UDP-Glucose: (1-->3)-beta-Glucan Synthases from Mung Bean and Cotton: Differential Effects of Ca and Mg on Enzyme Properties and on Macromolecular Structure of the Glucan Product

A re-examination of the kinetic properties of UDP-glucose: (1→3)-β-glucan (callose) synthases from mung bean seedlings (Vigna radiata) and cotton fibers (Gossypium hirsutum) shows that these enzymes have a complex interaction with UDP-glucose and various effectors. Stimulation of activity by micromolar concentrations of Ca²⁺ and millimolar concentrations of β-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V_max of the enzyme, and by lowering the apparent K_m for UDP-glucose from >1 millimolar to 0.2 to 0.3 millimolar. Mg²⁺ markedly enhances the affinity of the mung bean enzyme for Ca²⁺ but not for β-glucoside; with saturating Ca²⁺, Mg²⁺ only slightly stimulates further production of glucan. However, the presence of Mg²⁺ during synthesis, or NaBH₄ treatment after synthesis, changes the nature of the product from dispersed, alkali-soluble fibrils to highly aggregated, alkali-insoluble fibrils. Callose synthesized in vitro by the Ca²⁺, β-glucoside-activated cotton fiber enzyme, with or without Mg²⁺, is very similar in size to callose isolated from cotton fibers, but is a linear (1→3)-β-glucan lacking the small amount of branches at C-0-6 found in vivo. We conclude that the high degree of aggregation of the fibrils synthesized with Mg²⁺in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg²⁺ on the conformation of the enzyme. Rate-zonal centrifugation of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme.     

Susceptibility of UDP-Glucose:(1,3)-beta-Glucan Synthase to Inactivation by Phospholipases and Trypsin

UDP-glucose:(1,3)-β-glucan synthase from Beta vulgaris L. was rapidly inactivated by treatment with phospholipases C, D, and A₂. Enzyme activity could not be restored to the phospholipase-treated enzyme by the addition of phosphatidylethanolamine or other phospholipids. Membrane-bound and solubilized glucan synthase were also trypsin-labile with inactivation rates equal in the presence or absence of divalent cations or chelators. Gradual activity declines were observed in membranes incubated with divalent cations, but not with chelators.     

beta-Glucoside Activators of Mung Bean UDP-Glucose: beta-Glucan Synthase : I. Identification of an Endogenous beta-Linked Glucolipid Activator

Heat-stable activators of membranous β-glucan synthase have been isolated from the supernatant fraction of crude mung bean (Vigna radiata) extracts by DEAE-cellulose and silica-gel chromatography. One of the activators has been partially purified and characterized on the basis of susceptibility to various enzymes and by analysis of the products formed upon total acid hydrolysis, alkaline-methanolysis, and β-glucosidase digestion. This activator has the characteristics of a 1,2-dioleoyl diglyceride containing β-linked glucose residue(s) at the C-3 position. When expressed per mole of glucosyl residues, the maximal K_a value of the activator is estimated to be 25 micromolar. Both the intact glucosyl and fatty acid moiety are essential to the stimulatory effect of the activator.     

(1-->3)-beta-d-Glucan Synthase from Sugar Beet : II. Product Inhibition by UDP

The mode of inhibition of UDP, one of the products of the reaction catalyzed by (1→3)-β-d-glucan synthase in sugar beet (Beta vulgaris L.) was investigated. In the absence of added UDP, the enzyme, in the presence of Ca²⁺, Mg²⁺, and cellobiose, exhibited Michaelis-Menten kinetics and had an apparent K_m of 260 micromolar for UDP-glucose. Complex effects on the kinetics of the (1→3)-β-d-glucan synthase were observed in the presence of UDP. At high UDP-glucose concentrations, i.e. greater than the apparent K_m, UDP behaved as a competitive inhibitor with an apparent K_i of 80 micromolar. However, at low UDP-glucose concentrations, reciprocal plots of enzyme activity versus substrate concentration deviated sharply from linearity. This unusual effect of UDP is similar to that reported for fungal (1→3)-β-d-glucan synthase. However, papulacandin B, a potent inhibitor of this fungal enzyme, had no effect on the plant (1→3)-β-d-glucan synthase isolated from sugar beet petioles. The inhibitory effect of UDP was also compared with other known inhibitors of glucan synthases.     

