Enzymatic synthesis of a novel cyclic pentasaccharide consisting of alpha-D-glucopyranose with 6-alpha-glucosyltransferase and 3-alpha-isomaltosyltransferase

A novel cyclic pentasaccharide (CPS) and a branched cyclic pentasaccharide (6G-CPS) consisting of d-glucopyranose were synthesized with 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus N75. The structure of CPS was cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The other, 6G-CPS, had the structure cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-[alpha-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The formation of CPS was presumed to occur after the following four successive reactions: a 6-glucosyltransfer reaction with 6GT, a 4-glucosyltransfer reaction with 6GT, a 3-isomaltosyltransfer reaction with IMT, and a cyclization reaction with IMT.     

Enzymatic synthesis of a beta-D-galactopyranosyl cyclic tetrasaccharide by beta-galactosidases

The galactosyl transfer reaction to cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS) was examined using lactose as a donor and beta-galactosidases from Aspergillus oryzae and Bacillus circulans. The A. oryzae beta-galactosidase produced three galactosyl derivatives of CTS. The main galactosyl derivative produced by the A. oryzae enzyme was identified as 6-O-beta-D-galactopyranosyl-CTS, cyclo-[-->6)-alpha-D-Glcp-(1-->3)-[beta-D-Galp-(1-->6)]-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. The B. circulans beta-galactosidase also synthesized three galactosyl-transfer products to CTS. The structure of main transgalactosylation product was 3-O-beta-D-galactopyranosyl-CTS, cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[beta-D-Galp-(1-->3)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. These results showed that beta-galactosidase transferred galactose directly to the ring glucose residue of CTS.     

Penta-, hexa-, and heptasaccharide acceptor products of alternansucrase

In the presence of suitable acceptor molecules, dextransucrase makes a homologous series of oligosaccharides in which the isomers differ by a single glucosyl unit, whereas alternansucrase synthesizes one trisaccharide, two tetrasaccharides, etc. For the example of maltose as the acceptor, if one considers only the linear, unbranched possibilities for alternansucrase, the hypothetical number of potential products increases exponentially as a function of the degree of polymerization (DP). Experimental evidence indicates that far fewer products are actually formed. We show that only certain isomers of DP >4 are formed from maltose in measurable amounts, and that these oligosaccharides belong to the oligoalternan series rather than the oligodextran series. When the oligosaccharide acceptor products from maltose were separated by size-exclusion chromatography and HPLC, only one pentasaccharide was isolated. Its structure was alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Two hexasaccharides were formed in approximately equal quantities: alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc and alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Just one heptasaccharide was isolated from the reaction mixture, alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. We conclude that the enzyme is incapable of forming two consecutive alpha-(1-->3) linkages, and does not form products with more than two consecutive alpha-(1-->6) linkages. The distribution of products may be kinetically determined.     

Determination of the specificity of monoclonal antibodies against Schistosoma mansoni CAA glycoprotein antigen using neoglycoconjugate variants

The immunogenic O-glycan of circulating anodic antigen (CAA) is a high-molecular-mass polysaccharide with the unique -->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GalpNAc-(1--> repeating unit. To obtain information at the molecular level about the specificity of monoclonal antibodies against CAA, the immunoreactivity of two series of bovine serum albumin-coupled synthetic oligosaccharides related to the CAA O-glycan was monitored using ELISA and surface plasmon resonance spectroscopy. The importance of the axial hydroxyl group of beta-D-GalpNAc for antibody binding was investigated using the following series of analogues: beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->O); beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O); and beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O). In the second series of analogues, beta-D-Glcp6S-(1-->3)-beta-D-GalpNAc-(1-->O), beta-D-GalpNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), and beta-D-Glcp6S-(1-->3)-beta-D-Gal-pNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), the native beta-D-GlcpA moiety was replaced by beta-D-Glcp6S to evaluate the influence of the nature of the charge on antibody recognition. Comparison of the immunoreactivity of these series with that measured for conjugates containing corresponding synthetic CAA fragments showed that the antibody binding levels can be correlated to the antibody specificity to CAA fragments. For the most reactive antibodies, the structural changes chosen (beta-D-GalpNAc replaced by beta-D-GlcpNAc, and beta-D-GlcpA replaced by beta-D-Glcp6S) completely eradicated the binding.     

