Stereochemistry of the hydrogen abstraction from pyridoxamine phosphate catalyzed by alanine racemase of Bacillus stearothermophilus

Alanine racemase of Bacillus stearothermophilus catalyzes transamination as a side reaction. Stereospecificity for the hydrogen abstraction from C-4′ of pyridoxamine 5′-phosphate occurring in the latter half transamination was examined. Both apo-wild-type and apo-fragmentary alanine racemases abstracted approximately 20 and 80% of tritium from the stereospecifically-labeled (4′S)- and (4′R)-[4′-³H]PMP, respectively, in the presence of pyruvate. Alanine racemase catalyzes the abstraction of both 4′S- and 4′R-hydrogen like amino acid racemase with broad substrate specificity. However, R-isomer preference is a characteristic property of alanine racemase.     

Molecular pharmacology of the AMPA agonist, (S)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(S)-APPA] and the AMPA antagonist, (R)-APPA

The heterocyclic analogue of (S)-glutamic acid, (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid [(S)-AMPA] is a potent and selective AMPA receptor agonist, whereas the enantiomeric compound, (R)-AMPA, is virtually inactive. We have previously characterized (RS)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(RS)-APPA] as a partial AMPA receptor agonist showing about 60% of the efficacy of (RS)-AMPA. This partial agonism produced by (RS)-APPA is, however, only apparent, since resolution of (RS)-APPA has now been shown to provide the full AMPA receptor agonist, (S)-APPA, whereas (R)-APPA is a acid (non-NMDA) receptor antagonist showing preferential AMPA blocking effects. In agreement with classical theories for competitive interaction between agonists and antagonists, the efficacy of depolarizations produced by (S)-APPA in the rat cortical wedge preparation was shown to be progressively reduced with increasing molar ratios of (R)-APPA/(S)-APPA. These compounds and the competitive antagonists (RS)-2-amino-3-(3-carboxymethoxy-5-methyl-4-isoxazolyl)propionic acid [(RS)-AMOA], 6-cyano-7-nitroquinoxalin-2,3-dione (CNQX) and 6-nitro-7-sulfamoylbenzo(f)quinoxalin-2,3-dione (NBQX) were also tested in [³H]AMPA and [³H]CNQX binding systems, the latter ligand being used in the absence or presence of thiocyanate ions. On the basis of these studies it is suggested that (RS)-AMPA and the AMPA agonist (S)-APPA interact with a high-affinity receptor conformation, whereas the competitive antagonists (RS)-AMOA and (R)-APPA, derived from these agonists, preferentially bind to a low-affinity AMPA receptor conformation. The competitive antagonists, CNQX and NBQX which are structurally unrelated to (RS)-AMPA or (RS)-APPA, do not seem to discriminate between these two AMPA receptor conformations. The modified [³H]CNQX binding assay containing thiocyanate ions was shown to provide receptor affinity data for AMPA receptor agonists as well as antagonists, which correlate with the potencies of these compounds in the cortical wedge preparation. Using autoradiographic techniques, (S)- and (R)-APPA were shown to exhibit significantly different absolute potencies as inhibitors of [³H]AMPA binding in a number of regions of the rat brain.     

In Vivo Metabolism of the Vitamin D Analog, 22-Oxacalcitriol: Evidence for Side-chain Truncation and 17-Hydroxylation

After intravenous administration of the vitamin D₃ analog, 22-oxacalcitriol (OCT), to normal rats plasma metabolites were investigated by HPLC, GC-MS and LC-MS. Five side-chain oxidation metabolites, 24R(OH)OCT, 24S(OH)OCT, (25R)-26(OH)OCT, (25S)-26(OH)OCT and 24oxoOCT, were identified by comparison with the corresponding synthetic compounds. These side-chain oxidation metabolites were similar to those of calcitriol [1,25(OH)₂ vitamin D₃] described previously. Besides these five metabolites, two unique side-chain cleavage metabolites, 20S(OH)-hexanor-OCT and 17,20S(OH)₂-hexanor-OCT, were identified as main metabolites in plasma by GC-MS and LC-MS using a specific chemical reaction. Our studies suggest that OCT is extensively metabolized and circulates in blood as a number of metabolites as well as unchanged OCT. This metabolism includes both unique pathways of C₂₃-O₂₂ cleavage and 17-hydroxylation, in addition to the side-chain oxidation metabolites similar to those of 1,25-(OH)₂D₃.     

