Efficient Synthesis of Glycosyl Enaminoesters Directly from Glycosyl Azides

A convenient methodology has been developed for the synthesis of glycosylenaminoesters directly from glycosyl azides under hydrogenetion condition. Yields were moderate to good in all cases.

Expression of a bacterial xylanase in Trichoderma reesei under the egl2 and cbh2 glycosyl hydrolase gene promoters

Expression vectors were constructed for Trichoderma reesei using the promoters, secretion signals and the modular structure of the efficiently expressed and secreted cellulase enzymes EGL2 (Cel5A) and CBH2 (Cel6A) as a prelude to establishing a platform where a gene of interest can be expressed under several promoters simultaneously. The designs featured (i) EGL2sigpro (egl2 promoter and secretion signal), (ii) EGL2cbmlin (egl2 promoter, secretion signal, EGL2 cellulose binding module and linker), (iii) CBH2sigpro (cbh2 promoter and secretion signal) and (iv) CBH2cbmlin (cbh2 promoter, secretion signal, CBH2 cellulose binding module and linker). Recombinant vectors were introduced individually into the high protein-secreting T. reesei RUT-C30 strain to generate single-promoter transformants expressing the Dictyoglomus thermophilum xynB gene that encodes a thermophilic xylanase enzyme (XynB). Ten transformants producing XynB representing each of the four different types of vectors were selected for further testing and the highest XynB production was achieved from a transformant containing 1–2 copies of the EGL2cbmlin vector. Best xylanase producers did not show any particular pattern in terms of the number of gene copies and their mode of integration into the chromosomal DNA. Transformants generated with the cbmlin-type vectors produced multiple forms of XynB which were decorated with various N- and O-glycans. One of the O-glycans was identified as hexuronic acid, whose presence had not been observed previously in the glycosylation patterns of T. reesei.

Complex carbohydrates: 1. Conformational studies on some oligosaccharides related to N-glycosyl proteins which interact with concanavalin A

Empirical potential energy calculations have been carried out to determine the preferred conformations of some oligosaccharides having the trimannosidic core structure (Man3GlcNAc2) and which interact with concanavalin A. In the minimum energy conformations for the trimannosidic core the mannose residue on the Man α(1–6) arm comes close to one of the N-acetylglucosamine residues of the core. The addition of N-acetylglucosamine residues to the terminal mannose residues does not alter the preferred conformation of the trimannosidic core although it alters the relative preference of some of the higher energy conformations. The minimum energy conformation broadly agrees with available X-ray data. The presence of a bisecting N-acetylglucosamine residue on the middle mannose does not push the trimannosidic core to any new conformation but it does alter the relative preference for a particular conformation.

Synthesis of mixed glycosyl disulfides/selenenylsulfides using benzyltriethylammonium tetrathiomolybdate as a sulfur transfer reagent

An easy and mild method has been developed for the synthesis of mixed glycosyl disulfides/selenenylsulfides from glycosyl halides and diaryl/dialkyl dichalcogenides in the presence of benzyltriethylammonium tetrathiomolybdate (BnEt3N)(2)MoS4]. The salient feature of this method is the sulfur transfer from BnEt3N](2)MoS4 to form glycosyl disulfides which with excess tetrathiomolybdate further undergo exchange reaction with other dichalcogenides in a one-pot operation. (C) 2014 Elsevier Ltd. All rights reserved.

Predicted glycosyl transferase genes located outside the HEP island are required for formation of heterocyst envelope polysaccharide in Anabaena sp strain PCC 7120

During maturation, heterocysts form an envelope layer of polysaccharide, called heterocyst envelope polysaccharide (HEP), whose synthesis depends on a cluster of genes, the HEP island, and on an additional, distant gene, hepB, or a gene immediately downstream from hepB. We show that HEP formation depends upon the predicted glycosyl transferase genes all4160 at a third locus and alr3699, which is adjacent to hepB and is cotranscribed with it. Mutations in the histidine kinase genes hepN and hepK appear to silence the promoter of hepB and incompletely down-regulate all4160.

Functional gold nanoparticles for studying the interaction of lectin with glycosyl complex on living cellular surfaces

In this study. lectin-conjugated gold nanoparticles (GNPs) were prepared by standard biotin-streptavidin chemistry. The lectin-conjugated GNPs call be used as ail indicator for studying the interaction of lectin with glycosyl complex on living cellular Surfaces due to the high affinity of the lectin with saccharides. The interactions of two well-known lectins (Ricinus communis agglutinin and concanavalin A) and three different cell lines (HeLa, 293, and 293T) were selected here to establish this assay. Highly binding affinity of R. communis agglutinin with cells was demonstrated by conventional microscopic and UV-visible spectroscopic Studies. In addition, the binding process can be inhibited by galactose, giving further proof of the binding mechanism. (c) 2009 Elsevier Inc. All rights reserved.

