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1.
Org Biomol Chem ; 16(2): 228-238, 2018 01 03.
Article in English | MEDLINE | ID: mdl-29234770

ABSTRACT

A series of compounds associated with naturally occurring and biologically relevant glycans consisting of α-mannosides were prepared and analyzed using collision-induced dissociation (CID), energy-resolved mass spectrometry (ERMS), and 1H nuclear magnetic resonance spectroscopy. The CID experiments of sodiated species of disaccharides and ERMS experiments revealed that the order of stability of mannosyl linkages was as follows: 6-linked > 4-linked ≧ 2-linked > 3-linked mannosyl residues. Analysis of linear trisaccharides revealed that the order observed in disaccharides could be applied to higher glycans. A branched trisaccharide showed a distinct dissociation pattern with two constituting disaccharide ions. The estimation of the content of this ion mixture was possible using the disaccharide spectra. The hydrolysis of mannose linkages at 3- and 6-positions in the branched trisaccharide revealed that the 3-linkage was cleaved twice as fast as the 6-linkage. It was observed that the solution-phase hydrolysis and gas-phase dissociation have similar energetics.

2.
Nat Chem ; 6(1): 65-74, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24345949

ABSTRACT

Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.


Subject(s)
Carbohydrates/chemistry , Glycopeptides/chemistry , Mass Spectrometry/methods , Polysaccharides/chemistry , Amino Acid Sequence , Glycosyltransferases/chemistry , Humans , Molecular Sequence Data
3.
Biochemistry (Mosc) ; 76(7): 791-6, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21999540

ABSTRACT

Structure of the O-specific polysaccharide chain of the lipopolysaccharide (LPS) of Shewanella japonica KMM 3601 was elucidated. The initial and O-deacylated LPS as well as a trisaccharide representing the O-deacetylated repeating unit of the O-specific polysaccharide were studied by sugar analysis along with 1H and 13C NMR spectroscopy. The polysaccharide was found to contain a rare higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonic acid (a derivative of 4-epilegionaminic acid, 4eLeg). The following structure of the trisaccharide repeating unit was established: →4)-α-4eLegp5Ac7Ac-(2→4)-ß-D-GlcpA3Ac-(1→3)-ß-D-GalpNAc-(1→.


Subject(s)
O Antigens/chemistry , Shewanella/chemistry , Carbohydrate Sequence , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Shewanella/immunology , Sugar Acids/analysis
4.
Carbohydr Res ; 346(6): 872-6, 2011 May 01.
Article in English | MEDLINE | ID: mdl-21392735

ABSTRACT

The structure of the repeating unit of the O-antigen polysaccharide from Shigella flexneri provisional serotype 88-893 has been determined. (1)H and (13)C NMR spectroscopy as well as 2D NMR experiments were employed to elucidate the structure. The carbohydrate part of the hexasaccharide repeating unit is identical to the previously elucidated structure of the O-polysaccharide from S. flexneri prov. serotype Y394. The O-antigen of S. flexneri prov. serotype 88-893 carries 0.7 mol O-acetyl group per repeating unit located at O-2 of the 3-substituted rhamnosyl residue, as identified by H2BC and BS-CT-HMBC NMR experiments. The O-antigen polysaccharide is composed of hexasaccharide repeating units with the following structure: →2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap2Ac-(1→3)[α-D-Glcp-(1→2)-α-D-Glcp-(1→4)]-ß-D-GlcpNAc-(1→. Serological studies showed that type antigens for the two provisional serotypes are identical; in addition 88-893 expresses S. flexneri group factor 6 antigen. We propose that provisional serotypes Y394 and 88-893 be designated as two new serotypes 7a and 7b, respectively, in the S. flexneri typing scheme.


