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  • 1.
    Angles d'Ortoli, Thibault
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Sjöberg, Nils A.
    Vasiljeva, Polina
    Lindman, Jonas
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Bergenstråhle-Wohlert, Malin
    Wohlert, Jakob
    Temperature Dependence of Hydroxymethyl Group Rotamer Populations in Cellooligomers2015In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 119, no 30, p. 9559-9570Article in journal (Refereed)
    Abstract [en]

    Empirical force fields for computer simulations of carbohydrates are often implicitly assumed to be valid also at temperatures different from room temperature for which they were optimited: Herein, the temperature dependence of the hydroxymethyl group rotamer populations in short oligogaccharides is invegtigated using Molecular dynamics simulations and NMR spectroscopy. Two oligosaccharides, methyl beta-cellobioside and beta-cellotetraose were simulated using three different carbohydrate force fields (CHARMM C35, GLYCAM06, and GROMOS 56A(carbo)) in combination with different water models (SPC, SPC/E, and TIP3P) using replica exchange molecular dynamics simulations. For comparison, hydroxymethyl group rotamer populations were investigated for methyl beta-cellobioside and cellopentaose based- on measured NMR (3)J(H5,H6) coupling constants, in the latter case by using a chemical shift selective NMR-filter. Molecular dynamics simulations in combination with NMR spectroscopy show that the temperature dependence of the hydroxymethyl rotamer population in these short cellooligomers, in the range 263-344 K, generally becomes exaggerated in simulations when compared to experimental data, but also that it is dependent on simulation conditions, and most notably properties of the water model.

  • 2.
    Angles d'Ortoli, Thibault
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Synthesis of the tetrasaccharide glycoside moiety of Solaradixine and rapid NMR-based structure verification using the program CASPER2016In: Tetrahedron, ISSN 0040-4020, E-ISSN 1464-5416, Vol. 72, no 7, p. 912-927Article in journal (Refereed)
    Abstract [en]

    The major glycoalkaloid in the roots of Solanum laciniatum is Solaradixine having the branched tetrasaccharide beta-D-Glcp-(1 -> 2)-beta-D-Glcp-(1 -> 3)[alpha-L-Rhap-(1 -> 2)]-beta-D-Galp linked to O3 of the steroidal alkaloid Solasodine. We herein describe the synthesis of the methyl glycoside of the tetrasaccharide using a super-armed disaccharide as a donor molecule. A 2-(naphthyl)methyl protecting group was used in the synthesis of the donor since it was tolerant to a wide range of reaction conditions. The 6-O-benzylated-hexa-O-tert-butyldimethylsilyi-protected beta-D-Glcp-(1 -> 2)-beta-D-Glcp-SEt donor, which avoided 1,6-anydro formation, was successfully glycosylated at O3 of a galactoside acceptor molecule. However, subsequent glycosylation at O2 by a rhamnosyl donor was unsuccessful and instead a suitably protected alpha-L-Rhap(1 -> 2)-beta-D-Galp-OMe disaccharide was used as the acceptor molecule together with a super-armed beta-D-Glcp-(1 -> 2)-beta-D-Glcp-SEt donor in the glycosylation reaction, to give a tetrasaccharide in a yield of 55%, which after deprotection resulted in the target molecule, the structure of which was verified by the NMR chemical shift prediction program CASPER.

  • 3. Battistel, Marcos D.
    et al.
    Pendrill, Robert
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Freedberg, Daron I.
    Direct Evidence for Hydrogen Bonding in Glycans: A Combined NMR and Molecular Dynamics Study2013In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, no 17, p. 4860-4869Article in journal (Refereed)
    Abstract [en]

    We introduce the abundant hydroxyl groups of glycans as NMR handle's and structural probes to expand the repertoire of tools for structure function studies on glycans in solution. To this end, we present the facile detection and assignment of hydroxyl groups in a Wide range of sample concentrations (0.5-1700 mM) and temperatures, ranging from -5 to 25 degrees C.,We then exploit this information to directly detect hydrogen bonds, well-known for their importance in molecular structural determination through NMR. Via HSQC-TOCSY, we were able to determine the directionality; of these hydrogen bonds in sucrose Furthermore, by means Of molecular dynamics simulations in conjunction with NMR, we establish that one Out of the three detected hydrogen bonds arises from intermolecular interactions. This finding may shed light on glycan glycan interactions and glycan recognition by proteins.

