Biosynthesis of alkane-2, 3-diols: chemical synthesis of 3-hydroxy-[3-14C]octadecane-2-one and its reduction to [3-14C]octadecane-2, 3-diol in the uropygial glands of ring-necked pheasants (Phasianus colchicus).

Acyloin has been proposed to be an intermediate in the biosynthesis of long chain alkane-2,3-diols. In order to test this possibility, specifically labeled 3-hydroxyoctadecane-2-one (acyloin) was synthesized by coupling 2-methyl-1,3-dithiane with [1-¹⁴C]hexadecanal followed by cleaving of the thioketal. Injection of the synthetic 3-hydroxy [3-¹⁴C]octadecane-2-one into the uropygial gland of the ring-necked pheasant resulted in the formation of labeled octadecane-2,3-diol. Chemical degradation of this diol showed that all of the ¹⁴C was contained in C-3 of the diol showing direct conversion of acyloin to the diol. These observations support the hypothesis that alkane-2,3-diols might be biosynthesized by reduction of the acyloin derived from a condensation between hydroxyethyl thiamine pyrophosphate and fatty aldehyde. Gas-liquid chromatographic analysis of the alkane-2,3-diols, as their isopropylidene derivatives, of the pheasant strongly suggests that they are of the erythro-configuration; however, alkane-2,3-diol enzymatically formed from the racemic acyloin injected into the gland contained 59.5% erythro- and 40.5% threo-diastereoisomers. This distribution was identical to that produced by chemical reduction of the synthetic racemic acyloin. These results clearly show that the reduction step does not show a preference for either of the enantiomers of the acyloin and that the stereospecificity in diol biosynthesis probably resides in the condensation step.     

Evaluation of the efficiency of an assay procedure for gangliosides in human serum

An efficiency assessment of a ganglioside assay procedure was carried out on human serum gangliosides from healthy subjects of different sex and age. The analysis of the gangliosides, extracted with chloroform/methanol and purified by lipid partitioning, ion exchange column chromatographic separation and desalting procedures as described by Sennet al. (1989)Eur J Biochem 181: 657–62, was performed by HPTLC followed by densitometric quantification. The yield of the procedure, expressed as radioactivity recovery, was determined by adding GM3 ganglioside, tritium labelled at the sialic acid acetyl group and at the C3 position of sphingosine, to the lyophilized serum or by associating it with the serum lipoproteins. In spite of the fact that the extraction and purification procedures were performed exactly as described we found the radioactivity recovery to be variable (25–50%) and much lower than that proposed. Much of the radioactivity was found in the organic phase after lipid partitioning, whilst all the ganglioside purification steps led to some further loss. After the introduction of some modifications to the procedure the recovery improved, reaching 67–79%.The analyses on 33 samples of 5 ml showed a human serum ganglioside content of about 10 nmol ml^–1 (as corrected for the recovery), and confirmed that GM3 ganglioside is the main component of the total serum ganglioside mixture. Abbreviations: Ganglioside nomenclature is in accordance with Svennerholm (1980) [37] and the IUPAC-IUB Recommendations (1977, 1982) [38]. GM3, II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer; Cer, ceramide; Neu5Ac,N-acetyl-neuraminic acid;erythro-GM3, GM3 containingerythro-sphingosine;threo-GM3, GM3 containingthreo-sphingosine;erythro-C18 sphingosine, (2s,3R,4E)-2-amino-1,3-dihydroxy-octadecene;erythro-C20 sphingosine, (2S,3R,4E)-2-amino-1,3-dihydroxy-eicosene;threo-C18 sphingosine, (2S,3S,4E)-2-amino-1,3-dihydroxy-octadecene;threo-C20 sphingosine, (2S,3S,4E)-2-amino-1,3-dihydroxy-eicosene; DDQ, dichlorodicyano-benzoquinone.     

Enzymatic epimerization of d-erythro-dihydroneopterin triphosphate to l-threo-dihydroneopterin triphosphate

An enzyme has been discovered in Escherichia coli that catalyzes the conversion of the triphosphate ester of 2-amino-4-hydroxy-6-(d-erythro-1′,2′,3′-trihydroxypropyl)-7,8-dihydropteridine, (i.e. d-erythro-dihydroneopterin triphosphate) to an epimer of this compound, l-threo-dihydroneopterin triphophate. The enzyme, which is here named “d-erythro-dihydroneopterin triphosphate 2′-epimerase,” needs a divalent cation (Mg²⁺ or Mn²⁺ is most effective) for maximal activity. Its molecular weight is estimated at 87 000–89 000. Little or no activity can be detected if either the monophosphate or the phosphate-free form of the substrate is incubated with the enzyme. Evidence is presented to establish that all three phosphate residues of the substrate are retained in the product and that the product is of the l-threo configuration.     

