Discrimination between endotoxin and (1----3)-beta-D-glucan using turbidimetric kinetic assay with Limulus amebocyte lysate

A procedure for the Limulus amebocyte lysate (LAL) test discriminating between endotoxin and (1----3)-beta-D-glucan based on the turbidimetric kinetic method was proposed. Endotoxin and (1----3)-beta-D-glucan, which are elicitors of the activation of LAL, showed different reaction courses with this lysate. To analyze the difference in the reactions, two parameters, the maximum differential coefficient of the reaction (Dmax) and the reaction time required to obtain Dmax (Tp) were defined. The logarithmic plottings of Tp versus Dmax (Tp-Dmax plot) discriminated between endotoxin and (1----3)-beta-D-glucan. Endotoxin was measured with a standard curve plotting logarithmic endotoxin concentration versus Dmax (ET-Dmax plot). The endotoxin calculated from Dmax was less influenced by (1----3)-beta-D-glucan than that calculated from the usual gelation time. A small amount of endotoxin in a sample could be concealed by the addition of polymyxin B, which inhibited the activation of LAL by endotoxin. (1----3)-beta-D-glucan was measured without being affected by the presence of a small amount of endotoxin using LAL with polymyxin B. The following procedure is proposed as a LAL test to discriminate between endotoxin and (1----3)-beta-D-glucan. (1) Identify the main substance (endotoxin or (1----3)-beta-D-glucan) triggering the activation of LAL using the Tp-Dmax plot. (2) Use the appropriate method to measure the main substance: the ET-Dmax plot for endotoxin or the LAL with polymyxin B for (1----3)-beta-D-glucan.     

Separation of the hot water extracts of sclerotia of Sclerotinia sclerotiorum IFO 9395 into mitogenic and antitumor-active subfractions

The hot water extract of sclerotia of Sclerotinia sclerotiorum IFO 9395 (TSHW) was divided into representative fractions by ammonium sulfate and ethanol precipitations, and (1----3)-beta-D-glucanase treatment. The ammonium sulfate and ethanol precipitations gave a (1----3)-beta-D-glucan fraction (TSG) and a mannan fraction (TSM). After the degradation of (1----3)-beta-D-glucan in TSHW by (1----3)-beta-D-glucanase treatment, a water-insoluble protein fraction (EDP) and supernatant (EDS) were obtained. Among these fractions, the mitogenic and antitumor activities were mainly observed in EDP and TSG, respectively. On the other hand, the stimulatory effect on the reticuloendothelial system was mainly found in EDP and EDS, and a weak effect was observed in TSG. These findings suggest that the mitogenic and antitumor activities of TSHW were mainly due to the protein and (1----3)-beta-D-glucan, respectively, and that the mitogenic substance (EDP) is tightly bound to (1----3)-beta-D-glucan (TSG) in TSHW, accounting for its solubility in aqueous solution.     

Analysis of 8-aminonaphthalene-1,3,6-trisulfonic acid labelled N-glycans by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a fast and efficient analytical method which is now widely used in glycobiology for the separation and quantification of free or glycoprotein-released oligosaccharides. However, since identification by FACE of N-glycan structures is only based on their electrophoretic mobility after labelling with 8-aminonaphthalene-1,3, 6-trisulfonic acid (ANTS), co-migration of derived glycans on gel could occur which may result in erroneous structural assignments. As a consequence, a protocol was developed for the fast and efficient matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometric analysis of ANTS-labelled N-glycans. N-Glycans were isolated from plant and mammalian glycoproteins, reductively aminated with the charged fluorophore 8-aminonaphthalene-1, 3, 6-trisulfonic acid (ANTS) and separated using high resolution polyacrylamide gel electrophoresis. The ANTS-labelled glycans were eluted from FACE gel slices and then analysed by MALDI-TOF mass spectrometry in negative ion mode. Using 3-aminoquinoline containing 2.5 mM citrate NH(4)(+) as matrix, neutral N-linked N-glycans, as well as labelled sialylated oligosaccharides, were found to be easily detected in the 2-10 picomole range giving rise to ?M - H(-) ions.     

Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant

Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an alpha(1,3)- or alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the alpha(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the alpha(1,6)-linked Man of the natural substrate and the beta(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the beta(1,2)-linked GlcNAc. The structure is consistent with the approximately 80-fold preference of dGMII for the cleavage of substrates containing a nonreducing beta(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.     

