Stereochemistry of the Inhibition of δ‐Chymotrypsin with Optically Active Bicyclic Organophosphates: ³¹P‐NMR studies

Abstract

Abstract The inhibition of δ‐chymotrypsin with optically active, axially and equatorially substituted trans ‐3‐(2,4‐dini‐trophenoxy)‐2,4‐dioxa‐3λ 5 ‐phospbubicyclo[4.4.0]decan‐3‐ones ( = hexahydro‐4 H ‐1,3,2‐benzodioxaphosphorin 3‐oxides) was investigated. Their inhibitory power was determined by kinetic measurements, and the stereochemical course of the reaction of stoichiometric amounts of the enzyme and inhibitor was monitored with 31 P‐NMR spectroscopy at pH 7.8. The irreversible inhibitors show significant enantioselectivity (the (S p )‐enantiomer reacting faster) and yield diastereoisomeric, covalently phosphorylated derivatives of δ‐chymotrypsin. 31 P‐NMR Spectroscopic studies of the inhibition by the axially substituted inhibitor revealed for the racemic (±) ‐2a first a resonance at –4.4 ppm and later, while inhibition proceeded, a second one at –4.5 ppm. The reaction with optically active (+) ‐2a showed only one signal at –4.4 ppm and its enantiomer (–) ‐2a only one signal at –4.5 ppm. Using the equatorially substituted racemic epimer (±) ‐2b , we observed the main resonance at –5.3 ppm and two minor ones at –4.4 and –4.5 ppm. The optically active compound (+) ‐2b showed two peaks at –4.5 and –5.3 ppm, whereas its antipode (–) ‐2b revealed two signals at –4.4 and –5.3 ppm. Comparing the 31 P chemical shifts of the corresponding covalent phosphoserine derivatives 4a (‐5.7 ppm, axial) and 4b (‐4.5 ppm, equatorial) shows the inhibition with the axial compounds 2a to proceed via neat inversion of the configuration at the P‐atom, whereas the equatorial epimers 2b with a higher conformational flexibility seem to follow a different stereochemical pathway which results in both inversion and retention.