“Click” Chemistry, a New Approach to Familiar Reactions

Andrea MolengraftJanuary 26, 2005

“Click” Chemistry?

• As defined by K. B. Sharpless

– “‘Click’ chemistry…a set of powerful, virtually 100% reliable, selective reactions for the rapid synthesis of new compounds via heteroatom links (C-X-C)…Click chemistry is integral now to all research within theSharpless Lab.”

Borman, S. C & En. 2002, 80(6), 29.

“Click” Chemistry?• “Strategy for the rapid and efficient assembly of molecules with

diverse functionality…enabled by a few nearly perfect reactions, it guarantees reliable synthesis of the desired products in high yield and purity…”

– Brik, A.; Muldoon, J.; Lin, Y.; Elder, J. Goodsell, D. Olson, A.; Fokin, V.; Sharpless, B.; Wong, H. Chem. Bio. Chem. 2003, 4, 1246.

• “Designing powerful and selective reactions for an efficient synthesis of interesting compounds and combinatorial librariesthrough heteroatom links…” The Huisgen 1,3-dipolar cycloaddition of azides and alkynes is regarded as the ‘cream of the crop’ of concerted reactions…”

– Lober, S.; Rodriguez-Loaiza, P.; Gmeiner, P. Org. Lett. 2003, 5, 1753.

• “Synthetic appeal…high yields, simple reaction conditions, tolerance of oxygen and water, and simple product isolation...”

– Helms, B.; Mynar, J; Hawker, C.; Frechet, J. J. Am. Chem. Soc. 2004, 126, 15020.

Classes of “Click” Reactions

• Nucleophilic opening of highly strained rings– SN2 ring opening reactions– Epoxides, aziridines, cyclic sulfates, cyclic sulfamidates, aziridinium

ions

• “Protecting Group” Reactions– Reversible carbonyl chemistry– Acetals, ketals and their aza-analogs

• Cycloaddition Reactions– Hetero Diels-Alder, 1,3 dipolar cycloadditions involving heteroatoms

R

N3

R

N NN

R'

R'

R

N NNR'

H

H

H

+

1,4 triazole 1,5 triazole

+

Kolb, H.; Finn, M.; Sharpless, B. Angew. Chem. Int. Ed. 2001, 40, 2004.

Applications of Triazoles

• Agricultural– Fungicides– Herbicides– Antimicrobial

• Medicinal– Cytostatic– Virostatic– Antiinflamatory

• Industrial– Photostabilizers– Fluorescent whiteners– Optical brightening agents– Corrosion retardants

• Macromolecules– Drug delivery– Nanoscale electronics– Oligonucleotides

Tornoe, C.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.

Huisgen’s [1,3] Dipolar Cycloaddition

• Cycloaddition between azides and acetylenes to formtriazoles

R

N N N

R

N NN

R'

R'

R

N NNR'H

H

∆

H

R

NN

NR'

HR

NN

NH

R'

+

1,4 triazole 1,5 triazole

+

1 2

34

1 2

34

5

1:1

1 2 3

12

3

4

12 3

or

5

5

45

Huisgen, R. 1984. 1,3-dipolar cycloaddition- Introduction, survey, mechanism. In: Padwa, A.; ed. 1,3-dipolar Cycloaddition Chemistry (Vol. 1). Wiley. P. 1-176.

Regioselectivity of “Click” Chemistry

• Addition of Cu(I)-catalyst– “the champion “click” process…”

• Alkyne activation

Rostovtsev, V., Green, L., Fokin, V. Sharpless. B. Angew. Chem Int. Ed. 2002, 41, 2596. Li, Z.; Seo, T.; Ju, J. Tetrahedron. Lett. 2004, 45, 3143.

R

N N N

R

N NNR'

R'

H

H+

1,4 triazole

1 2 34

5 1 2

34

5[Cu]

Proposed catalytic cycle for Cu(I)-catalyzed ligation

Rostovtsev, V.; Green, L.;Fokin, V;Sharpless. B. Angew. Chem Int. Ed. 2002, 41, 2596.Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 16, 4467.Tornoe, C.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.

