Stereochemical specificity of organophosphorus acid anhydrolase toward p-nitrophenyl analogs of soman and sarin

Organophosphorus acid anhydrolase (OPAA) catalyzes the hydrolysis of p-nitrophenyl analogs of the organophosphonate nerve agents, sarin and soman. The enzyme is stereoselective toward the chiral phosphorus center by displaying a preference for the R(P)-configuration of these analogs. OPAA also exhibits an additional preference for the stereochemical configuration at the chiral carbon center of the soman analog. The preferred configuration of the chiral carbon center is dependent upon the configuration at the phosphorus center. The enzyme displays a two- to four-fold preference for the R(P)-enantiomer of the sarin analog. The k(cat)/K(m) of the R(P)-enantiomer is 250 M(-1) s(-1), while that of the S(P)-enantiomer is 110 M(-1) s(-1). The order of preference for the stereoisomers of the soman analog is R(P)S(C) > R(P)R(C) > S(P)R(C) > S(P)S(C). The k(cat)/K(m) values are 36,300 M(-1)s(-1), 1250 M(-1) s(-1), 80 M(-1) s(-1) and 5 M(-1) s(-1), respectively. The R(P)S(C)-isomer of the soman analog is therefore preferred by a factor of 7000 over the S(P)S(C)-isomer.     

Structural determinants of the substrate and stereochemical specificity of phosphotriesterase

Bacterial phosphotriesterase (PTE) catalyzes the hydrolysis of a wide variety of organophosphate nerve agents and insecticides. Previous kinetic studies with a series of enantiomeric organophosphate triesters have shown that the wild type PTE generally prefers the S(P)-enantiomer over the corresponding R(P)-enantiomers by factors ranging from 1 to 90. The three-dimensional crystal structure of PTE with a bound substrate analogue has led to the identification of three hydrophobic binding pockets. To delineate the factors that govern the reactivity and stereoselectivity of PTE, the dimensions of these three subsites have been systematically altered by site-directed mutagenesis of Cys-59, Gly-60, Ser-61, Ile-106, Trp-131, Phe-132, His-254, His-257, Leu-271, Leu-303, Phe-306, Ser-308, Tyr-309, and Met-317. These studies have shown that substitution of Gly-60 with an alanine within the small subsite dramatically decreased k(cat) and k(cat)/K(a) for the R(P)-enantiomers, but had little influence on the kinetic constants for the S(P)-enantiomers of the chiral substrates. As a result, the chiral preference for the S(P)-enantiomers was greatly enhanced. For example, the value of k(cat)/K(a) with the mutant G60A for the S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was 13000-fold greater than that for the corresponding R(P)-enantiomer. The mutation of I106, F132, or S308 to an alanine residue, which enlarges the small or leaving group subsites, caused a significant reduction in the enantiomeric preference for the S(P)-enantiomers, due to selective increases in the reaction rates for the R(P)-enantiomers. Enlargement of the large subsite by the construction of an H254A, H257A, L271A, or M317A mutant had a relatively small effect on k(cat)/K(a) for either the R(P)- or S(P)-enantiomers and thus had little effect on the overall stereoselectivity. These studies demonstrate that by modifying specific residues located within the active site of PTE, it is possible to dramatically alter the stereoselectivity and overall reactivity of the native enzyme toward chiral substrates.     

Catalytic properties of the PepQ prolidase from Escherichia coli

The PepQ prolidase from Escherichia coli catalyzes the hydrolysis of dipeptide substrates with a proline residue at the C-terminus. The pepQ gene has been cloned, overexpressed, and the enzyme purified to homogeneity. The k(cat) and k(cat)/K(m) values for the hydrolysis of Met-Pro are 109 s(-1) and 8.4 x 10(5)M(-1)s(-1), respectively. The enzyme also catalyzes the stereoselective hydrolysis of organophosphate triesters and organophosphonate diesters. A series of 16 organophosphate triesters with a p-nitrophenyl leaving group were assessed as substrates for PepQ. The S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was hydrolyzed with a k(cat) of 36 min(-1) and a k(cat)/K(m) of 710 M(-1)s(-1). The corresponding R(P)-enantiomer was hydrolyzed more slowly with a k(cat) of 0.4 min(-1) and a k(cat)/K(m) of 11 M(-1)s(-1). The PepQ prolidase can be utilized for the kinetic resolution of racemic phosphate esters. The PepQ prolidase was shown to hydrolyze the p-nitrophenyl analogs of the nerve agents GB (sarin), GD (soman), GF, and VX.     

Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues

The factors that govern the substrate reactivity and stereoselectivity of phosphotriesterase (PTE) toward organophosphotriesters containing various combinations of methyl, ethyl, isopropyl, and phenyl substituents at the phosphorus center were determined by systematic alterations in the dimensions of the active site. The wild type PTE prefers the S(P)-enantiomers over the corresponding R(P)-enantiomers by factors ranging from 10 to 90. Enlargement of the small subsite of PTE with the substitution of glycine and alanine residues for Ile-106, Phe-132, and/or Ser-308 resulted in significant improvements in k(cat)/K(a) for the R(P)-enantiomers of up to 2700-fold but had little effect on k(cat)/K(a) for the corresponding S(P)-enantiomers. The kinetic preferences for the S(P)-enantiomers were thus relaxed without sacrificing the inherent catalytic activity of the wild type enzyme. A reduction in the size of the large subsite with the mutant H257Y resulted in a reduction in k(cat)/K(a) for the S(P)-enantiomers, while the values of k(cat)/K(a) for the R(P)-enantiomers were essentially unchanged. The initial stereoselectivity observed with the wild type enzyme toward the chiral substrate library was significantly reduced with the H257Y mutant. Simultaneous alternations in the sizes of the large and small subsites resulted in the complete reversal of the chiral specificity. With this series of mutants, the R(P)-enantiomers were preferred as substrates over the corresponding S(P)-enantiomers by up to 500-fold. These results have demonstrated that the stereochemical determinants for substrate hydrolysis by PTE can be systematically altered through a rational reconstruction of the dimensions of the active site.     

Enzymes for the homeland defense: optimizing phosphotriesterase for the hydrolysis of organophosphate nerve agents

Phosphotriesterase (PTE) from soil bacteria is known for its ability to catalyze the detoxification of organophosphate pesticides and chemical warfare agents. Most of the organophosphate chemical warfare agents are a mixture of two stereoisomers at the phosphorus center, and the S(P)-enantiomers are significantly more toxic than the R(P)-enantiomers. In previous investigations, PTE variants were created through the manipulation of the substrate binding pockets and these mutants were shown to have greater catalytic activities for the detoxification of the more toxic S(P)-enantiomers of nerve agent analogues for GB, GD, GF, VX, and VR than the less toxic R(P)-enantiomers. In this investigation, alternate strategies were employed to discover additional PTE variants with significant improvements in catalytic activities relative to that of the wild-type enzyme. Screening and selection techniques were utilized to isolate PTE variants from randomized libraries and site specific modifications. The catalytic activities of these newly identified PTE variants toward the S(P)-enantiomers of chromophoric analogues of GB, GD, GF, VX, and VR have been improved up to 15000-fold relative to that of the wild-type enzyme. The X-ray crystal structures of the best PTE variants were determined. Characterization of these mutants with the authentic G-type nerve agents has confirmed the expected improvements in catalytic activity against the most toxic enantiomers of GB, GD, and GF. The values of k(cat)/K(m) for the H257Y/L303T (YT) mutant for the hydrolysis of GB, GD, and GF were determined to be 2 × 10(6), 5 × 10(5), and 8 × 10(5) M(-1) s(-1), respectively. The YT mutant is the most proficient enzyme reported thus far for the detoxification of G-type nerve agents. These results support a combinatorial strategy of rational design and directed evolution as a powerful tool for the discovery of more efficient enzymes for the detoxification of organophosphate nerve agents.     

