Reverse hydrolysis reaction of chitin deacetylase and enzymatic synthesis of beta-D-GlcNAc-(1-->4)-GlcN from chitobiose

We found that a chitin deacetylase from Colletotrichum lindemuthianum could acetylate free amino sugar residues into N-acetylated forms in the presence of 3.0 M sodium acetate. The result was analyzed using a beta-N-acetyl-hexosaminidase-coupled assay system with p-nitrophenyl 2-amino-2-deoxy-beta-D-glucopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta- D-glucopyranoside as the substrate, and the liberation of p-nitrophenol was observed as a consequence of enzymatic N-acetylation of the glucosamine residue at the nonreducing end of the substrate. The chitin deacetylase also acetylated chitobiose and chitotetraose as substrates, which was evidenced by the decrease in the amount of free amino sugar residues in the chitooligosaccharides. The reaction product of chitobiose after the acetylation reaction was exclusively 2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1-->4)-2-amino-2-deoxy-D-gluc ose [GlcNAcGlcN], the structure of which was determined by FABMS and NMR analyses. This study offers a novel method for enzymatic N-acetylation of amino sugars, and especially with chitobiose as substrate, a selectively N-acetylated product, GlcNAcGlcN, can be synthesized.     

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.     

An enzymatically produced novel cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->} (cyclic maltosyl-(1-->6)-maltose), from starch

A bacterial strain M6, isolated from soil and identified as Arthrobacter globiformis, produced a novel nonreducing oligosaccharide. The nonreducing oligosaccharide was produced from starch using a culture supernatant of the strain as enzyme preparation. The oligosaccharide was purified as a crystal preparation after alkaline treatment and deionization of the reaction mixture. The structure of the oligosaccharide was determined by methylation analysis, mass spectrometry, and (1)H and (13)C NMR spectroscopy, and it was demonstrated that the oligosaccharide had a cyclic structure consisting of four glucose residues joined by alternate alpha-(1-->4)- and alpha-(1-->6)-linkages. The cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->}, was found to be a novel oligosaccharide, and was tentatively called cyclic maltosyl-maltose (CMM). CMM was not hydrolyzed by various amylases, such as alpha-amylase, beta-amylase, glucoamylase, isoamylase, pullulanase, maltogenic alpha-amylase, and alpha-glucosidase, but hydrolyzed by isomalto-dextranase to give rise to isomaltose. This is the first report of the cyclic tetrasaccharide, which has alternate alpha-(1-->4)- and alpha-(1-->6)-glucosidic linkages.     

Mixed-linkage (1-->3,1-->4)-beta-D-glucan is a major hemicellulose of Equisetum (horsetail) cell walls

Mixed-linkage (1-->3,1-->4)-beta-d-glucan (MLG) is a hemicellulose reputedly confined to certain Poales. Here, the taxonomic distribution of MLG, and xyloglucan, especially in early-diverging pteridophytes, has been re-investigated. Polysaccharides were digested with lichenase and xyloglucan endoglucanase (XEG), which specifically hydrolyse MLG and xyloglucan, respectively. The oligosaccharides produced were analysed by thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) and alkaline peeling. Lichenase yielded oligo-beta-glucans from all Equisetum species tested (Equisetum arvense, Equisetum fluviatile, Equisetum scirpoides, Equisetum sylvaticum and Equisetum xtrachyodon). The major product was the tetrasaccharide beta-glucosyl-(1-->4)-beta-glucosyl-(1-->4)-beta-glucosyl-(1-->3)-glucose (G4G4G3G), which was converted to cellotriose by alkali, confirming its structure. Minor products included G3G, G4G3G and a nonasaccharide. By contrast, poalean MLGs yielded G4G3G > G4G4G3G > nonasaccharide > dodecasaccharide. No other pteridophytes tested contained MLG, including Psilotum and eusporangiate ferns. No MLG was found in lycopodiophytes, bryophytes, Chara or Nitella. XEG digestion showed that Equisetum xyloglucan has unusual repeat units. Equisetum, an exceedingly isolated genus whose closest living relatives diverged > 380 million years ago, has evolved MLG independently of the Poales. Equisetum and poalean MLGs share basic structural motifs but also exhibit clear-cut differences. Equisetum MLG is firmly wall-bound, and may tether neighbouring microfibrils. It is also suggested that MLG acts as a template for silica deposition, characteristic of grasses and horsetails.     

