1. Shane Mangold Kiessling Group February 14th 2008
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
1933- Dipolar nature of azide first recognized by Linus Pauling
1960- Mechanism of 1,3-dipolar cycloaddition of azides
and alkynes pioneered by Rolf Huisgen
2001- Copper catalyzed 1,3-Dipolar cycloaddition by Sharpless/Meldal
L. Pauling. Proc. Natl. Acad. Sci. USA 1933, 19, ; Huisgen, R. Angew. Chem. Int. Ed. 1963, 2,
Sharpless, K.B. et al. Angew. Chem. Int. Ed 2002, 41, ; Meldal,M.J. et al. J. Org. Chem. 2002, 67,

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 , 40,

4. Reactions that meet the “Click” Criteria
Kolb, H.C.; Finn, M.G.; Sharpless, B.K. Angew. Chem. Int. Ed , 40,

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
Rostovtsev et al. Angew. Chem. Int Ed. 2002, 41,

6. CuAAC Catalytic Cycle 23 kcal/mol 18 kcal/mol
Himo, F. et al. J. Am. Chem. Soc, 2005, 127,
Ahlquist, M., Fokin, V.V. Organometallics 2007, 26,

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,

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,

10. 1,2,3-Triazoles as Peptide Bond Isosteres
3.9 Å
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
5.1 Å
Kolb, H.C., Sharpless, B.K. Drug. Disc. Today. 2003, 8,

11. Retrosynthesis
Bock, V.D., et al. Org. Lett. 2006, 8,

12. Synthesis of Cyclic Tetrapeptide Analogue
Bock, et al. Org. Lett. 2006, 8,

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

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,

16. CuAAC of Multivalent Carbohydrate Peptide Conjugate
Wan, Q., Chen, J., Chen, G., Danishefsky, S.J. J. Org. Chem. 2006, 71,

17. Template-Assembled Oligosaccharide Epitope Mimics
2G12 antibody targets oligomannose cluster (Man-9) present on HIV-1 gp120
Recognizes terminal Man1-2Man 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,

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,

19. Synthesis of Man4
Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5,

20. Template Synthesis of Man-4 Cluster
Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5,

21. Synthetic Vaccine Conjugate
Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5,

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,

24. Synthesis of HIV Protease Inhibitor
Folkin, V.V. et al. J. Med. Chem. 2006, 49,

25. Synthesis of HIV Protease Inhibitor
Folkin, V.V. et al. J. Med. Chem. 2006, 49,

26. Inhibitor Optimization
Folkin, V.V. et al. J. Med. Chem. 2006, 49,

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,

29. Metabolic Oligosaccharide Engineering
Wong, C-H., et al. Proc. Natl. Acad. Sci. 2006, 103,

30. Intracellular Fucosylation
Fluorescent
probe
WGA-Dye
(Golgi Marker)
Overlay
Wong, C-H., et al. Proc. Natl. Acad. Sci. 2006, 103,

31. Chemoselective Functionalization of Antibiotics by Glycorandomization
Glycorandomization: Chemoenzymatic glycodiversity of natural product based scaffolds
Thorson, J.S. et al. Org. Lett. 2005, 7,

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,

33. Glycorandomization of Vancomycin
Thorson, J.S. et al. Org. Lett. 2005, 7,

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,

36. Transfer Printing of DNA Using Dendritic Architectures
Reinhoudt, D.A. et al. ChemBioChem, 2007, 8,

37. Synthesis of Alkyne Modified DNA Monomer
Reinhoudt, D.A. et al. ChemBioChem, 2007, 8,

38. Surface Patterning of ssDNA
Oxime Functionalized Template
CuAAC Functionalized Template
Reinhoudt, D.A. et al. J. Am. Chem. Soc. 2007, 129,

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,

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,
Mocharla, V.P. et al. Angew. Chem. Int. Ed. 2005, 44,
Whiting, M. et al. Angew. Chem. Int. Ed. 2006, 45,

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