Erythro-γ-hydroxyhomo-L-arginine: An amino acid from seed of Lonchocarpus costaricensis, and its preferential interaction with borate

A new non-protein amino acid, erythro-γ-hydroxyhomo-L-arginine has been isolated from seed of Lonchocarpus costaricensis by exploiting its property of interacting with borate ions. For structural comparisons, threo-γ-hydroxyhomo-L-arginine was isolated from seed of Lathyrus tingitanus and erythro-γ-hydroxyarginine from Vicia unijuga by novel procedures. The reasons for the interaction of borate with the erythro- but not the threo-forms of these amino acids are discussed.     

Oxidation of the erythro and threo forms of the phenolic lignin model compound 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol by laccases and model oxidants

Mixtures of equal amounts of the erythro and threo forms of the phenolic arylglycerol β-aryl ether 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol were oxidized (i) with laccases from Trametes versicolor, Agaricus bisporus, Myceliophthora thermophila and Rhus vernicifera, (ii) with laccase-mediator systems consisting of T. versicolor laccase and ABTS or HBT, and (iii) with various model oxidants including cerium(IV) ammonium nitrate (CAN), lignin peroxidase, Fenton’s reagent, and lead(IV) tetraacetate (LTA). All the laccases exhibited a similar preferential degradation of the threo form. The mediator ABTS counteracted the threo preference of laccase, but the mediator HBT did not affect it. The outer-sphere model oxidants CAN and lignin peroxidase showed a preferential degradation of the threo form. LTA and Fenton’s reagent did not exhibit any stereo-preference. The results suggest that laccases of different origin, primary structure, and redox potential behave as typical outer-sphere oxidants in their interaction with the diastereomers of the arylglycerol β-aryl ether.     

Studies on dehydro- -ascorbic acid 2-arylhydrazone 3-oximes: Conversion into substituted triazoles and isoxazolines

-threo-2,3-Hexodiulosono-1,4-lactone 2-(arylhydrazones) (2) were prepared by condensation of dehydro- -ascorbic acid with various arylhydrazines. Reaction of 2 with hydroxylamine gave the 2-(arylhydrazone) 3-oximes (3). On boiling with acetic anhydride, 3 gave 2-aryl-4-(2,3-di-O-acetyl- -threo-glycerol-l-yl)-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones (4). On treatment of 4 with liquid ammonia, 2-aryl-4-( -threo-glycerol-l-yl)-1,2,3-triazole-5-carboxamides (5) were obtained. Acetylation of 5 with acetic anhydride-pyridine gave the triacetates, and vigorous acetylation with boiling acetic anhydride gave the tetraacetyl derivatives. Periodate oxidation of 5 gave the 2-aryl-4-formyl-1,2,3-triazole-5-carboxamides (8), and, on reduction, 8 gave the 2-aryl-4-(hydroxymethyl)-1,2,3-triazole-5-carboxamides, characterized as the monoacetates and diacetates. Controlled reaction of 2 with sodium hydroxide, followed by neutralization, gave 3-( -threo-glycerol-l-yl)-4,5-isoxazolinedione 4-(arylhydrazones), characterized by their triacetates. Reaction of 2 with HBr-HOAc gave 5-O-acetyl-6-bromo-6-deoxy- -threo-2,3-hexodiulosono-1,4-lactone 2-(arylhydrazones); these were converted into 4-(2-O-acetyl-3-bromo-3-deoxy- -threo-glycerol-l-yl)-2-aryl-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones on treatment with acetic anhydride-pyridine.     

Biosynthesis of alkane-2,3-diols: enzymatic reduction of 3-hydroxyoctadecane-2-one to octadecane-2,3-diol by a NADPH (B-side) specific microsomal reductase from the uropygial glands of ring-necked pheasants (Phasianus colchicus).

