Formation of L-(+)-Tartaric Acid in Leaves of the Bean, Phaseolus vulgaris L.: Radioisotopic Studies with Putative Precursors

The biosynthetic pathway from D-glucose to L-(+)-tartaric acid(TA) in detached leaves of the bean, Phaseolus vulgaris L.,was studied in three cultivars, two of which were known to containTA and one of which lacked TA, with the aid of several putativeradiolabeled intermediates, namely D-[l-¹⁴C]glucose, D-[6-¹⁴C]glucose,D-[U-¹⁴C]glucose, D-[U-¹⁴C]gluconate, L-[U-¹⁴C]-ascorbic acid,L-[l-^l4C]idonate, D-xylo-5-[U-¹⁴C]hexulosonate, D-xylo-5-[l-¹⁴C]hexulosonate,D-xylo-5-[6-^l4C]hexulosonate and L-[U-^l4C]threonate. D-[U-¹⁴C]Glucoseand D-[U-^l4C]gluconate were converted to TA with low isotopicyield but this yield was further reduced when leaf tissues weresupplied with unlabeled D-gluconate or D-xylo-5-hexulosonate.D-xylo-5-[U-¹⁴C]Hexulosonate and D-xylo-5-[l-¹⁴C]hexulosonatewere good precursors of TA. D-xylo-5-[6-¹⁴C]Hexulosonate didnot furnish ¹⁴C to TA. Addition of a metabolic product of D-xylo-5-hexulosonate(which was labeled by D-xylo-5-[l-¹⁴C]hexulosonate but not byD-xylo-5-[6-¹⁴C]hexulosonate) to leaves labeled with D-xylo-5-[l-¹⁴C]hexulosonatedoubled the incorporation of ¹⁴C into TA. L-[U-¹⁴C]Ascorbicacid, L-[l-¹⁴C]idonate and L-[U-¹⁴C]threonate failed to producelabeled TA. A metabolic scheme to accommodate these observationsis presented. (Received October 21, 1988; Accepted March 29, 1989)     

Synthesis and reactions of some hydrazones of dehydro-l-ascorbic acid

l-threo-2,3-Hexodiulosono-1,4-lactone 2-(3-chlorophenylhydrazone) and 4- (2-acetoxyethylidene)-4-hydroxy-2,3-dioxobutano-1,4-lactone 2-(3-chlorophenylhydrazone) were prepared. The two geometric isomers of the corresponding bis(hydrazone) underwent an intramolecular rearrangement to 1-(3-chlorophenyl)- 3-(l-threo-glycerol-1-yl)-4,5-pyrazoledione 4-(3-chlorophenylhydrazone), which gave a tri-O-acetyl derivative upon acetylation and the anticipated formyl derivative upon periodate oxidation. Oxidation of the bis(hydrazone) with cupric chloride afforded the bicyclic compound 3,6-anhydro-3-C-(3-chlorophenylazo)-l- xylo-2-hexulosono-1,4-lactone 2-(3-chlorophenylhydrazone), whose acetylation afforded the mono-O-acetyl derivative.     

Studies on 3-epimeric l-2-hexulose phenylosazones. Structure and anomeric configuration of the 3,6-anhydro-osazone derivatives obtained from l-xylo- and l-lyxo-2-hexulose phenylosazone

Dehydration of the 3-epimeric 2-hexulose phenylosazones l-xylo-hexulose phenylosazone and l-lyxo-hexulose phenylosazone afforded 3,6-anhydro-l-lyxo-2-hexulose phenylosazone (2) as the preponderant isomer from both. The identity of 2 was obtained by t.l.c., and by acetylation followed by comparison of the products. Acetylation of 2 with acetic anhydride-pyridine afforded the di-O-acetyl derivative 4, and further acetylation gave the N-acetyldi-O-acetyl derivative 5. Refluxing of 2 with copper sulfate afforded a C-nucleoside analog, namely, 2-phenyl-4-α-l-threofuranosyl-1,2,3-osotriazole (6). The anomeric configuration was determined by n.m.r. spectroscopy. The stereochemical course of the dehydration process and the mass spectra of compounds 2, 4, 5, and 6 are discussed.     

