) was shaken for 15 min and, subsequently,

the solvent was removed in vacuo to leave a sticky orange residue behind. This crude

product was dry-loaded onto silica and purified by column chromatography twice

(SiO2, CH2Cl2/MeOH 9:1; SiO2, CH2Cl2/MeOH 24:1) to give 77 as a yellow oil.

121


Yield: 48.3 mg (0.180 mmol, 47%). Rf = 0.50 (SiO2, CH2Cl2/MeOH 9:1, detection UV).

MS (ESI+): m/z 291.03 (100, [M+Na]+). 1H NMR (200 MHz, CDCl3): δ 8.45 (br s, 1H),

6.64 (br s, 1H), 5.83–5.70 (m, 2H), 5.00–4.82 (m, 1H), 4.26–4.01 (m, 2H), 3.79 (s, 3H),

2.09 (s, 2H) ppm.

122


6.15 Boc-protected serines

Boc-D-cycloserine

ONH

OHN

Boc

74

74 was prepared by the literature procedure of Thorsteinsson et al. [130] and purified

according to the literature procedure of Wolfe et al. [129] With cooling in an ice bath,

triethylamine (863 mg, 1189 µl, 8.53 mmol, 1.0 equiv) was added dropwise to a solu-

tion of D-cycloserine (871 mg, 8.53 mmol, 1.0 equiv) in THF/H2O (1:1, v/v, 20 ml) and

stirred at 0 °C for 1 h. Then, a solution of di-tert-butyldicarbonate (1.86 g, 8.53 mmol,

1.0 equiv) in THF (8 ml) was added dropwise to the ice cold solution over the course

of 2 h. The reaction mixture was slowly warmed to room temperature and stirred

overnight before the solvent was removed in vacuo. The carefully dried, off-white,

sticky residue was dry-loaded onto silica and purified by a short column chromato-

graphy (SiO2, EtOAc/hexanes 2:1) to afford 74 as a fluffy white solid.

Yield: 925.5 mg (4.577 mmol, 54%). Rf = 0.38 (SiO2, EtOAc/hexanes 2:1, detection

UV). MS (ESI+): m/z 225.04 (100, [M+Na]+). 1H NMR (200 MHz, CDCl3): δ 5.11 (br

s, 1H, NH), 4.79 (t, 3J = 8.2 Hz, 1H), 4.67–4.51 (m, 1H), 4.09 (dd, 3J = 10.6 Hz, 3J = 8.2

Hz, 1H), 1.46 (s, 9H, BocCH3) ppm. 13C NMR (50.3 MHz, CDCl3): δ 171.1, 155.8, 81.0,

75.1, 52.9, 28.4 ppm. IR (FT-ATR): ν̃ 3314 (m, C(O)N-H), 2982 (w, CH3), 1716 (s, Boc

C=O), 1680 (s, C=O), 1539 (m), 1366 (m, C-(CH)3), 1252 (m), 1164 (s, C(O)-O) cm-1.

Boc-D-serine-N-chloromethylcarbonyl-OMe

ON

OHN

Boc

O

O

Cl75

75 was prepared by employing the conditions for the introduction of an (chlorometh-

oxy)carbamate protection group reported by Pothukanuri et al. [126] Under nitrogen,

a solution of 74 (82.3 mg, 0.407 mmol, 1.0 equiv) in dry CH2Cl2 (8 ml) was cooled

to -30 °C before dropwise addition of triethylamine (124.4 mg, 172 µl, 1.221 mmol,

3.0 equiv) and chloromethyl chloroformate (60.4 mg, 42 µl, 0.468 mmol, 1.15 equiv).

123

6.15 Boc-protected serines

The colourless solution was warmed to -20 °C and stirred at this temperature for 1

h . The reaction was quenched by cautious addition of H2O (5 ml) and acidified to

pH 3-4 with HCl (pH 2). After separation of the phases, the aqueous layer was ex-

tracted with EtOAc (3 × 20 ml), the combined organic phases dried over MgSO4 and

the solvents removed in vacuo. The crude product was dry-loaded onto silica and

purified by column chromatography (SiO2, hexane/EtOAc 2:1) to yield 75 as a sticky

white solid.

Yield: 67.7 mg (0.230 mmol, 56%). Rf = 0.25 (SiO2, hexane/EtOAc 2:1, detection UV).

MS (ESI+): m/z 317.12 (60, [M+Na]+), 349.03 (100, [M+Na+MeOH]+). 1H NMR (200

MHz, CDCl3): δ 5.88 (s, 2H, -CH2-), 5.06 (br s, 1H, NH), 4.84 (t, 3J = 8.0 Hz, 1H), 4.77–

4.60 (m, 1H), 4.22 (dd, 3J = 10.9 Hz, 3J = 7.9 Hz, 1H), 1.46 (s, 9H, BocCH3). 13C NMR

(50 MHz, CDCl3): δ 165.7, 145.6, 81.6, 73.1, 71.1, 53.5, 28.3 ppm. IR (FT-ATR): ν̃ 3425

(m, C(O)N-H), 3368 (m, C(O)N-H), 2918 (m, CH2), 2850 (w, CH3), 1787 (w, chlorozoc

C=O), 1760 (s, Boc C=O), 1698 (s, C=O), 1585 (s), 1441 (m), 1367 (m, C-(CH)3), 1283 (s),

1240 (s), 1157 (s, C(O)-O), 1096 (s), 962 (s), 873 (s) cm-1.

D-serine-O-amino-N-chloromethylcarbonyl-OMe

H2NOMe

O

O

HN O

O

Cl76

75 (18.6 mg, 0.062 mmol, 1.0 equiv) was dissolved in ice-cold HCl/MeOH (1.25 M,

5 ml), slowly warmed to room temperature and stirred overnight. The solvents were

removed in vacuo and the residue coevaporated with MeOH (2 × 50 ml) to obtain 76

as a colourless solid.

Yield: quantitative. MS (ESI+): m/z 227.05 (100, [M-Cl]+). 1H NMR (200 MHz, CDCl3):

δ 5.85 (s, 2H), 4.45–4.38 (m, 1H), 4.34–4.29 (m, 2H), 3.87 (s, 3H) ppm. 13C NMR

(50.3 MHz, CD3OD): δ 168.2, 157.3, 74.5, 71.9, 54.1, 53.1 ppm. IR (FT-ATR): ν̃ 2957 (m,

CH2), 1742 (s, C=O), 1570 (w), 1442 (m, CH2), 1364 (m, CH3), 1302 (s, C-Cl), 1236 (s),

1105 (s, C(=O)-O), 972 (m) cm-1.

124


6.16 Other serines and related molecules

Phenylpyrazolidinone hydrochloride (58)

O

O

NPh

NH2

58

A yellow solution of 1-phenyl-3-pyrazolidinone (1.0 g, 6.1 mmol, 1.0 equiv) in ice

cold HCl/MeOH (1.25 M, 150 ml) was allowed to warm slowly to room temperature

overnight. After removal of the solvents in vacuo, the remaining sticky yellow solid

was dissolved in chloroform (50 ml) and extracted with sat. NaHCO3 (3 × 50 ml).

The organic phase was dried over MgSO4 and the solvent removed in vacuo to give 58

as yellow oil.

Yield: 0.9 g (4.7 mmol, 77%). 1H NMR (250 MHz, CD3OD): δ 7.51–7.42 (m, 2H), 7.28

(dd, 3J = 7.8 Hz, 3J = 6.0 Hz, 3H), 3.76 (t, 3J = 6.6 Hz, 2H), 3.68 (s, 3H), 2.63 (t, 3J = 6.6

Hz, 2H) ppm.

