Carbohydrate Units Research Articles

Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway

Nogalamycin, an aromatic polyketide displaying high cytotoxicity, has a unique structure, with one of the carbohydrate units covalently attached to the aglycone via an additional carbon-carbon bond. The underlying chemistry, which implies a particularly challenging reaction requiring activation of an aliphatic carbon atom, has remained enigmatic. Here, we show that the unusual C5''-C2 carbocyclization is catalyzed by the non-heme iron α-ketoglutarate (α-KG)-dependent SnoK in the biosynthesis of the anthracycline nogalamycin. The data are consistent with a mechanistic proposal whereby the Fe(IV) = O center abstracts the H5'' atom from the amino sugar of the substrate, with subsequent attack of the aromatic C2 carbon on the radical center. We further show that, in the same metabolic pathway, the homologous SnoN (38% sequence identity) catalyzes an epimerization step at the adjacent C4'' carbon, most likely via a radical mechanism involving the Fe(IV) = O center. SnoK and SnoN have surprisingly similar active site architectures considering the markedly different chemistries catalyzed by the enzymes. Structural studies reveal that the differences are achieved by minor changes in the alignment of the substrates in front of the reactive ferryl-oxo species. Our findings significantly expand the repertoire of reactions reported for this important protein family and provide an illustrative example of enzyme evolution.

ChemInform Abstract: DNA Display of Glycoconjugates to Emulate Oligomeric Interactions of Glycans

AbstractReview: [63 refs.

Thiyl‐Radical Reactions in Carbo­hydrate Chemistry: From Thiosugars to Glycoconjugate Synthesis

AbstractThiyl‐radical mediated reactions have been extensively investigated for the preparation of carbohydrate derivatives. Due to the mild reaction conditions, high yields and tolerance to a wide range of functional groups, these reactions are ideally suited to the preparation of bioconjugates including glycopeptides and glycoproteins. This microreview covers the various synthetic strategies that have been employed for the preparation of thiosugars, glycoconjugates and glycodendrimers, employing the carbohydrate unit as both the carrier of the thiyl radical and also as the acceptor. Both the inter‐ and intramolecular thiol‐ene and ‐yne processes are presented and discussed. Future perspective and directions for this methodology in glycoscience are also examined.

Synthetic Receptors for the High-Affinity Recognition of O-GlcNAc Derivatives.

The combination of a pyrenyl tetraamine with an isophthaloyl spacer has led to two new water‐soluble carbohydrate receptors (“synthetic lectins”). Both systems show outstanding affinities for derivatives of N‐acetylglucosamine (GlcNAc) in aqueous solution. One receptor binds the methyl glycoside GlcNAc‐β‐OMe with K a≈20 000 m −1, whereas the other one binds an O‐GlcNAcylated peptide with K a≈70 000 m −1. These values substantially exceed those usually measured for GlcNAc‐binding lectins. Slow exchange on the NMR timescale enabled structural determinations for several complexes. As expected, the carbohydrate units are sandwiched between the pyrenes, with the alkoxy and NHAc groups emerging at the sides. The high affinity of the GlcNAcyl–peptide complex can be explained by extra‐cavity interactions, raising the possibility of a family of complementary receptors for O‐GlcNAc in different contexts.

Synthetic Receptors for the High‐Affinity Recognition of O‐GlcNAc Derivatives

AbstractThe combination of a pyrenyl tetraamine with an isophthaloyl spacer has led to two new water‐soluble carbohydrate receptors (“synthetic lectins”). Both systems show outstanding affinities for derivatives of N‐acetylglucosamine (GlcNAc) in aqueous solution. One receptor binds the methyl glycoside GlcNAc‐β‐OMe with Ka≈20 000 m−1, whereas the other one binds an O‐GlcNAcylated peptide with Ka≈70 000 m−1. These values substantially exceed those usually measured for GlcNAc‐binding lectins. Slow exchange on the NMR timescale enabled structural determinations for several complexes. As expected, the carbohydrate units are sandwiched between the pyrenes, with the alkoxy and NHAc groups emerging at the sides. The high affinity of the GlcNAcyl–peptide complex can be explained by extra‐cavity interactions, raising the possibility of a family of complementary receptors for O‐GlcNAc in different contexts.

