2014-2015

M2 recherche
Click Chemistry

Prof. Paul-Alain Jaffrs

Laboratoire de Chimie, UMR CNRS 6521
Web.: http://www-tmp.univ-brest.fr/phosvec/
Bureau : C123
E-mail : pjaffres@univ-brest.fr

Summary
1- Introduction
2- CuAAC
3- Copper Free AAC
4- Staudinger
5- Isocyanate / isothiocyante
6- Epoxide
7- Thiol-ene
8- Thiol-ynes
9- Novel developments
10- Conclusion

2
2

1- Introduction
Historique
The notion of Click reaction : introduced by Sharpless, Finn, Fokin in 2001

B. Sharpless
Scripps Research Institute

M.G. Finn
Georgia Institute
of Technology

V.V Fokin
Scripps Research Institute

M. Meldal
Coppenhagen

Nobel Price 2001

(Chemistry)

Kolb, H.C.; Finn, M.G.; Sharpless, B.K. Angew. Chem. Int. Ed. 2001, 40, 2004-2021.
Rostovtsev VV, Green LG, Fokin VV, Sharpless KB. Angew Chem Int Ed Engl. 2002, 41, 2596-9.
C. W. Tornoe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057.

1- Introduction
Historique

A click reaction must be modular,

wide in scope, high yielding, create
only inoffensive by-products (that
can be removed without
chromatography), are stereospecific,
simple to perform and that require
benign or easily removed solvent.
- Barry Sharpless

Kolb, H.C.; Finn, M.G.; Sharpless, B.K. Angew. Chem. Int. Ed. 2001, 40, 2004-2021.
Rostovtsev VV, Green LG, Fokin VV, Sharpless KB. Angew Chem Int Ed Engl. 2002, 41, 2596-9.

1- Introduction
Criteria for click reaction

be modular
be wide in scope
give very high chemical yields
generate only inoffensive byproducts
be stereospecific
have high atom economy.

The process would preferably:

have simple reaction conditions
use readily available starting materials and reagents
use no solvent or use a solvent that is benign or easily removed
(preferably water)
provide simple product isolation by non-chromatographic methods
(crystallisation or distillation)
Wikipedia, 17-10-2013 (selection)

2- CuAAC : copper catalyse Alkyne-Azide Cycloaddition

2-1- Cycloaddition de Huisgen

1933 Dipolar nature of azide first recognized by Linus Pauling

Azide

R N3

R N

R N N N

N N

R N N N

L. Pauling. Proc. Natl. Acad. Sci. USA 1933, 19, 860-867; Huisgen, R. Angew. Chem. Int. Ed. 1963, 2, 633-696

1960 Mechanism of 1,3-dipolar cycloaddition of azides and alkynes

pioneered by Rolf Huisgen
+ N3 R'

R''

N 1
N R'
N

80oC

5 R''

N 1
N R'
N
R'' 4
NH2

NH2
NH2
-

Cl
H+
N

N+

N+
Neat, 80oC, 6 days

N+

NH
N NH

6
HN

N3

HN

N
H

Kolb. J. Am. Chem. Soc. 2004, 126, 12809

NH
HN
N

6
1,5

1,4

2- CuAAC : copper catalyse Alkyne-Azide Cycloaddition

2-1- Cycloaddition de Huisgen

2001/2002 Copper catalyzed 1,3-Dipolar cycloaddition by Sharpless/Meldal

R''

N3 R'

Cu(I)
rt

N 1
N R'
N

Regioselectivity

R'' 4

Sharpless, K.B. et al. Angew. Chem. Int. Ed 2002, 41, 2596-2599; Meldal,M.J. et al. J. Org. Chem. 2002, 67, 3057-3064

2- CuAAC : copper catalyse Alkyne-Azide Cycloaddition

2-2- CuAAC Possible mechanism
N
N

R2

[CuLx]

R'

H+

H+

N
N
R

R2

R'

Direct

CuLx
R2

CuLx
N N N

R2
R2
N N N
R1

N N N

CuLx
RDS

18 kcal/mol
Himo, F. et al. J. Am. Chem. Soc, 2005, 127, 210-216.
Ahlquist, M., Fokin, V.V. Organometallics 2007, 26, 4389-4391.