Specific soman-hydrolyzing enzyme activity in a clonal neuronal cell culture

An enzymatic activity that specifically hydrolyzes the highly toxic organophosphorus anticholinesterase compound soman (pinacolyl methylphosphonofluoridate) has been identified and partially characterized in the clonal neuronal neuroblastoma-glioma hybrid NG108-15 cell line. Using the whole cell homogenate as the enzyme source and 1 mM substrate, the relative rate of hydrolysis of two other toxic anticholinesterase compounds sarin (isopropyl methylphosphonofluoridate) and tabun (ethyl-N-dimethyl phosphoramidocyanidate) is approximately one-tenth the rate of hydrolysis of soman, while DFP (diisopropyl phosphorofluoridate), paraoxon (p-nitrophenyl diethylphosphate), and a phosphinate PNMPP (p-nitrophenyl methyl (phenyl) phosphinate) are not hydrolyzed. Analysis of the kinetics of soman hydrolysis reveals two components of the enzyme activity with different affinities and reaction rates. Unlike previously reported enzymes of this type, this enzyme lacks chiral specificity and thus hydrolyzes both toxic and non-toxic soman stereoisomers at equal rates. The enzyme activity is stable at low temperature, found almost exclusively in the soluble fraction of these cells, and enhanced significantly by Mn2+ and by chemical differentiation of these cells in culture. The results suggest possible application of this enzyme for soman detection and/or detoxication, and use of the NG108-15 cell line to study the natural function(s) of enzymes of this type.     

Stereostructure-based differences in the interactions of cardiotoxic local anesthetics with cholesterol-containing biomimetic membranes

Amide-type pipecoloxylidide local anesthetics, bupivacaine, and ropivacaine, show cardiotoxic effects with the potency depending on stereostructures. Cardiotoxic drugs not only bind to cardiomyocyte membrane channels to block them but also modify the physicochemical property of membrane lipid bilayers in which channels are embedded. The opposite configurations allow enantiomers to be discriminated by their enantiospecific interactions with another chiral molecule in membranes. We compared the interactions of local anesthetic stereoisomers with biomimetic membranes consisting of chiral lipid components, the differences of which might be indicative of the drug design for reducing cardiotoxicity. Fluorescent probe-labeled biomimetic membranes were prepared with cardiolipin and cholesterol of varying compositions and different phospholipids. Local anesthetics were reacted with the membrane preparations at a cardiotoxically relevant concentration of 200 μM. The potencies to interact with biomimetic membranes and change their fluidity were compared by measuring fluorescence polarization. All local anesthetics acted on lipid bilayers to increase membrane fluidity. Chiral cardiolipin was ineffective in discriminating S(-)-enantiomers from their antipodes. On the other hand, cholesterol produced the enantiospecific membrane interactions of bupivacaine and ropivacaine with increasing its composition in membranes. In 40 mol% and more cholesterol-containing membranes, the membrane-interacting potency was S(-)-bupivacaine相似文献   

Stereoselective degradation of metalaxyl and metalaxyl-M in soil and sunflower plants.

A high proportion of agrochemicals are chiral compounds. Since stereoisomers often show different biological and physiological properties, the biological and metabolic responses to these compounds and their fate in the environment are expected to be different. In this work we investigate a possible stereo and/or enantioselective degradation in soil and plants (sunflower) of the fungicide Metalaxyl (rac-Metalaxyl) and the new compound Metalaxyl-M ((-)-(R)-Metalaxyl) and propose procedures for extraction, cleanup, chromatographic separation of enantiomers, and determination of the R : S ratio by using an HPLC chiral column. The degradation of the two stereoisomers of Metalaxyl proved to be enantioselective and dependent on the media: the (+)-(S)-enantiomer showed a faster degradation in plants, while the (-)-(R)-enantiomer showed a faster degradation in soil. In this study there was no evidence that racemization of Metalaxyl-M took place either in soil or in sunflowers.     

Inactivation of soman by beta-cyclodextrin

Soman (pinacolyl methylphosphonofluoridate), a mixture of four stereoisomers, is inactivated appreciably in Tris buffer, pH 7.40, mu = 0.155 at 25 degrees C by beta-cyclodextrin (cycloheptaamylose, beta-CD). Under these conditions, the dissociation constant Kd of the 1:1 complex formed by beta-CD and soman and the rate constant k2 for the phosphonylation of beta-CD by soman are (0.53 +/- 0.05)mM and (5.9 +/- 0.6) X 10(-2) min-1 respectively. It results that the inactivation of soman by the mono-anion of beta-CD is about 2,600 times faster than the hydrolysis of soman by the hydroxide ion. The inactivation of both P(-) isomers of soman by beta-CD proceeds apparently at the same rate but both P(+) isomers react more slowly. Thus the interaction is stereospecific. The inactivation of soman by beta-CD appears to be as fast in human plasma in vitro as in Tris buffer.     