NN

N

R1

R2

NN

N

R1

R2CuLn

CuLn

NR2 CuLnR1

N N NR2

NN

NCuLn

R1

R1 HR1

III

III

IV

NN

[CuLn]+DirectStepwise

V 1,4 triazoleR2

Rostovtsev, V.; Green, L.; Fokin, V.; Sharpless, B. Angew. Chem Int. Ed. 2002, 41, 2596.

Regioselectivity using Cu(I)

NN

N NNN

O

H

CuSO4 H2O (1mol%) Na ascorbate (5mol%) H2O: tBuOH (2:1), r.t., 8 hr

O H

91 %

+

12

3

45

1,4 substituted triazole

Cu catalyzed:

NN

N

O

H

neat, 92oC, 18 h

+NN

N

O H

12

3

45

NNN

HO

12

34

5

(1,4) (1,5)1.6 : 1

+

Thermal:

12

3

12

3

45

45

Rostovtsev, V.; Green, L.; Fokin, V.; Sharpless, B. Angew. Chem Int. Ed. 2002, 41, 2596.

Functional Group ToleranceNN

N PhO

O

NNN Ph

NNN Ph

OH

NN

N

Ph

NNN

NN

N NH

SO2NH2

NNN

NNN

OH

OOPh

NNN OH

Ph

NNN N

PhO

O

NNN

PhNEt2

NN N

OH

HO

HONNN

Ph

NHHN

NH2

NH

NH2HN

OHHO OH

H

H

H

R

NN

N

R'

H

92 %

93%

82%

84 %

91 %

88 %

88 %

84 %

88 %

90 %

94 %

4

+

[0.25-0.5] M

CuSO4 (1mol%)Na ascorbate (10 mol%)H2O: tBuOH12-24 hr.

12

3

5

Regioselectivity of “Click” Chemistry

• Addition of Cu(I)-catalyst– “the champion “click” process…”

• Alkyne activation

Rostovtsev, V.; Green, L.; Fokin, V.; Sharpless, B. Angew. Chem Int. Ed. 2002, 41, 2596. Li, Z.; Seo, T.; Ju, J. Tetrahedron Lett. 2004, 45, 3143.

R

N N N

R

N NNR'

R'

H

H+

1,4 triazole

1 2 34

5 1 2

34

5[Cu]

Li, Z.; Seo, T.; Ju, J. Tetrahedron Lett. 2004, 45, 3143.

Regioselectivity by Alkyne Activation

N3EtO

O

R' O

NNN

EtO

O

EtO

OEt

OO

NNN

EtO

O 94%

82%

NNN

EtO

O

OEt

O

NNN

EtO

OOMe

O

O SO

O

67%

+ H2O, rt, 6-12 h

OR''O

90%

85%

N/A

N/A

no [Cu] CuCl (0.1eq.), 1 h

81%

“Click” Applications in Macromolecules

• Dendrimer Synthesis

• Solid Support Chemistry– SPOS– SPPS

• DNA functionalization

Dendrimer Synthesis

Divergent-Growth

Convergent-Growth

Tomalia, I. Polymer J. 1985, 17, 117.Frechet, J.; Hawker, C. J. Amer. Chem. Soc. 1990, 112, 7638.

Dendrimer Synthesis

Wu, P.; Feldman, A.; Nugent, A.; Hawker, C.; Scheel, A.; Voit, B.; Pyun, J.; Frechet, J.; Sharpless, B.; Fokin, V.Angew. Chem. Int. Ed. 2004, 43, 3928.

RN

NX

Cl

NN

NR

X

Cl

NNN

R NN

NR

X

N3

NNN

R

X

Cl

N

+ 1. CuSO4 (5 mol %) Na ascorbate (10 mol %) H2O/tBuOH (1:1)

2. 1.5 equiv NaN3, acetone/H2O (4:1) 60oC, 1-3 h

[G-1]

2 equiv.+

3rd Generation [G-3]

1st Generation [G-1]

NN

N

X

N3

NNN

NN

NR

XNNN

RNN

NR

XNNN

R

NN

N

X

N3

NNN

2nd Generation [G-2]

