Photosynthetic preparation and characterization of 13C-labeled carbohydrates in agmenellum quadruplicatum

The blue-green alga Agmenellum quadruplicatum (strain PR6) has been used to prepare photobiosynthetically ¹³C-labeled d-glucose, 2-O-(α-d-glucopyranosyl)-glyceric acid (glucosylglycerate), 2-hydroxy-1-(hydroxymethyl)ethyl α-d-gluco-pyranoside (glucosylglycerol), and α-d-glueopyranosyl β-d-fructofuranoside (sucrose). When grown to a cell density of 4.4 g.L^-1 (dry weight) under nitrate-nitrogen limiting growth conditions for 120 h, the algal cells contained 38% of the dry-cell weight as(1 → 4)-α-d-glucan (amylose). About 1% of the dry-cell weight was glucosylglycerol, glucosylglycerate, and sucrose. Glutamate was obtained, together with carbohydrates of low molecular weight, when the cells were extracted with chloroform-methanol; d-glucose was recovered from the extracted cells by acid hydrolysis of the starch. The algae were grown by using 20 mol% [¹³C] carbon dioxide for preparation of labeled carbohydrates and for cellular component identification by whole-cell n.m.r. spectroscopy.     

3′,6′-anhydro-6-O-corynomycoloyl trehalose: Synthesis and identification as the impurity in synthetic cord factor preparations

Acylation of 2,3,4,2′,3′,4′-hexa-O-benzyl-6,6′-di-O-methanesulphonyl-α-α-trehalose (1) with a reduced amount of potassium corynomycolate yielded a mixture which consisted mainly of 2,3,4,2′,3′,4′-hexa-O-benzyl-6-O-corynomycoloyl-6′-O-methanesulphonyl-α,α-trehalose (2). Catalytic hydrogenolysis of 2 gave the mono-mesylate 4 which was converted into 3′,6′-anhydro-6-O-corynomycoloyl-α,α-trehalose (5) but treatment with sodium hydride. The structure of 5 was studied by mass-spectroscopy. Compound 5 was found to be identical with the byproduct obtained in the acylation of 6,6′-di-O-p-toluenesulphonyl-α,α-trehalose with potassium corynomycolate.     

The synthesis of 6-deoxy-6-fluoro-α,α-trehalose and related analogues

Selective acid-catalysed methanolysis of 2,3,2′,3′-tetra-O-benzyl-4,6:4′,6′-di-O-benzylidene-α,α-trehalose yielded the monobenzylidene derivative, which was converted into the 4,6-dimesylate. Selective nucleophilic displacement of the primary sulphonyloxy group then gave 2,3-di-O-benzyl-6-deoxy-6-fluoro-4-O-mesyl-α-d-glucopyranosyl 2,3-di-O-benzyl-4,6-O-benzylidene-α-d-glucopyranoside. Removal of the protecting groups then yielded 6-deoxy-6-fluoro-α,α-trehalose. In addition, 6-deoxy-6-fluoro-4-O-mesyl-α,α-trehalose and a derivative of 4-chloro-4,6-dideoxy-6-fluoro-α-d-galactopyranosyl α-d-glucopyranoside were also prepared from the same substrate. Iodide displacement of 2,3-di-O-benzyl-4,6-di-O-mesyl-α-d-glucopyranosyl 2,3-di-O-benzyl-4,6-di-O-mesyl-α-d-glucopyranoside afforded the 6-iodide and 6,6′-di-iodide in yields of 31 and 36%, respectively. Similarly, the 6-azide and 6,6′-diazide were isolated in yields of 17 and 21%, respectively.     

Stereochemistry of the hydrolysis of α,α-trehalose by trehalase,determined by using a labelled substrate

(1,1′-¹³C)α,α-Trehalose was obtained in 37% yield from the Pavia condensation of 2,3,4,6-tetra-O-benzyl-d-(1-¹³C)glucopyranose, in dichloromethane in the presence of trifluoromethanesulfonic anhydride, followed by the usual deprotection techniques. The hydrolysis of this substrate by cockchafer trehalase was monitored at 37° by using ¹³C-n.m.r. spectroscopy with short recording times. Equimolecular amounts of α- and β-d-glucopyranose are released simultaneously by the action of the enzyme. This result is consistent with a bimolecular substitution mechanism, taking into account previous results involving C-2 asymmetric participation in the catalytic step of hydrolysis of α,α-trehalose. For comparative evaluation of its accuracy, the usual polarimetric technique was also used for the determination of the anomeric configuration of the d-glucose released by the action of the enzyme on α,α-trehalose.     

