Synthesis of two photoreactive heterobifunctional reagents derived from hexanoic acid

The synthesis of two photoreactive heterobifunctional reagents derived from hexanoic acid is described. The compounds are succinimido 6-N-(4-azidobenzoyl)aminohexanoate (1c) and succinimido 6-mercapto-S-(4-azidothiophenyl)hexanoate (2c). Compound 1c was synthesized from benzyloxycarbonyl 4-aminobenzoic acid and 6-aminohexanoic acid benzyl ester. Compound 2c was obtained by disulfide exchange of dithiobis-4-aminobenzene with 6-mercaptohexanoic acid. The azido function was introduced by displacement of the corresponding diazonium salt and the active ester, by the mixed anhydride method. Both compounds were decomposed by ultraviolet irradiation. Phytohemagglutinin was modified by reaction with reagent 1c or 2c. Irradiation afforded polymeric lectin derivatives resulting from intermolecular cross-linking. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis analyses revealed the presence of Coomasie-positive bands of molecular weight 33000, 67000, 120000, 144000, and higher. Polymeric structures resulting from irradiation of phytohemagglutinin modified with compound 2c were cleaved by reduction with 2-mercaptoethanol or with dithiothreitol. Phytohemagglutinin modified by reaction with compound 1c or 2c retained its ability to stimulate pig spleen lymphocytes in vitro. Whereas the lectin treated with reagent 2c was active as the unmodified protein, the lectin treated with compound 1c was more active than unmodified lectin at nearly all the concentrations tested.