Enantioselective production of 2,2-dimethylcyclopropane carboxylic acid from 2,2-dimethylcyclopropane carbonitrile using the nitrile hydratase and amidase of Rhodococcus erythropolis ATCC 25544

The enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid was investigated in 53 Rhodococcus and Pseudomonas related strains. Rhodococcus erythropolis ATCC 25544 was selected as it showed the highest enantioselectivity. The enantioselectivity was due to the amidase activity in a two-step reaction involving nitrile hydratase. The enantiomeric excess of the amidase was highest at pH 7.0 and decreased significantly above 20 °C. For the enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid, the optimum reaction conditions of the cells were determined to be pH 7.0, 20 °C, and 10% (v/v) methanol and were the same as the optimum pH and temperature for the enantioselective conversion by the amidase. Under these conditions, the R. erythropolis ATCC 25544 cells, which harbored nitrile hydratase and amidase enzymes, produced 45 mM (S)-2,2-dimethylcyclopropane carboxylic acid from racemic 100 mM 2,2-dimethylcyclopropane carbonitrile with an 81.8% enantiomeric excess after 64 h.     

Esterified, optical pure (3S, 3′S)-astaxanthin from flowers of Adonis annua

The quantitative carotenoid composition of the red flower petals of Adonis annua is reported. Optically pure (3S, 3′S)-astaxanthin occurs both as a diester (64% of total carotenoid) and as a monoester (11%). The optical purity was determined by hydrolysis of the natural esters in the absence of oxygen and subsequent HPLC analysis of the paren -ketol esterified with (−)-camphanic acid. All non-animal sources hitherto examined synthesize pure 3S,3′S- or 3R,3′R-isomers of astaxanthin, whereas marine animal sources contain mixtures of all three optical isomers, including the meso form.     

Adenosine receptor agonists: Synthesis and biological evaluation of the diastereoisomers of 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA

Among the recently reported 2-(ar)alkynyl derivatives of 5′-N-ethylcarboxamidoadenosine (NECA), the (R,S)-2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA [(R,S)-PHPNECA or SCH 59761] was found to be a very potent agonist at A₁ and A_2A receptor subtypes, with a K_i of 2.5 nM and 0.9 nM, respectively. Furthermore, this compound showed an inhibitory activity on platelet aggregation 16-fold higher than NECA, being the most potent anti-aggregatory nucleoside reported so far. Since this compound bears a chiral carbon in the side chain, the diastereoisomer separation was undertaken both by chiral HPLC and by a stereospecific synthetic method. Binding assays have shown that the (S)-diastereomer is about fivefold more potent and selective than the (R)-diastereomer as agonist of the A_2A receptor subtype [(S)-PHPNECA, K_iA_2A = 0.5 nM; (R)-PHPNECA, K_iA_2A = 2.6 nM]. Functional studies indicated that (S)-PHPNECA possesses marked vasodilating activity and produces a relevant decrease in heart rate. Moreover, the (S)-diastereomer proved to be about ten times more potent than the (R)-diastereomer in inducing cardiovascular effects, in in vivo hemodynamic studies. However, the greatest difference between these two enantiomers resulted in the platelet aggregation test: in fact, the (R)-diastereomer displayed an inhibitory activity similar to that of NECA, whereas the (S)-diastereomer was 37-fold more active than NECA as an inhibitor of rabbit platelet aggregation, induced by ADP. These data suggest that (S)-PHPNECA could be a useful tool to investigate the mode of binding of agonists to the platelet adenosine receptor subtype.     

(3S,4S,5R)-3,4,5-trihydroxy-1-cyclohexene-carboxylic acid from Sequoiadendron giganteum

From the leafy lateral branchlets of Sequoiadendron giganteum, (3S,4S,5R)-3,4,5-trihydroxy-1-cyclohexenecarboxylic acid has been isolated. Its structure was proved spectroscopically.     

三种桑黄属真菌人工栽培子实体营养、药效成分及抗氧化活性分析评价

本研究通过测定人工段木栽培3年生杨树桑黄Sanghuangporus vaninii、鲍姆桑黄S. baumii、桑树桑黄S. sanghuang及野生桑树桑黄子实体的粗蛋白质、粗脂肪、粗纤维、氨基酸、多糖、三萜、总黄酮、总酚含量和抗氧化活性（ABTS和FRAP）,探究此3种栽培桑黄及野生桑树桑黄子实体的营养、药效成分及抗氧化活性差异。结果表明,供试材料在营养、药效成分及抗氧化活性上差异较大。其中,栽培鲍姆桑黄子实体粗纤维含量最低,多糖含量较高,总黄酮、总酚含量和ABTS、FRAP活性最高;栽培杨树桑黄粗纤维含量较低,粗脂肪含量最低,总黄酮、总酚、三萜含量较高,多糖含量最高;野生桑树桑黄粗蛋白、粗脂肪、总氨基酸含量最高,多糖含量较高,三萜含量最高,总黄酮、总酚含量和ABTS、FRAP活性最低;栽培桑树桑黄粗纤维含量较高,多糖、总黄酮、总酚、三萜含量和ABTS、FRAP均较低。栽培杨树桑黄、鲍姆桑黄和野生桑树桑黄在药效成分含量上各有所长。筛选到可结实、药效成分含量高的桑树桑黄菌株是可能的。桑黄优良品种选育是今后的工作重点。本研究为桑黄真菌资源开发利用提供了参考。     

