Here we present a synthetic route for solid phase synthesis of N-linked glycoconjugates containing high mannose oligosaccharides which allows the incorporation of useful functional handles on the N-terminus of asparagine. In this strategy, the C-terminus of an Fmoc protected aspartic acid residue is first attached to a solid phase support. The side chain of aspartic acid is protected by a 2-phenylisopropyl protecting group, which allows selective deprotection for the introduction of glycosylation. By using a convergent on-resin glycosylamine coupling strategy, an N-glycosidic linkage is successfully formed on the free side chain of the resin bound aspartic acid with a large high mannose oligosaccharide, Man8GlcNAc2, to yield N-linked high mannose glycosylated asparagine. The use of on-resin glycosylamine coupling provides excellent glycosylation yield, can be applied to couple other types of oligosaccharides, and also makes it possible to recover excess oligosaccharides conveniently after the on-resin coupling reaction. Useful functional handles including an alkene (p-vinylbenzoic acid), an alkyne (4-pentynoic acid), biotin, and 5-carboxyfluorescein are then conjugated onto the N-terminal amine of asparagine on-resin after the removal of the Fmoc protecting group. In this way, useful functional handles are introduced onto the glycosylated asparagine while maintaining the structural integrity of the reducing end of the oligosaccharide. The asparagine side chain also serves as a linker between the glycan and the functional group and preserves the native presentation of N-linked glycan which may aid in biochemical and structural studies. As an example of a biochemical study using functionalized high mannose glycosylated asparagine, a fluorescence polarization assay has been utilized to study the binding of the lectin Concanavalin A (ConA) using 5-carboxyfluorescein labeled high mannose glycosylated asparagine.
Solid Phase Synthesis; Glycosylated Asparagine; High Mannose; Glycosylamine; On-resin Coupling; Functionalized Oligosaccharide
•Synthesis of a matrix of sulfation-programmed GlcN–IdoA disaccharides.•Demonstrates effective synthesis using various GlcN donors and Ido acceptor.•Four homogeneous sulfation-varied heparin disaccharides used to obtain Raman and Raman ROA data.•Spectra provide indicative comparisons of signature sulfation bands for differing sulfation levels.•Data are used to provide comparisons/differences to native heparin spectra.
Synthesis of an array of differentially sulfated GlcN–IdoA disaccharides, accessible on good scale, directly from l-iduronate components is described. These are specifically directed to provide the sulfation variability at the key most common biologically relevant sulfation-variable l-IdoA O-2 and d-GlcN O-6 and amino sites of this heparin disaccharide. This sulfation-varied matrix has allowed the first evaluation of using Raman/ROA spectroscopy to characterize changes in spectra as a function of both site and level of sulfation with pure, defined heparin-related disaccharide species. This provides analysis of both similarities and differences to digest native heparin and this shows evidence of different types of changes in conformations and conformational freedom as a function of some specific sulfation changes at the disaccharide level. It is anticipated that this data set will open the way for applications to further site-specific sulfated saccharides and demonstrates the capability offered by Raman–ROA towards fingerprinting sulfation in heparin fragments.
Disaccharides; Heparin; Sulfated carbohydrates; Raman–ROA; Iduronic aid
Globo-series glycans are human cell-surface carbohydrates that include stem-cell
marker SSEA-4 and cancer-cell antigen Globo H. These two hexasaccharides differ only in
their terminal saccharide: N-acetylneuraminic acid in SSEA-4 and
l-fucose in Globo H. Herein, we evaluated the affinity of the monoclonal
antibodies α-SSEA-4 and α-GH for the glycans SSEA-4 and Globo H. Using
fluorescence polarization, we find that the two monoclonal antibodies have affinity for
their cognate glycanin the low nanomolar range, and have negligible affinity for the
non-cognate glycan. Using surface plasmon resonance, we find that each cognate affinity is
~20-fold greater if the glycanis immobilized on a surface rather than free in
solution. We conclude that the terminal saccharide plays a dominant role in the ability of
monoclonal antibodies to recognize these Globo-series glycans and that the extraordinary
specificity of these antibodies supports their use for identifying and sorting stem-cells
(α-SSEA-4) and as an agent in cancer immunotherapy (α-GH).
