Saccharide Complexes Research Articles

Light‐Driven Site‐Selective Glycosylation of Native Carbohydrates

Carbohydrates constitute the largest source of biomass on Earth, but their synthetic modification is highly challenging due to their high content of oxygen functionalities. The site‐ and stereoselective modification of native sugars is a definitive goal of glycochemistry research. Recent efforts to bypass the need for protecting groups, leveraging selective activation through photochemical mechanisms for site‐selective C‐C bond formation from native sugars, are likely to largely impact all glycochemistry‐related areas. Davis, Koh, and co‐workers have recently presented their use of photocatalysis to develop a “cap and glycosylate” approach for the site‐ and stereoselective C‐glycosylation of native sugars. The modernization of a direct radical functionalization of in situ formed thioglycoside using photocatalysis was used in the synthetic manipulation of unprotected carbohydrates. This allowed reaching complex saccharides, and post‐translational modification of proteins.

Light-Driven Site-Selective Glycosylation of Native Carbohydrates.

Carbohydrates constitute the largest source of biomass on Earth, but their synthetic modification is challenging due to their high content in oxygen functionalities. The site- and stereoselective modification of native sugars is a definite goal of glycochemistry research. Recent efforts to bypass the need for protecting groups, leveraging selective activation through photochemical mechanisms for site-selective C-C bond formation from native sugars, are likely to largely impact all glycochemistry-related areas. Davis, Koh, and co-workers have recently presented their use of photocatalysis to develop a "cap and glycosylate" approach for the site- and stereoselective C-glycosylation of native sugars. A modernized direct radical functionalization of in situ formed thioglycoside using photocatalysis was used in the synthetic manipulation of unprotected carbohydrates. This allowed reaching complex saccharides, and post-translational modification of proteins.

Direct radical functionalization of native sugars

Naturally occurring (native) sugars and carbohydrates contain numerous hydroxyl groups of similar reactivity1,2. Chemists, therefore, rely typically on laborious, multi-step protecting-group strategies3 to convert these renewable feedstocks into reagents (glycosyl donors) to make glycans. The direct transformation of native sugars to complex saccharides remains a notable challenge. Here we describe a photoinduced approach to achieve site- and stereoselective chemical glycosylation from widely available native sugar building blocks, which through homolytic (one-electron) chemistry bypasses unnecessary hydroxyl group masking and manipulation. This process is reminiscent of nature in its regiocontrolled generation of a transient glycosyl donor, followed by radical-based cross-coupling with electrophiles on activation with light. Through selective anomeric functionalization of mono- and oligosaccharides, this protecting-group-free ‘cap and glycosylate’ approach offers straightforward access to a wide array of metabolically robust glycosyl compounds. Owing to its biocompatibility, the method was extended to the direct post-translational glycosylation of proteins.

Research of saccharides and related biocomplexes: A review with recent techniques and applications.

Saccharides and biocompounds as saccharide (sugar) complexes have various roles and biological functions in living organisms due to modifications via nucleophilic substitution, polymerization, and complex formation reactions. Mostly, mono-, di-, oligo-, and polysaccharides are stabilized to inactive glycosides, which are formed in metabolic pathways. Natural saccharides are important in food and environmental monitoring. Glycosides with various functionalities are significant in clinical and medical research. Saccharides are often studied with the chromatographic methods of hydrophilic interaction liquid chromatography and anion exchange chromatograpy, but also with capillary electrophoresis and mass spectrometry with their on-line coupling systems. Sample preparation is important in the identification of saccharide compounds. The cases discussed here focus on bioscience, clinical, and food applications.

Ferrous-immobilized iron saccharide complexes for electro-synthesis of ammonia via NORR

Ferrous-immobilized iron saccharide complexes for electro-synthesis of ammonia via NORR

Capillary zone electrophoresis of lipoarabinomannan by multi-layered concentration.

