An acetylated galactomannoglucan from the stems of Dendrobium nobile Lindl

A water-soluble polysaccharide DNP-W2 composed of glucose, mannose, and galactose in the molar ratio of 6.1:2.9:2.0 had been isolated from the stems of Dendrobium nobile. Its molecular weight was 1.8 × 10⁴ Da determined by HPGPC. Structural features of DNP-W2 were investigated by a combination of chemical and instrumental analysis, including FTIR, GC, GC-MS, periodate oxidation-Smith degradation, methylation analysis, partial acid hydrolysis, and NMR spectroscopy. The results showed that DNP-W2 is a 2-O-acetylgalactomannoglucan and has a backbone consisting of (1→4)-linked β-d-Glcp, (1→6)-linked β-d-Glcp, and (1→4)-linked β-d-Manp, with branches at O-6 of (1→4)-linked β-d-Glcp and β-d-Manp. The branches are composed of α-d-Galp. The acetyl groups are substituted at O-2 of (1→4)-linked Manp. Preliminary tests in vitro reveals that DNP-W2 can stimulate ConA- and LPS-induced T and B lymphocyte proliferation.     

Structural characterization of a highly branched polysaccharide from the seeds of Plantago asiatica L.

In this paper, polysaccharides were extracted from the seeds of Plantago asiatica L. with hot water and separated into three fractions PLP-1 (18.9%), PLP-2 (52.6%) and PLP-3 (28.5%) by Sephacryl™ S-400 HR column chomatography. The main fraction PLP-2's structure was elucidated using oxalic acid hydrolysis, partial acid hydrolysis, methylation, GC, GC-MS, 1D and 2D NMR. PLP-2 was composed of Rha, Ara, Xyl, Man, Glc and Gal, in a molar ratio of 0.05:1.00:1.90:0.05:0.06:0.10. Its uronic acid was GlcA. PLP-2 was highly branched heteroxylan which consisted of a β-1,4-linked Xylp backbone with side chains attached to O-2 or O-3. The side chains consisted of β-T-linked Xylp, α-T-linked Araf, α-T-linked GlcAp, β-Xylp-(1 → 3)-α-Araf and α-Araf-(1 → 3)-β-Xylp, etc. Based on these results, the structure of PLP-2 was proposed.     

Isolation and characterization of poly- and oligosaccharides from the red microalga Porphyridium sp.

The current study forms part of an ongoing research effort focusing on the elucidation of the chemical structure of the sulfated extracellular polysaccharide of the red microalga Porphyridium sp. (UTEX 637). We report here on the chemical structure of a fraction separated from an acidic crude extract of the polysaccharide, as investigated by methylation analysis, carboxyl reduction-methylation analysis, desulfation-methylation analysis, partial acid hydrolysis, Smith degradation, together with 1D and 2D ¹H and ¹³C NMR spectroscopy. This fraction with a molar mass of 2.39 × 10⁵ g mol⁻¹ comprised d- and l-Gal, d-Glc, d-Xyl, d-GlcA, and sulfate groups in a molar ratio of 1.0:1.1:2.1:0.2:0.7. The almost linear backbone of the fraction is composed of (1→2)- or (1→4)-linked d-xylopyranosyl, (1→3)-linked l-galactopyranosyl, (1→3)-linked d-glucopyranosyl, and (1→3)-linked d-glucopyranosyluronic acid and comprises a possible acidic building unit:

[(2 or 4)-β-d-Xylp-(l→3)]_m-α-d-Glcp-(1→3)-α-d-GlcpA-(1→3)-l-Galp(l→

Attached to the backbone are sulfate groups and nonreducing terminal d-xylopyranosyl and galactopyranosyl residues, which occur at the O-6 positions of Glc-derived moieties in the main chain.     