Solubilization and Characterization of β-Mannosyltransferase from Pig Aorta

Glycosyltransferase in general play a very important role in the post-translational modification of number of biomolecules including glycoproteins. The glycosylation of the protein occurs in the endoplasmic reticulum and is carried out by at least 14 different types of membrane bound glycosyltransferases. It is now well established that glycosylation of protein occurs either at serine or threonine residues (O-glycosylation) or at asparagine residue (N-glycosylation) of proteins. To date there is no definite demonstration of the presence of O-mannosylation of proteins in higher eukaryotes. However, it is a common feature in yeast and other fungal strain, indicating that glycosyltransferases are species-specific as well as tissue-specific. In higher eukaryotes the Nglycosylation is the major post translational process, initiated by the synthesis of a large lipid oligosaccharide, Glc3-Man9-GlcNAc2-PP-Dolichol. This represents the immediate precursor of carbohydrate unit involved in the biosynthesis of glycoproteins. The large oligosaccharide is then transferred from the lipid oligosaccharide to the acceptor lipid by the oligosaccharide transferase. β-Mannosyltransferase is the key enzyme of the dolichol pathway that catalyzes the transfer of mannose to the chitobiose lipid (GlcNAc2-PP-Dol), acceptor substrate from GDP-mannose. This enzyme has been purified to near homogeneity and characterized from pig aorta tissue, using different chromatography techniques, and studying its properties.

Quantitative GSL-glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method

Glycosphingolipids (GSLs) are lipid molecules linked to carbohydrate units that form the plasma membrane lipid raft, which is clustered with sphingolipids, sterols, and specific proteins, and thereby contributes to membrane physical properties and specific recognition sites for various biological events. These bioactive GSL molecules consequently affect the pathophysiology and pathogenesis of various diseases. Thus, altered expression of GSLs in various diseases may be of importance for disease-related biomarker discovery. However, analysis of GSLs in blood is particularly challenging because GSLs are present at extremely low concentrations in serum/plasma. In this study, we established absolute GSL-glycan analysis of human serum based on endoglycoceramidase digestion and glycoblotting purification. We established two sample preparation protocols, one with and the other without GSL extraction using chloroform/methanol. Similar amounts of GSL-glycans were recovered with the two protocols. Both protocols permitted absolute quantitation of GSL-glycans using as little as 20 μl of serum. Using 10 healthy human serum samples, up to 42 signals corresponding to GSL-glycan compositions could be quantitatively detected, and the total serum GSL-glycan concentration was calculated to be 12.1-21.4 μM. We further applied this method to TLC-prefractionated serum samples. These findings will assist the discovery of disease-related biomarkers by serum GSL-glycomics.

Synthesis and Biological Evaluation of Cyanogenic Glycosides

GRAPHICAL ABSTRACTAn efficient procedure for the synthesis of cyanogenic glycosides with different carbohydrate units was developed. Amygdalin (3), prunasin (1), sambunigrin (2), and neoamygdalin (21) were prepared according to the elaborated method, and biological tests, including antifungal, antibacterial, and cytotoxic activities, were performed.

Cell wall components in torrefied softwood and hardwood samples

Torrefaction – the process of soft pyrolysis (200–300°С) in inert atmosphere – is considered to promote the usage of lignocellulosic biomass in various technologies. The initial raw material is not uniform in composition and we compared the effect of torrefaction on the samples of hardwood (birch) and softwood (pine). The major differences between the torrefied samples were observed between 225 and 250°С and were largely connected with different behavior of hemicelluloses. Monosaccharide analysis revealed the decrease in detectable xylose from 26% to 1% (250°С) of the raw sample in birch, and from 11% to 1%—in pine. Mannans were more resistant to degradation. Comparison of data from HPAEC, thermal analysis and IR-spectroscopy revealed that hemicelluloses are modified during torrefaction at 225–250°С, rather than fully degraded and removed from the sample. This may lead to considerable modification of wood properties, more pronounced in hardwoods. The relative content of aromatic structures went up during torrefaction, part of the effect was due to condensation of modified carbohydrate units. Index of cellulose crystallinity increased in torrefied samples. The content of cellulose in birch samples remained the same as in raw sample up to 250°С, while in pine it dramatically decreased after the torrefaction at 250°С. Torrefaction at 300°С made the samples of hardwood and softwood very much alike. The perspectives of usage of hardwoods and softwoods torrefied at different temperatures are discussed.