Fmoc-glycine-D-cycloserine (81)

ONH

HN

O

ONH

Fmoc

81

The procedure was adapted from a similar reaction reported by De et al. [132] Under

nitrogen and with cooling in an ice bath, oxalyl chloride (8.3 ml, 12.4 g, 100 equiv) was

added dropwise to Fmoc-Gly-OH (291 mg, 0.98 mmol, 1.0 equiv). The bright yellow

mixture was stirred for 2 h at room temperature until the whole starting material

dissolved and gas evolution ceased. Then, the volatiles were removed in vacuo to give

the acyl chloride as pale yellow solid. Also under nitrogen, D-cycloserine (100 mg,

0.98 mmol, 1.0 equiv) was suspended in dry CH2Cl2 (15 ml), before NMP (2.8 ml) and

N,O-bis(trimethylsilyl) acetamide (0.6 ml, 498 mg, 2.45 mmol, 2.5 equiv) were added

dropwise. After stirring at room temperature for 1 h, a clear colourless solution was

obtained, which was treated with pyridine (1.6 ml). Then, a solution of the acid

chloride in CH2Cl2 (6 ml) was added dropwise over the course of 15 min which

resulted in a colour change from yellow over orange to deep red. After stirring at

125


room temperature for 1.5 d, the solvents were removed in vacuo with gentle heating

(~50 °C). The dark orange residue was dissolved in EtOAc to give a yellow solution,

which was washed with aq. HCl (pH 2, 3 × 15 ml). The organic layer was dried over

MgSO4 and rotavapped to dryness to leave a yellow residue. This crude product was

dryloaded onto silica and purified by column chromatography (SiO2, CH2Cl2/MeOH,

50:1→ 15:1) to give 81 as a white foamy solid.


UV). 1H NMR (250 MHz, DMSO-D6): δ 8.45 (d, 3J = 7.6 Hz, 1H), 7.89 (d, 3J = 7.2 Hz,

2H), 7.72 (d, 3J = 7.5 Hz, 2H), 7.54 (t, 3J = 6.1 Hz, 1H), 7.47–7.28 (m, 4H), 4.83–4.61 (m,

1H), 4.50 (t, 3J = 8.4 Hz, 1H), 4.33–4.18 (m, 3H), 3.92 (dd, 3J = 9.8 Hz, 3J = 8.4 Hz, 1H),

3.66 (d, 3J = 6.1 Hz, 2H) ppm.

Fmoc-glycine-D-cycloserine-N(Me) (82)

ON

HN

O

ONH

Fmoc

82

Methyl iodide (0.26 ml, 0.6 g, 4.2 mmol, 1.3 equiv) was added dropwise to a slightly

yellow suspension of 81 (1.2 g, 3.1 mmol, 1.0 equiv) and K2CO3 (0.5 g, 3.9 mmol,

1.2 equiv) in acetone (35 ml). The reaction mixture was heated at reflux for 1 h,

filtered hot and finally the solvent was removed in vacuo. The obtained brown foamy

residue was dry-loaded onto silica and purified by column chromatography (SiO2,

CH2Cl2/MeOH 50:1→ 30:1) to afford 82 as a white foamy residue.

Yield: 0.8 g (2.1 mmol, 68%). Rf = 0.60 (SiO2, CH2Cl2/MeOH 9:1, detection UV).1H NMR (200 MHz, CDCl3): δ 7.76 (d, 3J = 6.9 Hz, 2H), 7.58 (d, 3J = 7.2 Hz, 2H),

7.45–7.35 (m, 2H), 7.32 (dd, 3J = 7.3 Hz, 4J = 1.4 Hz, 2H), 6.71 (br s, 1H), 5.45 (br s,

1H), 4.80–4.74 (m, 2H), 4.48–4.39 (m, 2H), 4.22 (t, J = 6.8 Hz, 1H), 4.03–3.84 (m, 3H),

3.21 (s, 3H) ppm.

Fmoc-gylcine-D-serine-O-aminomethyl-OMe·HCl (83)

NH

Fmoc

O

HN

O

O

NH

O

HCl

83

126


With cooling in an ice bath, 1.25 M HCl/MeOH (20 ml) was added dropwise to 82

(430 mg, 1.09 mmol). The clear colourless reaction mixture was allowed to slowly

warm to room temperature. After stirring overnight, the solvent was removed in

vacuo with gentle heating to 40 °C to leave a colourless sticky residue behind. The

crude product was dry-loaded onto silica and subjected to column chromatography

(SiO2, CH2Cl2/MeOH 50:1→ 9:1) to separate 83 from the byproduct Fmoc-Gly-OMe,

both being sticky, colourless solids.

Yield (83): 232 mg (0.54 mmol, 50%). Rf = 0.61 (SiO2, CH2Cl2/MeOH 50:1, detection

UV+I2 vapour). 1H NMR (200 MHz, CDCl3): δ 7.75 (d, 3J = 7.0 Hz, 2H), 7.58 (d, 3J =

7.2 Hz, 2H), 7.44–7.27 (m, 4H), 5.76 (br s, 1H), 4.73 (ddd, 3J = 7.6 Hz, 3J = 4.1 Hz, 3J =

3.2 Hz, 1H), 4.40 (d, J = 6.4 Hz, 2H), 4.28–4.15 (m, 1H), 4.11–3.83 (m, 4H), 3.72 (s, 3H),

2.57 (s, 3H) ppm.

Yield (Fmoc-Gly-OMe): 55 mg (0.176 mmol, 16%). Rf = 0.57 (SiO2, CH2Cl2/MeOH 9:1,

detection UV+I2 vapour). 1H NMR (200 MHz, CDCl3): δ 7.77 (d, 3J = 6.8 Hz, 2H), 7.60

(d, 3J = 7.2 Hz, 2H), 7.46–7.36 (m, 2H), 7.36–7.27 (m, 2H), 5.30 (s, 1H), 4.42 (d, 3J = 6.5

Hz, 2H), 4.32–4.17 (m, 1H), 4.00 (d, 3J = 5.6 Hz, 2H), 3.77 (s, 3H) ppm.

Fmoc-glycine-D-serine-O-aminomethyl-N-chloromethylcarbonyl-OMe (84)

NH

Fmoc

O

HN

O

O

N

O

O

O

Cl84

Under nitrogen, 83 (66.2 mg, 0.155 mmol, 1.0 equiv) was dissolved in dry CH2Cl2(1.5 ml) and the solution was cooled to -30 °C. Then, dry NEt3 (62 µl, 44.8 mg,

0.0443 mmol, 2.8 equiv) and chloromethyl chloroformate (15 µl, 22.2 mg, 0.172 mmol,

1.1 equiv) were added dropwise, before the reaction was warmed to -20 °C and stirred

at this temperature for 1 h until the reaction was completed (followed with TLC). The

reaction was quenched by addtion of water (1 ml) and acidified to pH 3-4 with aq.

HCl (pH 2). The two phases were separated and the aqueous layer extracted with

EtOAc (3 × 10 ml). The combined organic phases were dried over MgSO4 and rota-

vapped to dryness. The crude product was then dryloaded onto silica and purified

by column chromatography (SiO2, CH2Cl2/MeOH 50:1 → 24:1) to yield pure 84 as a

colourless, smelly sticky solid.

127


Yield: 57.1 mg (0.110 mmol, 71%). Rf = 0.43 (SiO2, CH2Cl2/MeOH 9:1, detection UV).1H NMR (200 MHz, CDCl3): δ 7.75 (d, J = 6.9 Hz, 2H), 7.60 (d, J = 7.2 Hz, 2H), 7.44–

7.28 (m, 4H), 5.70 (s, 2H), 5.62 (m, 1H), 4.80 (dt, J = 8.0 Hz, J = 3.2 Hz, 1H), 4.53–4.34

(m, 3H), 4.30–4.17 (m, 1H), 4.13–3.93 (m, 3H), 3.75 (s, 3H), 3.13 (s, 3H) ppm.

