Facile preparation of deuterium-labeled standards of indole-3-acetic acid (IAA) and its metabolites to quantitatively analyze the disposition of exogenous IAA in Arabidopsis thaliana

[2',2'-(2)H(2)]-indole-3-acetic acid ([2',2'-(2)H(2)]IAA) was prepared in an easy and efficient manner involving base-catalyzed hydrogen/deuterium exchange. 1-O-([2',2'-(2)H(2)]-indole-3-acetyl)-beta-D-glucopyranose, [2',2'-(2)H(2)]-2-oxoindole-3-acetic acid, and 1-O-([2',2'-(2)H(2)]-2-oxoindole-3-acetyl)-beta-D-glucopyranose were also successfully synthesized from deuterated IAA, and effectively utilized as internal standards in the quantitative analysis of IAA and its metabolites in Arabidopsis thaliana by using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The use of this technique shows that these metabolites were accumulated in the roots of Arabidopsis seedlings. Dynamic changes in the metabolites of IAA were observed in response to exogenous IAA, revealing that each metabolic action was regulated differently to contribute to the IAA homeostasis in Arabidopsis.     

Cooked Odor of Euphausia pacifica Hansen

Quinoxaline and benzimidazole derivatives obtained from L-rhamnose and L-fucose under deoxygenated, weakly acidic, heated conditions were studied using GLC, HPLC, and NMR.

Four quinoxalines and one benzimidazole were obtained from L-rhamnose (RHA-I, II, III, III′, and IV) and L-fucose (FUA-I, II, III, IV, and V) in an acidic solution (MeOH-AcOH-H₂I = 8 : 1 : 2) at 80°C. The total yield of the products as sugar was about 80% from either rhamnose or fucose.

The structure of RHA-I was (2′S)-2-methyl-3-(2′-hydroxypropyl)quinoxaline; RHA-II, (2′R,3′S)-2-(2′,3′-dihydroxybutyl)quinoxaline; RHA-III, (1′S,2′S,3′S)-2-(1′2′3′-trihydroxybutyl)quinoxaline[2-(L-arabino-1′,2′,3′-trihydroxybutyl)quinoxaline]; RHA-III′, 2-(L-ribo-1′,2′,3′-trihydroxybutyl)quinoxaline; and RHA-IV, 2-(L-manno-1′,2′,3′,4′-tetrahydroxypentyl)-benzimidazole, and the structure of FUA-I was the same as RHA-I; FUA-II, (2′S, 3′S)-2-(2′, 3′-dihydroxybutyl)quinoxaline; FUA-III, (1′R, 2′R, 3′S)-2-(1′,2′,3′-trihydroxybutyl)quinoxaline [2-(L-xylo-1′,2′,3′-trihydroxybutyl)quinoxaline; FUA-IV, 2-(L-lyxo-1′,2′,3′-trihydroxybutyl)-quinoxaline; and FUA-V, 2-(L-galacto-1′,2′,3′,4′-tetrahydroxypentyl)benzimidazole. These results suggest no significant difference for the pathways of quinoxaline and benzimidazole formation between L-rhamnose and L-fucose. Possible pathways are proposed for each sugar.     

EFFICIENT METHODS FOR THE SYNTHESIS OF [2-15N]GUANOSINE AND 2′-DEOXY[2-15N]GUANOSINE DERIVATIVES

The nucleophilic addition–elimination reaction of 2′,3′,5′-tri-O-acetyl-2-fluoro-O ⁶-[2-(4-nitrophenyl)ethyl]inosine (8) with [¹⁵N]benzylamine in the presence of triethylamine afforded the N ²-benzyl[2-¹⁵N]guanosine derivative (13) in a high yield, which was further converted into the N ²-benzoyl[2-¹⁵N] guanosine derivative by treatment with ruthenium trichloride and tetrabutyl-ammonium periodate. A similar sequence of reactions of 2′,3′,5′-tri-O-acetyl-2-fluoro-O ⁶-[2-(methylthio)ethyl]inosine (9) and the 6-chloro-2-fluoro-9-(β-D-ribofuranosyl)-9H-purine derivative (11), which were respectively prepared from guanosine, with potassium [¹⁵N]phthalimide afforded the N ²-phthaloyl [2-¹⁵N]guanosine derivative (15; 62%) and 9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-6-chloro-2-[¹⁵N]phthalimido-9H-purine (17; 64%), respectively. Compounds 15 and 17 were then efficiently converted into 2′,3′,5′-tri-O-acetyl[2-¹⁵N]guanosine. The corresponding 2′-deoxy derivatives (16 and 18) were also synthesized through similar procedures.     