Occurrence of [--> 3)-beta-D-Manp-(1 --> 4)-beta-D-Manp-(1 -->]n units in the antigenic polysaccharides from Leptospira biflexa serovar patoc strain Patoc I

In this study, we isolated three kinds of antigenic polysaccharide components (tentatively designed as AP-1-3) from cells of Leptospira biflexa serovar patoc strain Patoc I (L. biflexa patoc Patoc I) by the hot phenol-water procedure, followed by treatment with mild acid and column chromatography. Two of them (AP-1 and AP-2) were recovered from the phenol-soluble fraction whereas another (AP-3) was recovered from the aqueous fraction. All of them reacted toward an anti-L. biflexa serum and also cross-reacted in similar extents toward most of the other leptospiral antisera tested. Such immunoreactions were specifically inhibited by a beta-(1 --> 4)-linked mannobiose, but were not by any mono- and oligosaccharide tested. From their structural analyses including 1H and 13C NMR spectrometry, Smith degradation and methylation analysis, it was revealed that all of these antigenic polysaccharides had the same disaccharide unit --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Manp-(1 --> in their major polysaccharide parts, but they differed in the acyl substituents. Therefore it is most likely that such mannobiose unit is a candidate for the antigenic epitopes of L. biflexa polysaccharides.     

Effect of (1-->3)- and (1-->4)-linkages of fully sulfated polysaccharides on their anticoagulant activity

Chemically fully sulfated polysaccharides including xylan (-->4Xylbeta-(1-->4)Xylbeta1-->), amylose (-->4Glcalpha-(1-->4)Glcalpha1-->), cellulose (-->4Glcbeta-(1-->4)Glcbeta1-->), curdlan (-->3Glcbeta-(1-->3)Glcbeta1-->) and galactan (-->3Galbeta-(1-->3)Galbeta1-->), which have been isolated from Korean clam, were prepared, and their anticoagulant activity was investigated. The results strongly suggest that the activity might not be depending on anomeric configuration (alpha or beta) or monosaccharide species but on the glycosidic linkage, either (1-->3) or (1-->4). 1H NMR studies of these modified polysaccharides show that the neighboring sulfate groups at the C-2 and C-3 positions might have caused the conformational changes of each monosaccharide from 4C(1) to 1C(4). Furthermore, the effect of 6-sulfate residues on the anticoagulant activity was investigated using a specific desulfated reaction for the chemically fully sulfated polysaccharides. The 6-sulfate group is very important in determining anticoagulant activity of (1-->3)-linked polysaccharides, whereas the activity is not affected by presence or absence of the 6-sulfate group in (1-->4)-linked polysaccharides.     

Probing the substrate specificity of four different sialyltransferases using synthetic beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) (CH(2))7CH3 analogues general activating effect of replacing N-acetylglucosamine by N-propionylglucosamine

The acceptor specificities of ST3Gal III, ST3Gal IV, ST6Gal I and ST6Gal II were investigated using a panel of beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH(2))(7)CH(3) analogues. Modifications introduced at either C2, C3, C4, C5, or C6 of terminal D-Gal, as well as N-propionylation instead of N-acetylation of subterminal D-GlcN were tested for their influence on the alpha-2,3- and alpha-2,6-sialyltransferase acceptor activities. Both ST3Gal enzymes displayed the same narrow acceptor specificity, and only accept reduction of the Gal C2 hydroxyl function. The ST6Gal enzymes, however, do not have the same acceptor specificity. ST6Gal II seems less tolerant towards modifications at Gal C3 and C4 than ST6Gal I, and prefers beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc (LacdiNAc) as an acceptor substrate, as shown by replacing the Gal C2 hydroxyl group with an N-acetyl function. Finally, a particularly striking feature of all tested sialyltransferases is the activating effect of replacing the N-acetyl function of subterminal GlcNAc by an N-propionyl function.     