Unified picture for the conformation and stabilization of the O-glycosidic linkage in glycopeptide model structures

Glycoproteins play a central role in the immune response. In this study, we focus on the core of the common O-linked mucin-type glycopeptides. It has been observed that glycosylation stabilizes the protein in a stiffened, extended structure. We provide a unified picture for the conformation and stabilization of the O-glycosidic linkage using O-(2-acetylamino-2-deoxy-α- or -β-d-galacto- or -mannopyranosyl)-N-acetyl-l-serinamide model structures. We have calculated equilibrium geometries of the model structures with the B3LYP/6-31G(2df,p) method suggested in the Gaussian-4 theory. According to the relative energies, we confirm that the GalNAc-Ser linkage is more stable than its mannose analogues. The natural preference for the α-GalNAc-Ser over the β-GalNAc-Ser anomers can be explained by entropic effects. We explored the hydrogen bonding patterns on the carbohydrate unit calculating highly accurate dRPA@PBE0.25 and dRPA75 energies and found that in some cases, the acetamido group can be fixed by hydrogen bonding from the (O3Carb)H atom, but in most of the cases, it can rotate more freely. The torsion angles in the glycosidic linkage show that the linkage is stiffened more in the α-anomers and the most in the α-GalNAc-Ser structure because of the steric strains in the axial position and by two or three intramolecular hydrogen bonds. We also found that although, in our gas-phase model geometries, the peptide backbone prefers to be in a γ L-turn, a structural water molecule can stabilize a polyproline II helix of a proline-rich sequence, a β-sheet, or more likely random coils.

Probing multivalency in ligand-receptor-mediated adhesion of soft, biomimetic interfaces.

Many biological functions at cell level are mediated by the glycocalyx, a dense carbohydrate-presenting layer. In this layer specific interactions between carbohydrate ligands and protein receptors are formed to control cell–cell recognition, cell adhesion and related processes. The aim of this work is to shed light on the principles of complex formation between surface anchored carbohydrates and receptor surfaces by measuring the specific adhesion between surface bound mannose on a concanavalin A (ConA) layer via poly(ethylene glycol)-(PEG)-based soft colloidal probes (SCPs). Special emphasis is on the dependence of multivalent presentation and density of carbohydrate units on specific adhesion. Consequently, we first present a synthetic strategy that allows for controlled density variation of functional groups on the PEG scaffold using unsaturated carboxylic acids (crotonic acid, acrylic acid, methacrylic acid) as grafting units for mannose conjugation. We showed by a range of analytic techniques (ATR–FTIR, Raman microscopy, zeta potential and titration) that this synthetic strategy allows for straightforward variation in grafting density and grafting length enabling the controlled presentation of mannose units on the PEG network. Finally we determined the specific adhesion of PEG-network-conjugated mannose units on ConA surfaces as a function of density and grafting type. Remarkably, the results indicated the absence of a molecular-level enhancement of mannose/ConA interaction due to chelate- or subsite-binding. The results seem to support the fact that weak carbohydrate interactions at mechanically flexible interfaces hardly undergo multivalent binding but are simply mediated by the high number of ligand–receptor interactions.

DNA display of glycoconjugates to emulate oligomeric interactions of glycans.

Glycans (carbohydrate portion of glycoproteins and glycolipids) frequently exert their function through oligomeric interactions involving multiple carbohydrate units. In efforts to recapitulate the diverse spatial arrangements of the carbohydrate units, assemblies based on hybridization of nucleic acid conjugates have been used to display simplified ligands with tailored interligand distances and valences. The programmability of the assemblies lends itself to a combinatorial display of multiple ligands. Recent efforts in the synthesis and applications of such conjugates are discussed.

Type 1 diabetes mellitus and exercise in competitive athletes.

The number of patients with type 1 diabetes mellitus who are actively participating in competitive sports is increasing. Here, we aimed to assess individual experiences of competitive athletes with type 1 diabetes and to compare these experiences with current recommendations. A survey of 20 competitive athletes with type 1 diabetes, categorized as endurance (n=10) and non-endurance (n=10) athletes, was performed. Endurance and non-endurance athletes did not differ in gender distribution, age, body mass index, and known diabetes duration. Self-reported target blood glucose values prior to exercise were lower in non-endurance than in endurance athletes (195±34 vs. 137±28 mg/dl, P=0.001). The majority of all athletes experienced activity-induced hypo- and hyperglycemic events, independently of exercise type. However, endurance athletes used additional carbohydrate units to prevent activity-induced hypoglycemic events more frequently without monitoring their blood glucose levels than non-endurance athletes (50% vs. 0%, P=0.01). The reduction of the insulin dose on training and competition days compared to days without exercise was similar for endurance and non-endurance athletes. These results point to a very individual adaption of the athlete's therapy during training and competition. However, there are distinct differences in diabetes management between endurance and non-endurance athletes.