Cell-free preparations from the uropygial gland of ring-necked pheasant catalyzed the reduction of a synthetic R,S-mixture of 3-hydroxyl[3-¹⁴C]octadecane-2-one (acyloin) to a mixture of threo- and erythro-[3-¹⁴C]octadecane-2,3-diol, the final step in the postulated pathway for the biosynthesis of alkane-2,3-diols. The product of enzymatic reduction was identified by Chromatographic techniques and chemical degradation studies. The acyloin reductase showed a pH optimum near 4.0 and specificity for NADPH. With stereospecifically labeled [³H]NADPH, it was shown that acyloin reductase preferentially transferred hydride from the B-side of the nicotinamide ring to the acyloin. A typical Michaelis-Menten substrate saturation was observed for the acyloin and an apparent K_m of 70 μm was calculated from linear double reciprocal plots. Acyloin reductase was inhibited by thioldirected reagents such as p-chloromercuribenzoate and N-ethylmaleimide. Subcellular fractionation of the gland homogenates using sucrose density gradient centrifugation showed that acyloin reductase activity coincided with NADPH:cytochrome c reductase activity, strongly suggesting that acyloin reductase is localized in the microsomal membranes.     

Enzymatic epimerization of d-erythro-dihydroneopterin triphosphate to l-threo-dihydroneopterin triphosphate

An enzyme has been discovered in Escherichia coli that catalyzes the conversion of the triphosphate ester of 2-amino-4-hydroxy-6-(d-erythro-1′,2′,3′-trihydroxypropyl)-7,8-dihydropteridine, (i.e. d-erythro-dihydroneopterin triphosphate) to an epimer of this compound, l-threo-dihydroneopterin triphophate. The enzyme, which is here named “d-erythro-dihydroneopterin triphosphate 2′-epimerase,” needs a divalent cation (Mg²⁺ or Mn²⁺ is most effective) for maximal activity. Its molecular weight is estimated at 87 000–89 000. Little or no activity can be detected if either the monophosphate or the phosphate-free form of the substrate is incubated with the enzyme. Evidence is presented to establish that all three phosphate residues of the substrate are retained in the product and that the product is of the l-threo configuration.     

A new route to l-threo-3-[4-(methylthio)phenylserine], a key intermediate for the synthesis of antibiotics: recombinant low-specificity d-threonine aldolase-catalyzed stereospecific resolution

A new enzymatic resolution process was established for the production of l-threo-3-[4-(methylthio)phenylserine] (MTPS), an intermediate for synthesis of antibiotics, florfenicol and thiamphenicol, using the recombinant low-specificity d-threonine aldolase from Arthrobacter sp. DK-38. Chemically synthesized dl-threo-MTPS was efficiently resolved with either the purified enzyme or the intact recombinant Escherichiacoli cells overproducing the enzyme. Under the optimized experimental conditions, 100 mM (22.8 g l⁻¹) l-threo-MTPS was obtained from 200 mM (45.5 g l⁻¹) dl-threo-MTPS, with a molar yield of 50% and a 99.6% enantiomeric excess. Received: 2 September 1998 / Received revision: 27 October 1998 / Accepted: 29 November 1998     

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-Dicarboxycyclopropyl)glycine Binding in Rat Brain

Abstract: [(2S,2′R,3′R)-2-(2′,3′-[³H]Dicarboxycyclopropyl)glycine ([³H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K_D value of 180 ± 33 nM and a B_max of 780 ± 70 fmol/mg of protein. The nonspecific binding, measured using 100 µM LY354740, was <30%. NMDA, AMPA, kainate, l (?)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [³H]DCG IV binding up to 1 mM. However, several compounds inhibited [³H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1′S,2′S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1′S,2′S)-2-methyl-2-(2-carboxycyclopropyl)glycine > l -glutamate = ibotenate > quisqualate > (RS)-α-methyl-4-phosphonophenylglycine = l (+)-2-amino-3-phosphonopropionic acid > (S)-α-methyl-4-carboxyphenylglycine > (2S)-α-ethylglutamic acid > l (+)-2-amino-4-phosphonobutyric acid. N-Acetyl-l -aspartyl-l -glutamic acid inhibited the binding in a biphasic manner with an IC₅₀ of 0.2 µM for the high-affinity component. The binding was also affected by GTPγS, reducing agents, and CdCl₂. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPγS show that [³H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.     

