A Comparative Study of Bioorthogonal Reactions with Azides

1. A Comparative Study of Bioorthogonal Reactions with Azides Agard, N. J.; Baskin, J. M.; Prescher J. A.; Lo A.; Bertozzi C. R. ACS Chem. Biol.2006,10, 644. CHEM 258 Jodi Wyman

2. Overview • Biomolecule tagging • Bioorthogonal chemical reporters • Scope of labeling reactions • Protein and live cell labeling • Reaction guide

3. Green Fluorescent Protein (GFP) • Comprised of 238 amino acids • Isolated from jellyfish Aequorea victoria • Tripeptide Ser65-Tyr66-Gly67 (center of β–barrel) is the chromophore • Can be modified to fluoresce a variety of colors Tsien R.; Annu. Rev. Biochem.1998, 67, 509.

4. GFP Applications and Limitations • Application: • Monitoring proteins in living cells • Limitations: • Structure perturbation (very large) • Cannot be used for glycans, lipids, nucleic acids or other small metabolites

5. Bioorthogonal Chemical Reporters • Tags without direct genetic encoding • Small molecule reporter • Non-native, non-perturbing handles that can work in living cells • Can label biomolecules as long as biosynthetic pathway will tolerate modified precursors Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13.

6. Choosing a Bioorthogonal Chemical Reporter • Tolerated by cell machinery • Robust (avoid unwanted side reactions) • Rapid and selective labeling • Non-toxic (for use with live cells)

7. Bioorthogonal Chemical Reporter:Azide • Advantages: • 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)

8. Azide reacts with RPPh2 under mild conditions Internal electrophilic trap forms amide linkage Phosphines unreactive towards biological functional groups Staudinger Ligation Saxon, E.; Bertozzi, C. R. Science, 2000, 287, 2007.

9. Azide (1,3-dipole) can undergo reactions with activated alkynes Forms triazole adducts Performed at physiological conditions Fast but has high cellular toxicity Cu(I)-Catalyzed Azide-Alkyne Cycloaddition

10. Strain-promoted [3+2] Cycloaddition • Catalyst free [3+2] • Can be performed on surface of living cells • Increase reaction rate with addition of EWG on cyclooctyne

11. Improve Strain [3+2] Cycloaddition Kinetics • Strategies: • Remove phenyl ring • EWG next to alkyne

12. Synthesis of [3+2] Cycloaddition Probes

13. Synthesis of [3+2] Cycloaddition Probes

14. Kinetic Evaluation of Strained [3+2] Cycloaddition

15. Biotinylated Probes

16. Protein Labeling • Dehydrofolate reductase (DHFR) • Replaced methionine residues with azidohomoalanine • Noted: certain detergents used to solubilize the protein hindered click chemistry and Staudinger ligation

17. Protein Labeling Results • Top: Time analysis of 100μM of reagent • Bottom: Concentration dependence

18. Protein Labeling in Presence of Cell Lysate • Top: Labeled proteins detected by Western blot • Bottom: Total protein content determined by Ponceau S

19. Live Cell Labeling • Grew Jurkat cells with Ac4ManNAz which (expressed as SiaNAz on the cell surface)

20. Live Cell Labeling Varki, A.; Cummings, R. D.; Esko, J. D.; Freeze, H. H.; Stanley, P.; Bertozzi, C. R.; Hart, G. W.; Etzler, M. E. Essentials of Glycobiology, Second Ed., 2009, p. 686

21. Mean fluorescence intensity (MFI) Determined by flow cytometry Click chemistry resulted in significant cell death Live Cell Results

22. Toxicity in Live Cells

23. Specific Applications • Optimal reaction based on application • Staudinger ligation: Surfaces of cells and live organisms • Click chemistry: Proteomic samples • Strain-promoted [3+2]: Surfaces of cells

24. Summary • Bioorthogonal chemical reporters are powerful tools for tagging specific biomolecules • Azide is an easy and versatile reporter • A variety of chemical reactions to tag azide can be performed depending on desired sensitivity