N-glycomic Profiling as a Tool to Separate Rectal Adenomas from Carcinomas*

All human cells are covered by glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans. Most glycans are localized to cell surfaces and participate in events essential for cell viability and function. Glycosylation evolves during carcinogenesis, and therefore carcinoma-related glycan structures are potential cancer biomarkers. Colorectal cancer is one of the world's three most common cancers, and its incidence is rising. Novel biomarkers are essential to identify patients for targeted and individualized therapy. We compared the N-glycan profiles of five rectal adenomas and 18 rectal carcinomas of different stages by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Paraffin-embedded tumor samples were deparaffinized, and glycans were enzymatically released and purified. We found differences in glycosylation between adenomas and carcinomas: monoantennary, sialylated, pauci-mannose, and small high-mannose N-glycan structures were more common in carcinomas than in adenomas. We also found differences between stage I–II and stage III carcinomas. Based on these findings, we selected two glycan structures: pauci-mannose and sialyl Lewis a, for immunohistochemical analysis of their tissue expression in 220 colorectal cancer patients. In colorectal cancer, poor prognosis correlated with elevated expression of sialyl Lewis a, and in advanced colorectal cancer, poor prognosis correlated with elevated expression of pauci-mannose. In conclusion, by mass spectrometry we found several carcinoma related glycans, and we demonstrate a method of transforming these results into immunohistochemistry, a readily applicable method to study biomarker expression in patient samples.

Correlation between Molecular Weight and Branch Structure of Glycopolymers Stars and Their Binding to Lectins

It has been hypothesized that maximum binding of carbohydrates can be achieved when the distance between two carbohydrate units are equivalent to two binding sites of a lectin. Therefore, a library of glyco starpolymers was prepared using RAFT polymerization to study the interaction with Concanavalin A (Con A). The starpolymers had a block structure in each arm and were composed of poly(2-acryloylethyl-2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranoside) (PMEA) as the outer block and the inactive, but water-soluble, poly(2-hydroxyethyl acrylate) (PHEA) as the inner block. The sizes of the starpolymers were determined using dynamic light scattering and hydrodynamic diameters between 3 and 12 nm in buffer solution were measured, which match the distance between two binding pockets of 6.5 nm (Chem. Rev. 2002, 102, 555). Turbidity assay, precipitation assay, surface plasmon resonance and quartz crystal microbalance were employed to investigate the binding of the polymers with Con A. The effect of the molecular weight of PHEA, which is located in the core of the star, was found to contribute to chain stretching allowing the interaction with more Con A molecules. Longer PMEA blocks showed a lower binding efficiency per mannose functionality than shorter blocks. It was found that the initial diameter of the polymer in solution does not play a role in determining the binding efficiency. The measured amount of Con A conjugated per star polymer suggests that during the process the chains stretch significantly to accommodate a maximum amount of Con A.

Structure of FcγRI in complex with Fc reveals the importance of glycan recognition for high-affinity IgG binding

Fc gamma receptor I (FcγRI) contributes to protective immunity against bacterial infections, but exacerbates certain autoimmune diseases. The sole high-affinity IgG receptor, FcγRI plays a significant role in immunotherapy. To elucidate the molecular mechanism of its high-affinity IgG binding, we determined the crystal structure of the extracellular domains of human FcγRI in complex with the Fc domain of human IgG1. FcγRI binds to the Fc in a similar mode as the low-affinity FcγRII and FcγRIII receptors. In addition to many conserved contacts, FcγRI forms additional hydrogen bonds and salt bridges with the lower hinge region of Fc. Unique to the high-affinity receptor-Fc complex, however, is the conformation of the receptor D2 domain FG loop, which enables a charged KHR motif to interact with proximal carbohydrate units of the Fc glycans. Both the length and the charge of the FcγRI FG loop are well conserved among mammalian species. Ala and Glu mutations of the FG loop KHR residues showed significant contributions of His-174 and Arg-175 to antibody binding, and the loss of the FG loop-glycan interaction resulted in an ∼ 20- to 30-fold decrease in FcγRI affinity to all three subclasses of IgGs. Furthermore, deglycosylation of IgG1 resulted in a 40-fold loss in FcγRI binding, demonstrating involvement of the receptor FG loop in glycan recognition. These results highlight a unique glycan recognition in FcγRI function and open potential therapeutic avenues based on antibody glycan engineering or small molecular glycan mimics to target FcγRI for certain autoimmune diseases.

Templating carbohydrate-functionalised polymer-scaffolded dynamic combinatorial libraries with lectins.

