Chemical Synthesis of Δ-4,5 Unsaturated Heparan Sulfate Oligosaccharides for Biomarker Discovery.

A methodology is described that can provide heparan sulfate oligosaccharides having a Δ4,5-double bond, which are needed as analytical standards and biomarkers for mucopolysaccharidoses. It is based on chemical oligosaccharide synthesis followed by modification of the C-4 hydroxyl of the terminal uronic acid moiety as methanesulfonate. This leaving group is stable under conditions used to remove temporary protecting groups, O-sulfation, and hydrogenolysis. Treatment with NaOH results in elimination of the methanesulfonate and formation of a Δ4,5-double bond.

Heparan Sulfate Microarray Reveals That Heparan Sulfate-Protein Binding Exhibits Different Ligand Requirements.

Heparan sulfates (HS) are linear sulfated polysaccharides that modulate a wide range of physiological and disease-processes. Variations in HS epimerization and sulfation provide enormous structural diversity, which is believed to underpin protein binding and regulatory properties. The ligand requirements of HS-binding proteins have, however, been defined in only a few cases. We describe here a synthetic methodology that can rapidly provide a library of well-defined HS oligosaccharides. It is based on the use of modular disaccharides to assemble several selectively protected tetrasaccharides that were subjected to selective chemical modifications such as regioselective O- and N-sulfation and selective de-sulfation. A number of the resulting compounds were subjected to enzymatic modifications by 3-O-sulfotransferases-1 (3-OST1) to provide 3-O-sulfated derivatives. The various approaches for diversification allowed one tetrasaccharide to be converted into 12 differently sulfated derivatives. By employing tetrasaccharides with different backbone compositions, a library of 47 HS-oligosaccharides was prepared and the resulting compounds were used to construct a HS microarray. The ligand requirements of a number of HS-binding proteins including fibroblast growth factor 2 (FGF-2), and the chemokines CCL2, CCL5, CCL7, CCL13, CXCL8, and CXCL10 were examined using the array. Although all proteins recognized multiple compounds, they exhibited clear differences in structure-binding characteristics. The HS microarray data guided the selection of compounds that could interfere in biological processes such as cell proliferation. Although the library does not cover the entire chemical space of HS-tetrasaccharides, the binding data support a notion that changes in cell surface HS composition can modulate protein function.

De Novo Sequencing of Complex Mixtures of Heparan Sulfate Oligosaccharides.

Here, we describe the first sequencing method of a complex mixture of heparan sulfate tetrasaccharides by LC-MS/MS. Heparin and heparan sulfate (HS) are linear polysaccharides that are modified in a complex manner by N- and O-sulfation, N-acetylation, and epimerization of the uronic acid. Heparin and HS are involved in various essential cellular communication processes. The structural analysis of these glycosaminoglycans is challenging due to the lability of their sulfate groups, the high heterogeneity of modifications, and the epimerization of the uronic acids. While advances in liquid chromatography (LC) and mass spectrometry (MS) have enabled compositional profiling of HS oligosaccharide mixtures, online separation and detailed structural analysis of isomeric and epimeric HS mixtures has not been achieved. Here, we report the development and evaluation of a chemical derivatization and tandem mass spectrometry method that can separate and identify isomeric and epimeric structures from complex mixtures. A series of well-defined synthetic HS tetrasaccharides varying in sulfation patterns and uronic acid epimerization were analyzed by chemical derivatization and LC-MS/MS. These synthetic compounds made it possible to establish relationships between HS structure, chromatographic behavior and MS/MS fragmentation characteristics. Using the analytical characteristics determined through the analysis of the synthetic HS tetrasaccharide standards, an HS tetrasacharide mixture derived from natural sources was successfully sequenced. This method represents the first sequencing of complex mixtures of HS oligosaccharides, an essential milestone in the analysis of structure-function relationships of these carbohydrates.

Extension and validation of the GLYCAM force field parameters for modeling glycosaminoglycans.

Glycosaminoglycans (GAGs) are an important class of carbohydrates that serve critical roles in blood clotting, tissue repair, cell migration and adhesion, and lubrication. The variable sulfation pattern and iduronate ring conformations in GAGs influence their polymeric structure and nature of interaction. This study characterizes several heparin-like GAG disaccharides and tetrasaccharides using NMR and molecular dynamics simulations to assist in the development of parameters for GAGs within the GLYCAM06 force field. The force field additions include parameters and charges for a transferable sulfate group for O- and N-sulfation, neutral (COOH) forms of iduronic and glucuronic acid, and Δ4,5-unsaturated uronate (ΔUA) residues. ΔUA residues frequently arise from the enzymatic digestion of heparin and heparin sulfate. Simulations of disaccharides containing ΔUA reveal that the presence of sulfation on this residue alters the relative populations of 1H2 and 2H1 ring conformations. Simulations of heparin tetrasaccharides containing N-sulfation in place of N-acetylation on glucosamine residues influence the ring conformations of adjacent iduronate residues.