The present article describes a capillary zone electrophoresis method which relies on a multilayered water-alkali solvent stacking with online ionization to enhance detection of mannose, arabinose, and their oligosaccharides, which are used as the migration profile standards but are also the distinctive structural components of lipoarabinomannan. Lipoarabinomannan is detected in patients having tuberculosis. The capillary electrophoresis method with ionization of the whole saccharides without degradation in alkaline solution inside the capillary is based on the structural deprotonation of the molecules under ultrahigh pH conditions. The validation of the capillary electrophoresis parameters revealed that the 15-fold electrolyte-water-injection plug allowed detection of one-third lower concentrations than detected without online concentration. For the first time, the better detectability was seen especially for highly polymerized, but otherwise poorly ionized, arabinooctaose. The applicability of the method for detecting whole large biological saccharide complexes was confirmed by the glycolipid lipoarabinomannan. For the first time also, the migration of the indestructible lipoarabinomannan was detected together with oligosaccharides used representing the capping units, namely mannose, mannobiose, and mannotriose. The myo-inositol-phosphate-lipid unit was seen to migrate separately from the arabinomannan, since it was hydrolyzed from one lipoarabinomannan product under alkaline conditions in capillary electrophoresis.

Accumulation of Reducing Sugars by Small-leaved Linden Flowers (Tilia cordata Miller, 1768) in the Voronezh Region of Russia

Introduction. Small-leaved linden flowers are mainly used for aquatic extracts, and their pharmacological benefit is based on water-soluble polysaccharides.Aim. The aim of this study was to investigate the rate of accumulating all reducing sugars in small-leaved linden flowers, collected in agro- and urbo-cenoses of the Voronezh region.Materials and methods. 51 sites were selected for collecting flowers from the small-leaved linden, which is a widespread deciduous tree species in Russia. In order to determine the total level of reducing sugars in the samples, we measured glucose levels using the method described in Pharmacopeia article "Linden Flower". Correlation coefficients were analyzed to examine in detail the effect of the major pollutants (heavy metals and arsenic) on the accumulation of reducing sugars in small-leaved linden flowers.Results and discussion. All analyzed samples of medicinal plant raw materials were benign in terms of their reducing sugar levels. Samples collected in control (protected) areas contained reducing sugar levels 13.31 to 16.89 %, which is 6–8 times more than the lower numerical value established by the Pharmacopoeia article. In the agrocenoses of the region, the concentration of reducing sugars varied from 6.12 to 16.68 %, which is 3–8 times more than the value given in the Pharmacopoeia article. In the urbocenoses of the region, a lower level of reducing sugars was found compared to samples from protected areas (2.35–13.49 %). Correlation coefficients showed a noticeable negative impact of cadmium, chromium, cobalt, copper, zinc, as well as a moderate negative effect of lead and mercury on the accumulation of reducing sugars in the small-leaved linden flowers.Conclusion. The lowest concentrations of reducing sugars were detected in samples harvested along the streets of large cities in the region, and along highways, roads and railways. This suggests that anthropogenic factors might negatively impact the accumulation of reducing sugars in small-leaved linden flowers in these areas. At the same time, it is possible that saccharide complexes might have reacted with heavy metals instead of the complexing agent when levels of reducing sugars in the samples were quantitatively determined. This would have the effect of underrepresenting reducing sugar levels in the raw materials.

One-Pot Glycosylation Strategy for Rapid Access of Oligosaccharides with Wide Range of Molecular Diversity

: In carbohydrate chemistry, the synthesis of complex saccharides with well-defined structures is the most formidable process as it is quite strenuous to isolate carbohydrates in acceptable purity and amounts from natural sources. Therefore, complex saccharides with well-defined structures are often most conveniently accessed through chemical syntheses. This review mainly focuses on the methodologies for one-pot glycosylation into the complex glycans from the non-reducing end to reducing end and vice versa, orthogonal, preactivation based, photochemical as well as hybrid one-pot glycosylation. The main goal of this review in the carbohydrate community was to rapidly synthesize biologically relevant glycans that can be implemented for research in carbohydrate-based vaccine development, diagnostics, as well as drug discovery.

Combination of GC–MS and selective peak elimination procedures as a tool for characterization of complex saccharide mixtures – Application to pyrolysis bio-oils

In this study, a GC–MS method was developed for the quantification of saccharides in complex mixtures such as bio-oils and bio-oil aqueous phases produced by ablative pyrolysis of lignocellulosic biomass. The samples were first treated using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and the trimethylsilylated (more volatile) derivatives were analyzed by GC–MS. The method offers advantages of great separation capability and simultaneous identification of unknown peaks by comparison of the mass spectra and retention indices with extensive libraries available online. However, even with these tools at hand, the identification of several saccharide-resembling compounds can be challenging especially in such highly complex samples as pyrolysis bio-oils. For this reason, we devised a novel procedure, which eliminates certain saccharides depending on their specific chemical properties before subjecting the samples to the GC–MS analysis. The procedure was based on the combination of aniline treatment (elimination of reducing aldoses), and hydrolysis (elimination of anhydrosugars, glycosides, disaccharides and oligosaccharides). Based on the differences in chromatograms before and after the procedure, the unknown compounds were assigned into groups based on their susceptibility to each treatment. The combination of all methods above has allowed more accurate identification and quantification of saccharides, some of which were not as of today found in bio-oils.