Direct synthesis of 6-oxabicyclo[3.2.1]octane derivatives from deoxyinososes

In the presence of bases, even those (for example, pyridine) normally used for acylation reactions, 2 -(2,4,5/3)-2,3,4,-tribenzoyloxy-5-hydroxycyclohexanone (3) readily gives (2 -(2,4/3)-2,3,4-tribenzoyloxycyclohex-5-enone or aromatic products. Under acid conditions, efficient O-acylation and tetrahydropyranylation can be effected. The main product (60% isolated) formed on treatment of 3 with diazomethane is (1R,2S,3R,4S,5S)-2,3,4-tribenzoyloxy-1-hydroxy-6-oxabicyclo [3.2.1]octane (15); small proportions of the epimeric spiro-epoxides and 1-acetyl-6-benzoyloxy-7-methoxy-1H-indazole are also formed. On photobromination, the acetate (17) of 15 undergoes substitution at C-7 and, from the product, C-formyl-deoxyinositol derivatives are produced.     

Stereoselective synthesis of opine-type secondary amine car☐ylic acids by a new enzyme opine dehydrogenase use of recombinant enzymes

The substrate specificity of the recently discovered enzyme, opine dehydrogenase (ODH) fromArthrobacter sp. strain 1C for amino donors in the reaction that forms secondary amines using pyruvate as a fixed amino acceptor is examined. The enzyme was active toward short-chain aliphatic (S)-amino acids and those substituted with acyloxy, phosphonooxy, and halogen groups. The enzyme was named N-[1-(R)-(car☐yl)ethyl]-(S)-norvaline: NAD⁺ oxidoreductase (L-norvaline forming). Other substrates for the enzyme were 3-aminobutyric acid and (S)-phenylalaninol. Optically pure opine-type secondary amine car☐ylic acids were synthesized from amino acids and their analogs such as (S)-methionine, (S)-isoleucine, (S)-leucine, (S)-valine, (S)-phenylalanine, (S)-alanine, (S)-threonine, (S)-serine, and (S)-phenylalaninol, and -keto acids such as glyoxylate, pyruvate, and 2-oxobutyrate using the enzyme, with regeneration of NADH by formate dehydrogenase (FDH) fromMoraxella sp. C-1. The absolute configuration of the nascent asymmetric center of the opines was of the (R) stereochemistry with > 99.9% e.e. One-pot synthesis of N-[1-(R)-(car☐yl)ethyl]-(S)-phenylalanine from phenylpyruvate and pyruvate by using ODH, FDH, and phenylalanine dehydrogenase (PheDH) fromBacillus sphaericus, is also described.     

Fractal structure and conformational entropy of protein chain

The structures of 25 proteins arbitrarily chosen are investigated by fractal geometry, and their fractal dimensions (D_f) and conformational entropies S(N₀) are calculated by Havlin—Ben Avraham and Monte Carlo method, respectively. Comparison of the D_f and S(N₀) gives the relation: D_f = 1.532 - 3.00 × 10⁻⁴ S(N₀). The entropy data obtained by Monte Carlo method for the chain of random self-avoiding walks confirm the prediction of renormalization group: S(N₀) = 1.544N₀ + 0.1667 In N₀ + 0.1570 where N₀ is the number of residues in a protein chain. Both the D_f and S(N₀) reflect the conformational properties of a protein molecular chain. The idea resulting from the present communication suggests that the thermodynamic behaviours of proteins may be related to multifractals.     