Design of new enzyme stabilizers inspired by glycosides of hyperthermophilic microorganisms

In response to stressful conditions like supra-optimal salinity in the growth medium or temperature, many microorganisms accumulate low-molecular-mass organic compounds known as compatible solutes. In contrast with mesophiles that accumulate neutral or zwitterionic compounds, the solutes of hyperthermophiles are typically negatively charged. (2R)-2-(α-d-Mannopyranosyl)glycerate (herein abbreviated as mannosylglycerate) is one of the most widespread solutes among thermophilic and hyperthermophilic prokaryotes. In this work, several molecules chemically related to mannosylglycerate were synthesized, namely (2S)-2-(1-O-α-d-mannopyranosyl)propionate, 2-(1-O-α-d-mannopyranosyl)acetate, (2R)-2-(1-O-α-d-glucopyranosyl)glycerate and 1-O-(2-glyceryl)-α-d-mannopyranoside. The effectiveness of the newly synthesized compounds for the protection of model enzymes against heat-induced denaturation, aggregation and inactivation was evaluated, using differential scanning calorimetry, light scattering and measurements of residual activity. For comparison, the protection induced by natural compatible solutes, either neutral (e.g., trehalose, glycerol, ectoine) or negatively charged (di-myo-inositol-1,3′-phosphate and diglycerol phosphate), was assessed. Phosphate, sulfate, acetate and KCl were also included in the assays to rank the solutes and new compounds in the Hofmeister series. The data demonstrate the superiority of charged organic solutes as thermo-stabilizers of enzymes and strongly support the view that the extent of protein stabilization rendered by those solutes depends clearly on the specific solute/enzyme examined. The relevance of these findings to our knowledge on the mode of action of charged solutes is discussed.     

Melongosides n,o and p: steroidal saponins from seeds of solanum melongena

Three new saponins, melongosides N, O and P, have been isolated from the methanolic extract of seeds of Solanum melongena and their structures elucidated. Melongoside N is 3-O-[β-D-glucopyranosy l-(1 → 2)-β-D-glucopyranosyl]-26-O-(β-D-glucopyranosyl)-(25R)-5α-furostan-3β,22 α,26-triol, whereas melongoside O is 3-O-[β-D-glucopyranosyl-(1 → 2)β-D-glucopyranosyl]- 26-O-(β-D-glucopyranosyl)-(25R)-furost-5-en-3β,22α,26-triol and melongoside P is 3-O- [β-D-glucopyranosyl-(1 → 2)]-[α-L-rhamnopyranosyl-(1 → 3)]-β-D-glucopyranosyl)-26-O- (β-D-glucopyranosyl)-(25 R)-5α-furostan-3β,22α,26-triol.     

A Simple Transductional Method for Construction of Adenylate- cyclase or cAMP Receptor Protein-deficient Mutants of Escherichia coli K-12

The substrate specificity of α-d-xylosidase from Bacillus sp. No. 693–1 was further investigated. The enzyme hydrolyzed α-1,2-, α-1,3-, and α-1,4-xylobioses. It also acted on some heterooligosaccharides such as O-α-d-xylopyranosyl-(1→6)-d-glucopyranose, O-α-d-xylopyranosyl-(1→6)-O-β-d-glucopyranosyl-(1→4)-d-glucopyranose, O-α- d-xylopyranosyl-(1→6)-O-d-glucopyranosyl-(1→4)-O-[α-d-xylopyranosyl-(1→6)]-d-glucopyranose, and O-α-d-xylopyranosyl-(1→3)-l-arabinopyranose. The enzyme was unable to hydrolyze tamarinde polysaccharides although it could hydrolyze low molecular weight substrates with similar linkages.     

