Covalent modification of biomolecules through maleimide-based labeling strategies

Cyrille Sabot, Pierre-Yves Renard, Kevin Renault, and Jean Wilfried Fredy

Bioconjugate Chem., 2018
DOI: 10.1021/acs.bioconjchem.8b00252

Since their first use in bioconjugation more than 50 years ago, maleimides have become privileged chemical partners for the site selective modification of proteins through thio-Michael addition of biothiols and, to a lesser extent, via Diels‒Alder (DA) reactions in combination with biocompatible dienes. Prominent examples include immunotoxins and marketed maleimide-based antibody-drug conjugates (ADCs) such as Adcetris® used in cancer therapies. Among the keys to success is the availability of several maleimides N-functionalized by fluorophores, affinity tags, spin labels, pharmacophores, as well as their unique reactivity features in terms of selectivity or kinetics. However, maleimide conjugate reactions have long been thought to be irreversible, and it is only recently that systematic studies regarding their reversibility and stability towards hydrolysis have been reported. This review provides an overview of the diversity of applications of maleimides in bioconjugation, highlighting their strengths and weaknesses, which are being circumvented by recent strategies. Finally, the fluorescence quenching ability of maleimides was leveraged in the preparation of fluorogenic probes mainly involved in the specific detection of thiol analytes. A summary of reported structures, their photophysical features and relative efficiency are discussed in the last part of the review.