recent advances in copper catalyzed azide/alkyne cycloadditions: prototypical “click” reactions...

Recent Advances in Copper Catalyzed Azide/Alkyne Cycloadditions: Prototypical “Click” Reactions

Shane Mangold

Kiessling Group

February 14th 2008

2

Historical Perspective of Azide/Alkyne Cycloadditions

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

R'' R'N3 N NN

R'

R''

1

5

+80oC N NN

R'

R''

1

4

+

R N3 R N N N R N N NH2R N N N

1933- Dipolar nature of azide first recognized by Linus Pauling

1960- Mechanism of 1,3-dipolar cycloaddition of azidesand alkynes pioneered by Rolf Huisgen

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

R'' R'N3N N

NR'

R''

1

4

+ Cu(I)

rt

3

Defining a “Click” Chemistry Reaction

“ 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.

4

Reactions that meet the “Click” Criteria

Kolb, H.C.; Finn, M.G.; Sharpless, B.K. Angew. Chem. Int. Ed. 2001, 40, 2004-2021.

R

[O]

X

R

C=C Additions

HX

R Nuc

Catalyst

Nucleophilic Ring Opening

X = O, NR

R'

O

R''

N

Non-Aldol Carbonyl Chemistry

RO-NH2

[O]

RO

OR

Diels-Alder

OR

R

N

N

N

R

R'

Cu(I) catalyzed Huisgen1,3-dipolar cycloaddition

R-N3

Cu(I)

5

Copper Catalyzed Azide/Alkyne Cycloaddition (CuAAC)

• Thermodynamic and kinetically favorable (50 and 26 kcal/mol, respectively)

• Regiospecific

• Chemoselective

• 107 rate enhancement over non-catalyzed reaction

• Triazole stable to oxidation and acid hydrolysis

R'' R'N3

N NN

R'

R''

1

4

Cu(I)

+

Rostovtsev et al. Angew. Chem. Int Ed. 2002, 41, 2596-2599

6

CuAAC Catalytic Cycle

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

CuLxR'

N N N

R2

CuLx

HR'

CuLx

23 kcal/mol

18 kcal/mol

HR'

H+

CuLxR'

N N N

R2

CuLxR'

N N N

R2

CuLx

N N N

R1

R2

N NN

R CuLx

H+

N NN

R H

R2

R2

[CuLx]

RDS

7

CuAAC Chemistry Applications

• Peptide/Protein Modification

• Therapeutics

• Combinatorial Synthesis

• Polymer Functionalization

• Materials/Surface Chemistry

8

CuAAC as a Route to Cyclic Tetrapeptide Analogues

• Cyclic peptides important antimicrobial agents

• More stable to enzymatic degradation and better cellular uptake than linear chain form

• Conformational restriction allows better understanding of receptor-ligand interactions

• Difficult to synthesize due to strain energy of cyclization in transition state

Rich, D.H. et al. Tetrahedron 1988, 44, 685-695

N

NH

N

HN

O

O

O

HO

O

cyclo-[Pro-Val-Pro-Tyr]

9

Synthesis of Tetrapeptide Analogue cyclo-[Pro-Val-(triazole)-Pro-Tyr]

• Cyclo-[LPro-LVal-LPro-LTyr] is a tyrosinase inhibitor isolated from L. helveticus

• Previous attempts at synthesis had failed due to epimerization upon cyclization

• Hypothesize ring contraction mechanism of CuAAC may help promote cyclization

Van Maarseveen, J.H. et al. Org. Lett. 2006, 8, 919-922

N

NH

N

HN

O

O

O

HO

O

cyclo-[Pro-Val-Pro-Tyr]

N

N

N

HN

O

O

O

N

N

HO

cyclo-[Pro-Val-(Triazole)-Pro-Tyr]

10

1,2,3-Triazoles as Peptide Bond Isosteres

• Triazole and peptide bond both possess large dipole (5D, 3.7D, respectively)

• N2 and N3 lone pairs serve as hydrogen bond acceptors

• C distance comparable

• Triazole mimics planarity of amide bond

Kolb, H.C., Sharpless, B.K. Drug. Disc. Today. 2003, 8, 1128-1136.

