Structural investigation of cyclo-dioxo maleimide cross-linkers for acid and serum stability

• Published in: Organic & biomolecular chemistry 2017-11, Vol.15 (44), p.935-931
• Main Authors: Tobaldi, Elisabetta, Dovgan, Igor, Mosser, Michel, Becht, Jean-Michel, Wagner, Alain
• Format: Article
• Language: English
• Subjects: Biochemical characteristics; Biochemistry; Chemical Sciences; Conjugation; Crosslinking; Fluorescence; Fluorescence resonance energy transfer; Organic chemistry; Probes; Succinimide
• ISSN: 1477-0520; 1477-0539
• DOI: 10.1039/c7ob01757j

The biochemical characteristics of hetero-bifunctional cross-linkers used in bioconjugates are of essential importance to the desired features of the final adduct ( i.e. antibody-drug conjugates). These include stability in biological media, chemical and biological reactivities, cleavability under defined conditions, and solubility. In our previous work, we introduced a new amino-to-thiol linker, maleimidomethyl dioxane (MD), as an alternative to classical maleimide conjugation, with increased hydrophilicity and serum stability due to succinimidyl ring-opening. In this work, we investigate the generality of linkers containing a dioxo-ring with regard to their ability to self-hydrolyze and their surprising stability at a low pH. We synthesized four FRET probes which allowed us to address the stability of the dioxo-ring and to study the maleimide ring-opening and the thiol-exchange processes by means of detecting and measuring the generation of fluorescence. It was found that the ring expansion (from a 5- to a 6-membered ring) improved the stability of the probes in aqueous media, and the increase of the chain length between the dioxo-ring and the succinimide ring (from methylene to ethylene) decreased the rate of succinimidyl ring-opening. The size of the acetal ring and the length of the carbon chain strongly influence the serum stability of cyclo-dioxo-based amino-to-thiol coupling reagents.