The advent of antibody–drug conjugates as pharmaceuticals has fuelled a need for reliable methods of site-selective protein modification that furnish homogeneous adducts. Although bioorthogonal methods that use engineered amino acids often provide an elegant solution to the question of selective functionalization, achieving homogeneity using native amino acids remains a challenge. Here, we explore visible-light-mediated single-electron transfer as a mechanism towards enabling site- and chemoselective bioconjugation. Specifically, we demonstrate the use of photoredox catalysis as a platform to selectivity wherein the discrepancy in oxidation potentials between internal versus C-terminal carboxylates can be exploited towards obtaining C-terminal functionalization exclusively. This oxidation potential-gated technology is amenable to endogenous peptides and has been successfully demonstrated on the protein insulin. As a fundamentally new approach to bioconjugation this methodology provides a blueprint toward the development of photoredox catalysis as a generic platform to target other redox-active side chains for native conjugation.
Subscribe to Journal
Get full journal access for 1 year
only $13.33 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Boutureira, O. & Bernardes, G. J. L. Advances in chemical protein modification. Chem. Rev. 115, 2174–2195 (2015).
Krall, N., da Cruz, F. P., Boutureira, O. & Bernardes, G. J. L. Site-selective protein-modification chemistry for basic biology and drug development. Nat. Chem. 8, 103–113 (2016).
Sletten, E. M. & Bertozzi, C. R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew. Chem. Int. Ed. 48, 6974–6998 (2009).
Saxon, E. & Bertozzi, C. R. Cell surface engineering by a modified Staudinger reaction. Science 287, 2007–2010 (2000).
Junutula, J. R. et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 26, 925–932 (2008).
Lyon, R. P., Meyer, D. L., Setter, J. R. & Senter, P. D. Conjugation of anticancer drugs through endogenous monoclonal antibody cysteine resides. Methods Enzymol. 502, 123–138 (2012).
Baslé, E., Joubert, N. & Pucheault, M. Protein chemical modification on endogenous amino acids. Chem. Biol. 17, 213–227 (2010).
Miller, S., Janin, J., Lesk, A. M. & Chothia, C. Interior and surface of monomeric proteins. J. Mol. Biol. 196, 641–656 (1987).
Chen, X., Muthoosamy, K., Pfisterer, A., Neumann, B. & Weil, T. Site-selective lysine modification of native proteins and peptides via kinetically controlled labelling. Bioconjugate Chem. 23, 500–508 (2012).
Bader, B. et al. Bioorganic synthesis of lipid-modified proteins for the study of signal transduction. Nature 403, 223–226 (2000).
Rosen, C. B. & Francis, M. B. Targeting the N terminus for site-selective protein modification. Nat. Chem. Biol. 13, 697–705 (2017).
Romanini, D. W. & Francis, M. B. Attachment of peptide building blocks to proteins through tyrosine bioconjugation. Bioconjugate Chem. 19, 153–157 (2008).
Tilley, S. D. & Francis, M. B. Tyrosine-selective protein alkylation using π-allylpalladium complexes. J. Am. Chem. Soc. 128, 1080–1081 (2006).
Joshi, N. S., Whitaker, L. R. & Francis, M. B. A three-component Mannich-type reaction for selective tyrosine bioconjugation. J. Am. Chem. Soc. 126, 15942–15943 (2004).
Ban, H. et al. Facile and stable linkages through tyrosine: bioconjugation strategies with the tyrosine-click reaction. Bioconjugate Chem. 24, 520–532 (2013).
Antos, J. M., McFarland, J. M., Iavarone, A. T. & Francis, M. B. Chemoselective tryptophan labeling with rhodium carbenoids at mild pH. J. Am. Chem. Soc. 131, 6301–6308 (2009).
Antos, J. M. & Francis, M. B. Selective tryptophan modification with rhodium carbenoids in aqueous solution. J. Am. Chem. Soc. 126, 10256–10257 (2004).