Fmoc-glycine-D-aminoserine-N(Me)-Azoc methyl ester (85)

NH

Fmoc

O

HN

O

O

N

O

O

O

N385

Sodium azide (8.80 mg, 0.135 mmol, 1.5 equiv) was added as a solid to a solution

of 84 (46.4 mg, 0.089 mmol, 1.0 equiv) in DMF (1 ml). The reaction mixture was

stirred for 3 h at room temperature whereupon the colour changed from colourless

to slightly pink and the solution gets turbid due to the precipitation of NaCl. After

solvent removal the crude product was directly subjected to column chromatography

(SiO2, CH2Cl2/MeOH 50:1). The product (85) was eluted as 4th fraction and isolated

as colourless sticky solid.


UV+I2 vapour). 1H NMR (250 MHz, CDCl3): δ 7.76 (d, 3J = 7.2 Hz, 2H), 7.60 (d, 3J =

7.6 Hz, 2H), 7.45–7.27 (m, 4H), 5.54 (br s, 1H), 5.12 (s, 2H), 4.79 (dd, 3J = 7.8 Hz, 3J =

3.6 Hz, 1H), 4.50 (dd, 2J = 10.6 Hz, 3J = 2.8 Hz, 1H), 4.45–4.39 (m, 2H), 4.58–4.34 (m,

3H), 4.24 (t, J = 7.0 Hz, 1H), 4.11–3.95 (m, 3H), 3.76 (s, 3H), 3.14 (s, 3H) ppm.

128

7 Bibliography

[1] V. Ntziachristos, A. Leroy-Willig, B. Tavitian, Textbook of in vivo Imaging in Ver-

tebrates 1st ed., Wiley-Interscience, Chichester, 2007, xvi–xvii.

[2] R. Weissleder, U. Mahmood, Radiology 2001, 219, 316–333.

[3] H. Kobayashi, M. Ogawa, R. Alford, P. L. Choykeeter, Y. Urano, Chem. Rev. 2010,

110, 2620–2640.

[4] P. Armstrong, M. Wastie, A. Rockall, Diagnostic Imaging 6th ed., Wiley-

Interscience, Chichester, 2009, 1–16.

[5] C. A. Boswell, M. W. Brechbiel, Nucl. Med. Biol. 2007, 34, 757–778.

[6] J. T. Bushberg, J. A. Seibert, E. M. Leidholdt, J. M. Boone, The Essential Physics of

Medical Imaging 2nd ed., (Eds.: J.-R. John, A. Snyder, T. DeGeorge), Lippincott

Williams & Wilkins, Philadelphia, 2002, 13–16.

[7] C. H. Contag, in Molecular Imaging: Principles and Practice (Ed.: R. Weissleder,

B. D. Ross, A. Rehemtulla, S. S. Gambhir), PMPH-USA, Shelton, 1st ed., 2010,

118–138.

[8] R. Weissleder, V. Ntziachristos, Nat. Med. 2003, 9, 123–128.

[9] A. Oser, W. K. Roth, G. Valet, Nucleic Acids Res. 1988, 16, 1181–1196.

[10] J. Coates, P. G. Sammes, G. Yahioglu, R. M. West, A. J. Garman, J. Chem. Soc.,

Chem. Commun. 1994, 2311–2312.

[11] P. Gottlieb, E. Hazum, E. Tzehoval, M. Feldman, S. Segal, M. Fridkin, Biochem.

Biophys. Res. Commun. 1984, 119, 203–211.

[12] N. Loménie, D. Racoceanu, A. Gouaillard, Advances in Bio-Imaging: from Physics

to Signal Understanding Issues (Ed.: J. Kacprzyk), Springer, Berlin and Heidel-

berg, 2012, 37–54.

[13] E. Soini, I. Hemmila, P. Dahlen, Ann. Biol. Clin. 1990, 48, 567–571.

[14] R. A. Evangelista, A. Pollak, B. Allore, E. F. Templeton, R. C. Morton, E. P.

Diamandis, Clin. Biochem. 1988, 21, 173–178.

[15] E. Lopez, C. Chypre, B. Alpha, G. Mathis, Clin. Chem. 1993, 39, 196–201.

129

7 Bibliography

[16] G. Mathis, Clin. Chem. 1995, 41, 1391–1397.

[17] E. F. Dickson, A. Pollak, E. P. Diamandis, Photochem. Photobiol. 1995, 27, 3–19.

[18] V. Ntziachristos, A. G. Yodh, M. Schnall, B. Chance, Proc. Natl. Acad. Sci. U. S.

A. 2000, 97, 2767–2772.

[19] A. Corlu, R. Choe, T. Durduran, M. A. Rosen, M. Schweiger, S. R. Arridge, M. D.

Schnall, A. G. Yodh, Opt. Express 2007, 15, 6696–6716.

[20] H. Kobayashi, Y. Hama, Y. Koyama, T. Barrett, C. A. S. Regino, Y. Urano, P. L.

Choyke, Nano Lett. 2007, 7, 1711–1716.

[21] Y. Hama, Y. Koyama, Y. Urano, P. L. Choyke, H. Kobayashi, J. Invest. Dermatol.

2007, 127, 2351–2356.

[22] Y. Hama, Y. Urano, Y. Koyama, M. Kamiya, M. Bernardo, R. S. Paik, M. C.

Krishna, P. L. Choyke, H. Kobayashi, Neoplasia 2006, 8, 607–612.

[23] R. A. Sheth, R. Upadhyay, L. Stangenberg, R. Sheth, R. Weissleder, U. Mahmood,

Gynecol. Oncol. 2009, 112, 616–622.

[24] J.-C. G. Bünzli, Chem. Lett. 2009, 38, 104–109.

[25] D. Parker, J. A. G. Williams, J. Chem. Soc., Dalton Trans. 1996, 3613–3628.

[26] Y. M. W. Janssen, B. V. Houten, P. J. A. Borm, B. T. Mossman, Lab. Invest. 1993,

69, 261–274.

[27] T. Schaeffter, Prog. Drug. Res. 2005, 62, 15–81.

[28] K. Smith, The Science of Photobiology 1st ed., Plenum Press, New York, 1977.

[29] W. Mason, Fluorescent and Luminescent Probes for Biological Activity: A Practical

Guide to Technology for Quantitative Real-Time Analysis 2nd ed., Biological Tech-

niques Series, Academic Press, London, 1999, 17–39.

[30] A. Diaspro, Optical Fluorescence Microscopy: From the Spectral to the Nano Dimen-

sion 1st ed., Springer, Berlin and Heidelberg, 2011, 111–130.

[31] A. P. Neal, C. Schultz, in Protein Targeting with Small Molecules: Chemical Biology

Techniques and Applications (Ed.: H. Osada), Wiley-Interscience, Hoboken, 1st

ed., 2009, 91–113.

[32] N. C. Shaner, P. A. Steinbach, R. Y. Tsien, Nat. Methods 2005, 2, 905–909.

[33] K. Brejc, T. K. Sixma, P. A. Kitts, S. R. Kain, R. Y. Tsien, M. Ormo, S. J. Remington,

Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 2306–2311.

[34] L. Zhang, H. N. Patel, J. W. Lappe, R. M. Wachter, J. Am. Chem. Soc. 2006, 128,

4766–4772.

[35] D. M. Chudakov, S. Lukyanov, K. A. Lukyanov, Trends Biotechnol. 2005, 23, 605–

130

7 Bibliography

613.

[36] B. P. Aryal, D. E. Benson, J. Am. Chem. Soc. 2006, 128, 15986–15987.