R'

R2

CuLx

N N N
R'

CuLx
CuLx

2- CuAAC : copper catalyse Alkyne-Azide Cycloaddition

2-3- CuAAC Experimental conditions

Ph

Ph

CuSO4 5H2O 1mol%

Sodium Ascorbate 5mol%
H2O : tBuOH 4:1, RT, 8h

N3

Ph

O
Ph
91%

O
O

N N
N

Ph

N N
N

NH2
88%
OH N

HN

N
N NH
92%

HO

NH

N
N N

Ph

88%

Sharpless. Angew. Chem. Int. Ed. 2002, 41(14), 2596

N
N

OH
OH

H
94%

2- CuAAC : copper catalyse Alkyne-Azide Cycloaddition

2-3- CuAAC Experimental conditions

Ph

Ph

N3

CuSO4 5H2O 1mol%

Sodium Ascorbate 5mol%
H2O : tBuOH 4:1, RT, 8h

Ph

O
Ph
91%

Sodium ascorbate is a reducing agent

(red)
Cu(II)

Cu(I)

(ox)

(red)

10

2- CuAAC : copper catalyse Alkyne-Azide Cycloaddition

2-3- CuAAC Experimental conditions
pK = 4.1
-H+
+H+

Rq: Addition of a slight excess of sodium ascorbate prevents the

formation of oxidative homocoupling products.
11

2- CuAAC : copper catalyse Alkyne-Azide Cycloaddition

2-3- CuAAC Experimental conditions

Other conditions
CuI

CuBr(PPh3)3

An abnormal NHC complex of copper with 1,4-diphenyl-1,2,3-triazol-5-ylidene [CuCl(TPh)] efficiently catalyzed click reactions
of azides with alkynes to give 1,4-substituted 1,2,3-triazoles in excellent yields at room temperature with short reaction
times. CuCl(TPh) was particularly effective for the reaction between sterically hindered azides and alkynes.
12
T. Nakamura, T. Terashima, K. Ogata, S.-i. Fukuzawa, Org. Lett., 2011, 13, 620-623.

2- CuAAC : copper catalyse Alkyne-Azide Cycloaddition

2-4- RuAAC
1,5-regioisomer

Suggested mechanism

B. C. Boren, S. Narayan, L. K. Rasmussen, L. Zhang, H. Zhao, Z. Lin, G. Jia, V. V. Fokin,

J. Am. Chem. Soc., 2008, 130, 8923-8930.

13

2- CuAAC : copper catalyse Alkyne-Azide Cycloaddition

2-4- RuAAC
Rq : possibility to use di-substituted alkyne
Different regioisomer
with CuAAC

Not possible with CuAAC

Rq : Active catalysts : RuCp*L2Cl

RuAAC is not as robust as the CuAAC with respect to functional group

tolerance and reaction conditions compatibility.

The catalyst should not be mixed with the azide in the absence of alkyne
(side reaction below dimerization of RN3 to produce a characteristic
green product).
R-N3

14

2- CuAAC : copper catalyse Alkyne-Azide Cycloaddition

2-5- Exemples
Exemples
Synthesis of polymers

D. J. V. C. van Steenis, O. R. P. David, G. P. F. van Strijdonck, J. H. van Maarseveen, J. N. H. Reek, Chem. Commun.
2005, 4333.

