Rates of thrombin acylation and deacylation upon reaction with low molecular weight acylating agents, carbamylating agents and carbonylating agents Original Research Article

Acylated derivatives of thrombin have been made using low molecular weight acylating agents, carbamylating agents and carbonylating agents. The compounds used to acylate the active site serine include isatoic anhydrides, benzoxazinones, benzylisocyanate, N-(benzylcarbonyloxy)succinimide and p-(dimethylamino)benzoylimidazolide. The rates of acylation and deacylation were determined. The best overall inhibitors of thrombin are 2-ethoxy-4H-3,1-benzoxazin-4-one, isatoic anhydride and tert-butyl-2,4-dioxo-2H-3,1-benzoxazine-1(4H)-acetate, which have k2K1; values of 52,700 M−1s−1, 48,900 M−1s−1 and 5400 M−1s−1, respectively. The carbamyl derivative of thrombin formed with benzylisocyanate had the slowest rate of deacylation (2.3 × 10−7 s−1), while the ester derivative formed with 2-(N,N-dimethylamino)methylimino-4H-3,1-benzoxazin-4-one had the fastest rate of deacylation (1.9 × 10−4 s−1)