DIASTEREOMERIC PHOSPHONATE ESTER ADDUCTS OF CHYMOTRYPSIN - P-31-NMR MEASUREMENTS

Generation of diastereomeric phosphonate ester adducts of chymotrypsin was evidenced for the first time by P-31 NMR and spectrophotometric k inetic measurements. P-31 NMR signals were recorded for 4-nitrophenyl 2-propyl methylphosphonate (IMN) at 32.2 ppm and for its hydrolysis pr oduct at 26.3 ppm downfield from phosphoric acid. The inhibition of al pha-chymotrypsin at pH > 8.0 by the faster reacting enantiomer of IMN or 2-propyl methylphosphonochloridate (IMCl), or other phosphonate est er analogs of these compounds, all caused a approximately 6.0 ppm down field shift of the P-31 signal to the 39-40 ppm region. IMN, when appl ied below the stoichiometric amount of chymotrypsin, under the same co nditions, generated two signals, at 39. 0 and at 37.4 ppm. Scans accum ulated in hourly intervals showed the decomposition of both diastereom ers, with approximate half-lives of 12 h at pH 8.0 and 22-degrees-C, i nto a species with a resonance at 35.5 ppm. The most likely reaction t o account for the appearance of this new peak is the enzymic dealkylat ion of the isopropyl group from the covalently bound phosphonate ester . We base this conclusion mostly on the similarity of the upfield shif t to the hydrolysis of phosphonate esters. Contrary to experience with phosphate ester adducts of serine proteases, no signal was detected h igher than 25.0 ppm downfield from phosphoric acid for several phospho nate ester adducts of chymotrypsin and in no case did the resonance fo r the adduct shift further downfield in the course of the experiments.