[37] D. B. Cordes, S. Gamsey, B. Singaram, Angew. Chem., Int. Ed. 2006, 45, 3829–

3832.

[38] X. Wang, M. J. Ruedas-Rama, E. A. H. Hall, Anal. Lett. 2007, 40, 1497–1520.

[39] I. L. Medintz, H. T. Uyeda, E. R. Goldman, H. Mattoussi, Nat. Mater. 2005, 4,

435–446.

[40] J.-H. Park, L. Gu, G. von Maltzahn, E. Ruoslahti, S. N. Bhatia, M. J. Sailor, Nat.

Mater. 2009, 8, 331–336.

[41] F. Yang, L. G. Moss, G. N. Phillips, Nat. Biotechnol. 1996, 14, 1246–1251.

[42] Picture source: http://www.photonics.com/images/spectra/features/2007/May/Quantum

Dots_Fig5_CoreShell.jpg (last access 21st Jan 2013).

[43] L. D. Lavis, R. T. Raines, ACS Chem. Biol. 2008, 3, 142–155.

[44] Z. Zhu, Dyes Pigm. 1995, 27, 77–111.

[45] A. Loudet, K. Burgess, Chem. Rev. 2007, 107, 4891–4932.

[46] H. E. Katerinopoulos, Curr. Pharm. Des. 2004, 10, 3835–3852.

[47] C. C. Woodroofe, M. H. Lim, W. Bu, S. J. Lippard, Tetrahedron 2005, 61, 3097–

3105.

[48] W.-C. Sun, K. R. Gee, D. H. Klaubert, R. P. Haugland, J. Org. Chem. 1997, 62,

6469–6475.

[49] F. Wang, X. Liu, Chem. Soc. Rev. 2009, 38, 976–989.

[50] B. Alpha, J. M. Lehn, G. Mathis, Angew. Chem. 1987, 99, 259–261.

[51] B. Alpha, R. Ballardini, V. Balzani, J. M. Lehn, S. Perathoner, N. Sabbatini, Pho-

tochem. Photobiol. 1990, 52, 299–306.

[52] J. M. Lehn, Acc. Chem. Res. 1978, 11, 49–57.

[53] B. Alpha, V. Balzani, J. M. Lehn, S. Perathoner, N. Sabbatini, Angew. Chemie 1987,

99, 1310–1311.

[54] E. Brucher, Z. Baranyai, G. Tircso, RSC Drug Discovery Ser. 2012, 15, 208–260.

[55] J.-C. Bünzli, C. Piguet, Chem. Soc. Rev. 2005, 34, 1048–1077.

[56] S. Comby, J.-C. G. Bünzli 2007, Vol. 37, pp. 217–470.

[57] V. Balzani, F. Scandola, in Supramolecular Photochemistry, Ellis Horwood,

Chichester, Ch. 12, 1991.

[58] S. Weissmann, J. Chem. Phys. 1942, 10, 214–221.

131

7 Bibliography

[59] J. Zhou, Z. Liub, F. Li, Chem. Soc. Rev. 2012, 41, 1323–1349.

[60] M. H. V. Werts, in Lanthanide Luminescence - Photophysical, analytical and biological

aspects (Ed.: O. Wolfbeiss, P. Hänninen, H. Härmä), Springer, Berlin-Heidelberg,

Springer Series on Fluorescence, 2011, 133–160.

[61] K. Matsumoto, T. Nojima, H. Sano, K. Majima, Macromol. Symp. 2002, 186, 117–

121.

[62] H. Bazin, M. Preaudat, E. Trinquet, G. Mathis, Spectrochim. Acta, Part A 2001,

57A, 2197–2211.

[63] E. Soini, H. Kojola, Clin. Chem. 1983, 29, 65–68.

[64] I. Hemmila, S. Dakubu, V. M. Mukkala, H. Siitari, T. Lovgren, Anal. Biochem.

1984, 137, 335–343.

[65] J.-C. Bünzli, S. V. Eliseeva, J. Rare Earths 2010, 28, 824–842.

[66] S. Faulkner, S. J. A. Pope, B. P. Burton-Pye, Appl. Spectrosc. Rev. 2005, 40, 1–31.

[67] M. D. Ward, Coord. Chem. Rev. 2007, 251, 1663–1677.

[68] M. H. V. Werts, Sci. Prog. 2005, 88, 101–131.

[69] V. Bulach, F. Sguerra, M. W. Hosseini, Coord. Chem. Rev. 2012, 256, 1468–1478.

[70] A. D’Aleo, F. Pointillart, L. Ouahab, C. Andraud, O. Maury, Coord. Chem. Rev.

2012, 256, 1604–1620.

[71] F. Wang, D. Banerjee, Y. Liu, X. Chen, X. Liu, Analyst 2010, 135, 1839–1854.

[72] F. Auzel, C. R. Acad. Sci., Ser. A B 1966, 263B, 819–821.

[73] F. Auzel, C. R. Seances Acad. Sci., Ser. B 1966, 262B, 1016–1019.

[74] V. Ovsyankin, P. Feofilov, Zh. Eksp. Teor. Fiz. 1966, 4, 471–474.

[75] F. Auzel, Chem. Rev. 2004, 104, 139–173.

[76] D. E. Achatz, R. Ali, O. S. Wolfbeis, in Luminescence Applied in Sensor Science (Ed.:

L. Prodi, M. Montalti, N. Zaccheroni), Springer, Topics in Current Chemistry,

2011, 29–50.

[77] T. Soukka, T. Rantanen, K. Kuningas, Ann. N. Y. Acad. Sci. 2008, 1130, 188–200.

[78] G.-L. Law, in Tomorrow’s chemistry today - Concepts in nanoscience, organic materials

and environmental chemistry (Ed.: B. Pignataro), Wiley-VCH, Weinheim, 2nd ed.,

2008, 161–184.

[79] K.-L. Wong, W.-M. Kwok, W.-T. Wong, D. L. Phillips, K.-W. Cheah, Angew.

Chem., Int. Ed. 2004, 43, 4659–4662.

[80] K.-L. Wong, G.-L. Law, W.-M. Kwok, W.-T. Wong, D. L. Phillips, Angew. Chem.,

132

7 Bibliography

Int. Ed. 2005, 44, 3436–3439.

[81] X. Xiao, J. P. Haushalter, G. W. Faris, Opt. Lett. 2005, 30, 1674–1676.

[82] N. Tancrez, C. Feuvrie, I. Ledoux, J. Zyss, L. Toupet, H. L. Bozec, O. Maury, J.

Am. Chem. Soc. 2005, 127, 13474–13475.

[83] L. Aboshyan-Sorgho, C. Besnard, P. Pattison, K. R. Kittilstved, A. Aebischer,

J.-C. G. Bünzli, A. Hauser, C. Piguet, Angew. Chem. Int. Ed. 2011, 50, 4108–4112.

[84] J. L. Kropp, M. W. Windsor, J. Chem. Phys. 1963, 39, 2769–2770.



[87] R. S. Dickins, D. Parker, A. S. de Sousa, J. A. G. Williams, Chem. Commun. 1996,

697–698.

[88] A. Beeby, I. M. Clarkson, R. S. Dickins, S. Faulkner, D. Parker, L. Royle, A. S.

de Sousa, J. A. G. Williams, M. Woods, J. Chem. Soc., Perkin Trans. 2 1999, 493–

504.

[89] J. C. Rodriguez-Ubis, B. Alpha, D. Plancherel, J. M. Lehn, Helv. Chim. Acta 1984,

67, 2264–2269.

[90] S. Faulkner, A. Beeby, M. C. Carrie, A. Dadabhoy, A. M. Kenwright, P. G.

Sammes, Inorg. Chem. Commun. 2001, 4, 187–190.