3.9 Å

5.1 Å

H2NNH

COOH

R1

O R2

H2N

N

NN

R1

COOH

R2

11Bock, V.D., et al. Org. Lett. 2006, 8, 919-922

Retrosynthesis

Pathway "A"

H2NN

O

NNN O

N

CO2H

OBn

N

N

N

HN

O

O

O

NN

BnO

Triazole Formation:Pathway "B"

Peptide BondFormation Pathway "A"

Pathway "B"

N3N

NH

N

O O

BnO

O

N3

O

N

CO2tBu

BocHNN

O

BnO

12Bock, et al. Org. Lett. 2006, 8, 919-922

Synthesis of Cyclic Tetrapeptide Analogue

BocHNN

O

OBn

(1)

N3

O

N

CO2tBu

H2NN

O

NNN O

N

CO2H

OBn

N

N

N

HN

O

O

O

NN

BnO

1) CuI, DIPEA5:1 MeCN:THF

2) TFA: CH2Cl274%

(2)

1 + 2

no product formation

1 + 2EDCI, HOBt, DIPEA

N3N

NH

N

O O

BnO

O

N

N

N

HN

O

O

O

NN

BnOCuBr, DBU

Toluene, 70%

Pathway A

Pathway B

DCM, 70%

13Bock, V.D. et al. Org. Biomol. Chem., 2007, 5, 971-975

Tyrosinase Inhibition

Compound Tyrosinase Activity IC50 / mM

Cyclo-[Pro-Tyr-Pro-Val] 1.5

Triazole analogue 2 0.6

Triazole analogue 3 0.5

Triazole analogue 4 1.6

N

NH

NO

O

HO

N

N

NO

O

NN

HON

NN

O NNN

N

NH

N

HN

O

O

O

HO

O

N

N

N

HN

O

O

O

NN

HO

cyclo-[Pro-Tyr-Pro-Val] 2 3 4

14

Outline

• Peptide/Protein Modification– Peptide Macrocyclization

• Therapeutics – Multivalent carbohydrate vaccines

• Inhibitors

• Chemoenzymatic Functionalization

• Materials Science/Polymers

15

Anticancer Vaccines Through Extended Cycloaddition Chemistry

• To exploit antitumor immune response, induce antibodies against carbohydrate antigens

• Protein Scaffold upon which

carbohydrates are attached is important for eliciting antibody production

• Drawback is that monovalent carbohydrate/antibody interactions are weak

Wan, Q., Chen, J., Chen, G., Danishefsky, S.J. J. Org. Chem. 2006, 71, 8244-8249

Glycopeptide N3 Peptide

HN

O

Glycopeptide NNN

Peptide

HN

O

m

m

+

CuAAC

GlycopeptideHN

SHO

PeptideN

O

OO

n

PeptideN

O

OO

NH

O S

ConjugationLow Yielding

n

Glycopeptide

16Wan, Q., Chen, J., Chen, G., Danishefsky, S.J. J. Org. Chem. 2006, 71, 8244-8249.

CuAAC of Multivalent Carbohydrate Peptide Conjugate

OOH

HO

O

NHAc

OH

AcNH HN

O

N3

NH

OHN

NH

HN

NH

O

O

O

OHN

NH

HN

O

O

NH

OH

O

O

NH

NH2

HNHN O

HNO

HNO

Ala-Lys-Arg-Tyr-Lys-Phe-Ala-Lys-Ser-Ala

O

O

O

Cu nanoparticle, PBS buffer, 65%

OOH

HO

O

NHAc

OH

AcNH HN

O

NN

N

NNN

OOH

OHO

NHAc

OH

AcNH HN

O

NNN

OOH

HOO

NHAc

OH

AcNH HN

O

HN

NH2

OH

O

O

17

Template-Assembled Oligosaccharide Epitope Mimics

• 2G12 antibody targets oligomannose cluster (Man-9) present on HIV-1 gp120

• Recognizes terminal Man1-2Man residues

• Man-4 had comparable affinity to the antibody as that of Man-9 moeity

Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540.

OHO

HO

HO OH

OHO

HO

HO O

OHO

HO

HO O

OHO

HO

HO OH

OHO

HO

HO O

OHO

HO

HO OH

OHO

HO

HO O

OHO

HO O

O

OHO

OHO

O

HOAcNH

O

HONHAc

HN

OH OH

OOO

Man-9

18

Template-Assembled Oligosaccharide Epitope Mimics

• Cyclic decapeptide shown to be better immunogen than linear form

• T-helper peptide previously shown to increase immunogenicity of conjugate

• Synthesize template consisting of decapeptide conjugated with T-helper peptide epitopes for IgG antibody production.

Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540.

