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Selective N-Terminal Cysteine Protein Modification with Cyclopropenones

preprint
submitted on 26.08.2020, 14:13 and posted on 27.08.2020, 07:25 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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