Direct and stereoselective synthesis of 1,3-cis-3- arylsulphonaminodeoxydisaccharides and oligosaccharides

The 3-aminoglycosides are ubiquitous in biologically important classes of glycoconjugates and naturally occurring oligosaccharides. Despite the rapid growth in the development of synthetic method of 3-amino glycosides, the current state-of-the art suffers from limited substrate scope, low yields, long reaction times, and anomeric mixtures. This work presents a novel direct method for the synthesis of 1,3-cis-3-arylsulphonaminodeoxydisaccharides and oligosaccharides via α-selective glycosylation and hydroamination of glycal in a one-pot manner. This efficient multicomponent reaction methodology provides ready access to 1,3-cis-3-arylsulphonaminodeoxydisaccharides and oligosaccharides and allows derivatization by variation of each component.     

Synthesis and self-assembly of "tree-like" amphiphilic glycopolypeptides

Novel synthetic tree-like oligosaccharides-grafted-polypeptides were prepared by using Huisgen 1,3-dipolar cycloaddition between poly(γ-benzyl-L-glutamate)-block-poly(propargylglycine) and two different oligosaccharides, dextran or hyaluronan. By direct solubilisation in water, these tree-like glycopeptides spontaneously form very small assemblies with sizes below 50 nm and low polydispersity.     

Capillary zone electrophoresis for the quantitation of oligosaccharides formed through the action of chitinase.

Capillary zone electrophoresis with fluorescence detection was used to analyze the products formed by chitinase acting on N-acetylchitooligosaccharide-fluorescent conjugates. Six oligosaccharides of the structure [N-acetylglucosamine(1----4)]n (where n = 1-6) were conjugated to 7-amino-1,3-naphthalene disulfonic acid by reductive amination. Each oligosaccharide-fluorescent conjugate was purified by preparative gradient polyacrylamide gel electrophoresis, semi-dry electrotransfer to a positively-charged nylon membrane and recovered by washing the membrane with salt solution. The products formed by treating each oligosaccharide-fluorescent conjugate with chitinase were analyzed by capillary zone electrophoresis. The chitinase treatment hexasaccharide-fluorescent conjugate was also examined kinetically to study the action pattern of this enzyme.     

Identification of alpha-galactosyl epitope mimetics through rapid generation and screening of C-linked glycopeptide library

A general methodology has been established for rapid generation and screening of combinatorial glycopeptide library and subsequent mass spectrometric sequencing to identify the mimetics of Galalpha(1,3)Gal epitopes. Using this approach, several active glycopeptide sequences were recognized and found to inhibit the binding of human natural anti-Gal antibodies with comparable IC(50)s to synthetic Galalpha(1,3)Gal oligosaccharides. The most active glycopeptides detected from the library included Gal-Tyr-Trp-Arg-Tyr, Gal-Thr-Trp-Arg-Tyr, and Gal-Arg-Trp-Arg-Tyr. These glycopeptides showed higher affinities to anti-Gal antibodies than known Galalpha(1,3)Gal peptide mimetics, such as DAHWESWL and SSLRGF. Our results suggest that, by combining a peptide sequence (the "functional" mimic part) with a terminal alpha-linked galactose moiety (the "structural" mimic part), the resulting glycopeptide could be a very good Galalpha(1,3)Gal mimetic. Analysis of these active glycopeptides provided first-hand information regarding the binding site of anti-Gal antibodies to facilitate the structurally based design of more potent and stable inhibitors.     

Synthesis of sugar arrays in microtiter plate

1,3-Dipolar cycloadditions between azides and alkynes were exploited to attach oligosaccharides to a C(14) hydrocarbon chain that noncovalently binds to the microtiter well surface. Synthesis of sugar arrays was performed on a micromolar scale in situ in the microtiter plate. As a model study, the beta-galactosyllipid 5 was displayed on a 4-micromol scale. Formation of product was confirmed via ESI-MS, and the yield was determined via chemical and biological assays. Several complex carbohydrates (6-16) were also displayed in microtiter plates and successfully screened with various lectins. Moreover, sialyl Lewis x (17) was synthesized via the enzymatic fucosylation of a precursor displayed in the plate. Studies on inhibition of this biotransformation have been carried out, and the IC(50) value found for the known inhibitor 20 was consistent with previous studies in solution.     