NN

N

XNNN

NN

NR

XNNN

RNN

NR

XNNN

R

NN

N

XNNN

NN

NR

XNNN

RNN

NR

XNNN

R

1. CuSO4 (5 mol %) Na ascorbate (10 mol %) H2O/tBuOH (1:1)2. 1.5 equiv NaN3, acetone/H2O (4:1) 60oC, 1-3 h

> 95%

> 90%

Dendrimer Synthesis

ClO

O

NCl

O

Cl

SN

OO

N3HO

N3

O

ButO NH

N3

O

N3HO

N3O

ON3

Azides:

Alkynes:

OO

O

O

O

O

N

N

O O

OO

N

N

N

O

OO

branching "cores"

Central "cores"

Dendrimer Synthesis

• 4th generation dendrimer synthesis

Wu, P.; Feldman, A.; Nugent, A.; Hawker, C.; Scheel, A.; Voit, B.; Pyun, J.; Frechet, J.; Sharpless, B.; Fokin, V.Angew. Chem. Int. Ed. 2004, 43, 3928.

N3 NCl

O

N

N

N

O

OO

NN

N

X

N3

NNN

NN

N

XNNN

NN

NR

X

NNN

RNN

NR

XNNN

R

NN

N

XNNN

NN

NR

XNNN

RNN

NR

X

NNN

R

+

3rd Generation [G-3]

Cu(I)r.t., 30 h

Wu, P.; Feldman, A.; Nugent, A.; Hawker, C.; Scheel, A.; Voit, B.; Pyun, J.; Frechet, J.; Sharpless, B.; Fokin, V.Angew. Chem. Int. Ed. 2004, 43, 3928.

92 % yield

NN NO

N NN

ON

NN

N

O

N

NN

NO

N

N NN

NO

NNN

NNN

NN

N

NNN

NN N

O N

N NN

O

N

NNN

N

O

N NNN

N N

NNN

N NN

OO

NNN

O

NN

N NO

N

N NN

O

N

NNN

N

O

N NN

NN N

NNN

N NN

NNN

ONN

N N

ON NNN

NO

NNN

NNN

N NN

NN

N

NNN O

N

NNN

ONN

N N

ON

NNN

NO

NNN

NNN

N NN

NN

N

NN

N

O

N

NNN

ON

NN N

NO

NNN

NN

N

NN

N

NNN

Dendronized linear polymers

• Three main pathways– I “grafting-to”

– II “grafting-from”

– III incorporate dendrons into the monomer

Helms, B.; Mynar,J; Hawker, C.; Frechet, J. J. Am. Chem. Soc. 2004, 126, 15020.

+

Ochiai, B.; Tomalia, I.; Endo, T. Macromol. Chem. Phys. 2001, 202, 3099.Helms, B.; Mynar, J.; Hawker, C.; Frechet, J. J. Am. Chem. Soc. 2004, 126, 15020.

Dendronized linear polymers

BrTMS

H TMS TMS

KOH, H2OTHF, MeOH

H

H

OO

N3O

O

N NN

+AIBN60oC, 24 h

n nPd/Cl2(PPh3)2CuI, Et3N

poly vinylacetylene(PV acet)

+

1st generation dendrimer

CuSO4 5H2O (5 mol %) Na ascorbate (10 mol %)1:1 THF/H2O

n n

PV acet

Quantitative yield

“Click” Applications in Macromolecules

• Dendrimer Synthesis

• Solid Support Chemistry– SPOS– SPPS

• DNA functionalization

“Click” Chemistry Using a Solid Support

• Solid Phase Organic Synthesis (SPOS)

• Solid Phase Peptide Synthesis (SPPS)

N3

Lober, S.; Rodriguez-Loaiza, P.; Gmeiner, P. Org. Lett. 2003, 5, 1753.

SPOS: Solid Phase Organic Synthesis

NH2 HO2C

CHOAr N

H

CHOAr

O

N3

CHOAr

NCHO

Ar

N N

+

+

BAL Resin:

"Click" Resin

SPOS: Library of Tertiary Amines

Lober, S.; Gmeiner, P. Tetrahedron, 2004, 60, 8699.

