Conjugation of HS-oligopeptides with polymeric branched chain polypeptidescontaining multiple amino groups

For the preparation of bioconjugates containing polymeric polypeptides with well-defined structure and composition, we systematically studied 3-(2-pyr idyldithio)propionic acid N-hydroxy-succinimide ester (SPDP). SPDP as amino - and thiol-reactive heterobifunctional coupling agent is mainly used for p rotein-based conjugates, and very little data are available on its applicat ion for the modification of polymers. In this communication, we describe th e effect of polymer/oligopeptide structure and of the reaction condition on the incorporation of oligopeptides with free thiol group (CAVKDEL, CTGRGDS P) into polymeric polypeptides possessing multiple amino groups. For these studies, linear poly[L-lysine] with free E-amino groups and its XAK-type br anched polypeptide derivatives {poly[Lys(X-i-DL-Ala(m))] (i < 1, m similar to 3, XAK)} either with polycationic character {X = emptyset (AK), X = Ser (SAK)} or with amphoteric nature {X = Glu (EAK)} were utilized. First, the polymers were modified with SPDP under various conditions, and the degree o f substitution was determined. We found that the efficacy of the nucleophil ic substitution of NH2 groups with SPDP depended not only on the pH and the concentration of the coupling reagent but also on the polymer composition, mainly on the pK(a) of the branch-terminal amino group of the polymers. SP DP-modified polymeric polypeptides were reacted with the HS-oligopeptides, and the effects of polymer/oligopeptide structure as well as the reaction c onditions (pH, peptide/(SSP)polymer molar ratio) on the composition of the product; were evaluated. The results suggest that acidic pH is more favorab le for the thiol-disulphide exchange, and the side chain composition of the polymers had a pronounced effect while the chemical structure of oligopept ides had only moderate influence on the average degree of substitution.