Preventive effect of 20(S)-ginsenoside Rg3 against lipopolysaccharide-induced hepatic and renal injury in rats

The preventive effect of 20(S)-ginsenoside Rg₃ (20(S)-Rg₃) on lipopolysaccharide (LPS)-induced oxidative tissue injury in rats was investigated in this study. The elevated serum nitrite/nitrate, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and creatinine levels in LPS-treated control rats were significantly decreased following 15 consecutive days of 20(S)-Rg₃ administration. In addition, thiobarbituric acid-reactive substance levels in the serum, liver and kidney were dose-dependently lower in 20(S)-Rg₃-treated groups than in the LPS-treated control group. The nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) protein expressions in the liver and kidney were significantly increased by LPS treatment. However, the 20(S)-Rg₃ administrations significantly decreased these protein expressions except for HO-1 in the liver. On the other hand, in the kidney, oral administration of 20(S)-Rg₃ showed a tendency to reduce NF-κB and iNOS protein expressions and also significantly reduced the elevated COX-2 and HO-1 protein expressions at a dose of 10 mg/kg body weight/day. All these results suggest the preventive effect of 20(S)-Rg₃ against LPS-induced acute oxidative damage in the liver and kidney and the preventive effect of 20(S)-Rg₃ administration against LPS toxicity was thought to be more predominant in the liver than kidney.     

Enzymatic resolution of (R, S)-Naproxen in water-saturated ionic liquid

A water-saturated ionic liquid has been exploited for resolution of (R, S)-Naproxen by lipase-catalyzed hydrolysis to enhance the conversion and facilitate product recovery. From the enantioselectivity and activity of lipase, water-saturated [bmim]PF₆ (1-butyl-3-methylimidazolium hexafluorophosphate) was selected as the best reaction medium. To prevent the dissolution of lipase in the ionic liquid, a weakly polar, amorphous multiporous silica YWG-C₆H₅ was used as a support for immobilization. The production of (S)-Naproxen was initially performed in a batch reactor containing 20 mL of substrate solution. After 72 h reaction, 98.2% enantiomeric excess of the (S)-Naproxen was obtained with 28.3% hydrolysis conversion. The unconventional solvent properties of ionic liquids have been exploited in reaction medium recycling, product recovery and water recruiting schemes. In a repetitive batch reaction system, the immobilized lipase could be repeatedly used for 5 times with only a slight reduction in reaction conversion.     

Gene cloning of an NADPH-dependent menadione reductase from Candida macedoniensis, and its application to chiral alcohol production

The gene encoding an NADPH-dependent menadione reductase of Candida macedoniensis AKU4588 was cloned and sequenced. A 1035 bp nucleotide fragment (mer) was confirmed to be the gene encoding the enzyme based on the agreement of N-terminal and internal amino acid sequences. The mer encodes 345 amino acid residues, and the deduced amino acid sequence shows high similarity with those of hypothetical proteins from Debaryomyces, Candida and Saccharomyces, and ketoreductase from Zygosaccharomyces. It includes NADPH-binding motif GXXGXXA in its N-terminal region. These findings suggest that the enzyme belongs to the dihydroflavonol-4-reductase superfamily. An expression vector, pETMER, which contains the full length of the mer, was constructed. Escherichia coli cells harboring pETMER exhibits a 127-fold increase in specific menadione-reducing activity under the control of T7 promoter as compared with that of C. macedoniensis.

The asymmetric reduction of 4-chloro-3-oxobutanoate ethyl ester to (S)-4-chloro-3-hydroxybutanoate ethyl ester (CHBE) with E. coli cells, in which both the mer and the glucose dehydrogenase gene were co-expressed, as a catalyst was investigated. The (S)-CHBE formed amounted to 1680 mM (281 mg/ml), the molar yield being 92.2%. The optical purity of the product was 91.6% enantiomeric excess for the (S)-isomer. The calculated turnover number of NADP⁺ added to CHBE formed was 12,900 mol/mol.     

Total syntheses of (+/-)-ovalicin, C4(S *)-isomer, and its C5-analogs and anti-trypanosomal activities

Total syntheses of (±)-ovalicin, its C4(S^*)-isomer 44, and C5-side chain intermediate 46 were accomplished via an intramolecular Heck reaction of (Z)-3-(tert-butyldimethylsilyloxy)-1-iodo-1,6-heptadiene and a catalytic amount of palladium acetate. Subsequent epoxidation, dihydroxylation, methylation, and oxidation led to (3S^*,5R^*,6R^*)-5-methoxy-6-(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]octan-4-one (2), a reported intermediate. The addition of a side chain with cis-1-lithio-1,5-dimethyl-1,4-hexadiene (27) followed by oxidation afforded (±)-ovalicin. The functional group manipulation afforded a number of regio- and stereoisomers, which allow the synthesis of analogs for bioevaluation. The structure of 44 was firmly established via a single-crystal X-ray analysis. The stereochemistry at C4 generated from the addition reactions of alkenyllithium with ketones 2, 40, and 45 is dictated by C6-alkoxy functionality. Anti-trypanosomal activities of various ovalicin analogs and synthetic intermediates were evaluated, and C5-side chain analog, 46, shows the strongest activity. Compound 44 shows antiproliferative effect against HL-60 tumor cells in vitro. Compounds 46 and a precursor, (3S^*,4R^*,5R^*,6R^*)-5-methoxy-4-[(E)-(1′,5′-dimethylhexa-1′,4′-dienyl)]-6-(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]octan-4-ol (28), may be explored for the development of anti-parasitic drugs.     