Synthesis of a group of diosgenyl saponins with combined use of glycosyl trichloroacetimidate and thioglycoside donors

With the combined use of glycosyl trichloroacetimidates and thioglycosides, a group of natural diosgenyl saponins (1-6) are efficiently synthesized, in either a stepwise or a 'one-pot' manner. The trichloroacetimidate is employed as an efficient temporary hydroxy protecting group in glycosylation with the glycosyl trichloroacetimidate. The intermolecular alkylthio-group transfer is demonstrated to be a common side-reaction during glycosylation with thioglycosides.

Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease.

In common with many other plasma membrane glycoproteins of eukaryotic origin, the promastigote surface protease (PSP) of the protozoan parasite Leishmania contains a glycosyl-phosphatidylinositol (GPI) membrane anchor. The GPI anchor of Leishmania major PSP was purified following proteolysis of the PSP and analyzed by two-dimensional 1H-1H NMR, compositional and methylation linkage analyses, chemical and enzymatic modifications, and amino acid sequencing. From these results, the structure of the GPI-containing peptide was found to be Asp-Gly-Gly-Asn-ethanolamine-PO4-6Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol-1-PO4-(1-alkyl-2-acyl-glycerol). The glycan structure is identical to the conserved glycan core regions of the GPI anchor of Trypanosoma brucei variant surface glycoprotein and rat brain Thy-1 antigen, supporting the notion that this portion of GPIs are highly conserved. The phosphatidylinositol moiety of the PSP anchor is unusual, containing a fully saturated, unbranched 1-O-alkyl chain (mainly C24:0) and a mixture of fully saturated unbranched 2-O-acyl chains (C12:0, C14:0, C16:0, and C18:0). This lipid composition differs significantly from those of the GPIs of T. brucei variant surface glycoprotein and mammalian erythrocyte acetylcholinesterase but is similar to that of a family of glycosylated phosphoinositides found uniquely in Leishmania.

Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses

Xylan, a hemicellulosic component of the plant cell wall, is one of the most abundant polysaccharides in nature. In contrast to dicots, xylan in grasses is extensively modified by alpha-(1,2)- and alpha-(1,3)-linked arabinofuranose. Despite the importance of grass arabinoxylan in human and animal nutrition and for bioenergy, the enzymes adding the arabinosyl substitutions are unknown. Here we demonstrate that knocking-down glycosyltransferase (GT) 61 expression in wheat endosperm strongly decreases alpha-(1,3)-linked arabinosyl substitution of xylan. Moreover, heterologous expression of wheat and rice GT61s in Arabidopsis leads to arabinosylation of the xylan, and therefore provides gain-of-function evidence for alpha-(1,3)-arabinosyltransferase activity. Thus, GT61 proteins play a key role in arabinoxylan biosynthesis and therefore in the evolutionary divergence of grass cell walls.

RNA interference suppression of genes in glycosyl transferase families 43 and 47 in wheat starchy endosperm causes large decreases in arabinoxylan content

The cell walls of wheat (Triticum aestivum) starchy endosperm are dominated by arabinoxylan (AX), accounting for 65% to 70% of the polysaccharide content. Genes within two glycosyl transferase (GT) families, GT43 (IRREGULAR XYLEM9 [IRX9] and IRX14) and GT47 (IRX10), have previously been shown to be involved in the synthesis of the xylan backbone in Arabidopsis, and close homologs of these have been implicated in the synthesis of xylan in other species. Here, homologs of IRX10 TaGT47_2 and IRX9 TaGT43_2, which are highly expressed in wheat starchy endosperm cells, were suppressed by RNA interference (RNAi) constructs driven by a starchy endosperm-specific promoter. The total amount of AX was decreased by 40% to 50% and the degree of arabinosylation was increased by 25% to 30% in transgenic lines carrying either of the transgenes. The cell walls of starchy endosperm in sections of grain from TaGT43_2 and TaGT47_2 RNAi transgenics showed decreased immunolabeling for xylan and arabinoxylan epitopes and approximately 50% decreased cell wall thickness compared with controls. The proportion of AX that was water soluble was not significantly affected, but average AX polymer chain length was decreased in both TaGT43_2 and TaGT47_2 RNAi transgenics. However, the long AX chains seen in controls were absent in TaGT43_2 RNAi transgenics but still present in TaGT47_2 RNAi transgenics. The results support an emerging picture of IRX9-like and IRX10-like proteins acting as key components in the xylan synthesis machinery in both dicots and grasses. Since AX is the main component of dietary fiber in wheat foods, the TaGT43_2 and TaGT47_2 genes are of major importance to human nutrition.

