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Site-selective tyrosine bioconjugation via photoredox catalysis for native-to-bioorthogonal protein transformation

Nature Chemistry volume 13, pages 902–908 (2021)Cite this article

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The growing prevalence of synthetically modified proteins in pharmaceuticals and materials has exposed the need for efficient strategies to enable chemical modifications with high site-selectivity. While genetic engineering can incorporate non-natural amino acids into recombinant proteins, regioselective chemical modification of wild-type proteins remains a challenge. Herein, we use photoredox catalysis to develop a site-selective tyrosine bioconjugation pathway that incorporates bioorthogonal formyl groups, which subsequently allows for the synthesis of structurally defined fluorescent conjugates from native proteins. A water-soluble photocatalyst, lumiflavin, has been shown to induce oxidative coupling between a previously unreported phenoxazine dialdehyde tag and a single tyrosine site, even in the presence of multiple tyrosyl side chains, through the formation of a covalent C–N bond. A variety of native proteins, including those with multiple tyrosines, can successfully undergo both tyrosine-specific and single-site-selective labelling. This technology directly introduces aldehyde moieties onto native proteins, enabling rapid product diversification using an array of well-established bioorthogonal functionalization protocols including the alkyne–azide click reaction.

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Fig. 1: The photoredox-catalysed merger of native protein site-selective bioconjugation with bioorthogonal methodology.
Fig. 2: Tyrosine microenvironments.
Fig. 3: Proposed reaction mechanism for site-selective amination on tyrosine residues.
Fig. 4: Diversification of photoredox tyrosine amination products via bioorthogonal aldehyde-click functionalization.

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The data supporting the findings of this study are available within the article and its Supplementary Information.

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Acknowledgements

Research was primarily supported as a part of BioLEC, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Basic Energy Sciences under award DE-SC0019370 for all research efforts directed towards bioconjugation studies and spectroscopic analyses. We acknowledge K. Saw, H.H. Shwe and the use of Princeton’s Imaging and Analysis Center, which is partially supported by the Princeton Center for Complex Materials, a National Science Foundation/Materials Research Science and Engineering Centers programme (DMR-1420541). We also acknowledge V. G. Vendavasi and the use of Princeton’s Biophysics Core Facility. We thank T. W. Muir and members of the Muir Laboratory for their advice and analytical support. Finally, we thank T.F. Brewer for additional discussions and advice.

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Author notes

  1. These authors contributed equally: Beryl X. Li, Daniel K. Kim.

Authors and Affiliations

  1. Merck Center for Catalysis at Princeton University, Princeton, NJ, USA

    Beryl X. Li, Daniel K. Kim, Steven Bloom & David W. C. MacMillan

  2. Pharmaceutical Candidate Optimization, Research and Development, Bristol-Myers Squibb Company, Princeton, NJ, USA

    Richard Y.-C. Huang

  3. Discovery Chemistry, Research and Development, Bristol-Myers Squibb Company, Princeton, NJ, USA

    Jennifer X. Qiao & William R. Ewing

  4. Department of Chemistry, Princeton University, Princeton, NJ, USA

    Daniel G. Oblinsky & Gregory D. Scholes

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Contributions

B.X.L., D.K.K. and S.B. performed and analysed the bioconjugation experiments. R.Y.-C.H. performed and analysed the mass spectrometry experiments. D.G.O. performed and analysed the transient absorption spectroscopy experiments. D.K.K., B.X.L., S.B. and D.W.C.M. designed the experiments. D.K.K., B.X.L. and D.W.C.M. prepared this manuscript. G.D.S., J.X.Q. and W.R.E. provided helpful discussions.

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Correspondence to David W. C. MacMillan.

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Supplementary information

Supplementary Information

Experimental details, intact mass spectrometry product characterization, fluorescence assay details, reagent synthesis procedures, NMR spectra, LC-MS/MS spectra and ultrafast spectroscopy details.

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Li, B.X., Kim, D.K., Bloom, S. et al. Site-selective tyrosine bioconjugation via photoredox catalysis for native-to-bioorthogonal protein transformation. Nat. Chem. 13, 902–908 (2021). https://doi.org/10.1038/s41557-021-00733-y

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