PK

K

KG

P

K

KKG

T - H e l p e rT - H e l p e r Mannose

1-2

1-3

1-2

1-2

1-2

1-3

1-2

1-2

1-3

1-2

1-2

1-3

19Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540.

Synthesis of Man4

OOBz

HOO

O N3

OOPh

TMSOTf, DCM, 77%

2) 80% AcOHNaOMe/MeOH

OBzO

BzO

BzO OH

OBzO

BzO

BzO O

OAll

OBzO

BzO

BzO OAc

OBzO

BzO

BzO O

OBzO

BzO O

BzO

OAll

OBzO

BzO

BzO OAc

OBzO

BzO

BzO O

OBzO

BzO O

BzO

O

NH

CCl3

OBzOBzO

OAc

BzOO

NH

CCl3

TMSOTf, DCM, 82%

PdCl2, MeOH1)

2) CCl3CN, DBU

OHO

HO

HO OH

OHO

HO

HO O

OHO

HO

HO O

OHO

OH

O

HO

OO N3

Man4

1)

76% (2 steps)

20Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540.

Template Synthesis of Man-4 Cluster

NNN

R NNN

R

NNN

R

NNN

R

PK

K

KG

P

K

KKG

NHNH

BocHNNHBoc

NH

HNO

O OO

PK

K

KG

P

K

KKG

NHNH

BocHNNHBoc

NH

HNO

O OO

PK

K

KG

P

K

KKG

NH2H2N

BocHNNHBoc

H2N

NH2

PK

K

KG

P

K

KKG

DdeDde

BocHNNHBoc

Dde

Dde

2% Hydrazine, DMF

84%

Propynoic Acid, DCC

77%

Man-4

CuSO4, Sodium AscorbatetBuOH:H2O (1:1); 90%

21Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540

Synthetic Vaccine Conjugate

OO

N3

O

ON

O

O

0.5 M NaHCO3, ACN:MeOH, 90%

NNN

R NNN

R

NNN

R

NNN

R

PK

K

KG

P

K

KKG

NHNH

H2NNH2

NH

HNO

O OO O

NH

T-helper

CuSO4, Sodium AscorbatetBuOH:H2O (1:1), 70%

HN

HN

R2

R2

T-helper

T-helperR2 = O

ON

O NN

O

R = Man-4

O

O

O

O

OO

N3N3

NNN

R NNN

R

NNN

R

NNN

R

PK

K

KG

P

K

KKG

NHNH

HNHN

NH

HNO

O OO

NNN

R NNN

R

NNN

R

NNN

R

PK

K

KG

P

K

KKG

NHNH NH

HNO

O OO

Kd = 2.64 MFully synthetic Vaccine

Man4

Kd = 2 mM

Template withSingle Mannose Kd > 20 M

Man9

Kd = 1.9 mM

22

Outline

• Protein Molecular Architecture– Peptide Macrocyclization

• Multivalent Architecture – Vaccine Conjugates

• Inhibitors– Combinatorial Chemistry

• Chemoenzymatic Functionalization

• Materials Science/Polymers

23

Inhibitors of HIV-Protease by CuAAC

• HIV-Protease cleaves proteins to yield active HIV virus

• Amprenavir is HIV-protease inhibitor used clinically since 1997.

• Develop Amprenavir analogue using CuAAC for combinatorial screening

Folkin, V, V. et al. J. Med. Chem. 2006, 49, 7697-7710

O NH

O

NS

OH

PhO O

NH2

O

Amprenavir

R1 NH

N

O R2NN

R4

R1 X H2N R4

ON3

R2

R3

R3

24Folkin, V.V. et al. J. Med. Chem. 2006, 49, 7697-7710

Synthesis of HIV Protease Inhibitor

Ph

BocHN

O

NOMe

1) BnMgCl, THF 1) MsCl, Et3N, DCM Ph

NHBoc

Ph

N3

Ph

NHBoc

O

H BnMgCl/CuBrDMS, THF 1) MsCl, Et3N, DCM Ph

NHBoc

Ph

N3

Ph

NHBoc

Ph

OH

Ph

NHBoc

Ph

OH

2) NaBH4, MeOH, -20oC 2) NaN3, DMF

2) NaN3, DMF

Ph

HN

Ph

N3

1) TFA/DCM

2) cyclopentyl chloroformateTEA, Toluene,75% (2 steps)

O

O

Ph

HN

Ph

N3

O

O

dr: 90:10 anti:syn

dr: 80:20 syn:anti

79% (2 steps)

60%

60% (2 steps)

54% (2 steps)