cancer-cell antigen; globo-series glycan; Globo H; monoclonal antibody; SSEA-4; stem-cell marker
O-Polysaccharides (OPS) were isolated from purified Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharides by mild-acid hydrolysis and gel-permeation chromatography. 1-D and 2-D 1H and 13C NMR spectroscopy experiments revealed that the OPS antigens were unbranched heteropolymers with the following structures:
Collectively, our results demonstrate that the predominant OPS antigens expressed by B. pseudomallei and B. mallei isolates are structurally more complex than previously described and provide evidence that different capping residues are used by these closely related pathogens to terminate chain elongation. Additionally, they confirm that Burkholderia thailandensis and B. pseudomallei express OPS antigens that are essentially identical to one another.
Burkholderia pseudomallei; Burkholderia mallei; Burkholderia thailandensis; Lipopolysaccharide; O-polysaccharide; Structure
A highly convergent and efficient synthesis of a new sialyl Lewisx (sLex) mimic, which was predicted by computational studies to fulfil the spacial requirements for a selectin antagonist, has been developed. With a β2,3-amino acid residue L-galactose (bioisostere of the L-fucose moiety present in the natural sLex) and succinate are linked, leading to a mimic of sLex that contains all the required pharmacophores, namely the 3- and 4-hydroxy group of L-fucose, the 4- and 6-hydroxy group of D-galactose and the carboxylic acid of N-acetylneuraminic acid. The key step of the synthesis involves a tandem reaction consisting of a N-deprotection and a suitable O→N intramolecular acyl migration reaction which is promoted by cerium ammonium nitrate (CAN). Finally, the new sialyl Lewisx mimic was biologically evaluated in a competitive binding assay.
sLex; Selectin antagonist; β3-Amino acid; Glycomimetic
We are interested in the development of surfactants derived from
hemicellulosic biomass, as they are potential components in pharmaceuticals,
personal care products and other detergents. Such surfactants should exhibit low
toxicity in mammalian cells. In this study we synthesized a series of alkyl or
fluoroalkyl β-xylopyranosides from azides and an alkyne using the
copper-catalyzed azide-alkyne (CuAAC) “Click” reaction in 4 steps
from xylose. The purified products were evaluated for both their surfactant
properties, and for their biocompatibility. Unlike other carbohydrate-based
surfactants, liquid-crystalline behavior was not observed by differential
scanning calorimetry. The triazole-containing β-xylopyranosides with
short (6 carbons) and long (>12 carbons) chains exhibited no toxicity at
concentrations ranging from 1 to 1000 μM. Triazole-containing
β-xylopyranosides with 8, 10 or 12 carbons caused toxicity
via apoptosis, with CC50 values ranging from
26-890 μM. The two longest chain compounds did form stable monolayers at
the air-water interface over a range of temperatures, although a brief
transition to an unstable monolayer was observed.
Click chemistry; Huisgen cycloaddition; carbohydrate surfactants; Langmuir monolayers; cytotoxicity
The structures of three complex saponins from the fruit pods of Blighia sapida have been elucidated and their 1H and 13C NMR spectra assigned employing a variety of one- and two-dimensional NMR techniques without degradative chemistry. The saponins have either four or six monosaccharide units linked to a triterpene aglycone. High-resolution, proton-coupled-HSQC spectra were important for determining both the identities of the intact monosaccharide units and coupling constants in strongly coupled proton spin systems. These NMR experiments will prove crucial as the complexity of saponin structures reaches the limit that can be determined solely by NMR.
Ackee; Blighia sapida; Coupled-HSQC; Triterpene glycosides; Saponins
O-Specific polysaccharides of Brucella contain two antigenic determinants, called A and M. Most of the strains express epitope A with a small amount of epitope M, whereas B. melitensis strain 16M expresses longer polymer consisting mostly of M-type epitopes. Proposed explanation was that epitope A is defined by 1–2-linked homopolymer of N-formylperosamine (Rha4NFo), while epitope M is a pentasaccharide with four 2- and one 3-substituted Rha4NFo. We reinvestigated both types of structures by 2D NMR and showed that M-epitope is a tetrasaccharide, missing one of the 2-linked Rha4NFo as compared to the previously proposed structure. Polysaccharide from B. melitensis 16M contains a fragment of 1–2-lnked polymer, capped with M-type polymer. Other strains contain one or two M-type units at the non-reducing end of the 1–2-linked O-chain.