N3

NN N

O

OO

O

ArCHO

NN N

ArCHO

= polystyrene

(Backbone Amide Linker)

(Regenerative Michael Acceptor)

Library of amides

Library of tertiary amines

"click"

"click"

REM

BAL

Lober, S.; Gmeiner, P. Tetrahedron, 2004, 60, 8699.

SPOS: Library of Tertiary Amines• Triazolylmethyl acrylate (TMA) Resin

Cl N3 NN N

O

O

NN N

O

ON

BnCl

Cl N

O

O

NaN3, DMSO, 70oC, 48 h CuI,DMF, THF,DIPEA

35oC, 10 h

benzylmethylamineDMFrt, 16 h

1. DMSO, rt, 16h2. TEA, DMF,rt, 16 h

TMA Resin

Cl

ClBr

68%

+TMA Resin

66%

• Parallel synthesis: TMA & REM Resins

47 (>90)53 (91)p-NitrobenzylN-Phenyl-piperazinee

75 (>90)79 (93)AllylN-Phenyl-piperazined

73 (>90)75 (92)CH2CO2MeTetrahydro-isochinolinec

63 (>90)77 (92)p-NitrobenzylTetrahydro-isochinolineb

88 (>90)82 (95)AllylTetrahydro-isochinolinea

REMyield (purity)

TMA yield (purity)

R’’NRR’

Lober, S.; Gmeiner, P. Tetrahedron, 2004, 60, 8699.

NN N

O

O

O

O

NH

tetrahydro-isochinoline

NHN

N-phenyl piperazine

NN N

O

O

NN N

O

O

NRR'H-NRR' NRR'R''1. R''Br2. cleavage

a-eTMA Resin

SPOS: Selective Receptor Ligands

Lober, S.; Rodriguez-Loaiza, P.; Gmeiner, P. Org. Lett. 2003, 5, 1753.

N3 ArCHO

NN N

ArCHO

= polystyrene

SPOS by BAL

Library of lead amides

"click"

CHO

O

R

R'N

N N

N

CHO

NN N

FAMT FIMT

FAMT: Formyl Aryloxy Methyl Triazole

FIMT: Formyl Indole Methyl Triazole

Lober, S.; Rodriguez-Loaiza, P.; Gmeiner, P. Org. Lett. 2003, 5, 1753.

SPOS: Selective Receptor Ligands

CHO

R'

R

OH

CHO

O

R

R'

CHO

O

R

R'N

N N

N3

N

N

N

CHO N

CHO

NN N

Br

Br

DMF, K2CO3,70oC, 24 h

THF, DIPEA,CuI35oC, 16 h

1. THF, rt to 60oC, 1 h2. NaOH, H2O rt, 0.5 h

FAMT

FIMT

+

+ THF, DIPEA,CuI35oC, 16 h

Lober, S.; Rodriguez-Loaiza, P.; Gmeiner, P. Org. Lett. 2003, 5, 1753.

SPOS: Selective Receptor Ligands

O

CHOO

a

CHOO

b

OCHO

c

O

OO

O

CHOO

O

d

O

CHO

e

O

O

6 (>95) 67 (>95)9 (>95)94 (>95)9 (>95)

f

94 (>95)

N

CHO

Yield (purity) of g

CHO

O

R

R'N

N N

N

CHO

NN N

NN N

ArN

NBn

OS

HN

NBn

OS

f

1. 4-amino-1-benzylpiperdine, Na(OAc)3BH2. benzothiophene-2-carboxylic acid DIC, HOAt, DIPEA, DMF, rt, 16 h

TFA (2% in CH2Cl2)rt, 2 h

a-e

g

Preparation of Amide Library using FIMT Resins

74% (99%)

60% (99%)

90% (99%)

60% (97%)

A5

72% (95%)

53% (96%)

90% (96%)

53% (90%)

A4

78% (95%)

93% (93%)

93% (93%)

58% (90%)

A3

74% (91%)

88% (97%)

91% (98%)

50% (77%)

A2

73% (85%)

90% (94%)

92% (94%)

55% (85%)

A1B4B3B2B1

50-93 % yield77-99 % purity

N N

CN

NEt N

N

N N S

MeO

OMe

R =

R' =A1 A2 A3 A4 A5

B1 B2 B3 B4

N

CHO

NN N

N NN

N

N

R'

R

O

R' NH

RO1. A(1-5)-NH2, Na(OAc)2BH,

CH2Cl2, rt, 16 h2. B(1-4)-CO2H, TFFH, DIPEA, DMF, rt, 24 h

TFA (2% in CH2Cl2)rt, 16 h

f

“Click” Applications in Macromolecules

• Dendrimer Synthesis

• Solid Support Chemistry– SPOS– SPPS

• DNA functionalization

Tornoe, C.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.