25

Synthesis of HIV Protease Inhibitor

Ki of Amprenavir = 19 nM

Folkin, V.V. et al. J. Med. Chem. 2006, 49, 7697-7710

R

CuSO4, Cu(s)t-BuOH/H2O (1:1) 50oC

(36 Alkynes)

> 90% conversion

1)

2)

R

CuSO4, Cu(s)t-BuOH/H2O (1:1) 50oC

(36 Alkynes)

> 90% conversion

1)

2)

Ph

HN

Ph

N3

O

O

Ph

HN

Ph

N3

O

O

Ph

HN

Ph

N

O

O

Ph

HN

Ph

O

O

NN

NNN

R

R

Ki = 23 nM

Ph

HN

Ph

NNN

O

O

N N

Cl

26

Inhibitor Optimization

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Folkin, V.V. et al. J. Med. Chem. 2006, 49, 7697-7710

1) n-BuLi (2 eq), THF, -78oC

2) (CH2O)n

Ph

HN

Ph

NNN

N N

ClOH

Ki = 8 nM

O

O

HO

H Ph

HN

Ph

NNN

N N

Cl

Ki = 23 nM

O

O

27

Outline

• Protein Molecular Architecture– Peptide Macrocyclization

• Multivalent Architecture – Vaccine Conjugates

• Inhibitors– Combinatorial

• Chemoenzymatic Functionalization– Metabolic Engineering– Antibiotic Derivatization

• Polymers/Materials Science

28

Glycoproteomic Probes for Imaging of Fucosylated Glycans in vivo

• Develop probe that is fluorescently active when undergoing reaction, whereas unreacted reagent remains traceless

• Fluorescent signal of naphthalimides modulated by electron donating properties of triazole

• Incorporate azidofucose analog into glycoproteins using biosynthetic pathway

Wong, C.H. et al. Proc. Natl. Acad. Sci. 2006, 103, 12371-12376

NO O

O

OH

OR2

OHHO

N3

R2 = glycoprotein

NO O

N

N N fucose OR2

strongly fluorescent

non-fluorescent

29

Metabolic Oligosaccharide Engineering

Wong, C-H., et al. Proc. Natl. Acad. Sci. 2006, 103, 12371-12376.

L-fucose

glycoconjugatesubstrateFucTs

ON3

ON3

ON3

1-P

ON3

GDP

ON3

GDP

glycoconjugate ON

glycoconjugateON3

NN

Golgi

30

Intracellular Fucosylation

Fluorescentprobe

WGA-Dye(Golgi Marker)

Overlay

Wong, C-H., et al. Proc. Natl. Acad. Sci. 2006, 103, 12371-12376

31

Chemoselective Functionalization of Antibiotics by Glycorandomization

• Glycorandomization: Chemoenzymatic glycodiversity of natural product based scaffolds

Thorson, J.S. et al. Org. Lett. 2005, 7, 1513-1515

OHO

HO

OHOH

N3

OHO

HO

OROH

N3

activated sugar

OHO

HO

OOH

N3

Add antibioticand enzyme

OHO

HO

OOH

NCuAAC

NN

R'

randomized library ofAntibiotic Derivatives

R'

Add activating groupand enzyme

non-natural substrate

glycosylated antibiotic

antibiotic

antibiotic

32

Glycorandomization of Vancomycin

• Vancomycin: glycosylated natural product isolated from the bacteria Amycolatopsis orientalis

• Last defense against infections caused by methicillin-resistant Gram-positive bacteria such as Stapholococcus aureas

• Chemical and chemoenzymatic alterations to vancomycin impact both molecular target and organism specificity vancomycin

Hubbard, B.K., Walsh, C.T. Angew. Chem. Int. Ed. 2003, 42, 730-765

O

NH

O

HN

OHN

O

NH

O

NH2

O O

HN

OH

NH

HO

NH

HO2C

O

O

OCl

Cl

HO

OH

OH

O

HO

HO

O

O

HO

OH

NH2

33

Glycorandomization of Vancomycin

Thorson, J.S. et al. Org. Lett. 2005, 7, 1513-1515

Twice as potent as Vancomycin

O

NH

O

HN

OHN

O

NH

OHO

HO

OH O

N3

NH2

O O

HN

OH

NH

HO

NH

HO2C

O

O

OCl

Cl

HO

OH

OH

R

(24 Alkynes)

