Synthesis of Fenthion Sulfoxide and Fenoxon Sulfoxide Enantiomers: Effect of Sulfur Chirality on Acetylcholinesterase Activity Rama Sarma V. S. Gadepalli, ² John M. Rimoldi,* ,² Frank R. Fronczek, ‡ Mae Nillos, § Jay Gan, § Xin Deng, § Gabriela Rodriguez-Fuentes, § and Daniel Schlenk* ,§ Department of Medicinal Chemistry and EnVironmental Toxicology Research Program, UniVersity of Mississippi, Mississippi 38677, Department of Chemistry, Louisiana State UniVersity, Baton Rouge, Louisiana 70803, and Department of EnVironmental Sciences, UniVersity of California, RiVerside, California 92521 ReceiVed July 6, 2006 Earlier reports have demonstrated that recombinant flavin-containing monooxygenase 1 (FMO1) catalyzes the oxidation of the organophosphate pesticide fenthion to (+)-fenthion sulfoxide in a stereoselective fashion. In order to elucidate the absolute configuration of the sulfoxide metabolite produced, we established an efficient synthesis of both enantiomers of fenthion sulfoxide, which were transformed into chiral fenoxon sulfoxides using a two-step protocol. The use of chiral oxidants, namely, N-(phenylsulfonyl)(3,3-dichlorocamphoryl) oxaziridines, afforded enantioenriched fenthion sulfoxides with high ee (>82%) from the parent sulfide. Single recrystallizations afforded chiral fenthion sulfoxides with >99% ee, measured by chiral HPLC analysis. The absolute configuration of the (+)-sulfoxide generated from fenthion metabolism by FMO1 was determined to be ( R)-(+)-fenthion sulfoxide, confirmed by X-ray crystallographic analysis of the (S)-(-)-antipode. Inhibition of human recombinant (hrAChE) and electric eel (eeAChE) acetylcholinesterase were assayed with fenthion, fenoxon, and the racemates and enantiomers of fenthion sulfoxide and fenoxon sulfoxide. Results revealed stereoselective inhibition with (R)-(+)-fenoxon sulfoxide when compared with that of (S)-(-)-fenoxon sulfoxide (IC 50 of 6.9 and 6.5 μM vs 230 and 111 μM in hrAChE and eeAChE, respectively). Fenthion sulfoxide (R or S enantiomers) did not present anti-AChE properties. Although the stereoselective sulfoxidation of fenthion to (R)-(+)- fenthion sulfoxide by FMO represents a detoxification pathway, the results of this study support the notion that subsequent oxidative desulfuration of (R)-(+)-fenthion sulfoxide (in ViVo) may represent a critical bioactivation pathway, resulting in the production of (R)-(+)-fenoxon sulfoxide, a potent AChE inhibitor. Introduction Organophosphate (OP) insecticides are a structurally diverse class of compounds that have virtually replaced the environ- mentally persistent organochlorine pesticides and represent the largest group of insecticides marketed worldwide (1, 2). OP insecticides exert their principal biological effect by phospho- rylation of the enzyme acetylcholinesterase (AChE), resulting in subsequent accumulation of acetylcholine and continuous stimulation of the nervous system (3). Fenthion (O,O-dimethyl- O-[4-(methylthio)-m-tolyl] phosphorothioate) (1) is a broad- spectrum insecticide with contact, stomach, and respiratory actions used to control insect and bird pests (4, 5) and is classified by the U.S. Environmental Protection Agency as a restricted use pesticide because of its toxic effects in birds, reptiles, and fish. Upon uptake by organisms, fenthion undergoes oxidative metabolism to primary and secondary metabolites, with either enhanced or reduced potency to AChE, under the mediation of cytochrome P450 (P450) and flavin-containing monooxygenases (FMO). Two major pathways include oxidative desulfuration of the phosphorothioate and sulfoxidation of the thioether group (6-9). In Vitro and in ViVo studies demonstrated that fenthion is biotransformed to fenthion sulfoxide (2) and fenoxon (3) in liver microsomes of fish and rats (7-9). Residue analyses in animals and plants indicate the formation of several principal metabolites and include fenthion sulfoxide (2), fenoxon (3), fenoxon sulfoxide (4), and the corresponding sulfones (10- 12). Fenthion also experiences nonenzymatic transformation including photodegradation to the sulfoxide, a relatively stable oxidation product in the environment (13). Fenoxon sulfoxide is also susceptible to nonenzymatic hydrolysis, representing a key detoxification mechanism (14). Because of the formation of an asymmetric sulfur center, enzyme-catalyzed sulfoxidation can produce an enantiomeric enrichment of fenthion sulfoxide or fenoxon sulfoxide (15). Stereoselective oxidations by P450 and FMO have long been recognized in other organophosphates (16). Similar to other organophosphates, fenthion was predominantly converted into (+)-fenthion sulfoxide when incubated with human tissue microsomes (6). However, information regarding the biological activity and/or toxicity of the enantiomers as well as other metabolites of fenthion is scarce. Furnes and Schlenk reported that (+)- and (-)-fenthion sulfoxides had similar potency toward AChE inhibition, which was less than the potency of fenthion, indicating detoxification (6). In contrast, the LD 50 of fenthion sulfoxide (125 mg/kg) is nearly half that of fenthion (220 mg/ kg) (17). Therefore, the objective of this study was to evaluate * Corresponding author. Phone: 1-662-915-5119. Fax: 1-662-915-5638. E-mail: jrimoldi@olemiss.edu (J.M.R.). Phone: 1-951-827-2018. Fax: 1-951-827-3993. E-mail: daniel.schlenk@ucr.edu (D.S.). ² University of Mississippi. ‡ Louisiana State University. § University of California, Riverside. 10.1021/tx060153l CCC: $37.00 © xxxx American Chemical Society PAGE EST: 5.2 Published on Web 01/25/2007