SPPS: Cu(I) catalysis on Solid Support

FGFG

O

FmocHN

FGFG

O

FGFG=Phe-Gly-Phe-Gly = HMBA-PEGA800

CHCNCH2Cl2 THF tolueneN,N-dimethylformamide N-ethyldiisopropylamine

Solvents:

Quantitative conversions & purities: 75-95%

Tornoe, C.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.

SPPS: Cu(I) catalysis on Solid Support

All conversions > 95%*

FGFG

OFmocHN

FGFG

OFmocHN

NN N

ROH

1. R-N3, DIPEA,Cu(I) 20% piperidine/DMF2. 0.1 M NaOH (aq)

a-e

FGFG=Phe-Gly-Phe-Gly = HMBA-PEGA800

H2N N3

FmocHN N3

OH

O

N3 N3SGAL

NR90% 79% 78% 75%

a b c d e

Si

SPPS:Cu(I) catalysis on Solid Support

Tornoe, C.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.

O 1. RN3DIPEA,Cu(I)2. 0.1M NaOH (aq.)

FGFG

O

NN N

HO

O OH

> 95% conversion> 75-95 % purity

OH

O

N3

OH

O

OH

O

OH

ON3

N3

OH

O

N3

OH

O

N3N3OH

ON3

OH

O

N3

OH

ON3

NH2N3

N3

NH2

NH2N3

SN3

N3

93 % 90 %

84 %HN

NH

NH2

NH2N3

OH

O

N3

75 %

95 %

NR

Si N3

SPPS: Cu(I) catalysis on Solid Support

• Peptide Protecting Group Compatibility

88Arg(Pmc)90Asn(Trt)

> 95Trp(Boc)> 95Asp (tBu)

> 95Lys(Boc)> 95Tyr (tBu)

85Met> 95Thr(tBu)

81Cys(Trt)> 95Pro

80His(Trt)> 95Ala

% purityXxx% purityXxx

Tornoe, C.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.

> 95% conversion

Triphenylmethyl (Trt)

O

O

t-Butyloxycarbonyl (Boc)

OSO

O

2,2,5,7,8-pentamethyl-chroman-6-sulphonyl (Pmc)

O1.

DIPEA,Cu(I)2. 0.1M NaOH (aq.)

Xxx FG

OHN3

O

O

Xxx FG OHNN N

HO

O

Tornoe, C.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.

SPPS: Cu(I) catalysis on Solid Support

• Scale Up

NH

HN

O

O

O

O

HN

OPh

H2N

NN N

NH

HN

OH

HO

O

O

O

O

Ph

NN N

NH

HN

OH

O

O

OPh

N3

HON3

DIPEA,CuI0.1M NaOH (aq)

DIPEA,CuI0.1M NaOH (aq)

79% 87%

O

“Click” Applications in Macromolecules

• Dendrimer Synthesis

• Solid Support Chemistry– SPOS– SPPS

• DNA functionalization

“Click” Chemistry in Biology

• Construction of fluorescent oligonucleotides for DNA sequencing

• Biological Inhibitors

• In-situ “Click” approach

DNA Sequencing

• Modified Oligonucleotides• Introducing additional functional groups

in DNA– Staudinger reaction

– Limitations: aqueous conditions required hydrolyze intermediate aza-ylide

Seo, T.; Li, Z.; Ruparel H.; Ju J. J. Org Chem. 2003, 68, 609.Saxon, E.; Bertozzi C. Science, 2002, 287, 2007.

R3P: N3 R' R3P N R' H2O H2N R'OP

R3+ +

Seo, T.; Li, Z.; Ruparel H.; J. Org Chem. 2003, 68, 609.