Chromosomal Location of Genes Encoding Barley (1-->3, 1-->4)-beta-Glucan 4-Glucanohydrolases

Preparations of DNA from wheat (Triticum aestivum, cv Chinese Spring), barley (Hordeum vulgare, cv Betzes) and six euplasmic wheat-barley addition lines were digested to completion with restriction endonucleases and the products probed by Southern blot analysis using a cDNA-encoding barley (1→3, 1→4)-β-glucanase isoenzyme II. It is shown that one of the barley (1→3, 1→4)-β-glucanase genes is located on chromosome 1.     

Inhibition and Ultraviolet-Induced Chemical Modification of UDP-Glucose:(1,3)-beta-Glucan (Callose) Synthase by Chlorpromazine : Mechanism of Chlorpromazine Binding to the Plant Plasma Membrane

UDP-glucose:(1,3)-β-glucan (callose) synthase (CS) from storage tissue of red beet (Beta vulgaris L.) was strongly inhibited by the phenothiazine drug chlorpromazine (CPZ). In the absence of ultraviolet irradiation, CPZ was a noncompetitive inhibitor with 50% inhibitory concentration values for plasma membrane and solubilized CS of 100 and 90 μm, respectively. Both the Ca²⁺- and Mg²⁺- stimulated components of CS activity were affected. CPZ inhibition was partially alleviated at saturating levels of Ca²⁺, but not Mg²⁺, suggesting that CPZ interferes with the Ca²⁺-binding site of CS. Binding experiments with [¹⁴C]CPZ, however, showed strong non-specific partitioning of CPZ into the plasma membrane, providing evidence that perturbation of the membrane environment is probably the predominant mode of inhibition. Ultraviolet irradiation at 254 nm markedly enhanced CPZ inhibition, with complete activity loss following exposure to 4 μm CPZ for 2 min. Inhibition followed a pseudo-first order mechanism with at least three CPZ binding sites per CS complex. Under these conditions, [³H]CPZ was covalently incorporated into plasma membrane preparations by a free radical mechanism; however, polypeptide labeling profiles showed labeling to be largely nonspecific, with many polypeptides labeled even at [³H]CPZ levels as low as 1 μm, and with boiled membranes. Although CPZ is one of the most potent known inhibitors of CS, its use as a photolabel will require a homogeneous CS complex or establishment of conditions that protect against the interaction of CPZ with specific binding sites located on various polypeptide components of the CS complex.     

UDP-Glucose: (1,3)-beta-Glucan Synthase from Daucus carota L. : Characterization, Photoaffinity Labeling, and Solubilization

The membrane-bound UDP-glucose-β-(1,3)-glucan synthase from Daucus carota L. was characterized and a solubilization procedure was developed. The enzyme exhibited maximal activity in the presence of 0.75 millimolar Ca²⁺, 0.5 millimolar EGTA, and 5 millimolar cellobiose at pH 7.5 and 30°C at 1 millimolar UDPG. Reaction products were confirmed to be (1,3)-linked glucan. Polypeptides of 150, 57, and 43 kilodaltons were labeled with the photoactivatible affinity label 5-azido-uridine 5′-β-[³²P] diphosphateglucose. Labeling of the 150 and 57 kilodalton polypeptides was completely protected against by 1 millimolar non-radioactive UDPG suggesting that one or both of these polypeptides may represent the UDPG binding subunit of glucan synthase. Carrot glucan synthase was solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in the absence of divalent cations and chelators; however, the percentage of enzyme which could be solubilized showed variability with membrane source. With microsomal membranes, up to 80% of the enzyme was released with 0.7% CHAPS. Solubilized enzyme was stable for at least 9 hours at 4°C. When more highly purified membrane fractions were isolated from sucrose step gradients a slightly different picture emerged. Activity from the 20/30% interface (Golgi and tonoplast enriched) was readily solubilized and expressed. Activity from the 30/40% interface (plasma membrane enriched) was also solubilized; however, it was necessary to add heat inactivated microsomes to assay mixtures for full activity to be expressed. A requirement for endogenous activators is suggested.     

beta-Furfuryl-beta-Glucoside: An Endogenous Activator of Higher Plant UDP-Glucose:(1-3)-beta-Glucan Synthase : Biological Activity, Distribution, and in Vitro Synthesis

