Maleimide-mediated methodologies are among the most used in
bioconjugation. Due to fast reactions and high selectivity towards
cysteine residues in
proteins, a large variety of maleimide heterobifunctional reagents are used for the preparation of targeted therapeutics, assemblies for studying proteins in their biological context, protein-based microarrays, or proteins immobilisation. For instance,
antibody-drug conjugates, are constituted of three main components: a
monoclonal antibody, a cytotoxic drug, and a linker molecule often containing a maleimide group, which conjugates the drug through thiols or dienes to the antibody. Attaching the other end of the polyethylene chain to a bead or solid support allows for easy separation of protein from other molecules in solution, provided these molecules do not also possess thiol groups. Maleimide-functionalised polymers and liposomes exhibit enhanced ability to adhere to mucosal surfaces (
mucoadhesion) due to the reactions with thiol-containing mucins. This could be applicable in the design of dosage forms for transmucosal drug delivery. The retro-Michael reactions resulting in maleimide-thiol adducts require precise control. The targeting ability of drugs containing the adducts can be easily hindered or lost due to their instability in vivo. The instability is mainly attributed to the formation of the thiosuccinimide which might be involved in thiol exchange reaction with glutathione. B-elimination reaction follows, resulting in off-target activity and a loss of efficacy of the drugs. No general method exist for stabilizing thioesters, such as thiosuccinimides, so that their off-target effects can be eliminated in drugs. Problems associated with thiol exchange can be mitigated by hydrolyzing the thiosuccinimide, which prevents elimination of the maleimide-thiol bond. The process of ring-opening hydrolysis requires special catalysts and bases, which may not be biocompatible and lead to harsh conditions. Alternatively, cysteines in the positively charged environment or an electron-withdrawing group enable the thiosuccinimide ring to undergo self-hydrolysis. Another problem with hydrolysis arises if it is applied to
N-alkyl-substituted derivatives instead of the N-aryl-substituted derivatives because they hydrolyze at a rate that’s too slow to yield consistently stable adducts. ==Technological applications==