DNA Sequencing

• Fluorescent ss DNA sequencing

Br OEt

O

N3 OH

O

N3 O

ON

O

O

HNN3

O

H2N

GTT TTC CCA GTC ACG ACG-3'

GTT TTC CCA GTC ACG ACG-3'

EDC, CH2Cl2

96%

Azide labeled DNA

1. NaN3, DMSO2. NaOH, MeOH, H2O3. HCl

OO NHO

DNA Sequencing

Seo, T.; Li, Z.; Ruparel H.; J. Org Chem. 2003, 68, 609.

HNN3

O

O O

ON

O

OO

OH

HO2C H2N

O O

HN

O

OH

O2C

O O

ONH

O

OH

O2C

NN N N

H

OO O

ONH

O

OH

O2C

NN

N NH

O

GTT TTC CCA GTC ACG ACG-3'

+

DMSO/ H2O80'C, 72 h

GTT TTC CCA GTC ACG ACG-3'

GTT TTC CCA GTC ACG ACG-3'

+

DMFNaHCO3

91%

Biological Inhibitors

Brik, A.; Muldoon, J.; Lin, Y.; Elder, J. Goodsell, D. Olson, A.; Fokin, V.; Sharpless, B.; Wong, H. Chem. Bio. Chem. 2003, 4, 1246.

• HIV-1 protease (HIV-1 PR)

OHN

O

O

OHN

O

NOH

SO

O

O

OHN

O

OH

N3 OHN

O

OH

N3O

NaN3, EtOH/H2O 60oC, 2 h

1. 4 N HCl/dioxane2. (S)-3-tetrahydrofuranyl N-oxysuccinimidyl carbonate, Et3N

1. 4 N HCl/dioxane2. TfN3, H2O/CH2Cl2/MeOH, rt.

1. i-BuNH2, MeOH2. p-methoxybenenesulfonyl chloride,K2CO3, CH3CN, 3 h.

83%

76%

N3 NOH

SO

O

O

Brik, A.; Muldoon, J.; Lin, Y.; Elder, J. Goodsell, D. Olson, A.; Fokin, V.; Sharpless, B.; Wong, H. Chem. Bio. Chem.2003, 4, 1246.

Biological Inhibitors

OH

NH

OO

R=

HN

O

NH

Boc

HN O

OO

0N H

N

O

1 2

3 4

17 +/- 139 +/- 1G48V

52 +/- 246 +/- 2V82A

24 +/- 119 +/- 1V82F

13 +/- 0.56 +/- 0.5HIV PR

21Enzyme

IC50 [nM]

N3 NOH

SO

O

O

OHN

O

OH

N3O O

HN

O

OH

NON N

R

N N

OHSO

O

O

NNR

R H

R H

1.H2O,tBuOH (1:1) Cu+/Cu++

1.H2O,tBuOH (1:1) Cu+/Cu++

In Situ “Click” Chemistry

PERFECT FIT Model of acetylcholinesterase inhibitor.

Lewis, W.; Green, L.; Grynszpan, F.; Radic, Z.; Carlier, P.; Taylor, P.; Finn, M.; Sharpless, B. Angew chemie. Int. Ed, 2002, 41, 1054.

In-Situ “Click” Chemistry

Lewis, W.; Green, L.; Grynszpan, F.; Radic, Z.; Carlier, P.; Taylor, P.; Finn, M.; Sharpless, B. Angew chemie. Int. Ed, 2002, 41, 1054.

N

HNN3

HCl

N

HN

HCl

NN3

H2N NH2

N

H2N NH2

H

H

n

1 (n=2-6)

3 (n=1-3) 4 (m=6-8)

2 (m=2-6)

m

nm

• Enzyme Templating– Inhibitor for acetylcholinesterase

80oC6 days

N

H2N NH2

NN

N

NH

N

N

H2N NH2

NN

N

NH

N

(1,5) (1,4)

Why “Click” Chemistry?

• Functional group tolerance• Aqueous conditions• Shorter reaction time• High yield• High purity• Regiospecificity

Acknowledgments

• Dr. Jackson• Dr. Borhan• Dr. Odom• Jackson & Miller Research Group