Enthalpy change on mixing a couple of S- and R-enantiomers of some chiral compounds at 298.15 K

Enthalpy change on the mixing of R- and S-enantiomers of chiral liquid compounds such as dimethyl malate (1), methyl 3-hydroxylbutanoate (2), 2-butanol (3), ethyl 4-chloro-3-hydroxylbutanoate (4), 1,3,3-trimethylbicycle-[2.2.1]heptan-2-one (5), 3,7-dimethyl-6-octenal (6), and 8-bromo-2,6-dimethyl-2-octene (7) is measured over the entire range of mole fractions at 298.15 K, albeit very small values. The mixing of chiral liquids of R-1 + S-1, R-2 + S-2, R-3 + S-3, R-6 + S-6, and R-7 + S-7 produces enthalpic destabilization over the entire range of mole fractions, while that of R-4 + S-4 and R-5 + S-5 shows enthalpic stabilization over entire compositions. Enthalpy change on mixing at an equimolar concentration and the intermolecular interaction obtained by the molecular mechanics calculations show a linear correlation, except for a few compounds measured.     

Insights from crystal structures into the opposite effects on RNA affinity caused by the S- and R-6'-methyl backbone modifications of 3'-fluoro hexitol nucleic acid

Locked nucleic acid (LNA) analogues with 2',4'-bridged sugars show promise in antisense applications. S-5'-Me-LNA has high RNA affinity, and modified oligonucleotides show weakened immune stimulation in vivo. Conversely, an R-5'-methyl group dramatically lowers RNA affinity. To test the effects of S- and R-6'-methyl groups on 3'-fluoro hexitol nucleic acid (FHNA) stability, we synthesized S- and R-6'-Me-FHNA thymidine and incorporated them into oligo-2'-deoxynucleotides. As with LNA, S-6'-Me is stabilizing whereas R-6'-Me is destabilizing. Crystal structures of 6'-Me-FHNA-modified DNAs explain the divergent consequences for stability and suggest convergent origins of these effects by S- and R-6'-Me (FHNA) [-5'-Me (LNA and RNA)] substituents.     

Stereoselectivity and stereospecificity of the alpha,beta-dihydroxyacid dehydratase from Salmonella typhimurium.

1. In addition to the known 2R,3R- and 2R, 3S-2,3-dihydroxy-3-methylpentanoic acids (DHI), the 1S,3S- and sS,DR-isomers were prepared. 2S-2,3-Dihydroxy-3-methylbutanoic acid (DHV) was also prepared in addition to the known 2R-isomer. 2. The six dihydroxy acids were examined for their ability to promote the growth of isoleucine-valine (ilv)-requiring strains of Salmonella typhimurium and to serve as substrates for the alpha,beta-dihydroxyacid dehydratase of the same organism. 3. Only 2R,3R-2,3-dihydroxy-3-methylpentanoic and 2R-2,3-dihydroxy-3-methylbutanoic acids supported growth of the ilv strains of S. typhimurium. 4. alpha,beta-Dihydroxyacid dehydratase utilized the three isomers with the 2R-configuration as substrates but not those with the 2S-configuration. 5. In an additional growth study that utilized the 3R- and 3S-isomers of 3-methyl-2-oxopentanoic acid, the alpha-keto acid analogue of isoleucine, only the 3S-isomer supported growth. 6. It is concluded that the mechanism of action of the dehydratase is stereospecific in that the proton that is attached to C-3 of the substrate occupies the same steriochemical position as the departing hydroxyl group (Fig. 6).     

Bisanthraquinone glycosides of Hypericum perforatum with binding inhibition to CRH-1 receptors

Four new bisanthraquinone glycosides, S-(+)-skyrin-6-O-beta-glucopyranoside (1), R-(-)-skyrin-6-O-beta-glucopyranoside (2), S-(+)-skyrin-6-O-beta-xylopyranoside (3) and S-(+)-skyrin-6-O-beta-alpha-arabinofuranoside (4), have been isolated from an ethanol-water (1:1, v/v) dry extract of the aerial parts of Hypericum perforatum L. The structures were elucidated by spectroscopic methods, mainly NMR and mass spectrometry. Circular dichroism was used to determine their axial stereochemistry revealing 1 and 2 to be atropisomers. 1 and 2 inhibited [125I]sauvagine binding to corticotropin releasing hormone (CRH-1) receptors.     

