Bioconjugation through mesitylene thiol alkylation

The design and generation of complex multifunctional macromolecular structures by bioconjugation is a hot topic due to increasing interest in conjugates with therapeutic applications. In this regard, the development of efficient, selective, and safe conjugation methods is a major objective. In this report, we describe the use of the bis(bromomethyl)benzene scaffold as a linker for bioconjugation with special emphasis on antibody conjugation. We first performed the monothioalkylation of 1,3,5-tris(bromomethyl)benzene, which rendered the reactive dibromotrimethylbenzyl derivatives to be used in thiol bis-alkylation. Next, we introduced into the linker either a bis(Cys)-containing peptide or anti-CD4 and -CD13 monoclonal antibodies, previously subjected to partial reduction of disulfide bonds. Mass spectrometry, UV–vis spectra, and SDS-PAGE experiments revealed that this bis-alkylating agent for bioconjugation preserved both antibody integrity and antibody–antigen binding affinity, as assessed by flow cytometry. Taken together, our results show that the mesitylene scaffold is a suitable linker for thiol-based bioconjugation reactions. This linker could be applicable in the near future for the preparation of antibody drug conjugates.I.R-T. thanks the Generalitat de Catalunya for a predoctoral fellowship. This work was funded in part by the following: the Ministerio de Economia y Competitividad (MINECO) and European Regional Development’s funds (ERDF) - Programa INNPACTO, project MarinMab (IPT-2012-0198-090000), the Generalitat de Catalunya (2014 SGR 137), and the Institute for Research in Biomedicine Barcelona (IRB Barcelona) (Spain), and the National Research Foundation (NRF) (Blue Sky’s Research Programme # 110960) (South Africa).Peer Reviewe