Synthesis of New 1,3-Thiazolecarbaldehydes

H-Lithiation and Br-lithiation reactions of 1,3-thiazole were studied in order to obtain new thiazole derivatives. Four isomeric chloromethyl derivatives of 1,3-thiazole containing a protected aldehyde group like 2-(1,3-dioxolan-2-yl)-5-(chloromethyl)-1,3-thiazole, 5-(1,3-dioxolan-2-yl)-2-(chloromethyl)-1,3-thiazole, 4-(1,3-dioxolan-2-yl)-2-(chloromethyl)-1,3-thiazole, and 2-(1,3-dioxolan-2-yl)-4-(chloromethyl)-1,3-thiazole were synthesized. Their nucleophilic substitution reactions with dimethylamine and sodium methylthiolate were studied. New aldehydes of 1,3-thiazole series of low-molecular weight were obtained.     

β(1,3) Branched heptadeca- and linear hexadeca-saccharides possessing an aminoalkyl group as a strong ligand to dectin-1

In this report, we describe the convergent synthesis of β(1,3) oligosaccharides containing an aminoalkyl group. The branched heptadecasaccharide and linear hexadecasaccharide acted as ligands of dectin-1 whose binding affinity was only 10-fold weaker than that of natural SPG and exhibited dectin-1 agonist activity.     

Prompt chemoenzymatic synthesis of diverse complex-type oligosaccharides and its application to the solid-phase synthesis of a glycopeptide with Asn-linked sialyl-undeca- and asialo-nonasaccharides

We describe herein the preparation of 24 pure asparagine-linked oligosaccharides (Asn-oligosaccharides) from asparagine-linked biantennary complex-type sialylundecasaccharide [(NeuAc-alpha-2,6-Gal-beta-1,4-GlcNAc-beta-1,2-Man-alpha-1,6/1,3-)(2)-Man-beta-1,4-GlcNAc-beta-1,4-GlcNAc-beta-1-asparagine, 2] obtained from egg yolk. Our synthetic strategy aimed at adapting branch specific exo-glycosidases digestion (beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase and alpha-D-mannosidase) of the individual asialo-branch after preparation of monosialyloligosaccharides obtained from 2 by acid hydrolysis of NeuAc. In order to perform branch specific exo-glycosidase digestion, isolation of pure monosialyloligosaccharides obtained was essential. However, isolation of two kinds of monosialyloligosaccharides are difficult by HPLC due to their highly hydrophilic nature. Therefore, we examined chemical protection with hydrophobic protecting (Fmoc and benzyl) groups. These chemical protection enabled us to separate the monosialyloligosaccharides by use of a HPLC column (ODS) on synthetic scales. Using these pure monosialiloligosaccharides enable us to obtain 24 Asn-linked oligosaccharides (100 mg scale) within a few weeks by branch specific exo-glycosidase digestions (alpha-D-neuraminidase, beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase and alpha-D-mannosidase). In addition, solid-phase synthesis of glycopeptide having Asn-linked sialyl-undeca- and asialo-nonasaccharides thus obtained, was also performed on an acid labile HMPA-PEGA resin.     

Introducing porous graphitized carbon liquid chromatography with evaporative light scattering and mass spectrometry detection into cell wall oligosaccharide analysis

Separation and characterization of complex mixtures of oligosaccharides is quite difficult and, depending on elution conditions, structural information is often lost. Therefore, the use of a porous-graphitized-carbon (PGC)-HPLC-ELSD-MSⁿ-method as analytical tool for the analysis of oligosaccharides derived from plant cell wall polysaccharides has been investigated. It is demonstrated that PGC-HPLC can be widely used for neutral and acidic oligosaccharides derived from cell wall polysaccharides. Furthermore, it is a non-modifying technique that enables the characterization of cell wall oligosaccharides carrying, e.g. acetyl groups and methylesters. Neutral oligosaccharides are separated based on their size as well as on their type of linkage and resulting 3D-structure. Series of the planar β-(1,4)-xylo- and β-(1,4)-gluco-oligosaccharides are retained much more by the PGC material than the series of β-(1,4)-galacto-, β-(1,4)-manno- and α-(1,4)-gluco-oligosaccharides. Charged oligomers such as α-(1,4)-galacturonic acid oligosaccharides are strongly retained and are eluted only after addition of trifluoroacetic acid depending on their net charge. Online-MS-coupling using a 1:1 splitter enables quantitative detection of ELSD as well as simple identification of many oligosaccharides, even when separation of oligosaccharides within a complex mixture is not complete. Consequently, PGC-HPLC-separation in combination with MS-detection gives a powerful tool to identify a wide range of neutral and acidic oligosaccharides derived from various cell wall polysaccharides.     