Inhibition of Clostridium butyricum by 1,3-propanediol and diols during glycerol fermentation

1,3-Propanediol inhibition during glycerol fermentation to 1,3-propanediol by Clostridium butyricum CNCM 1211 has been studied. The initial concentration of the 1,3-propanediol affected the growth of the bacterium more than the glycerol fermentation. μ _max was inversely proportional to the initial concentration of 1,3-propanediol (0–65 g l⁻¹). For glycerol at 20 g l⁻¹, the growth and fermentation were completely stopped at an initial 1,3-propanediol concentration of 65 g l⁻¹. However, for an initial 1,3-propanediol concentration of 50 g l⁻¹ and glycerol at 70 g l⁻¹, the final concentration (initial and produced) of 1,3-propanediol reached 83.7 g l⁻¹(1.1 M), with complete consumption of the glycerol. Therefore, during the fermentation, the strain tolerated a 1,3-propanediol concentration higher than the initial inhibitory concentration (65 g l⁻¹). The addition of 1,2-propanediol or 2,3-butanediol (50 g l⁻¹) in the presence of glycerol (50–100 g l⁻¹), showed that 2-diols reduced the μ _max in a similar way to 1,3-propanediol. The measurement of the osmotic pressure of glycerol solutions, diols and diol/glycerol mixtures did not indicate any differences between these compounds. The hypothesis of diol inhibition was discussed. Taking into account the strain tolerance of highly concentrated 1,3-propanediol during fermentation, the fermentation processes for optimising production were considered. Received: 15 November 1999 / Revision received: 1 February 2000 / Accepted: 4 February 2000     

Synthesis and Biological Activity of 1,2-Dithiolanes and 1,2-Dithianes Bearing a Nitrogen-containing Substituent

A series of 1,2-dithiolanes, 1,2-dithianes and related compounds bearing a nitrogen-containing substituent were synthesized and their pesticidal activity was tested. A new general synthetic route to 1,2-dithiolanes was established from 1,3-diols. A variation in the position and character of the nitrogen atom is shown to be allowable to some extent for promoting insecticidal activity, unlike the case of sulfur atoms. Most compounds showed acaricidal activity, the strongest being displayed by cis-3,5-bis(dimethylaminomethyl)-1,2-dithiolane.     

l-threo- and l-erythro-2-amino-3-hydroxyhex-4-ynoic acids: New amino acids from Tricholomopsis rutilans

2-Amino-3-hydroxyhex-4-ynoic acid, reported previously from Tricholomopsis rutilans, was shown to be a mixture of its threo- and erythro-forms. They were separated from each other and characterized by elementary analysis, optical rotation, TLC, IR, NMR spectra, catalytic hydrogenation, and by chemical synthesis. Their configurations were determined by the comparison of their hydrogenation products with known threo- and erythro-2-amino-3-hydroxyhexanoic acids.     

Synthesis of All Four Possible Stereoisomers of 1-Phenyl-2,3-butanediol and Both Enantiomers of 3-Hydroxy-4-phenyl-2-butanone to Determine the Absolute Configuration of the Natural Constituents

Enantioselective syntheses of all the possible stereoisomers of 1-phenyl-2,3-butanediol (erythro isomer 1 and threo isomer 2) and of 3-hydroxy-4-phenyl-2-butanone 3, the odor components of wisteria flowers, was accomplished via Sharpless asymmetric epoxydation. The absolute configurations of 1–3 were determined by an HPLC analysis of the corresponding MTPA esters of synthetic samples.     

Adaptive Use of N2-Dimethyl-Substituted Pterins by Cultures of Crithidia fasciculata1