The synthesis of 3-amino-3-deoxy-α-d-glucopyranosyl α-d-glucopyranoside (3-amino-3-deoxy-α,α-trehalose

The preparation of 2,3-di-O-benzoyl-4,6-O-benzylidene-α-d-glucopyranosyl-2-O-benzoyl-4,6-O-benzylidene-α-d-ribo-hexopyranosid-3-ulose (3) from 4,6:4′,6′-di-O-benzylidene-α,α-trehalose (1) via the 2,3,2′-tribenzoate 2 has been improved. Reduction of 3 with sodium borohydride gave 2-O-benzoyl-4,6-O-benzylidene-α-d-allopyranosyl 2,3-di-O-benzoyl-4,6-O-benzylidene-α-d-glucopyranoside (4), which was converted into the methanesulfonate 5 and trifluoromethanesulfonate 6. Displacement of the sulfonic ester group in 6 with lithium azide was very facile and afforded a high yield of 3-azido-2-O-benzoyl-4,6-O-benzylidene-3-deoxy-α-d-glucopyranosyl 2,3-di-O-benzoyl-4,6-O-benzylidene-α-d-glycopyranoside (7), whereas similar displacement in 5 proceeded sluggishly, giving a lower yield of 7 together with an unsaturated disaccharide (8). The azido sugar 7 was converted by conventional reactions into the analogous 2,3,2′-triacetate 9, the corresponding 2,3,2′-triol 10, and deprotected 3-azido-3-deoxy-α-d-glucopyranosyl α-d-glucopyranoside (11). Hydrogenation of 11 over Adams' catalyst furnished crystalline 3-amino-3-deoxy-α,α-trehalose hydrochloride (12), the overall yield from 3 being 35%.     

Identifizierung von selaginose und deren verbreitung in der gattung Selaginella

A new trisaccharide selaginose was isolated from Selaginella kraussiana and identified as 2-O-α-d-Glucopyranosyl-α,α-trehalose. This compound occurs only in two distinct groups of species of the subgenus Heterophyllum.     

Studies on the stereoselective synthesis of a protected α-d-Gal-(1→2)-d-Glc fragment

A novel 1,2-cis stereoselective synthesis of protected α-d-Gal-(1→2)-d-Glc fragments was developed. Methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-α-d-glucopyranoside (13), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-d-glucopyranoside (15), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-β-d-glucopyranoside (17), and methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-β-d-glucopyranoside (19) were favorably obtained by coupling a new donor, isopropyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-1-thio-β-d-galactopyranoside (2), with acceptors, methyl 3-O-benzoyl-4,6-O-benzylidene-α-d-glucopyranoside (4), methyl 3,4,6-tri-O-benzoyl-α-d-glucopyranoside (5), methyl 3-O-benzoyl-4,6-O-benzylidene-β-d-glucopyranoside (8), and methyl 3,4,6-tri-O-benzoyl-β-d-glucopyranoside (12), respectively. By virtue of the concerted 1,2-cis α-directing action induced by the 3-O-allyl and 4,6-O-benzylidene groups in donor 2 with a C-2 acetyl group capable of neighboring-group participation, the couplings were achieved with a high degree of α selectivity. In particular, higher α/β stereoselective galactosylation (5.0:1.0) was noted in the case of the coupling of donor 2 with acceptor 12 having a β-CH₃ at C-1 and benzoyl groups at C-4 and C-6.     

Systematic Synthesis of Sulfur-containing p-Nitrophenyl α-Maltopentaoside Derivatives for a Differential Assay of Human α-Amylases

For use in a differential assay of human α-amylases, a variety of 6⁵-S-substituted p-nitrophenyl α-maltopentaoside derivatives (6-54) were systematically synthesized via the key intermediate, p-nitrophenyl O-(2,3-di-O-acetyl-6-S-acetyl-4-O-benzoyl-6-thio-α-D-glucopyranosyl)-(1 →4)-tris[O-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1→4)]-2,3,6-tri-O-acetyl-α-D-glucopyranoside (4), which was easily prepared from p-nitrophenyl α-maltopentaoside (G5P) in four steps. The sulfoxide and sulfone derivatives were prepared by oxidizing the corresponding sulfides with m-chloroperbenzoic acid.     