  • 4. Both, P.
    et al.
    Green, A. P.
    Gray, C. J.
    Sardzik, R.
    Voglmeir, J.
    Fontana, Carolina
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Austeri, M.
    Rejzek, M.
    Richardson, D.
    Field, R. A.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Flitsch, S. L.
    Eyers, C. E.
    Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing2014In: Nature Chemistry, ISSN 1755-4330, E-ISSN 1755-4349, Vol. 6, no 1, p. 65-74Article in journal (Refereed)
    Abstract [en]

    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-a-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.

  • 5. Chassagne, Pierre
    et al.
    Fontana, Carolina
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Guerreiro, Catherine
    Gauthier, Charles
    Phalipon, Armelle
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Mulard, Laurence A.
    Structural Studies of the O-Acetyl-Containing O-Antigen from a Shigella flexneri Serotype 6 Strain and Synthesis of Oligosaccharide Fragments Thereof2013In: European Journal of Organic Chemistry, ISSN 1434-193X, E-ISSN 1099-0690, no 19, p. 4085-4106Article in journal (Refereed)
    Abstract [en]

    Extensive analysis by NMR spectroscopy of the delipidated lipopolysaccharide of Shigella flexneri serotype 6 strain MDC 2924-71 confirmed the most recently reported structure of the O-antigen repeating unit as {4)--D-GalpA-(13)--D-GalpNAc-(12)--L-Rhap3Ac/4Ac-(12)--L-Rhap-(1}, and revealed the non-stoichiometric acetylation at O-3C/4C. Input from the CASPER program helped to ascertain the fine distribution of the three possible patterns of O-acetylation. The non-O-acetylated repeating unit (ABCD) corresponded to about 2/3 of the population, while 1/4 was acetylated at O-3C (3AcCDAB), and 1/10 at O-4C (4AcCDAB). Di- to tetrasaccharides with a GalpA residue (A) at their reducing end were synthesized as their propyl glycosides following a multistep linear strategy relying on late-stage acetylation at O-3C. Thus, the 3C-O-acetylated and non-O-acetylated targets were synthesized from common protected intermediates. Rhamnosylation was most efficiently achieved by using imidate donors, including at O-4 of a benzyl galacturonate acceptor. In contrast, a thiophenyl 2-deoxy-2-trichloroacetamido-D-galactopyranoside precursor was preferred for chain elongation involving residue B. Final Pd/C-mediated deprotection ensured O-acetyl stability. All of the target molecules represent parts of the O-antigen of S. flexneri 6, a prevalent serotype. Non-O-acetylated oligosaccharides are also fragments of the Escherichia coli O147 O-antigen.

  • 6. Chen, Mo
    et al.
    Pendrill, Robert
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Brady, John W.
    Wohlert, Jakob
    Molecular Dynamics Simulations of the Ionic Liquid 1-n-Butyl-3-Methylimidazolium Chloride and Its Binary Mixtures with Ethanol2014In: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 10, no 10, p. 4465-4479Article in journal (Refereed)
    Abstract [en]

    Room temperature ionic liquids (ILs) of the imidazolium family have attracted much attention during the past decade for their capability to dissolve biomass. Besides experimental work, numerous compuational studies have been concerned with the physical properties of both neat ILs and their interactions with different solutes, in particular, carbohydrates. Many classical force fields designed specifically for ILs have been found to yield viscosities that are too high for the liquid state, which has been attributed to the fact that the effective charge densities are too high due to the lack of electronic polarizability. One solution to this problem has been uniform scaling of the partial charges by a scale factor in the range 0.6-0.9, depending on model. This procedure has been shown to improve the viscosity of the models, and also to positively affect other properties, such as diffusion constants and ionic conductivity. However, less attention has been paid to how this affects the overall thermodynamics of the system, and the problems it might create when the IL models are combined with other force fields (e.g., for solutes). In the present work, we employ three widely used IL force fields to simulate 1-n-buty1-3-methyl-imidazolium chloride in both the crystal and the liquid state, as well as its binary mixture with ethanol. Two approaches are used: one in which the ionic charge is retained at its full integer value and one in which the partial charges are uniformly reduced to 85%. We investigate and calculate crystal and liquid structures, molar heat capacities, heats of fusion, self-diffusion constants, ionic conductivity, and viscosity for the neat IL, and ethanol activity as a function of ethanol concentration for the binary mixture. We show that properties of the crystal are less affected by charge scaling compared to the liquid. In the liquid state, transport properties of the neat IL are generally improved by scaling, whereas values for the heat of fusion are unaffected, and results for the heat capacity are ambiguous. Neither full nor reduced charges could reproduce experimental ethanol activities for the whole range of compositions.