Toward the synthesis of fine chemicals from lactose: preparation of d-xylo and l-lyxo-aldohexos-5-ulose derivatives

The transformation of (5R)-2,6-di-O-benzyl-5-C-methoxy-β-d-galactopyranosyl-(1→4)-2,3:5,6-di-O-isopropylidene-aldehydo-d-glucose dimethyl acetal (8) into partially protected derivatives of d-xylo- and l-lyxo-aldohexos-5-ulose has been reported, applying appropriate epimerisation methods to its 3′-O- and 4′-O-protected alcoholic derivatives.     

The structure and properties of the products of reaction between 3,4,6-tri-O-acetyl-2-deoxy-2-nitroso-α-d-galactopyranosyl chloride and pyrazole

Dimeric 3,4,6-tri-O-acetyl-2-deoxy-2-nitro-α-d-galactopyranosyl chloride reacts with pyrazole in acetonitrile to give 1-(3,4,6-tri-O-acetyl-2-deoxy-2-hydroxyimino-α-d-lyxo-, -β-d-lyxo-, and -β-d-xylo-hexopyranosyl)pyrazole. The stereospecificity of the reaction depends on the temperature and its duration. Transformations of the type α-d-lyxo-←β-d-lyxoα β-d-xylo have been observed. The condensation products were modified at C-2 or C-3. The following derivatives have thus been obtained: 1-(α-d-galacto-, 2-acetamido-2-deoxy-α-d-galacto-, -α-d-talo-, and -α-d-xylo-hexo-pyranosyl)pyrazole, (Z)- and (E)-1-(3-azido-2,3-dideoxy-2-hydroxyimino-α- and -β-d-lyxo- and -α-d-xylo-hexopyranosyl)pyrazole, 1-(3-acetamido-2-acetoxyimino-4,6-di-O-acetyl-2,3-dideoxy-α- and -β-d-lyxo-hexopyranosyl)pyrazole, as well as (Z)- and (E)-1-(2,3-dideoxy-2-hydroxyimino-α-d-threo-hexopyranosyl)pyrazoles.     

Synthesis of C-glycopyranosylfuran derivatives by reaction of dialdehydes with cyanoacetamide

The reaction of diglycol- and thiodiglycol-aldehyde (1a,b) with cyanoacetamide yields cis-3,5-diacetoxy-4-carbamoyl-4-cyano-tetrahydropyran (2a) and -tetrahydrothiopyran (2b). When this reaction is applied to (2S)-2-(3-ethoxycarbonyl-2-methyl-5-furyl)-3,5-dihydroxy-1,4-dioxane (1c), (2S)-3,5-dihydroxy-2-(3-methoxycarbonyl-2-methyl-5-furyl)-1,4-dioxane (1d), and (2S,3R,5S)-2-(3-acetyl-2-methyl-5-furyl)-3,5-dihydroxy-1,4-dioxane (1e), 5-(3-carbamoyl-3-cyano-3-deoxy-β-d-xylo-pentopyranosyl)-3-ethoxycarbonyl-2-methylfuran (2c), 5-(2,4-di-O-acetyl-3-carbamoyl-3-cyano-3-deoxy-β-d-xylo-pentopyranosyl)-3-methoxycarbonyl-2-methylfuran (2e), and 3-acetyl-5-(2,4-di-O-acetyl-3-carbamoyl-3-cyano-3-deoxy-β-d-xylo-pentopyranosyl)-2-methylfuran (2f), respectively, are formed with (4S,5S)-4-carbamoyl-4-cyano-2-(3-ethoxycarbonyl-2-methyl-5-furyl)-5-hydroxy-5,6-dihydropyran (3a) and (4S,5S)-4-carbamoyl-4-cyano-5-hydroxy-2-(3-methoxycarbonyl-2-methyl-5-furyl)-5,6-dihydropyran (3b) as minor products. The dehydration of 2a,b, 5-(2,4-di-O-acetyl-3-carbamoyl-3-cyano-3-deoxy-β-d-xylo-pentopyranosyl)-3-ethoxycarbonyl-2-methylfuran (2d), 2e, and 2f yields cis-3,5-diacetoxy-4,4-dicyano-tetrahydropyran and -tetrahydrothiopyran (2l,m), and the 5-(2,4-di-O-acetyl-3,3-dicyano-3-deoxy-β-d-erythro-pentopyranosyl) derivatives (2n–p) of 3-ethoxycarbonyl-2-methylfuran, 3-methoxycarbonyl-2-methylfuran, and 3-acetyl-2-methylfuran, respectively.     

