Synthesis of a natural oligosaccharide antibiotic active against Helicobacter pylori

An oligosaccharide active against Helicobacter pylori was synthesized in a highly efficient manner for the first time. The anti-H. pylori oligosaccharide structure is a core-2 branched-type oligosaccharide with a characteristic alpha-N-acetylglucosamine at the nonreducing end. The oligosaccharide was synthesized from the nonreducing end to the reducing end, with an N-benzyl-2,3-oxazolidinone-carrying glycosyl donor used to introduce an alpha-N-acetylglucosamine at the nonreducing end. Complete chemoselective activation of a bromo sugar in the presence of a thioglycoside acceptor was achieved, and the use of 2,6-dimethylphenyl thioglycoside prevented the aglycon transfer observed when the corresponding phenyl thioglycoside is used as an acceptor.     

A facile and novel route to the antigenic branched phosphoglycan of the protozoan Leishmania major parasite

A novel approach for the iterative synthesis of the antigenic branched phosphoglycan of the protozoan parasite Leishmania major is presented that exploits remarkable dual selectivity in the functionalization of d-lactal, providing flexibility to extend the PG chain either towards the reducing or nonreducing end.     

Synthesis of novel cyclic oligosaccharides: beta-1,6-thio-linked cycloglucopyranosides

[structure: see text] A protocol for the synthesis of novel cyclic beta-1,6-S-linked glucopyranosides is developed. The key intermediate is a linear thiooligosaccharide bearing an iodo group at C-6 of the nonreducing sugar and a thioacetyl group at the anomeric center of the reducing end sugar. The crucial macrocyclization step was achieved through base-promoted intramolecular S(N)2 glycosylation in remarkably high yields (92-95%) and with well-controlled stereochemistry.     

Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases

Fungal-derived, copper-dependent polysaccharide monooxygenases (PMOs), formerly known as GH61 proteins, have recently been shown to catalyze the O(2)-dependent oxidative cleavage of recalcitrant polysaccharides. Different PMOs isolated from Neurospora crassa were found to generate oxidized cellodextrins modified at the reducing or nonreducing ends upon incubation with cellulose and cellobiose dehydrogenase. Here we show that the nonreducing end product formed by an N. crassa PMO is a 4-ketoaldose. Together with isotope labeling experiments, further support is provided for a mechanism involving oxygen insertion and subsequent elimination to break glycosidic bonds in crystalline cellulose.     

First total synthesis of a GPI-anchored peptide

A GPI-anchored dipeptide of sperm CD52 antigen was prepared through a convergent synthesis. First, the dipeptide with its C-terminus free and the GPI with its nonreducing end phosphoethanolamine bearing a free amino group were synthesized separately. Then, the two building blocks were coupled with use of EDC/HOBt as the condensation reagent. Finally, the GPI-anchored peptide was deprotected to give the target molecule 1.     

Synthesis of galactofuranose-containing acceptor substrates for mycobacterial galactofuranosyltransferases

The major structural component of the cell wall in Mycobacterium tuberculosis, infection by which causes tuberculosis, is the mycolyl-arabinogalactan (mAG) complex. This large glycoconjugates has at its core a backbone of approximately 30 D-galactofuranose (Gal(f)) residues that are linked to peptidoglycan by way of a linker disaccharide containing L-rhamnose and 2-acetamido-2-deoxy-D-glucose. Recent studies have supported a model of galactan biosynthesis in which the entire structure is assembled by the action of two bifunctional galactofuranosyltransferases. These biochemical investigations were made possible, in part, by access to a panel of oligosaccharide fragments of the mAG complex (1-12), the synthesis of which we describe here. An early key finding in this study was that the iodine-promoted cyclization of galactose diethyl dithioacetal (19) in the presence of an alcohol solvent led to the formation Gal(f) glycosides contaminated with no pyranoside isomer, thus allowing the efficient preparation of furanoside derivatives of this monosaccharide. The synthesis of disaccharide targets 1, 2, 11 and 12 proceeded without difficulty through the use of thioglycoside donors and octyl glycoside acceptors, both carrying benzoyl protection. In the synthesis of the tri- and tetrasaccharides 3-6, we explored routes in which the molecule was assembled from the reducing to nonreducing end, and the reverse. The latter approach was found to be preferable for the preparation of 6, and in the case of 3 and 4, this strategy allowed the development of efficient one-pot methods for their synthesis. We have also carried out the first synthesis of three mAG fragments (8-10) consisting of the linker disaccharide further elaborated with one, two or three Gal(f) residues. A key step in the synthesis of these target compounds was the coupling of a protected linker disaccharide derivative (58) with a mono-, di-, or trigalactofuranosyl thioglycoside (17, 54, or 53, respectively).     

