Chemical synthesis of (1 → 2)-α-D-mannopyranan

The polymerization of 1,2-anhydro-3,4,6-tri-O-benzyl-β-D -mannopyranose proceeds in the presence of Lewis acids, cationic coordination catalysts, and strong bases. Debenzylation of the products yields oligomeric saccharides or low polymers. Polymerization in toluene by means of potassium alkoxide complexed with crown ethers leads to essentially stereoregular (1 → 2)-α-D -mannopyranan. The original derivatives have been characterized by optical rotation, viscosity, molecular weight, gel permeation chromatography, and spectrometry. The free polysaccharides have been characterized by optical rotation, molecular weight, and ¹H- and ¹³C-nmr spectrometry and compared to yeast mannan hydrolysate oligomers.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-β- -galactopyranoside and methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-O-β- -galactopyranosyl-(1→6)-β- -galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α- -galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β- -galactopyranoside (4) gave a fully acetylated (1→6)-β- -galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α- -galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β- -galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β- -galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

Sequential synthesis and C-N.m.r. spectra of methyl β-glycosides of (1→4)-β-d-xylo-oligosaccharides

The reaction of 2,3-di-O-acetyl-4-O-benzyl-α,β-d-xylopyranosyl bromide (2) with methyl 2,3-di-O-acetyl-β-d-xylopyranoside gave methyl O-(2,3-di-O-acetyl-4-O-benzyl-β-d-xylopyranosyl)-(1→4)-2,3-di-O-acetyl-β-d-xylopyranoside (22). Catalytic hydrogenolysis of 22 exposed HO-4′ which was then condensed with 2. This sequence of reactions was repeated three more times to afford, after complete removal of protecting groups, a homologous series of methyl β-glycosides of (1→4)-β-d-xylo-oligosaccharides. ¹³C-N.m.r. spectra of the synthetic methyl β-glycosides (di- to hexa-saccharide) are presented together with data for six other, variously substituted, homologous series of (1→4)-d-xylo-oligosaccharides.     

Isolation and characterization of a (1 → 3)-β-glucan endohydrolase from germinating barley (Hordeum vulgare): amino acid sequence similarity with barley (1 → 3, 1 → 4)-β-glucanases

A (1 → 3)-β-glucan 3-glucanohydrolase (EC 3.2.1.39) has been purified approx. 190-fold from extracts of germinating barley. The enzyme has an apparent M_r 32 000, a pI of 8.6, and a pH optimum of 5.6. Analysis of hydrolysis products released from the (1 → 3)-β-glucan, laminarin, shows that the enzyme is an endohydrolase. Sequence analysis of the 46 NH₂-terminal amino acids of the (1 → 3)-β-glucanase reveals 54% positional identity with barley (1 → 3,1 → 4)-β-glucanases (EC 3.2.1.73) and suggests a common evolutionary origin for these two classes of β-glucan endohydrolases. The barley (1 → 3)-β-glucanase also exhibits significant similarity with a (1 → 3)-β-glucanase from tobacco.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-O-β- -galactopyranosyl-(1→6)-3-deoxy-3-fluoro -β- -galactopyranoside and related n.m.r. studies

Sequential tritylation, benzoylation, and detritylation of methyl 3-deoxy-3-fluoro-β- -galactopyranoside gave crystalline methyl 2,4-di-O-benzoyl-3-deoxy-3-fluoro-β- -galactopyranoside (9), which was used as the initial nucleophile in the synthesis of the target oligosaccharide (16). Treatment of 9 with 2,3,4-tri-O-benzoyl-6-O-bromoacetyl-α- -galactopyranosyl bromide gave the corresponding disaccharide derivative 13, having a selectively removable blocking group at O-6′. Debromoacetylation of 13 afforded the disaccharide nucleophile 14 which, when treated with 2,4,6-tri-O-benzoyl-3-deoxy-3-fluoro-α- -galactopyranosyl bromide, gave the fully protected trisaccharide 15. Debenzoylation of 15 gave the title glycoside 16. Condensation reactions were performed with silver trifluoromethane-sulfonate as a promoter in the presence of sym-collidine under base-deficient conditions, and gave excellent yields of the desired β-(trans)-products. Analyses of the ¹H- and ¹³C-n.m.r. spectra, as well as determination of the J_CF and J_HF coupling constants, were made by using various one- and two-dimensional n.m.r. techniques.     