Transglycosylation of glycosyl residues to cyclic tetrasaccharide by Bacillus stearothermophilus cyclomaltodextrin glucanotransferase using cyclomaltodextrin as the glycosyl donor

Cyclomaltodextrin glucanotransferase (EC 2.4.1.19, abbreviated as CGTase) derived from Bacillus stearothermophilus produced a series of transfer products from a mixture of cyclomaltohexaose and cyclic tetrasaccharide (cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->], CTS). Of the transfer products, only two components, saccharides A and D, remained and accumulated after digestion with glucoamylase. The total combined yield of the saccharides reached 63.4% of total sugars, and enzymatic and instrumental analyses revealed the structures of both saccharides. Saccharide A was identified as 4-mono-O-alpha-glucosyl-CTS, [-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->], and sachharide D was 4,4'-di-O-alpha-glucosyl-CTS, [-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. These structures led us to conclude that the glycosyltransfer catalyzed by CGTase was specific to the C4-OH of the 6-linked glucopyranosyl residues in CTS.     

Synthesis of 3-O-beta-N-acetylglucosaminyl cyclic tetrasaccharide through a lysozyme-catalyzed transfer reaction

Egg white lysozyme was found to catalyze the transfer of N-acetylglucosamine to cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS). Structural analysis showed that the transfer product was 3-O-beta-N-acetylglucosaminyl CTS, cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[beta-GlcNAc-(1-->3)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. This branched saccharide is anticipated to be a model compound of the sugar chains of glycoproteins.     

Synthesis of two isomeric pentasaccharides,the possible repeating unit of the beta-glucan from the micro fungus Epicoccum nigrum Ehrenb. ex Schlecht

Two isomeric pentasaccharides, beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp (I) and beta-D-Glcp-(1-->6)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)]-beta-D-Glcp (II), the possible repeating unit of the beta-glucan from the micro fungus Epicoccum nigrum Ehrenb. ex Schlecht, were synthesized as their 4-methoxyphenyl glycosides in a regio- and stereoselective manner. The pentasaccharide I was obtained from 3-O-selective glycosylation of 4-methoxyphenyl 4,6-O-benzylidene-beta-D-glucopyranoside (12) with 2,3,4,6-tetra-O-benzoyl-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 (6) followed by acetylation, debenzylidenation, and 6-O-selective glucosylation with 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl trichloroacetimidate (1), and then by deprotection. The pentasaccharide II was obtained from 3-O-selective coupling of 12 with 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)-2,4-di-O-acetyl-3-O-allyl-alpha-D-glucopyranosyl trichloroacetimidate (10) followed by acetylation, debenzylidenation, and 6-O-selective glycosylation with 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (11), and finally by deprotection.     

Synthesis of galactose-containing analogues of (1-->6)-branched (1-->3)-glucohexaose and its lauryl glycoside

Coupling of the trisaccharide acceptor either 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 (13) or lauryl 2,4,6-tri-O-acetyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,5-di-O-acetyl-alpha-D-glucopyranoside (15) with the trisaccharide donor 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-galactopyranosyl trichloroacetimidate (12) gave alpha-linked hexasaccharides 14 and 16, respectively, while coupling of either 13 or 15 with trisaccharide donor 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-galactopyranosyl trichloroacetimidate 17 did not afford any hexasaccarides. The analogues of the immunomodulator beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-beta-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp (1) was obtained by deprotection of 14 and 16.     

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.     

Transglycosylation reactions of Bacillus stearothermophilus maltogenic amylase with acarbose and various acceptors

