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

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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 RP 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 SP enantiomer, and to determine accurately Km and kcat values for the individual isomers, two approaches were used to obtain the corresponding SP and RP 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 SP and RP 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 SP configuration of the tested surrogates was established for the enantiomer with the more potent anti-AChE activity, with SP/RP inhibition ratios of 10–100, whereas the RP isomers of the O-ethyl and O-n-propyl were hydrolyzed by wt rePON1 about 600- and 70-fold faster, respectively, than the SP counterpart. Wt rePON1-induced RP/SP hydrolysis ratios for the O-cyclohexyl and O-i-propyl analogs are estimated to be ≫1000. The various SP 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.

Introduction

Organophosphate ester hydrolases (OPH) displaying multiple turnover, such as bacterial phosphotriesterases (PTE) and mammalian serum paraoxonases (PON1), have been demonstrated to have the capacity to serve as potential prophylactic drugs to counteract in vivo intoxication by nerve agents and organophosphate-based pesticides [1], [2], [3], [4], [5]. A significant obstacle in applying OPHs as antidotes against nerve agent intoxication is their preference for hydrolysis of the less-toxic isomers of nerve agents with the RP absolute configuration at the phosphorous center [6], [7], [8], [9]. All nerve agents are racemic mixtures due to the chiral phosphorous atom, with the more potent anti-AChE agents, i.e., the toxic isomers, being assigned the absolute configuration S around the P atom (SP) [10]. It should be pointed out, however, that the assignment of the SP configuration to the toxic isomers of sarin (GB), soman (GD), cyclosarin (GF) and VX (all liquids) has been suggested on the basis of deductive stereo-chemistry, and only indirect structural evidence exists to support this contention [11].

The combined application of computational design and directed evolution of mammalian PON1 has the potential to evolve a variant with reversed stereo-preference and kinetic properties that will qualify it to serve as a realistic candidate for prophylactic of nerve agent intoxications. The need to screen huge libraries of mutants, and to rapidly identify and select variants evolved to hydrolyze the toxic isomers, as well as the call for accurate biochemical characterization of kcat and Km of both the SP and RP enantiomers, necessitate the synthesis of the individual fluorogenic optical isomers. The synthesis and biochemical properties of racemic O-alkyl methylphosphonates containing coumarin analogs as the fluorogenic leaving group have already been described [12], [13], [14]. In order to qualify as catalytic scavenger drug candidates, rePON1 variants should be capable of hydrolyzing the toxic OP isomers at kcat/Km  5 × 107 M−1 min−1 (unpublished calculations). To achieve the desired specificity and efficacy towards the toxic isomers when reacting with racemic nerve agents, it was, therefore, necessary to develop stereo-specific methods for the synthesis of appropriate SP and RP fluorescent surrogates for screening of libraries, for improved selection, and for accurate biochemical characterization of promising catalytic variants.

In this report we describe the synthesis, purity, structural determination and biochemical properties of optical isomers of nerve agents in which the leaving groups fluoride (G agents) and N,N-dialkylaminoethanthiolo (V agents) were replaced by the fluorescent moiety, 3-cyano-7-hydroxy-4-methylcoumarin (CHMC) (Fig. 1). The SP enantiomers were utilized to screen libraries by fluorescence-activated cell sorting and by direct monitoring of the release of the CHMC leaving group by crude lysates in 96-well plates.

Section snippets

Chemicals and enzymes

3-Cyano-7-hydroxy-4-methylcoumarine (CHMC) and l-proline methyl ester hydrochloride were purchased from Aldrich (USA) and used as obtained. Methylphosphonyl dichloride (CH3POCl2) was prepared according to Moedritzer and Miller [15]. Recombinant human AChE (hAChE) was purchased from Sigma (USA) and Torpedo californica AChE (TcAChE) was purified as previously described [16]. Wt rePON1 is variant G3C9 of the gene shuffling products of four mammalian PON1s [17], and its H115W mutant were produced

Stereo-specific synthesis via construction of pairs of diastereoisomers

The separated pair of diastereoisomers which was obtained by reacting CH3POCl2 in a one-pot synthesis with l-proline methyl ester, followed by reaction with CHMC (Fig. 1A), was found by X-ray diffraction to possess the same SC configuration at the chiral proline carbon. The SP and RP configurations at the phosphorous atom were established, and the purified diastereoisomers designated as I-SPSC and I-RPSC, respectively (Table 1). X-ray diffraction showed that reaction with either primary or

Conclusions

The individual stereoisomers of several O-alkyl methylphosphonylated coumarin analogs of nerve agents were prepared by a stereo-specific synthesis protocol, and by an enzymatic procedure that utilized the racemic mixture of the fluorogenic surrogates and a rePON1 mutant that hydrolyze preferentially the RP isomer. The high enantiomeric purity, and the low level of free chromophore suggests that the SP chiral analogs of nerve agents are suitable for screening of large rePON1 libraries for

Conflict of interest

The authors declare that there are no conflicts of interests. (It should be noted that a patent application was submitted with respect to the generation of the rePON1 variants mentioned in this paper.)

Acknowledgements

Financial support by the NIH CounterACT Program (1U54NS058183) and the Defense Threat Reduction Agency (HDTRA 1-07-C-0024) to DST and JLS is gratefully acknowledged.

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