Les glycosaminoglycanes (GAG) sont une classe importante d'hydrates de carbone qui jouent un rôle crucial dans la coagulation sanguine, la réparation des tissus, la migration et l'adhérence cellulaires, et la lubrification. La disposition variable des groupes sulfate et la conformation du cycle de l'iduronate des GAG influent sur leur structure polymérique et sur la nature des interactions. Dans la présente étude, nous caractérisons divers GAG disaccharidiques et tétrasaccharidiques semblables à l'héparine par RMN et modélisation de dynamique moléculaire en vue de contribuer à la détermination de paramètres pour les GAG dans le champ de force GLYCAM06. Les éléments additionnels au champ de force comprennent les paramètres et les charges associés à un groupe sulfate transférable lors de la O-sulfatation et de la N-sulfatation, les formes neutres (COOH) des acides iduronique et glucuronique et les résidus uronate Δ4,5-insaturés (ΔUA). Des résidus ΔUA sont souvent formés lors de la digestion enzymatique de l'héparine et du sulfate d'héparine. Des modélisations de disaccharides contenant des ΔUA révèlent que la présence de groupes sulfate sur ces résidus modifie les populations relatives des conformations de cycle 1H2 et 2H1. Les modelisations de tétrasaccharides à base d'héparine présentant une N-sulfatation au lieu d'une N-acétylation des résidus glucosamine influent sur les conformations de cycle des résidus iduronate adjacents. [Traduit par la Rédaction].

High-field asymmetric-waveform ion mobility spectrometry and electron detachment dissociation of isobaric mixtures of glycosaminoglycans.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is shown to be capable of resolving isomeric and isobaric glycosaminoglycan negative ions and to have great utility for the analysis of this class of molecules when combined with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and tandem mass spectrometry. Electron detachment dissociation (EDD) and other ion activation methods for tandem mass spectrometry can be used to determine the sites of labile sulfate modifications and for assigning the stereochemistry of hexuronic acid residues of glycosaminoglycans (GAGs). However, mixtures with overlapping mass-to-charge values present a challenge, as their precursor species cannot be resolved by a mass analyzer prior to ion activation. FAIMS is shown to resolve two types of mass-to-charge overlaps. A mixture of chondroitin sulfate A (CSA) oligomers with 4-10 saccharides units produces ions of a single mass-to-charge by electrospray ionization, as the charge state increases in direct proportion to the degree of polymerization for these sulfated carbohydrates. FAIMS is shown to resolve the overlapping charge. A more challenging type of mass-to-charge overlap occurs for mixtures of diastereomers. FAIMS is shown to separate two sets of epimeric GAG tetramers. For the epimer pairs, the complexity of the separation is reduced when the reducing end is alkylated, suggesting that anomers are also resolved by FAIMS. The resolved components were activated by EDD and the fragment ions were analyzed by FTICR-MS. The resulting tandem mass spectra were able to distinguish the two epimers from each other.

Fluorous supported modular synthesis of heparan sulfate oligosaccharides.

The modular synthesis of heparan sulfate fragments is greatly facilitated by employing an anomeric aminopentyl linker protected by a benzyloxycarbonyl group modified by a perfluorodecyl tag, which made it possible to purify highly polar intermediates by fluorous solid phase extraction. This tagging methodology made it also possible to perform repeated glycosylations to drive reactions to completion.

ELECTRON DETACHMENT DISSOCIATION OF SYNTHETIC HEPARAN SULFATE GLYCOSAMINOGLYCAN TETRASACCHARIDES VARYING IN DEGREE OF SULFATION AND HEXURONIC ACID STEREOCHEMISTRY.

