Facile synthesis of cleistetroside-2, a partially acetylated oligorhamnoside from Cleistopholis glauca and patens

A tetrasaccharide, dodecanyl 4-O-acetyl-alpha-l-rhamnopyranosyl-(1-->3)-2,4-di-O-acetyl-alpha-l-rhamnopyranosyl-(1-->3)-4-O-acetyl-alpha-l-rhamnopyranosyl-(1-->4)-alpha-l-rhamnopyranoside (cleistetroside-2), was synthesized via '2+2' convergent strategy. Sequential regioselective 3-O-glycosylation of isopropyl 1-thio-alpha-l-rhamnopyranoside (4) with 4-O-acetyl-2,3-O-isopropylidene-alpha-l-rhamnopyranosyl trichloroacetimidate (8), and isopropyl 4-O-acetyl-2,3-O-isopropylidene-alpha-l-rhamnopyranosyl-(1-->3)-2,4-di-O-acetyl-alpha-l-1-thio-rhamnopyranoside (10) with dodecanyl 4-O-acetyl-alpha-l-rhamnopyranosyl-(1-->4)-2,3-O-isopropylidene-alpha-l-rhamnopyranoside (12), greatly facilitate the target availability.     

The structure of the O-antigen of Escherichia coli O116:K+:H10

The primary structure of the O-antigen of Escherichia coli O116:K+:H10 was shown by monosaccharide analysis, a partial hydrolysis study and by 1D and 2D 1H and 13C NMR spectroscopy to be composed of linear pentasaccharide repeating units with the structure: -->6)-alpha-D-GlcpNAc-(1-->4)-alpha-D-GalpNAc-(1-->4)-alpha-D-GalpA++ +-(1-->3)- beta-D-GlcpNAc-(1-->2)-beta-D-Quip4NAc-(1-->.     

Syntheses of a series of lacto-N-neotetraose clusters using a carbosilane dendrimer scaffold

4-Pentenyl (2,3,4,6-tetra-O-acetyl-beta-d-galactopyranosyl)-(1-->4)-(3,6-di-O-acetyl-2-deoxy-2-phthalimido-beta-d-glucopyranosyl)-(1-->3)-(2,6-di-O-benzoyl-beta-d-galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzoyl-beta-d-glucopyranoside (4) was synthesized by regioselective glycosylation of 4-pentenyl (2,6,-di-O-benzoyl-beta-d-galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzoyl-beta-d-glucopyranoside and (2,3,4,6-tetra-O-acetyl-beta-d-galactopyranosyl)-(1-->4)-3,6-di-O-acetyl-2-deoxy-2-phthalimido-beta-d-glucopyranosyl chloride. By conversion of the protecting groups followed by thioacetylation, 4 was transformed into the corresponding lacto-N-neotetraose derivative, 5-(acetylthio)pentenyl (2,3,4,6-tetra-O-acetyl-beta-d-galactopyranosyl)-(1-->4)-O-(3,6-di-O-acetyl-2-acetamido-2-deoxy-beta-d-glucopyranosyl)-(1-->3)-(2,4,6-di-O-acetyl-beta-d-galactopyranosyl)-(1-->4)-2,3,6-tri-O-acetyl-beta-d-glucopyranoside (6). The lacto-N-neotetraose derivative 6 was introduced into carbosilane dendrimer cores of three shapes, and three kinds of new carbosilane dendrimers peripherally functionalized by lacto-N-neotetraose were obtained.     

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.     