Synthesis of gel

N3

N3

Cu(I)

D. D. Diaz, K. Rajagopal, E. Strable, J. Schneider, M. G. Finn, J. Am. Chem. Soc. 2006, 128, 6056;

15

2- CuAAC : copper catalyse Alkyne-Azide Cycloaddition

2-5- Exemples

Exemple
Synthesis of dendrimers : divergent syntesis

Cu(I)

V. V. Fokin et al., Macromolecules 2005, 38, 3663.

16

2- CuAAC : copper catalyse Alkyne-Azide Cycloaddition

2-5- Exemples

Exemple
Synthesis of dendrimers : convergente syntesis

Cu(I)

J. W. Lee, B.-K. Kim, J. H. Kim, W. S. Shin, S.-H. Jin, J. Org. Chem. 2006, 71, 4998.

17

3- Copper free Alkyne-Azide Cycloaddition

3-1- Introduction

Toxicity of copper
Reactive
Oxygen
Species
(ROS)

Toxicity

Rq
 The nature of ligand (L) influence the toxicity.
 The nature of ligand (L) modify the reduction potential Cu(II/I)
 The nature of ligand (L) modify cell uptake

18

3- Copper free Alkyne-Azide Cycloaddition

3-2- Bio-compatible copper complexes
 Cu(histidine)2

Low toxicity

High
toxicity

L-Histidine
Kennedy et al., J Am Chem Soc. 2011, 133, 17993-8001.

19

3- Copper free Alkyne-Azide Cycloaddition

3-2- Bio-compatible copper complexes
 Cu(histidine)2

Mannosamine
(Incorporation into
glycan glycoprotein)
Kennedy et al., J Am Chem Soc. 2011, 133, 17993-8001.

Viable cells only with Cu(Hist)2

O
S

Biotine

H
HN

H
NH
O
Tetrameric structure
of streptavidin with
2 bound biotins

Steptavidin

Streptavidine/biotine :
dissociation constant (Kd) on
the order of 1014 mol/L
Wikipedia 21-10-2013

Huh7.5 cells

20

3- Copper free Alkyne-Azide Cycloaddition

3-2- Bio-compatible copper complexes
 Cu(histidine)2

Mannosamine
(Incorporation into
glycan glycoprotein)
Kennedy et al., J Am Chem Soc. 2011, 133, 17993-8001.

 Syntheis of non-natural saccharides

O
HO
HO
HO

1) NaOMe/MeOH
2) (ClCH2CO)2O

NH2 HCl
O
OH
O

HO
HO
HO

N3

HN
O

Ac2O, DMAP,
Pyridine
95% Yield

OH

HO
HO
HO

Cl

HN

NaN3, DMF
reflux

O
59% 2 Steps

OH
O
AcO
AcO
AcO

N3

HN
O

OAc
21

3- Copper free Alkyne-Azide Cycloaddition

3-2- Bio-compatible copper complexes
 Cu-BTTES

P. Wu et al.,
J. Am. Chem. Soc., 2010, 132, 16893-16899.

22

3- Copper free Alkyne-Azide Cycloaddition

3-2- Bio-compatible copper complexes
 Cu-BTTES

P. Wu et al.,
J. Am. Chem. Soc., 2010, 132, 16893-16899.

Toxicity : importance of the ligand

Low toxicity
With ligand
High toxicity
Without ligand

Cell growth curve after click chemistry treatment.

23

3- Copper free Alkyne-Azide Cycloaddition

3-2- Bio-compatible copper complexes
 Cu-BTTES

P. Wu et al.,
J. Am. Chem. Soc., 2010, 132, 16893-16899.

Fucosylation of glycan : use GDP-fucose as substrate

 Two biosynthetic ways to produce GDP-fucose
% of bioproduction
of GDP-fucose

90 %

10 %

Alkyne-based
synthetic equivalent
24
GDP-fucose (GuanosineDiPhosphate - GDP)

3- Copper free Alkyne-Azide Cycloaddition

3-2- Bio-compatible copper complexes
 Cu-BTTES

P. Wu et al.,
J. Am. Chem. Soc., 2010, 132, 16893-16899.

Alexa Fluor 488-azide

Micro-injection in
zebrafish embryo
Rq : GDP-Fuc-N3 : toxic

Goal : track the fucosilated glycan after embryo developement

25

3- Copper free Alkyne-Azide Cycloaddition

3-3- Reactive substrates Real copper free

Context
 Classical Huisgen cycloaddition : require thermal activation
R''

