title: Asymmetric fluorogenic organophosphates for the development of active organophosphate hydrolases with reversed stereoselectivity.
abstract: In order to enhance the enzymatic detoxification rate of organophosphorus (OP) nerve agents we have searched for more active variants of recombinant mammalian paraoxonase (PON1). We have previously identified three key positions in PON1 that affect OP hydrolysis: Leu69, Val346 and His115, that significantly enhance the hydrolysis of cyclosarin (GF), soman, chlorpyrifos-oxon (ChPo), O-isopropyl-O-(p-nitrophenyl)methylphosphonate (IMP-pNP) and diisopropyl fluorophosphate (DFP). GC/FPD analysis compared to residual AChE inhibition assay displayed stereoselective hydrolysis of GF, soman and IMP-pNP, indicating that wild type PON1 and its variant V346A are more active toward the less toxic P(+) optical isomer. In order to obtain new PON1 variants with reversed stereoselectivity, displaying augmented activity toward the more toxic isomer P(-) of nerve agents, we synthesized new asymmetric fluorogenic OPs (Flu-OPs). Six Flu-OPs were prepared containing either ethyl (E), cyclohexyl (C) or pinacolyl (P) alkyl radicals attached to methyl-phosphonyl (MP) moiety analogous to the structure of VX, GF and soman, respectively. The fluorescent moieties are either 3-cyano-4-methyl-7-hydroxy coumarin (MeCyC) or 1,3-dichloro-7-hydroxy-9,9-dimethyl-9H-acridin-2-one (DDAO). The kinetics of AChE and BChE inhibition by these new Flu-OPs display k(i) values 8.5x10(4) to 8.5x10(7) and 5x10(4) to 2x10(6)M(-1)min(-1), respectively. EMP-MeCyC and EMP-DDAO are the most active inhibitors of AChE whereas CMP-MeCyC and CMP-DDAO are better inhibitors of BChE than AChE, indicating accommodation of bulky cyclohexyl group inside the active site of BChE. PMP-MeCyC and PMP-DDAO are the least active inhibitors of both AChE and BChE. CMP-MeCyC and CMP-DDAO were significantly detoxified only by the five-site mutations PON1 variant L69V/S138L/S193P/N287D/V346A. Degradation kinetics of Flu-OPs measured by increase in absorbance of the released fluorogenic group was fit by a two exponential function, indicating faster hydrolysis of the less toxic optical isomer. Interestingly, wt PON1 caused only 50% degradation of racemic EMP-MeCyC, CMP-MeCyC and CMP-DDAO indicating complete hydrolysis of P(+) isomer. This remarkable stereoselectivity was used for the enzymatic separation of the P(-) isomer of CMP-MeCyC. The bimolecular rate constant k(i) for human AChE inhibition by the isolated P(-) isomer of CMP-MeCyC is five-fold larger than that of its P(+) isomer. The marked preference of wt PON1 toward P(+) stereo-isomer of CMP-MeCyC and CMP-DDAO renders their P(-) stereo-isomers suitable for the selection of new OP hydrolase variants with reversed stereoselectivity.
authors.affiliation: Division of Medicinal Chemistry, Israel Institute for Biological Research, PO Box 19, Ness Ziona 74100, Israel. gabia@iibr.gov.il
authors.forename: Gabi
authors.initials: G
authors.lastname: Amitai
authors.forename: Rellie
authors.initials: R
authors.lastname: Adani
authors.forename: Guy
authors.initials: G
authors.lastname: Yacov
authors.forename: Shelly
authors.initials: S
authors.lastname: Yishay
authors.forename: Shai
authors.initials: S
authors.lastname: Teitlboim
authors.forename: Liat
authors.initials: L
authors.lastname: Tveria
authors.forename: Osnat
authors.initials: O
authors.lastname: Limanovich
authors.forename: Moshe
authors.initials: M
authors.lastname: Kushnir
authors.forename: Haim
authors.initials: H
authors.lastname: Meshulam
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry; D000818:Animals; D043303:Aryldialkylphosphatase / Q000737:chemistry* / Q000235:genetics; D002091:Butyrylcholinesterase / Q000737:chemistry; D002800:Cholinesterase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry; D002801:Cholinesterase Reactivators / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006736:Horses; D006801:Humans; D006868:Hydrolysis; D015394:Molecular Structure; D009943:Organophosphorus Compounds / Q000138:chemical synthesis / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D002800:Cholinesterase Inhibitors; D002801:Cholinesterase Reactivators; D005456:Fluorescent Dyes; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D000110:Acetylcholinesterase; D002091:Butyrylcholinesterase; D043303:Aryldialkylphosphatase