Transcriptome analysis of Actinoplanes utahensis reveals molecular signature of saccharide impact on acarbose biosynthesis.

Different carbon sources lead to differential acarbose production in Actinoplanes. To uncover the underlying differentiation in the context of genes and pathways, we performed transcriptome sequencing of Actinoplanes utahensis ZJB-03852 grown on different saccharides, such as glucose, maltose, or the saccharide complex consisting of glucose plus maltose. The differentially expressed genes were classified into GO (gene ontology) terms and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways for functional annotations. Key enriched modules were uncovered. Our data revealed that both maltose and its complex with glucose gave improved acarbose titer. Sugar transportation, cytochrome oxidase, protein synthesis and amino acid metabolism modules were enriched under the saccharide complex condition, while ferritin metabolism gene expressions were enriched in the glucose medium. Our results provided the foundation for uncovering the mechanism of carbon source on acarbose production in A. utahensis.

Probabilistic identification of saccharide moieties in biomolecules and their protein complexes

The chemical composition of saccharide complexes underlies their biomedical activities as biomarkers for cardiometabolic disease, various types of cancer, and other conditions. However, because these molecules may undergo major structural modifications, distinguishing between compounds of saccharide and non-saccharide origin becomes a challenging computational problem that hinders the aggregation of information about their bioactive moieties. We have developed an algorithm and software package called “Cheminformatics Tool for Probabilistic Identification of Carbohydrates” (CTPIC) that analyzes the covalent structure of a compound to yield a probabilistic measure for distinguishing saccharides and saccharide-derivatives from non-saccharides. CTPIC analysis of the RCSB Ligand Expo (database of small molecules found to bind proteins in the Protein Data Bank) led to a substantial increase in the number of ligands characterized as saccharides. CTPIC analysis of Protein Data Bank identified 7.7% of the proteins as saccharide-binding. CTPIC is freely available as a webservice at (http://ctpic.nmrfam.wisc.edu).

IR action spectroscopy of glycosaminoglycan oligosaccharides

Glycosaminoglycans (GAGs) are a physio- and pharmacologically highly relevant class of complex saccharides, possessing a linear sequence and strongly acidic character. Their repetitive linear core makes them seem structurally simple at first glance, yet differences in sulfation and epimerization lead to an enormous structural diversity with only a few GAGs having been successfully characterized to date. Recent infrared action spectroscopic experiments on sulfated mono- and disaccharide ions show great promise. Here, we assess the potential of two types of gas-phase action spectroscopy approaches in the range from 1000 to 1800 cm−1 for the structural analysis of complex GAG oligosaccharides. Synthetic tetra- and pentasaccharides were chosen as model compounds for this benchmark study. Utilizing infrared multiple photon dissociation action spectroscopy at room temperature, diagnostic bands are largely unresolved. In contrast, cryogenic infrared action spectroscopy of ions trapped in helium nanodroplets yields resolved infrared spectra with diagnostic features for monosaccharide composition and sulfation pattern. The analysis of GAGs could therefore significantly benefit from expanding the conventional MS-based toolkit with gas-phase cryogenic IR spectroscopy.Graphical abstract

New Rabbit Erythrocyte Specific Mycelial Lectins from Fusarium sp. with Complex Saccharide Specificity

Eight Fusarium sp. namely, F. acutatum, F. globosum, F. graminearum, F. lactis, F. nivale, F. proliferatum, F. pseudoanthophilum and F. robustum were screened for the presence of lectins by hemagglutination activity using human ABO, porcine, ovine, goat and rabbit erythrocytes. Mycelial extracts of all the fungal cultures except F. graminearum displayed unique lectin activity with only rabbit erythrocytes. Enzymatic treatment of rabbit erythrocytes with neuraminidase has significantly enhanced the titre of all the lectin positive extracts of fungal cultures. In contrast, most of the lectins showed a decline in lectin activity with protease treated rabbit erythrocytes. Saccharide specificity studies have shown that majority of the lectins were inhibitory towards O-acetyl sialic acids. None of the lectins from Fusarium sp. were inhibited by dextran, meso-inositol and N-acetyl-D-glucosamine. Most of the fungal cultures displayed highest hemagglutination activity during 10th day of growth in broth cultures. The unique saccharide specificity of Fusarium sp. lectins can be used for elucidating their clinical role in glycobiology research.