o-nitrophenyl 6-deoxy-3-O-(6-deoxy-β-d-xylo-hex-5-enopyranosyl)-β-d-xylo-hex-5-enopyranoside,the major product of β-d-glucosidase action on o-nitrophenyl 6-deoxy-β-d-xylo-hex-5-enopyranoside

Incubation of o-nitrophenyl 6-deoxy-β-d-xylo-hex-5-enopyranoside (1) with emulin β-d-glucosidase gave, instead of the expected 6-deoxy-d-xylo-hexos-5-ulose (3), o-nitrophenyl 6-deoxy-3-O-(6-deoxy-β-d-xylo-hex-5-enopyranosyl)-β-d-xylo-hex-5-enopyranoside (2) in high yield (≈90% under optimal conditions). The structure of 2 was established from spectroscopic data and by correlation with compounds synthesised definitively. The specificity of the transfer reaction is discussed as an argument for an acceptor or aglycon binding-site.     

Radioimmunoassay for biopterin in body fluids and tissues

Specific antibodies against l-erythro-biopterin have been prepared in rabbits using the conjugates to bovine serum albumin. The antiserum against l-erythro-biopterin distinguished among l-erythro-tetrahydro- or 7,8-dihydro-biopterin, the other three stereoisomers of biopterin, d-erythro-neopterin, folic acid, and other synthetic pteridines. Using the specific antiserum against l-erythro-biopterin, a radioimmunoassay has been developed to measure the biopterin concentrations in urine, serum, cerebrospinal fluid, and tissues. The conjugate of l-erythro-biopterin with tyramine, 4-hydroxy-2-[2-(4-hydroxyphenyl)ethylamino]-6-(l-erythro-1,2-dihydroxypropyl)pteridine (BP-TYRA), was synthesized and labeled with ¹²⁵I as the labeled ligand for the radioimmunoassay. BP-¹²⁵I-TYRA had similar binding affinity as the natural l-erythro-biopterin and was thus permitted to establish a highly sensitive radioimmunoassay for biopterin. The limit of sensitivity of the radioimmunoassay with BP-¹²⁵I-TYRA as labeled ligand was 0.5 pmol. The total concentration of biopterins, i.e., biopterin, 7,8-dihydro-, quinonoid dihydro and tetrahydrobiopterins, in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, whereas iodine oxidation under alkaline conditions gave the concentration only of the former two. Biopterin in urine could be measured directly using 1 μl of urine, but a pretreatment with a small Dowex 50-H⁺ column was required for serum, cerebrospinal fluid, and brain tissues.     

γ-Irradiation-induced ring-opening of polycrystalline cycloamylose hydrates

The chemical modifications induced in polycrystalline cycloamylose hydrates during γ-irradiation have been investigated by using g.l.c-m.s. to analyse the monosaccharide mixtures formed on hydrolysis. Unchanged substrate and material retaining the original cyclic structure were removed by precipitation prior to hydrolysis, and the products therefore reflect the effect of the radical-induced opening of the cycloamylose ring structure. The following products were identified: glucose and glucono-1, 5-lactone (1), 4-deoxy-xylo-hexose (2), arabinose (3), ribose (4), 2-deoxy-erythro-pentose (5), 3-deoxy-erythro-hexos-4-ulose (6), xylo-hexos-5-ulose (7), 6-deoxy-xylo-hexos-5-ulose (8), 5-deoxy-xylo-hexodialdose (9), 2,6-dideoxyhexos-5-ulose (10), xylose (11), 5-deoxypentose (12), 3-deoxypentulose (13), erythrose (14), and threose (15). Products 1-9 appear to be terminals of the “anhydroglucose” chain. Established free-radical reactions, typical for carbohydrates. are invoked to account for these products.     

