The inhibition of S-chymotrypsin with optically active, axially and equatorially substituted frans-3-(2,4-dinitrophenoxy)-2,4-dioxa-325-phosphabicyclo[4.4.O]decan-3-ones ( = hexahydro-4H-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 "P-NMR spectroscopy at pH 7.8. The irreversible inhibitors show significant enantioselectivity (the (S,)-enantiomer reacting faster) and yield diastereoisomeric, covalently phosphorylated derivatives of 6-chymotrypsin.31P-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 -4.4ppm 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 31P chemical shifts of the corresponding covalent phosphoserine derivatives 4a (-5.7 ppm, axial) and 4b (-4.5ppm, 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.

Investigation of the inhibition of δ‐chymotrypsin with the four novel, optically active, axially and equatorially substituted cis‐3‐(2,4‐dinitrophenoxy)‐2,4‐dioxa‐3λ5‐phosphabicyclo[4.4.0]decan‐3‐ones (= 3‐(2,4‐dinitrophenoxy)hexahydro‐4H‐1,3,2‐benzodioxaphosphorin 2‐oxides) showed only the equatorially substituted enantiomer (−)‐4b to be an irreversible inhibitor of the enzyme, and (−)‐4b at pH 7.8 revealed a quickly rising resonance at −2.49 ppm assigned to the hydrolysis product 8 and later, while inhibition proceeded, a second one at −4.08 ppm. attributed to the δ‐chymotrypsin adduct 7 (Scheme 3). Comparision of the latter signal with the 31P‐NMR chemical shifts of the covalent phosphoserine model compounds (−)‐6a (−5.67 ppm, axial substitution) and (+)‐6b (−4.02 ppm, equatorial substitution) suggests that the inhibition proceeded via neat retention of the configuration at the P‐atom of (−)‐4b yielding the equatorially substituted covalent Ser195 adduct 7.

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Investigation of the inhibition of 6-chymotrypsin with the four novel, optically active, axially and equatorially substituted cis-3-(2,4-dinitrophenoxy)-2,4-dioxa-3~.s-phosphabicyclo[4.4.0]de~an-3-ones ( = 3-(2,4-dinitrophenoxy)hexahydro-4H-l,3,2-benzodioxaphosphorin 2-oxides) showed only the equatorially substituted enantiomer (-)-4b to be an irreversible inhibitor of the enzyme. 3'P-NMR Spectroscopic monitoring of the inhibition of stoichiometric amounts of the enzyme and (-)-4b at pH 7.8 revealed a quickly rising resonance at -2.49 ppm assigned to the hydrolysis product 8 and later, while inhibition proceeded, a second one at -4.08 ppm, attributed to the 6-chymotrypsin adduct 7 (Scheme 3). Comparison of the latter signal with the 31P-NMR chemical shifts of the covalent phosphoserine model compounds (-)-6a ( -5.67 ppm, axial substitution) and (+)-6b (-4.02 ppm, equatorial substitution) suggests that the inhibition proceeded via neat retention of the configuration at the P-atom of (-)-4b yielding the equatorially substituted covalent Ser19' adduct 7.Introduction. -In the course of our current program concerning the synthesis of conformationally restricted organophosphates as inhibitors of serine hydrolases and the investigation of the regio-and stereochemistry of the inhibition reaction, we have recently reported on the enantiomeric trans-3-(2,4-dinitrophenoxy)-2,4-dio~a-3~~-phosphabicyclo[4.4.0]decan-3-ones ( = 3-(2,4-dinitrophenoxy)hexahydro-4H-1,3,2-benzodioxa-

1997 magnetic resonance, nuclear quadrupole resonance magnetic resonance, nuclear quadrupole resonance (organic substances) K 2560 -026Stereochemistry of the Inhibition of δ-Chymotrypsin with Optically Active Bicyclic Organophosphates: 31P NMR Studies.--(GANCI, W.; MEIER, E. J. M.; MERCKLING, F. A.; PRZIBILLE, G.; RINGEISEN, U.; RUEEDI, P.; Helv.