Crystalline structure of mangiferin, a C-glycosyl-substituted 9H-xanthen-9-one isolated from the stem bark of Mangifera indica

The crystalline structure of mangiferin (= 2-beta-D-glucopyranosyl-1,3,6,7-tetrahydroxy-9H-xanthen-9-one; 1), a biologically active xanthenone C-glycoside, isolated from the stem bark of Mangifera indica (Anacardiaceae), was unambiguously determined by single-crystal X-ray diffraction (XRD). The crystal structure is summarized as follows: triclinic, P1, a = 7.6575(5), b = 11.2094(8), c = 11.8749(8) angstrom, alpha = 79.967(5), beta = 87.988(4), gamma = 72.164(4)degrees, V = 955.3(1) angstrom(3), and Z = 2. The structure also shows two molecules in the asymmetric unit cell and five crystallization H2O molecules. The packing is stabilized by several intermolecular H-bonds involving either the two symmetry-independent mangiferin molecules 1a and 1b, or the H2O ones.

C-glycosyl flavones from Peperomia blanda

The methanol extract from aerial parts of the Peperomia blanda (Piperaceae) yielded two C-glycosyl-flavones. Their structures were elucidated on the basis of extensive spectroscopic analysis, including 1D and 2D NMR, chemical transformation and comparison with the related known compounds. The structure of the new flavonoids were established as 4`-methoxy-vitexin 7-O-beta-D-xylopyranoside (1) (7-O-beta-D-xylopyranosyl-8-C-beta-D-glucopyranosyl-4`-methoxy-apigenin) and vicenin-2 (2). The antioxidant activity of both compounds was investigated using the DPPH assay. Both compounds showed only modest activity, with IC50 values of 357.2 mu M for 1, and 90.5 mu M for 2. (C) 2008 Elsevier B.V. All rights reserved.

A glycosyl hydrolase family 16 gene is responsible for the endogenous production of β-1,3-glucanases within decapod crustaceans

To identify the gene responsible for the production of a β-1,3-glucanase (laminarinase) within crustacea, a glycosyl hydrolase family 16 (GHF16) gene was sequenced from the midgut glands of the gecarcinid land crab, Gecarcoidea natalis and the freshwater crayfish, Cherax destructor. An open reading frame of 1098bp for G. natalis and 1095bp for C. destructor was sequenced from cDNA. For G. natalis and C. destructor respectively, this encoded putative proteins of 365 and 364 amino acids with molecular masses of 41.4 and 41.5kDa. mRNA for an identical GHF16 protein was also expressed in the haemolymph of C. destructor. These putative proteins contained binding and catalytic domains that are characteristic of a β-1,3-glucanase from glycosyl hydrolase family 16. The amino acid sequences of two short 8-9 amino acid residue peptides from a previously purified β-1,3-glucanase from G. natalis matched exactly that of the putative protein sequence. This plus the molecular masses of the putative proteins matching that of the purified proteins strongly suggests that the sequences obtained encode for a catalytically active β-1,3-glucanase. A glycosyl hydrolase family 16 cDNA was also partially sequenced from the midgut glands of other amphibious (Mictyrisplatycheles and Paragrapsus laevis) and terrestrial decapod species (Coenobita rugosus, Coenobita perlatus, Coenobita brevimanus and Birgus latro) to confirm that the gene is widely expressed within this group. There are three possible hypothesised functions and thus evolutionary routes for the β-1,3-glucanase: 1) a digestive enzyme which hydrolyses β-1,3-glucans, 2) an enzyme which cleaves β-1,3-glycosidic bonds within cell walls to release cell contents or 3) an immune protein which can hydrolyse the cell walls of potentially pathogenic micro-organisms.

Mild and efficient hydrolysis of thioglycosides to glycosyl hemiacetals using N-iodosaccharin

A convenient methodology has been developed for the mild hydrolysis of thioglycosides to the corresponding hemiacetals using N-iodosaccharin without any requirement of co-activator. Most of the functional groups used for the protecting group manipulation of carbohydrates are remain unaffected under the reaction condition.