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.     

A concise and practical synthesis of antigenic globotriose, alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-Glc

A concise and practical synthesis of the antigenic globotriose, alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-Glc (13), was achieved by coupling of a monosaccharide donor, 3-O-allyl-2-O-benzoyl-4,6-O-benzylidene-alpha-D-galactopyranosyl trichloroacetimidate (4) with a disaccharide acceptor, p-methoxyphenyl 2,3,6-tri-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranoside (8), followed by deprotection. In spite of the existence of a C-2-ester substituent capable of neighboring-group participation in the donor, the coupling gave exclusively the alpha-linkage in satisfactory yield. The acceptor 8 was readily obtained from selective 3-O-benzoylation of the galactosyl ring of p-methoxyphenyl 2,6-di-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranoside (7), which was prepared from p-methoxyphenyl beta-D-lactoside (5) via isopropylidenation, benzoylation, and deisopropylidenation. Donor 4 was obtained from p-methoxylphenyl 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranoside (1) via selective 4,6-di-O-debenzoylation, oxidative removal of 1-O-MP, benzylidenation, and trichloroacetimidate formation.     

Type I arabinogalactan contains beta-D-Galp-(1-->3)-beta-D-Galp structural elements

Arabinogalactan type I from potato was partially degraded by endo-galactanase from Aspergillus niger. High-performance anion-exchange chromatography revealed that several of the oligomeric degradation products eluted as double peaks. To investigate the nature of these products, the digest was fractionated by Bio-Gel P2 chromatography. The pool that contained tetramers was treated with a beta-D-Galp-(1-->4)-specific galactosidase from Bifidobacterium adolescentis to obtain a dimer with deviating linkage type, which was further purified by BioGel P2 chromatography. By obtaining all (1)H and (13)C chemical shifts and the presence of intra residual scalar coupling (HMBC) it could be concluded that the dimer contained a beta-(1-->3)-linkage instead of the expected beta-(1-->4)-linkage. Using the same NMR techniques as for the dimer, it was found that the pool of tetramers consisted of the following two galactose tetramers: beta-Galp-(1-->4)-beta-Galp-(1-->4)-beta-Galp-(1-->4)-alpha/beta-Galp-OH and beta-Galp-(1-->4)-beta-Galp-(1-->4)-beta-Galp-(1-->3)-alpha/beta-Galp-OH. The fact that the deviating beta-(1-->3)-linked galactose was found at the reducing end of the dimer showed that this deviating linkage is present within the backbone. The beta-(1-->3)-galactosyl interruption appeared to be a common structural feature of type I arabinogalactans with a frequency ranging from approximately 1 in 160 (potato, soy, citrus) to 1 in 250 (onion).     

Synthesis of methyl alpha-D-glucopyranosyl-(1-->4)-alpha-D-galactopyranoside and methyl alpha-D-xylo-hex-4-ulopyranosyl-(1-->4)-alpha-D-galactopyranoside

The syntheses of methyl alpha-D-glucopyranosyl-(1-->4)-alpha-D-galactopyranoside (1) and methyl alpha-D-xylo-hex-4-ulopyranosyl-(1-->4)-alpha-D-galactopyranoside (4) are reported. The keto-disaccharide 4 is of interest in our design, synthesis, and study of pectate lyase inhibitors. The key step in the syntheses was the high-yielding, stereospecific formation of methyl 4,6-O-benzylidene-2',3'-di-O-benzyl-alpha-D-glucopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-alpha-D-galactopyranoside (15), which was accomplished by reacting 2,3-di-O-benzyl-4,6-O-benzylidene-D-glucopyranosyl trichloroacetimidate (10) with methyl 2,3,6-tri-O-benzyl-alpha-D-galactopyranoside (14) in the presence of a catalytic amount of tert-butyldimethylsilyl trifluoromethane sulfonate (TMSOTF). Compound 15 was either hydrogenolyzed to yield disaccharide 1 or treated with NaBH3CN-HCl in 1:1 tetrahydrofuran-ether to yield methyl 2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-alpha-D-galactopyranoside (2). The free 4'-OH of compound 2 was oxidized to a carbonyl group by a Swern oxidation, and the protecting groups were removed by hydrogenolysis to yield keto-disaccharide 4. These synthetic pathways were simple, yet high yielding.     