Synthesis of 5-deoxy-d-xylo-hexose and 5-deoxy-l-arabino-hexose,and their conversion into adenine nucleosides

Anti-Markovnikov hydration of the olefinic bond of 5,6-dideoxy-1,2-O-isopropylidene-3-O-p-tolylsulfonyl-α- d-xylo-hex-5-enofuranose (4) and methyl 5,6-dideoxy-2,3-di-O-p-tolylsulfonyl-α-l-arabino-hex-5-enofuranoside (11) by the addition of iodine trifluoroacetate, followed by hydrogenation in the presence of a Raney nickel catalyst in ethanol containing triethylamine, afforded 5-deoxy-1,2-O-ísopropylidene-3-O-p-tolylsulfonyl-α-d-xylo-hexofuranose (6) and methyl 5-deoxy-2,3-di-O-p-tolylsulfonyl-α-d-arabino-hexofuranoside (14), respectively. 5-deoxy-d-xylo-hexose and 5-deoxy-l-arabino-hexose were prepared from 6 and 14, respectively, by photolytic O-detosylation and acid hydrolysis. Syntheses of 9-(5-deoxy-β-d-xylo-hexofuranosyl)-adenine and 9-(5-deoxy-α-l-arabino-hexofuranosyl)adenine are also described. Application of the sodium naphthalene procedure, for O-detosylation, to 11 is reported in connection with an alternative synthetic route to methyl 5-deoxy-α-l-arabino- hexofuranoside.     

Benzeneboronates of acyclic triols

Structures have been assigned to the benzeneboronates of glycerol, DL-butane-1,2,4-triol, L-erythro-butane-1,2,3-triol, and L-arabino-, ribo-, and xylo-pentane-2,3,4-triols. All, except the benzeneboronate of xylo-pentane-2,3,4-triol, are mixtures of isomers. The abundance of the isomers has been related to conformational effects.     

Chiral analysis of methylphenidate and dextromoramide by capillary electrophoresis

Capillary electrophoretic methods have been developed to separate the enantiomers of methylphenidate (MPH) and dextromoramide. For MPH separation was achieved with heptakis (2,6-di-O-methyl)-β-cyclodextrin (DMCD) as chiral selector in a 100 mM phosphoric acid buffer adjusted to pH 3.0 with triethanolamine. Commercial samples of d,l-erytho-MPH HCl and d,l-threo-MPH HCl were analysed using the method. There was no evidence of the presence of d,l-threo-MPH HCl in d,l-erytho-MPH HCl and vice versa. The ratio of the enantiomers was determined for each diastereoisomer. Hydroxypropyl-β-cyclodextrin was the chiral selector of choice for the chiral separation of the enantiomers of moramide. The separation which gave a resolution of about 3.5 was achieved in 4 min using only a 6 cm of length of capillary. In a sample of dextro-R-moramide tartrate only a small quantity (4.9% w/w) of levo-S-moramide was detected with this method.     

Studies on dehydro-l-ascorbic acid 3-oxime 2-phenylhydrazone: Conversion into substituted triazoles

l-threo-2,3-Hexodiulosono-1,4-lactone 3-oxime 2-(phenylhydrazone) (1) gave 2-(p-bromophenyl)-4-(l-threo-1,2,3-trihydroxypropyl)-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone (2), and this gave a diacetyl and a dibenzoyl derivative. On treatment of 2 with liquid ammonia, methylamine, or dimethylamine, the corresponding triazole-5-carboxamides (5–7) were obtained. Periodate oxidation of 5 gave 2-(p-bromophenyl)-4-formyl-1,2,3-triazole-5-carboxamide (10), and, on reduction, 10 gave 2-(p-bromophenyl)-4-(hydroxymethyl)-1,2,3-triazole-5-carboxamide, characterized as its monoacetate. Condensation of 10 with phenylhydrazine gave the triazole hydrazone. Acetonation of 2 gave the isopropylidene derivative. Reaction of 2 with HBr-HOAc gave 4-(l-threo-2-O-acetyl-3-bromo-1,2-dihydroxypropyl)-2-(p-bromophenyl)-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone. Similar treatment of 1 with HBr-HOAc gave 5-O-acetyl-5-bromo-6-deoxy-l-threo-2,3-hexodiulosono-1,4-lactone 3-oxime 2-(phenylhydrazone). This was converted into 4-(l-threo-2-O-acetyl-3-bromo-1,2-dihydroxypropyl)-2-phenyl-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone on treatment with boiling acetic anhydride. On reaction of 1 with benzoyl chloride in pyridine, dehydrative cyclization occurred, with the formation of 4-(l-threo-2,3-dibenzoyloxy-1-hydroxypropyl)-2-phenyl-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone, which was converted into the amide on treatment with ammonia.     

