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).     

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.     

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.     

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.     

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.     

Remote control of alpha- or beta-stereoselectivity in (1-->3)-glucosylations in the presence of a C-2 ester capable of neighboring-group participation

In (1-->3)-glucosylation the glycosyl bond originally present in either donor or acceptor is shown to control the stereoselectivity of the forthcoming bond, i.e., the newly formed glycosidic linkage has the opposite anomeric configuration of that of either the donor or acceptor. Therefore, with alpha-(1-->3)-linked disaccharides with nonreducing ends that have the 3-OH free as the acceptor and an acetylated glucosyl trichloroacetimidate as the donor, or with an alpha-(1-->3)-linked acetylated disaccharide trichloroacetimidate as the donor and a glucoside with 3-OH free as the acceptor, beta-linked trisaccharides were obtained. Meanwhile, with beta-(1-->3)-linked disaccharides that have nonreducing ends with the 3-OH free as the acceptor and an acetylated glucosyl trichloroacetimidate as the donor, or with a beta-(1-->3)-linked acetylated disaccharide trichloroacetimidate as the donor and a glucoside with the 3-OH free as the acceptor, alpha-linked trisaccharides were obtained in spite of the C-2 neighboring group participation.     

Theory and computation show that Asp463 is the catalytic proton donor in human endoplasmic reticulum alpha-(1-->2)-mannosidase I

It has been difficult to identify the proton donor and nucleophilic assistant/base of endoplasmic reticulum alpha-(1-->2)-mannosidase I, a member of glycoside hydrolase Family 47, which cleaves the glycosidic bond between two alpha-(1-->2)-linked mannosyl residues by the inverting mechanism, trimming Man(9)GlcNAc(2) to Man(8)GlcNAc(2) isomer B. Part of the difficulty is caused by the enzyme's use of a water molecule to transmit the proton that attacks the glycosidic oxygen atom. We earlier used automated docking to conclusively determine that Glu435 in the yeast enzyme (Glu599 in the corresponding human enzyme) is the nucleophilic assistant. The commonly accepted proton donor has been Glu330 in the human enzyme (Glu132 in the yeast enzyme). However, for theoretical reasons this conclusion is untenable. Theory, automated docking of alpha-d-(3)S(1)-mannopyranosyl-(1-->2)-alpha-d-(4)C(1)-mannopyranose and water molecules associated with candidate proton donors, and estimation of dissociation constants of the latter have shown that the true proton donor is Asp463 in the human enzyme (Asp275 in the yeast enzyme).     

Concise syntheses of arabinogalactans with beta-(1-->6)-linked galactopyranose backbones and alpha-(1-->3)- and alpha-(1-->2)-linked arabinofuranose side chains

4-methoxyphenyl glycosides of 2,3'-bis-alpha-L-arabinofuranosyl branched beta-D-(1-->6)-linked galactopyranosyl tetraose (16), 3',2'-bis-alpha-L-arabinofuranosyl branched beta-D-(1-->6)-linked galactopyranosyl hexaose (27), and a twentyose (42) consisting of beta-(1-->6)-linked D-galactopyranosyl pentadecaoligosaccharide backbone with alpha-L-arabinofuranosyl side chains alternately attached at C-2 and C-3 of the middle galactose residue of each consecutive beta-(1-->6)-linked galactotriose unit of the backbone, were synthesized with isopropyl 3-O-allyl-2,4-di-O-benzoyl-1-thio-beta-D-galactopyranoside (6), 2,3,4,6-tetra-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate (7), 2,3,5-tri-O-benzoyl-alpha-L-arabinofuranosyl trichloroacetimidate (12), 6-O-acetyl-2,3,4-tri-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate (17), 4-methoxyphenyl 2,3,4-tri-O-benzoyl-beta-D-galactopyranoside (19), and 2,6-di-O-acetyl-3,4-di-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate (28) as the key synthons. Condensation of 6 with 7 gave the disaccharide donor 8, and subsequent condensation of 8 with 4-methoxyphenyl 2,3,4-tri-O-benzoyl-beta-D-galactopyranosyl-(1-->6)-2-O-acetyl-3,4-di-O-benzoyl-beta-D-galactopyranoside (9) followed by selective deacetylation afforded the tetrasaccharide acceptor 11. Coupling of 11 with 12 gave the pentasaccharide 13, its deallylation followed by coupling with 12, and debenzoylation gave the hexasaccharide 16 with beta-(1-->6)-linked galactopyranose backbone and 2- and 3'-linked alpha-L-arabinofuranose side chains. The octasaccharide 27 was similarly synthesized, while the twentyoside 42 was synthesized with tetrasaccharides 33 or 24 as the donors and 23, 36, 38, and 40 as the acceptors by consecutive couplings followed by deacylation.     