Modulation of sodium pumps by steroidal saponins

Costus spicatus, used in Brazilian traditional medicine to expel kidney stones, contains steroidal saponins with different chemical characteristics. In spite of its popular utilization as potent diuretic, no scientific reports correlate this activity with the chemical constituents of the extract. Therefore, two steroidal saponins (3 beta,22 alpha,25R)-26-(beta-D-glucopyranosyloxy)-2-methoxyfurost-5-en-3-yl O-D-apio-beta-D-furanosyl-(1-->2)-O-[6-deoxy-alpha-L-mannopyranosyl-(1-->4)]-beta-D-glucopyranoside (1) and (3 beta,22 alpha,25R)-spirostan-3-yl O-D-apio-beta-D-furanosyl-(1-->2)-O-[6-deoxy-alpha-L-mannopyranosyl-(1-->4)]-beta-D-glucopyranoside (1a), were isolated from the rhizomes of this plant and their effects on the Na+-ATPase and (Na+ + K+)-ATPase activities of the proximal tubule from pig kidney were evaluated. It was observed that 1 and 1a inhibit specifically the Na+-ATPase activity.     

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.     

A new steroidal saponin from AGAVE brittoniana and its biphasic effect on the Na+-ATPase activity

A new steroidal saponin, 3-{(O-6-deoxy-a-L-mannopyranosyl-(1 --> 4)-O-beta-D-glucopyranosyl-(1 --> 3)-O-[O-beta-D-glucopyranosyl-(1 --> 3)-beta-D-glucopyranosyl-(1 --> 2)]-O-beta-D-glucopyranosyl-(1 --> 4)-beta-D-galactopyranosyl)oxy}-6-hydroxy-(3beta,5alpha,6alpha,25R)-spirostan-12-one, was isolated from Agave brittoniana Trel. The structure was determined by extensive NMR spectroscopy studies and chemical conversions. Its effects on the Na+-ATPase and (Na+ + K+)-ATPase activities of the proximal tubule from pig kidney were evaluated. It was observed that this steroidal saponin exerts a biphasic effect on the Na+-ATPase activity. It is concluded that the effect of the aqueous extract as a diuretic is due, at least in part, to the action of saponin on the ouabain-insensitive Na+-ATPase.     

Synthesis of (R)-2,3-epoxypropyl(1-->3)-beta-D-pentaglucoside

The title pentasaccharide was synthesized via a 2+3 strategy. The disaccharide donor, 3-O-acetyl-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (8), was obtained by selective coupling of allyl 2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranoside with 3-O-acetyl-2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (4), followed by deallylation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor, allyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranoside (12), was prepared by coupling of allyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranoside with 4, followed by deacetylation. Condensation of 8 with 12, followed by epoxidation, and deprotection, gave the target pentaoside.     

Synthesis of a novel asparagine-linked heptasaccharide structure via p-methoxybenzyl-assisted beta-mannosylation

Synthesis of a core heptasaccharide asparagine N4-[alpha-D-mannopyranosyl-(1 --> 6)-[(alpha-D-mannopyranosyl)-(1 --> 3)]-[(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 --> 2)]-(beta-D-mannopyranosyl)-(1 --> 4)-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 --> 4)-[(alpha-L-fucopyranosyl)-(1 --> 6)]-2-acetamido-2-deoxy-beta-D-glucopyranosyl]-L-asparagine (1a) found from CHO glycosylation mutant cell LEC 14 is described. The structure of 1a is highly novel in terms of the presence of an extra GlcNAc residue linked to the 2-position of beta-linked mannose. The synthesis was performed using p-methoxybenzyl-assisted intramolecular aglycon delivery as the key transformation. 4,6-O-TIDPS-protected thiomannoside methyl 2-O-p-methoxybenzyl-4,6-O-(1,1,3,3-tetraisopropyl)disiloxanylid ene-3-O-trimethylsilyl-1-thio-alpha-D-mannopyranoside was adopted for this particular purpose, which afforded beta-mannoside p-methoxyphenyl 2,3-O-(p-methoxybenzylidene)-4,6-O-(1,1,3,3-tetraisopropyl)+ ++disiloxanylidene-beta-D-mannopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranoside stereoselectively in 75% yield.     

Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a

We have synthesized several ganglio-oligosaccharide structures using glycosyltransferases from Campylobacter jejuni. The enzymes, alpha-(2-->3/8)-sialyltransferase (Cst-II), beta-(1-->4)-N-acetylgalactosaminyltransferase (CgtA), and beta-(1-->3)-galactosyltransferase (CgtB), were produced in large-scale fermentation from Escherichia coli and further characterized based on their acceptor specificities. 2-Azidoethyl-glycosides corresponding to the oligosaccharides of GD3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GM2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GD2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), and GM1 (beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) were synthesized in high yields (gram-scale). In addition, a mammalian alpha-(2-->3)-sialyltransferase (ST3Gal I) was used to sialylate GM1 and generate GD1a (alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) oligosaccharide. We also cloned and expressed a rat UDP-N-acetylglucosamine-4'epimerase (GalNAcE) in E. coli AD202 cells for cost saving in situ conversion of less expensive UDP-GlcNAc to UDP-GalNAc.     

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.     

Structure of an acidic O-specific polysaccharide of the marine bacterium Pseudoalteromonas sp. KMM 634

An acidic O-specific polysaccharide containing D-glucuronic acid (D-GlcA), 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid (D-GlcNAc3NAcA), 2,3-diacetamido-2,3-dideoxy-D-mannuronoyl-L-alanine (D-ManNAc3NAcA6Ala), and 2-acetamido-2,4, 6-trideoxy-4-[(S)-3-hydroxybutyramido]-D-glucose (D-QuiNAc4NAcyl) was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Pseudoalteromonas sp. KMM 634 followed by gel-permeation chromatography. The polysaccharide was cleaved selectively with a new solvolytic agent, trifluoromethanesulfonic acid, to give a disaccharide and a trisaccharide with D-GlcNAc3NAcA at the reducing end. The borohydride-reduced oligosaccharides and the initial polysaccharide were studied by GLC-MS and 1H- and 13C-NMR spectroscopy, and the following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established: -->3)-alpha-D-QuipNAc4Ac4NAcyl-(1-->4)-beta-D-ManpNAc3NAcA6Ala+ ++-(1-->4)-b eta-D-GlcpNAc3NAc3NAcA-(1-->4)-beta-D-GlcpA-(1-->.     

Analysis of the interaction between lectins and tetra- and tri-saccharide mimics of the Sd(a) determinant by surface plasmon resonance detection

The binding properties of a spacer-linked synthetic Sd(a) tetrasaccharide beta-D-GalpNAc-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (1), two tetrasaccharide mimics beta-D-Galp-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (2) and beta-D-GlcpNAc-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (3), and two trisaccharide mimics beta-D-GalpNAc-(1-->4)-3-O-(SO(3)H)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (4) and beta-D-GalpNAc-(1-->4)-3-O-(CH(2)COOH)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (5) with lectins from Dolichos biflorus (DBL), Maackia amurensis (MAL), Phaseolus limensis (PLL), Ptilota plumosa (PPL), Ricinus communis 120 (RCL120) and Triticum vulgaris (wheat germ agglutinin, WGA) have been investigated by surface plasmon resonance (SPR) detection. MAL, PPL, RCL120 and WGA did not display any binding activity with compounds 1-5. However, DBL and PLL, both exhibiting GalNAc-specificity, showed strong binding activity with compounds 1, 4 and 5, and 1, 3, 4 and 5, respectively. The results demonstrate that SPR is a very useful analysis system for identifying biologically relevant oligosaccharide mimics of the Sd(a) determinant.     

Chemoenzymatic synthesis of the sialyl-alpha-(2-->3')-lactosamine trisaccharide with a 3-aminopropyl group as a spacer at the reducing end

The trisaccharide, 3-aminopropyl 5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid-(2-->3)-beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside has been synthesized chemoenzymatically for the first time. First, the acceptor, 3-aminopropyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside was synthesized in a conventional chemical manner, and then it was coupled with CMP-sialic acid using alpha-(2-->3)-(N)-sialyltransferase to afford the desired trisaccharide by an enzymatically stereocontrolled manner.     

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.     

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+.     