Synthesis of some pyrazole derivatives having l-threo and d-erythro side chains

The mixed bis(arylhydrazones) of l-threo-2,3-hexodiulosono-1,4-lactone rearrange into pyrazolediones. Mono- and bis-(arylhydrazones) of isoascorbic acid were prepared; the latter are present in two forms that afford the same pyrazoledione. Acetylation, benzoylation, and periodate oxidation of these pyrazolediones were studied, and some condensation products from the pyrazole aldehyde were prepared. Some of the i.r. and mass-spectral data were discussed.     

Synthesis of the reported structure of homocereulide and its vacuolation assay

Homocereulide, isolated from marine bacterium Bacillus cereus, is an analog of emetic toxin cereulide. There is no report on its structure determination and involvement in B. cereus-associated food poisoning. Homocereulide is a cyclic dodecadepsipeptide composed of l-O-Val-l-Val-d-O-Leu-d-Ala and l-O-allo-Ile-d-Val-d-O-Leu-d-Ala. Here, we synthesized homocereulide using liquid phase fragment condensation. The NMR spectrum of synthesized homocereulide confirmed the intended structure and LC-MS results were consistent with natural products. Morphological evaluation using HEp-2 cells showed higher toxicity with homocereulide (1.39?nM) than cereulide (3.95?nM). Though cereulide is the main component in broth culture, homocereulide is also likely involved in B. cereus-associated food poisoning.     

Synthesis of l-homophenylalanine with immobilized enzymes

A bi-enzyme process for the synthesis of l-homophenylalanine (l-HPA) from N-carbamoyl-d-homophenylalanine with immobilized N-acylamino acid racemase (racemase) and immobilized N-carbamoyl-l-amino acid amidohydrolase (l-N-carbamoylase) was demonstrated in this study. Upon covalent immobilization on Eupergit C, the operational pH range and temperature range were markedly broadened. The broadening of the range of operation pH bridges the gap between the optimal reaction pHs of the two free enzymes and thus makes possible the utilization of both enzymes in a single reactor. Under optimal conditions, the immobilized racemase and the immobilized l-N-carbamoylase exhibited a specific activity of 0.79 U/mg protein and 2.91 U/mg protein, respectively. The immobilized racemase had a lower activity retention but a significantly higher operation stability compared to the immobilized l-N-carbamoylase. The racemization activity of the immobilized racemase remained essentially unchanged after 40 cycles; the hydrolysis activity of the immobilized l-N-carbamoylase dropped by 40% after 14 cycles. In batch operation, quantitative conversion of N-carbamoyl-d-homophenylalanine to l-HPA with immobilized enzymes was achieved. However, the low stability of the immobilized l-N-carbamoylase complicated the development of repeated-batch or continuous processes. In continuous process, a stoichiometric excess of l-N-carbamoylase was used to extend the operation time of the system. The bi-enzyme process is a promising alternative for the synthesis of l-HPA from racemate of N-carbamoyl-d,l-homophenylalanine.     