[91] M. J. Cook, A. P. Lewis, G. S. G. McAuliffe, V. Skarda, A. J. Thomson, J. L.

Glasper, D. J. Robbins, J. Chem. Soc., Perkin Trans. 2 1984, 1293–1301.

[92] J. M. Lehn, C. O. Roth, Helv. Chim. Acta 1991, 74, 572–578.

[93] C. Doffek, N. Alzakhem, M. Molon, M. Seitz, Inorg. Chem. 2012, 51, 4539–4545.

[94] J. M. Lehn, G. Mathis, B. Alpha, R. Deschenaux, E. Jolu, Heterocycle complexes

with rare earth metals as fluorescent labels and their preparation (EP 321353 A1), 1989.

[95] F. Havas, N. Leygue, M. Danel, B. Mestre, C. Galaup, C. Picard, Tetrahedron 2009,

65, 7673–7686.

[96] I. Bkouche-Waksman, J. Guilhem, C. Pascard, B. Alpha, R. Deschenaux, J. M.

Lehn, Helv. Chim. Acta 1991, 74, 1163–70.

[97] U. Maheswari Palanisamy, K. Lappalainen, M. Sfregola, S. Barends, P. Gamez,

U. Turpeinen, I. Mutikainen, P. van Wezel Gilles, J. Reedijk, Dalton Trans. 2007,

3676–3683.

[98] T. Rode, E. Breitmaier, Synthesis 1987, 574–575.

[99] G. R. Newkome, W. E. Puckett, G. E. Kiefer, V. D. Gupta, Y. Xia, M. Coreil, M. A.

Hackney, J. Org. Chem. 1982, 47, 4116–4120.

133

7 Bibliography

[100] N. Psychogios, J.-B. Regnouf-de Vains, H. M. Stoeckli-Evans, Eur. J. Inorg. Chem.

2004, 2514–2523.

[101] F. Bottino, M. Di Grazia, P. Finocchiaro, F. R. Fronczek, A. Mamo, S. Pappalardo,

J. Org. Chem. 1988, 53, 3521–3529.

[102] M. Y. Alyapyshev, V. A. Babain, N. E. Borisova, R. N. Kiseleva, D. V. Safronov,

M. D. Reshetova, Mendeleev Commun. 2008, 18, 336–337.

[103] A. J. Blake, N. R. Champness, P. V. Mason, C. Wilson, Acta Crystallogr., Sect. C:

Cryst. Struct. Commun. 2007, C63, m280–m282.

[104] Z. Wang, J. Reibenspies, R. J. Motekaitis, A. E. Martell, J. Chem. Soc., Dalton

Trans. 1995, 1511–1518.

[105] C. Bischof, J. Wahsner, J. Scholten, S. Trosien, M. Seitz, J. Am. Chem. Soc. 2010,

132, 14334–14335.

[106] M. P. O. Wolbers, F. C. J. M. Van Veggel, B. H. M. Snellink-Ruel, J. W. Hofstraat,

F. A. J. Guerts, D. N. Reinhoudt, J. Chem. Soc., Perkin Trans. 2 1998, 2141–2150.

[107] V. L. Ermolaev, E. B. Sveshnikova, Chem. Phys. Lett. 1973, 23, 349–354.

[108] T. Forster, Ann. Physik [6 Folge] 1948, 2, 55–75.

[109] J. Mathieu, A. Marsura, Synth. Commun. 2003, 33, 409–414.

[110] J. Agrawal, R. Hodgson, Organic Chemistry of Explosives Wiley-Interscience,

Chichester, 2007, 17–18.

[111] C. J. Moody, J. L. O’Connell, Chem. Commun. 2000, 1311–1312.

[112] N. Alzakhem, Stable Lanthanide Cryptates as Building Blocks for Solid Phase Peptide

Synthesis - Synthesis, Functionalization and Photophysical Properties, Dissertation,

Ruhr-Universität Bochum, 2013.

[113] V. M. Mukkala, J. J. Kankare, Helv. Chim. Acta 1992, 75, 1578–1592.

[114] Z. Zhou, G. H. Sarova, S. Zhang, Z. Ou, F. T. Tat, K. M. Kadish, L. Echegoyen,

D. M. Guldi, D. I. Schuster, S. R. Wilson, Chem.–Eur. J. 2006, 12, 4241–4248.

[115] M. Karramkam, F. Hinnen, F. Vaufrey, F. Dolle, J. Labelled Compd. Radiopharm.

2003, 46, 979–992.

[116] E. Delfourne, F. Darro, N. Bontemps-Subielos, C. Decaestecker, J. Bastide,

A. Frydman, R. Kiss, J. Med. Chem. 2001, 44, 3275–3282.

[117] M. Hapke, H. Staats, I. Wallmann, A. Luetzen, Synthesis 2007, 2711–2719.

[118] S. Caron, N. M. Do, J. E. Sieser, Tetrahedron Lett. 2000, 41, 2299–2302.

[119] J. Wahsner, Isotopencodierte Lanthanoid-Kryptate für die quantitative Proteomik, Mas-

ter thesis, Ruhr-Universität Bochum, 2011.

134

7 Bibliography

[120] P. F. Van Swieten, M. A. Leeuwenburgh, B. M. Kessler, H. S. Overkleeft, Org.

Biomol. Chem. 2005, 3, 20–27.

[121] J. M. Baskin, C. R. Bertozzi, QSAR Comb. Sci. 2007, 26, 1211–1219.

[122] R. Garrett, C. Grisham, Biochemistry 4th ed., Brooks/Cole Cengage Learning,

Boston, 2007, 103–105.

[123] R. Lundblad, Chemical Reagents for Protein Modification 3rd ed., CRC Press, Boca

Raton, 2010, 269–279.

[124] R. Reid, Peptide and Protein Drug Analysis Drugs and the Pharmaceutical Sciences

Series, Marcel Dekker, New York, 1999, 260–262.

[125] T. Bissot, R. Parry, D. Campbell, J. Am. Chem. Soc. 1957, 79, 796–800.

[126] S. Pothukanuri, N. Winssinger, Org. Lett. 2007, 9, 2223–2225.

[127] M. F. Gordeev, G. W. Luehr, H. C. Hui, E. M. Gordon, D. V. Patel, Tetrahedron

1998, 54, 15879–15890.

[128] J. C. Howard, J. Org. Chem. 1981, 46, 1720–1723.

[129] S. Wolfe, M.-C. Wilson, M.-H. Cheng, G. V. Shustov, C. I. Akuche, Can. J. Chem.

2003, 81, 937–960.

[130] T. Thorsteinsson, M. Masson, T. Jarvinen, T. Nevalainen, T. Loftsson, Chem.

Pharm. Bull. 2002, 50, 554–557.

[131] A. G. Griesbeck, S. Bondock, J. Lex, J. Org. Chem. 2003, 68, 9899–9906.

[132] P. De, G. Koumba Yoya, P. Constant, F. Bedos-Belval, H. Duran, N. Saffon,

M. Daffe, M. Baltas, J. Med. Chem. 2011, 54, 1449–1461.

[133] U. Sreenivasan, R. K. Mishra, R. L. Johnson, J. Med. Chem. 1993, 36, 256–263.

[134] W. Fuller, V. Yalamoori, in Methods of organic chemistry (Houben-Weyl), Vol. E 22a

(Ed.: M. Goodman, A. Felix, L. Moroder, C. Toniolo), Georg Thieme Verlag,

Stuttgart, 4. ed., 2004.

[135] A. Prox, F. Weygand, Peptides, Proc. Eur. Peptide Symp. 8th, Noordwijk, Neth. 1967,

158–172.