Synthesis of LeLe oligosaccharide fragments and efficient one-step deprotection

We describe here the synthesis of two oligosaccharide fragments of the tumor associated carbohydrate antigen Le^aLe^x. While the linear lacto-N-triose I: β-d-Galp-(1→4)-β-d-GlcNAcp-(1→3)-β-d-Galp-OMe is a known compound, this is the first reported preparation of the branched tetrasaccharide β-d-GlcNAcp-(1→3)-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-β-d-GlcNAcp-OMe. Our synthetic schemes involved using an N-trichloroacetylated trichloroacetimidate glucosaminyl donor activated with excess TMSOTf at 0 °C for glycosylation at O-3 of galactosyl residues and that of trichloroacetimidate galactosyl donors activated with excess BF₃·OEt₂ to glycosylate either O-3 or O-4 of glucosamine residues. The fucosylation at O-3 of the glucosamine acceptor was accomplished using a thiofucoside donor activated with copper(II) bromide and tetrabutylammonium bromide. Thus, syntheses of the protected tri- and tetrasaccharides were achieved easily and efficiently using known building blocks. Of particular interest, we also report that these protected oligosaccharides were submitted to dissolving metal conditions (Na-NH₃) to provide in one single step the corresponding deprotected compounds. Under these conditions all protecting groups (O-acyl, benzylidene, benzyl, and N-trichloroacetyl) were efficiently cleaved. The work-up procedure for such reactions usually involves quenching with excess methanol and then neutralization with acetic acid. In our work the neutralization was carried out using acetic anhydride rather than acetic acid to ensure N-acetylation of the glucosamine residue. Both fully deprotected compounds were then simply purified and desalted by gel permeation chromatography on a Biogel P2 column eluted with water.     

Synthesis and cytotoxic activity of the N-acetylglucosamine-bearing triterpenoid saponins

Fourteen ursolic acid and oleanolic acid saponins with N-acetyl-β-d-glucosamine, and (1→4)-linked and (1→6)-linked N-acetyl-β-d-glucosamine oligosaccharide residues were synthesized in a convergent manner. The structures of all compounds were confirmed by ¹H NMR and ¹³C NMR spectroscopy and by mass spectrometry, and their cytotoxic activities were assayed in three cancer cell lines. Only oleanolic acid-3-yl β-d-GluNAc showed significant cytotoxicity against HL-60 and BGC-823.     

Step-wise enzymatic preparation and structural characterization of singly and doubly substituted arabinoxylo-oligosaccharides with non-reducing end terminal branches

Shearzyme (GH10 endo-1,4-β-d-xylanase) and two different α-l-arabinofuranosidases (AXH-m and AXH-d3) were used stepwise to manufacture arabinoxylo-oligosaccharides (AXOS) with α-l-Araf (1→2)-monosubstituted β-d-Xylp residues or α-l-Araf (1→2)- and (1→3) doubly substituted β-d-Xylp residues from wheat arabinoxylan (AX) in a rather straightforward way. Four major AXOS (d-I, d-II, m-I and m-II) were formed in two separate hydrolyses. The AXOS were purified and the structures were confirmed using TLC, HPAEC-PAD, MALDI-TOF-MS and 1D and 2D NMR spectroscopy. The samples were identified as d-I: α-l-Araf-(1→2)-[α-l-Araf-(1→3)]-β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-d-Xylp, d-II: α-l-Araf-(1→2)-[α-l-Araf-(1→3)]-β-d-Xylp-(1→4)-d-Xylp, m-I: α-l-Araf-(1→2)-β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-d-Xylp and m-II: α-l-Araf-(1→2)-β-d-Xylp-(1→4)-d-Xylp. To our knowledge, this is the first report on structural ¹H and ¹³C NMR analysis of xylobiose-derived AXOS d-II and m-II. The latter compound has not been reported previously. The doubly substituted AXOS were produced for the first time in good yields, as d-I and d-II corresponded to 11.8 and 5.6 wt% of AX, respectively. Singly α-l-Araf (1→2)-substituted AXOS could also be prepared in similar yields by treating the doubly substituted AXOS further with AXH-d3.     

Synthesis and absolute structures of Mycoplasma pneumoniae β-glyceroglycolipid antigens

Just recently, a pair of β-glycolipids was isolated from the cell membrane of Mycoplasma pneumoniae as a mixture of the two compounds. They are the major immunodeterminants of this pathogenic Mycoplasma and indicate high medicinal potential. They have a β-(1→6)-linked disaccharide structure close to each other; one has β-d-galactopyranoside (β-Gal-type 1) at the non-reducing terminal, and another has β-d-glucopyranoside (β-Glc-type 2). In the present study, the first stereoselective synthesis was conducted for each of the two β-glycolipids 1 and 2. ¹H NMR and TLC-immunostaining studies of the synthetic compounds enable us to establish the absolute structures having the β-(1→6)-linked disaccharides at the glycerol sn-3 position.     