The compound 6-(L-erythro-1,2′,3′-trihydroxypropyl)pterin, at a concentration of 50 pg/ml (“L-erythro-neopteria”), supports half-maximal growth of Crithidia fasciculata; biopterin at a concentration of 30 pg/ml is shown to yield similar growth. N²-dimethyl-6-(L-erythro-1′,2′,3′-trihydroxypropyl)pterin (A) was inactive even at 100 ng/ml. Synergism was observed with the N²-dimethylamino derivative (A) in the presence of suboptimal biopterin, its activity then being of the order of L-erythro-neopterin. In contrast, the stereoisomeric N²-dimethyl-6-(D-erythro-1′,2′,3′-trihydroxypropyl)pterin (“dimethyl-D-erythro-neopterin”) and its 3′-mono-phosphate only slightly enhanced growth under similar conditions but both threo-isomers had no supplementary activity. Biopterin-induced growth was slowed by 6-(D-erythro1′,2′,3′-trihydroxypropyl)pterin (D-neopterin); the threo-isomers had no such effect. An adaptive demethylation capacity by growing cultures and competition of biopterin uptake by D-neopterin seems likely. The report of the occurrence in Euglena of N₂-dimethyl-6-(L-threo-1′,2′,3′-trihydroxypropyl)pterin and its 3′-mono-phosphate adds further interest to our observations.     

Isolation and Characterization of a Cyclic Phosphate of Neopterin and Other 6-Alkylpterin Compounds Enzymatically Synthesized from GTP by Cell-free Extracts of Serratia indica

A cell-free system for the biosynthesis of l-threo-neopterin, a growth factor for protozoan, Crithidia fasciculata from guanosine-5′-triphosphate (GTP) was obtained from extracts of Serratia indica IFO 3759. This preparation catalyzed the production of a specific pteridine from GTP, which was isolated and characterized as a cyclic phosphate of neopterin (cNP). Among the other products, l-threo-neopterin, as the Crithidia factor, 6-hydroxymethylpterin, and erythro-neopterin were tentatively identified. Requirements for the synthesis of these products include GTP, Mg²⁺, and disodium phosphate. Fluorescence formation was inhibited by purine nucleotides.

When a disodium phosphate was included in the reaction system, cNP and erythro-neopterin were effectively synthesized from GTP. On the other hand, when the phosphate was omitted 6-hydroxymethylpterin was formed.

The possible biosynthetic process of l-threo-neopterin was discussed.     

Evaluation of the Kinetics of Hydrolysis of Monoamino Analogues of 2′- or 3′-Deoxyadenosine and of 9-(2-Deoxy-β-D-Threo-pentofuranosyl)Adenine or 9-(3-Deoxy-β-D-threo-pentofuranosyl)Adenine by Liquid Chromatography

Abstract

Liquid chromatography was used to follow the degradation of monoamino analogues of 2′- or 3′-deoxyadenosine and of 9-(2-deoxy-β-D-threo-pentofuranosyl) adenine or 9-(3-deoxy-β-D-threo-pentofuranosyl) adenine in buffers of different pH and constant ionic strength (μ). Comparison of stabilities of some of the compounds under study with those of corresponding hydroxyl analogues showed that at acid pH the aminated compounds are more stable than the corresponding hydroxyl compounds. The higher stability associated with the presence of an amino group in the sugar is explained in function of pK_a values, which were determined by ¹³C NMR.     

Formation of an Unexpected 2-Deoxy-α-D-Threo-Pentofuranosyl Azide by Reaction of O2,3′-Anhydro-5′-O-trityl-2′-deoxycytidine with Lithium Azide

Abstract

Reaction of 0²,3′-anhydro-5′-0-trityl-2′-deoxycytidine (1) with LiN₃s in DMF resulted in the formation of 1-(3-azido-2,3-dideoxy-5-0-trityl-β-D-erythro-pentofuranosyl) cytosine (2) and 3-0-(4-amino-1,3-pyrimidin-2-yl)-5-0-trityl-2-deoxy-α-D-threo-pentofuranosyl azide (3) (2:3 = 1:1) in 88% yield. Compound 3 was deprotected with 80% aqueous AcOH yielding 4     

The separation and synthesis of lipidic 1,2- and 1,3-diols from natural phenolic lipids for the complexation and recovery of boron

A study has been made of the semi-synthesis of 1,3-diols (anacardic alcohols) from natural phenolic lipid resources from Anacardium occidentale and Anacardium giganteum which have given C15 and C11 derivatives, respectively. An isomeric 1,3-diol (isoanacardic alcohol) has been obtained from cardanol separated from technical cashew nut-shell liquid. Homologous 1,3-diols have been synthesised from a range of synthetic 2-alkyl-, 3-alkyl- and 4-alkylphenols and from 6-alkylsalicylic acids. The natural 1,2-diol, urushiol, from Rhus vernicifera has been purified. All these lipidic compounds have been studied for their complexation and the potential recovery of boron as boric acid.