The shikimate pathway. V. Fluorine-containing analogues of 3-deoxy-D-arabino hept-2-ulosonate-7-phosphate (DAHP)

(3R) and (3S) 3-deoxy-3-fluoro-7-phospho-D-arabino hept-2-ulosonic acids (3R and 3S-3F-DAHP) the 3-fluoro analogues of DAHP were synthesized from the corresponding 2-deoxy-2-fluoro hexose-6-phosphates. 3R- and 3S-3F-DAHP were tested as substrates for 3-dehydroquinate synthetase from E. coli. Determination of kinetic parameters showed that their apparent Km and Vm were in the same order of magnitude for these two compounds. Further conversion of 3R- and 3S-3F-DAHP into (6R) and (6S) 6-fluoro dehydroshikimate and (6R) and (6S) 6-fluoro shikimate, respectively, was investigated and results are discussed.     

An Efficient Chemoenzymatic Synthesis of S-(-)-Fenpropimorph

First enantioselective synthesis of S-(-)-1-[3-(4-tert-butylphenyl)-2-methyl]propyl-cis-3,5-dimethylmorpholine (6), biologically active enantiomer of the systematic fungicide fenpropimorph, is reported. It comprises reacting 4-tert-butylbenzylbromide with methyldiethylmalonate, decarbethoxylation of 2 into racemic 3-(4-tert-butylphenyl)-2-methylpropionic acid ethylester (3) in DMSO in the presence of alkali, then Pseudomonas sp. lipase catalyzed kinetic resolution of racemic 3 into S-(+)-acid (4), base-catalyzed racemization and recycling of the R-(-)-ester 3, acylation of cis-3,5-dimethylmorpholine, and final reduction of the intermediary amide 5 to provide enantiomerically pure S-(-)-6.     

Chiral separation and determination of R-(-)- and S-(+)-baclofen in human plasma by high-performance liquid chromatography

The method presented here is a high-performance liquid chromatography (HPLC)-UV detection method for the determination of baclofen R-(-)- and S-(+)-enantiomers in human plasma using a chiral separation technique. Baclofen enantiomers were extracted from human plasma with a reversed-phase solid-phase extraction (SPE) cartridge. The extract was then injected onto a HPLC system with a UV detection system set at 220 nm. The separation was achieved by using a 150x4.6 mm, 5 microm Phenomenex chirex 3216 chiral column with a mobile phase consisting of 0.4 mM CuSO(4) in acetonitrile-20 mM sodium acetate (17:83). The calibration curves were linear for both R-(-)- and S-(+)-enantiomers of baclofen in the concentration range of 20-5000 ng/ml. The average regressions were 0.9980 and 0.9991 for R-(-)- and S-(+)-baclofen, respectively. Inter-day precision was 3.3-5.2% for R-(-)-baclofen and 3.5-3.9% for S-(+)-baclofen at a concentration range of 60-4000 ng/ml. Intra-day precisions were 0.6-4.4 and 0.5-3.5% for R-(-)-baclofen and S-(+)-baclofen, respectively. The average extraction recovery was 81.6% for R-(-)-baclofen, 83.0% for S-(+)-baclofen and 94.0% for the internal standard (p-aminobenzoic acid). The limit of quantitation for both R-(-)- and S-(+)-baclofen in human plasma was 20 ng/ml. The method is simple and easy to operate with accuracy and reproducibility and it is suitable for pharmacokinetic studies.     

Synthesis and pharmacological evaluation of the stereoisomers of 3-carba cyclic-phosphatidic acid

Cyclic phosphatidic acid (CPA) is a naturally occurring analog of lysophosphatidic acid (LPA) in which the sn-2 hydroxy group forms a five-membered ring with the sn-3 phosphate. Here, we describe the synthesis of R-3-CCPA and S-3-CCPA along with their pharmacological properties as inhibitors of lysophospholipase D/autotaxin, agonists of the LPA(5) GPCR, and blockers of lung metastasis of B16-F10 melanoma cells in a C57BL/6 mouse model. S-3CCPA was significantly more efficacious in the activation of LPA(5) compared to the R-stereoisomer. In contrast, no stereoselective differences were found between the two isomers toward the inhibition of autotaxin or lung metastasis of B16-F10 melanoma cells in vivo. These results extend the potential utility of these compounds as potential lead compounds warranting evaluation as cancer therapeutics.     