Physicochemical and biological properties of 2-O-α-d-galactosyl-cyclomaltohexaose (α-cyclodexterin) and -cyclomaltoheptaose (β-cyclodextrin)

The physicochemical and biological properties of the new branched cyclomaltooligosaccharides (cyclodextrins; CDs), 2-O-α-d-galactosyl-cyclomaltohexaose (2-O-α-d-galactosyl-α-cyclodextrin, 2-Gal-αCD) and 2-O-α-d-galactosyl-cyclomaltoheptaose (2-O-α-d-galactosyl-β-cyclodextrin, 2-Gal-βCD), were investigated. The formation of inclusion complexes of 2-Gal-CDs with various kinds of guest compounds (clofibrate, cholesterol, cholecalciferol, digitoxin, digitoxigenin, and prostaglandin A₁) was examined by a solubility method, and the results were compared with those of non-branched CDs and other 6-O-glycosyl-CDs such as 6-O-α-d-galactosyl-CDs, 6-O-α-d-glucosyl-CDs, and 6-O-α-maltosyl-CDs. The inclusion abilities of 2-Gal-αCD for clofibrate and prostaglandin A₁, and 2-Gal-βCD for clofibrate, cholecalciferol, cholesterol, and digitoxigenin were markedly weaker than those of non-branched CD and other 6-O-glycosyl-CDs in each series, probably because of a steric hindrance caused by the α-(1→2)-galactoside linkage. The hemolytic activities of 2-Gal-CDs on human erythrocytes were the lowest among each CD series, and the compounds showed negligible cytotoxicity towards Caco-2 cells up to at least 100 mM.     

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.     

Synthesis of potassium (2R)-2-O-α-d-glucopyranosyl-(1→6)-α-d-glucopyranosyl-2,3-dihydroxypropanoate a natural compatible solute

Ethyl 6-O-acetyl-2,3,4-tribenzyl-1-thio-d-glucopyranoside, as a mixture of anomers, was employed for the stereoselective synthesis of the potassium salt of (2R)-2-O-α-d-glucopyranosyl-(1→6)-α-d-glucopyranosyl-2,3-dihydroxypropanoic acid (α-d-glucosyl-(1→6)-α-d-glucosyl-(1→2)-d-glyceric acid, GGG), a recently isolated compatible solute. The α-anomer was by far the major product of both glycosylation reactions using NIS/TfOH as activator.     

Isolation of tyrosinase and melanogenesis inhibitory flavonoids from Juniperus chinensis fruits

ABSTRACT

A new biflavonoid, amentoflavone-7-O-β-D-glucoside, and thirteen known flavonoids were isolated from the fruits of Juniperus chinensis using a bioactivity-guided method and their tyrosinase inhibitory effects were tested using a mushroom tyrosinase bioassay. Two isolates, hypolaetin-7-O-β-D-glucoside and quercetin-7-O-α-L-rhamnoside, were found to reduce tyrosinase activity at a concentration of 50 μM. Quercetin-7-O-α-L-rhamnoside attenuated cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells. Molecular docking simulation revealed that quercetin-7-O-α-L-rhamnoside inhibits tyrosinase activity by hydrogen bonding with residues His85, His244, Thr261, and Gly281 of tyrosinase.

Abbreviations: EtOH, ethanol; CH2Cl2, dichloromethane; EtOAc, ethylacetate; n-BuOH, n-butanol; MeOH, metanol; CHCl3,chloroform; DMSO, dimethylsulfoxide; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; α-MSH, α-melanocyte stimulating hormone; L-DOPA, L-3, 4-dihydroxyphenylalanine     

A New Enzyme “Nitrile Hydratase” which Degrades Acetonitrile in Combination with Amidase

The stereoselective synthesis of the 1-O-α-d-ghicopyranosides and 1-O-α-d-cellobiosides of 3-deoxy-2(R)- and 2(S)-glycerols to determine the complete stereochemistry of rhynchosporoside, which is a host selective phytotoxin from Rhynchosporium secalis, is described in detail.     