Syntheses of Some Derivatives of Glycosyl Pantothenic Acids,Analogues of Growth Factor for MLF Bacteria

A growth factor (TJF) for a malo-lactic fermentation bacterium (Leuconostoc sp.) has been found to be 4′-O-(β-D-glucopyranosyl)-D-pantothenic acid with structural and synthetical studies. Now other 4′-O-glycosides (β-D-ribofuranosyl, α-D-glucopyranosyl, β-D-galacto-pyranosyl, β-maltosyl and β-cellobiosyl) and 2′,4′-O-di-β-D-glucopyranoside of DL-pantothenic acid, and 4′-O-β-D-glucopyranoside of DL-pantethine were synthesized to examine their biological activities. The improved syntheses of TJF were also examined.     

Screening of the regioselectivity of acetyl xylan esterase from Bacillus pumilus as a catalyst for the deacetylation of glycoside acetates

Regioselective deacetylations of nine glycosides catalyzed by acetyl xylan esterase from Bacillus pumilus have been studied. The glycosides were methyl and benzyl glycosides of the tetraacetates of α-D-glucopyranose, α-D-galactopyranose and α-D-mannopyranose, and the methyl glycosides of tetra-O-acetyl-β-D-glucopyranose, tetra-O-acetyl-β-D-galactopyranose and tetra-O-acetyl-α-D-glucopyranose. The kinetics of successive deacetylations was monitored by GLC and 21 sugar acetates have been identified.     

Antitumor Activities and Immunochemical Properties of the Cell-wall Polysaccharides from Aureobasidium pullulans

Delipidated cell walls from Aureobasidium pullulans were fractionated systematically.

The cell surface heteropolysaccharide contains D-mannose, D-galactose, D-glucose, and D-glucuronic acid (ratio, 8.5:3.9:1.0:1.0). It consists of a backbone of (1→6)-α-linked D-mannose residues, some of which are substituted at O-3 with single or β-(1→6)-linked D-galactofuranosyl side chains, some terminated with a D-glucuronic acid residue, and also with single residues of D-glucopyranose, D-galactopyranose, and D-mannopyranose.

This glucurono-gluco-galactomannan interacted with antiserum against Elsinoe leucospila, which also reacted with its galactomannan, indicating that both polysaccharides contain a common epitope, i.e., at least terminal β-galactofuranosyl groups and also possibly internal β-(1→6)-linked galactofuranose residues.

It was further separated by DEAE-Sephacel column chromatography to gluco-galactomannan and glucurono-gluco-galactomannan.

The alkali-extracted β-D-glucan was purified by DEAE-cellulose chromatography to afford two antitumor-active (1→3)-β-D-glucans. One of the glucans (M_r, 1–2 × 10⁵) was a O-6-branched (1→3)-β-D-glucan with a single β-D-glucosyl residue, d.b., 1/7, and the other (M_r, 3.5–4.5 × 10⁵) had similar branched structure, but having d.b., 1/5. Side chains of both glucans contain small proportions of β-(1→6)-and β-(1→4)-D-glucosidic linkages.     

Enzyme-Substrate Complex of p-Hydroxybenzoate Hydroxylase; One of the Activated Intermediates of the Overall Reaction

The transglucosidation reaction of brewer’s yeast α-glucosidase was examined under the co-existence of l-sorbose and phenyl-α-glucoside. As the transglucosidation products, three kinds of new disaccharide were chromatographically isolated. It was presumed that these disaccharides consisting of d-glucose and l-sorbose were 1-O-α-d-glucopyranosyl-l-sorbose ([α]_D+89.0), 3-O-α-d-glucopyranosyl-l-sorbose ([α]_D+69.1) and 4-O-α-d-glucopyranosyl-l-sorbose ([α]_D+81.0). The principal product formed in the enzyme reaction was 1-O-α-d-glucopyranosyl-l-sorbose.     

