Aged and non-aged pyrenebutyl-containing organophosphoryl conjugates of chymotrypsin: Preparation and comparison by 31P-NMR spectroscopy

Homologous pairs of non-aged and aged pyrene-containing phosphoryl conjugates of chymotrypsin were prepared in order to characterize by NMR and optical spectroscopy putative differences in the conformation of non-aged and aged organophosphoryl conjugates of serine hydrolases. Pyrenebutyl-O-P(O)(OC 2H 5)F and pyrenebutyl-O-P(O)(OC 2H 5)Cl were used to obtain the non-aged form pyrenebutyl-O-P(O)(OC 2H 5)-Cht, whereas pyrenebutyl-O-P(O)Cl 2, pyrenebutyl-O-P(O)( p-nitrophenoxy) Cl, and pyrenebutyl-O-P(O)( p-nitrophenoxy) 2 were used to produce the aged conjugate pyrenebutyl-O-P(O)(O −)-Cht. These ligands bind covalently to the active site of serine hydrolases. The absorption spectra of both the non-aged and aged conjugates fitted approximately a 1:1 stoichiometry of bound organophosphate and enzyme in the non-aged and aged conjugates. Pyrenebutyl-O-P (O)(OC 2H 5)-Cht could be reactivated by pyridine-3-aldoxime methiodide, whereas no reactivation was observed for the similarly treated pyrenebutyl-O-P(O)(O −)-Cht. The 31P-NMR and reactivation data taken together strongly support the hypothesis that the aged form of the OP-Cht conjugate contains a P-O − bond. These results provide a partial interpretation for the known resistance of the aged conjugates of serine hydrolases to reactivation.