It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an alpha-(1-->6) glycosidic linkage and the formation of isoacarbose. The addition of a number of different carbohydrates to the digest gave transfer products in which PTS was primarily attached alpha-(1-->6) to D-glucose, D-mannose, D-galactose, and methyl alpha-D-glucopyranoside. With D-fructopyranose and D-xylopyranose, PTS was linked alpha-(1-->5) and alpha-(1-->4), respectively. PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose, lactose, and gentiobiose. Lesser amounts of alpha-(1-->3) and/or alpha-(1-->4) transfer products were also observed for these carbohydrate acceptors. The major transfer product to sucrose gave PTS linked alpha-(1-->4) to the glucose residue. alpha,alpha-Trehalose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4). Maltitol gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the glucopyranose residue. Raffinose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the D-galactopyranose residue. Maltotriose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the nonreducing end glucopyranose residue. Xylitol gave PTS linked alpha-(1-->5) as the major product and D-glucitol gave PTS linked alpha-(1-->6) as the only product. The structures of the transfer products were determined using thin-layer chromatography, high-performance ion chromatography, enzyme hydrolysis, methylation analysis and 13C NMR spectroscopy. The best acceptor was gentiobiose, followed closely by maltose and cellobiose, and the weakest acceptor was D-glucitol.     

Characterization of novel structures of mannosylinositolphosphorylceramides from the yeast forms of Sporothrix schenckii.

Novel structures of glycoinositolphosphorylceramide (GIPC) from the infective yeast form of Sporothrix schenckii were determined by methylation analysis, mass spectrometry and NMR spectroscopy. The lipid portion was characterized as a ceramide composed of C-18 phytosphingosine N-acylated by either 2-hydroxylignoceric acid (80%), lignoceric (15%) or 2,3-dihydroxylignoceric acids (5%). The ceramide was linked through a phosphodiester to myo-inositol (Ins) which is substituted on position O-6 by an oligomannose chain. GIPC-derived Ins oligomannosides were liberated by ammonolysis and characterized as: Manpalpha1-->6Ins; Manpalpha1-->3Manpalpha1-->6Ins; Manpalpha1-->6Manpalpha1-->3Manpalpha1-->3Manpalpha1-->6Ins; Manpalpha1-->2Manpalpha1-->6Manpalpha1-->3Manpalpha1-->3Manpalpha1-->6Ins. These structures comprise a novel family of fungal GIPC, as they contain the Manpalpha1-->6Ins substructure, which has not previously been characterized unambigously, and may be acylated with a 2,3 dihydroxylignoceric fatty acid, a feature hitherto undescribed in fungal lipids.     

Synthesis of a mannose nonasaccharide existing in the exopolysaccharide of Cryphonectria parasitica

alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)[alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)]-alpha-D-Manp-(1-->6)-[alpha-D-Manp-(1-->2)]-alpha-D-Manp, existing in the exopolysaccharide of Cryphonectria parasitica was synthesized as its allyl glycoside in a regio- and stereoselective manner.     

Synthesis of oligosaccharide derivatives related to those from sanqi, a Chinese herbal medicine from Panax notoginseng

Oligosaccharide derivatives from sanqi, a Chinese herbal medicine derived from Panax notoginseng, methyl beta-D-galactopyranosyl-(1-->3)-[alpha-L-arabinofuranosyl-(1-->6)]-alpha-D-galactopyranoside, diosgenyl beta-D-galactopyranosyl-(1-->3)-[alpha-L-arabinofuranosyl-(1-->6)]-alpha-D-galactopyranoside, and methyl beta-D-galactopyranosyl-(1-->3)-[alpha-L-arabinofuranosyl-(1-->6)]-alpha-D-galactopyranosyl-(1-->4)-beta-D-galactopyranosyl-(1-->3)-[alpha-L-arabinofuranosyl-(1-->6)]-alpha-D-galactopyranoside, were synthesized under standard glycosylation conditions. An unexpected alpha-(1-->4) linkage was formed predominantly in the presence of neighboring participation group during regioselective synthesis of hexasaccharide via 3+3 strategy.     

Isolation and structural analysis of ajugose from Vigna mungo L

The hexasaccharide ajugose, alpha-D-galactopyranosyl-(1-->6)-alpha-D-galactopyranosyl-(1-->6)-O-alpha-D-galactopyranosyl-(1-->6)-alpha-D-galactopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1<-->2)-beta-D-fructofuranoside, generally uncommon in legumes, was detected in the seeds of Vigna mungo L. by TLC and paper chromatography. Ajugose was then isolated by silica gel chromatography and its structure was established by acid and enzymatic hydrolysis, fast atom bombardment mass spectrometry and both one- and two-dimensional 1H and 13C NMR techniques.     