Trypanocidal structure-activity relationship for cis- and trans-methylpluviatolide

The trypanocidal activity of racemic mixtures of cis- and trans-methylpluviatolides was evaluated in vitro against trypomastigote forms of two strains of Trypanosoma cruzi, and in the enzymatic assay of T. cruzi gGAPDH. The cytotoxicity of the compounds was assessed by the MTT method using LLC-MK2 cells. The effect of the compounds on peroxide and NO production were also investigated. The mixture of the trans stereoisomers displayed trypanocidal activity (IC50 approximately 89.3 microM). Therefore, it was separated by chiral HPLC, furnishing the (+) and (-)-enantiomers. Only the (-)-enantiomer was active against the parasite (IC50 approximately 18.7 microM). Despite being inactive, the (+)-enantiomer acted as an antagonistic competitor. Trans-methylpluviatolide displayed low toxicity for LLC-MK2 cells, with an IC50 of 6.53 mM. Furthermore, methylpluviatolide neither inhibited gGAPDH activity nor hindered peroxide and NO production at the evaluated concentrations.     

Stereoselective and species-dependent kinetics of reboxetine in mouse and rat

Reboxetine, (RS)-2-[(RS)-α-(2-ethoxyphenoxy)benzyl]morpholine methanesulphonate, is a racemic compound and consists of a mixture of the (R,R)- and (S,S)-enantiomers. In this study, brain and plasma levels of both enantiomers were determined in mice and rats after oral administration of reboxetine at doses (1.1 mg/kg, mouse; 20 mg/kg, rat) twice the respective ED₅₀ values in the antireserpine test. Plasma and brain concentrations of each enantiomer were measured up to 6 h postdosing using an HPLC method with fluorimetric detection after derivatization with a chiral agent (FLEC). In mice and rats, brain and plasma levels of the (R,R)-enantiomer were always higher than those of the (S,S)-enantiomer. After normalization for dose, the mean AUC_0-tz values of both the (R,R)- and (S,S)-enantiomers in mouse brain were about 23 and 32 times higher than in rat brain, respectively. In plasma, the corrected mean AUC_0-tz values were about 5 (R,R) and 10 (S,S) times higher in mice than in rats. These results provide evidence for the higher bioavailability and/or lower clearance of both enantiomers in mice than in rats, and for a higher penetration of both enantiomers into mouse brain compared to rat brain. © 1995 Wiley-Liss, Inc.     

Specific soman-hydrolyzing enzyme activity in a clonal neuronal cell culture

An enzymatic activity that specifically hydrolyzes the highly toxic organophosphorus anticholinesterase compound soman (pinacolyl methylphosphonofluoridate) has been identified and partially characterized in the clonal neuronal neuroblastoma-glioma hybrid NG108-15 cell line. Using the whole cell homogenate as the enzyme source and 1 mM substrate, the relative rate of hydrolysis of two other toxic anticholinesterase compounds sarin (isopropyl methylphosphonofluoridate) and tabun (ethyl-N-dimethyl phosphoramidocyanidate) is approximatelt one-tenth the rate of hydrolysis of soman, while DFP (diisopropyl phosphorofluoridate), paraoxon (p-nitrophenyl diethylphosphate), and a phosphinate PNMPP (p-nitrophenyl methyl (phenyl) phosphinate) are not hydrolyzed. Analysis of the kinetics of soman hydrolysis reveals two components of the enzyme activity with different affinities and reaction rates. Unlike previously reported enzymes of this type, this enzyme lacks chiral specificity and thus hydrolyzes both toxic and non-toxic soman stereoisomers at equal rates. The enzyme activity is stable at low temperature, found almost exclusively in the soluble fraction of these cells, and enhanced significantly by Mn²⁺ and by chemical differentiation of these cells in culture. The results suggest possible application of this enzyme for soman detection and/or detoxication, and use of the NG108-15 cell line to study the natural function(s) of enzymes of this type.     

