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

Abstract

The amidine-containing α-aminoalkylphosphonofluoridate 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 trypsin-like enzymes. It inhibits trypsin and thrombin with second-order rate constants (kobs/[I]) of 2.6×105M−1s−1 and 1.0×105M−1s−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 t1/2 of 4.5s at pH 7.5.