+ N3 R'

80oC

N 1
N R'
N
5 R''

N 1
N R'
N
R'' 4

 Alternative : activated substrates to enhance chemical reactivity

26

3- Copper free Alkyne-Azide Cycloaddition

3-3- Reactive substrates Real copper free
 activated substrates to enhance chemical reactivity

G. Wittig, A. Krebs.
Chem. Ber. 1961, 94, 3260-3275.

Z. Li, T. Seo, J. Ju.

Tetrahedron Lett. 2004, 45, 3143-3146.

Carolyn Bertozzi
Proc Natl Acad Sci U S A. 2007 ,104, 16793
J. M. Nolte
ChemBioChem 2007, 8, 1504-1508.

27

3- Copper free Alkyne-Azide Cycloaddition

3-4- Strained Alkyne
Concept

sp hybridized C
; but 160angles
Due to the cyclic structure

Enhance the reactivity

C. R. Bertozzi. J. Am. Chem. Soc. 2004, 126, 15046-15047.

28

3- Copper free Alkyne-Azide Cycloaddition

3-4- Strained Alkyne
Kinetic of cycloaddition (relative rate)

Krel = 1
R. Bertozzi.
J. Am. Chem. Soc.
2004, 126, 15046

Krel = 3
C. R. Bertozzi.
ACS Chem. Biol.
2006, 1, 644-648.

Krel = 1.5
C. R. Bertozzi.
Org. Lett.
2008, 10, 3097-3099.
Rq : More water
soluble

Krel = 60
C. R. Bertozzi.
Proc. Natl. Acad. Sci. U.S.A.
2007, 104, 16793-16797.

Krel = 450

Adjacent aryl
groups

Rq : limited
solubility

 Synthesis of fluorine derivative : difficult, expensive

 Adjacent aryl groups also increase the reactivity of alkyne.
29

3- Copper free Alkyne-Azide Cycloaddition

3-4- Strained Alkyne
Adjacent aryl group s: synthesis

L. S. Campbell-Verduyn, PhD-thesis

30

3- Copper free Alkyne-Azide Cycloaddition

3-4- Strained Alkyne
Commercial compounds

1 mg : 21

10 mg : 55

5 mg : 47

Dibenzocyclooctyne-fluor 488

1 mg : 68
31

4- Staudinger ligation
4-1- Introduction
 Staudinger (Nobel Laureate -1953) and Meyer first reported in 1919 that azides react
smoothly with triaryl phosphines to form iminophosphoranes after elimination of
nitrogen

aza-ylide
iminophosphoranes
 Reactivity of iminophosphoranes

32

4- Staudinger ligation
4-2- Application in biology
 Used for the production of bioconjugate : Saxon and Bertozzi, 2000
 Azides are ideal for bioorthogonal chemical reporter strategies.
Small
Stable in physiological conditions
Have metabolic precursors compatible with existing cellular machinery
Not found in many natural species
Reacts only with soft nucleophiles (highly selective)
 Azide-functionnlized natural substrates can be incorporated in bio-synthesis of
peptide, polysaccharides etc

Saxon, E. and Bertozzi, C. Cell surface engineering by a modified Staudinger reaction. Science, 2000, 287:2007-10

33

4- Staudinger ligation
4-2- Application in biology

Saxon, E. and Bertozzi, C. Cell surface engineering by a modified Staudinger reaction. Science, 2000, 287:2007-10

34

4- Staudinger ligation
4-2- Application in biology

Concept

Zhang et al., Molecules, 2013, 18, 7145

35

4- Staudinger ligation
4-3- Application in biology - Example

Bertozzi. Nature. 2004, 430, 873

36

4- Staudinger ligation
4-4- Traceless Staudinger Ligation

Incorporation of
triphenylphosphine oxide
moiety

Traceless Staudinger Ligation = Without the incorporation of triphenylphosphine oxide

in the final conjugate

37

4- Staudinger ligation
4-4- Traceless Staudinger Ligation
 Goal : amide bond formation but without the incorporation of the unnatural
phosphine oxide moiety in the final product.
 Developed by Bertozzi and Raines simultaneously
 The auxiliary phosphine reagent can be cleaved from the final product after the ligation

Saxon, E.; Armstrong, C.R.; Bertozzi, C.R. Org. Lett. 2000, 2, 2141.
Nilsson, B.L.; Kiessling, L.L.; Raines, R.T. Org. Lett. 2000, 2, 1939.