Mutant barley (1-->3,1-->4)-beta-glucan endohydrolases with enhanced thermostability

The similar three-dimensional structures of barley (1-->3)-beta-glucan endohydrolases and (1-->3,1-->4)-beta-glucan endohydrolases indicate that the enzymes are closely related in evolutionary terms. However, the (1-->3)-beta-glucanases hydrolyze polysaccharides of the type found in fungal cell walls and are members of the pathogenesis-related PR2 group of proteins, while the (1-->3,1-->4)-beta-glucanases function in plant cell wall metabolism. The (1-->3)-beta-glucanases have evolved to be significantly more stable than the (1-->3,1-->4)-beta-glucanases, probably as a consequence of the hostile environments imposed upon the plant by invading microorganisms. In attempts to define the molecular basis for the differences in stability, eight amino acid substitutions were introduced into a barley (1-->3,1-->4)-beta-glucanase using site-directed mutagenesis of a cDNA that encodes the enzyme. The amino acid substitutions chosen were based on structural comparisons of the barley (1-->3)- and (1-->3,1-->4)-beta-glucanases and of other higher plant (1-->3)-beta-glucanases. Three of the resulting mutant enzymes showed increased thermostability compared with the wild-type (1-->3,1-->4)-beta-glucanase. The largest increase in stability was observed when the histidine at position 300 was changed to a proline (mutant H300P), a mutation that was likely to decrease the entropy of the unfolded state of the enzyme. Furthermore, the three amino acid substitutions which increased the thermostability of barley (1-->3,1-->4)-beta-glucanase isoenzyme EII were all located in the COOH-terminal loop of the enzyme. Thus, this loop represents a particularly unstable region of the enzyme and could be involved in the initiation of unfolding of the (1-->3,1-->4)-beta-glucanase at elevated temperatures.     

Separation of Galfbeta1-->XGlcNAc and Galpbeta1-->XGlcNAc (X = 3, 4, and 6) as the alditols by high-pH anion-exchange chromatography and thin-layer chromatography: characterization of mucins from Trypanosoma cruzi

The O-linked N-acetylglucosamine oligosaccharides in the mucins of Trypanosoma cruzi may contain galactofuranose or galactopyranose, depending on the strain, one of the components being the disaccharide. Since galactofuranose is a site for antibody recognition, it is desirable to have a sensitive method for the detection of the galactofuranosyl structures. In this paper, we present procedures for the separation of Galfbeta1-->XGlcNAc and Galpbeta1-->XGlcNAc (X = 3, 4, and 6) as the corresponding alditols by high-pH anion-exchange chromatography with pulse amperometric detection. All the isomeric disaccharides could be resolved on a CarboPac PA-10 column, the galactofuranose-containing disaccharides being more retained in the column. GlcNAcol and Galfbeta1-->4(Galpbeta1-->6)GlcNAcol could be analyzed in the same run. The compounds could also be separated by thin-layer chromatography on silica gel 60, a convenient method for analysis of the radiolabeled alditols obtained by reductive beta-elimination in the presence of NaB(3)H(4). Both methods were applied for the analysis of the O-linked sugars in the mucins of T. cruzi CL 14 and revealed that they contained only N-acetylglucosamine and the disaccharide Galpbeta1-->4GlcNAc.     

