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

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

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
Compound
Reaction rate (M-1s-1) 12
4.3x10-3 1
2.4x10-3 8
1.3x10-3 13
1.2x10-3

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

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