Fermentable hexose production from corn stalks and wheat straw with combined supercritical and subcritical hydrothermal technology

Lignocellulosic wastes, including corn stalks and wheat straw, were pretreated and hydrolyzed with combined supercritical and subcritical hydrothermal technology. Soluble sugars were collected by pre-washing the crushed materials before hydrolysis. The effects of solid–liquid ratio, temperature, and reaction time on oligosaccharide production were investigated and the optimum supercritical conditions were found to be 20 mg/2.5 ml water, 384 °C, 17 s for corn stalks and 20 mg/2.5 ml water, 384 °C, 19 s for wheat straw. Subsequent subcritical processing of the hydrolyzate (with or without the water extract) from supercritical treatment was guided by a previous analysis of cellulose hydrolysis kinetics. The highest yield of fermentable hexoses from corn stalks (27.4% of raw material) was obtained at 280 °C, 27 s, and from wheat straw (6.7% of raw material) at 280 °C, 54 s. This study provides novel key parameters for fermentable hexose production from lignocellulosic feedstocks using combined supercritical and subcritical hydrothermal treatment.     

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.     

Mass spectrometric quantification of neutral and sialylated N-glycans from a recombinant therapeutic glycoprotein produced in the two Chinese hamster ovary cell lines

Quality control and assurance of glycan profiles of a recombinant glycoprotein from lot to lot is a critical issue in the pharmaceutical industry. To develop an easy and simple quantitative and qualitative glycan profile method based on matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), the modification with Girard’s reagent T (GT) was exploited. Because GT-derivatized quantification of oligosaccharides using MALDI-TOF MS is possible only with neutral glycans, sialylated glycans are not subjected to quantitative analysis with MALDI-TOF MS. To solve this problem, mild methyl esterification and subsequent GT derivatization were employed, enabling us to perform rapid qualitative and quantitative analysis of sialylated and neutral N-linked oligosaccharides using MALDI-TOF MS. This modified method was used in the comparative quantification of N-glycans from the recombinant therapeutic glycoprotein expressed in two different Chinese hamster ovary (CHO) cell lines. The percentages of sialylated N-glycans to total were 22.5 and 5.2% in CHO-I and CHO-II cells, respectively, resulting in a significant difference in the biological activity of the recombinant glycoprotein.     

Enhanced stereoselectivity of α-mannosylation under thermodynamic control using trichloroacetimidates

O-Specific polysaccharides of Vibrio cholerae O1, serotypes Inaba and Ogawa, consist of α-(1→2)-linked N-(3-deoxy-l-glycero-tetronyl)perosamine (4-amino-4,6-dideoxy-d-mannose). The blockwise synthesis of larger fragments of such O-PSs involves oligosaccharide glycosyl donors that contain a nonparticipating 2-O-glycosyl group at the position vicinal to the anomeric center where the new glycosidic linkage is formed. Such glycosyl donors may bear at C-4 either a latent acylamino (e.g., azido) or the 3-deoxy-l-glycero-tetronamido group. While monosaccharide glycosyl donors, even those bearing a nonparticipating group at O-2 (e.g., methyl), and the 4-N-(3-deoxy-l-glycero-tetronyl) side chain form α-linked oligosaccharides with excellent stereoselectivity, α-mannosylation with analogous oligosaccharide donors in this series is adversely affected by the presence of the side chain. Consequently, the unwanted β-product is formed in a considerable amount. Conducting the reaction at elevated temperature under thermodynamic control substantially enhances formation of the α-linked oligosaccharide. This effect is much more pronounced when glycosyl trichloroacetimidates, rather than thioglycosides or glycosyl chlorides, are used as glycosyl donors.     

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.     

Novel fructopyranose oligosaccharides isolated from fermented beverage of plant extract

Four oligosaccharides containing a fructopyranosyl residue have been found from fermented beverage of plant extract and isolated from the beverage using carbon-Celite column chromatography and preparative high performance liquid chromatography. Structure confirmation of the saccharides was provided by methylation analysis, MALDI-TOF-MS and NMR measurements. These saccharides were identified as oligosaccharides of fructopyranoside series; β-d-fructopyranosyl-(2→6)-d-fructofuranose (1), β-d-fructopyranosyl-(2→1)-d-fructopyranose (2), β-d-fructopyranosyl-(2→1)-β-d-fructofuranosyl-(2↔1)-α-d-glucopyranoside (3), and β-d-fructopyranosyl-(2→6)-α-d-glucopyranosyl-(1↔2)-β-d-fructofuranoside (4). Saccharides 3 and 4 among novel saccharides 1, 3, and 4 were named ‘pyrano-1-kestose (pyrano-isokestose)’ and ‘pyrano-neokestose’, respectively.     