Structural analysis of two 2-deoxy analogues of α- and β-KDO and the methyl α- and β-glycosides of KDO, and determination of their metal-ion-binding properties

Ammonium 2,6-anhydro-3-deoy- -glycero- -talo-octonate (1), a potent inhibitor of the enzyme CMP-KDO synthetase, its C-2 epimer 2, and the methyl β-(3) and α-glycoside (4) of KDO were studied by ¹H- and ¹³C-n.m.r. spectroscopy. Compound 1 was also analysed by X-ray crystallography. Each compound adopted a ⁵C₂ chair conformation with the side chain equatorial. The preponderant side-chain conformation of 1 in solution was the same as that in the crystal and was stabilised by an intramolecular hydrogen bond from HO-8 to the carboxylate group. This hydrogen bond appeared to be present also in 3. However, the side-chain conformation of 2 and 4 was different from that in 1 and 3. The metal-ion-binding properties, determined on the basis of the line-broadening effects of Mn²⁺ on the ¹³C-n.m.r. signals, showed that the carboxylate group was involved in the binding with O-8 in 1 and 3 and with O-6 and O-8 in 2 and 4.     

Medicinal importance of fungal β-(1→3), (1→6)-glucans

Non-cellulosic β-glucans are now recognized as potent immunological activators, and some are used clinically in China and Japan. These β-glucans consist of a backbone of glucose residues linked by β-(1→3)-glycosidic bonds, often with attached side-chain glucose residues joined by β-(1→6) linkages. The frequency of branching varies. The literature suggests β-glucans are effective in treating diseases like cancer, a range of microbial infections, hypercholesterolaemia, and diabetes. Their mechanisms of action involve them being recognized as non-self molecules, so the immune system is stimulated by their presence. Several receptors have been identified, which include: dectin-1, located on macrophages, which mediates β-glucan activation of phagocytosis and production of cytokines, a response co-ordinated by the toll-like receptor-2. Activated complement receptors on natural killer cells, neutrophils, and lymphocytes, may also be associated with tumour cytotoxicity. Two other receptors, scavenger and lactosylceramide, bind β-glucans and mediate a series of signal pathways leading to immunological activation. Structurally different β-glucans appear to have different affinities toward these receptors and thus generate markedly different host responses. However, the published data are not always easy to interpret as many of the earlier studies used crude β-glucan preparations with, for the most part, unknown chemical structures. Careful choice of β-glucan products is essential if their benefits are to be optimized, and a better understanding of how β-glucans bind to receptors should enable more efficient use of their biological activities.     

Solution conformation of laminaribioside and (1 → 3)-β-D-glucan from optical rotation

A chiroptical method of conformational analysis is applied to linear (1 → 3)-β-D -glucans and the dimeric analogues β- and α-laminaribioside. The method is based on a recently developed semiempirical calculational model for saccharide optical activity. We conclude that disaccharide conformational energy maps in the literature represent the effective potential energy surface in aqueous solution well. The positive optical rotation observed with long chains in dilute alkaline solution is not characteristic of any single–chain conformation, and must reflect chain association.     

Synthesis, and carbon-13 n.m.r. study, of O-α- -rhamnopyranosyl-(1→3)-O-α- -rhamnopyranosyl-(1→2)- -rhamnopyranose and O-α- -rhamnopyranosyl-(1→3)-O-α- -rhamnopyranosyl (1→3)- -rhamnopyranose, constituents of bacterial, cell-wall polysaccharides

O-α- -Rhamnopyranosyl-(1→3)- -rhamnopyranose (19) and O-α- -rhamnopyranosyl-(1→2)- -rhamnopyranose were obtained by reaction of benzyl 2,4- (7) and 3,4-di-O-benzyl-α- -rhamnopyranoside (8) with 2,3,4-tri-O-acetyl-α- -rhamnopyranosyl bromide, followed by deprotection. The per-O-acetyl α-bromide (18) of 19 yielded, by reaction with 8 and 7, the protected derivatives of the title trisaccharides (25 and 23, respectively), from which 25 and 23 were obtained by Zemplén deacetylation and catalytic hydrogenolysis, With benzyl 2,3,4-tri-O-benzyl-β- -galactopyranoside, compound 18 gave an ≈3:2 mixture of benzyl 2,3,4-tri-O-benzyl-6-O-[2,4-di-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-α- -rhamnopyranosyl]-β- -galactopyranoside and 4-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-β- -rhamnopyranose 1,2-(1,2,3,4-tetra-O-benzyl-β- -galactopyranose-6-yl (orthoacetate). The downfield shift at the α-carbon atom induced by α- -rhamnopyranosylation at HO-2 or -3 of a free α- -rhamnopyranose is 7.4-8.2 p.p.m., ≈1 p.p.m. higher than when the (reducing-end) rhamnose residue is benzyl-protected (6.6-6.9 p.p.m.). α- -Rhamnopyranosylation of HO-6 of gb- -galactopyranose deshields the C-6 atom by 5.7 p.p.m. The 1 2-orthoester ring structure [O₂,C(me)OR] gives characteristic resonances at 24.5 ±0.2 p.p.m. for the methyl, and at 124.0 ±0.5 p.p.m. for the quaternary, carbon atom.     

