The chemical synthesis of polysaccharides : Part III. Synthesis of 6-O-α-D-mannopyranosyl-D-mannose and 4-O-α-D-mannopyranosyl-D-mannose. Isolation of A (1→6)-linked D-mannan

When 1,2,3,4-tetra-O-acetyl-α-D-mannopyranose was fused with a catalytic amount of toluene-p-sulphonic acid, 6-O-α-D-mannopyranosyl-D-mannose and 4-O-α-D-mannopyranosyl-D-mannose were isolated after deacetylation of the reaction mixture. No β-D-linked disaccharide was detected in the reaction mixture. When the corresponding β-D-tetra-acetate was fused with zinc chloride as catalyst, higher oligomers were formed, and a D-mannan was isolated and shown to be mainly an α-(1→6)-linked polymer having d.p. of 10. With 5% of zinc chloride, the α-D-tetra-acetate showed oligosaccharide formation, and yielded a smaller proportion of a (1→6)-linked D-mannan.     

C-methylation of sucrose: synthesis of 6- and 6′-C-methylsucrose

2,3,4,6,1′,3′,4′-Hepta-O-benzylsucrose, obtained by acid-catalysed hydrolysis of the 6′-O-trityl derivative, was oxidised with the Pfitzner-Moffatt reagent and the product was alkylated with methylmagnesium iodide. Removal of the protecting groups then gave a mixture of diastereomers, namely 7-deoxy-β-d-altro and -α-l-galacto-hept-2-ulofuranosyl α-d-glucopyranoside. Application of this reaction sequence to 2,3,4,1′,3′,4′,6′-hepta-O-benzylsucrose afforded β-d-fructo-furanosyl 7-deoxy-dl-glycero-α-d-gluco-heptopyranoside.     

Biosynthesis,characterisation and direct high-performance liquid chromatographic analysis of gemfibrozil 1-O-β-acylglucuronide

Gemfibrozil 1-O-β-acylglucuronide was purified from the urine of a volunteer administered gemfibrozil, and an isocratic reversed-phase HPLC method was developed for its direct measurement. Quantitation of gemfibrozil and gemfibrozil 1-O-β-acylglucuronide was carried out from plasma, following extraction from acidified specimens into ethyl acetate, on a 5-μm CN reversed-phase column with a mobile phase (pH 3.5) containing acetonitrile, tetrabutylammonium sulphate and distilled water, using fluorescence detection at 284 nm excitation and 316 nm emission. Calibration curves were linear for both compounds over a concentration range of 0.1 to 40 mg/l, with intra-assay coefficients of variation <5% at concentrations of 20.0, 2.0 and 0.2 mg/l, and inter-assay coefficients of variation <10%. No degradation of gemfibrozil 1-O-β-acylglucuronide was detected as a result of the analytical procedure. However, a preliminary application of the method indicates that gemfibrozil acylglucuronide is chemically unstable undergoing intra-molecular rearrangement and hydrolysis under physiological conditions.     

Parallel synthesis and nucleic acid binding properties of C(6')-α-functionalized bicyclo-DNA

Two novel bicyclo-T nucleosides carrying a hydroxyl or a carboxymethyl substituent in C(6')-α-position were prepared and incorporated into oligodeoxynucleotides. During oligonucleotide deprotection the carboxymethyl substituent was converted into different amide substituents in a parallel way. T(m)-measurements showed no dramatic differences in both, thermal affinity and mismatch discrimination, compared to unmodified oligonucleotides. The post-synthetic modification of the carboxymethyl substituent allows in principle for a parallel preparation of a library of oligonucleotides carrying diverse substituents at C(6'). In addition, functional groups can be placed into unique positions in a DNA double helix.     

Isolation and structural analyses of positional isomers of 61,6m-di-O-α-d-mannopyranosyl-cyclomaltooctaose (m=2–5) and 6-O-α-(n-O-α-d-mannopyranosyl)-α-d-mannopyranosyl-cyclomaltooctaose (n=2, 3, 4, and 6)

Eight positional isomers of 6¹,6^m-di-O-α-d-mannopyranosyl-cyclomaltooctaose (γCD) (m=2–5) and 6-O-α-(n-O-α-d-mannopyranosyl)-d-mannopyranosyl-γCD (n=2, 3, 4, and 6) in a mixture of products from γCD and d-mannose by condensation reaction of α-mannosidase from jack bean were isolated by HPLC. The structures of four isomers of 6-O-α-(n-O-α-d-mannopyranosyl)-d-mannopyranosyl-γCD were elucidated by NMR spectroscopy. On the other hand, four positional isomers of 6¹,6^m-di-O-α-d-mannopyranosyl-γCD were determined by LC–MS analysis of degree of polymerization of the branched oligosaccharides produced by enzymatic degradation with bacterial saccharifying α-amylase (BSA), and combination of BSA and glucoamylase. Similarly cyclomaltodextrin glucanotransferase also digested these isomers.     

Expression of the Arabidopsis G-protein GPα1: purification and characterisation of the recombinant protein

The Arabidopsis G subunit, GP1, was expressedwithin Escherichia coli by co-transformation with the expressionvector and the dnaY gene which encodes tRNA^Arg _AGA/AGG. Isolation of the recombinant GP1 in a highly pureform could be achieved by a combination of anion exchange and dyeaffinity chromatography or by a single step affinity procedure viachromatography on 4-amino-anilido-GTP agarose. The recombinant proteinyielded by both procedures was highly active and bound GTPS withan apparent K_d in the nM range. GTPS binding wasstimulated two-fold in the presence of Zn²⁺ compared with that inthe presence of Mg²⁺, Mn²⁺ or Ca²⁺.Abbreviations: 4aaGTP, 4-amino-anilido-GTP; GTPS,guanosine- 5-(3-O-thiotriphosphate), PMSF,phenylmethylsulphonyl fluoride; PVDF, polvinylidene fluoride;rGP1, recombinant GP1     

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 A(1)) 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 A(1), 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 100mM.     

