Nonreducing End Glucosamine Research Articles

Convergent synthesis of tetra- and penta-saccharide fragments of dimeric Lewis X

The convergent synthesis of tetra- and penta-saccharide fragments of the TACA dimeric Lex is described. The synthetic strategy relied on the preparation of a protected GlcNTCA-(1,3)-Gal-(1,4)-GlcNAc trisaccharide diol free at O-3 of both glucosamine residues. Key steps in the preparation of this diol involved glycosylation at O-4 of N-acetylglucosamine using activation of a trichloroacetimidate with BF3·Et2O at 40 °C, removal of the non-reducing end O-3’ chloroacetate with thiourea, and glycosylation with a N-trichloroacetamido glucosamine trichloroacetimidate donor. After conversion to the diol acceptor, the trisaccharide was selectively fucosylated at the nonreducing end under NIS/TMSOTf activation, or di-fucosylated under CuBr2/Bu4NBr activation. The protected tetra- and pentasaccharides were then efficiently deprotected under dissolving metal conditions and the nonreducing end glucosamine residues were N-acetylated during the reaction work up. The deprotected compounds will be used as soluble competitors to characterize the epitopes recognized by anti-polymeric Lex antibodies.

Orthoesters formation leading to mismatched Helferich glycosylations at O-3 of N-trichloroacetylated glucosamine residues

Using trisaccharide diol acceptors displaying two glucosamine residues free at O-3, we observed that α-l-fucosylation with α armed donor proceeded smoothly at the most accessible N-trichloroacetyl nonreducing end glucosamine residue. In contrast, glycosylations with peracetylated glycosyl bromide donors activated under Helferich conditions seemed to proceed preferentially or exclusively at the more sterically hindered N-acetylated reducing end unit. Thus, we concluded that disarmed donors were mismatched at O-3 of the N-trichloroacetylated glucosamine residue regardless of α or β configuration of the glycosidic bond formed and d or l configuration of the donor. Interestingly orthoester formation occurred in some cases at this position while they were not observed at the reducing end unit. Conversion of the nonreducing end trichloroacetamido to an acetamido allowed the Helferich catalyzed galactosylation to occur at both positions and revealed the impact of the N-trichloroacetamido on the mismatched glycosylations. Changing the activation conditions from the mild Lewis acid Hg(CN)2 to the stronger acid AgOTf revealed that in fact β-d-galactosylation at the less hindered N-trichloroacetylated residue was kinetically favored over that at the reducing end residue. Isolation of equal amounts of orthoester at this position suggested that it was formed first but that the strong AgOTf Lewis acid was able to promote rearrangement to the β-d-galactosidic bond. These results shed additional light on the apparent mismatch of disarmed glycosyl donors with hydroxyl groups deemed more accessible. Depending on electronic factors imposed by the acceptor and activation conditions, transient unstable orthoester formation may explain in some cases why these donors appear mismatched with the most accessible hydroxyl groups which are otherwise glycosylated by armed donors.

Distribution of Heparan Sulfate Oligosaccharides in Murine Mucopolysaccharidosis Type IIIA.

Heparan sulfate (HS) catabolism begins with endo-degradation of the polysaccharide to smaller HS oligosaccharides, followed by the sequential action of exo-enzymes to reduce these oligosaccharides to monosaccharides and inorganic sulfate. In mucopolysaccharidosis type IIIA (MPS IIIA) the exo-enzyme, N-sulfoglucosamine sulfohydrolase, is deficient resulting in an inability to hydrolyze non-reducing end glucosamine N-sulfate esters. Consequently, partially degraded HS oligosaccharides with non-reducing end glucosamine sulfate esters accumulate. We investigated the distribution of these HS oligosaccharides in tissues of a mouse model of MPS IIIA using high performance liquid chromatography electrospray ionization-tandem mass spectrometry. Oligosaccharide levels were compared to total uronic acid (UA), which was used as a measure of total glycosaminoglycan. Ten oligosaccharides, ranging in size from di- to hexasaccharides, were present in all the tissues examined including brain, spleen, lung, heart, liver, kidney and urine. However, the relative levels varied up to 10-fold, suggesting different levels of HS turnover and storage. The relationship between the di- and tetrasaccharides and total UA was tissue specific with spleen and kidney showing a different disaccharide:total UA ratio than the other tissues. The hexasaccharides showed a stronger correlation with total UA in all tissue types suggesting that hexasaccharides may more accurately reflect the storage burden in these tissues.