Synthesis and Physicochemical Properties of 2′-Deoxy- 2′,2″-difluoro-β-D-ribofuranosyl and 2′-Deoxy-2′,2″- difluoro-α-D-ribofuranosyl Oligonucleotides

Abstract

We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (T _m) and structural analysis (CD spectra) of 2′-deoxy-2′,2″-difluoro-α-D-ribofuranosyl and 2′-deoxy-2′,2″-difluoro-β-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.     

Riboside and Ribotide of 5(or 3)-Aminopyrazole-4-carboxamide

During the investigation for dephosphorylation of 4-hydroxy-1-β-D-ribofuranosylpyrazolo-[3,4-d] pyrimidine 5′-phosphate, it was found that the compound was converted to an unknown substance by alkaline hydrolysis for 3 hr at 140°C. The structure of the substance was assigned to be 5-amino-1-β-D-ribofuranosylpyrazole-4-carboxamide 5′-phosphate. 5(or3)-Amino- pyrazole-4-carboxamide and its riboside were also obtained from 4-hydroxypyrazolo [3,4-d] pyrimidine and its riboside, respectively, under the similar conditions.

5-Amino-1-β-D-ribofuranosyipyrazole-4-carboxamide and 5-amino-1-β-D-ribofuranosyl- pyrazole-4-carboxamide 5′-phosphate are new compounds.     

Production of 2-Phenylethanol in Roses as the Dominant Floral Scent Compound from L-Phenylalanine by Two Key Enzymes,a PLP-Dependent Decarboxylase and a Phenylacetaldehyde Reductase

We investigated the biosynthetic pathway for 2-phenylethanol, the dominant floral scent compound in roses, using enzyme assays. L-[²H₈] Phenylalanine was converted to [²H₈] phenylacetaldehyde and [²H₈]-2-phenylethanol by two enzymes derived from the flower petals of R. ‘Hoh-Jun,’ these being identified as pyridoxal-5′-phosphate-dependent L-aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). The activity of rose petal AADC to yield phenylacetaldehyde was nine times higher toward L-phenylalanine than toward its D-isomer, and this conversion was not inhibited by iproniazid, a specific inhibitor of monoamine oxidase. Under aerobic conditions, rose petal AADC stoichiometrically produced NH₃ together with phenylacetaldehyde during the course of decarboxylation and oxidation, followed by the hydrolysis of L-phenylalanine. Phenylacetaldehyde was subsequently converted to 2-phenylethanol by the action of PAR. PAR showed specificity toward several volatile aldehydes.     

Glycosidase-catalyzed Deoxy Oligosaccharide Synthesis. Practical Synthesis of Monodeoxy Analogs of Ethyl β-Thioisomaltoside Using Aspergillus niger α-Glucosidase

Enzymatic transglycosylation using four possible monodeoxy analogs of p-nitrophenyl α-D-glucopyranoside (Glcα-O-pNP), modified at the C-2, C-3, C-4, and C-6 positions (2D-, 3D-, 4D-, and 6D-Glcα-O-pNP, respectively), as glycosyl donors and six equivalents of ethyl β-D-thioglucopyranoside (Glcβ-S-Et) as a glycosyl acceptor, to yield the monodeoxy derivatives of glucooligosaccharides were done. The reaction was catalyzed using purified Aspergillus niger α-glucosidase in a mixture of 50 mM sodium acetate buffer (pH 4.0)/CH₃CN (1: 1 v/v) at 37°C. High activity of the enzyme was observed in the reaction between 2D-Glcα-O-pNP and Glcβ-S-Et to afford the monodeoxy analogs of ethyl β-thiomaltoside and ethyl β-thioisomaltoside that contain a 2-deoxy α-D-glucopyranose moiety at their glycon portions, namely ethyl 2-deoxy-α-D-arabino-hexopyranosyl-(1,4)-β-D-thioglucopyranoside and ethyl 2-deoxy-α-D-arabino-hexopyranosyl-(1,6)-β-D-thioglucopyranoside, in 6.72% and 46.6% isolated yields (based on 2D-Glcα-O-pNP), respectively. Moreover, from 3D-Glcα-O-pNP and Glcβ-S-Et, the enzyme also catalyzed the synthesis of the 3-deoxy analog of ethyl β-thioisomaltoside that was modified at the glycon α-D-glucopyranose moiety, namely ethyl 3-deoxy-α-D-ribo-hexopyranosyl-(1,6)-β-D-thioglucopyranoside, in 23.0% isolated yield (based on 3D-Glcα-O-pNP). Products were not obtained from the enzymatic reactions between 4D- or 6D-Glcα-O-pNP and Glcβ-S-Et.     