The structure of the carbohydrate backbone of the lipooligosaccharide from the halophilic bacterium Arcobacter halophilus

A novel oligosaccharide was isolated and identified from the lipooligosaccharide fraction of the halophilic marine bacterium Arcobacter halophilus. The complete structure was achieved by chemical analysis, 2D NMR spectroscopy, and MALDI mass spectrometry as the following:

α-Glc-(1→7)-α-Hep-(1→5)-α-Kdo4P-(2→6)-β-GlcN4P-(1→6)-α-GlcN1P.     

Exploration of the use of an acylsulfonamide safety-catch linker for the polymer-supported synthesis of hyaluronic acid oligosaccharides

The synthesis of hyaluronic acid oligosaccharides on polyethylene glycol (PEG) using an acylsulfonamide linker has been explored. Hyaluronic acid is a challenging synthetic target that usually involves the condensation of highly disarmed glucuronic acid building blocks. Amine-ended PEG monomethyl ether was efficiently functionalized with a hydroxyl-terminated acylsulfonamide linker. Suitably protected d-glucosamine (GlcN) and d-glucuronic acid (GlcA) monosaccharide building blocks were coupled to the polymer acceptor using the trichloroacetimidate glycosylation method. The sulfonamide safety-catch linker enables simultaneous cleavage of the monosaccharide from the polymer and orthogonal functionalization for further (bio)-conjugation of the sugar sample. Subsequent glycosylation of PEG-bound glycosyl acceptor to generate hyaluronic acid oligosaccharide chain failed. Model glycosylation experiments in solution and on soluble support using the same unreactive acceptors and donors allows for the synthesis of an orthogonally protected hyaluronic acid disaccharide and suggest that the encountered difficulties could be attributed to the presence of the N-acylsulfonamide.     

Dilute liquid crystals used to enhance residual dipolar couplings may alter conformational equilibrium in oligosaccharides

The solution structures of a trisaccharide and a pentasaccharide containing the Lewis(x) motif were determined by two independent approaches using either dipolar cross-relaxation (NOE) or residual dipolar coupling (RDC) data. For the latter, one-bond 13C[bond](1)H RDC enhanced by two different mineral liquid crystals were used alone. Home-written programs were employed firstly for measuring accurately the coupling constants in the direct dimension of non-decoupled HSQC experiments, secondly for transforming each RDC data set into geometrical restraints. In this second program, the complete molecular structure was expressed in a unique frame where the alignment tensor is diagonal. Assuming that the pyranose rings are rigid, their relative orientation is defined by optimizing the glycosidic torsion angles. For the trisaccharide, a good agreement was observed between the results of both approaches (NOE and RDC). In contrast, for the pentasaccharide, strong discrepancies appeared, which seem to result from interactions between the pentasaccharide and the mesogens, affecting conformational equilibrium. This observation is of importance, as it reveals that using simultaneously NOE and RDC can be hazardous as the former represent 99% of the molecules free in solution, whereas the latter correspond to less than 1% of the structure bound to the mesogen.     

Synthesis of Lewis X trisaccharide analogues in which glucose and rhamnose replace N-acetylglucosamine and fucose,respectively

Two analogues of the Le(x) trisaccharide, alpha-L-Fucp-(1-->3)-[beta-D-Galp-(1-->4)]-D-Glcp were synthesized as allyl glycosides. In these derivatives either only the N-acetylglucosamine is replaced by glucose or both the N-acetylglucosamine and the fucosyl residue are replaced by glucose and rhamnose, respectively. Our synthetic scheme used armed beta-thiophenyl fuco- and rhamnoside glycosyl donors that were prepared anomerically pure from the corresponding alpha-glycosyl bromides. The protecting groups were chosen to allow access to the fully deprotected trisaccharides without reduction of the allyl glycosidic group. These analogues will be used as soluble antigens in binding experiments with anti-Le(x) antibodies and can also be conjugated to a carrier protein and used as immunogens. In the course of this synthetic work, we also describe the use of reversed-phase HPLC to purify key protected trisaccharide intermediates prior to their deprotection.