| Autor: |
Istrate A; Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, United Kingdom., Geeson MB; Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, United Kingdom., Navo CD; Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 800, 48160 Derio, Spain., Sousa BB; Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal., Marques MC; Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal., Taylor RJ; Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, United Kingdom., Journeaux T; Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, United Kingdom., Oehler SR; Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland., Mortensen MR; Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland., Deery MJ; Cambridge Centre for Proteomics, Gleeson Building, University of Cambridge, Tennis Court Road, CB2 1QR Cambridge, United Kingdom., Bond AD; Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, United Kingdom., Corzana F; Departamento de Química, Universidad de La Rioja, Centro de Investigación en Síntesis Química, 26006 Logroño, Spain., Jiménez-Osés G; Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 800, 48160 Derio, Spain.; Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Spain., Bernardes GJL; Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, United Kingdom.; Instituto de Medicina Molecular, João Lobo Antunes, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal. |
| Abstrakt: |
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. |