Brucella; LPS; structure; NMR; MS
Bacterial lipopolysaccharides (LPSs) are ubiquitous molecules that are prominent components of the outer membranes of most gram-negative bacteria. Genetic and structural characterizations of Francisella LPS have revealed substantial differences when compared to more commonly studied LPSs of the Enterobacteriaceae. This review discusses both the general characteristics and the unusual features of Francisella LPS.
Francisella tularensis; Lipopolysachharide; Lipid A; O-antigen; Kdo hydrolase; Structure; Virulence
The structures of a series of large oligosaccharides derived from acharan sulfate were characterized. Acharan sulfate is an unusual glycosaminoglycan isolated from the giant African snail, Achatina fulica. Oligosaccharides from decasaccharide to hexadecasaccharide were enzymatically prepared using heparin lyase II and purified. Capillary electrophoresis and gel electrophoresis confirmed the purity of these oligosaccharides. Their structures, determined by ESI-MS and NMR, were consistent with the major repeating sequence in acharan sulfate, →4)-α-d-GlcNpAc-(1→4)-α-l-IdoAp2S-(1→, terminated by 4-linked α-d-GlcNpAc residue at the reducing end and by 4,5-unsaturated pyranosyluronic acid 2-sulfate at the non-reducing end.
Acharan sulfate; Oligosaccharides; Structural determination
Proliferation of pulmonary artery smooth muscle cells (PASMCs) appears to play a significant role in chronic pulmonary hypertension. The proliferation of PASMCs is strongly inhibited by some commercial heparin preparations. Heparin fragments were prepared by periodate treatment, followed by sodium borohydride reduction, to enhance potency. The tributylammonium salt of this fragmented heparin was O-acylated with hexanoic anhydride. Gradient polyacrylamide gel electrophoresis showed that the major heparin fragment contained eight disaccharide units. NMR analysis showed that approximately one hexanoyl group per disaccharide residue was present. The O-hexanoyl heparin fragments were assayed for growth inhibitory effect on bovine PASMCs in culture. This derivative was found to be more effective in growth inhibition of bovine PASMCs in culture than the heparin from which it was derived. In the future, it is envisioned that this or similar derivatives may be an effective treatment for pulmonary hypertension.
O-Hexanoyl heparin; Pulmonary artery smooth muscle cell proliferation
A methyl ester of hyaluronan in which the carboxyl groups were fully esterified was prepared using trimethylsilyl diazomethane. This derivative, while not depolymerized by hyaluronan lyases or hyaluronan hydrolases, was a substrate for both chondroitin ACI lyase (EC 220.127.116.11) from Flavobacterium heparinum and chondroitin ACII lyase (EC 18.104.22.168) from Arthrobacter aurescens. The major product isolated in these depolymerization reactions was methyl α-l-threo-hex-4-enepyranosyluronate-(1→3)-2-acetamido-2-deoxy-α,β-d-glucopyranoside as determined by 1H NMR spectroscopy and MALDITOF mass spectrometry.
Methyl ester of hyaluronan; Chondroitin AC lyase; Chondroitinase; Flavobacterium heparinum; Arthrobacter aurescens
The anomeric composition of D-fructose in dimethyl sulfoxide changes when the solution is heated from room temperature to 150 °C, with a small increase in the α-furanose form at the expense of the β-pyranose tautomer. Additionally, a small amount of α-pyranose form was also observed at 150 °C. A mechanism is proposed for the dehydration of D-fructose to 5-hydroxymethylfurfural in DMSO at 150 °C, where the solvent acts as the catalyst. A key intermediate in the reaction was identified as (4R,5R)-4-hydroxy-5-hydroxymethyl-4,5-dihydrofuran-2-carbaldehyde by using 1H and 13C NMR spectra of the sample during the reaction.