  • 7.
    Engström, Olof
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Muñoz, Antonio
    Illescas, Beatriz M.
    Martin, Nazario
    Ribeiro-Viana, Renato
    Rojo, Javier
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Investigation of glycofullerene dynamics by NMR spectroscopy2015In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 13, no 32, p. 8750-8755Article in journal (Refereed)
    Abstract [en]

    Glycofullerenes, in which carbohydrate molecules are attached via a linker to a [60]fullerene core, facilitate spherical presentation of glyco-based epitopes. We herein investigate the dynamics of two glycofullerenes, having 12 and 36 mannose residues at their periphery, by NMR translational diffusion and quantitative C-13 relaxation studies employing a model-free approach for their interpretation. The sugar residues are shown to be highly flexible entities with S-2 < 0.2 in both compounds. Notably, the larger glycofullerene with longer linkers shows faster internal dynamics and higher flexibility than its smaller counterpart. The dynamics and flexibility as well as the slower translational diffusion of the larger glycofullerene, thereby favoring rebinding to a receptor, may together with its spatial extension explain why it is better than the smaller one at blocking the DC-SIGN receptor and inhibiting the infection by pseudotyped Ebola virus particles.

  • 8.
    Fontana, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Kovacs, Helena
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    NMR structure analysis of uniformly 13C-labeled carbohydrates2014In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 59, no 2, p. 95-110Article in journal (Refereed)
    Abstract [en]

    In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of C-13-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly C-13-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-C-13)-sucrose, 342 Da] and one compound of medium molecular weight (C-13-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, similar to 10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The C-13 resonances are traced using C-13-C-13 correlations from homonuclear experiments, such as (H)CC-CT-COSY, (H)CC-NOESY, CC-CT-TOCSY and/or virtually decoupled (H)CC-TOCSY. Based on the assignment of the C-13 resonances, the H-1 chemical shifts are derived in a straightforward manner using one-bond H-1-C-13 correlations from heteronuclear experiments (HC-CT-HSQC). In order to avoid the (1) J (CC) splitting of the C-13 resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either C-13 or H-1 detected experiments, namely CC-CT-COSY, band-selective (H)CC-TOCSY, HC-CT-HSQC-NOESY or long-range HC-CT-HSQC. However, due to the short T-2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the H-1-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the C-13-enriched polysaccharide were assigned by using HC-H2BC spectra. The assignment of the N-acetyl groups with N-15 at natural abundance was completed by using HN-SOFAST-HMQC, HNCA, HNCO and C-13-detected (H)CACO spectra.

  • 9.
    Fontana, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Li, Shengyu
    Yang, Zhennai
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Structural studies of the exopolysaccharide from Lactobacillus plantarum C88 using NMR spectroscopy and the program CASPER2015In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 402, p. 87-94Article in journal (Refereed)
    Abstract [en]

    Some lactic acid bacteria, such as those of the Lactobacillus genus, have the ability to produce exopolysaccharides (EPSs) that confer favorable physicochemical properties to food and/or beneficial physiological effects on human health. In particular, the EPS of Lactobacillus plantarum C88 has recently demonstrated in vitro antioxidant activity and, herein, its structure has been investigated using NMR spectroscopy and the computer program CASPER (Computer Assisted Spectrum Evaluation of Regular polysaccharides). The pentasaccharide repeating unit of the O-deacetylated EPS consists of a trisaccharide backbone, -> 4)-alpha-DGalp-(1 -> 2)-alpha-D-Glcp-(1 -> 3)-beta-D-Glcp-(1 ->, with terminal D-Glc and D-Gal residues (1.0 and 0.8 equiv per repeating unit, respectively) extending from O3 and O6, respectively, of the -> 4)-alpha-D-Galp-(1 -> residue. In the native EPS an O-acetyl group is present, 0.85 equiv per repeating unit, at O2 of the alpha-linked galactose residue; thus the repeating unit of the EPS has the following structure: -> 4)[beta-D-Glcp-(1 -> 3)][beta-D-Galp-(1 -> 6)]alpha-D-Galp2Ac-(1 -> 2)-alpha-D-Glcp-(1 -> 3)-beta-D-Glcp-(1 ->. These structural features, and the chain length (similar to 10(3) repeating units on average, determined in a previous study), are expected to play an important role in defining the physicochemical properties of the polymer.