Glycosaminoglycan (GAG) carbohydrates provide a challenging analytical target for structural determination due to their polydisperse nature, non-template biosynthesis, and labile sulfate modifications. The resultant structures, although heterogeneous, contain domains which indicate a sulfation pattern or code that correlates to specific function. Mass spectrometry, in particular electron detachment dissociation Fourier transform ion cyclotron resonance (EDD FT-ICR MS), provides a highly sensitive platform for GAG structural analysis by providing cross-ring cleavages for sulfation location and product ions specific to hexuronic acid stereochemistry. To investigate the effect of sulfation pattern and variations in stereochemistry on EDD spectra, a series of synthetic heparan sulfate (HS) tetrasaccharides are examined. Whereas previous studies have focused on lowly sulfated compounds (0.5-1 sulfate groups per disaccharide), the current work extends the application of EDD to more highly sulfated tetrasaccharides (1-2 sulfate groups per disaccharide) and presents the first EDD of a tetrasaccharide containing a sulfated hexuronic acid. For these more highly sulfated HS oligomers, alternative strategies are shown to be effective for extracting full structural details. These strategies inlcude sodium cation replacement of protons, for determining the sites of sulfation, and desulfation of the oligosaccharides for the generation of product ions for assigning uronic acid stereochemistry.

A synthetic heparan sulfate oligosaccharide library reveals the novel enzymatic action of D-glucosaminyl 3-O-sulfotransferase-3a.

Heparan sulfate (HS) glucosaminyl 3-O-sulfotranferases sulfate the C3-hydroxyl group of certain glucosamine residues on heparan sulfate. Six different 3-OST isoforms exist, each of which can sulfate very distinct glucosamine residues within the HS chain. Among these isoforms, 3-OST1 has been shown to play a role in generating ATIII-binding HS anticoagulants whereas 3-OST2, 3-OST3, 3-OST4 and 3OST-6 have been shown to play a vital role in generating gD-binding HS chains that permit the entry of herpes simplex virus type 1 into cells. 3-OST5 has been found to generate both ATIII- and gD-binding HS motifs. Previous studies have examined the substrate specificities of all the 3-OST isoforms using HS polysaccharides. However, very few studies have examined the contribution of the epimer configuration of neighboring uronic acid residues next to the target site to 3-OST action. In this study, we utilized a well-defined synthetic oligosaccharide library to examine the substrate specificity of 3-OST3a and compared it to 3-OST1. We found that both 3-OST1 and 3-OST3a preferentially sulfate the 6-O-sulfated, N-sulfoglucosamine when an adjacent iduronyl residue is located to its reducing side. On the other hand, 2-O-sulfation of this uronyl residue can inhibit the action of 3-OST3a on the target residue. The results reveal novel substrate sites for the enzyme actions of 3-OST3a. It is also evident that both these enzymes have promiscuous and overlapping actions that are differentially regulated by iduronyl 2-O-sulfation.

Negative electron transfer dissociation Fourier transform mass spectrometry of glycosaminoglycan carbohydrates.

Electron transfer through gas phase ion-ion reactions has led to the widespread application of electron- based techniques once only capable in ion trapping mass spectrometers. Although any mass analyzer can in theory be coupled to an ion-ion reaction device (typically a 3-D ion trap), some systems of interest exceed the capabilities of most mass spectrometers. This case is particularly true in the structural characterization of glycosaminoglycan (GAG) oligosaccharides. To adequately characterize highly sulfated GAGs or oligosaccharides above the tetrasaccharide level, a high resolution mass analyzer is required. To extend previous efforts on an ion trap mass spectrometer, negative electron transfer dissociation coupled with a Fourier transform ion cyclotron resonance mass spectrometer has been applied to increasingly sulfated heparan sulfate and heparin tetrasaccharides as well as a dermatan sulfate octasaccharide. Results similar to those obtained by electron detachment dissociation are observed.

Multivariate analysis of electron detachment dissociation and infrared multiphoton dissociation mass spectra of heparan sulfate tetrasaccharides differing only in hexuronic acid stereochemistry.

The structural characterization of glycosaminoglycan (GAG) carbohydrates by mass spectrometry has been a long-standing analytical challenge due to the inherent heterogeneity of these biomolecules, specifically polydispersity, variability in sulfation, and hexuronic acid stereochemistry. Recent advances in tandem mass spectrometry methods employing threshold and electron-based ion activation have resulted in the ability to determine the location of the labile sulfate modification as well as assign the stereochemistry of hexuronic acid residues. To facilitate the analysis of complex electron detachment dissociation (EDD) spectra, principal component analysis (PCA) is employed to differentiate the hexuronic acid stereochemistry of four synthetic GAG epimers whose EDD spectra are nearly identical upon visual inspection. For comparison, PCA is also applied to infrared multiphoton dissociation spectra (IRMPD) of the examined epimers. To assess the applicability of multivariate methods in GAG mixture analysis, PCA is utilized to identify the relative content of two epimers in a binary mixture.