In a recent paper (P Ohana, DP Delmer, JC Steffens, DE Matthews, R Mayer, M Benziman [1991] J Biol Chem 266: 13472-13475), we described the purification and structural characterization of β-furfuryl-β-glucoside (FG), an endogenous activator of plant UDP-glucose:(1→3)-β-glucan (callose) synthase. In the present report, we provide evidence that FG specifically stimulates callose synthase. The effects of FG on the kinetic properties of callose synthase were studied, and we ascertained that FG, or at least a very similar compound, is present in other plant systems. Chemically synthesized α-furfuryl-β-glucoside also stimulates callose synthase, exhibiting a slightly higher K_a of 80 micromolar, compared with 50 micromolar for FG. In addition, we have identified and partially characterized an enzyme that catalyzes the synthesis of FG using β-furfuryl alcohol and UDP-glucose as substrates. A model for the regulation of callose synthesis in vivo, involving changes in intracellular compartmentation of FG and Ca²⁺, is proposed.     

beta-Glucoside Activators of Mung Bean UDP-Glucose: beta-Glucan Synthase : II. Comparison of Effects of an Endogenous beta-Linked Glucolipid with Synthetic n-Alkyl beta-d-Monoglucopyranosides

n-Alkyl (C₆-C₁₂) β-d-monoglucopyranosides have been found to be highly potent activators of mung bean β-glucan synthase in vitro, increasing the V_max of the enzyme as much as 60-fold and with K_a values as low as 10 micromolar. Activation is highly specific for the β-linked terminal glucose residue; other alkyl glycosides such as, octyl-α-glucoside, dodecyl β-maltoside, 6-lauryl sucrose, 6-lauryl glucose, which lack this structure, are ineffective as activators. Based on the similarities in their structure and effects on β-glucan synthesis under a variety of conditions, it is proposed that the alkyl β-glucosides are structural analogs of the native glucolipid activator of β-glucan synthase isolated from mung bean extracts.     

Rapid Enrichment of CHAPS-Solubilized UDP-Glucose: (1,3)-beta-Glucan (Callose) Synthase from Beta vulgaris L. by Product Entrapment : Entrapment Mechanisms and Polypeptide Characterization

Rapid enrichment of CHAPS-solubilized UDP-glucose:(1,3)-β-glucan (callose) synthase from storage tissue of red beet (Beta vulgaris L.) is obtained when the preparation is incubated with an enzyme assay mixture, then centrifuged and the enzyme released from the callose pellet with a buffer containing EDTA and CHAPS (20-fold purification relative to microsomes). When centrifuged at high speed (80,000g), the enzyme can also be pelleted in the absence of substrate (UDP-Glc) or synthesis of callose, due to nonspecific aggregation of proteins caused by excess cations and insufficient detergent in the assay buffer. True time-dependent and substrate-dependent product-entrapment of callose synthase is obtained by low-speed centrifugation (7,000-11,000g) of enzyme incubated in reaction mixtures containing low levels of cations (0.5 millimolar Mg²⁺, 1 millimolar Ca²⁺) and sufficient detergent (0.02% digitonin, 0.12% CHAPS), together with cellobiose, buffer, and UDP-Glc. Entrapment conditions, therefore, are a compromise between preventing nonspecific precipitation of proteins and permitting sufficient enzyme activity for callose synthesis. Further enrichment of the enzyme released from the callose pellet was not obtained by rate-zonal glycerol gradient centrifugation, although its sedimentation rate was greatly enhanced by inclusion of divalent cations in the gradient. Preparations were markedly cleaner when product-entrapment was conducted on enzyme solubilized from plasma membranes isolated by aqueous two-phase partitioning rather than by gradient centrifugation. Product-entrapped preparations consistently contained polypeptides or groups of closely-migrating polypeptides at molecular masses of 92, 83, 70, 57, 43, 35, 31/29, and 27 kilodaltons. This polypeptide profile is in accordance with the findings of other callose synthase enrichment studies using a variety of tissue sources, and is consistent with the existence of a multi-subunit enzyme complex.     

Synthesis of beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->3)]-beta-D-GlcpOLauryl, an oligosaccharide with anti-tumor activity

A concise and effective synthesis of lauryl heptasaccharide 17 was achieved from the key intermediates lauryl 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2,4-di-O-benzoyl-beta-D-glucopyranoside (10) and isopropyl 2,4,6-tri-O-acetyl-3-O-allyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-1-thio-beta-D-glucopyranoside (15). The key trisaccharide glycosyl acceptor 10 was constructed by coupling 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl trichloroacetimidate (3) with lauryl 6-O-acetyl-2,4-di-O-benzoyl-beta-D-glucopyranoside (9), followed by deacetylation. The thioglycoside donor 15 was obtained by condensation of 2,4,6-tri-O-acetyl-3-O-allyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (11) with isopropyl 4,6-O-benzylidene-1-thio-beta-D-glucopyranoside (12), followed by debenzylidenation and acetylation. A bioassay of the inhibition of S180 noumenal tumors showed that lauryl heptasaccharide 17 could be employed as a potential agent for cancer treatment.     