38

4- Staudinger ligation
4-4- Traceless Staudinger Ligation
 Mercaptomethylenediphenylphosphine

Nilsson, B.L.; Kiessling, L.L.; Raines, R.T. Org. Lett. 2000, 2, 1939.
39

4- Staudinger ligation
4-4- Traceless Staudinger Ligation
 Phenol-phosphine
O
R2

R2
O
PPh2

-N N N
+
R

OH O
PPh2
R

-O
O

R
-N
P+ Ph
Ph

H2O

H
N

R2
O

Saxon, E.; Armstrong, C.R.; Bertozzi, C.R. Org. Lett. 2000, 2, 2141.

O-

R2
R
N
P+ Ph
Ph

Ph
Ph O
P+
N
R2
R

40

4- Staudinger ligation
4-5- Examples
Context: Dynamic alterations in cell surface glycosylation occur in numerous biological processes that involve
cellcell communication and cell migration. We report here imaging of cell surface glycosylation in live mice
using double click chemistry.

Diels Alder
2ndClick

Mice were injected i.p. daily for 3 days

with peracetylated azido-labeled Nacetylgalactosamine

Copper free Huisgen

1st Click

Staudinger ligation
41

Brindle et al., Bioconjugate Chem., 2013, 24, 924-941

4- Staudinger ligation
4-5- Examples
Context: Dynamic alterations in cell surface glycosylation occur in numerous biological processes that involve
cellcell communication and cell migration. We report here imaging of cell surface glycosylation in live mice
using double click chemistry.

2ndClick

1st Click

Imagerie des tumeurs

42
Brindle et al., Bioconjugate Chem., 2013, 24, 924-941

5- Isocyanate / isothiocyante
5-1- Synthesis of isocyanate and isothiocyanante

 Phosgene route

Diphosgene
Aliphatic
or
Aromatic
amine
hydrochloride

Non
Nucleophilic
amine

Eckstein et al., J. Org. Chem., 1996, 61, 3883

43

5- Isocyanate / isothiocyante
5-1- Synthesis of isocyanate and isothiocyanante

 Curtius rearrangement

Diphenylphosphoryl azide

44

5- Isocyanate / isothiocyante
5-1- Synthesis of isocyanate and isothiocyanante
Synthesis of isothiocyanante

 With CS2

 With thiophosgene

45

5- Isocyanate / isothiocyante
5-2- Reactivity of isocyanate and isothiocyanante

Isocyanate
More reactive
Use to produce polymers
 With diol : polyurethanes
Polyaddition

Tolune diisocyanate
(2,6 TDI)

Polyurethanes
(carbamates)

 With diamine: polyurea

O
O
HN

O
H
C
H

HN
NH

NH

HN

H
C
H

NH

HN
NH

Polyurea

46

5- Isocyanate / isothiocyante
5-2- Reactivity of isocyanate and isothiocyanante

isothiocyanante
 Less reactive than isocyanate
 Compatible with biologic materials and environment

Acharya et al., Bioconjugate Chem., 2008, 19, 1352-13620

47

5- Isocyanate / isothiocyante
5-2- Reactivity of isocyanate and isothiocyanante

isothiocyanante
 Less reactive than isocyanate
 Compatible with biologic materials and environment

pH 8.5

Acharya et al., Bioconjugate Chem., 2008, 19, 1352-13620

48

6- Epoxide
6-1- Synthesis of epoxide

If a ketone is present, BaeyerVilliger reaction may occur.

