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Selective N-Terminal Cysteine Protein Modification with Cyclopropenones

preprint
submitted on 26.08.2020 and posted on 27.08.2020 by Alena Istrate, Claudio D. Navo, Bárbara B. Sousa, Marta C. Marques, Michael Deery, Andrew Bond, Francisco Corzana, Gonzalo Jiménez-Osés, Gonçalo Bernardes

Protein conjugates are valuable tools to create therapeutics, such as antibody-drug conjugates, or to study biological processes. Despite a number of protein conjugation strategies having been developed over recent years, the ability to modify one specific amino acid on a protein in the presence of other side chains with similar reactivity remains a challenge. We used the reaction between a monosubstituted cyclopropenone (CPO) probe and the 1,2-aminothiol of an N-terminal cysteine to give a stable 1,4-thiazepa-5-none linkage under mild, biocompatible conditions. The method relies on the ability of cyclopropenones to ring-open after sequential sulfhydryl and α-amine conjugation to be truly specific for N-terminal cysteine. We show that our CPO probes selectively label N-terminal cysteine containing peptides and proteins even in the presence of internal, solvent-exposed cysteines, which can be subsequently modified by using conventional cysteine modification reagents. The ability to distinguish and specifically target N-terminal cysteine residues on a protein will facilitate the construction of elaborate multi-labelled bioconjugates.

Funding

Fundação para a Ciência e a Tecnologia, Investigator IF/00624/2015 to G.J.L.B.

Spanish Ministry of Science and Innovation (MCI) co-financed with FEDER funds (RTI2018-099592-B-C21 to F.C., and RTI2018-099592-B-C22 to G.J.O.)

Swiss National Science Foundation (Early Postdoc.Mobility P2BEP3_175253 to A.I.)

G.J.L.B. is a Royal Society University Research Fellow (URF\R\180019)

History

Email Address of Submitting Author

gb453@cam.ac.uk

Institution

Department of Chemistry, University of Cambridge

Country

United Kingdom

ORCID For Submitting Author

0000-0001-6594-8917

Declaration of Conflict of Interest

The authors declare no competing financial interest.

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