Efficient Method for the Elongation of the N‐Acetylglucosamine Unit by Combined Use of Chitinase and β‐Galactosidase

A novel strategy for the regio‐ and stereoselective synthesis by two enzymatic steps of oligosaccharides having an N‐acetylglucosamine unit at the nonreducing end was developed. The first step involves a chitinase‐catalyzed highly selective β‐N‐acetyllactosamination of an oligosaccharide acceptor with a 4,5‐dihydrooxazole derivative of N‐acetyllactosamine as the glycosyl donor. The usage of a transition‐state‐analogue substrate for the chitinase under basic conditions allows the reaction to proceed only in the synthetic direction while suppressing hydrolysis of the product in aqueous media. Several chitinase mutants also catalyzed the glycosylation efficiently under neutral conditions. The second step is a regioselective cleavage of the glycosidic bond between the terminal galactose unit and the adjacent N‐acetylglucosamine unit by the action of a β‐galactosidase. This constitutes a very useful method to add an N‐acetylglucosamine unit to the nonreducing end of chito‐ and cello‐oligosaccharide derivatives in a regio‐ and stereoselective manner.     

Identification of reducing and nonreducing neutral carbohydrates by laser‐enhanced in‐source decay (LEISD) MALDI MS

In this work, laser‐enhanced in‐source decay (LEISD) technique of matrix‐assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI‐FT‐ICR‐MS) was used to distinguish reducing and nonreducing carbohydrates. Interestingly, easier cleavage of (1 → 2)‐linked glycosidic bonds for nonreducing carbohydrates containing D‐fructofuranosyl units was observed in MALDI‐FT‐ICR‐MS, which was in agreement with the result of theoretical calculation by the software package Gaussian 09. Importantly, no cross‐ring cleavage of fructofuranosyl residues was detected in the LEISD spectra of nonreducing carbohydrates. LEISD method therefore offers an attractive alternative for fast and efficient differentiation of reducing and nonreducing carbohydrates, and the positions of nonreducing monosaccharide residues in a carbohydrate chain could be easily speculated. Copyright © 2013 John Wiley & Sons, Ltd.     

The first total synthesis of a highly branched arabinofuranosyl hexasaccharide found at the nonreducing termini of mycobacterial arabinogalactan and lipoarabinomannan

[reaction--see text] The first total synthesis of the arabinofuranosyl hexasaccharide present at the nonreducing termini of mycobacterial arabinogalactan and lipoarabinomannan is reported. The oligosaccharide was prepared as its methyl glycoside via a route that is both highly efficient and convergent. Addition of two beta-D-arabinofuranosyl residues simultaneously in high yield and with excellent stereocontrol was the key step of the synthesis.     

Mass spectrometry characterization of the glycation sites of bovine insulin by tandem mass spectrometry

Bovine insulin was glycated under hyperglycemic reducing conditions and in nonreducing conditions. Purification through HPLC allowed isolating glycated forms of insulin and a novel triglycated form (6224.5 Da) was purified. Endoproteinase Glu-C digestion combined with mass spectrometry (MALDI-TOF/TOF) allowed determining the exact location of the glycation sites in each of the isolated glycated insulins. For the first time, a triglycated form of insulin was isolated and characterized accordingly to its glycation sites. These glucose binding sites were identified as the N-terminals of both chains (Gly1 and Phe1) and residue Lys29 of B-chain. Moreover, in diglycated insulin we found the coexistence of one specie glycated at the N-terminals of both chains (Gly1 and Phe1) and another specie containing the two glucitol adducts in B-chain (Phe1 and Lys29). Also, in monoglycated insulin generated in reducing and nonreducing conditions, one specie glycated at Phe1 and another specie glycated at Lys29, both B-chain residues coexist.     