Synthesis of divalent beta-(1-->6)-branched (1-->3)-glucohexaose and trivalent beta-(1-->6)-branched (1-->3)-glucotriose

Hexaose, beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp, based dimers were synthesized by twofold glycosidation of the hexaosyl trichloroacetimidate with hexylene 1,6-diol, diethylene glycol and triethylene glycol, respectively. Meanwhile, a triose, beta-1D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp, based trimer was obtained by glycosidation of the triosyl trichloroacetimidate with a glycerol-derived triol scaffold.     

An enzymatically produced novel cyclomaltopentaose cyclized from amylose by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}

A bacterial strain AM7, isolated from soil and identified as Bacillus circulans, produced two kinds of novel cyclic oligosaccharides. The cyclic oligosaccharides were produced from amylose using a culture supernatant of the strain as the enzyme preparation. The major product was a cyclomaltopentaose cyclized by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}. The other minor product was cyclomaltohexaose cyclized by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}. We propose the names isocyclomaltopentaose (ICG5) and isocyclomaltohexaose (ICG6) for these novel cyclic maltooligosaccharides having one alpha-(1-->6)-linkage. ICG5 was digested by alpha-amylase derived from Aspergillus oryzae, cyclomaltodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus, and maltogenic alpha-amylase. On the other hand, ICG6 was digested by CGTase from B. stearothermophilus and B. circulans, and maltogenic alpha-amylase. This is the first report of enzymatically produced cyclomaltopentaose and cyclomaltohexaose, which have an alpha-(1-->6)-linkage in their molecules.     

(1-->3)-beta-d-Glucan Synthase from Sugar Beet : I. Isolation and Solubilization

A (1→3)-β-glucan synthase has been isolated from petiole tissue of sugar beet (Beta vulgaris L.). Enzyme activity is associated with a membrane fraction with a density of 1.03 grams per cubic centimeter when subjected to isopycnic density gradient centrifugation in Percoll. The reaction product was determined to be a linear (1→3)-β-glucan by methylation analysis and by glucanase digestion. (1→3)-β-Glucan synthase activity is markedly stimulated by Ca²⁺; activation is half-maximal at about 50 micromolar Ca²⁺ and is nearly saturated at 100 micromolar. Other divalent cations tested, Mg²⁺, Mn²⁺, and Sr²⁺, also stimulate enzyme activity but are less effective. Enzyme activity was also stimulated up to 12-fold by β-glucosides. Sirofluor, the fluorochrome from aniline blue, inhibited enzyme activity 95% when included at 1 millimolar. The enzyme was solubilized in Zwittergent 3-14; 85% of total enzyme activity was solubilized in 0.03% detergent and the optimal detergent-to-protein ratio was 0.3 at 3 milligrams per milliliter protein.     

Identification of bound water molecules in the cyclic tetrasaccharide, cyclo-{-->6}-alpha-d-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->)

A structural characterization of bound water molecules in the cyclic tetrasaccharide, cyclo-{-->6}-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->), was carried out by NMR spectroscopy. H-1', 2'-OH, H-3', and 4'-OH of the 3-O-glycosylated residue and H-1 of the 6-O-glycosylated residue were found to cross-relax with protons of bound waters using the double-pulsed field-gradient spin-echo ROESY experiment. In the crystal structure, one water molecule is located in the center of the plate, and its temperature factor is very low, indicating that this water molecule is an intrinsic component.     

A practical synthesis of alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-[alpha-D-Glcp-(1-->3)]-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp, an O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c

An O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c, alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-[alpha-D-Glcp-(1-->3)]-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp was synthesized as its methyl glycoside. Acetylation of allyl 4,6-O-benzylidene-alpha-D-mannopyranoside, followed by debenzylidenization and benzoylation gave allyl 2,3-di-O-acetyl-4,6-di-O-benzoyl-alpha-D-mannopyranoside (3), and subsequent deacetylation of 3 with CH(3)COCl-MeOH gave the monosaccharide acceptor 4. Condensation of isopropyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-glucopyranoside (6) with 4 selectively afforded the alpha-(1-->3)-linked disaccharide 7. Condensation of 7 with the (1-->3)-linked disaccharide donor 9, followed by deallylation and trichloroacetimidation, afforded the tetrasaccharide donor 12. Coupling of 12 with disaccharide acceptor 13, followed by debenzylation and deacylation, furnished the target heterohexasaccharide 16.     