Separation, structure characterization, conformation and immunomodulating effect of a hyperbranched heteroglycan from Radix Astragali

A water soluble polysaccharide (RAP) was isolated and purified from Radix Astragali and its structure was elucidated by monosaccharide composition, partial acid hydrolysis and methylation analysis, and further supported by FT-IR, GC-MS and ¹H and ¹³C NMR spectra, SEM and AFM microscopy. Its average molecular weight was 1334 kDa. It was composed of Rha, Ara, Glc, Gal and GalA in a molar ratio of 0.03:1.00:0.27:0.36:0.30. The backbone consisted of 1,2,4-linked Rhap, α-1,4-linked Glcp, α-1,4-linked GalAp6Me, β-1,3,6-linked Galp, with branched at O-4 of the 1,2,4-linked Rhap and O-3 or O-4 of β-1,3,6-linked Galp. The side chains mainly consisted of α-T-Araf and α-1,5-linked Araf with O-3 as branching points, having trace Glc and Gal. The terminal residues were T-linked Araf, T-linked Glcp and T-linked Galp. Morphology analysis showed that RAP took random coil feature. RAP exhibited significant immunomodulating effects by stimulating the proliferation of human peripheral blood mononuclear cells and enhancing its interleukin production.     

Improved procedures for the selective chemical fragmentation of rhamnogalacturonans

The structural characterization of branched rhamnogalacturonans (RGs) requires the availability of methods that selectively cleave the Rhap-(1→4)-α-GalAp linkage and thereby generate oligosaccharide fragments that are suitable for mass spectrometric and NMR spectroscopic analyses. Enzymic cleavage of this linkage is often ineffective, especially in highly branched RGs. Therefore, we have developed an improved chemical fragmentation method based on β-elimination of esterified 4-linked GalpA residues. At least 85% of the carboxyl groups of the GalA residues in Arabidopsis thaliana seed mucilage RG is esterified using methyl iodide or 3-iodopropanol in Me₂SO containing 8% water and 1% tetrabutylammonium fluoride. However, β-elimination fragmentation at pH 7.3 and 120 °C is far more extensive with hydroxypropyl-esterified RG than with methyl-esterified RG. The non-reducing 4-deoxy-β-l-threo-hex-4-enepyranosyluronic acid residue formed by the β-elimination reaction is completely removed by treatment with aqueous N-bromosuccinimide, thereby simplifying the structural characterization of the chemically generated oligoglycosyl fragments. This newly developed procedure was used to selectively fragment the branched RG from peppergrass seed mucilage. The products were characterized using MALDI-TOF mass spectrometry, glycosyl residue composition analysis, and 1 and 2D NMR spectroscopy. Our data show that the most abundant low-molecular weight fragments contained a backbone rhamnose residue substituted at O-4 with a single sidechain, and suggest that peppergrass seed mucilage RG is composed mainly of the repeating unit 4-O-methyl-α-d-GlcpA-(1→4)-β-d-Galp-(1→4)-[→4)-α-d-GalpA-(1→2)-]-α-l-Rhap-(1→.     

A dual role for the lex2 locus: identification of galactosyltransferase activity in non-typeable Haemophilus influenzae strains 1124 and 2019

Lipopolysaccharide (LPS) of Haemophilus influenzae comprises a conserved tri-l-glycero-d-manno-heptosyl inner-core moiety (l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-[β-d-GlcIp-(1→4)]-l-α-d-Hepp-(1→5)-α-Kdop) to which addition of β-d-Glcp to O-4 of GlcI in serotype b strains is controlled by the gene lex2B. In non-typeable H. influenzae strains 1124 and 2019, however, a β-d-Galp is linked to O-4 of GlcI. In order to test the hypothesis that the lex2 locus is involved in the expression of β-d-Galp-(1→4-β-d-Glcp-(1→ from HepI, lex2B was inactivated in strains 1124 and 2019, and LPS glycoform populations from the resulting mutant strains were investigated. Detailed structural analyses using NMR techniques and electrospray-ionisation mass spectrometry (ESIMS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESIMSⁿ on permethylated dephosphorylated OS, indicated both lex2B mutant strains to express only β-d-Glcp extensions from HepI. This provides strong evidence that Lex2B functions as a galactosyltransferase adding a β-d-Galp to O-4 of GlcI in these strains, indicating that allelic polymorphisms in the lex2B sequence direct alternative functions of the gene product.     