Purification and Characterization of an Intracellular α-D-Xylosidase II from Aspergillus flavus MO-5

α-D-Xylosidase II activity from Aspergillus flavus MO-5 was increased roughly 5- to 10-fold by use of xylose instead of methyl α-D-xylopyranoside (α-MX) as a carbon source.

The enzyme was purified to an electrophoretically pure state by successive chromatography on Q-Sepharose, Phenyl Superose, PL-SAX, and TSK-gel G3000SWXL. The purified enzyme hydrolyzed isoprimeverose [α-D-xylopyranosyl-(1→6)-D-glucopyranose] and p-nitrophenyl α-D-xylopyranoside (α-p-NPX), but not α-MX or xyloglucan oligosaccharide. The apparent K_m and V_max of the enzyme for α-p-NPX and isoprimeverose were 0.97 mM and 28.0 µmol/min/mg protein, and 47.62 mM and 2.0 µmol/min/mg protein, respectively. This enzyme had an apparent molecular weight of 67,000 by SDS-polyacrylamide gel electrophoresis and 180,000 by gel filtration chromatography (TSK-gel G3000SWXL).

The enzyme showed the highest activity at pH 6.0 and 40°C, and was stable in the pH range from 6.0 to 7.0 and at the temperatures up to 40°C. The activity was inhibited by Cu²⁺, Zn²⁺, Hg²⁺, p-CMB, SDS, Fe³⁺, and N-ethylmaleimide.

This enzyme had nothing in common with α-D-xylosidase I and four α-D-xylosidases reported already.     

Levoglucosan Dehydrogenase Involved in the Assimilation of Levoglucosan in Arthrobacter sp. I-552

A levoglucosan (1,6-anhydro-β-D-glucopyranose)-using bacterium, isolated from soil, was identified. It was shown to belong to the genus Arthrobacter and tentatively named Arthrobacter sp. I-552. A novel enzyme catalyzed the dehydrogenation of levoglucosan to form 1,6-anhydro-β-D-ribo-hexopyranos-3-ulose (3-keto levoglucosan), using NAD⁺ as an electron acceptor, i.e. NAD⁺: 1,6-anhydro-β-D-glucopyranose oxidoreductase (trivial name: levoglucosan dehydrogenase). This enzyme was purified and characterized. A possible reaction scheme for the glucose formation was proposed. This pathway for levoglucosan use is distinct from those in yeast and fungi.     

Oligonucleotides Containing Disaccharide Nucleosides: Synthesis,Physicochemical, and Substrate Properties

Abstract

The efficient synthesis of oligonucleotides containing 2′-O-β-D-ribofuranosyl (and β-D-ribopyranosyl)nucleosides, 2′-O-α-D-arabinofuranosyl (and α-L-arabinofuranosyl)nucleosides, 2′-O-β-D-erythrofuranosylnucleosides, and 2′-O-(5′-amino-5-deoxy-β-D-ribofuranosyl)nucleosides have been developed.     

EFFICIENT SYNTHESIS OF D-[1′-13C]-RIBONUCLEOSIDES FOR INCORPORATION INTO OLIGO-RNA

Syntheses of the monomer building blocks used for the solid-phase synthesis of specifically 1′-¹³C-labeled oligoribonucleotides from the D-[1-¹³C]ribose is presented. Procedure has been used for the selective formation of 2′-O-silylated ribonucleosides. After 5′-O-dimethoxytritylation, the synthesis of D-[1′-¹³C] ribonucleoside phosphoramidites has been achieved.     