Subject(s)
O Antigens/chemistry , Shigella flexneri/chemistry , Carbohydrate Sequence , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Serotyping
5.
Chemistry ; 7(14): 3069-77, 2001 Jul 16.
Article in English | MEDLINE | ID: mdl-11495434

ABSTRACT

The trisaccharide beta-D-Glcp-(1-->2)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-OMe has been investigated by molecular dynamics (MD) simulations and NMR experiments in water. 13C spin-lattice (T1) and spin-spin (T2) relaxation times, together with 1H,13C NOE data were measured at two magnetic field strengths (9.4 and 14.1 T) in a 277 K D2O solution. Relaxation data interpreted by means of the model-free formalism revealed a rigid (S2 approximately 0.9) oligosaccharide tumbling in solution. 1H,1H Cross-relaxation rates were determined at 600 MHz by 1D DPFGSE NOESY and T-ROESY experiments, which provided high quality data and subsequently proton-proton distances within the trisaccharide. The presence of anti conformers at both torsions of a glycosidic linkage is demonstrated for the first time. MD simulations were carried out to facilitate analysis of the NOE data. In total, 15 simulations-starting from five different conformational states--were performed, with production runs of up to 10 ns, resulting in 83 ns of oligosaccharide dynamics in water. anti Conformers were populated to different degrees in the simulations, especially at the phi2 torsion angle. By combining the results from the NOE experiments and the MD simulations, the anti conformers at the (1-->2)-linkage were quantified as 7% anti-phi2 and 2% anti-psi2, revealing a highly flexible trisaccharide in which large conformational changes occur. From the MD simulations, interresidue hydrogen bonding, from HO2" to O2 or O3, was significantly populated (approximately 40%) in both of the anti conformational states. The contentious issue over rigidity versus flexibility in oligosaccharides has thus been thoroughly examined, showing that the dynamics should be taken into account for a relevant description of the molecular system.


Subject(s)
Oligosaccharides/chemistry , Carbohydrate Conformation , Carbon Isotopes , Glycosides/chemistry , Magnetic Resonance Spectroscopy/methods , Protons , Water/chemistry
6.
J Mol Graph Model ; 19(3-4): 338-42, 396-7, 2001.
Article in English | MEDLINE | ID: mdl-11449573

ABSTRACT

Molecular dynamics simulations have been performed of the disaccharide alpha-D-Manp-(1-->3)-beta-D-Glcp-OMe in two different solvents, namely in methanol and in dimethyl sulfoxide. The conformation of the disaccharide is similar to that previously determined in water. The three-dimensional structure around the solute was investigated by geometric hydrogen bonding criteria, radial distribution functions, coordination number analysis, residence times for hydrogen bonds, and spatial distribution functions. Differences and similarities between methanol and the aprotic dimethyl sulfoxide as solvent are analyzed.


Subject(s)
Carbohydrate Conformation , Computer Simulation , Disaccharides/chemistry , Models, Molecular , Dimethyl Sulfoxide , Hydrogen Bonding , Methanol , Solutions , Thermodynamics
7.
J Magn Reson ; 151(1): 136-41, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11444948

ABSTRACT

Double-quantum heteronuclear local field NMR is performed on a sample of a 13C2-labeled disaccharide, in which the two 13C spins are located on opposite sides of the glycosidic linkage. The evolution of the double-quantum coherences is found to be consistent with the solid-state conformation of the molecule, as previously determined by X-ray diffraction. The dependence of the double-quantum evolution on the glycosidic torsional angles is examined by using a graphical molecular manipulation program interfaced to a numerical spin simulation module.


Subject(s)
Disaccharides/chemistry , Glycosides/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Computer Simulation , Magnetic Resonance Spectroscopy , Molecular Sequence Data
8.
Carbohydr Res ; 333(4): 263-9, 2001 Jul 19.
Article in English | MEDLINE | ID: mdl-11454333

ABSTRACT

We have studied the interaction between the Vibrio cholerae O139 specific phage JA1, belonging to the Podoviridae family, and the capsular polysaccharide (CPS) of the parent strain from which the phage was isolated. Upon incubation of the JA1 phage with the CPS, oligosaccharides were isolated and purified. The oligosaccharides derived from one (shown below) and two repeating units of the CPS were characterized using NMR spectroscopy, mass spectrometry and sugar analysis (structure: see text). The cleavage was found to occur by beta-elimination at the 4-substituted alpha-linked galacturonic acid, which results in a 4-deoxy-beta-L-threo-hex-4-enopyranosyl uronic acid group (Sug). The enzyme associated with the JA1 phage responsible for the depolymerization of the V. cholerae O139 CPS is thus a lyase.