Synthesis of methyl glycosides of β-(1→6)-linked -galactobiose, galactotriose, and galactotetraose having a 3-deoxy-3-fluoro-β- -galactopyranoside end-residue

Methyl 2,4-di-O-acetyl-3-deoxy-3-fluoro-β- -galactopyranoside was synthesized by sequential tritylation, acetylation, and detritylation of methyl 3-deoxy-3-fluoro-β- -galactopyranoside, and used as the initial nucleophile in the synthesis of methyl β-glycosides of (1→6)-β- -galacto-biose, -triose (20), and -tetraose (22) having a 3-deoxy-3-fluoro-β- -galactopyranoside end-residue. The extension of the oligosaccharide chais, to form the internal units in 20 and 22, was achieved by use of 2,3,4-tri-O-acetyl-6-O-bromoacetyl-α- -galactopyranosyl bromide as a glycosyl donor, and mercuric cyanide or silver triflate as the promotor. While fewer by-products were formed in the reactions involving mercuric cyanide, the reactions catalyzed by silver triflate were stereospecific and yielded only the desired β (trans) products.     

Chemical synthesis of a dextran model,poly-α-(1 → 6)-anhydro-D-glucopyranose

Poly-α-(1 → 6)-anhydro-D -glucopyranose has been synthesized by the phosphorus pentafluoride-catalyzed polymerization of l,6-anhydro-2,3,4-tri-O-benzyl-β-D -glucopyranose and subsequent debenzylation. Physical characterization establishes its high polymeric character and stereoregularity.     

Chemical synthesis of polysaccharides. 7. Enzymatic hydrolysis of (1 → 6)-α-DL-glucopyranan (DL-dextran)

Hydrolysis of (1 → 6)-α-DL -glucopyranan (synthetic DL -dextran) by an endo-dextranase from a Penicillium species was examined in an acetate buffer solution (pH 5.3) at 37°C. Three samples of different tacticities (isotactic dyad content, 55, 63, and 72%) were employed with a clinical dextran for comparison. Colorimetric determination of the reducing end units of the saccharides produced during hydrolysis showed that the maximum degrees of hydrolysis based on the D-glucose units, (D.H.)_D, for the DL -dextrans were 21.4, 27.8, and 33.0% in the order of increasing isotacitic dyad content, whereas the (D.H.)_D value for the clinical dextran was 51.9%. A statistical treatment of the enzymatic hydrolysis is proposed to interpret the experimental results.     

Conformational analysis and molecular dynamics simulation of α-(1 → 2) and α-(1 → 3) linked rhamnose oligosaccharides: Reconciliation with optical rotation and NMR experiments

Molecular mechanics and dynamics calculations were carried out on the disaccharides α-L-Rhap-(1 → 2)-α-L-Rhap-(1 → OMe) (1) and α-L-Rhap-(1 → 3)-α-L-Rhap-(1 OMe) (2), and the trisaccharide α-L-Rhap-(1 → 2)-α-L-Rhap-(1 → 3)-α-L-Rhap-(1 → OMe) (3). The semiflexible conformational behavior of these molecules was characterized by the occupation of a combination of different glycosidic linkage and side-chain conformational positions whose relative occupations were sensitive to dielectric screening. Molecular dynamics simulations of the trisaccharide 3 showed little difference between the linkage conformations in the trisaccharide and the component disaccharides 1 and 2. Experimental optical rotation data of 1 and 2 were obtained as a function of temperature in varying solvents. The molecular models were combined with the semiempirical theory of Stevens and Sathyanarayana to yield calculated optical rotations. Interpretation of the data of both 1 and 2 implied that a combination of conformations, both in glycosidic and side-chain positions, could explain the experimental data. Solvents effects were important in influencing the conformational mix and averaged optical rotation. Three-bond heteronuclear coupling constants ³J_{C, H} were obtained for the glycosidic linkages of 1 and 2 in D₂O and DMSO. Analysis of the coupling constants with a Karplus curve showed that small reductions in the glycosidic torsion angles of the conformations of the models used here of ca. 10°–15° in ϕ and 5°–10° in ψ were required to give better agreement with experiment; a combination of conformations for both 1 and 2 was consistent with the data. There was a negligible influence on the coupling constants of 1 on changing the solvent from D₂O to DMSO. © 1997 John Wiley & Sons, Inc.     