Mitigation of Lead-Induced Neurotoxicity by the Naringin: Erythrocytes as Neurons Substitute Markers

This study aimed to investigate the effect of lead (Pb) on neuronal nitric oxide synthase (nNOS) activity using erythrocytes as neurons surrogate markers. Moreover, the protective effect of naringin (NAR) against lead acetate (PbAc)-induced neurotoxicity was investigated. Human erythrocytes were incubated with l-arginine (l-Arg), N ^ω-nitro-l-Arginine methyl ester ( l-NAME), NAR, PbAc, PbAc + l-Arg, PbAc + NAR, or PbAc + l-Arg +NAR. The present results revealed that incubation of erythrocytes with PbAc inhibited NOS activity and decreased nitrite levels as an index for nitric oxide (NO) production to values similar that of l-NAME as known NOS inhibitor. Likewise, PbAc induced a significant decrease in activities of ATPases and acetylcholinesterase compared to control cells. Furthermore, PbAc exposure significantly increased protein carbonyl content (PCC) and malondialdehyde (MDA) levels while significantly decrease the levels of reduced glutathione (GSH). On the contrary, incubation of erythrocytes with PbAc in the presence of l-Arg + NAR synergistically ameliorated the investigated parameters compared to erythrocytes incubated with PbAc alone. These data suggest that NAR can restore NO bioavailability in a situation of Pb-induced cellular damage. This attributed to antioxidant activity and restoration NOS activity.     

Synthesis of 3,4-anhydro-1-deoxy-3-C-methyl-d-hexulose derivatives

Epoxidation of (E)-1,3,4-trideoxy-5,6-O-isopropylidene-3-C-methyl-d-glycero-hex-3-enulose by alkaline hydrogen peroxide gave a mixture of 3,4-anhydro-1-deoxy-5,6O-isopropylidene-3-C-methyl-d-arabino- (2) and -d-xylo-hexulose (3) that was resolved by chromatography. From the reaction of 2 with 3-chloroperbenzoic acid, the Baeyer-Villiger rearrangement product (2R)-2-O-acetyl-2,3-anhydro-1-deoxy-4,5-O-isopropylidene-d-eythro-pentulose hydrate was isolated. The structures and configurations of the above products were established on the basis of chemical transformations and anlytical and spectroscopic data.     

On the ring transformation of hydrazine derivatives of l-ascorbic acid into nitrogen heterocyclic derivatives

l-hreo-2,3-hexodiulosono-1,4-lactone 2-(p-methoxyphenylhydrazone) (1) was condensed with arylhydrazines to give mixed bishydrazones, whose acetylation gave the corresponding di-O-acetyl derivatives. The hydrazone 1 undergoes elimination of one molecule of water per molecule during, the acetylation, and gives 4-(2-acetoxy- ethylidene)-4-hydroxy-2,3-dioxobutano-1,4-lactone 2-(p-methoxyphenylhydrazone), which reacts with methylhydrazine, via a ring transformation process, to give 1-methyl-3-(L-methylpyrazolin-3-yl)-4,5-pyrazoledione 4-(p-methoxyphenylhydrazone). Alkali rearranged the mixed bishydrazones to 1-aryl-3-(l-threo-glycerol-1-yl)-4,5- pyrazoledione 4-(p-methoxyphenylhydrazones), which gave triacetyl and tribenzoyl derivatives, and, upon periodate oxidation, afforded 1-aryl-3-formyl-4,5- pyrazolediones 4-(p-methoxyphenylhydrazones) that gave the corresponding phenylhydrazones. The n.m.r. and mass spectra of some of these derivatives have been investigated.     

Purification and characterization of l-allo-threonine aldolase from Aeromonas jandaei DK-39

l-allo-Threonine aldolase (l-allo-threonine acetaldehyde-lyase), which exhibited specificity for l-allo-threonine but not for l-threonine, was purified from a cell-free extract of Aeromonas jandaei DK-39. The purified enzyme catalyzed the aldol cleavage reaction of l-allo-threonine (K_m=1.45 mM, V_max=45.2 μmol min⁻¹ mg⁻¹). The activity of the enzyme was inhibited by carbonyl reagents, which suggests that pyridoxal-5′-phosphate participates in the enzymatic reaction. The enzyme does not act on either l-serine or l-threonine, and thus it can be distinguished from serine hydroxy-methyltransferase (l-serine:tetrahydrofolate 5,10-hydroxy-methyltransferase, EC 2.1.2.1) or l-threonine aldolase (EC 4.1.2.5).