Kinetic mechanism and enantioselectivity of halohydrin dehalogenase from Agrobacterium radiobacter

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the reversible intramolecular nucleophilic displacement of a halogen by a hydroxyl group in vicinal haloalcohols, producing the corresponding epoxides. The enzyme displays high enantioselectivity toward some aromatic halohydrins. To understand the kinetic mechanism and enantioselectivity of the enzyme, steady-state and pre-steady-state kinetic analysis was performed with p-nitro-2-bromo-1-phenylethanol (PNSHH) as a model substrate. Steady-state kinetic analyses indicated that the k(cat) of the enzyme with the (R)-enantiomer (22 s(-1)) is 3-fold higher than with the (S)-enantiomer and that the K(m) for the (R)-enantiomer (0.009 mM) is about 45-fold lower than that for the (S)-enantiomer, resulting in a high enantiopreference for the (R)-enantiomer. Product inhibition studies revealed that HheC follows an ordered Uni Bi mechanism for both enantiomers, with halide as the first product to be released. To identify the rate-limiting step in the catalytic cycle, pre-steady-state experiments were performed using stopped-flow and rapid-quench methods. The results revealed the existence of a pre-steady-state burst phase during conversion of (R)-PNSHH, whereas no such burst was observed with the (S)-enantiomer. This indicates that a product release step is rate-limiting for the (R)-enantiomer but not for the (S)-enantiomer. This was further examined by doing single-turnover experiments, which revealed that during conversion of the (R)-enantiomer the rate of bromide release is 21 s(-1). Furthermore, multiple turnover analyses showed that the binding of (R)-PNSHH is a rapid equilibrium step and that the rate of formation of product ternary complex is 380 s(-1). Taken together, these findings enabled the formulation of an ordered Uni Bi kinetic mechanism for the conversion of (R)-PNSHH by HheC in which all of the rate constants are obtained. The high enantiopreference for the (R)-enantiomer can be explained by weak substrate binding of the (S)-enantiomer and a lower rate of reaction at the active site.     

Asymmetrical ligand binding by abscisic acid 8'-hydroxylase

Abscisic acid (ABA), a plant stress hormone, has a chiral center (C1') in its molecule, yielding the enantiomers (1'S)-(+)-ABA and (1'R)-(-)-ABA during chemical synthesis. ABA 8'-hydroxylase (CYP707A), which is the major and key P450 enzyme in ABA catabolism in plants, catalyzes naturally occurring (1'S)-(+)-enantiomer, whereas it does not recognize naturally not occurring (1'R)-(-)-enantiomer as either a substrate or an inhibitor. Here we report a structural ABA analogue (AHI1), whose both enantiomers bind to recombinant Arabidopsis CYP707A3, in spite of stereo-structural similarity to ABA. The difference of AHI1 from ABA is the absence of the side-chain methyl group (C6) and lack of the alpha,beta-unsaturated carbonyl (C2'C3'-C4'O) in the six-membered ring. To explore which moiety is responsible for asymmetrical binding by CYP707A3, we synthesized and tested ABA analogues that lacked each moiety. Competitive inhibition was observed for the (1'R) enantiomers of these analogues in the potency order of (1'R,2'R)-(-)-2',3'-dihydro-4'-deoxo-ABA (K(I)=0.45 microM)>(1'R)-(-)-4'-oxo-ABA (K(I)=27 microM)>(1'R)-(-)-6-nor-ABA and (1'R,2'R)-(-)-2',3'-dihydro-ABA (no inhibition). In contrast to the (1'R)-enantiomers, the inhibition potency of the (1'S)-analogues declined with the saturation of the C2',C3'-double bond or with the elimination of the C4'-oxo moiety. These findings suggest that the C4'-oxo moiety coupled with the C2',C3'-double bond is the significant key functional group by which ABA 8'-hydroxylase distinguishes (1'S)-(+)-ABA from (1'R)-(-)-ABA.     