Subject(s)
Bacteriophages/enzymology , Lyases/metabolism , Polysaccharides, Bacterial/metabolism , Polysaccharides/metabolism , Vibrio cholerae/chemistry , Carbohydrate Sequence , Hydrolysis , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Polymers/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
9.
Carbohydr Res ; 333(2): 179-83, 2001 Jul 03.
Article in English | MEDLINE | ID: mdl-11448680

ABSTRACT

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O77 has been determined. Sugar and methylation analysis together with 1H and 13C NMR spectroscopy were the main methods used. The PS is composed of tetrasaccharide repeating units with the following structure:-->2)-alpha-D-Manp-(1-->2)-beta-D-Manp-(1-->3)-alpha-D-GlcpNAc-(1-->6)-alpha-D-Manp-(1-->


Subject(s)
Escherichia coli/chemistry , O Antigens/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Escherichia coli/metabolism , Magnetic Resonance Spectroscopy , Methylation , Molecular Sequence Data
10.
Carbohydr Res ; 331(3): 285-90, 2001 Apr 12.
Article in English | MEDLINE | ID: mdl-11383898

ABSTRACT

The exocellular polysaccharide S-7, a heteropolysaccharide from Azotobacter indicus var. myxogenes has been studied using methylation analysis, Smith degradation, partial acid hydrolysis, NMR spectroscopy and mass spectrometry as the principal methods. It is concluded that the repeating unit has the following structure: [structure: see text] The absolute configuration of the deoxyhexuronic acid was deduced from 1H NMR chemical shifts and is most likely D. Approximately two O-acetyl groups per repeating unit are present, one of which is presumably on the Rha residue. The structure bears great resemblance to another polysaccharide, recently studied, produced by Sphingomonas paucimobilis I-886.


Subject(s)
Hexuronic Acids/analysis , Monosaccharides/analysis , Polysaccharides, Bacterial/chemistry , Azotobacter/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Hydrolysis , Magnetic Resonance Spectroscopy , Mass Spectrometry , Methylation , Molecular Sequence Data , Molecular Structure
11.
Chemistry ; 7(8): 1750-8, 2001 Apr 17.
Article in English | MEDLINE | ID: mdl-11349917

ABSTRACT

Proton-proton cross-relaxation rates have been measured for the trisaccharide beta-D-Glcp-(l --> 2)[beta-D-Glcp-(1 --> 3)]alpha-D-Glcp-OMe in D2O as well as in D2O/[D6]DMSO 7:3 solution at 30 degrees C by means of one-dimensional NMR pulsed field gradient 1H,1H NOESY and TROESY experiments. Interatomic distances for the trisaccharide in D2O were calculated from the cross-relaxation rates for two intraresidue and three interglycosidic proton pairs, using the isolated spin-pair approximation. In the solvent mixture one intraresidue and three interglycosidic distances were derived without the use of a specific molecular model. In this case the distances were calculated from the cross-relaxation rates in combination with "model-free" motional parameters previously derived from 13C relaxation measurements. The proton-proton distances for interglycosidic pairs were compared with those averaged from Metropolis Monte Carlo and Langevin Dynamics simulations with the HSEA, PARM22, and CHEAT95 force fields. The crystal structure of the trisaccharide was solved by analysis of X-ray data. Interresidue proton pairs from the crystal structure and those observed by NMR experiments were similar. However, the corresponding proton-proton distances generated by computer simulations were longer. For the (1 --> 2) linkage the glycosidic torsion angles of the crystal structure were found in a region of conformational space populated by all three force fields, whereas for the (1 --> 3) linkage they occupied a region of low population density, as seen from the simulations.


