Sulfosuccinimidyl 4-(N-maleimidomethyl)-1-cyclohexane carboxylate as a bifunctional immobilization agent. Optimization of the coupling conditions

Immobilization of biologically active components on supports has become an important tool in biological research. The use of a spacer is often necessary to avoid steric hindrance and in order to retain biological activity. A spacer arm, exhibiting heterobifunctional character with an N-hydroxysulfosuccinimide (S-NHS) active ester linked to a maleimide moiety by an aliphatic chain can be used to link antigenic peptides to functionalized polystyrene microspheres in very selective two-step procedures. To study the kinetic parameters in aqueous buffers of such procedures, reactions of sulfosuccinimidyl 4-(N-maléimidométhyl)-1-cyclohexane carboxylate (S-SMCC) have been carried out. Alkylation of thiol groups by maleimide has been demonstrated to be a fast, specific and strongly pH dependent reaction which follows second order kinetics in the pH range 5.5–8.5. The dependence of the rate constant on the thiolate anion concentration has shown that, as with most alkylating agents, the reactive species are the thiolate anions. This alkylation was characterized by the equation v=kS−[RS−][S-SMCC] with, at 20.5°C, kS−=8.54±0.91×105 l mol−1 min−1. Hydrolysis of the active ester was shown to be the most important competing reaction and its rate constant kH has been measured. Above pH=8, hydrolysis is first order in [OH−], and characterized by the equation kH=kOH[OH−] with, at 20.5°C, kOH=12200±700 l mol−1 min−1. Reaction of active esters with primary amines is strongly linked to the concentration of non-protonated amine (free amine). Of the rates investigated, the rate of aminolysis was observed to increase more rapidly than the rate of hydrolysis with increasing pH or temperature. The reaction with nitrogen nucleophiles is first order in free amine concentration and the rate constant kA remains constant whatever the excess of nucleophile: kA=34700±750 l mol−1 min−1, at 20.5°C. All the constants concerning the reaction of the active ester are summarized in the equation: (kobs−kH)=kA×[amine]free. Alkylation of thiols by the maleimide group and reaction of active ester with amines have been shown to be very fast. Considering these results and the calculated constants, optimized coupling conditions and experimental procedures are proposed.