49

6- Epoxide
6-2- Reactivity of epoxide
 With thiol

Hoyle et al., Chem. Soc. Rev., 2010, 39, 1355-1387

50

6- Epoxide
6-2- Reactivity of epoxide
 With primary amine

Love et al., PNAS, 2010, 107, 1864-1869

 With secondary amine

M. R. Saidi, Org. Lett., 2005, 7, 3649-3651

51

6- Epoxide
6-2- Reactivity of epoxide
 With thiol

Hoyle et al., Chem. Soc. Rev., 2010, 39, 1355-1387

52

6- Epoxide
6-2- Examples
 With amine

90 %
L. D. S. Yadav et al., Synthesis, 2012, 2353-2358.

53

7- Thiol-ene
7-1- Thiol-Michael addition
 With a base

 With a nucleophile

Nu : Phosphine (better than amine as catalyst)

In water : use of TCEP : Tris(2-carboxyethyl)phosphine)

TCEP

54

7- Thiol-ene
7-1- Thiol-Michael addition

M.G. Finn et al., J. Am. Chem. Soc., 2009, 131, 9986-94

55

7- Thiol-ene
7-2- Thiol-radical click

Initiation : Photochemical, Thermal, Redox

 Photochemical : DMPA (2,2-Dimethoxy-2-phenylacetophenone)

DMPA

 Thermal: AIBN

Decomposition above 65C

56

7- Thiol-ene
7-2- Thiol-radical click

Propagation and chain transfer

57

7- Thiol-ene
7-2- Thiol-radical click

Example

h, 60 min

DMPA

General procedure for thiol-ene reaction.

The thiol-ene reaction between Gn(OHx)-Enez and cysteamine
hydrochloride (3.0 eq to each ene moiety) was carried out using
2,2-dimethoxy-2-phenylacetophenone (0.05 eq. to each ene
moiety) as photo-initiator in methanol. The reaction mixture was
sparged with dry nitrogen for about 20 mins and then exposed to
the handheld UV-lamp (exc = 365nm) for 60 minutes. Triethylamine and
water were then added to the reaction mixture until the pH of the
solution became 10 (to neutralize the hydrochloride salt and
obtain free amines). Methanol and excess triethylamine were
then removed under reduced pressure.

C.J. Hawker et al., ChemComm, 2010, 46, 1556-1558

58

8- Thiol-yne
8-1- Mechanism

59

8- Thiol-yne
8-2- Experimental conditions
Xu et al.,
ChemComm., 2011, 47, 7509-7511

Xu et al.,
ChemComm., 2011, 47, 3930-3932

Shiu et al.,
ChemEur J., 2009, 15, 3839-3850

60

8- Thiol-yne
8-2- Examples

Lowe et al., J. Am. Chem. Soc., 2009, 131, 5751-5753

61

8- Thiol-yne
8-2- Examples

Wendeln et al., Langmuir, 2010, 26, 15966-15971

62

9- Novel developments

Tyrosine-Click reaction

C.F. Barbas, Bioconjugate Chem., 2013, 24, 520-532

63

9- Novel developments

Diels Alder reaction

84%
furane

maleimide

64

9- Novel developments

Diels Alder reaction
Dienophile (maleimide)

Diene (furane)
NHS, DCC, 1,4-dioxane, RT
puis amine + NaHCO3, H2O, 50C

 Reactions for the synthesis of PEG-amine

65

9- Novel developments

Diels Alder reaction
 Reactions for the synthesis of maleimide-PEG (A)

84%
furane

maleimide

Mitsunobu

Retro-DA
66

9- Novel developments

Diels Alder reaction
 Reactions for the synthesis of maleimide-PEG (A)
Direct Mitsunobu reaction
with maleimide : low yield

84%
furane

maleimide

Mitsunobu

Retro-DA
67

10- Conclusion

 Many reactions available

 Many application in material sciences, biology etc..
 New reaction are welcome
 A good strategy to produce complex molecules

68