Structural confirmation of oligosaccharides newly isolated from sugar beet molasses

ABSTRACT: BACKGROUND: Sugar beet molasses is a viscous by-product of the processing of sugar beets into sugar. The molasses is known to contain sucrose and raffinose, a typical trisaccharide, with a well-established structure. Although sugar beet molasses contains various other oligosaccharides as well, the structures of those oligosaccharides have not been examined in detail. The purpose of this study was isolation and structural confirmation of these other oligosaccharides found in sugar beet molasses. RESULTS: Four oligosaccharides were newly isolated from sugar beet molasses using high-performance liquid chromatography (HPLC) and carbon-Celite column chromatography. Structural confirmation of the saccharides was provided by methylation analysis, matrix-assisted laser desorption/ionaization time of flight mass spectrometry (MALDI-TOF-MS), and nuclear magnetic resonance (NMR) measurements. CONCLUSION: The following oligosaccharides were identified in sugar beet molasses: beta-D-galactopyranosyl-(1- > 6)-beta-D-fructofuranosyl-(2 <-> 1)-alpha-D-glucopyranoside (named beta-planteose), alpha-D-galactopyranosyl-(1- > 1)-beta-D-fructofuranosyl-(2 <-> 1)-alpha-D-glucopyranoside (named1-planteose), alpha-D-glucopyranosyl-(1- > 6)-alpha-D-glucopyranosyl-(1 <-> 2)-beta-D-fructofuranoside (theanderose), and beta-D-glucopyranosyl-(1- > 3)-alpha-D-glucopyranosyl-(1 <-> 2)-beta-D-fructofuranoside (laminaribiofructose). 1-planteose and laminaribiofructose were isolated from natural sources for the first time.     

Synthesis and antitumor activities of mitomycin C (1----3)-beta-D-glucan conjugate.

The conjugate of mitomycin C (MMC) with linear (1----3)-beta-D-glucan from Alcaligenes faecalis var. myxogenes IFO 13140 was synthesized and its antitumor activities investigated. The conjugate (MMC-carboxymethylated linear (1----3)-beta-D-glucan (CMPS)) was obtained by treatment of CMPS with MMC in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. In vitro cytotoxicity of MMC-CMPS against L1210 leukemia cells was similar to that of MMC. In i.p.-i.p. system in vivo against P388 leukemia in mice, the maximum increase of MMC-CMPS conjugate in life span (ILSmax) was higher than that of MMC but the therapeutic index was reduced. However, the antitumor activity of MMC-CMPS conjugate against subcutaneously implanted sarcoma 180 solid tumor in mice by i.p. administration was similar to that of MMC at a dose of 1.5 mg eq MMC/kg/d x 7 and the reduction of the number of leukocytes caused by MMC was suppressed by attaching MMC to CMPS. In addition, on assay using serum of sarcoma 180 solid tumor-bearing mice with injection of MMC-CMPS conjugate, a drastic loss of tumor cells and an increase in polymorphonuclear leukocytes (PMN) were observed. This result suggested that MMC-CMPS conjugate induced tumor-regressing factor similar to CMPS.     

Automated Solid‐Phase Synthesis of a β‐(1,3)‐Glucan Dodecasaccharide

β‐Glucans are a group of structurally heterogeneous polysaccharides found in bacteria, fungi, algae and plants. β‐(1,3)‐D ‐Glucans have been studied in most detail due to their impact on the immune system of vertebrates. The studies into the immunomodulatory properties of these glucans are typically carried out with isolates that contain a heterogeneous mixture of polysaccharides of different chain lengths and varying degrees of branching. In order to determine the structure–activity relationship of β‐(1,3)‐glucans, access to homogeneous, structurally‐defined samples of these oligosaccharides that are only available through chemical synthesis is required. The syntheses of β‐glucans reported to date rely on the classical solution‐phase approach. We describe the first automated solid‐phase synthesis of a β‐glucan oligosaccharide that was made possible by innovating and optimizing the linker and glycosylating agent combination. A β‐(1,3)‐glucan dodecasaccharide was assembled in 56 h in a stereoselective fashion with an average yield of 88 % per step. This automated approach provides means for the fast and efficient assembly of linker‐functionalized mono‐ to dodecasaccharide β‐(1,3)‐glucans required for biological studies.     