Iterative synthesis of Leishmania phosphoglycans by solution,solid-phase,and polycondensation approaches without involving any glycosylation

A general strategy (solution, solid-phase, and polycondensation) for the synthesis of antigenic phosphoglycans (PG) of the protozoan parasite Leishmania is presented. Phosphoglycans constitute the variable structural and functional domain of major cell-surface lipophosphoglycan (LPG) and secreted proteophosphoglycan (PPG), the molecules involved in infectivity and survival of the Leishmania parasite inside human macrophages. We have shown that the chemically labile, anomerically phosphodiester-linked phosphoglycan repeats can be assembled in an iterative and efficient manner from a single key intermediate, without involving any glycosylation steps. Furthermore, the phosphoglycan chain can be extended toward either the nonreducing (6'-OH) or the reducing (1-OH) end. We also describe a new and efficient solid-phase methodology to construct phosphoglycans based on design and application of a novel cis-allylphosphoryl solid-phase linker that enabled the selective cleavage of the first anomeric-phosphodiester linkage without affecting any of the other internal anomeric-phosphodiester groups of the growing PG chain on the solid support. The strategy to construct larger phosphoglycans in a one-pot synthesis by polycondensation of a single key intermediate is also described, enabling CD spectrometric measurements to show the helical nature of phosphoglycans. Our versatile synthetic approach provides easy access to Leishmania phosphoglycans and the opportunity to address key immunological, biochemical, and biophysical questions pertaining to the phosphoglycan family (LPG and PPG) unique to the parasite.     

Acid-catalyzed methanolysis of tri-O-methylamylose and tri-O-methylcellulose

Past experimental evidence has indicated that the acid-catalyzed hydrolysis of polysaccharides does not proceed randomly, and it has been suggested that hydrolysis is more rapid for the glycosidic bonds by which the nonreducing endgroups are attached. To test this hypothesis, amylose and cellulose were permethylated and subjected to methanolysis. It was found that in both the methanolysis of tri-O-methylamylose and tri-O-methylcellulose the production of methyl 2,3,4,6-tetra-O-methyl-α,β-D -glucopyranoside was complete before the production of methyl 2,3,6-tri-O-methyl-α,β-D -glucoside was finished. Since the former compound could only come from the original nonreducing end units and the latter from all other units, these results were interpreted as giving support to the idea of a preferential scission of the bonds at the nonreducing ends, even though the release of original end units was not complete until 70–85% of the glycosidic bonds had been cleaved. It was concluded that methanolysis proceeds by a modification of the hydrolysis mechanism and that methanolysis is therefore a poor model for hydrolysis.     

Structural Analysis and Aggregation Propensity of Reduced and Nonreduced Glycated Insulin Adducts

The milieu within pancreatic β cells represents a favorable environment for glycation of insulin. Therefore, in this study, insulin samples were individually subjected to glycation under reducing and nonreducing conditions. As monitored by ortho-phthalaldehyde and fluorescamine assays, the reduced glycated insulin adduct demonstrates extensively higher level of glycation than the nonreduced glycated counterpart. Also, gel electrophoresis experiments suggest a significant impact of glycation under a reducing system on the level of insulin oligomerization. Furthermore, reduced and nonreduced glycated insulin adducts respectively exhibit full and partial resistance against dithiothreitol-induced aggregation. The results of thioflavin T and Congo red assays suggest the existence of a significant quantity of amyloid-like entities in the sample of reduced glycated insulin adduct. Both fluorescence and far-ultraviolet circular dichroism studies respectively suggest that the extents of unfolding and secondary structural alteration were closely correlated to the level of insulin glycation. Moreover, the surface tension of two glycated insulin adducts was inversely correlated to their glycation extents and to the quantity of exposed hydrophobic patches. Overall, the glucose-modified insulin molecules under reducing and nonreducing systems display different structural features having significant consequences on aggregation behaviors and surface tension properties. The particular structural constraints of glycated insulin may reduce the binding interaction of this hormone to its receptor which is important for both insulin function and clearance.     