HPLC and NMR methods for the quantitation of the (R)-enantiomer in (−)-(S)-timolol maleate drug raw materials

HPLC and ¹H-NMR methods for the quantitation of the (R)-enantiomer in (?)-(S)-timolol maleate were developed and validated. The HPLC method requires a 25 cm × 4.6 mm 5 μm Chiracel OD-H (cellulose tris-3,5-dimethylphenylcarbamate) column, a mobile phase of 0.2% (v/v) diethylamine and 4% (v/v) isopropanol in hexane at a flow rate of 1 ml/min and UV detection at 297 nm. A system suitability test was devised to verify the separation of the (R)- and (S)-enantiomers of timolol from other drug-related impurities. The NMR method requires the use of a high-field NMR spectrometer (>360 MHz) and a chiral solvating agent, (?)-(R)-2,2,2-trifluoro-1-(9-anthrylethanol) (R-TFAE). The limits of quantitation were 0.05% and 0.2% (m/m) for HPLC and NMR, respectively. The methods were applied to the determination of the (R)-enantiomer in eight lots of raw material. The results for the two methods were in very good agreement, with results ranging from 0.1 to 4.1% (m/m) by HPLC and none detected to 4.3% (m/m) by NMR. The USP method for specific rotation was found to be unsuitable for detecting the presence of low levels of the (R)-enantiomer in (?)-(S)-timolol maleate. © 1994 Wiley-Liss, Inc.     

Stereospecificity of fatty acid 2-hydroxylase and differential functions of 2-hydroxy fatty acid enantiomers

FA 2-hydroxylase (FA2H) is an NAD(P)H-dependent enzyme that initiates FA α oxidation and is also responsible for the biosynthesis of 2-hydroxy FA (2-OH FA)-containing sphingolipids in mammalian cells. The 2-OH FA is chiral due to the asymmetric carbon bearing the hydroxyl group. Our current study performed stereochemistry investigation and showed that FA2H is stereospecific for the production of (R)-enantiomers. FA2H knockdown in adipocytes increases diffusional mobility of raft-associated lipids, leading to reduced GLUT4 protein level, glucose uptake, and lipogenesis. The effects caused by FA2H knockdown were reversed by treatment with exogenous (R)-2-hydroxy palmitic acid, but not with the (S)-enantiomer. Further analysis of sphingolipids demonstrated that the (R)-enantiomer is enriched in hexosylceramide whereas the (S)-enantiomer is preferentially incorporated into ceramide, suggesting that the observed differential effects are in part due to synthesis of sphingolipids containing different 2-OH FA enantiomers. These results may help clarify the mechanisms underlying the recently identified diseases associated with FA2H mutations in humans and may lead to potential pharmaceutical and dietary treatments. This study also provides critical information to help study functions of 2-OH FA enantiomers in FA α oxidation and possibly other sphingolipid-independent pathways.     

Reaction intermediate analogues for mandelate racemase: interaction between Asn 197 and the alpha-hydroxyl of the substrate promotes catalysis

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelic acid, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, carboxylate-, phosphonate-, and hydroxamate-containing substrate and intermediate analogues were examined for their ability to inhibit MR. Comparison of the competitive inhibition constants revealed that an alpha-hydroxyl function is required for recognition of the ligand as an intermediate analogue. Two intermediate analogues, alpha-hydroxybenzylphosphonate (alpha-HBP) and benzohydroxamate, were bound with affinities approximately 100-fold greater than that observed for the substrate. Furthermore, MR bound alpha-HBP enantioselectively, displaying a 35-fold higher affinity for the (S)-enantiomer relative to the (R)-enantiomer. In the X-ray structure of mandelate racemase [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., and Petsko, G. A. (1994) Biochemistry 33, 635-643], the alpha-hydroxyl function of the competitive inhibitor (S)-atrolactate is within hydrogen bonding distance of Asn 197. To demonstrate the importance of the alpha-hydroxyl function in intermediate binding, the N197A mutant was constructed. The values of k(cat) for N197A were reduced 30-fold for (R)-mandelate and 179-fold for (S)-mandelate relative to wild-type MR; the values of k(cat)/K(m) were reduced 208-fold for (R)-mandelate and 556-fold for (S)-mandelate. N197A shows only a 3.5-fold reduction in its affinity for the substrate analogue (R)-atrolactate but a 51- and 18-fold reduction in affinity for alpha-HBP and benzohydroxamate, respectively. Thus, interaction between Asn 197 and the substrate's alpha-hydroxyl function provides approximately 3.5 kcal/mol of transition-state stabilization free energy to differentially stabilize the transition state relative to the ground state.     

