NHSestermaleimide

NHSestermaleimide is a class of heterobifunctional crosslinkers that incorporate an N-hydroxysuccinimide (NHS) ester on one end and a maleimide group on the other. This arrangement enables sequential conjugation: the NHS ester reacts with primary amines, such as lysine residues or N-terminal amines on proteins, forming stable amide bonds; the exposed maleimide group then reacts with thiols, typically cysteine residues, via a Michael-type addition to form a stable thioether linkage. The two reactive ends allow selective linking of amine-containing molecules to thiol-containing partners under mild, aqueous conditions.

Commercial derivatives of NHSestermaleimide often feature a chemical spacer between the NHS and maleimide groups. Well-known examples include crosslinkers that combine an NHS ester with a maleimide moiety, such as SMCC and Sulfo-SMCC, which differ mainly in solubility and spacer design. These reagents are widely used in bioconjugation to assemble protein conjugates, antibody-drug conjugates, and surface-immobilized biomaterials, as well as in the preparation of labeled biomolecules for imaging or detection. PEG-containing variants can improve solubility and reduce steric hindrance.

Handling and storage require consideration of NHS ester hydrolysis in water, which reduces reactivity. Reagents are typically stored as dry solids at low temperature (often -20°C) and used in anhydrous or anhydrous-like solvents or buffers at near-neutral pH. Reactions should be planned to minimize exposure to moisture and light. Safety concerns include irritation from NHS esters and potential sensitization, so appropriate protective equipment and waste disposal practices are recommended.

See also: NHS ester chemistry, maleimide chemistry, heterobifunctional crosslinkers, SMCC.