A practical synthesis of beta-D-GlcA-(1-->3)-beta-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-D-Xyl,a part of the common linkage region of a glycosaminoglycan

A practical synthesis of beta-D-GlcA-(1-->3)-beta-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-beta-D-Xyl-(1-->OMe) was achieved by coupling of methyl 2,3,4-tri-O-acetyl-alpha-D-glucopyranosyluronate trichloroacetimidate with a trisaccharide acceptor. The trisaccharide acceptor was obtained by condensation of 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate with methyl 2,3-di-O-benzoyl-beta-D-xylopyranoside, followed by deallylation. The beta-(1-->3)-linked disaccharide was prepared readily with p-methoxyphenyl 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranoside as the key synthon. The alpha-(1-->3)-linkage was formed in considerable amount with galactose mono- and disaccharide trichloroacetimidate donors with C-2 neighboring group participation.     

Enantioseparation properties of (1-->6)-alpha-D-glucopyranan and (1-->6)-alpha-D-mannopyranan tris(phenylcarbamate)s as chiral stationary phases in HPLC

Synthetic polysaccharides, (1-->6)-alpha-D-glucopyranan (3a) and (1-->6)-alpha-D-mannopyranan (3b), were prepared by the cationic ring-opening polymerization of 1,6-anhydro-2,3,4-tri-O-allyl-beta-D-glucopyranose (1a) and 1,6-anhydro-2,3,4-O-6-allyl-beta-D-mannopyranose (1b), followed by the cleavage of the allyl ether linkage of 2,3,4-tri-O-allyl-(1-->6)-alpha-D-glucopyranan (2a) and 2,3,4-tri-O-allyl-(1-->6)-alpha-D-mannopyranan (2b), respectively. 2,3,4-Tris-O-(3,5-dimethylphenylcarbamoyl)- and 2,3,4-tris-O-(3,5-dichlorophenylcarbamoyl)-(1-->6)-alpha-D-glucopyranan (CSP-1 and CSP-2, respectively) and 2,3,4-tris-O-(3,5-dimethylphenylcarbamoyl)- and 2,3,4-tris-O-(3,5-dichlorophenylcarbamoyl)-(1-->6)-alpha-D-mannopyranan (CSP-3 and CSP-4, respectively) were prepared by the reaction of 3 with the corresponding 3,5-disubstituted phenylisocyanates and the chiral recognition abilities of CSP-1-4 as chiral stationary phases (CSPs) in high-performance liquid chromatography (HPLC) were evaluated. Racemic compounds such as trans-cyclopropanedicarboxylic acid dianilide (9), 1,2,2,2-tetraphenylethanol (10), flavanone (11), Tr?ger's base (12), benzoin (13), and cobalt(III) tris(acetylacetonate) (14) were efficiently resolved using CSP-1-4. For comparison among CSPs, the chiral recognition properties of the (1-->6)-alpha-D-glucopyranan CSPs were different from that of the (1-->6)-alpha-D-mannopyranan CSPs, and CSP-4 exhibited the highest chiral recognition ability among the CSPs. The resolution factors of 12 and 14 were 0.42 and 0.56 for CSP-1, 0.32 and 2.16 for CSP-2, 1.80 and 0.84 for CSP-3, and 2.31 and 8.26 for CSP-4, respectively.     

Purification and characterization of cyclic maltosyl-(1-->6)-maltose hydrolase and alpha-glucosidase from an Arthrobacter globiformis strain