Presence of 1→3-linked 2-O-β-d-xylopyranosyl-α-l-arabinofuranosyl side chains in cereal arabinoxylans

The presence of a fairly uncommon side chain 2-O-β-d-xylopyranosyl-α-l-arabinofuranosyl in arabinoxylans (AX) from eight different cereal by-products was investigated, using ¹H NMR spectroscopy and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) after Shearzyme^® (GH10 endo-1,4-β-d-xylanase) hydrolysis. This disaccharide side group was present in significant amounts in AX extracted from corn cobs and barley husks. For the first time, it was also detected in AX from oat spelts and rice husks, and in lesser amounts in wheat straw AX. Arabinoxylo-oligosaccharide (AXOS) containing the 2-O-β-d-Xylp-α-l-Araf side chain was purified from the oat spelt AX hydrolysate and the structure was fully analyzed using 1D and 2D NMR spectroscopy. The AXOS was identified as β-d-Xylp-(1→2)-α-l-Araf-(1→3)-β-d-Xylp-(1→4)-d-Xyl. To our knowledge, such a structure with 2-O-β-d-Xylp-α-l-Araf attached to the O-3 of the nonreducing end of xylobiose has not been described previously. New information on substitution of AX from various cereal by-products was obtained by combining NMR and enzyme-assisted HPAEC-PAD analysis.     

Enzymatic synthesis of unique sialyloligosaccharides using marine bacterial α-(2→3)- and α-(2→6)-sialyltransferases

We investigated the acceptor substrate specificities of marine bacterial α-(2→3)-sialyltransferase cloned from Photobacterium sp. JT-ISH-224 and α-(2→6)-sialyltransferase cloned from Photobacterium damselae JT0160 using several saccharides as acceptor substrates. After purifying the enzymatic reaction products, we confirmed their structure by NMR spectroscopy. The α-(2→3)-sialyltransferase transferred N-acetylneuraminic acid (Neu5Ac) from cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) to the β-anomeric hydroxyl groups of mannose (Man) and α-Manp-(1→6)-Manp, and α-(2→6)-sialyltransferase transferred N-acetylneuraminic acid to the 6-OH groups of the non-reducing end galactose residues in β-Galp-(1→3)-GlcpNAc and β-Galp-(1→6)-GlcpNAc.     

Structure of the O-polysaccharide of the lipopolysaccharide of Pragia fontium 97U116

The O-polysaccharide of Pragia fontium 97U116 was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar analysis along with 1D and 2D ¹H and ¹³C NMR spectroscopy. The following structure of the pentasaccharide-repeating unit was established: →2)-α-d-Galf-(1→3)-α-l-Rhap2Ac^I-(1→4)-α-d-GlcpNAc^I-(1→2)-α-l-Rhap^II-(1→3)-β-d-GlcpNAc^II-(1→     

Structural characterization and antioxidant properties of an exopolysaccharide produced by the mangrove endophytic fungus Aspergillus sp. Y16

A homogeneous exopolysaccharide, designated As1-1, was obtained from the culture medium of the mangrove endophytic fungus Aspergillus sp. Y16 and purified by anion-exchange and gel-permeation chromatography. Results of chemical and spectroscopic analyses, including one- and two-dimensional nuclear magnetic resonance (1D and 2D NMR) spectroscopy showed that As1-1 was mainly composed of mannose with small amounts of galactose, and that its molecular weight was about 15 kDa. The backbone of As1-1 mainly consists of (1 → 2)-linked α-d-mannopyranose units, substituted at C-6 by the (1 → 6)-linked α-d-mannopyranose, (1→)-linked β-d-galactofuranose and (1→)-linked β-d-mannopyranose units. As1-1 possessed good in vitro antioxidant activity as evaluated by scavenging assays involving 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide radicals. The investigation demonstrated that As1-1 is an exopolysaccharide different from those of other marine microorganisms, and could be a potential antioxidant and food supplement.     

Studies on the chemical structure and antitumor activity of an exopolysaccharide from Rhizobium sp. N613

The molecular structure of the rhizobium exopolysaccharide (REPS) was analyzed by enzymolysis, periodate oxidation, and Smith degradation, and by IR and NMR spectroscopy. The results indicated that REPS was a β-glucan with a backbone of β-d-(1→4)-linked glucose residues and branches of β-d-(1→6)-linked glucose residues. The branch was attached to the main chain at the 6-O-position. The molar ratio of 1→4 and 1→6 was 2:1. The terminal C3 of the (1→6)-Glc branch had an O-acetyl group. The molecular weight was estimated to be 35 kDa by Sephadex G-100 column chromatography. The antitumor activity of REPS was evaluated in mice bearing sarcoma 180, hepatoma 22, and Ehrlich ascites carcinoma tumor, respectively. At doses of 10-60 mg/kg, it was observed that tumor formation decreased significantly (P <0.01), but the relative spleen and thymus weight, the phagocytic function of monocytes, lymphocyte proliferation, and serum hemolysis antibody increased significantly (P <0.05). Results of these studies demonstrated that the REPS polysaccharide possessed antitumor activity.     