Microscale synthesis of isotopically labeled R-[6-xH]N5,N10-methylene-5,6,7,8-tetrahydrofolate as a cofactor for thymidylate synthase

A one-pot synthesis of isotopically labeled R-[6-xH]N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4F) is presented, where x=1, 2, or 3 represents hydrogen, deuterium, or tritium, respectively. The current procedure offers high-yield, high-purity, and microscale-quantity synthesis. In this procedure, two enzymes were used simultaneously in the reaction mixture. The first was Thermoanaerobium brockii alcohol dehydrogenase, which stereospecifically catalyzed a hydride transfer from C-2-labeled isopropanol to the re face of oxidized nicotinamide adenine dinucleotide phosphate to form R-[4-xH]-labeled reduced nicotinamide adenine dinucleotide phosphate. The second enzyme, Escherichia coli dihydrofolate reductase, used the xH to reduce 7,8-dihydrofolate (H2F) to form S-[6-xH]5,6,7,8-tetrahydrofolate (S-[6-xH]H4F). The enzymatic reactions were followed by chemical trapping of S-[6-xH]H4F with formaldehyde to form the final product. Product purification was carried out in a single step by reverse phase high-pressure liquid chromatography separation followed by lyophilization. Two analytical methods were developed to follow the reaction progress. Finally, the utility of the labeled cofactor in mechanistic studies of thymidylate synthase is demonstrated by measuring the tritium kinetic isotope effect on the enzyme's second order rate constant.     

Tracer studies of the interconversion of R- and S-methylmalonic semialdehydes in man.

Two human subjects were given separate oral doses of sodium [2H6]isobutyrate and [methyl-2H3]thymine and the labelling patterns of urinary metabolites were determined. Ingestion of deuterated isobutyrate resulted in the excretion of 2H5-labelled S-3-hydroxyisobutyric acid, formed on the direct catabolic pathway, and of S- and R-[2H4]-3-hydroxyisobutyric acids, formed by the reduction of S- and R-methylmalonic semialdehydes respectively. Only the R-enantiomer of urinary 3-hydroxyisobutyric acid was labelled by thymine. This labelling pattern indicates a flow from S- to R-methylmalonic semialdehyde, suggesting that the R-enantiomer is the substrate of methylmalonic semialdehyde dehydrogenase.     

Synthesis and characterization of stereoisomers of 5,6-dihydro-5,6-dihydroxy-thymidine

Six products were isolated by reverse phase HPLC from the reaction of thymidine with osmium tetroxide. Four of the products were identified as stereoisomers of 5,6-dihydro-5,6-dihydroxy-thymidine (TG). The absolute configurations of these four compounds (from the shortest to the longest HPLC retention times) were determined by two-dimensional nuclear magnetic resonance spectroscopy to be (-)-trans-5S,6S-, (+)-trans-5R,6R-, (-)-cis-5R,6S-, and (+)-cis-5S,6R-5,6-dihydro-5,6-dihydroxy-thymidine. The other two products were dimers with unknown linking sites. Parameters of the mass and nuclear magnetic resonance spectra are reported and discussed.     

Serotonergic and dopaminergic involvement in the mechanism of action of R-(−)-2, 5-dimethoxy-4-bromoamphetamine (DOB) in cats

Intraperitoneally administered R-(?)- and S-(+)- enantiomers of 2,5-dimethoxy-4-bromoamphetamine were evaluated for their ability to induce head-body shake, limb flick and abortive grooming behaviors in cats. The R-(?)-enantiomer was consistently more effective than the S-(+)-isomer in all three behavioral measures. Dose-response relationships were evident for head-body shakes and limb flicks for both enantiomers, but reliable abortive grooming responses appeared only after the higher doses of R-(?)-DOB. Cinanserin and methysergide pretreatments effectively antagonized the induction of head-body shakes and limb flicks by 0.1 mg/kg R-(?)-DOB. In addition, haloperidol pretreatment significantly antagonized the appearance of these behaviors suggesting that dopaminergic as well as serotonergic stimulation is involved in the elicitation of these cat behaviors by R-(?)-DOB.     

Enzymic generation of chiral acetates. A quantitative evaluation of their configurational assay.