Cyclic maltosyl-maltose [CMM, cyclo-[-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]], a novel cyclic tetrasaccharide, has a unique structure. Its four glucose residues are joined by alternate alpha-1,4 and alpha-1,6 linkages. CMM is synthesized from starch by the action of 6-alpha-maltosyltransferase from Arthrobacter globiformis M6. Recently, we determined the mechanism of extracellular synthesis of CMM, but the degrading pathway of the saccharide remains unknown. Hence we tried to identify the enzymes involved in the degradation of CMM to glucose from the cell-free extract of the strain, and identified CMM hydrolase (CMMase) and alpha-glucosidase as the responsible enzymes. The molecular mass of CMMase was determined to be 48.6 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and 136 kDa by gel filtration column chromatography. The optimal pH and temperature for CMMase activity were 6.5 and 30 degrees C. The enzyme remained stable from pH 5.5 to 8.0 and up to 25 degrees C. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates, but it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes the alpha-1,6-linkage of CMM. The molecular mass of alpha-glucosidase was determined to be 60.1 kDa by SDS-PAGE and 69.5 kDa by gel filtration column chromatography. The optimal pH and temperature for alpha-glucosidase activity were 7.0 and 35 degrees C. The enzyme remained stable from pH 7.0 to 9.5 and up to 35 degrees C. alpha-Glucosidase degraded maltosyl-maltose to glucose via panose and maltose as intermediates, but it did not degrade CMM. Furthermore, when CMMase and alpha-glucosidase existed simultaneously in a reaction mixture containing CMM, glucose was detected as the final product. It was found that CMM was degraded to glucose by the synergistic action of CMMase and alpha-glucosidase.     

Synthesis and biological activities of octyl 2,3-di-O-sulfo-alpha-L-fucopyranosyl-(1-->3)-2-O-sulfo-alpha-L-fucopyranosyl-(1-->4)-2,3-di-O-sulfo-alpha-L-fucopyranosyl-(1-->3)-2-O-sulfo-alpha-L-fucopyranosyl-(1-->4)-2,3-di-O-sulfo-beta-L-fucopyranoside

Octyl 2,3-di-O-sulfo-alpha-L-fucopyranosyl-(1-->3)-2-O-sulfo-alpha-L-fucopyranosyl-(1-->4)-2,3-di-O-sulfo-alpha-L-fucopyranosyl-(1-->3)-2-O-sulfo-alpha-L-fucopyranosyl-(1-->4)-2,3-di-O-sulfo-beta-L-fucopyranoside, a fucosyl pentasaccharide with a regular structure resembling the repeating unit of a natural sulfated fucan, was chemically synthesized using a convergent '2+3' strategy. Regioselective 3-O-silylation of beta-thiofucopyranoside and AgOTf-catalyzed glycosylation of the protected glycosyl trichloroacetimidate facilitated a one-pot trisaccharide synthesis. The synthesized target compound showed good antitumor activity in vivo, and promising anticoagulant activity in vitro.     

Sulfated beta-(1-->4)-galacto-oligosaccharides and their effect on angiogenesis

Sulfated beta-(1-->4)-galacto-oligosaccharides were prepared from an arabino-galacto-rhamno-galacturonan from Lupinus polyphyllus Lindl. by successive partial hydrolysis and SO3-pyridine sulfation in DMF. The resulting oligosaccharide polysulfates were analyzed by analytical GPC and the sulfate content was determined by ion chromatography. DP 5 and higher showed a pronounced antiangiogenic effect with scores of 0.9-1.2 for DP 7-9 using the CAM-assay. An interaction with the fibroblast growth factor FGF-2 was noticed for DP 4-12 depending on the degree of sulfation using the FGF-2-trypsin assay.     

Synthesis and stability assay of 4-methylumbelliferyl (1-->3)-beta-D-pentaglucoside

The synthesis and stability of 4-methylumbelliferyl (1 --> 3)-beta-D-pentaglucoside 3 are described. The (1 --> 3)-beta-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method. The acid-solubilized (1 --> 3)-beta-D-oligoglucosides were prepared by partial acid hydrolysis of glucan. The peracetylated (1 --> 3)-beta-D-pentaglucoside 1 was obtained by isolation of peracetylated (1 --> 3)-beta-D-oligoglucoside mixture. The peracetylated 4-methylumbelliferyl (1 --> 3)-beta-D-pentaglucoside 2 was synthesized by treating compound 1 with the 4-methylumbelliferone and a Lewis acid (SnCl4) catalyst. NaOMe in dry methanol was used for the deacetylation of the blocked derivative, to give the target compound 3 in an overall yield of 35%. Activity assays with beta-glucosidase indicated that compound 3 was much more stable than the corresponding pentasaccharide.