Subject(s)
Trisaccharides/chemistry , Algorithms , Computer Simulation , Crystallography, X-Ray , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Conformation , Molecular Structure , Monte Carlo Method , Structure-Activity Relationship
12.
Carbohydr Res ; 331(2): 219-24, 2001 Mar 22.
Article in English | MEDLINE | ID: mdl-11322737

ABSTRACT

A lipopolysaccharide (LPS) was isolated by hot phenol-water extraction from Helicobacter pylori strain D4 and found to contain no fucosylated poly-N-acetyllactosamine chain typical of most H. pylori strains studied but a homopolymer of D-glycero-D-manno-heptose (DD-Hep). The heptan attached to a core oligosaccharide was released by mild acid degradation of the LPS, and the following structure of the trisaccharide-repeating unit was established by chemical methods and 1H and 13C NMR spectroscopy: --> 2)-D-alpha-D-Hepp-(1 --> 3)-D-alpha-D-Hepp-(1 --> 3)-D-alpha-D-Hepp-(1 -->. 1H NMR spectroscopy performed on small amounts of the intact LPS revealed the presence of the same polysaccharide in LPS of H. pylori strains D2 and D5, but not strain D10.


Subject(s)
Helicobacter pylori/chemistry , Heptoses/chemistry , Lipopolysaccharides/chemistry , Magnetic Resonance Spectroscopy
13.
Biochemistry ; 40(12): 3623-8, 2001 Mar 27.
Article in English | MEDLINE | ID: mdl-11297429

ABSTRACT

The three-dimensional structure of a cyclic enterobacterial common antigen (ECA) having four trisaccharide repeating units has been investigated by NMR spectroscopy and molecular dynamics simulations. Three different NMR parameters were determined: (a) (1)H,(1)H cross-relaxation rates from NOE experiments were used for determination of proton-proton distances; (b) trans-glycosidic (3)J(C,H) scalar coupling constants analyzed via a Karplus-type relationship provided information on torsion angles; and (c) (1)H,(13)C one-bond dipolar couplings obtained in a dilute liquid-crystalline medium were interpreted in terms of the orientational order and molecular conformations. The molecular dynamics simulations of the dodecasaccharide were performed with explicit water and counterions, which are important factors that strongly influence molecular conformation. Subsequently, the results from computer simulation were used to generate a three-dimensional structure of the cyclic ECA which is consistent with the experimental NMR parameters.


Subject(s)
Antigens, Bacterial/chemistry , Plesiomonas/chemistry , Plesiomonas/immunology , Carbohydrate Conformation , Carbohydrate Sequence , Computer Simulation , Glucans/chemistry , Models, Chemical , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular/methods , Polysaccharides/chemistry , Protons , Solutions , Thermodynamics , Trisaccharides/chemistry
14.
Eur J Biochem ; 268(8): 2239-45, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11298740

ABSTRACT

The structure of the O-antigen polysaccharide from Escherichia coli O172 has been determined. In combination with sugar analysis, NMR spectroscopy shows that the polysaccharide is composed of pentasaccharide repeating units. Sequential information was obtained by mass spectrometry and two-dimensional NMR techniques. An O-acetyl group was present as 0.7 equivalent per repeating unit. Treatment of the O-deacetylated polysaccharide with aqueous 48% hydrofluoric acid rendered cleavage of the phosphodiester in the backbone of the polymer and the pentasaccharide isolated after gel permeation chromatography was structurally characterized. Subsequent NMR experiments on polymeric materials revealed the structure of the repeating unit of the O-polysaccharide from E. coli O172 as:-->P-4)-alpha-D-Glcp-(1-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D- GlcpNAc-(1-->3)-alpha-L-FucpNAc-(1-->4)-alpha-D-Glcp6Ac-(1-->