1. R-Acetate was generated enzymically from R-acetate in the sequence acetate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. It was concluded that the formation of malate on malate synthase involves the operation of a normal isotopic effect combined with inversion of configuration. The malate synthase kH/k2H was determined as 3.7 +/- 0.5 by a method which yields results independently of the stereochemical purity of the chiral acetates used initially. 2. R-Acetate was also generated from R-acetate in the sequence acetate leads to citrate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. The conclusion is the same as given above, but refers to the formation of citrate on the re-synthase. 3. 2S,3R-[2-2H1,3-2H1,3H1]Malate and 2S,3S-[2-2H1,3-2H1]malate were prepared from 2S-[2,3-2H3]malate by treatment with fumarase in tritiated water and normal water, respectively. It was assumed that these malate specimens were pure with respect to chirality as generated by isotopic labelling. 4. These two malate specimens were partially converted (about 9%) to acetates in conditions where no racemization at the level of transiently formed oxaloacetate occurred. That no racemization took place was demonstrated experimentally. Oxidative enzymic hydrolysis of 2S,3R-[2-2H1,3-2H1,3H1]malate in normal water and of 2S,3S-[2-2H1,3-2H1]malate in tritiated water produced S-[2H1,3H1]acetate and R-[2H1,3H1]acetate, respectively. 5. The isolated R-[2H1,3H1]acetate and S-[2H1,3H1]acetate on configurational analysis yielded malates which in the presence of fumarase retained 79.7 +/- 0.7% and 20.3 +/- 0.9%, respectively, of their total tritium content. The symmetric deviation from the 50% value found with [3H1]acetate strengthens the conclusion that stereochemically pure chiral acetates were analyzed. The malate synthase kH/k2H was determined from the data of this study as 3.9 +/- 0.2. 6. The average of the values given under paragraphs 1 and 5 for the isotopic discrimination on malate synthase corresponds to kH/k2H=3.8 +/- 0.1. It was concluded that the configurational analysis of stereochemically pure R-[2H1,3H1]acetate and S-[2H1,3H1]acetate yields malates which in the presence of fumarase retain 79 +/- 2% and 21 +/- 2%, respectively, of their total tritium content. Hence, a deviation of 29 +/- 2% from the 50% value represents the actual amplitude of the configurational assay. 7. Outlines are given for an enzymic generation of chiral acetates in preparative scale.     

Syntheses and highly enantioselective fluorescent recognition of α-aminocarboxylic acid anions using chiral oxacalix[2]arene[2]bisbinaphthes

The triazine-based bisbinaphthyl crown ethers oxacalix[2]arene[2]bisbinaphthes R-1, R-2, R-3 and S-1, S-2, S-3 were synthesized. The interactions of these compounds with various α-aminocarboxylic acid anions were studied. The crown ethers were found to carry out highly enantioselective fluorescent recognition of α-aminocarboxylic acid anions. It is observed that within a certain concentration range, one enantiomer of the chiral α-aminocarboxylic acid anions can increase the fluorescence intensity of the crown ethers by fivefold to sixfold, whereas the other enantiomer scarcely enhances the fluorescence. Such unusually high enantioselective responses make these crown ethers very attractive as fluorescent sensors in determining the enantiomeric composition of α-aminocarboxylic acid anions.     

Assay of physiological levels of 2,3-butanediol diastereomers in blood and urine by gas chromatography-mass spectrometry

We present an assay for 2,3-butanediol by gas chromatography-mass spectrometry of its trimethylsilyl ethers. 2R,3R- and/or 2S,3S-2,3-butanediol and meso-2,3-butanediol are quantitated with corresponding internal standards of [2,3-2H2]butanediol. Limits of detection are 1 and 0.1 microM for split and splitless injections, respectively. Blood concentrations of 2,3-butanediol in nonalcoholics are 0.5 +/- 0.3 (SD) microM for 2R,3R- and/or 2S,3S-2,3-butanediol and 0.8 +/- 0.4 microM for meso-2,3-butanediol (n = 9). Two hours after alcohol ingestion, blood levels had risen in eight of nine subjects to 1.2 +/- 0.7 microM for 2R,3R-/2S,3S-2,3-butanediol and to 1.2 +/- 0.6 microM for meso-2,3-butanediol. Baseline urinary excretion of 2,3-butanediol is 0.4 +/- 0.2 mumol/mmol creatinine for 2R,3R-/2S,3S-2,3-butanediol and 0.9 +/- 0.5 mumol/mmol creatinine for meso-2,3-butanediol.     

Structural requirements for hybridization at the 5'-position are different in α-l-LNA as compared to β-D-LNA

The synthesis and biophysical evaluation of R and S-5'-Me-α-l-LNA nucleoside phosphoramidites and modified oligo-2'-deoxyribonucleotides is reported. Synthesis of the nucleoside phosphoramidites was accomplished in multi-gram quantities starting from diacetone glucose. The 5'-methyl group in the S configuration was introduced by reacting the sugar 5'-aldehyde with MeMgBr. Synthesis of the R-5'-Me isomer was accomplished from the S-5'-Me nucleoside by a late stage inversion using Mitsunobu conditions. Evaluation of the modified oligonucleotides in thermal denaturation experiments revealed that R-5'-Me-α-l-LNA showed similar RNA affinity as α-l-LNA while the S-5'-Me analog was less stabilizing. This result is in contrast to the β-d-series where the S-5'-Me isomer showed LNA-like affinity for RNA while the R-5'-Me group completely reversed the stabilization effect on duplex thermostability.     

