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

1. 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, 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

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.

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, 2596-2599

6. CuAAC Catalytic Cycle 23 kcal/mol 18 kcal/mol Himo, F. et al. J. Am. Chem. Soc, 2005, 127, 210-216. Ahlquist, M., Fokin, V.V. Organometallics2007, 26, 4389-4391.

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. Tetrahedron1988, 44, 685-695

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

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, 1128-1136.

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

12. Synthesis of Cyclic Tetrapeptide Analogue Bock, et al. Org. Lett. 2006, 8, 919-922

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

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

16. CuAAC of Multivalent Carbohydrate Peptide Conjugate Wan, Q., Chen, J., Chen, G., Danishefsky, S.J. J. Org. Chem. 2006, 71, 8244-8249.

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, 1529-1540.

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.

19. Synthesis of Man4 Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540.

20. Template Synthesis of Man-4 Cluster Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540.

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

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

24. Synthesis of HIV Protease Inhibitor Folkin, V.V. et al. J. Med. Chem. 2006, 49, 7697-7710

25. Synthesis of HIV Protease Inhibitor Folkin, V.V. et al. J. Med. Chem. 2006, 49, 7697-7710

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

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

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

30. Intracellular Fucosylation Fluorescent probe 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

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

33. Glycorandomization of Vancomycin Thorson, J.S. et al. Org. Lett. 2005, 7, 1513-1515

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

36. Transfer Printing of DNA Using Dendritic Architectures Reinhoudt, D.A. et al. ChemBioChem, 2007, 8, 1997-2002

37. Synthesis of Alkyne Modified DNA Monomer Reinhoudt, D.A. et al. ChemBioChem, 2007, 8, 1997-2002

38. Surface Patterning of ssDNA Oxime Functionalized Template CuAAC Functionalized Template Reinhoudt, D.A. et al. J. Am. Chem. Soc.2007, 129, 11593-11599

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

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 Mocharla, V.P. et al. Angew. Chem. Int. Ed. 2005, 44, 116-120 Whiting, M. et al. Angew. Chem. Int. Ed. 2006, 45, 1435-1439

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. 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 Acknowledgements