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 becom e an important tool in biological research. The use of a spacer is oft en necessary to avoid steric hindrance and in order to retain biologic al activity. A spacer arm, exhibiting heterobifunctional character wit h an N-hydroxysulfosuccinimide (S-NHS) active ester linked to a maleim ide moiety by an aliphatic chain can be used to link antigenic peptide s to functionalized polystyrene microspheres in very selective two-ste p procedures. To study the kinetic parameters in aqueous buffers of su ch procedures, reactions of sulfosuccinimidyl 4-(N maleimidomethyl)-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 kinetic s 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 a gents, the reactive species are the thiolate anions. This alkylation w as characterized by the equation v=k(s-)[RS-][S-SMCC] with, at 20.5 de grees C, k(s-)=8.54+/-0.91x10(5) l mol(-1) min(-1). Hydrolysis of the active ester was shown to be the most important competing reaction and its rate constant k(H) has been measured. Above pH=8, hydrolysis is f irst order in [OH-], and characterized by the equation k(H)=k(OH)[OH-] with, at 20.5 degrees C, k(OH)=12200+/-700 l mol(-1) min(-1). Reactio n of active esters with primary amines is strongly linked to the conce ntration of non-protonated amine (free amine). Of the rates investigat ed, 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 k(A), remains constant whatever the excess of n ucleophile: k(A)=31700+/-750 l mol(-1) min(-1), at 20.5 degrees C. All the constants concerning the reaction of the active ester are summari zed in the equation: (X-obs-k(H))=k(A) x [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 ca lculated constants, optimized coupling conditions and experimental pro cedures are proposed. (C) 1998 Elsevier Science B.V.