[136] T. Wieland, G. Lueben, H. Ottenheym, J. Faesel, J. X. De Vries, A. Prox,

J. Schmid, Angew. Chem., Int. Ed. Engl. 1968, 7, 204–208.

[137] R. R. Burgess, in Methods in Enzymology, Vol. 463 (Guide to protein purification)

(Ed.: M. P. Burgess, Richard R. Deutscher), Elsevier/Academic Press, Oxford,

2nd ed., 2009.

[138] S. Rastogi, Biochemistry 3rd ed., Tata McGraw-Hill, Delhi, 2010.

[139] L. J. Farrugia, J. Appl. Crystallogr. 1999, 32, 837–838.

135

7 Bibliography

[140] A. Altomare, G. Cascarano, C. Giacovazzo, A. Guagliardi, J. Appl. Crystallogr.

1993, 26, 343–50.

[141] G. M. Sheldrick, in Institut für Anorganische Chemie der Universität Göttingen.

[142] H. Duerr, K. Zengerle, H. P. Trierweiler, Z. Naturforsch., B: Chem. Sci. 1988, 43,

361–367.

[143] S. Caron, N. M. Do, J. E. Sieser, Tetrahedron Lett. 2000, 41, 2299–2302.

[144] L. Della Ciana, W. J. Dressick, A. Von Zelewsky, J. Heterocycl. Chem. 1990, 27,

163–165.

136

Appendix

A Data for crystal structures

Compound 16

Identification code 6,6’-bis(aminomethyl)-2,2’-bipyridinetrihydrobromide trihydrate

Empirical formula C12H19Br3N4OFormula weight 475.04Temperature 173(2)Wavelength 0.71073 Å

Crystal system, space group monoclinic P2(1)/nUnit cell dimensions a = 11.377(9) Å alpha = 90 deg.

b = 6.818(5) Å beta = 104.657(15) deg.c = 22.214(19) Å gamma = 90 deg.

Volume 1667(2) Å-3

Z, Calculated density 4 1.893 Mg/m3

Absorption coefficient 7.261 mm−1

F(000) 928Crystal size 0.200 x 0.200 x 0.200 mm

Theta range for data collection 2.97 to 25.00 deg.Limiting indices -13<=h<=13, -8<=k<=8, -26<=l<=26Reflections collected / unique 13687 / 2924 [R(int) = 0.1790]

Completeness to theta = 25.00 99.6%Absorption correction NoneRefinement method Full-matrix least-squares on F2

Data / restraints / parameters 2924 / 0 / 217Goodness-of-fit on F2 1.002Final R indices [I>2sigma(I)] R1 = 0.0403, wR2 = 0.1024

R indices (all data) R1 = 0.0442, wR2 = 0.1055Largest diff. peak and hole 1.121 and -0.718 e.Å-3

i


Table 7.1: Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (A2 x 103) for

16. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.

x y z U(eq)

Br(1) 6177(1) 7095(1) 2433(1) 25(1)Br(2) 2231(1) 403(1) 1796(1) 25(1)Br(3) -4832(1) 18223(1) -885(1) 29(1)N(3) -54(3) 7251(4) 1081(2) 16(1)O(1) 2227(3) 5190(5) 1702(2) 25(1)C(3) 2404(4) 7053(5) 82(2) 19(1)N(4) 1558(3) 7163(4) 399(2) 17(1)N(1) -799(4) 7262(7) 2607(2) 25(1)N(2) 3803(3) 7225(5) 1126(2) 26(1)C(8) -494(3) 7519(5) 459(2) 17(1)

C(11) 42(4) 7534(5) -562(2) 23(1)C(12) 2143(4) 7157(5) -562(2) 25(1)C(13) -2453(4) 8036(6) 647(2) 23(1)C(14) -1729(4) 7879(5) 232(2) 20(1)C(15) -1955(4) 7753(6) 1284(2) 22(1)C(16) -47(4) 6976(6) 2161(2) 25(1)C(17) -734(3) 7357(5) 1500(2) 17(1)C(19) 386(4) 7398(5) 77(2) 16(1)C(20) 3707(4) 6820(6) 466(2) 27(1)C(26) 933(4) 7390(6) -893(2) 26(1)

Table 7.2: Bond lengths [Å] for 16

Bond lengths [Å]

N(3)-C(17) 1.354(5)N(3)-C(8) 1.357(5)C(3)-N(4) 1.330(5)C(3)-C(12) 1.389(6)C(3)-C(20) 1.519(6)N(4)-C(19) 1.354(5)N(1)-C(16) 1.478(6)N(2)-C(20) 1.469(6)C(8)-C(14) 1.389(6)C(8)-C(19) 1.470(6)C(11)-C(19) 1.377(5)C(11)-C(26) 1.398(7)C(12)-C(26) 1.395(6)C(13)-C(14) 1.387(6)C(13)-C(15) 1.398(6)C(15)-C(17) 1.377(6)C(16)-C(17) 1.502(5)

Symmetry transformations used to generate equivalent atoms

ii

Appendix

Table 7.3: Bond lengths [A] and angles [deg] for 16

Angle [deg]

C(17)-N(3)-C(8) 124.4(4)N(4)-C(3)-C(12) 123.2(4)N(4)-C(3)-C(20) 116.3(4)C(12)-C(3)-C(20) 120.5(4)C(3)-N(4)-C(19) 118.4(4)N(3)-C(8)-C(14) 118.1(4)N(3)-C(8)-C(19) 116.8(3)C(14)-C(8)-C(19) 125.0(3)C(19)-C(11)-C(26) 118.8(4)C(3)-C(12)-C(26) 118.3(4)C(14)-C(13)-C(15) 120.3(4)C(13)-C(14)-C(8) 119.2(4)C(17)-C(15)-C(13) 119.6(4)N(1)-C(16)-C(17) 112.8(4)N(3)-C(17)-C(15) 118.2(4)N(3)-C(17)-C(16) 114.6(4)C(15)-C(17)-C(16) 127.2(4)N(4)-C(19)-C(11) 122.6(4)N(4)-C(19)-C(8) 115.1(3)C(11)-C(19)-C(8) 122.3(4)N(2)-C(20)-C(3) 110.9(4)

C(12)-C(26)-C(11) 118.8(4)

iii


Table 7.4: Anisotropic displacement parameters (A2 x 103) for 16. The anisotropic displacement factor

exponent takes the form: -2 π2 [ h2 a*2 U11 + ... + 2 h k a* b* U12 ]

U11 U22 U33 U23 U13 U12

Br(1) 24(1) 30(1) 24(1) 2(1) 9(1) 1(1)Br(2) 29(1) 22(1) 22(1) -1(1) 3(1) -2(1)Br(3) 31(1) 35(1) 21(1) 3(1) 6(1) -6(1)N(3) 15(2) 16(2) 18(2) 0(1) 3(1) -1(1)O(1) 36(2) 20(2) 20(2) 3(1) 9(1) 2(1)C(3) 23(2) 15(2) 23(2) -2(1) 12(2) 0(1)N(4) 18(2) 15(2) 19(2) 2(1) 8(1) 1(1)N(1) 26(2) 34(2) 16(2) 0(2) 7(2) -3(2)N(2) 21(2) 26(2) 28(2) 7(2) 4(2) 1(2)C(8) 22(2) 11(2) 18(2) -3(1) 5(2) -5(1)

C(11) 29(2) 19(2) 17(2) 1(2) 2(2) -2(2)C(12) 37(3) 17(2) 25(2) -2(2) 16(2) -3(2)C(13) 15(2) 25(2) 25(2) -2(2) 0(2) 0(2)C(14) 20(2) 20(2) 18(2) 0(2) -1(2) -1(1)C(15) 22(2) 26(2) 21(2) -3(2) 10(2) -1(2)C(16) 20(2) 38(2) 19(2) 2(2) 7(2) 3(2)C(17) 20(2) 14(2) 19(2) 0(1) 9(2) -1(1)C(19) 22(2) 12(2) 16(2) -1(1) 6(2) -2(1)C(20) 21(2) 31(2) 32(2) 3(2) 11(2) 4(2)C(26) 38(3) 21(2) 18(2) -3(2) 8(2) -4(2)

iv

Appendix

Compound 69

Identification code acetylcycloserine

Empirical formula C5H8N2O3Formula weight 144.13Temperature 173(2) KWavelength 0.71075 Å

Crystal system, space group orthorhombic P212121Unit cell dimensions a = 4.985(11) Å alpha = 90 deg.

b = 9.67(2) Å beta = 90 deg.c = 13.62(3) Å gamma = 90 deg.