Enantioselective and diastereoselective separation of synthetic pyrethroid insecticides on a novel chiral stationary phase by high-performance liquid chromatography

A novel chiral packing material for high-performance liquid chromatography (HPLC) was prepared by connecting (R)-1-phenyl-2-(4-methylphenyl) ethylamine (PTE) amide derivative of (S)-isoleucine to aminopropyl silica gel through 2-amino-3,5-dinitro-1-carboxamido-benzene unit. This chiral stationary phase was applied to the enantioselective and diastereoselective separation of five pyrethroid insecticides by HPLC under normal phase condition. To achieve satisfactory baseline separation an optimization of the variables of mobile phase composition was required. The two enantiomers of fenpropathrin and four stereoisomers of fenvalerate were baseline separated using hexane-1,2-dichloroethane-2-propanol as mobile phase. The results show that the enantioselectivity of CSP is better than Pirkle type 1-A column for these compounds. Only partial separations for the cypermethrin and cyfluthrin stereoisomers were observed. Seven peaks and eight peaks were observed for cypermethrin and cyfluthrin, respectively. The elution orders were assigned by using different stereoisomer-enriched products.     

Structure of hybrid backbone methylphosphonate DNA heteroduplexes: effect of R and S stereochemistry.

Methyl phosphonate oligonucleotides have been used as antisense and antigene agents. Substitution of a methyl group for oxygen in the phosphate ester backbone introduces a new chiral center. Significant differences in physical properties and hybridization abilities are observed between the R(p) and S(p) diastereomers. Chirally pure methylphosphonate deoxyribooligonucleotides were synthesized, and the solution structures of duplexes formed between a single strand heptanucleotide methylphosphonate, d(Cp(Me)Cp(Me)Ap(Me)Ap(Me)Ap(Me)Cp(Me)A), hybridized to a complementary octanucleotide, d(TpGpTpTpTpGpGpC), were studied by NMR spectroscopy. Stereochemistry at the methylphosphonate center for the heptanucleotide was either RpRpRpRpRpRp (R(p) stereoisomer) or RpRpRpSpRpRp (S(p) stereoisomer, although only one of the six methylphosphonate centers has the S(p) stereochemistry). The results show that the methylphosphonate strands in the heteroduplexes exhibit increased dynamics relative to the DNA strand. Substitution of one chiral center from R(p) to S(p) has a profound effect on the hybridization ability of the methylphosphonate strand. Sugars in the phosphodiester strand exhibit C(2)(') endo sugar puckering while the sugars in the methyl phosphonate strand exhibit an intermediate C(4)(') endo puckering. Bases are well stacked on each other throughout the duplex. The hybridization of the methylphosphonate strand does not perturb the structure of the complementary DNA strand in the hetero duplexes. The sugar residue 5' to the S(p) chiral center shows A-form sugar puckering, with a C(3)(')-endo conformation. Minor groove width in the R(p) stereoisomer is considerably wider, particularly at the R(p) vs S(p) site and is attributed to either steric interactions across the minor groove or poorer metal ion coordination within the minor groove.     

Direct detection of stereospecific soman hydrolysis by wild-type human serum paraoxonase

Human serum paraoxonase 1 (HuPON1; EC 3.1.8.1) is a calcium-dependent six-fold beta-propeller enzyme that has been shown to hydrolyze an array of substrates, including organophosphorus (OP) chemical warfare nerve agents. Although recent efforts utilizing site-directed mutagenesis have demonstrated specific residues (such as Phe222 and His115) to be important in determining the specificity of OP substrate binding and hydrolysis, little effort has focused on the substrate stereospecificity of the enzyme; different stereoisomers of OPs can differ in their toxicity by several orders of magnitude. For example, the C+/-P- isomers of the chemical warfare agent soman (GD) are known to be more toxic by three orders of magnitude. In this study, the catalytic activity of HuPON1 towards each of the four chiral isomers of GD was measured simultaneously via chiral GC/MS. The catalytic efficiency (k(cat)/K(m)) of the wild-type enzyme for the various stereoisomers was determined by a simultaneous solution of hydrolysis kinetics for each isomer. Derived k(cat)/K(m) values ranged from 625 to 4130 mm(-1).min(-1), with isomers being hydrolyzed in the order of preference C+P+ > C-P+ > C+P- > C-P-. The results indicate that HuPON1 hydrolysis of GD is stereoselective; substrate stereospecificity should be considered in future efforts to enhance the OPase activity of this and other candidate bioscavenger enzymes.