Global targeting of functional tyrosines using sulfur-triazole exchange chemistry

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Covalent probes serve as valuable tools for global investigation of protein function and ligand binding capacity. Despite efforts to expand coverage of residues available for chemical proteomics (e.g., cysteine and lysine), a large fraction of the proteome remains inaccessible with current activity-based probes. Here, we introduce sulfur-triazole exchange (SuTEx) chemistry as a tunable platform for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can furnish sulfonyl probes with ~5-fold enhanced chemoselectivity for tyrosines over other nucleophilic amino acids to investigate more than 10,000 tyrosine sites in lysates and live cells. We discover that tyrosines with enhanced nucleophilicity are enriched in enzymatic, protein–protein interaction and nucleotide recognition domains. We apply SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites. Collectively, we describe SuTEx as a biocompatible chemistry for chemical biology investigations of the human proteome.

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Fig. 1: Development of sulfur-triazole exchange (SuTEx) chemistry for chemical proteomics.
Fig. 2: Functional tyrosine profiling in proteomes and live cells.
Fig. 3: SuTEx-enabled discovery of intrinsically nucleophilic tyrosines in human cell proteomes.
Fig. 4: Tuning SuTEx probes for tyrosine chemoselectivity in cell proteomes.
Fig. 5: Triazole LG enhances phenol reactivity of sulfonyl probes in solution.
Fig. 6: Chemical phosphotyrosine-proteomics by SuTEx.

Data availability

All data produced or analyzed for this study are included in the published article (and its Supplementary Information files) or are available from the corresponding author on reasonable request. Crystallographic data for small molecules has been deposited in the Cambridge Crystallographic Data Centre and have been assigned the following deposition numbers HHS-465 (CCDC 1954297), HHS-475 (CCDC 1954298) and HHS-483 (CCDC 1954299). These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via

Code availability

All code is available upon reasonable request from the corresponding author.


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We thank all members of the Hsu Laboratory and colleagues at the University of Virginia for helpful discussions and review of the manuscript. We thank M. Ross for his assistance with mass spectrometry experiments and data analysis. We thank S. Campbell for assistance with molecular biology experiments. We thank D. Dickie for assistance with small molecule crystallography studies. We thank R. Rumana for assistance with cell culture studies. This work was supported by the University of Virginia (start-up funds to K.-L.H.), University of Virginia Cancer Center (A.H.L. and K.-L.H), National Institutes of Health grant nos. GM801868 (T.B.W.), GM007055 (J.W.B.), CA009109 (A.L.B.) and DA043571 (K.-L.H.), the Schiff Foundation (K.-L.H.), the Wagner Fellowship (A.L.B), the National Science Foundation Graduate Research Fellowship (grant no. 2018255830 to R.L.M.) and the U.S. Department of Defense (no. W81XWH-17-1-0487 to K.-L.H.).

Author information

H.S.H., E.K.T., A.L.B., J.W.B. and K.-L.H. conceived of the project, designed experiments and analyzed data. H.S.H. and E.K.T. performed mass spectrometry experiments and data analysis. A.L.B. wrote software and performed bioinformatics analysis. H.S.H., J.W.B. and K.Y. synthesized compounds. J.W.B. expressed proteins, conducted inhibition studies and performed biochemical assays. E.K.T. and J.W.B. conducted cellular studies. A.H.L. assisted with compound synthesis and characterization. T.B.W., A.M.C. and R.L.M. performed site-directed mutagenesis and assisted with cloning and expression of proteins. H.S.H., E.K.T., A.L.B., J.W.B. and K.-L.H. wrote the manuscript.

Correspondence to Ku-Lung Hsu.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–31

Reporting Summary

Supplementary Dataset 1

Supplementary Note

Synthetic Procedures

X-ray crystal structures of compounds 2, 3, and 6

Individual CIF files for HHS-465, HHS-475 and HHS-483 crystal structures combined into a single CIF file.

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Hahm, H.S., Toroitich, E.K., Borne, A.L. et al. Global targeting of functional tyrosines using sulfur-triazole exchange chemistry. Nat Chem Biol (2019) doi:10.1038/s41589-019-0404-5

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