Convergent Preparation of DimLex Hexasaccharide Analogues

AbstractWe report the convergent preparation of four hexasaccharides to be used towards the synthesis of a safe anticancer vaccine based on the tumor‐associated carbohydrate antigen dimLex. A common trisaccharide intermediate was first synthesized as a precursor to the tetrasaccharide backbones. These tetrasaccharides were then converted into diol acceptors and fucosylated at O‐3 of both glucosamine residues. The conditions required to promote fucosylation at O‐3 of the reducing end glucosamine units led to some loss of the fucosyl units already introduced at O‐3 of the nonreducing end glucosamine residues. Despite this hurdle, optimization of the glycosylation conditions provided the desired hexasaccharides in sufficient quantities to prepare the final analogues. Propyl glycosides and cysteamine adducts were prepared easily in two steps from the protected allyl glycoside hexasaccharides, and a one‐step deprotection under metal‐dissolving conditions was key to our deprotection strategy.

The Structural Basis of Substrate Recognition in an exo-β- d-Glucosaminidase Involved in Chitosan Hydrolysis

Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with β-galactosidase activity ( Escherichia coli LacZ), β-glucuronidase activity ( Homo sapiens GusB), and β-mannosidase activity ( Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-β- d-glucosaminidase ( exo-chitosanase) activity. Analysis of a product complex (1.85 Å resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role of E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural β-1,4- d-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-β- d-glucosaminide synthetic substrate provide insight into interactions in the + 1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.

Characterization of Sulfated Oligosaccharides in Mucopolysaccharidosis Type IIIA by Electrospray Ionization Mass Spectrometry

Heparan sulfate is a linear glycosaminoglycan with considerable structural diversity that binds a myriad of growth factors and proteins that play pivotal roles in a variety of biological processes. We have investigated the structural complexity of partially degraded fragments of heparan sulfate in mucopolysaccharidosis type IIIA in which there is a defect in heparan sulfate catabolism. Mono- to hexadecasaccharides were isolated from the urine of a mucopolysaccharidosis IIIA patient and shown to have non-reducing end glucosamine N-sulfate residues, reflecting the catabolic deficiency in heparan N-sulfatase (sulfamidase) activity. The use of nitrous acid digestion (pH 1.5) combined with separation by reverse-phase high-performance liquid chromatography and analysis by electrospray ionization-mass spectrometry identified multiple forms of these oligosaccharides with some N-acetylated glucosamine residues and one to three sulfates per disaccharide. Furthermore, we demonstrated that each oligosaccharide existed in multiple sulfated forms. Many structural isomers were present, suggesting a complex mixture of oligosaccharides present in the urine as a consequence of a defect in heparan sulfate degradation.

Chemoenzymatic synthesis of thio-nod factor intermediates — Enzymatic transfer of glucosamine on thiochitobiose derivatives

