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

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

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.

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

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.

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)

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)

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.

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

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

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

Synthesis of [3+2] Cycloaddition Probes

Synthesis of [3+2] Cycloaddition Probes

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

Biotinylated Probes

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

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

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

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

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

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

Toxicity in Live Cells

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

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