Modular synthesis of heparan sulfate oligosaccharides for structure-activity relationship studies.

Although hundreds of heparan sulfate binding proteins have been identified and implicated in a myriad of physiological and pathological processes, very little information is known about the ligand requirements for binding and mediating biological activities by these proteins. This difficulty results from a lack of technology for establishing structure-activity relationships, which in turn is due to the structural complexity of natural heparan sulfate (HS) and difficulties of preparing well-defined HS oligosaccharides. To address this deficiency, we developed a modular approach for the parallel combinatorial synthesis of HS oligosaccharides that utilizes a relatively small number of selectively protected disaccharide building blocks, which can easily be converted into glycosyl donors and acceptors. The utility of the modular building blocks has been demonstrated by the preparation of a library of 12 oligosaccharides, which has been employed to probe the structural features of HS for inhibiting the protease, BACE-1. The complex variations in activity with structural changes support the view that important functional information is embedded in HS sequences. Furthermore, the most active derivative provides an attractive lead compound for the preparation of more potent compounds, which may find use as a therapeutic agent for Alzheimer's disease.

One-pot synthesis of oligosaccharides by combining reductive openings of benzylidene acetals and glycosylations.

Combining triflic acid-promoted glycosylations of trichloroacetimidates with reductive opening of benzylidene acetals with triflic acid and triethylsilane as one-pot procedures provides access to a wide range of disaccharides and 2,4- and 3,4-branched trisaccharides.

Chemo-enzymatic synthesis of C-9 acetylated sialosides.

A chemo-enzymatic synthesis of [(5-acetamido-9-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid)-(2-->3)-O-(beta-D-galactopyranosyl)-(1-->3)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)]-l-serine acetate (1) has been accomplished by a regioselective chemical acetylation of Neu5Ac (2) to give 9-O-acetylated sialic acid 3, which was enzymatically converted into CMP-Neu5,9Ac(2) (4) employing a recombinant CMP-sialic acid synthetase from Neisseria meningitis [EC 2.7.7.43]. The resulting compound was then employed for the enzymatic glycosylation of the C-3' hydroxyl of chemically prepared glycosylated amino acid 10 using recombinant rat alpha-(2-->3)-O-sialyltransferase expressed in Spodooptera frugiperda [EC 2.4.99.4] to give, after deprotection of the N(alpha)-benzyloxycarbonyl (CBz)-protecting group of serine, target compound 1. The N(alpha)-CBz-protected intermediate 11 can be employed for the synthesis of glycolipopeptides for immunization purposes.

Spin-labeled analogs of CMP-NeuAc as NMR probes of the alpha-2,6-sialyltransferase ST6Gal I.

Structural data on mammalian proteins are often difficult to obtain by conventional NMR approaches because of an inability to produce samples with uniform isotope labeling in bacterial expression hosts. Proteins with sparse isotope labels can be produced in eukaryotic hosts by using isotope-labeled forms of specific amino acids, but structural analysis then requires information from experiments other than nuclear Overhauser effects. One source of alternate structural information is distance-dependent perturbation of spin relaxation times by nitroxide spin-labeled analogs of natural protein ligands. Here, we introduce spin-labeled analogs of sugar nucleotide donors for sialyltransferases, specifically, CMP-TEMPO (CMP-4-O-[2,2,6,6-tetramethylpiperidine-1-oxyl]) and CMP-4carboxyTEMPO (CMP-4-O-[4-carboxy-2,2,6,6-tetramethylpiperidinine-1-oxyl]). An ability to identify resonances from active site residues and produce distance constraints is illustrated on a (15)N phenylalanine-labeled version of the structurally uncharacterized, alpha-2,6-linked sialyltransferase, ST6Gal I.

NMR structural characterization of substrates bound to N-acetylglucosaminyltransferase V.

