Matos, Maria J.Navo, Claudio D.Hakala, TuuliFerhati, XhentiGuerreiro, AnaHartmann, DavidBernardim, BarbaraSaar, Kadi L.Compañón, IsmaelCorzana, FranciscoKnowles, Tuomas P. J.Jiménez‐Osés, GonzaloBernardes, Gonçalo J. L.2022-05-102022-05-102019Angew Chem Int Ed Engl. 2019 May 13;58(20):6640-66441433-7851http://hdl.handle.net/10451/52854© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Quaternized vinyl- and alkynyl-pyridine reagents were shown to react in an ultrafast and selective manner with several cysteine-tagged proteins at near-stoichiometric quantities. We have demonstrated that this method can effectively create a homogenous antibody-drug conjugate that features a precise drug-to-antibody ratio of 2, which was stable in human plasma and retained its specificity towards Her2+ cells. Finally, the developed warhead introduces a +1 charge to the overall net charge of the protein, which enabled us to show that the electrophoretic mobility of the protein may be tuned through the simple attachment of a quaternized vinyl pyridinium reagent at the cysteine residues. We anticipate the generalized use of quaternized vinyl- and alkynyl-pyridine reagents not only for bioconjugation, but also as warheads for covalent inhibition and as tools to profile cysteine reactivity.engAntibody-drug conjugatesBioconjugationCysteineMicrofluidicsProtein modificationjournal article10.1002/anie.2019014051521-3773