Improved Targeting of the Flanks of a DNA Stem Using α-Oligodeoxynucleotides.-The Enhanced Effect of an Intercalator

Abstract

2′-Deoxy-5′-0-(4,4′-dimethoxytrityl)-5-methyl-N ⁴-(1-pyrenylmethyl)-α-cytidine (5) was prepared by reaction of 1-pyrenylmethylamine with an appropriate protected 4-(l,2,4-triazolyl)-α-thymidine derivative 3 which was synthesized from 5-O-DMT protected α-thymidine 1. Aminolysis of 3 afforded 3′-O-acetyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (8). Benzoylation of 8 and removal of acetyl afforded N ⁴-benzoyl-2-deoxy-5–0-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (10). The amidites of compounds 5and 10 were prepared and used in α-oligonucleotide synthesis. DNA three-way junction (TWJ) is stabilized when an α-ODN is used for targeting the dangling flanks of the stem in a DNA hairpin. Further stabilization of the TWJ is observed when 5 is inserted into the α-ODN at the junction region.

     

Practical Synthesis of the Disaccharide Epitope,D-Galactopyranosyl-α-1,3-D- galactopyranose,by using 1,2;5,6-Di-O-cyclohexylidene-α-D-galactofuranose as the Glycosyl Acceptor

D-Galactosyl-α-1,3-D-galactopyranose (1) was chemically prepared in a good yield by coupling phenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactopyranoside (5) or 2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl bromide (8) with 1,2:5,6-di-O-cyclohexylidene-α-D-galactofuranose (3) with subsequent de-O-benzylation and de-O-cyclohexylidenation of the resulting protected α-1,3-disaccharide.     

Synthesis of stereoisomers of antithrombotic nipecotamides

The stereoisomers of α,α′-bis[3-(N,N-diethylcarbamoyl)-piperidino]-p-xylene ( 1 ) were synthesized. Rac ethyl nipecotate was resolved by diastereomeric (-)-D - and (+)-L-tartrate salt formation. The enantiomeric esters were hydrolyzed to the corresponding nipecotic acids, which were then converted into t-BOC derivatives. Treatment of the latter with diethylamine/isobutyl chloroformate and removal of the t-BOC protecting group afforded (R)- and (S)-N,N-diethylnipecotamides. Condensation of the latter with α,α′-dibromo-p-xylene gave (R,R)- and (S,S)- 1 . The meso-diastereomer was obtained by stereospecific synthesis in addition to our earlier procedure involving fractional crystallization of the diastereomeric mixture obtained by synthesis. The latter was resolved earlier into 1A , 1B , and 1C using chiral high-performance liquid chromatography (HPLC). Based on the stereospecific synthesis now achieved, 1A and 1B are assigned the configurations, (R,R) and (S,S) respectively, and 1C is assigned the meso configuration. The (R,S) structure of the latter is also confirmed by X-ray crystallography. © 1995 Wiley-Liss, Inc.     

Dilignol glycosides from needles of Picea abies

(2R,3R)-2 3-Dihydro-2-(4′-hydroxy-3′-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-5-benzofuranpropanol 4′-O-β-d-glucopyranoside [dihydrodehydrodiconiferyl alcohol glucoside], (2R,3R)-2 3-dihydro-7-hydroxy-2-(4′-hydroxy-3′-methoxyphenyl)-3-(hydroxymethyl)-5-benzofuranpropanol 4′-O-β-d-glucopyranoside and 4′-O-α-l-rhamnopyranoside, 1-(4′-hydroxy-3′-methoxyphenyl)-2- [2″-hydroxy-4″-(3-hydroxypropyl)phenoxy]-1, 3-propanediol 1-O-β-d-glucopyranoside and 4′-O-β-d-xylopyranoside, 2,3-bis[(4′-hydroxy-3′-methoxyphenyl)-methyl]-1,4-butanediol 1-O-β-d-glucopyranoside [(?)-seco-isolariciresinol glucoside] and (1R,2S,3S)-1,2,3,4-tetrahydro-7-hydroxy-1-(4′-hydroxy-3′-methoxyphenyl)-6-methoxy-2 3-naphthalenedimethanol α²-O-β-d-xylopyranoside [(?)-isolariciresinol xyloside] have been isolated from needles of Picea abies and identified.