Structural investigation of a fucoidan containing a fucose-free core from the brown seaweed, Hizikia fusiforme

A fucoidan, obtained from the hot-water extract of the brown seaweed, Hizikia fusiforme, was separated into five fractions by DEAE Sepharose CL-6B and Sepharose CL-6B column chromatography. All five fractions contained predominantly fucose, mannose and galactose and also contained sulfate groups and uronic acid. The fucoidans had MWs from 25 to 950 kDa. The structure of fraction F32 was investigated by desulfation, carboxyl-group reduction, partial hydrolysis, methylation analysis and NMR spectroscopy. The results showed that the sugar composition of F32 was mainly fucose, galactose, mannose, xylose and glucuronic acid; sulfate was 21.8%, and the MW was 92.7 kDa. The core of F32 was mainly composed of alternating units of -->2)-alpha-D-Man(1--> and -->4)-beta-D-GlcA(1-->, with a minor portion of -->4)-beta-D-Gal(1--> units. The branch points were at C-3 of -->2)-Man-(1-->, C-2 of -->4)-Gal-(1--> and C-2 of -->6)-Gal-(1-->. About two-thirds of the fucose units were at the nonreducing ends, and the remainder were (1-->4)-, (1-->3)- and (1-->2)-linked. About two-thirds of xylose units were at the nonreducing ends, and the remainder were (1-->4)-linked. Most of the mannose units were (1-->2)-linked, and two-thirds of them had a branch at C-3. Galactose was mainly (1-->6)-linked. The absolute configurations of the sugar residues were alpha-D-Manp, alpha-L-Fucp, alpha-D-Xylp, beta-D-Galp and beta-D-GlcpA. Sulfate groups in F32 were at C-6 of -->2,3)-Man-(1-->, C-4 and C-6 of -->2)-Man-(1-->, C-3 of -->6)-Gal-(1-->, C-2, C-3 or C-4 of fucose, while some fucose had two sulfate groups. There were no sulfate groups in either the GlcA or xylose residues.     

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

alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[alpha-D-Manp-(1-->6)]-D-Glcp and alpha-D-Manp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)[-alpha-D-Manp-(1-->6)]-D-Glcp were synthesized in a regio- and stereoselective way as the mannose-containing analogues of the immunomodulating 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.     

Oxidation and metal-ion affinities of a novel cyclic tetrasaccharide

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 oxidized in high yield to a dicarboxylic acid, cyclo-(-->6)-alpha-D-Glcp-(1-->3)-alpha-D-GlcpA-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-GlcpA-(1-->). The parent and oxidized compound were then screened for the ability to form stable complexes with 20 metal cations. Ion-exchange thin-layer chromatography was utilized to survey binding in aqueous and 50% methanolic solutions. The screening identified Pb2+, Fe2+ and Fe3+ as forming strong metal chelates with the oxidized cyclic tetrasaccharide. The stoichiometry of the oxidized cyclic tetrasaccharide and Pb2+ complex was determined to be 1:1 using aqueous gel-permeation chromatography. Perturbations between the free and complexed structure were examined using NMR spectroscopy. Molecular simulations were used to identify a probable structure of oxidized cyclic tetrasaccharide complexed with Pb2+.     

Enzymatic synthesis of a 2-O-alpha-D-glucopyranosyl cyclic tetrasaccharide by kojibiose phosphorylase

The glucosyl transfer reaction of kojibiose phosphorylase (KPase) from Thermoanaerobacter brockii ATCC35047 was examined using cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->} (CTS) as an acceptor. KPase produced four transfer products, saccharides 1-4. The structure of a major product, saccharide 4, was 2-O-alpha-d-glucopyranosyl-CTS, cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-[alpha-d-Glcp-(1-->2)]-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->}. The other transfer products, saccharides 1-3, were 2-O-alpha-kojibiosyl-, 2-O-alpha-kojitriosyl-, and 2-O-alpha-kojitetraosyl-CTS, respectively. These results showed that KPase transferred a glucose residue to the C-2 position at the ring glucose residue of CTS. This enzyme also catalyzed the chain-extending reaction of the side chain of 2-O-alpha-d-glycopyranosyl-CTS.     