Synthesis of N-acetyl Glucosamine Analogs as Inhibitors for Hyaluronan Biosynthesis

Elevated hyaluronan expression is a hallmark of many types of cancer. Therefore, inhibition of hyaluronan biosynthesis can potentially slow the growth of tumor cells. Herein, we explore a chain termination strategy to reduce hyaluronan synthesis by tumor cells. Several analogs of glucosamine were prepared, which contained modifications at the C-3 positions. These analogs can possibly cap the nonreducing end of a growing hyaluronan chain, thus lowering the amount of hyaluronan synthesized. Upon incubation with pancreatic cancer cells, a fluorine-containing glucosamine analog was found to exhibit significant inhibitory activities of hyaluronan synthesis. Furthermore, it drastically reduced the proliferation of cancer cells.

Supplemental materials are available for this article. Go to the publisher's online edition of Journal of Carbohydrate Chemistry to view the supplemental file.     

The stereochemical dependence of unimolecular dissociation of monosaccharide-glycolaldehyde anions in the gas phase: a basis for assignment of the stereochemistry and anomeric configuration of monosaccharides in oligosaccharides by mass spectrometry via a key discriminatory product ion of disaccharide fragmentation, m/z 221

Mass spectrometry of hexose-containing disaccharides often yields product ions of m/z 221 in the negative ion mode. Using a Paul trap, isolation and collision-induced dissociation of the m/z 221 anions yielded mass spectra that easily differentiated their stereochemistry and anomeric configuration, for all 16 stereochemical variants. The ions were shown to be glycopyranosyl-glycolaldehydes through chemical synthesis of their standards. The stereochemistry dramatically affected fragmentation which was dependent on four relative stereochemical arrangements: (1) the relationship between the hydroxyl group at position 2 and the anomeric configuration, (2) a cis relationship of the anomeric position and positions 2 and 3 (1,2,3-cis), (3) a 1,2 trans-2,3 cis relationship, and (4) the relationship between the hydroxyl group at position 4 and the anomeric configuration. After labeling the reducing carbonyl oxygen of a series of disaccharides with 18O to mass-discriminate between their monosaccharide components, it was demonstrated that m/z 221 anions are comprised of an intact nonreducing sugar glycosidically linked to a 2-carbon aglycon derived from the reducing sugar, irrespective of the linkage position between monosaccharides. This enabled the location of the intact sugar to be assigned to the nonreducing side of a glycosidic linkage. Detailed studies of experimental factors necessary for reproducibility demonstrated that the unique mass spectrum for each m/z 221 anion could be obtained from month-to-month through the use of an internal energy-input calibrant ion that ensured reproducible energy deposition into the ions. The counterparts to these ions for the 2-acetamido-2-deoxyhexoses were m/z 262 anions, and the anomeric configuration and stereochemistry of these anions could also be reproducibly discriminated for N-acetylglucosamine and N-acetylgalactosamine. The fragmentation patterns of m/z 221 anions provide a firm reproducible basis for assignment of sugar stereochemistries in the gas phase.     

An improvement in the bending ability of a hinged trisaccharide with the assistance of a sugar-sugar interaction

Hinged di- and trisaccharides incorporating 2,4-diamino-beta-D-xylopyranoside as a hinge unit (Hin) were synthesized. Bridging of the diamino group of Hin by carbonylation or chelation to a metal ion results in a conformational change from (4)C1 to (1)C4, which in turn causes a bending of the oligosaccharides. In this study, the bending abilities of the hinged oligosaccharides were compared, in terms of the reactivities toward carbonylation and chelation. Di- or trisaccharides containing a 6-O-glycosylated mannopyranoside or galactopyranoside at their reducing ends had bending abilities similar to that of the Hin monosaccharide, probably because there were neither attractive nor repulsive interactions between the reducing and nonreducing ends. However, when Hin was attached at O2 of methyl mannopyranoside (Man alphaMe), the bending ability was dependent on the nonreducing sugar and the reaction conditions. Typically, a disaccharide--Hin beta(1,2)Man alphaMe--was difficult to bend under all the tested reaction conditions, and the bent population in the presence of Zn(II) was only 4%. On the other hand, a trisaccharide--Man alpha(1,3)Hin beta(1,2)Man alphaMe--was bent immediately after the addition of Zn(II) or Hg(II), and the bent population reached 75%, much larger than those of all the other hinged trisaccharides ever tested (<40%). This excellent bending ability suggests an attractive interaction between the reducing and nonreducing ends. The extended conformation was recovered by the addition of triethylenetetramine, a metal ion chelator. Reversible, quick, and efficient bending of the hinged trisaccharide was thus achieved.     

