Conjugation using maleimide functionality is a widely used technique. Antibody can be modified to contain maleimide functionality which can then react with compounds containing sulfhydryl/thiol groups to form a stable thioether linkage. For information on maleimide modified antibody, refer to the section antibodies/antibody modification for conjugation.
PDP PE derivatives generate thioether linkages with maleimide modified antibodies. The double bond of maleimide undergos an additional reaction with thiol forming a stable thioether bonds. One of the carbon adjacent to the maleimide double bond undergoes a nucleophilic attack by the thiolate anion to generate the additional product. Maleimide reactions are specific for thiols in the pH range of 6.5-7.5. At pH 7.0 the reaction of the maleimide with sulfhydryls proceeds at a rate 1000 times greater than its reaction with amines. At higher pH values, some cross reactivity with amino groups takes place. When sufficient quantities of –SH are being alkylated, the reaction may be followed specrophotometrically by the decrease in absorbance at 300 nm as the double bond reacts and disappears.
After the sulfhydryl has been added to the maleimide the ring opening reaction happens faster as the pH becomes higher. Also, hydrolysis depends on the type of functional chemical group next to maleimide. For instance SMCC provides increased stability to maleimide hydrolysis probabaly due to its stearic effects and its lack of aromatic character. However, an adjacent phenyl ring of MBS allows much greater rates of hydrolysis to occur.