A conceptually new approach to the design of macromolecular receptors for lectins is outlined. Carbohydrate-functionalised Polymer-Scaffolded Dynamic Combinatorial Libraries (PS-DCLs) have been prepared in aqueous solution by the reversible conjugation of carbohydrates possessing acylhydrazide functionalities in their aglycone on to an aldehyde-functionalised polymer scaffold. PS-DCLs have been shown to undergo compositional change in response to the addition of lectin templates, with polymer scaffolds preferentially incorporating carbohydrate units which recognise the lectin added. This compositional change has been shown to generate polymers of significantly enhanced affinity for the lectin added, with enhancements in free energy of binding in the range of 5.2-8.8 kJ mol(-1) observed. Experiments indicate that these enhancements are not only as a consequence of increased display of the preferred carbohydrate upon the polymer scaffold, but that templation also reorganises key residues into strategic positions in order to interact more strongly with the target.

In-situ annotation of carbohydrate diversity, abundance, and degradability in highly complex mixtures using NMR spectroscopy.

Many functions of carbohydrates depend on the detection of short structural motifs, approximately up to hexasaccharide length, by receptors or catalysts. This study investigates the usefulness of state-of-the-art (1)H-(13)C nuclear-magnetic-resonance (NMR) spectroscopy for characterizing the diversity, abundance, and degradability of such short structural motifs in plant-derived carbohydrates. Assignments of carbohydrate signals for (1)H-(13)C NMR spectra of beer, wine, and fruit juice yield up to >130 assignments in situ, i.e. in individual samples without separation or derivatization. More than 500 structural motifs can be resolved over a concentration range of ~10(3)in experiments of a few hours duration. The diversity of carbohydrate units increases according to power laws at lower concentrations for both cereal and fruit-derived samples. Simple graphs resolve the smaller overall contribution of more diverse but less abundant motifs to carbohydrate biomass. Susceptibility to enzymatic degradation is probed simultaneously for hundreds of structural motifs to reduce uncertainties in the identification of motifs and to probe enzyme specificity with assigned motifs. Several properties of fundamental biotechnological importance can thus be assessed in situ by heteronuclear NMR methods.

Structural determination of Streptococcus pneumoniae repeat units in serotype 41A and 41F capsular polysaccharides to probe gene functions in the corresponding capsular biosynthetic loci

We report the repeating unit structures of the native capsular polysaccharides of Streptococcus pneumoniae serotypes 41A and 41F. Structural determinations yielded six carbohydrate units in the doubly branched repeating unit to give the following structure for serotype 41A: [Display omitted] The structure determinations were motivated (1) by an ambition to help close the remaining gaps in S. pneumoniae capsular polysaccharide structures, and (2) by the attempt to derive functional annotations of carbohydrate active enzymes in the biosynthesis of bacterial polysaccharides from the determined structures. An activity present in 41F but not 41A is identified as an acetyltransferase acting on the rhamnopyranosyl sidechain E. The genes encoding the formation of the six glycosidic bonds in serogroup 41 were determined from the capsular polysaccharide structures of serotype 41A, 41F, and genetically related serotypes, in conjunction with corresponding genomic information and computational homology searches. In combination with complementary information, NMR spectroscopy considerably simplifies the functional annotation of carbohydrate active enzymes in the biosynthesis of bacterial polysaccharides.

Synthesis of Chiral Imidazolium Salts from a Carbohydrate and Their Application in Pd-Catalyzed Suzuki–Miyaura Reaction

A series of chiral 1-(acetylated glucopyranosyl)-3-substituted-imidazolium salts [3-substitute = n-butyl (1a), 3-bromopropyl (1b), 2-chloromethyl benzyl (1c), and 4-chloromethyl benzyl (1d)] have been synthesized. Preliminary catalytic studies show that these imidazolinium salts are remarkably efficient in Pd-catalyzed Suzuki–Miyaura reaction. Functionalized aryl boronic acids reaction with aryl halides (including aryl iodides, aryl bromides and activated aryl chlorides) using environmentally friendly conditions (ethanol aqueous and ambient). The excellent isolate yields reveal that the bulky carbohydrate unit is promising for the construction of highly active transition-metal catalyst. Four C-1 bonded sugar-containing chiral imidazolium salts 1a–d were synthesized and they all exhibited excellent catalytic activity in Pd-catalyzed Suzuki reactions. Pd/1a is most effective for the coupling of functionalized arylboronic acids with arylhalides including activated arylchlorides in ethanol aqueous under air.