O-Methylated mannogalactan from the microalga Coccomyxa mucigena, symbiotic partner of the lichenized fungus Peltigera aphthosa

A structural study of the carbohydrates from Coccomyxa mucigena, the symbiotic algal partner of the lichenized fungus Peltigera aphthosa, was carried out. It produced an O-methylated mannogalactan, with a (1 → 6)-linked β-galactopyranose main-chain partially substituted at O-3 by β-Galp, 3-OMe-α-Manp or α-Manp units. There were no similarities with polysaccharides previously found in the lichen thallus of P. aphthosa. Moreover, the influence of lichenization in polysaccharide production by symbiotic microalgae and the nature of the photobiont in carbohydrate production in lichen symbiosis are also discussed.     

The O-antigen of Salmonella enterica O13 and its relation to the O-antigen of Escherichia coli O127

The following structure of the O-polysaccharide (O-antigen) of Salmonella enterica O13 was established by chemical analyses along with 2D ¹H and ¹³C NMR spectroscopy:→2)-α-l-Fucp-(1→2)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→The O-antigen of S. enterica O13 was found to be closely related to that of Escherichia coli O127, which differs only in the presence of a GalNAc residue in place of the GlcNAc residue and O-acetylation. The location of the O-acetyl groups in the E. coli O127 polysaccharide was determined. The structures of the O-polysaccharides studied are in agreement with the DNA sequence of the O-antigen gene clusters of S. enterica O13 and E. coli O127 reported earlier.     

β-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides

Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously over-expressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-β-d-galactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLM-family type, consisting of a small subunit of 35 kDa and a large subunit of 72 kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 °C, respectively. The enzyme is most stable in the pH range of 6.5-8.0. The K_m, k_cat and k_cat/K_m values for oNPG and lactose are 0.9 mM, 92 s⁻¹, 130 mM⁻¹ s⁻¹ and 29 mM, 98 s⁻¹, 3.3 mM⁻¹ s⁻¹, respectively. The L. plantarum β-galactosidase possesses a high transgalactosylation activity and was used for the synthesis of prebiotic galacto-oligosaccharides (GOS). The resulting GOS mixture was analyzed in detail, and major components were identified by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as capillary electrophoresis. The maximal GOS yield was 41% (w/w) of total sugars at 85% lactose conversion (600 mM initial lactose concentration). The enzyme showed a strong preference for the formation of β-(1→6) linkages in its transgalactosylation mode, while β-(1→3)-linked products were formed to a lesser extent, comprising ∼80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were β-d-Galp-(1→6)-d-Lac, accounting for 34% of total GOS, and β-d-Galp-(1→6)-d-Glc, making up 29% of total GOS.     

Synthesis of trisaccharides containing internal galactofuranose O-linked in Trypanosoma cruzi mucins

The trisaccharides β-d-Galf-(1→2)-β-d-Galf-(1→4)-d-GlcNAc (5) and β-d-Galp-(1→2)-β-d-Galf-(1→4)-d-GlcNAc (6) constitute novel structures isolated as alditols when released by reductive β-elimination from mucins of Trypanosoma cruzi (Tulahuen strain). Trisaccharides 5 and 6 were synthesized employing the aldonolactone approach. Thus, a convenient d-galactono-1,4-lactone derivative was used for the introduction of the internal galactofuranose and the trichloroacetimidate method was employed for glycosylation reactions. Due to the lack of anchimeric assistance on O-2 of the galactofuranosyl precursor, glycosylation studies were performed under different conditions. The nature of the solvent strongly determined the stereochemical course of the glycosylation reactions when the galactofuranosyl donor was substituted either by 2-O-Galp or 2-O-Galf.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain Escherichia coli O82

The structure of the O-antigen polysaccharides (PS) from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain E. coli O82 have been determined. Component analysis and ¹H, ¹³C, and ³¹P NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by ¹H, ¹³C-heteronuclear multiple-bond correlation, and ¹H, ¹H-NOESY experiments. d-GroA as a substituent is linked via its O-2 in a phosphodiester-linkage to O-6 of the α-d-Glcp residue. The PS is composed of tetrasaccharide repeating units with the following structure:→4)-α-d-Glcp6-(P-2-d-GroA)-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→3)-β-d-GlcpNAc-(1→Cross-peaks of low intensity from an α-d-Glcp residue were present in the NMR spectra and spectral analysis indicates that they originate from the terminal residue of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. Enzyme immunoassay using specific anti-E. coli O82 rabbit sera showed identical reactivity to the LPS of the two strains, in agreement with the structural analysis of their O-antigen polysaccharides.