Subject(s)
Escherichia coli/metabolism , O Antigens/chemistry , Shiga Toxin/chemistry , Carbohydrate Sequence , Chromatography, High Pressure Liquid , Escherichia coli/chemistry , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Sequence Data , Phosphorylation
15.
Carbohydr Res ; 329(2): 465-9, 2000 Nov 03.
Article in English | MEDLINE | ID: mdl-11117331

ABSTRACT

A viscous extracellular polysaccharide produced by Lactobacillus helveticus K16 has been investigated. Sugar and methylation analysis, 1H and 13C NMR spectroscopy revealed that the polysaccharide is composed of a hexasaccharide repeating unit. The sequence of sugar residues was determined by use of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear multiple bond connectivity experiments. The structure of the repeating unit of the exopolysaccharide from L. helveticus K16 is as follows: carbohydrate sequence [see text].


Subject(s)
Lactobacillus/chemistry , Polysaccharides, Bacterial/chemistry , Carbohydrate Sequence , Gas Chromatography-Mass Spectrometry , Magnetic Resonance Spectroscopy , Methylation , Molecular Sequence Data , Viscosity
16.
J Magn Reson ; 147(2): 266-72, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11097818

ABSTRACT

One-dimensional nuclear magnetic resonance techniques were applied to the conformational investigation of a disaccharide. More specifically, nuclear Overhauser enhancements (NOEs) of protons on either side of the glycosidic bond have been used to determine the conformation of the disaccharide alpha-l-Rhap-(1 --> 2)-alpha-l-Rhap-OMe. A modified GOESY sequence, incorporating selective excitation and pulsed field gradient enhancement, was developed and used to accurately measure small NOE signals of interest. These experiments were named M-GOESY, for modified GOESY, and the data they provided were used to calculate internuclear distances in the disaccharide molecule. The accuracy of the M-GOESY measurements was enhanced by elimination of indirect effects, or spin diffusion, by selective inversion(s) of either the intermediate magnetization or the source and target magnetization during the mixing time. Results of this study indicate that the alpha-l-Rhap-(1 --> 2)-alpha-l-Rhap-OMe disaccharide molecule exists primarily in one conformation, with the glycosidic torsion angle psi approximately -30 degrees based on past molecular dynamics simulations.


Subject(s)
Disaccharides/chemistry , Magnetic Resonance Spectroscopy/methods , Mathematics , Molecular Conformation
17.
Carbohydr Res ; 329(1): 227-32, 2000 Oct 20.
Article in English | MEDLINE | ID: mdl-11086704

ABSTRACT

The acidic polysaccharide from Serratia marcescens serogroup O14:K12 was analyzed by means of chemical studies and NMR spectroscopy and its repeating unit structure found to be carbohydrate sequence [see text] O-Acetyl groups are proposed to be present in non-stoichiometric amounts on O-6 on one of the hexose residues in the main chain.


Subject(s)
Bacterial Capsules/chemistry , Polysaccharides/chemistry , Serratia marcescens/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Polysaccharides, Bacterial/chemistry , Sequence Analysis , Serotyping
20.
Carbohydr Res ; 326(2): 113-9, 2000 Jun 02.
Article in English | MEDLINE | ID: mdl-10877094

ABSTRACT

A viscous extracellular polysaccharide produced by Lactobacillus helveticus Lb161 isolated from raw milk has been investigated. Sugar and methylation analysis, and 1H and 13C NMR spectroscopy revealed that the polysaccharide is composed of a heptasaccharide repeating unit. The sequence of sugar residues was determined by use of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear multiple bond connectivity experiments. The structure of the repeating unit of the exopolysaccharide from L. helveticus Lb161 is as follows: carbohydrate structure [see text]. The polysaccharide contains approximately 0.6 equivalents of O-acetyl group per repeating unit (not located).


Subject(s)
Lactobacillus/chemistry , Polysaccharides/chemistry , Animals , Carbohydrate Sequence , Carbohydrates/chemistry , Electron Spin Resonance Spectroscopy , Gas Chromatography-Mass Spectrometry , Magnetic Resonance Spectroscopy , Methylation , Milk/microbiology , Models, Chemical , Molecular Sequence Data
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