Stereochemistry and kinetic isotope effects in the bovine plasma amine oxidase catalyzed oxidation of dopamine.

The stereochemistry of the bovine plasma amine oxidase catalyzed oxidation of 2-(3,4-dihydroxyphenyl)-ethylamine (domapine) has been investigated by comparing 3H/14C ratios of 3,4-dibenzyloxyphenethyl alcohols, derived from 3,4-dihydroxyphenylacetaldehydes, to starting dopamines chirally labeled at C-1 and C-2. The oxidation of [2RS-3H]-, [2R-3H]-, and [2S-3H]dopamine leads to products which have retained 53, 59, and 47% of their tritium. Similarly, oxidation of [1RS-3H]-, [1R-3H]-, and [1S-3H]dopamine leads to an 80, 80, and 92% retention of tritium. The configurational purity of tritium at C-2 of dopamine and C-1 of the dopamine precursor 3-methoxy-4-hydroxyphenethylamine has been confirmed employing dopamine-beta-hydroxylase (specific for the pro-R hydrogen at C-2) and pea seedling amine oxidase (specific for the pro-S hydrogen at C-1). In addition, chromatographically resolved isozymes of bovine plasma amine oxidase have been demonstrated to lead to the same stereochemical result as pooled enzyme fractions. We have been able to rule out carbon interchange and tritium transfer in the ethylamine side chain of dopamine as the source of the apparent nonstereospecificity. Estimated primary tritium isotope effects are 1 for [2-3H]dopamines and 5--6 and 26--34 for [1R-3H]- and [1S-3H]dopamine, respectively. We propose the presence of alternate dopamine binding modes, characterized by absolute but opposing stereochemistries and differential primary tritium isotope effects at C-1.     

Comparison of interactions of R-(+)- and S-(-)-isomers of beta-adrenergic partial agonists, befunolol and carteolol, with high affinity site of beta-adrenoceptors in isolated rabbit ciliary body and guinea-pig taenia caeci.

The stereoselectivities of beta-adrenergic partial agonists for the high affinity binding site of beta-adrenoceptors in the rabbit ciliary body and the guinea-pig taenia caeci were studied. The pA2 values of the S-(-)-isomers of befunolol and carteolol against S-(-)-isoprenaline, which were calculated from the shift of each concentration - response curve in increasing cyclic AMP levels, were significantly larger than those of the R-(+)-isomers in the guinea-pig taenia caeci, while the pA2 values of the S-(-)-isomers were not significantly larger than those of the R-(+)-isomers in the rabbit ciliary body. The pK1 values determined from the binding experiments were in good agreement with the pA2 values from the increases in cyclic AMP levels. These results suggest that the high affinity binding site of beta-adrenoceptors in the guinea-pig taenia caeci may be able to discriminate stereoselectively between the R-(+)- and S-(-)-isomers, while in the rabbit ciliary body there is no stereo-selectivity between the two enantiomers.     

Synthesis and receptor binding of oxytocin analogs containing conformationally restricted amino acids

Summary We report the solid-phase synthesis and receptor-binding properties of eleven oxytocin analogs (Mpa-Xxx-Ile-Gln-Asn-Cys-Sar-Arg-Gly-NH₂) containing non-coded amino acids in position 2: D-α- and L-α-(2-indanyl)glycine, R,S-6-methoxy-2-aminotetralin-2-carboxylic acid, D- and L-pentafluorophenylalanine, D,L-2,4-dimethylphenylalanine, D,L-2,4,6-trimethylphenylalanine, R,R- and S,S-1,2,3,4-tetrahydro-1-methyl-β-carboline-3-carboxylic acid and R- and S-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid. Some of these amino acid analogs (2-indanylglycine and D-pentafluorophenylalanine) were earlier successfully applied for the synthesis of potent bradykinin antagonists [1, 2]. Their receptor bindings were tested on isolated guinea-pig uterus, rat liver and rat kidney inner medulla plasma membranes. The extent of binding of the peptides to the oxytocin receptor was in several cases was even higher than that of the parent hormone (oxytocin). However, the real pharmacological value of these analogs can be evaluated only afterin vivo measurements of their inhibition of uterine motor activity.