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.     

First total synthesis of alpha-(2-->3)/alpha-(2-->6)-disialyl lactotetraosyl ceramide and disialyl Lewis A ganglioside as cancer-associated carbohydrate antigens

The first total synthesis of alpha-(2-->3)/alpha-(2-->6)-disialyl lactotetraosyl (DSLc4) ceramide and alpha-(2-->3)/alpha-(2-->6)-disialyl Lewis A (DSLe(a)) ganglioside as cancer-associated antigens is described. The suitably protected lactotriose (Lc3) derivatives were successively glycosylated with sialic acid, sialyl-alpha-(2-->3)-D-galactose and/or L-fucose donors in a regio- and stereo-selective manner, to give the protected type I hexa- and hepta-saccharides, respectively, which were then converted to the target gangliosides by the introduction of ceramide and subsequent complete deprotection.     

Automated docking of alpha-(1-->4)- and alpha-(1-->6)-linked glucosyl trisaccharides and maltopentaose into the soybean beta-amylase active site

The Lamarckian genetic algorithm of AutoDock 3.0 was used to dock alpha-maltotriose, methyl alpha-panoside, methyl alpha-isopanoside, methyl alpha-isomaltotrioside, methyl alpha-(6(1)-alpha-glucopyranosyl)-maltoside, and alpha-maltopentaose into the closed and, except for alpha-maltopentaose, into the open conformation of the soybean beta-amylase active site. In the closed conformation, the hinged flap at the mouth of the active site closes over the substrate. The nonreducing end of alpha-maltotriose docks preferentially to subsites -2 or +1, the latter yielding nonproductive binding. Some ligands dock into less optimal conformations with the nonreducing end at subsite -1. The reducing-end glucosyl residue of nonproductively-bound alpha-maltotriose is close to residue Gln194, which likely contributes to binding to subsite +3. In the open conformation, the substrate hydrogen-bonds with several residues of the open flap. When the flap closes, the substrate productively docks if the nonreducing end is near subsites -2 or -1. Trisaccharides with alpha-(1-->6) bonds do not successfully dock except for methyl alpha-isopanoside, whose first and second glucosyl rings dock exceptionally well into subsites -2 and -1. The alpha-(1-->6) bond between the second and third glucosyl units causes the latter to be improperly positioned into subsite +1; the fact that isopanose is not a substrate of beta-amylase indicates that binding to this subsite is critical for hydrolysis.     

Molecular dynamics simulations of a cyclic-beta-(1-->2) glucan containing an alpha-(1-->6) linkage as a 'molecular alleviator' for the macrocyclic conformational strain