N-Acetylglucosaminyltransferase V (GnT-V) is an enzyme involved in the biosynthesis of asparagine-linked oligosaccharides. It is responsible for the transfer of N-acetylglucosamine (GlcNAc) from the nucleotide sugar donor, uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), to the 6 position of the alpha-1-6 linked Man residue in N-linked oligosaccharide core structures. GnT-V up-regulation has been linked to increased cancer invasiveness and metastasis and, appropriately, targeted for drug development. However, drug design is impeded by the lack of structural information on the protein and the way in which substrates are bound. Even though the catalytic domain of this type II membrane protein can be expressed in mammalian cell culture, obtaining structural information has proved challenging due to the size of the catalytic domain (95 kDa) and its required glycosylation. Here, we present an experimental approach to obtaining information on structural characteristics of the active site of GnT-V through the investigation of the bound conformation and relative placement of its ligands, UDP-GlcNAc and beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-GlcpOOctyl. Nuclear magnetic resonance (NMR) spectroscopy experiments, inducing transferred nuclear Overhauser effect (trNOE) and saturation transfer difference (STD) experiments, were used to characterize the ligand conformation and ligand-protein contact surfaces. In addition, a novel paramagnetic relaxation enhancement experiment using a spin-labeled ligand analogue, 5'-diphospho-4-O-2,2,6,6-tetramethylpiperidine 1-oxyl (UDP-TEMPO), was used to characterize the relative orientation of the two bound ligands. The structural information obtained for the substrates in the active site of GnT-V can be useful in the design of inhibitors for GnT-V.

Trisaccharide mimetics of the aminoglycoside antibiotic neomycin.

A highly convergent approach for the chemical synthesis of eight structurally related trisaccharides that contain 3 to 5 amino groups has been described. Fourier-transformation ion cyclotron resonance mass spectrometry (FT-ICR MS) has been employed to determine the dissociation constants (Kd) for the binding of the trisaccharides to a prototypical fragment of 16S ribosomal RNA. A compound that contained a 4,6-dideoxy-4-amino-beta-D-glucopyranoside moiety at C-3 displayed binding in the low micromolar range. It was found that small structural changes of the saccharides resulted in large differences in affinity. The described structure-activity relationship is expected to be valuable for the development of novel antibiotics that target rRNA.

Glycosidic torsional motions in a bicelle-associated disaccharide from residual dipolar couplings.

An analysis of torsional motions about glycosidic bonds in a disaccharide is undertaken using residual dipolar coupling measurements and selective immobilization of the reducing end sugar to provide a suitable motional reference. The immobilization is accomplished by using the short chain of an alkyl glycoside to anchor the disaccharide to a bilayer medium aligned in magnetic field. Motions about the beta-(1-4) linkage of the n-butyl-4-O-beta-d-galactopyranosyl-alpha-d-mannopyranoside are shown to be substantial (+/-40 degrees ) and in good agreement with predictions of a fully solvated molecular dynamics simulation.

Disaccharide mimetics of the aminoglycoside antibiotic neamine.

A highly convergent approach has been employed for the facile synthesis of a library of 24 disaccharides that are alpha(1-3), beta(1-3), alpha(1-4), or beta(1-4) linked and contain 2-4 amino groups. Fourier-transformation ion cyclotron resonance mass spectrometry (FT-ICR MS) has been used to determine dissociation constant (Kd) values for the binding of the disaccharides to a prototypical fragment of 16S ribosomal RNA. Several derivatives bound with affinities similar to that of neamine. Structure-activity relationships have revealed the substitution pattern that is important for high-affinity binding. The compounds described here are unique lead compounds for the design of novel aminoglycoside antibiotics.

The design and synthesis of a selective inhibitor of fucosyltransferase VI.

Inversion of configuration of the C-2[prime or minute] hydroxyl of methyl N-acetyllactosamine was accomplished by a two-step procedure involving oxidation to a ketone followed by reduction with NaBH(4). After deprotection, the resulting derivative was examined as a substrate for [small alpha]-(2,6)- and [small alpha]-(2,3)-sialyltransferase and fucosyltransferase III, IV, V and VI. It was found that none of these enzymes could glycosylate. However, it showed exquisite selectivity for inhibition of fucosyltransferase VI. The kinetic data support an unusual mechanism in which the inhibitor can bind to the GDP-fucose complex as well as another enzyme form.

Glycosyltransferase activity can be selectively modulated by chemical modifications of acceptor substrates.

A range of N-acetyllactosamine derivatives, which are modified by a wide range of functionalities at C-2(') and C-6, have been synthesised and the kinetic parameters of transfer catalysed by recombinant alpha-2,6-sialyltransferase and alpha-1,3-fucoyltransferase VI determined. Several of the chemical modifications led to selective modulate the activity the enzymes and offer promising lead compounds for the development of oligosaccharide primers for selective metabolic inhibition of oligosaccharide biosynthesis.