Volume 657(2) Å-3


Absorption coefficient 0.121 mm−1F(000) 304Crystal size 0.36 x 0.12 x 0.12 mm


Completeness to theta = 24.98 99.4 %Max. and min. transmission 0.9855 and 0.9572Refinement method Full-matrix least-squares on F2


R indices (all data) R1 = 0.0440, wR2 = 0.0954Absolute structure parameter 0(2)Largest diff. peak and hole 0.190 and -0.167 e.Å-3

v



xxx. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.

x y z U(eq)

O(1) 622(3) 9497(2) 1577(1) 33(1)O(2) 4515(4) 7204(2) 3257(1) 35(1)N(3) 2461(4) 6568(2) 2700(1) 27(1)N(4) 4435(4) 8813(2) 860(2) 25(1)O(5) 1302(4) 6237(2) 1113(1) 38(1)C(6) 2698(4) 6770(2) 1749(2) 23(1)C(7) 2247(4) 9600(2) 894(2) 24(1)C(8) 5005(4) 7781(2) 1599(2) 24(1)C(9) 1840(6) 10611(3) 77(2) 34(1)

C(10) 5470(5) 8303(2) 2633(2) 30(1)

Table 7.6: Bond lengths [Å] for acetylcycloserine

Bond lengths [Å]

O(1)-C(7) 1.237(3)O(2)-N(3) 1.415(3)O(2)-C(10) 1.441(3)N(3)-C(6) 1.316(4)N(3)-H(3) 0.8800N(4)-C(7) 1.330(4)N(4)-C(8) 1.446(3)N(4)-H(1) 0.86(3)O(5)-C(6) 1.225(3)C(6)-C(8) 1.523(4)C(7)-C(9) 1.496(4)

C(8)-C(10) 1.515(5)C(8)-H(8) 1.0000

C(9)-H(9A) 0.9800C(9)-H(9B) 0.9800C(9)-H(9C) 0.9800

C(10)-H(10A) 0.9900C(10)-H(10B) 0.9900


vi

Appendix

Table 7.7: Bond lengths [A] and angles [deg] for acetylcycloserine

Angle [deg]

N(3)-O(2)-C(10) 104.2(2)C(6)-N(3)-O(2) 113.4(2)C(6)-N(3)-H(3) 123.3O(2)-N(3)-H(3) 123.3C(7)-N(4)-C(8) 122.1(2)C(7)-N(4)-H(1) 117.9(18)C(8)-N(4)-H(1) 118.9(18)O(5)-C(6)-N(3) 125.6(2)O(5)-C(6)-C(8) 127.1(2)N(3)-C(6)-C(8) 107.22(19)O(1)-C(7)-N(4) 121.2(2)O(1)-C(7)-C(9) 121.5(2)N(4)-C(7)-C(9) 117.3(2)

N(4)-C(8)-C(10) 116.6(2)N(4)-C(8)-C(6) 112.9(2)C(10)-C(8)-C(6) 101.8(2)N(4)-C(8)-H(8) 108.4C(10)-C(8)-H(8) 108.4C(6)-C(8)-H(8) 108.4

C(7)-C(9)-H(9A) 109.5C(7)-C(9)-H(9B) 109.5

H(9A)-C(9)-H(9B) 109.5C(7)-C(9)-H(9C) 109.5

H(9A)-C(9)-H(9C) 109.5H(9B)-C(9)-H(9C) 109.5O(2)-C(10)-C(8) 104.6(2)

O(2)-C(10)-H(10A) 110.8C(8)-C(10)-H(10A) 110.8O(2)-C(10)-H(10B) 110.8C(8)-C(10)-H(10B) 110.8

H(10A)-C(10)-H(10B) 108.9

vii


Table 7.8: Anisotropic displacement parameters (A2 x 103) for acetylcycloserine. The anisotropic dis-

placement factor exponent takes the form: -2 π2 [ h2 a*2 U11 + ... + 2 h k a* b* U12 ]

U11 U22 U33 U23 U13 U12

O(1) 33(1) 32(1) 36(1) 4(1) 11(1) 7(1)O(2) 48(1) 35(1) 22(1) 6(1) -8(1) -16(1)N(3) 30(1) 28(1) 24(1) 3(1) -2(1) -10(1)N(4) 28(1) 28(1) 19(1) 6(1) 7(1) 4(1)O(5) 47(1) 38(1) 27(1) -1(1) -11(1) -8(1)C(6) 27(1) 19(1) 23(1) 0(1) -2(1) 5(1)C(7) 25(1) 21(1) 26(1) -1(1) 1(1) 0(1)C(8) 23(1) 25(1) 24(1) 4(1) 2(1) 4(1)C(9) 44(2) 31(1) 28(1) 5(1) -5(1) 5(1)C(10) 34(1) 31(1) 25(1) 5(1) -3(1) -8(1)

viii

Appendix

Compound 70

Identification code N-methyl-acetylcycloserine

Empirical formula C6 H10 N2 O3Formula weight 158.16Temperature 293(2) KWavelength 0.71075 Å

Crystal system, space group orthorhombic P212121Unit cell dimensions a = 5.2295(9) Å alpha = 90 deg.

b = 8.3281(15) Å beta = 90 deg.c = 17.934(3) Å gamma = 90 deg.

Volume 781(2) Å-3


Absorption coefficient 0.109 mm−1F(000) 336Crystal size 0.49 x 0.06 x 0.04 mm


Completeness to theta = 24.98 99.6 %Max. and min. transmission 0.9962 and 0.9483Refinement method Full-matrix least-squares on F2


R indices (all data) R1 = 0.0592, wR2 = 0.1042Absolute structure parameter 1(2)Largest diff. peak and hole 0.133 and -0.152 e.Å-3

ix



xxx. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.

x y z U(eq)

C(1) 461(8) 4094(5) 5584(2) 64(1)C(2) 993(5) 4683(3) 4815(1) 46(1)C(3) 3652(5) 6369(3) 4026(1) 44(1)C(4) 1435(5) 7134(3) 3613(1) 43(1)C(5) 35(7) 7082(5) 2279(2) 60(1)C(6) 4642(5) 5167(4) 3456(2) 53(1)N(1) 2949(4) 5730(3) 4738(1) 47(1)N(2) 1589(4) 6684(3) 2905(1) 49(1)O(1) -243(4) 4244(2) 4272(1) 61(1)O(2) -209(4) 8028(2) 3865(1) 63(1)O(3) 3749(4) 5724(3) 2746(1) 57(1)

Table 7.10: Bond lengths [Å] for N-methyl-acetylcycloserine

Bond lengths [Å]

C(1)-C(2) 1.490(4)C(1)-H(2) 0.99(4)C(1)-H(3) 0.92(5)C(1)-H(1) 1.00(5)C(2)-O(1) 1.224(3)C(2)-N(1) 1.351(3)C(3)-N(1) 1.432(3)C(3)-C(4) 1.516(4)C(3)-C(6) 1.521(4)C(3)-H(5) 0.90(3)C(4)-O(2) 1.223(3)C(4)-N(2) 1.326(3)C(5)-N(2) 1.425(3)C(5)-H(8) 1.00(4)C(5)-H(6) 0.98(4)C(5)-H(7) 0.96(4)C(6)-O(3) 1.434(3)C(6)-H(9) 1.01(3)