Regioselective synthesis of p-nitrophenyl glycosides of beta-D-galactopyranosyl-disaccharides by transglycosylation with beta-D-galactosidases

The beta-D-galactosidase from porcine liver induced regiospecific transglycosylation of beta-D-galactose from beta-D-Gal-OC6H4NO2-o to OH-6 of, respectively, p-nitrophenyl glycoside acceptors of Gal, GlcNAc and GalNAc to afford beta-Gal-(1-->6)-alpha-Gal-OC6H4NO2-p, beta-Gal-(1--> 6)-beta-Gal-OC6H4NO2-p, beta-Gal-(1-->6)-alpha-GalNAc-OC6H4NO2-p, beta-Gal-(1-->6)-beta-GalNAc-OC6H4NO2-p, beta-Gal-(1-->6)-alpha-GlcNAc-OC6H4NO2-p, and beta-Gal-(1-->6)-beta-GlcNAc-OC6H4NO2-p. The enzyme showed much higher transglycosylation activity for the alpha-glycoside acceptors than the corresponding beta-glycoside acceptors. The regioselectivity of the beta-D-galactosidase from Bacillus circulans ATCC 31382 greatly depended on the nature of the acceptor. When alpha-D-GalNAc-OC6H4NO2-p and alpha-D-GlcNAc-OC6H4NO2-p were used as acceptors, the enzyme showed high potency for regioselective synthesis of beta-Gal-(1-->3)-alpha-GalNAc-OC6H4NO2-p and beta-Gal-(1-->3)-alpha-GlcNAc-OC6H4NO2-p in high respective yields of 75.9 and 79.3% based on the acceptors added. However, replacement of beta-D-Gal-OC6H4NO2-p by beta-D-GalNAc-OC6H4NO2-p did change the direction of galactosylation. The enzyme formed regioselectively beta-Gal-(1-->6)-beta-Gal-OC6H4NO2-p with (beta-Gal-1-->(6-beta-Gal-1-->)n6-beta-Gal-OC6H4NO2-p, n = 1-4). No beta-(1-->3)-linked product was detected during the reaction. Use of the two readily available beta-D-galactosidases facilitates the preparation of (1-->3)- and (1-->6)-linked disaccharide glycosides of beta-D-Gal-GalNAc and beta-D-Gal-GlcNAc.     

Syntheses of arabinogalactans consisting of beta-(1-->6)-linked D-galactopyranosyl backbone and alpha-(1-->3)-linked L-arabinofuranosyl side chains

Two arabinogalactosyl nonasaccharides, beta-D-Galp-(1-->6)-[alpha-L-Araf-(1-->3)]-beta-D-Galp-(1-->6)-beta-D-Galp-(1-->6)-beta-D-Galp-(1-->6)-[alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->3)]-beta-D-Galp-(1-->6)-beta-D-Galp and beta-D-Galp-(1-->6)-[alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->3)]-beta-D-Galp-(1-->6)-beta-D-Galp-(1-->6)-beta-D-Galp-(1-->6)-[alpha-L-Araf-(1-->3)]-beta-D-Galp-(1-->6)-beta-D-Galp, were synthesized as their 4-methoxyphenyl glycosides with 2,3,4,6-tetra-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate (1), 6-O-acetyl-2,3,4-tri-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate (14), 4-methoxyphenyl 3-O-allyl-2,4-di-O-benzoyl-beta-D-galactopyranoside (2), 4-methoxyphenyl 2,3,4-tri-O-benzoyl-beta-D-galactopyranoside (5), 2,3,5-tri-O-benzoyl-alpha-L-arabinofuranosyl trichloroacetimidate (8), and 2,3,5-tri-O-benzoyl-alpha-L-arabinofuranosyl-(1-->5)-2,3-di-O-benzoyl-alpha-L-arabinofuranosyl trichloroacetimidate (11), as the key synthons. The tetra- (10) and pentasaccharide donor (13), and the tetra- (20) and pentasaccharide acceptor (22) were synthesized based on these synthons through simple transformations. Coupling of 22 with 10, and coupling of 20 with 13 and subsequent deacylation gave nonasaccharides 24 and 26, respectively, consisting of beta-(1-->6)-linked glactopyranosyl backbone and alpha-(1-->3)-linked arabinofuranosyl side chains of different size.