Fractionation of mono- and disaccharides via nanofiltration: Influence of pressure, temperature and concentration

During the enzymatic biotransformation of lactose to galactooligosaccharides, complex saccharide solutions are generated. In most cases, solely a fraction of these saccharides is desired. Therefore, four industrial relevant nanofiltration membranes were tested regarding their suitability to fractionate mono- and disaccharides. The most promising membrane was selected for further experiments. With this membrane additional parameters, namely transmembrane pressure difference ΔpTM, filtration temperature ϑ and saccharide concentration cS, were varied under steady state conditions. Saccharide concentrations in all retentate and permeate samples were analyzed by HPLC-ELSD. The criteria of flux J, rejection RS, solute flux JS, fractionation purity ΔRS and efficiency QJS were evaluated in a complex food matrix at an industrial relevant scale. The interaction between ΔpTM and ϑ and their influence on ΔRS and QJS was further investigated by performing a multiple linear regression analysis with a quadratic model. The results suggest that for the chosen membrane and saccharides, trade-offs between purity and efficiency are necessary depending on the overall aim of the process. In the case that high purities need to be achieved, combinations of either low pressures and temperatures or high pressures and temperatures are promoting, while only increasing one parameter decreases the purity of the fractions. In order to achieve a high process efficiency high pressures are promoting.

Noncovalent Complexation of Saccharides with Ions and Isomer Discrimination for Saccharides

Noncovalent Complexation of Saccharides with Ions and Isomer Discrimination for Saccharides

"One-Pot" Protection, Glycosylation, and Protection-Glycosylation Strategies of Carbohydrates.

Carbohydrates, which are ubiquitously distributed throughout the three domains of life, play significant roles in a variety of vital biological processes. Access to unique and homogeneous carbohydrate materials is important to understand their physical properties, biological functions, and disease-related features. It is difficult to isolate carbohydrates in acceptable purity and amounts from natural sources. Therefore, complex saccharides with well-defined structures are often most conviently accessed through chemical syntheses. Two major hurdles, regioselective protection and stereoselective glycosylation, are faced by carbohydrate chemists in synthesizing these highly complicated molecules. Over the past few years, there has been a radical change in tackling these problems and speeding up the synthesis of oligosaccharides. This is largely due to the development of one-pot protection, one-pot glycosylation, and one-pot protection-glycosylation protocols and streamlined approaches to orthogonally protected building blocks, including those from rare sugars, that can be used in glycan coupling. In addition, new automated strategies for oligosaccharide syntheses have been reported not only for program-controlled assembly on solid support but also by the stepwise glycosylation in solution phase. As a result, various sugar molecules with highly complex, large structures could be successfully synthesized. To summarize these recent advances, this review describes the methodologies for one-pot protection and their one-pot glycosylation into the complex glycans and the chronological developments associated with automated syntheses of oligosaccharides.

Sequencing of glycosaminoglycans with potential to interrogate sequence-specific interactions

Technologies to sequence nucleic acids/proteins are widely available, but straightforward methodologies to sequence complex polysaccharides are lacking. We here put forward a strategy to sequence glycosaminoglycans, long linear polysaccharides involved in many biochemical processes. The method is based on the covalent immobilization and (immuno)chemical characterization of only those size-separated saccharides that harbor the original reducing end of the full-length chain. Using this methodology, the saccharide sequence of the chondroitin sulfate chain of the proteoglycan bikunin was determined. The method can be performed in any standard biochemical lab and opens studies to the interaction of complex saccharide sequences with other biomolecules.

Metabolic response of Agrobacterium tumefaciens 5A to arsenite.