The conformational preferences of a cyclic osmoregulated periplasmic glucan of Ralstonia solanacearum (OPGR), which is composed of 13 glucose units and linked entirely via beta-(1-->2) linkages excluding one alpha-(1-->6) linkage, were characterized by molecular dynamics simulations. Of the three force fields modified for carbohydrates that were applied to select a suitable one for the cyclic glucan, the carbohydrate solution force field (CSFF) was found to most accurately simulate the cyclic molecule. To determine the conformational characteristics of OPGR, we investigated the glycosidic dihedral angle distribution, fluctuation, and the potential energy of the glucan and constructed hypothetical cyclic (CYS13) and linear (LINEAR) glucans. All beta-(1-->2)-glycosidic linkages of OPGR adopted stable conformations, and the dihedral angles fluctuated in this energy region with some flexibility. However, despite the inherent flexibility of the alpha-(1-->6) linkage, the dihedral angles have no transition and are more rigid than that in a linear glucan. CYS13, which consists of only beta-(1-->2) linkages, is somewhat less flexible than other glycans, and one of its linkages adopts a higher energy conformation. In addition, the root-mean-square fluctuation of this linkage is lower than that of other linkages. Furthermore, the potential energy of glucans increases in the order of LINEAR, OPGR, and CYS13. These results provide evidence of the existence of conformational constraints in the cyclic glucan. The alpha-(1-->6)-glycosidic linkage can relieve this constraint more efficiently than the beta-(1-->2) linkage. The conformation of OPGR can reconcile the tendency for individual glycosidic bonds to adopt energetically favorable conformations with the requirement for closure of the macrocyclic ring by losing the inherent flexibility of the alpha-(1-->6)-glycosidic linkage.     

Molecular characterization of a novel glucosyltransferase from Lactobacillus reuteri strain 121 synthesizing a unique,highly branched glucan with alpha-(1-->4) and alpha-(1-->6) glucosidic bonds

Lactobacillus reuteri strain 121 produces a unique, highly branched, soluble glucan in which the majority of the linkages are of the alpha-(1-->4) glucosidic type. The glucan also contains alpha-(1-->6)-linked glucosyl units and 4,6-disubstituted alpha-glucosyl units at the branching points. Using degenerate primers, based on the amino acid sequences of conserved regions from known glucosyltransferase (gtf) genes from lactic acid bacteria, the L. reuteri strain 121 glucosyltransferase gene (gtfA) was isolated. The gtfA open reading frame (ORF) was 5,343 bp, and it encodes a protein of 1,781 amino acids with a deduced M(r) of 198,637. The deduced amino acid sequence of GTFA revealed clear similarities with other glucosyltransferases. GTFA has a relatively large variable N-terminal domain (702 amino acids) with five unique repeats and a relatively short C-terminal domain (267 amino acids). The gtfA gene was expressed in Escherichia coli, yielding an active GTFA enzyme. With respect to binding type and size distribution, the recombinant GTFA enzyme and the L. reuteri strain 121 culture supernatants synthesized identical glucan polymers. Furthermore, the deduced amino acid sequence of the gtfA ORF and the N-terminal amino acid sequence of the glucosyltransferase isolated from culture supernatants of L. reuteri strain 121 were the same. GTFA is thus responsible for the synthesis of the unique glucan polymer in L. reuteri strain 121. This is the first report on the molecular characterization of a glucosyltransferase from a Lactobacillus strain.     

Syntheses of beta-(1-->6)-branched beta-(1-->3)-linked d-galactans that exist in the rhizomes of Atractylodes lancea DC

Effective syntheses of galactose hepta-, octa-, nona-, and decasaccharides that exist in the rhizomes of Atractylodes lancea DC were achieved with 2,3,4,6-tetra-O-benzoyl-alpha-d-galactopyranosyl trichloroacetimidate (1), 4-methoxyphenyl 2,3,4-tri-O-benzoyl-beta-d-galactopyranoside (2), 6-O-acetyl-2,3,4-tri-O-benzoyl-alpha-d-galactopyranosyl trichloroacetimidate (5), 4-methoxyphenyl 6-O-acetyl-2,4-di-O-benzoyl-beta-d-galactopyranoside (22), and 4-methoxyphenyl 2,4,6-tri-O-benzoyl-beta-d-galactopyranoside (26) as the key synthons. Coupling of 2 with 1, followed by oxidative cleavage of 1-OMP and subsequent trichloroacetimidate formation gave the beta-(1-->6)-linked disaccharide donor 4. Condensation of 2 with 5 and subsequent selective deacetylation by methanolysis produced the beta-(1-->6)-linked disaccharide acceptor 7. Reaction of 7 with 4, oxidative cleavage of 1-OMP, and trichloroacetimidate formation produced the tetrasaccharide donor 9. The penta- (15), the hexa- (17), and the heptasaccharide donor 19 were synthesized similarly. Meanwhile, treatment of 1 with 22 yielded beta-(1-->3)-linked disaccharide 23 and alpha-(1-->3)-linked disaccharide 25. Oxidative cleavage of 1-OMp of 23 followed by trichloroacetimidate formation produced the disaccharide donor 24. Coupling of 26 with 24, again, gave beta-linked 27 and alpha-linked 29. Selective 6-O-deacetylation of 27 afforded the trisaccharide acceptor 28. TMSOTf-promoted condensation 28 of with the tetra- (9), penta- (15), hexa-(17), and heptasaccharide donor 19, followed by deprotection, gave the target compounds.     