D-fructose; 5-Hydroxymethylfurfural; Dehydration
The α-D-glucopyranosyl-(1→5)-substituted methyl glycosides of 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo), 3-deoxy-α-D-lyxo-hept-2-ulosonic acid (Kdh) and D-glycero-α-D-talo-oct-2-ulosonic acid (Ko) were prepared using orthogonally protected glycosyl acceptor derivatives via glycosylation with a torsionally disarmed 4,6-O-benzylidene protected trifluoroacetimidate glucosyl donor followed by global deprotection. The related 6-O-phosphoryl-α-D-glucopyranosyl-(1→5)-substituted Kdo and Kdh derivatives were derived from a benzylidene-protected glucosyl intermediate using phosphoramidite-based and phosphoryl chloride promoted phosphorylation steps, respectively. The deprotected disaccharides serve as ligands to study lectin binding of Acinetobacter lipopolysaccharide core oligosaccharides.
lipopolysaccharide; Kdo; Ko; oligosaccharide synthesis; Acinetobacter
•Synthesis of Acinetobacter LPS fragments with orthogonal protecting pattern.•First synthesis of Ko-glycosides substituted at position 5.•α-Selective glucosylation using benzylidene trifluoroacetimidate donor.•Regioselective phosphorylation at C-6 of a glucosyl substituent.
The α-d-glucopyranosyl-(1→5)-substituted methyl glycosides of 3-deoxy-α-d-manno-oct-2-ulosonic acid (Kdo), 3-deoxy-α-d-lyxo-hept-2-ulosonic acid (Kdh), and d-glycero-α-d-talo-oct-2-ulosonic acid (Ko) were prepared using orthogonally protected glycosyl acceptor derivatives via glycosylation with a torsionally disarmed 4,6-O-benzylidene protected trifluoroacetimidate glucosyl donor followed by global deprotection. The related 6-O-phosphoryl-α-d-glucopyranosyl-(1→5)-substituted Kdo and Kdh derivatives were derived from a benzylidene-protected glucosyl intermediate using phosphoramidite and phosphoryl chloride-based phosphorylation steps, respectively. The deprotected disaccharides serve as ligands to study lectin binding of Acinetobacter lipopolysaccharide core oligosaccharides.
Lipopolysaccharide; Kdo; Ko; Oligosaccharide synthesis; Acinetobacter
Self-assembled monolayers (SAMs) of α-D-Gal-(1→4)-β-D-Gal-(1→4)-β-D-Glc-mercaptooctane (globotriose, Gb3-C8-SH) were prepared both as single-component SAMs and as mixed SAMs with either octanethiol (OCT) or 8-mercapto-3,6-dioxaoctanol (HO-PEG2-SH), on flat gold and on nanoporous gold (NPG) electrodes. The binding of soybean agglutinin (SBA) to the globotriose (Gb3) unit in the SAMs was then studied using electrochemical impedance spectroscopy (EIS), which is a label free method found to be quite sensitive to SAM composition and to the differences in SAM structure on NPG versus on flat Au. The affinity of SBA to the mixed SAM of HO-PEG2-SH and Gb3-C8-SH on NPG is found to be greater on NPG than on flat gold, and indicates a potential advantage for NPG as a substrate. The SAMs of HO-PEG2-SH were found to resist protein adsorption on either NPG or flat gold. The non-specific adsorption of SBA to OCT SAMs on flat Au was observed in EIS by the increase in charge transfer resistance; whereas, the increase seen on the NPG surface was smaller, and suggests that EIS measurements on NPG are less affected by non-specific protein adsorption. Atomic force microscopy (AFM) images of the SBA binding to mixed SAM of HO-PEG2-SH and Gb3-C8-SH on NPG showed a greater number of proteins on top of the OCT containing SAMs.
Phytochemical investigation of the MeOH extract of Dioscorea villosa root resulted in the isolation of two new bidesmosidic cholestane steroid glycosides, dioscoreavillosides A and B (1 and 2). In addition, the extract yielded 12 previously known furostane and spirostane steroid glycosides (3-14), along with diosgenin (15). Compounds 3-7, 9, 14, and 15 were isolated for the first time from D. villosa. The structures of the isolated compounds were determined using spectroscopic and chemical methods including 1D and 2D NMR. The antimicrobial action of most of these compounds was tested against five fungal and five bacterial strains.