Candida antarctica lipase B catalysed amidation of pyroglutamic acid derivatives. A reaction survey

Pyroglutamic acid esters, both (S)- and (R)-enantiomers, have been studied as substrates of the Candida antarctica lipase B catalyzed amidation in anhydrous organic solvents. They behaved as very good substrates when primary amines or ammonia were used as nucleophiles, affording the corresponding secondary and primary amides, respectively, but did not react with secondary amines. The reaction was enantioselective for the (R)-enantiomer of chiral amines although little kinetic difference was observed between (S)- and (R)-pyroglutamates as acyl donors. As an example of an infrequent reaction, free (S)-pyroglutamic acid may also act as a substrate of the reaction, but is much less reactive than its esters.     

On the mechanism of biosynthesis of leukotrienes and related compounds

[10D-³H; 3-¹⁴C]- and [10L-³H; 3-¹⁴C]arachidonic acids were incubated with human polymorphonuclear leukocytes and with human platelets. Leukotriene B₄ and 5(S),12(S)-dihydroxy-6trans,8cis,10trans,14-cis-eicosatetraenoic acid (5,12-DHETE) were isolated and the ³H/¹⁴C ratios determined. It could be concluded that the 10D (pro-R)-hydrogen is eliminated in the conversion of 5(S)-hydroperoxy-6trans,8cis,11cis,14cis-eicosatetraenoic acid into leukotriene A₄ whereas in the conversion of arachidonic acid into 5,12-DHETE the 10L (pro-S)-hydrogen is lost. Incubation of the doubly labeled arachidonic acids with human platelets confirmed and extended previous data on the stereochemistry of the hydrogen removal from C-10 during the conversion into 12(S)-hydroperoxy-5cis,8cis,10trans,14cis-eicosatetraenoic acid, i.e., the 10L (pro-S)-hydrogen is eliminated and the 10D (pro-R)-hydrogen retained.     

Catalytic properties of cytochrome P-450scc from bovine and porcine adrenocortical mitochondria: effect of Tween20 concentration

Cholesterol side-chain cleavage activities of cytochrome P-450ssc purified from bovine adrenocortical mitochondria were measured for various substrates, including cholesterol, 20[S]-hydroxycholesterol, 22[R]-hydroxycholesterol and 20[R], [R]-dihydroxycholesterol, in the reconstituted enzyme system at various Tween20 concentrations. The side-chain cleavage activity for cholesterol showed more than 10-fold enhancement upon addition of 0.1% Tween20, compared with that without the detergent. Addition of Tween20 did not cause any enhancement of the side-chain cleavage activities for 20[S]-hydroxycholesterol and 22[R]-hydroxycholesterol; rather, it resulted in an inhibition of the activities. The side-chain cleavage activity for 20[R],22[R]-dihydroxycholesterol showed a very high value even without the detergent. As the stimulatory effect of Tween20 was only specific for cholesterol, Tween20 seemed to enhance the rate of access of cholesterol to cytochrome P-450scc. These results are consistent with the suggestion that a transfer of substrate, cholesterol, in mitochondrial inner membrane, to the substrate-binding site of cytochrome P-450scc is the rate-limiting step in the cholesterol side-chain cleavage reaction.     

Monoclonal antibodies for the detection and quantitation of the endogenous plant growth regulator, abscisic acid

Monoclonal antibodies (mAB) have been produced which recognize the physiologically active 2-cis-(S-form of the endogenous plant growth regulator, abscisic acid (ABA). Cross-reaction with the ABA-catabolites, phaseic and dihydrophaseic acid, is negligible, and (R)-ABA, 2-trans-ABA, the ABA-conjugate, ABA-β-D-glucopyranosyl ester, as well as the putative ABA precursor, xanthoxin, are totally unreactive. In addition to being very specific, the mAB exhibit high affinites for 2-cis-(S)-ABA: the K values were 7.9 × 10⁹ l/mol and 3.7 × 10⁹ l/mol for antibodies from two different clones. By mAB-radioimmunoassay (RIA), 4 pg 2-cis-(S)-ABA (99.5% confidence level) can be detected. mAB-RIA can be used to quantitate ABA directly in unprocessed plant extracts.