Solution properties of antitumor sulfated derivative of alpha-(1-->3)-D-glucan from Ganoderma lucidum

Four fractions of a water-insoluble alpha-(1-->3)-D-glucan GL extracted from fruiting bodies of Ganoderma lucidum were dissolved in 0.25 M LiCl/DMSO, and then reacted with sulfur trioxide-pyridine complex at 80 degrees C to synthesize a series of water-soluble sulfated derivatives S-GL. The degree of substitution of DS was measured by using IR infrared spectra, elemental analysis, and 13C NMR to be 1.2-1.6 in the non-selective sulfation. Weight-average molecular weight Mw and intrinsic viscosity [eta] of the sulfated derivatives S-GL were measured by multi-angle laser light scattering and viscometry. The Mw value (2.4 x 10(4)) of sulfated glucan S-GL-1 was much lower than that (44.5 x 10(4)) of original alpha-(1-->3)-D-glucan GL-1. The Mark-Houwink equation and average value of characteristic ratio C(infinity) for the S-GL in 0.2 M NaCl aqueous solution at 25 degrees C were found to be: [eta] = 1.32 x 10(-3) Mw(1.06) (cm3 g(-1)) and 16, respectively, in the Mw range from 1.1 x 10(4) to 2.4 x 10(4). It indicated that the sulfated derivatives of the alpha-(1-->3)-D-glucan in the aqueous solution behave as an expanded chain, owing to intramolecular hydrogen bonding or interaction between charge groups. Interestingly, two sulfated derivatives synthesized from the alpha-(1-->3)-D-glucan and curdlan, a beta-(1-->3)-D-glucan, all had significant higher antitumor activity against Ehrlich ascites carcinoma (EAC) than the originals. The effect of expanded chains of the sulfated glucan in the aqueous solution on the improvement of the antitumor activity could not be negligible.     

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.     

Efficient chemoenzymatic synthesis of globotriose and its derivatives with a recombinant alpha-(1-->4)-galactosyltransferase

A truncated alpha-(1-->4)-galactosyltransferase (LgtC) gene from Neisseria meningitidis was cloned. The recombinant glycosyltransferase was expressed in Escherichia coli BL21 (DE3) strain with high specific activity (5 units/mg protein). Its acceptor specificity was carefully characterized. Then the purified enzyme was utilized in highly efficient syntheses of globotriose and a variety of alpha-(1-->4)-galactosylated derivatives as potential antibacterial agents.     

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.     

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.     

Modified mannose disaccharides as substrates and inhibitors of a polyprenol monophosphomannose-dependent alpha-(1-->6)-mannosyltransferase involved in mycobacterial lipoarabinomannan biosynthesis

A panel of alpha-(1-->6)-linked mannose disaccharides (5-8) in which the 2'-OH group has been replaced, independently, by deoxy, fluoro, amino, and methoxy functionalities has been synthesized. Evaluation of these compounds as potential substrates or inhibitors of a polyprenol monophosphomannose-dependent alpha-(1-->6)-mannosyltransferase involved in mycobacterial LAM biosynthesis demonstrated that the enzyme is somewhat tolerant substitution at this site. The enzyme recognizes the disaccharides with groups similar or smaller in size than the native hydroxyl (6-8), but not the disaccharide with the more sterically demanding methoxy group (5). The 2'-OH appears not form a critical hydrogen bonding interaction with the protein as the 2'-deoxy analog is a substrate for the enzyme.