Dioscorea villosa; Dioscoreaceae; Wild yam; Cholestane steroid; Dioscoreavilloside A; Dioscoreavilloside B
A “clickable” disaccharide was prepared by treating the aldehyde precursor with hydroxylamine, followed by the catalytic hydrogenation and diazotransfer reaction. This disaccharide was successfully applied to the elongation of the backbone construction of ultralow molecular weight (ULMW) heparins using two bacterial glycosyl transferases, N-acetyl glucosaminyl transferase from Escherichia coli K5 (KfiA) and heparosan synthase-2 (pmHS2) from Pasteurella multocida.
Heparan sulfate; Heparin; Heparosan; Depolymerization; Azido-clickable acceptor; Enzymatic glycosylation
The conversion of UDP-α-d-gdlucuronic acid to UDP-α-d-xylose and UDP-α-d-apiose by a bifunctional potato enzyme UDP-apiose/UDP-xylose synthase was studied using real-time nuclear magnetic resonance (NMR) spectroscopy. UDP-α-d-glucuronic acid is converted via the intermediate uridine 5′-β-l-threo-pentapyranosyl-4″-ulose diphosphate to UDP-α-d-apiose and simultaneously to UDP-α-d-xylose. The UDP-α-d-apiose that is formed is unstable and is converted to α-d-apio-furanosyl-1,2-cyclic phosphate and UMP. High-resolution real-time NMR spectroscopy is a powerful tool for the direct and quantitative characterization of previously undetected transient and labile components formed during a complex enzyme-catalyzed reaction.
Apiose; UDP-apiose; UDP-xylose synthase; NMR spectroscopy; Stocsy
•First non-substrate analogue inhibitor of the trypanosome GPI pathway.•Active against recombinant enzyme and cell-free system.•Low molecular weight and good ligand efficiency.
The zinc-metalloenzyme GlcNAc-PI de-N-acetylase is essential for the biosynthesis of mature GPI anchors and has been genetically validated in the bloodstream form of Trypanosoma brucei, which causes African sleeping sickness. We screened a focused library of zinc-binding fragments and identified salicylic hydroxamic acid as a GlcNAc-PI de-N-acetylase inhibitor with high ligand efficiency. This is the first small molecule inhibitor reported for the trypanosome GPI pathway. Investigating the structure activity relationship revealed that hydroxamic acid and 2-OH are essential for potency, and that substitution is tolerated at the 4- and 5-positions.
GPI; Trypanosoma brucei; Hydroxamic acid; Inhibitor; N-Deacetylase
Carbapenem resistant Klebsiella pneumoniae (CRKP) are isolated with increasing frequency, especially from immunocompromized patients. The capsular polysaccharide (CPS) types of CPKP were not determined. Investigation of two CRKP isolates from a 2011 outbreak at the Clinical Center, the National Institutes of Health, identified a new capsular type shared by the two isolates, similar to K. pneumonia K19 and K34 but structurally different than any published K. pneumoniae CPS repeating unit:
The LPS of the two isolates was found to have no O-specific polysaccharide and the chemical structure of the core oligosaccharides agreed with published data.
If this structure type will be prevalent among CPKP isolates, our findings could facilitate rapid diagnosis and help develop new therapeutic solutions to this antibiotic resistant pathogen.
capsular polysaccharide; LPS; structure; KPC; CRKP
Altered carbohydrate metabolism in cancer cells was first noted by Otto Warburg more than 80 years ago. Upregulation of genes controlling the glycolytic pathway under normoxia, known as the Warburg effect, clearly differentiates malignant from non-malignant cells. The resurgence of interest in cancer metabolism aims at a better understanding of the metabolic differences between malignant and non-malignant cells and the creation of novel therapeutic and diagnostic agents exploiting these differences.
Modified d-glucose and d-mannose analogs were shown to interfere with the metabolism of their respective monosaccharide parent molecules and are potentially clinically useful anticancer and diagnostic agents.