Synthesis of β-D-Fructopyranosyl-(2→6)-D-glucopyranose from D-Glucose and D-Fructose by a Thermal Treatment

The synthesis is reported of β-D-fructopyranosyl-(2→6)-D-glucopyranose that had previously been isolated from a fermented plant extract as a new saccharide. A disaccharide was predominately formed from an equal amount of D-glucose and D-fructose under melting conditions at 140 °C for 60 to 90 min. This saccharide was isolated from the reaction mixture by carbon-Celite column chromatography and preparative HPLC, and was confirmed to be β-D-fructopyranosyl-(2→6)-D-glucopyranose by TOF-MS and NMR analyses.     

Syntheses of a Neobiosamine C Derivative and Its Analogs

Methyl 2,5-di-O-p-nitrobenzoyl-β-d-ribofuranoside was prepared via methyl 2,3-O-ethoxyethylidene-β-d-ribofuranoside from d-ribose. It was condensed with 3,4,6-tri-O-acetyl-2-deoxy-2-(2′,4′-dinitroanilino)-α-d-glucopyranosyl bromide and 3,4-di-O-acetyl-2,6-dideoxy-2-(2′,4′-dinitroanilino)-6-phthalimido-α-d-glucopyranosyl bromide by a modified Königs-Knorr reaction to give neobiosamine analogs. The condensation reaction gave α-glucosides as the minor product, and the corresponding β-glucoside as the major product.     

Glycolipids Isolated from Spirulina maxima: Structure and Fatty Acid Composition

Several glycolipids were isolated from Spirulina maxima, an edible blue-green algae, by systematic fractionation with different solvents. Structural investigation by using methylation, GC-MS, and enzymic techniques indicated that the major glycolipids are O-β-d-galactosyl-(1→l′)-2′, 3′-di-O-acyl-d-glycerol, O-α-d-galactosyl-(l-→6)-O-β-d-galactosyl-(1→l′)-2′,3′-di-O-acyl-d-glycerol and 6-sulfo-O-α-quinovosyl-(l→l′)-2′, 3′-di-O-acyl-d-glycerol. Main fatty acid components of these glycolipids were identified as palmitic acid and linoleic or linolenic acid. Based on-these fatty acid compositions, Spirulina glycolipids were compared with those in higher plants.     

Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80°C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70°C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and α-D-glucose-1-phosphate. The K _m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k _cat was 5.4 s^-1. In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-β-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s^-1) k _cat followed by 6-deoxy-D-glucose (17 s^-1) and 2-deoxy-D-glucose (16 s^-1). The natural substrate, D-glucose with the k _cat of 8.0 s^-1 had the highest (1.1×10⁴ M^-1 s^-1) k _cat/K _m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, α-D-glucose-1-phosphate, at higher concentrations.     

Preparation of [1-13C]D-Glucose 6-Phosphate from [13C]Methanol and D-Ribose 5-Phosphate with Methylotrophic Enzymes

[¹³C]Formaldehyde was selectively incorporated into the C-1 position of D-fructose 6-phosphate by condensation with D-ribulose 5-phosphate catalyzed by a partially purified enzyme system for formaldehyde fixation in Methylomonas aminofaciens 77a. Much of the [1-¹³C]D-fructose 6-phosphate produced in this reaction was converted to [1-¹³C]D-glucose 6-phosphate by the addition of glucose-6-phosphate isomerase. A fed-batch reaction with periodic additions of the substrates afforded 56.2 g/liter D-glucose 6-phosphate and 26.8g/liter D-fructose 6-phosphate. When [¹³C]methanol was used as the C₁-donor, the yield of [1-¹³C]D-glucose 6-phosphate was high when alcohol oxidase was added. The optimum conditions for sugar phosphate production in the fed-batch reaction gave 45.6g/liter [1-¹³C]D-glucose 6-phosphate and 16.4g/liter [1-¹³C]D-fructose 6-phosphate in 165min. The molar yield of the total sugar phosphates to methanol added was 95%. The addition of H₂O₂ and catalase to the reaction system supplied molecular oxygen for methanol oxidation to formaldehyde by alcohol oxidase.