Synthesis of beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3) linked bond with antitumor activity

A beta-(1-->6)-branched beta-(1-->3)-glucohexaose, present in many biologically active polysaccharides from traditionally herbal medicines such as Ganoderma lucidum, Schizophyllum commune and Lentinus edodes, was synthesized as its lauryl glycoside 32, and its analogues 18, 20 and 33 containing an alpha-(1-->3) linked bond were synthesized. It is interesting to find that coupling of a 3,6-branched acylated trisaccharide trichloroacetimidate donor 9 with 3,6-branched acceptors 13 and 16 with 3'-OH gave the alpha-(1--> 3)-linked hexasaccharides 17 and 19, respectively, in spite of the presence of C-2 ester capable of neighboring group participation. However, coupling of 9 with 4-methoxyphenyl 4,6-O-benzylidene-beta-D-glucopyranoside (27) selectively gave beta-(1-->3)-linked tetrasaccharide 28. Simple chemical transformation of the tetrasaccharide 28 gave acylated tetrasaccharide trichloroacetimidate 29. Coupling of 29 with lauryl (1-->6)-linked disaccharide 26 with 3-OH gave beta-(1-->3)-linked hexasaccharide 30 as the major product. Bioassay showed that in combination with the chemotherapeutic agent cyclophospamide (CPA), the hexaose 18 at a dose of 0.5-1mg/kg substantially increased the inhibition of S(180) for CPA, but decreased the toxicity caused by CPA. Some of these oligosaccharides also inhibited U(14) noumenal tumor in mice effectively.     

Synthesis of the tetrasaccharide alpha-D-Glcp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp recognized by calreticulin/calnexin

The title compound as its methyl glycoside was efficiently synthesized using a block synthesis approach. Halide-assisted glycosidations between 6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl iodide and ethyl 2-O-acetyl-4,6-di-O-benzyl-1-thio-alpha-D-mannopyranoside using triphenylphosphine oxide as promoter yielded, with complete alpha-selectivity, a disaccharide building block in high yield. The perbenzylated derivative of this proved to be an excellent donor affording 88% of the protected target tetrasaccharide in an NIS/AgOTf-promoted coupling to a known methyl dimannoside acceptor. Deprotection through catalytic hydrogenolysis then gave the target compound in 47% overall yield.     

Synthesis of alpha-Manp-(1-->2)-alpha-Manp-(1-->3)-alpha-Manp-(1-->3)-Manp, the tetrasaccharide repeating unit of Escherichia coli O9a, and alpha-Manp-(1-->2)-alpha-Manp-(1-->2)-alpha-Manp-(1-->3)-alpha-Manp-(1-->3)-Manp, the pentasaccharide repeating unit of E. coli O9 and Klebsiella O3

The tetrasaccharide repeating unit of Escherichia coli O9a, alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-D-Manp, and the pentasaccharide repeating unit of E. coli O9 and Klebsiella O3, alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-D-Manp, were synthesized as their methyl glycosides. Thus, selective 3-O-allylation of p-methoxyphenyl alpha-D-mannopyranoside via a dibutyltin intermediate gave p-methoxyphenyl 3-O-allyl-alpha-D-mannopyranoside (2) in good yield. Benzoylation (-->3), then removal of 1-O-methoxyphenyl (right arrow4), and subsequent trichloroacetimidation afforded the 3-O-allyl-2,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (5). Condensation of 5 with methyl 4,6-O-benzylidene-alpha-D-mannopyranoside (6) selectively afforded the (1-->3)-linked disaccharide 7. Benzoylation of 7, debenzylidenation, benzoylation, and deallylation gave methyl 2,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-mannopyranoside (11) as the disaccharide acceptor. Coupling of 11 with (1-->2)-linked mannose disaccharide donor 17 or trisaccharide donor 21, followed by deacylation, furnished the target tetrasaccharide and pentasaccharide, respectively.     

Synthesis of mannose-containing analogues of (1-->6)-branched (1-->3)-glucohexaose

Coupling of the trisaccharide acceptor 2,4,6-tri-O-acetyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-5-O-acetyl-1,2-O-isopropylidene-alpha-D-glucofuranose (2) with the trisaccharide donor 2,3,4,6-tetra-O-benzoyl-alpha-D-annopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (1) gave an alpha-linked hexasaccharide 3, while coupling of 2 with the trisaccharide donor 2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (7) produced alpha- 8 and beta-linked 12 hexasaccharides in a ratio of 3:2. Deprotection of 3, 8, and 12 afforded the analogues of the immunomodulator beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-D-Glcp (A).