The chemoenzymatic syntheses of thioanalogues of nodulation factors in which the nonreducing end glucosamine residue is available for the introduction of the fatty acid moiety at the free NH2group are reported. We are describing the chemical synthesis of UDP-GlcNH2and its use in the enzymatic transfer of GlcNH2by the bovine galactosyltransferase (EC 2.4.1.90) onto O-4 of the nonreducing end N-acetylglucosamine residues of chitobiose, thiochitobiose, and allyl thiochitobioside. The enzymatic reactions on chitobiose and thiochitobiose were followed by TLC and MALDI MS and showed about 50% conversion of the disaccharides to the desired products. However, these reducing trisaccharides could not be obtained totally free of salts and degraded on ion exchange chromatography. Thus, we investigated the enzymatic transfer on the nonreducing allyl thiochitobioside analogue. We describe here the chemical synthesis of this thiodisaccharide and the enzymatic transfer of GlcNH2at O-4 of its nonreducing end glucosamine residue to give the desired allyl thiotrisaccharide. This thiotrisaccharide was obtained pure in 41% yield and was characterized by1H NMR (HSQC) and HRMS.Key words: nodulation factors, synthesis, enzymatic transfer, thiooligosaccharides, UDP-glucosamine.

Structural analysis of Francisella tularensis lipopolysaccharide.

The structure of the lipid A and core region of the lipopolysaccharide (LPS) from Francisella tularensis (ATCC 29684) was analysed using NMR, mass spectrometry and chemical methods. The LPS contains a beta-GlcN-(1-6)-GlcN lipid A backbone, but has a number of unusual structural features; it apparently has no substituent at O-1 of the reducing end GlcN residue in the lipid part in the major part of the population, no substituents at O-3 and O-4 of beta-GlcN, and no substituent at O-4 of the Kdo residue. The largest oligosaccharide, isolated after strong alkaline deacylation of NaBH4 reduced LPS had the following structure: where Delta-GalNA-(1-3)-beta-QuiNAc represents a modified fragment of the O-chain repeating unit. Two shorter oligosaccharides lacking the O-chain fragment were also identified. A minor amount of the disaccharide beta-GlcN-(1-6)-alpha-GlcN-1-P was isolated from the same reaction mixture, indicating the presence of free lipid A, unsubstituted by Kdo and with phosphate at the reducing end. The lipid A, isolated from the products of mild acid hydrolysis, had the structure 2-N-(3-O-acyl4-acyl2)-beta-GlcN-(1-6)-2-N-acyl1-3-O-acyl3-GlcN where acyl1, acyl2 and acyl3 are 3-hydroxyhexadecanoic or 3-hydroxyoctadecanoic acids, acyl4 is tetradecanoic or (minor) hexadecanoic acids. No phosphate substituents were found in this compound. OH-1 of the reducing end glucosamine, and OH-3 and OH-4 of the nonreducing end glucosamine residues were not substituted. LPS of F. tularensis exhibits unusual biological properties, including low endoxicity, which may be related to its unusual lipid A structure.

Active conformations of glycosaminoglycans. NMR determination of the conformation of heparin sequences complexed with antithrombin and fibroblast growth factors in solution.

Binding to proteins usually induces perturbation of nuclear magnetic resonances of ligand molecules. Using sensitive nuclear magnetic resonance (NMR) spectroscopy techniques, these perturbations have been measured for heparin oligosaccharides in aqueous solution in the presence of proteins and the NMR data have been used to characterize the three-dimensional (3D) structure of the oligosaccharides in the bound state. The pentasaccharide corresponding to the active site of heparin/heparan sulfate for antithrombin (AT) adopts in the complex with the protein a conformation different from that in the absence of the protein. A notable difference involves the 2-O-sulfated iduronic acid (IdoA2S) residue, which is driven to adopt an exclusively skew-boat @affil2: 2S 0 form in the complex. In addition, complexing induces a change in the geometry around the glycosidic linkage between the nonreducing end glucosamine and the adjacent glucuronic acid residue as compared with the free state. NMR and molecular modeling data also indicate that the 2-O-sulfate group in the IdoA2S residue is not directly involved in binding to AT. This suggests that its role is mainly that of affecting the conformational equilibrium of this residue, leading to a 3D structure of pentasaccharide in the bound state that meets the stereochemical requirements of the receptor and results in high-affinity binding to the protein. On the other hand, NMR studies of heparin tetrasaccharides in the presence of fibroblast growth factors FGF-1 and FGF-2 indicate that FGF binding stabilizes the @affil1: 1C 4 conformation of the IdoA2S residue directly involved in binding. These studies also confirm the crucial role of the 6-O-sulfate group on at least one glucosamine residue in the formation of the complex with FGF-1 but not with FGF-2.