Small angle x-ray scattering study of local structure and collapse transition of (1,3)-beta-D-glucan-chitosan gels

Scleroglucan is a (1,3)-beta-D-glucan polysaccharide produced by the fungus Sclerotium. Dissolved in water at room temperature it adopts a linear, rigid, triple helical structure. Gelation of scleroglucan can be obtained by Schiff-base formation between partly periodate oxidized scleroglucan and the primary amine groups of chitosan. The scleraldehyde-chitosan gels exhibit a collapse transition when exposed to volume fractions of isopropanol, Wp, larger than 65%. The aim of the present study is to provide structural information concerning the local polymer distribution and the collapse transition in (1,3)-beta-D-glucan-chitosan gels. Small angle x-ray scattering was used to investigate solutions and gels of scleroglucan in water, as well as in an aqueous mixture containing 65% isopropanol. The results reveal that in aqueous solution, the polysaccharide scleroglucan chains have an approximately cylindrical cross section of external diameter close to 17 A. The gels display the same local structure, but form clusters on a longer distance scale. For the collapsed gels in the water-isopropanol mixture, partial phase separation occurs in which ordered domains of approximate size of 110 A develop. This study indicates that local ordering in liquid-crystalline-type domains is a possible molecular mechanism contributing to the collapse of gels composed of semiflexible polymers. The triple helical structure of the molecule appears not to be conserved in the majority phase in this solvent, but it is conserved in the liquid crystalline domains.     

Glycoside Synthesis with Anomeric 1-N-Glycobiosyl-1,2,3-triazoles1

Abstract

1,3-Dipolar cycloaddition of the acetylated 1,2-trans- and 1,2-cis-cellobiosyl, -lactosyl, -maltosyl, and -melibiosyl azides with various acetylenedicarboxylic acid esters gave the corresponding 1-N-glycobiosyl-1,2,3-triazoles (1a,b,c-8a,b,c) which have been used as glycosyl donors for the synthesis of oligosaccharides (15-17) and an anthracycline type antibiotic (18).     

Protecting‐Group‐Free One‐Pot Synthesis of Glycoconjugates Directly from Reducing Sugars

The conversion of sugars into glycomimetics typically involves multiple protecting‐group manipulations. The development of methodology allowing the direct aqueous conversion of free sugars into glycosides, and mimics of oligosaccharides and glycoconjugates in a high‐yielding and stereoselective process is highly desirable. The combined use of 2‐azido‐1,3‐dimethylimidazolinium hexafluorophosphate and the Cu‐catalyzed Huisgen cycloaddition allowed the synthesis of a range of glycoconjugates in a one‐step reaction directly from reducing sugars under aqueous conditions. The reaction, which is completely stereoselective, may be applied to the convergent synthesis of triazole‐linked glycosides, oligosaccharides, and glycopeptides. The procedure provides a method for the one‐pot aqueous ligation of oligosaccharides and peptides bearing alkyne side chains.     

Structure and antitumor activity of the less-branched derivatives of an alkali-soluble glucan isolated from Omphalia lapidescens. (Studies on fungal polysaccharide. XXXVIII).

The structure and antitumor activity of Smith-type degradation products (OL-2-I, OL-2-II and OL-2-III) of an alkali-soluble glucan, OL-2, isolated from a crude fungal drug "Leiwan" (Omphalia lapidescens) were investigated. Methylation analysis suggested that OL-2-I was a (1----3)-beta-D-glucan with approximately one branch at every three main chain glucosyl units at each C-6 position; OL-2-II was a (1----3)-beta-D-glucan with approximately one branch at twenty four main chain glucosyl units at each C-6 position (number of all main chain glucosyl units is on average). OL-2-I, OL-2-II and OL-2-III which were Smith-type degradation products of OL-2, showed potent antitumor activity against the solid form of sarcoma 180 in ICR mice. These results indicated that the degree of beta-linked branching at position 6 was remarkably related to the antitumor activity.