Synthesis and kinetic studies of an amidine-containing phosphonofluoridate: a novel potent inhibitor of trypsin-like enzymes

The amidine-containing alpha-amino alkylphosphonofluoridate 3 (Cbz-(4-AmPhGly)(P)(OPh) (F)) is a very potent inhibitor of trypsin-like enzymes. It was prepared by hydrolyzing the corresponding phosphonate diphenyl ester 4 followed by reaction of fluoride with the phosphonochloridate prepared from the intermediate phosphonic acid monoester 5. Compound 3 is the most potent amidine-containing organophosphorus inhibitor yet reported for trypsinlike enzymes. It inhibits trypsin and thrombin with second-order rate constants (k(obs)/[I]) of 2.6 x 10(5) M-1 s(-1) and 1.0 x 10(5) M-1 s(-1), respectively, showing a 130-fold and a 1250-fold rate enhancement over the corresponding diphenyl ester (4). It also inactivates trypsin 2 orders of magnitude more potently than simple phosphonofluoridates such as DFP,(1) Sarin and soman. The phosphonofluoridate 3 does not inhibit other serine proteases such as porcine pancreatic elastase (PPE) and the esterase acetylcholinesterase (AChE). The phosphonofluoridate 3 is hydrolyzed rapidly in buffer solution and has a t(1/2) of 4.5 s at pH 7.5. (C) 1998 Elsevier Science Ltd. All rights reserved.