A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome).

Mucopolysaccharidosis type III A (MPS III A, Sanfilippo syndrome) is a rare, autosomal recessive, lysosomal storage disease characterized by accumulation of heparan sulfate secondary to defective function of the lysosomal enzyme heparan N- sulfatase (sulfamidase). Here we describe a spontaneous mouse mutant that replicates many of the features found in MPS III A in children. Brain sections revealed neurons with distended lysosomes filled with membranous and floccular materials with some having a classical zebra body morphology. Storage materials were also present in lysosomes of cells of many other tissues, and these often stained positively with periodic-acid Schiff reagent. Affected mice usually died at 7-10 months of age exhibiting a distended bladder and hepatosplenomegaly. Heparan sulfate isolated from urine and brain had nonreducing end glucosamine- N -sulfate residues that were digested with recombinant human sulfamidase. Enzyme assays of liver and brain extracts revealed a dramatic reduction in sulfamidase activity. Other lysosomal hydrolases that degrade heparan sulfate or other glycans and glycosaminoglycans were either normal, or were somewhat increased in specific activity. The MPS III A mouse provides an excellent model for evaluating pathogenic mechanisms of disease and for testing treatment strategies, including enzyme or cell replacement and gene therapy.

Synthesis of S-Linked Thiooligosaccharide Analogues of Nodulation Factors. 2.1 Synthesis of an Intermediate Thiotrisaccharide

An S-linked thiotrisaccharide analogue of chitin that carries different N-protecting groups at the reducing and nonreducing end glucosamine residues was prepared as an intermediate for the synthesis of thioanalogues of nodulation factors. 1,6-Anhydro-2-azido-3-O-benzoyl-2-deoxy-β-d-glucopyranose (3) was prepared either through the nucleophilic displacement of the 4-triflate galacto analogue 1 with potassium chloroacetate or via the selective acylation of the analogous gluco diol 4. Condensation of 3 with the N-phthalimido trichloroacetimidate 9 led to the disaccharide 10, which was converted in four steps to the glucosyl bromide 15. Nucleophilic displacement of the anomeric bromide by a 4-thiolate derivative of glucosamine (16) bearing a trichloroethoxycarbamate at C-2 was performed in anhydrous oxygen-free THF and led to the desired thiotrisaccharide precursor of thioanalogues of nodulation factors. Alternatively, the disaccharide 10 was prepared by regioselective glycosylation of the 1,6-anhydro diol 4 ...

Three unusual modifications, a D-arabinosyl, an N-methyl, and a carbamoyl group, are present on the Nod factors of Azorhizobium caulinodans strain ORS571.

Azorhizobium caulinodans strain ORS571 is a symbiont of the tropical legume Sesbania rostrata. Upon nod gene induction with naringenin, strain ORS571 secretes into the culture medium Nod factors that morphologically change the host plant--in particular, deformed root hairs (Hai/Had) and meristematic foci are formed at the basis of lateral roots. The latter infrequently develop further into nodule-like structures. The azorhizobial Nod factors are chitin tetramers or pentamers, N-acylated at the nonreducing-end glucosamine with either vaccenic acid (C18:1) or stearic acid (C18:0). They, thus, resemble the previously described Nod factors from (brady)rhizobia. The backbone lipooligosaccharide is substituted with unusual modifications, presumably involved in host-specificity determination. There is a D-arabinose branch on the reducing end and an N-methyl and O-carbamoyl substitution on the nonreducing end of the oligosaccharide chain. The previously identified nod gene nolK may be involved in the synthesis of a D-arabinose derivative. The nodS gene product is probably responsible for the N-methylation of Nod factors.