The multiple activities of polyphosphate kinase of Escherichia coli and their subunit structure determined by radiation target analysis

Polyphosphate kinase (PPK), the principal enzyme required for the synthesis of inorganic polyphosphate (polyP) from ATP, also exhibits other enzymatic activities, which differ significantly in their biochemical optima and responses to chemical agents. These several activities include: polyP synthesis (forward reaction), nATP --> polyP(n) + nADP (Equation 1); ATP synthesis from polyP (reverse reaction), ADP + polyP(n) --> ATP + polyP(n - 1) (Equation 2); general nucleoside-diphosphate kinase, GDP + polyP(n) --> GTP + polyP(n - 1) (Equation 3); linear guanosine 5'-tetraphosphate (ppppG) synthesis, GDP + polyP(n) --> ppppG + polyP(n - 2) (Equation 4); and autophosphorylation, PPK + ATP --> PPK-P + ADP (Equation 5). The Mg(2+) optima are 5, 2, 1, and 0.2 mM, respectively, for the activities in Equations 1, 2, 3, and 4. Inorganic pyrophosphate inhibits the activities in Equations 1 and 3 but stimulates that in Equation 4. The kinetics of the activities in Equations 1, 2, and 3 are highly processive, whereas the transfer of a pyrophosphoryl group from polyP to GDP (Equation 4) is distributive and demonstrates a rapid equilibrium, random Bi-Bi catalytic mechanism. Radiation target analysis revealed that the principal functional unit of the homotetrameric PPK is a dimer. Exceptions are a trimer for the synthesis of ppppG (Equation 4) and a tetrameric state for the autophosphorylation of PPK (Equation 5) at low ATP concentrations. Thus, the diverse functions of this enzyme involve different subunit organizations and conformations. The highly conserved homology of PPK among 18 microorganisms was used to determine important residues and conserved regions by alanine substitution, by site-directed mutagenesis, and by deletion mutagenesis. Of 46 single-site mutants, seven exhibit none of the five enzymatic activities; in one mutant, ATP synthesis from polyP is reduced relative to GTP synthesis. Among deletion mutants, some lost all five PPK activities, but others retained partial activity for some reactions but not for others.     

Active-site mapping of a Populus xyloglucan endo-transglycosylase with a library of xylogluco-oligosaccharides

Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16-34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites -1 and +1). Donor binding is dominated by the higher affinity of the XXXG moiety (G=Glcbeta(1-->4) and X=Xylalpha(1-->6)Glcbeta(1-->4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with k(cat)/K(m) values for the donor substrates showed that a GG-unit on negative and -XXG on positive subsites are the minimal requirements for activity. Subsites -2 and -3 (for backbone Glc residues) and +2' (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal=Galbeta(1-->4)) is the best donor substrate with a "blocked" nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.     

Facile synthesis of a pentasaccharide mimic of a fragment of the capsular polysaccharide of Streptococcus pneumoniae type 15C

A pentasaccharide mimic of a fragment of the capsular polysaccharide of Streptococcus pneumoniae type 15C beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[alpha-D-Galp-(1-->2)-beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->OCH2CH2N3) (1) was synthesized in a regio- and stereoselective manner. The 2-azidoethyl-spacered pentasaccharide mimic 1 can be used to construct a neoglycoconjugate antigen.     

Triterpenoid saponins from Ardisia mamillata

Two saponins were isolated from the roots of Ardisia mamillata HANCE. Their structures were established on the basis of MALDI-TOFMS, 1H, 13C NMR and 2D NMR (COSY, HOHAHA, HETCOR, HMBC and ROESY) spectra, and on chemical evidence, to be ardisimamilloside A, 3-O-[alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosyl-(1 --> 4)-[beta-D-glucopyranosyl-(1 --> 2)]-alpha-L-arabinopyranosyl]-3beta, 16alpha,28alpha-trihydroxy-13beta,28-epoxy-oleanan+ ++-30-al; and ardisimamilloside B, 3-O-[alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosyl-(1 --> 4)-[beta-D-glucopyranosyl-( 1 --> 2)]-alpha-L-arabinopyranosyl]-beta3-hydroxy-13beta,28- epoxy-oleanan-16-oxo-30-al.