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Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents

[Image: see text] Protein conjugates are valuable tools for studying biological processes or producing therapeutics, such as antibody–drug conjugates. Despite the development of several protein conjugation strategies in recent years, the ability to modify one specific amino acid residue on a protein...

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Autores principales: Istrate, Alena, Geeson, Michael B., Navo, Claudio D., Sousa, Barbara B., Marques, Marta C., Taylor, Ross J., Journeaux, Toby, Oehler, Sebastian R., Mortensen, Michael R., Deery, Michael J., Bond, Andrew D., Corzana, Francisco, Jiménez-Osés, Gonzalo, Bernardes, Gonçalo J. L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2022
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9490850/
https://www.ncbi.nlm.nih.gov/pubmed/35658467
http://dx.doi.org/10.1021/jacs.2c02185
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author Istrate, Alena
Geeson, Michael B.
Navo, Claudio D.
Sousa, Barbara B.
Marques, Marta C.
Taylor, Ross J.
Journeaux, Toby
Oehler, Sebastian R.
Mortensen, Michael R.
Deery, Michael J.
Bond, Andrew D.
Corzana, Francisco
Jiménez-Osés, Gonzalo
Bernardes, Gonçalo J. L.
author_facet Istrate, Alena
Geeson, Michael B.
Navo, Claudio D.
Sousa, Barbara B.
Marques, Marta C.
Taylor, Ross J.
Journeaux, Toby
Oehler, Sebastian R.
Mortensen, Michael R.
Deery, Michael J.
Bond, Andrew D.
Corzana, Francisco
Jiménez-Osés, Gonzalo
Bernardes, Gonçalo J. L.
author_sort Istrate, Alena
collection PubMed
description [Image: see text] Protein conjugates are valuable tools for studying biological processes or producing therapeutics, such as antibody–drug conjugates. Despite the development of several protein conjugation strategies in recent years, the ability to modify one specific amino acid residue on a protein in the presence of other reactive side chains remains a challenge. We show that monosubstituted cyclopropenone (CPO) reagents react selectively with the 1,2-aminothiol groups of N-terminal cysteine residues to give a stable 1,4-thiazepan-5-one linkage under mild, biocompatible conditions. The CPO-based reagents, all accessible from a common activated ester CPO-pentafluorophenol (CPO-PFP), allow selective modification of N-terminal cysteine-containing peptides and proteins even in the presence of internal, solvent-exposed cysteine residues. This approach enabled the preparation of a dual protein conjugate of 2×cys-GFP, containing both internal and N-terminal cysteine residues, by first modifying the N-terminal residue with a CPO-based reagent followed by modification of the internal cysteine with a traditional cysteine-modifying reagent. CPO-based reagents enabled a copper-free click reaction between two proteins, producing a dimer of a de novo protein mimic of IL2 that binds to the β-IL2 receptor with low nanomolar affinity. Importantly, the reagents are compatible with the common reducing agent dithiothreitol (DTT), a useful property for working with proteins prone to dimerization. Finally, quantum mechanical calculations uncover the origin of selectivity for CPO-based reagents for N-terminal cysteine residues. The ability to distinguish and specifically target N-terminal cysteine residues on proteins facilitates the construction of elaborate multilabeled bioconjugates with minimal protein engineering.
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spelling pubmed-94908502022-09-22 Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents Istrate, Alena Geeson, Michael B. Navo, Claudio D. Sousa, Barbara B. Marques, Marta C. Taylor, Ross J. Journeaux, Toby Oehler, Sebastian R. Mortensen, Michael R. Deery, Michael J. Bond, Andrew D. Corzana, Francisco Jiménez-Osés, Gonzalo Bernardes, Gonçalo J. L. J Am Chem Soc [Image: see text] Protein conjugates are valuable tools for studying biological processes or producing therapeutics, such as antibody–drug conjugates. Despite the development of several protein conjugation strategies in recent years, the ability to modify one specific amino acid residue on a protein in the presence of other reactive side chains remains a challenge. We show that monosubstituted cyclopropenone (CPO) reagents react selectively with the 1,2-aminothiol groups of N-terminal cysteine residues to give a stable 1,4-thiazepan-5-one linkage under mild, biocompatible conditions. The CPO-based reagents, all accessible from a common activated ester CPO-pentafluorophenol (CPO-PFP), allow selective modification of N-terminal cysteine-containing peptides and proteins even in the presence of internal, solvent-exposed cysteine residues. This approach enabled the preparation of a dual protein conjugate of 2×cys-GFP, containing both internal and N-terminal cysteine residues, by first modifying the N-terminal residue with a CPO-based reagent followed by modification of the internal cysteine with a traditional cysteine-modifying reagent. CPO-based reagents enabled a copper-free click reaction between two proteins, producing a dimer of a de novo protein mimic of IL2 that binds to the β-IL2 receptor with low nanomolar affinity. Importantly, the reagents are compatible with the common reducing agent dithiothreitol (DTT), a useful property for working with proteins prone to dimerization. Finally, quantum mechanical calculations uncover the origin of selectivity for CPO-based reagents for N-terminal cysteine residues. The ability to distinguish and specifically target N-terminal cysteine residues on proteins facilitates the construction of elaborate multilabeled bioconjugates with minimal protein engineering. American Chemical Society 2022-06-05 2022-06-15 /pmc/articles/PMC9490850/ /pubmed/35658467 http://dx.doi.org/10.1021/jacs.2c02185 Text en © 2022 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Istrate, Alena
Geeson, Michael B.
Navo, Claudio D.
Sousa, Barbara B.
Marques, Marta C.
Taylor, Ross J.
Journeaux, Toby
Oehler, Sebastian R.
Mortensen, Michael R.
Deery, Michael J.
Bond, Andrew D.
Corzana, Francisco
Jiménez-Osés, Gonzalo
Bernardes, Gonçalo J. L.
Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents
title Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents
title_full Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents
title_fullStr Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents
title_full_unstemmed Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents
title_short Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents
title_sort platform for orthogonal n-cysteine-specific protein modification enabled by cyclopropenone reagents
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9490850/
https://www.ncbi.nlm.nih.gov/pubmed/35658467
http://dx.doi.org/10.1021/jacs.2c02185
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