Stereo-specific synthesis of analogs of nerve agents and their utilization for selection and characterization of paraoxonase (PON1) catalytic scavengers

Abstract

Fluorogenic organophosphate inhibitors of acetylcholinesterase (AChE) homologous in structure to nerve agents provide useful probes for high throughput screening of mammalian paraoxonase (PON1) libraries generated by directed evolution of an engineered PON1 variant with wild-type like specificity (rePON1). Wt PON1 and rePON1 hydrolyze preferentially the less-toxic R(P) enantiomers of nerve agents and of their fluorogenic surrogates containing the fluorescent leaving group, 3-cyano-7-hydroxy-4-methylcoumarin (CHMC). To increase the sensitivity and reliability of the screening protocol so as to directly select rePON1 clones displaying stereo-preference towards the toxic S(P) enantiomer, and to determine accurately K(m) and k(cat) values for the individual isomers, two approaches were used to obtain the corresponding S(P) and R(P) isomers: (a) stereo-specific synthesis of the O-ethyl, O-n-propyl, and O-i-propyl analogs and (b) enzymic resolution of a racemic mixture of O-cyclohexyl methylphosphonylated CHMC. The configurational assignments of the S(P) and R(P) isomers, as well as their optical purity, were established by X-ray diffraction, reaction with sodium fluoride, hydrolysis by selected rePON1 variants, and inhibition of AChE. The S(P) configuration of the tested surrogates was established for the enantiomer with the more potent anti-AChE activity, with S(P)/R(P) inhibition ratios of 10-100, whereas the R(P) isomers of the O-ethyl and O-n-propyl were hydrolyzed by wt rePON1 about 600- and 70-fold faster, respectively, than the S(P) counterpart. Wt rePON1-induced R(P)/S(P) hydrolysis ratios for the O-cyclohexyl and O-i-propyl analogs are estimated to be >>1000. The various S(P) enantiomers of O-alkyl-methylphosphonyl esters of CHMC provide suitable ligands for screening rePON1 libraries, and can expedite identification of variants with enhanced catalytic proficiency towards the toxic nerve agents.

Fig.1

Stereo-specific synthesis of R_P- and S_P-O-alkyl methylphosphonyl-CHMC esters from methylphosphonyl dichloride [CH₃P(O)Cl₂], L-proline methyl ester, and 3-cyano-7-hydroxy-4-methylcoumarin (CHMC).The indicated absolute configuration was determined by X-ray diffraction of the corresponding crystals (Tables1&2) and by enzymatic hydrolysis (Fig. 3). Alcoholysis was catalyzed by 1 M H₂SO₄.

Fig.2

Hydrolysis of 0.014 mM racemic EMP-CHMC. Panel A: □- □, 0.1 M NaF; ○-○, 0.05 M NaF; •-•, 0.03 μM wt rePON1; ▪- ▪, 0.012 μM 3B3. Panel B: ○-○, 0.05 M, NaF; ▪-▪, 0.048 μM 3D8; •-•, 0.035 μM H115W

Fig. 3

Determination of the optical purity of the enantiomers of EMP-CHMC (Panels A & B) and of nPrMP-CHMC (Panels C & D) by use of mutants 3B3 (○-○) and 3D8 (▪-▪). The optical purities of S_P-CMP-CHMC and R_P-iPrMP-CHMC are illustrated in panels E and F, respectively. In all cases, 3D8 was added after termination of CHMC release in presence of 3B3. The latter hydrolyzes exclusively the R_P isomer, whereas 3D8 hydrolyzes both the S_P and R_P enantiomers (Fig. 2B)