Wide-spread abundance in soil and water, coupled with high toxicity have put arsenic at the top of the list of environmental contaminants. Early studies demonstrated that both concentration and the valence state of inorganic arsenic (arsenite, As(III) vs. arsenate As(V)) can be modulated by microbes. Using genetics, transcriptomic and proteomic techniques, microbe-arsenic detoxification, respiratory As(V) reduction and As(III) oxidation have since been examined. The effect of arsenic exposure on whole-cell intracellular microbial metabolism, however, has not been extensively studied. We combined LC-MS and 1 H NMR to quantify metabolic changes in Agrobacterium tumefaciens (strain 5A) upon exposure to sub-lethal concentrations of As(III). Metabolomics analysis reveals global differences in metabolite concentrations between control and As(III) exposure groups, with significant perturbations to intermediates shuttling into and cycling within the TCA cycle. These data are most consistent with the disruption of two key TCA cycle enzymes, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. Glycolysis also appeared altered following As(III) stress, with carbon accumulating as complex saccharides. These observations suggest that an important consequence of As(III) contamination in nature will be to alter microbial carbon metabolism at the microbial community level and thus has the potential to foundationally impact all biogeochemical cycles in the environment.

Effects of fruit juice concentrate production on individual anthocyanin species in elderberry

SummaryElderberry concentrate is widely used for food colouring. Its production includes several processing steps, which influence pigment composition, thus affecting the quality of final product. In this study, qualitative and quantitative changes in anthocyanin components were followed during the production of elderberry juice concentrate using Haschberg and Samocco varieties. Juice concentration resulted in more than five‐fold concentration increase in anthocyanins; however, clarification, filtration and evaporation steps caused slight but continuous decrease in anthocyanins relative to the dry matter of the product. Pectolytic enzymatic treatment was found to be one of the most critical processing steps that affects anthocyanin yield during production, and this effect on yield can be variety‐dependent. Additionally, two condensed dimeric anthocyanin species generated after heat treatment are reported for the first time to be present in elderberry juice concentrate. Our results suggest that cyanidin‐based anthocyanin conjugates with more complex saccharide moieties are more robust during production.

Saprophytic and pathogenic fungi in the Ceratocystidaceae differ in their ability to metabolize plant-derived sucrose.

BackgroundProteins in the Glycoside Hydrolase family 32 (GH32) are carbohydrate-active enzymes known as invertases that hydrolyse the glycosidic bonds of complex saccharides. Fungi rely on these enzymes to gain access to and utilize plant-derived sucrose. In fungi, GH32 invertase genes are found in higher copy numbers in the genomes of pathogens when compared to closely related saprophytes, suggesting an association between invertases and ecological strategy. The aim of this study was to investigate the distribution and evolution of GH32 invertases in the Ceratocystidaceae using a comparative genomics approach. This fungal family provides an interesting model to study the evolution of these genes, because it includes economically important pathogenic species such as Ceratocystis fimbriata, C. manginecans and C. albifundus, as well as saprophytic species such as Huntiella moniliformis, H. omanensis and H. savannae.ResultsThe publicly available Ceratocystidaceae genome sequences, as well as the H. savannae genome sequenced here, allowed for the identification of novel GH32-like sequences. The de novo assembly of the H. savannae draft genome consisted of 28.54 megabases that coded for 7 687 putative genes of which one represented a GH32 family member. The number of GH32 gene family members appeared to be related to the ecological adaptations of these fungi. The pathogenic Ceratocystis species all contained two GH32 family genes (a putative cell wall and a putative vacuolar invertase), while the saprophytic Huntiella species had only one of these genes (a putative cell wall invertase). Further analysis showed that the evolution of the GH32 gene family in the Ceratocystidaceae involved transposable element-based retro-transposition and translocation. As an example, the activity of a Fot5-like element likely facilitated the assembly of the genomic regions harbouring the GH32 family genes in Ceratocystis.ConclusionsThis study provides insight into the evolutionary history of the GH32 gene family in Ceratocystidaceae. Our findings suggest that transposable elements shaped the evolution of the GH32 gene family, which in turn determines the sucrolytic activities and related ecological strategies of the Ceratocystidaceae species that harbour them. The study also provides insights into the role of carbohydrate-active enzymes in plant-fungal interactions and adds to our understanding of the evolution of these enzymes and their role in the life style of these fungi.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0550-7) contains supplementary material, which is available to authorized users.