Enantioselective and Reversible Inhibition of Trypsin and .alpha.-Chymotrypsin by Phosphonate Esters

Abstract

Trypsin is inactivated by the levorotatory enantiomers (most likely PS) of 4-nitrophenyl 4-H-, 4-CH3-,4-OCH3-, and 4-Cl-phenacyl methylphosphonates (PMNs) with second-order rate constants between 231 and 884 M-1 s-1. 4-NO2-PMN hydrolyzes before inhibiting the enzyme. The second-order rate constants for the inactivation of alpha-chymotrypsin by the levorotatory enantiomers of the five PMNs are between 37,000 and 770,000 M-1 s-1, and those for the dextrorotatory enantiomers are between 400 and 640 M-1 s-1; the enantioselectivity is 90-1880. Specific rotation [alpha]22D of the faster-reacting enantiomer of 4-CH3-PMN with trypsin and alpha-chymotrypsin is -30 +/- 6 degrees. 31P NMR of the adducts shows a signal at 41.0 ppm, 10 ppm downfield from the parent compound. Results of molecular mechanics and dynamics calculations show that the principal interactions are between the phosphonyl group and constituents of the oxyanion hole and between the aromatic fragment and residues in the binding regions of the enzymes. Trypsin activity returns from its phenacyl methylphosphonyl adducts on the hour time scale and in reversed order to the rates of inactivation within the series. Recovery of alpha-chymotrypsin activity from the adducts formed with the (-) enantiomers is on a slower time scale still, whereas its recovery from the adducts formed with the (+) enantiomers is on the second to minute time scale. The data support a mechanism of reactivation involving rate-determining intramolecular displacement of Ser by the carbonyl hydrate of the phenacyl moiety. The pH-rate profiles for trypsin reactivation from its adducts indicate involvement of an ionizable group with pKa approximately 8.0. The pH dependence and solvent isotope effects are small in most cases. The compounds demonstrate favorable properties for controllable and temporary modulation of enzyme activity.

Computer-Aided Analysis and Design of Phosphonic and Phosphinic Enzyme Inhibitors as Potential Drugs and Agrochemicals

“…Structures of both enantiomers bound to trypsin, -chymotrypsin and thrombin were optimised and analysed. Interaction of P S diastereomeric adduct was found to be favoured by about 3 kcal/mol in case of trypsin and chymotrypsin, which is consistent with experimental data showing that inactivation of these enzymes is given only by P S stereoisomer [72]. Inhibition of thrombin is less stereoselective, which was attributed to two different conformations of 39 bound with similar interaction energy in the active site [73].…”

“…(8)) and its analogues (40)(41)(42)(43) towards serine proteases [72,73]. Similarly to diphenyl esters, this class of inhibitors binds to enzyme irreversibly forming ester bond with serine hydroxylic residue, a process which is accompanied with release of p-nitrophenol.…”

Broadly Distributed Chemical Reactivity of Natural Antibodies Expressed in Coordination with Specific Antigen Binding Activity

“…CRA III, which contains a weaker leaving group than CRA II did not form detectable adducts with IgG (the presence of methoxy-leaving groups reduces the electrophilicity of the phosphorus atom, and methoxy-containing phosphonate diesters are reported to bind weakly with certain serine proteases) (for review see Ref. 21). Increasing formation of covalent CRA I adducts with IgG and trypsin was evident as a FIG.…”

The phospha-Michael addition of dimethyl- and diphenylphosphites to the η1-N-maleimidato ligand: Inhibition of serine hydrolases by half-sandwich metallocarbonyl azaphosphonates

“…These compounds inhibit to a lesser extent the serine protease chymotrypsin (CT) with IC 50 ranging from 1 lM to 1 mM depending of the alkyl chain length [18]. Other phosphonates containing a p-nitrophenyl group were also shown to irreversibly inhibit trypsin, CT and BChE [19,20]. More recently, van Koten and coll.…”