One such agent, 2-deoxy-d-glucose (2-DG), has been extensively studied in vitro and in vivo and also clinically evaluated. Studies clearly indicate that 2-DG has a pleiotropic mechanism of action. In addition to effectively inhibiting glycolysis, 2-DG has also been shown to affect protein glycosylation. In order to better understand its molecular mechanism of action, we have designed and synthesized deuterated molecular probes to study 2-DG interference with d-glucose and d-mannose metabolism using mass spectrometry. We present here the synthesis of all desired probes: 2-deutero-d-glucose, 2-deutero-d-mannose, 6-deutero-d-glucose, 6-deutero-d-mannose, and 2-deutero-2-deoxy-d-glucose as well as their complete chemical characterization.
2-Deutero-d-glucose; 2-Deutero-d-mannose; 6-Deutero-d-glucose; 6-Deutero-d-mannose; 2-Deutero-2-deoxy-d-glucose; Metabolic probes
Brucella is an animal and human pathogen that expresses several virulence factors required for host cell invasion and intracellular survival. It produces LPS with unusually low toxicity, which hamper the detection of bacteria by the host immune system and thus provides resistance against intracellular antimicrobial mechanisms of the host. By chemical and spectroscopic methods we determined the structure of LPS core and of a non-repetitive oligosaccharide fragment at the reducing end of the O-specific polysaccharide. These data should be useful for understanding biological role of the Brucella LPS.
Brucella; LPS; structure; NMR; MS
•Aqueous 10 μs simulations of N-linked mannosyl cores and sialyl Lewis (sLe) antennae are validated.•Sequence dependent glycosidic linkage and pyranose ring μs motions are implicated in bioactivity.•Stacked pyranoses in sLea and sLex are predicted to be atypically rigid on μs timescales.•In a 25 μs simulation of sLex, all known conformers were sampled within the initial 10 μs of dynamics.•Unbiased 10 μs simulations are proposed as a route to systematic and accurate glycomic 3D-analysis.
The human glycome comprises a vast untapped repository of 3D-structural information that holds the key to glycan recognition and a new era of rationally designed mimetic chemical probes, drugs, and biomaterials. Toward routine prediction of oligosaccharide conformational populations and exchange rates at thermodynamic equilibrium, we apply hardware-accelerated aqueous molecular dynamics to model μs motions in N-glycans that underpin inflammation and immunity. In 10 μs simulations, conformational equilibria of mannosyl cores, sialyl Lewis (sLe) antennae, and constituent sub-sequences agreed with prior refinements (X-ray and NMR). Glycosidic linkage and pyranose ring flexing were affected by branching, linkage position, and secondary structure, implicating sequence dependent motions in glycomic functional diversity. Linkage and ring conformational transitions that have eluded precise quantification by experiment and conventional (ns) simulations were predicted to occur on μs timescales. All rings populated non-chair shapes and the stacked galactose and fucose pyranoses of sLea and sLex were rigidified, suggesting an exploitable 3D-signature of cell adhesion protein binding. Analyses of sLex dynamics over 25 μs revealed that only 10 μs were sufficient to explore all aqueous conformers. This simulation protocol, which yields conformational ensembles that are independent of initial 3D-structure, is proposed as a route to understanding oligosaccharide recognition and structure–activity relationships, toward development of carbohydrate-based novel chemical entities.
Conformation analysis; Glycome; Microsecond timescale; Kinetics; Molecular dynamics; Puckering
O-polysaccharide (OPS) was isolated from purified Burkholderia thailandensis E264 lipopolysaccharide by mild-acid hydrolysis and gel-permeation chromatography. Glycosyl composition and methylation analyses along with 1D and 2D 1H and 13C NMR spectroscopy experiments revealed that the OPS antigen was an unbranched heteropolymer with the following structure:
Collectively, these results suggest that B. thailandensis OPS is structurally more complex than B. pseudomallei OPS and provide evidence of the signal used by B. thailandensis to terminate chain elongation.
Burkholderia thailandensis; Burkholderia pseudomallei; Lipopolysaccharide; O-polysaccharide; NMR; Structure