BIOL 53

A chromogenic thiol-conjugated maleimide was synthesized. Using UV spectroscopy, the maleimido group was found to be susceptible to hydrolysis but not fragmentation at neutral pH, leading to heterogeneity. Kinetic analyses of several potential catalysts revealed that the molybdate anion was an efficient catalyst, and could serve to increase the uniformity of maleimide conjugates.

Using NMR spectroscopy, the stability of isostructural hydrazones and an oxime was compared. The hydrolysis of adducts of pivalaldehyde with methylhydrazine, methylhydrazide, and methoxyamine was monitored in deuterated buffers (pD 5.0 - 9.0). Each hydrolysis reaction was performed in the presence of excess deuterated formaldehyde as a trap to drive the reaction to completion. The hydrolysis of each adduct was found to be catalyzed by acid. The first-order rate constants for the hydrolysis of the oxime were found to be several orders of magnitude lower than those for the hydrazones. The data suggests a detailed mechanism for the hydrolysis reactions and guide the choice of linkage for stable bioconjugation.