C(6)-H(10) 0.95(3)N(1)-H(4) 0.84(3)N(2)-O(3) 1.413(3)


x

Appendix

Table 7.11: Bond lengths [A] and angles [deg] for acetylcycloserine

Angle [deg]

C(2)-C(1)-H(2) 116(2)C(2)-C(1)-H(3) 115(3)H(2)-C(1)-H(3) 109(3)C(2)-C(1)-H(1) 113(2)H(2)-C(1)-H(1) 107(3)H(3)-C(1)-H(1) 94(3)O(1)-C(2)-N(1) 120.7(2)O(1)-C(2)-C(1) 122.6(3)N(1)-C(2)-C(1) 116.6(2)N(1)-C(3)-C(4) 113.3(2)N(1)-C(3)-C(6) 116.2(2)C(4)-C(3)-C(6) 102.0(2)N(1)-C(3)-H(5) 107.7(16)C(4)-C(3)-H(5) 108.7(15)C(6)-C(3)-H(5) 108.6(16)O(2)-C(4)-N(2) 124.6(2)O(2)-C(4)-C(3) 127.8(2)N(2)-C(4)-C(3) 107.6(2)N(2)-C(5)-H(8) 108(2)N(2)-C(5)-H(6) 108(2)H(8)-C(5)-H(6) 111(3)N(2)-C(5)-H(7) 110(2)H(8)-C(5)-H(7) 106(3)H(6)-C(5)-H(7) 114(3)O(3)-C(6)-C(3) 105.8(2)O(3)-C(6)-H(9) 110.8(17)C(3)-C(6)-H(9) 108.0(17)

O(3)-C(6)-H(10) 105(2)C(3)-C(6)-H(10) 111(2)H(9)-C(6)-H(10) 116(3)C(2)-N(1)-C(3) 121.7(2)C(2)-N(1)-H(4) 117(2)C(3)-N(1)-H(4) 122(2)C(4)-N(2)-O(3) 113.73(19)C(4)-N(2)-C(5) 130.8(2)O(3)-N(2)-C(5) 115.3(2)N(2)-O(3)-C(6) 105.27(18)

xi


Table 7.12: Anisotropic displacement parameters (A2 x 103) for acetylcycloserine. The anisotropic dis-

placement factor exponent takes the form: -2 π2 [ h2 a*2 U11 + ... + 2 h k a* b* U12 ]

U11 U22 U33 U23 U13 U12

C(1) 80(2) 58(2) 53(2) 14(2) -3(2) -8(2)C(2) 47(1) 46(1) 45(1) 4(1) -10(1) 0(1)C(3) 40(1) 51(2) 42(1) 0(1) -6(1) -3(1)C(4) 47(1) 42(1) 40(1) 3(1) 4(1) 1(1)C(5) 60(2) 82(2) 39(2) 13(2) -4(1) 13(2)C(6) 40(1) 67(2) 52(2) -3(1) -6(1) 9(1)N(1) 49(1) 58(1) 34(1) 1(1) -13(1) -10(1)N(2) 47(1) 64(2) 37(1) 0(1) 2(1) 20(1)O(1) 61(1) 67(1) 54(1) 1(1) -19(1) -16(1)O(2) 73(1) 67(1) 49(1) 1(1) 10(1) 27(1)O(3) 52(1) 78(1) 42(1) -7(1) 3(1) 24(1)

xii

Appendix

B Publication list

Publications with peer review process

A1. Understanding the quenching effects of aromatic C-H- and C-D-oscillators in near-IR

lanthanoid luminescence

C. Doffek, N. Alzakhem, C. Bischof, J. Wahsner, T. Gueden, J. Lügger, C. Platas-

Iglesias, M. Seitz*, J. Am. Chem. Soc. 2012, 134, 16413-16423.

A2. Anomalous reversal of aromatic C-H and C-D quenching efficiencies in luminescent

praseodymium cryptates

J. Scholten, G.A. Rosser, J. Wahsner, N. Alzakhem, C. Bischof, F. Stog, A. Beeby,

M. Seitz*, J. Am. Chem. Soc. 2012, 134, 13915-13917.

A3. The dependence of the photophysical properties on the number of 2,2’-bipyridine units in

a series of luminescent Europium and Terbium cryptates

N. Alzakhem, C. Bischof, M. Seitz*, Inorg. Chem. 2012, 51, 9343-9349

A4. Quantification of C-H quenching in near-IR luminescent ytterbium and neodymium

cryptates

C. Bischof, J. Wahsner, J. Scholten, S. Trosien, M. Seitz* , J. Am. Chem. Soc. 2010,

132, 14334-143353.

A5. A [4+2] mixed ligand approach to ruthenium DNA metallointercalators [Ru(tpa)(N-

N)](PF6)2 using a tris(2-pyridylmethyl)amine (tpa) capping ligand

S. Seeberg (nee Kraft), C. Bischof, A. Loos, S. Braun, N. Jafarova, U. Schatz-

schneider*, J. Inorg. Biochem. 2009, 103, 1126-1134.

A6. [N,N’-Bis(salicylidene)-1,2-phenylenediamine]metal complexes with cell death promoting

properties

A. Hille, I. Ott, A. Kitanovic, I. Kitanovic, H. Alborzina, E. Lederer, S. Wölfl,

N. Metzler-Nolte, S. Schäfer, W.S. Sheldrick, C. Bischof, U. Schatzschneider, R.

Gust*, J. Biol. Inorg. Chem. 2009, 14, 711-725.

The following above mentioned publications have evolved from my doctoral disserta-

tion: A1, A2, A3 and A4.

xiii

B Publication list

Publications without peer review process

Conference talks

B1. C. Bischof: Selective deuteration of Lehn cryptands and its influence on near-IR lumin-

escent lanthanides. Utrecht University and IMPRS Joint Symposium on Chemical

Biology in Utrecht, Netherlands. May 2011.

B2. C. Bischof: Quantification of C-H quenching in near-IR luminescent lanthanide crypt-

ates. 7th Coordination Chemistry Meeting in Stuttgart, Germany. March 2011.

B3. C. Bischof: Synthesis of ruthenium CO releasing molecules and their behaviour un-

der physiological conditions. 12th JCF Spring Symposium in Goettingen, Germany.

March 2010.

B4. C. Bischof: Synthesis and characterisation of ruthenium complexes with photolabile CO

and NO ligands. 4th workshop of DFG research group 630 (“Biological function of

organometallic compounds”) in Bergisch Gladbach, Germany. October 2007.

Conference posters

C1. C. Bischof: UnClicking biorelevant bonds with a chemical trigger. Challenges in Or-

ganic Chemistry and Chemical Biology (ISACS7) in Edinburgh, Scotland. June

2012.

C2. C. Bischof: Quantification of C-H quenching in near-IR luminescent lanthanide crypt-

ates. 242nd ACS National Meeting in Denver/CO, USA. September 2011.

C3. C. Bischof: Selective Deuteration of Lanthanoid Bipyridine Cryptates. 39th Interna-

tional Conference on Coordination Chemistry in Adelaide, Australia. July 2010.

The following above mentioned publications have evolved from my doctoral disserta-

tion: B1, B2, C1, C2 and C3

xiv

Documents

Building blocks for multinulear near-IR luminescent ... luminescent lanthanide complexes Caroline Bischof Ruhr-Universität Bochum Faculty of Chemistry and Biochemistry Building blocks