Enzymatic Synthesis of α‐d‐Xylosylated Malto‐oligosaccharides by Phosphorylase‐catalyzed Xylosylation

This paper describes the enzymatic synthesis of α‐d‐xylosylated malto‐oligosaccharides by phosphorylase‐catalyzed xylosylation of maltotetraose. When the xylosylation was carried out using α‐d‐xylose‐1‐phosphate as a glycosyl donor in the presence of phosphorylase, xylosylated oligosaccharides were produced with high conversion. α‐d‐Xylosyl‐(1→4)‐maltotetraose was isolated as the main product. Glucoamylase‐catalyzed reaction of the isolated material revealed that one α‐xyloside unit is positioned at the nonreducing end.     

Gas-Phase Fragmentation Studies of Biotinylated,Hexaethylene Glycol–Spacered Oligosaccharides—Molecular Probes—Using Electrospray Mass Spectrometry on a Hybrid High-Resolution Mass Spectrometer

The electrospray ionization high-resolution mass spectra of biotinylated hexaethylene glycol–spacered molecular probes bearing biologically relevant carbohydrate moieties in positive and negative modes were recorded and interpreted. Collisionally induced decay mass spectra (positive mode) revealed different patterns depending on the charge of the parent ion, attached cations (or ions), the composition, and the sequence of carbohydrate fragments. The most intense peaks (two series) originated from the sequential cleavage of glycoside bonds resulting in charge location on the reducing end (Y series observed for all of the test compounds) or nonreducing end (B series). Hexaethylene glycol chain fragmentation giving rise to the cleavage of the C–O bond remote from the biotin moiety was observed. Other fragment ions lighter than the above by a difference of (C₂H₄O) _n were absent or much smaller. Similar fragmentation was found for all of the nonsulfated biotinylated glycosides with the hexaethylene glycol spacer thus demonstrating that this type of fragmentation was characteristic of such molecular probes. Similar cleavages along with biotin moiety decay via the elimination of H₂S and H₂CS were observed for negative ions in the collisionally induced decay mass spectra of sulfated and neutral molecular probes.     

One-Pot Synthesis of Asymmetrically Difunctionalized Oligomaltosides by Cyclodextrin Ring Opening

The synthesis of pure difunctionalized hexa-, hepta- and octamaltosides was performed by one-pot chemical reaction from perbenzoylated cyclodextrin. Oligomaltosides with azide, propargyl or allyl on reducing end and an unprotected hydroxyl group on non-reducing end were obtained from perbenzoylated α-, β- and γ-cyclodextrin with 12 to 48 % yields.     

Deciphering the mode of action of the processive polysaccharide modifying enzyme dermatan sulfate epimerase 1 by hydrogen–deuterium exchange mass spectrometry

Distinct from template-directed biosynthesis of nucleic acids and proteins, the enzymatic synthesis of heterogeneous polysaccharides is a complex process that is difficult to study using common analytical tools. Therefore, the mode of action and processivity of those enzymes are largely unknown. Dermatan sulfate epimerase 1 (DS-epi1) is the predominant enzyme during the formation of iduronic acid residues in the glycosaminoglycan dermatan sulfate. Using recombinant DS-epi1 as a model enzyme, we describe a tandem mass spectrometry-based method to study the mode of action of polysaccharide processing enzymes. The enzyme action on the substrate was monitored by hydrogen–deuterium exchange mass spectrometry and the sequence information was then fed into mathematical models with two different assumptions of the mode of action for the enzyme: processive reducing end to non-reducing end, and processive non-reducing end to reducing end. Model data was scored by correlation to experimental data and it was found that DS-epi1 attacks its substrate on a random position, followed by a processive mode of modification towards the non-reducing end and that the substrate affinity of the enzyme is negatively affected by each additional epimerization event. It could also be shown that the smallest active substrate was the reducing end uronic acid in a tetrasaccharide and that octasaccharides and longer oligosaccharides were optimal substrates. The method of using tandem mass spectrometry to generate sequence information of the complex enzymatic products in combination with in silico modeling can be potentially applied to study the mode of action of other enzymes involved in polysaccharide biosynthesis.