CuI, MeOH/H2O70oC, 12h

OHO

HO

OH O

N

N

N

RR = COOH

OHOHO

OH

N3

O P

O

O

OHOHO

OH

N3

O

Thiamine Pyrophosphate

Nucleotidyltransferase

vancomycin aglycon

GtfE

NH

O

ON

O

OH

OP

O

O

P O

O

OO

34

Outline

• Protein Molecular Architecture– Peptide Macrocyclization

• Multivalent Architecture – Vaccine Conjugates

• Inhibitors– Combinatorial

• Chemoenzymatic Functionalization– Metabolic Engineering– Antibiotic Derivatization

• Polymers/Materials Science– Surface Patterning with Dendritic Scaffolds

35

DNA Microarrays Using CuAAC

• DNA microarrays (DNA chips) useful for large scale parallel analysis of gene expression

• Chemistry used for immobilization is limited by cross-reactivity on surface

• Efficiency and Bioorthogonality of CuAAC could overcome existing limitations of immobilization

Reinhoudt, D.A. et al. ChemBioChem, 2007, 8, 1997-2002

Create ssDNA or RNA library

hybridize to surface

Add complementaryDNA strand with dye

36

Transfer Printing of DNA Using Dendritic Architectures

Reinhoudt, D.A. et al. ChemBioChem, 2007, 8, 1997-2002

N N

NH3

NH3

H3N

H3N

PDMS

Si

N N

NH3

NH3

H3N

H3N

N

NN

DNA

N

NN

DNA

N

NN

DNA

OSi

OSi

O

Si

O

N3 N3 N3 N3 N3

Add alkyne modified ssDNA

PDMS

Si

OSi

OSi

O

Si

O

Add Azide Coated Glass

N N

NH3

NH3

H3N

H3N

PDMS

Si

OSi

OSi

O

Si

O

1) Add Cu(I)

3) Wash away unbounddendrimer

2) Remove PDMS Stamp

37

Synthesis of Alkyne Modified DNA Monomer

Reinhoudt, D.A. et al. ChemBioChem, 2007, 8, 1997-2002

NH

O

ON

O

OTBDMS

TBDMSO

I

TMS NH

O

ON

O

OTBDMS

TBDMSO

TMS

PdCl2(PPh3)3

CuI, DIPEA, 92%

1) TBAF

2) DMTrCl, pyridineDMAP, 55% (2 steps)

NH

O

ON

O

OH

DMTrO

NP

O

Cl

CN

THF, DIPEA, 70%

NH

O

ON

O

O

DMTrO

PO

NN

ssDNA

38Reinhoudt, D.A. et al. J. Am. Chem. Soc. 2007, 129, 11593-11599

Surface Patterning of ssDNA

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QuickTime™ and aTIFF (Uncompressed) decompressor

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Oxime Functionalized Template CuAAC Functionalized Template

39

Future Directions: Target Guided Synthesis (TGS)

• Target Guided synthesis uses enzyme for assembling its own inhibitors in situ

• Kinetically controlled approach by irreversible formation of product

• Chemoselectivity of azide/alkyne reaction eliminates byproducts that may alter enzyme

• In situ generated inhibitors separated by LCMS and re-synthesized for Ki determination

Krasinski, A. et al. J. Am. Chem. Soc. 2005, 127, 6686-6692

N3

N3N3

Enzyme

Inhibited Enzyme

N3

N NN

Add enzyme

40

Future Directions

• Target Guided Synthesis has created the most potent inhibitors of HIV Protease, Acetylcholine esterase, and Carbonic Anhydrase known.

• May lead to a revolution in drug discovery

Manetsch, R. et al. J. Am. Chem. Soc. 2004, 126, 12809-12818

Whiting, M. et al. Angew. Chem. Int. Ed. 2006, 45, 1435-1439

Mocharla, V.P. et al. Angew. Chem. Int. Ed. 2005, 44, 116-120

41

Conclusions

• Stepwise, non-concerted mechanism accounts for 1,4 regiospecificity

• Chemoselectivity of azide/alkyne cycloaddition allows for bioorthogonal conjugation and combinatorial screening

• Electronic properties of triazole serve as peptide bond mimics and modulate fluorescence of dyes

• High thermodynamic stability of triazole offers superior control for surface functionalization

42

Acknowledgements

• Laura Kiessling• Hans Reich • Kathleen Myhre• Kiessling Lab Members

Practice Talk Attendees• Chris Shaffer• Christie McGinnis• Emily Dykhuizen• Raja Annamalai• Chris Brown• Katie Garber• Margaret Wong• Aim Tongpenyai• Becca Splain