Physicochemical properties of (1 → 6)-branched (1 → 3)-β-D-glucans. 1. Physical dimensions estimated from hydrodynamic and electron microscopic data

The physical dimensions of several (1 → 6) branched (1 → 3) -β-D -glucan samples obtained from different organisms and their derivatives have been studied by electron microscopy, light scattering measurements, viscometry, and gel permeation chromatography. The electron micrographs indicate that in most samples these biopolymers are adequately described as linear worm-like coils. A sample reconstituted from alkaline media appeared as a blend of the linear, circular, and aggregated polymer morphologies. The average mass per unit length, M_L = M_w/L_w for the macroscopically linear samples, was estimated to be 2100 ± 200 g mol^?1 nm^?1. The parameter m_L was determined from the contour lengths obtained by electron microscopy and the molecular weight by light scattering measurements. The observed M_L was consistent with the triple-helical structure reported from x-ray diffraction studies and observed degree of side-chain substitution. From the molecular snapshots shown in the electron micrographs, the persistence lengths of these β-D -glucans were determined to be 140 ± 30 nm. The experimentally determined intrinsic viscosities were consistent with these estimates of M_L and persistence length. Comparison of the molecular weight distributions obtained from gel permeation chromatography and those deduced from the electron micrographs indicates that number and weight average contour lengths are more reliable than z and z + 1 averages. © 1993 John Wiley & Sons, Inc.     

Three-dimensional features of the bacterial polysaccharide (1 → 4)-β-D-glucuronan: A molecular modeling study

The mutant strain M5N1 CS of Rhizobium meliloti produces, in a Rhizobium complete medium supplemented with fructose and sucrose, a partially acetylated homopolymer of D -glucuronic acid residues linked β-(1 → 4). This polysaccharide forms thermoreversible gels with monovalent salts and thermally stable gels with divalent salts. In order to define the different levels of structural characterization, modeling simulations were performed for both the regular (1 → 4)-β-D -glucuronan and the acetylated derivatives. This required the evaluation of the accessible conformational space for the 16 disaccharides. Detailed conformational analysis was accomplished using the flexible residue of the MM3 molecular mechanics procedure and the results were used to access the configurational statistics of representative polysaccharide chains. Within the potential energy surfaces calculated for each disaccharide, several low energy conformers can be identified. When these conformations are extrapolated to regular polysaccharide structures, they generate polymers with right- and left-handed chirality along with a 2-fold axis. This later arrangement (n = 2, h = 5.16 Å) closely corresponds to that derived from a fiber x-ray diffraction investigation. The insertion of acetyl groups induces changes in the helical features of the polymer. As for the simulation of the configurational properties of (1 → 4)-β-D -glucuronan, an extended disordered chain having a persistence length of 105 Å (corresponding to 22 monomers) is predicted. This agrees with previous conclusions derived from solution study. The inclusion of varying amounts of acetyl groups only slightly perturbs the calculated persistence length. © 1998 John Wiley & Sons, Inc. Biopoly 45: 165–175, 1998     

Experimental chiroptical verification of linkage flexibility in methyl 3-O-(α-D-mannopyranosyl)-α-D-mannopyranoside

Vacuum UV CD spectra of methyl 3-O-(α-D -mannopyranosyl)-α-D -mannopyranoside in D₂O and as a cast film were obtained in the 145–200 nM region. The disaccharide solution CD per residue is nearly identical to that of the monosaccharide solution CD, and to the monosaccharide film CD. Conversely, the disaccharide film spectrum exhibits a strong positive CD linkage contribution in the 160–170 nm range, which is consistent with the known crystal conformation under the aegis of previously determined sector rules. The close similarity between the monosaccharide and disaccharide solution spectra, therefore, reflects conformational averaging in which the net linkage contribution is approximately zero. The present observation of significant solution linkage flexibility confirms previous conclusions based on optical rotation, as well as conclusions of others based on nmr data. Moreover, when combined with those earlier results, the present work demonstrates the population of at least three distinct potential energy wells on the disaccharide ϕ, ψ potential energy surface. © 1996 John Wiley & Sons, Inc.     

Enzymatic degradation of β(1 → 4) xylan single crystals

The enzymatic degradation of β(1 → 4) xylan single crystals with xylanases was investigated by electron microscopy and electron diffraction. The enzyme attack takes place at the edge of the crystals and progresses towards their centers. This is consistent with an endo-enzyme mechanism, where the enzyme interacts essentially with the accessible xylan chains located at the crystal periphery.