Seki, Y. et al. Transition metal-free tryptophan-selective bioconjugation of proteins. J. Am. Chem. Soc. 138, 10798–10801 (2016).
Lin, S. et al. Redox-based reagents for chemoselective methionine bioconjugation. Science 355, 597–602 (2017).
Zuo, Z. & MacMillan, D. W. C. Decarboxylative arylation of α-amino acids via photoredox catalysis: a one-step conversion of biomass to drug pharmacophore. J. Am. Chem. Soc. 136, 5257–5260 (2014).
Chu, L., Ohta, C., Zuo, Z. & MacMillan, D. W. C. Carboxylic acids as a traceless activation group for conjugate additions: a three-step synthesis of (±)-pregabalin. J. Am. Chem. Soc. 136, 10886–10889 (2014).
Noble, A. & MacMillan, D. W. C. Photoredox-mediated α-vinylation of α-amino acids and N-aryl amines. J. Am. Chem. Soc. 136, 11602–11605 (2014).
Zuo, Z. et al. Merging photoredox with nickel catalysis: coupling of α-carboxyl sp3-carbons with aryl halides. Science 345, 437–440 (2014).
Galicia, M. & González, F. J. Electrochemical oxidation of tetrabutylammonium salts of aliphatic carboxylic acids in acetonitrile. J. Electrochem. Soc. 149, D46–D50 (2002).
Hu, Q.-Y., Berti, F. & Adamo, R. Towards the next generation of biomedicines by site-selective conjugation. Chem. Soc. Rev. 45, 1691–1719 (2016).
McGrath, N. A., Andersen, K. A., Davis, A. K. F., Lomax, J. E. & Raines, R. T. Diazo compounds for the bioreversible esterification of proteins. Chem. Sci. 6, 752–755 (2015).
Rajagopalan, T. G., Stein, W. H. & Moore, S. The inactivation of pepsin by diazoacetylnorleucine methyl ester. J. Biol. Chem. 241, 4295–4297 (1966).
Delpierre, G. R. & Fruton, J. S. Specific inactivation of pepsin by a diazo ketone. Proc. Natl Acad. Sci. USA 56, 1817–1822 (1966).
Totaro, K. A. et al. Systematic investigation of EDC/sNHS-mediated bioconjugation reactions for carboxylated peptide substrates. Bioconj. Chem. 27, 994–1004 (2016).
Noble, A., McCarver, S. J. & MacMillan, D. W. C. Merging photoredox and nickel catalysis: decarboxylative cross-coupling of carboxylic acids with vinyl halides. J. Am. Chem. Soc. 137, 624–627 (2015).
Slutskyy, Y. & Overman, L. E. Generation of the methoxycarbonyl radical by visible-light photoredox catalysis and its conjugate addition with electron-deficient olefins. Org. Lett. 18, 2564–2567 (2016).
MacDonald, J. I., Munch, H. K., Moore, T. & Francis, M. B. One-step site-specific modification of native proteins with 2-pyridinecarboxyaldehydes. Nat. Chem. Biol. 11, 326–331 (2015).
Novak, M., Miller, A., Bruice, T. C. & Tollin, G. The mechanism of flavin 4a substitution which accompanies photolytic decarboxylation of α-substituted acetic acids. Carbanion vs. radical intermediates. J. Am. Chem. Soc. 102, 1465–1467 (1980).
Islam, S. D. M., Penzkofer, A. & Hegemann, P. Quantum yield of triplet formation of riboflavin in aqueous solution and of flavin mononucleotide bound to the LOV1 domain of Phot1 from Chlamydomonas reinhardtii. Chem. Phys. 291, 97–114 (2003).
Lu, C. et al. Riboflavin (VB2) photosensitized oxidation of 2′-deoxyguanosine-5′-monophosphate (dGMP) in aqueous solution: a transient intermediates study. Phys. Chem. Chem. Phys. 2, 329–334 (2000).
Bortolamei, N., Isse, A. A. & Gennaro, A. Estimation of standard reduction potentials of alkyl radicals involved in atom transfer radical polymerization. Electrochim. Acta 55, 8312–8318 (2010).
