ABSTRACT
Previous studies within our group have yielded a class of cinnamoyl-based competitive reversible inhibitors for tissue transglutaminase (TG2), with Ki values as low as 1.0 µM (compound CP4d). However, due to the electrophilic nature of their alkene moiety, this class of inhibitors is susceptible to nucleophilic attack by glutathione, a key element in cellular metabolism and toxicity response. To address this issue, we made several modifications to the inhibitor scaffold, ultimately showing that a bis(triazole) scaffold increased resistance to nucleophilic attack, with compound 27d being the most potent (Ki = 10 µM). In the process of reducing reactivity, we also prepared a new class of inhibitors, replacing the alkene of CP4d with an alkyne, leading to a significant increase in potency for compound 22b (Ki = 420 nM).
ABSTRACT
Maleimide groups are used extensively in bioconjugation reactions, but limited kinetic information is available regarding their thiol addition and hydrolysis reactions. We prepared a series of fluorogenic coumarin maleimide derivatives that differ by the substituent on their maleimide CâC bond. Fluorescence-based kinetic studies of the reaction with ß-mercaptoethanol (BME) yielded the second-order rate constants (k2), while pH-rate studies from pH 7 to 9 gave base-catalyzed hydrolysis rate constants (kOH). Linear free-energy relationships were studied through the correlation of log k2 and log kOH to both electronic (σ(+)) and steric (Es(norm)) parameters of the CâC substituent. These correlations revealed the thiol addition reaction is primarily sensitive to the electronic effects, while steric effects dominate the hydrolysis reaction. These mechanistic studies provide the basis for the design of novel bioconjugation reactants or fluorogenic labeling agents.