Conformational analysis of methyl 6-O-[(R)- and (S)-1-carboxyethyl]-α-D-galactopyranoside by MM and Langevin dynamics simulations

The conformational space of methyl 6-O-[(R)- and (S)- 1-carboxyethyl]-α-D-galactopyranoside has been investigated. A grid search employing energy minimization at each grid point over the three major degrees of freedom, namely φ, ψ and ω, identified low energy regions. The R-isomer shows five low energy conformers within ca. 1 kcal mol−1 of the global energy minimum. The S-isomer has two conformers within a few tenths of a kcal mol−1 of the global energy minimum. Langevin dynamics simulations have been have been performed at 300 K for 30 ns of each isomer. The φ dihedral angle has as its major conformer (g−1) for the R-isomer whereas it is the (g+) conformer for the S-isomer. For the ψ dihedral angle the (t) conformer has the highest population for both isomers. The dihedral angle ω has the (g+) conformer most highly populated, both for the R- and S-isomer. The above five and two conformational states for the R- and S-isomers, respectively, make up 90% in each case of the populated states during the Langevin dynamics (LD) simulations. Rate constants for the ω dihedral angle have been calculated based on a number correlation function. Three bond homo- and heteronuclear, i.e. proton and carbon-13, coupling constants have been calculated from the dynamics trajectories for comparison to experimental values. The heteronuclear coupling constant H2′,C6 has been measured for the S-isomer and found to be 3.3 Hz. The J value calculated from the LD simulations, namely 2.6 Hz, is in fair agreement with experiment. A comparison to the X-ray structure of the R-isomer shows that the conformation of the crystalline compound occupies the low energy region most highly populated as a single R-conformer (30%) during the LD simulations. This revised version was published online in August 2006 with corrections to the Cover Date.     

Total synthesis of apigenin-4'-yl 2-O-(p-coumaroyl)-β-D-glucopyranoside

The first total synthesis of apigenin-4′-yl 2-O-(p-coumaroyl)-β-d-glucopyranoside, which exhibits good inhibitory activity against xanthine oxidase (XO), was accomplished in seven steps from a 1,2-blocked sugar unit and natural apigenin. A unique allyl protecting group, a phase-transfer-catalyzed (PTC) regioselective coupling reaction, and robustness in large-scale preparation are the merits of this synthetic strategy.     

Synthesis of 7-(β-D-Glucopyranosyl)-6-Thiotheophylline and 7-(α-D-Arabinopyranosyl)-6-Thiotheopwlline. Conformational Study of the Peracetyl Derivatives.

Abstract

The synthesis of two 7-glycosyl-6-thiotheophylline nucleosides where the sugar moieties are β-D-glucose (1b) and α-D-arabinose (2b) is reported. The syn-anti equilibrium of the peracetyl derivatives was studied by the line-shape and the ¹H-NMR nOe methods, and molecular mechanics analysis.     

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.     

Crystal structure and conformation of 3-O-(2,3-anhydro-4-deoxy-α-l-lyxo-hexopyranosyl)-1,2:5,6-di-O-isopropylidene-α-d-glucofuranose

A single crystal of 3-O-(2,3-anhydro-4-deoxy-α-l-lyxo-hexopyranosyl)-1,2:5,6-di-O-isopropylidene-α-d-glucofuranose (1), obtained from its 6-acetate, has been investigated by X-ray diffraction methods. Compound 1 crystallises in the orthorhombic system, space group P2₁2₁2P₁, with cell dimensions a = 8.556(1), b = 12.303(1), and c = 18.397(1) Å. An almost ideal half-chair conformation ⁵H_o was found for the 2,3-anhydropyranose moiety of 1.     

Isolation and molecular characterisation of the gene encoding eburicol 14α-demethylase (CYP51) fromPenicillium italicum

TheCYP51 gene encoding eburicol 14α-demethylase (P450_14DM) was cloned from a genomic library of the filamentous fungal plant pathogenPenicillium italicum, by heterologous hybridisation with the corresponding gene encoding lanosterol 14α-demethylase from the yeastCandida tropicalis. The nucleotide sequence of a 1739-bp genomic fragment and the corresponding cDNA clone comprises an open reading frame (ORF) of 1545 bp, encoding a protein of 515 amino acids with a predicted molecular mass of 57.3 kDa. The ORF is interrupted by three introns of 60, 72 and 62 bp. The C-terminal part of the protein includes a characteristic haem-binding domain, HR2, common to all P450 genes. The deducedP. italicum P450_14DM protein and the P450_14DM proteins fromCandida albicans, C. tropicalis andSaccharomyces cerevisiae share 47.2, 47.0 and 45.8% amino acid sequence identity. Therefore, the cloned gene is classified as a member of theCYP51 family. Multiple copies of a genomic DNA fragment ofP. italicum containing the cloned P450 gene were introduced intoAspergillus niger by transformation. Transformants were significantly less sensitive to fungicides which inhibit P450_14DM activity, indicating that the cloned gene encodes a functional eburicol 14α-demethylase.