Huvaere, K. & Skibsted, L. H. Light-induced oxidation of tryptophan and histidine. Reactivity of aromatic N-heterocycles toward triplet-excited flavins. J. Am. Chem. Soc. 131, 8049–8060 (2009).
Harriman, A. Further comments on the redox potentials of tryptophan and tyrosine. J. Phys. Chem. 91, 6102–6104 (1987).
Stubbe, J. & van der Donk, W. A. Protein radicals in enzyme catalysis. Chem. Rev. 98, 705–762 (1998).
Sikorska, E. et al. Spectroscopy and photophysics of lumiflavins and lumichromes. J. Phys. Chem. A 108, 1501–1508 (2004).
Koziol, J. Studies on flavins in organic solvents – I. Spectral characteristics of riboflavin, riboflavin tetrabutyrate, and lumichrome. Photochem. Photobiol. 5, 41–54 (1966).
Garbaccio, R. M. in Comprehensive Organic Synthesis II 2nd edn, Vol. 1 (eds Knochell, P. & Molander, G. A.) Ch. 9, 438–462 (Elsevier, 2014).
Ravelli, D., Zema, M., Mella, M., Fagnoni, M. & Albini, A. Benzoyl radicals from (hetero)aromatic aldehydes. Decatungstate photocatalyzed synthesis of substituted aromatic ketones. Org. Biomol. Chem. 8, 4158–4164 (2010).
Wagner, A. & Koniev, O. Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation. Chem. Soc. Rev. 44, 5495–5551 (2015).
Uchida, K. & Stadtman, E. R. Modification of histidine residues in proteins by reaction with 4-hydroxynonenal. Proc. Natl Acad. Sci. USA 89, 4544–4548 (1992).
Wang, Y., Luo, Y. & Zhang, R. Investigation on insulin tyrosine modification mediated by peroxynitrite. In Proc. IEEE/ICME Int. Conf. Complex Chem. Engineering Beijing, Beijing, 1813–1816 (IEEE, 2007).
Lindsay, D. G. & Shall, S. The acetylation of insulin. Biochem. J. 121, 737–745 (1971).
The authors acknowledge financial support provided by the NIHGMS (R01 01GM093213-04) and gifts from Merck and BMS. D.K.K. acknowledges the Deutsche Forschungsgemeinschaft (DFG) for a postdoctoral fellowship (KO 4867/2-1). The authors thank T. Muir, Z. Brown, R. Thompson and members of the Muir Laboratory for their advice and analytical support. The authors also thank I. Pelczer and K. Conover for assistance with NMR spectroscopy.
The authors declare no competing financial interests.
About this article
Cite this article
Bloom, S., Liu, C., Kölmel, D. et al. Decarboxylative alkylation for site-selective bioconjugation of native proteins via oxidation potentials. Nature Chem 10, 205–211 (2018). https://doi.org/10.1038/nchem.2888
Direct Decarboxylative Allylation and Arylation of Aliphatic Carboxylic Acids Using Flavin-Mediated Photoredox Catalysis
European Journal of Organic Chemistry (2020)
Radical Aza-Cyclization of α-Imino-oxy Acids for Synthesis of Alkene-Containing N-Heterocycles via Dual Cobaloxime and Photoredox Catalysis
Organic Letters (2020)
Visible‐Light‐Promoted C(sp 3 )−H Alkylation by Intermolecular Charge Transfer: Preparation of Unnatural α‐Amino Acids and Late‐Stage Modification of Peptides
Angewandte Chemie (2020)
Selective Modification of Tryptophan Residues in Peptides and Proteins Using a Biomimetic Electron Transfer Process
Journal of the American Chemical Society (2020)
C( sp 3 )−C( sp 3 ) Cross‐Coupling of Alkyl Bromides and Ethers Mediated by Metal and Visible Light Photoredox Catalysis
Advanced Synthesis & Catalysis (2020)