  • 10.
    Fontana, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Lundborg, Magnus
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Weintraub, Andrej
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Rapid structural elucidation of polysaccharides employing predicted functions of glycosyltransferases and NMR data: Application to the O-antigen of Escherichia coli O592014In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 24, no 5, p. 450-457Article in journal (Refereed)
    Abstract [en]

    A computerized method that uses predicted functions of glycosyltransferases (GTs) in conjunction with unassigned NMR data has been developed for the structural elucidation of bacterial polysaccharides (PSs). In this approach, information about the action of GTs (consisting of possible sugar residues used as donors and/or acceptors, as well as the anomeric configuration and/or substitution position in the respective glycosidic linkages) is extracted from the Escherichia coli O-antigen database and is submitted, together with the unassigned NMR data, to the CASPER program. This time saving methodology, which alleviates the need for chemical analysis, was successfully implemented in the structural elucidation of the O-antigen PS of E. coli O59. The repeating unit of the O-specific chain was determined using the O-deacylated PS and has a branched structure, namely, -> 6)[alpha-d-GalpA3Ac/4Ac-(1 -> 3)]-alpha-d-Manp-(1 -> 3)-alpha-d-Manp-(1 -> 3)-beta-d-Manp-(1 -> 3)-alpha-d-GlcpNAc-(1 ->. The identification of the O-acetylation positions was efficiently performed by comparison of the H-1,C-13 HSQC NMR spectra of the O-deacylated lipopolysaccharide and the lipid-free PS in conjunction with chemical shift predictions made by the CASPER program. The side-chain d-GalpA residue carries one equivalent of O-acetyl groups at the O-3 and O-4 positions distributed in the LPS in a 3:7 ratio, respectively. The presence of O-acetyl groups in the repeating unit of the E. coli O59 PS is consistent with the previously proposed acetyltransferase WclD in the O-antigen gene cluster.

  • 11.
    Fontana, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Weintraub, Andrej
    Karolinska Institute.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Facile Structural Elucidation of Glycans Using NMR Spectroscopy Data and the Program CASPER: Application to the O-Antigen Polysaccharide of Escherichia coli O1552013In: ChemPlusChem, ISSN 2192-6506, Vol. 78, no 11, p. 1327-1329Article in journal (Refereed)
    Abstract [en]

    The program CASPER was successfully employed to rapidly elucidate a new O-antigen polysaccharide structure (obtained from a strain of Escherichia coli serogroup O155), using solelyunassigned NMR spectroscopy data as input information. Thus, what is considered the most tedious and time-consuming part of the structural elucidation process has been reduced from several hours (or even days) of manual interpretation to about four minutes of automated analysis.

  • 12.
    Fontana, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Weintraub, Andrej
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Structural Elucidation of the O-Antigen Polysaccharide from Escherichia coli O1812015In: ChemistryOpen, ISSN 2191-1363, Vol. 4, no 1, p. 47-55Article in journal (Refereed)
    Abstract [en]

    Shiga-toxin-producing Escherichia coli (STEC) is an important pathogen associated to food-borne infection in humans; strains of E.coli O181, isolated from human cases of diarrhea, have been classified as belonging to this pathotype. Herein, the structure of the O-antigen polysaccharide (PS) from E.coli O181 has been investigated. The sugar analysis showed quinovosamine (QuiN), glucosamine (GlcN), galactosamine (GalN), and glucose (Glc) as major components. Analysis of the high-resolution mass spectrum of the oligosaccharide (OS), obtained by dephosphorylation of the O-deacetylated PS with aqueous 48% hydrofluoric acid, revealed a pentasaccharide composed of two QuiNAc, one GlcNAc, one GalNAc, and one Glc residue. The H-1 and (CNMR)-C-13 chemical shift assignments of the OS were carried out using 1D and 2D NMR experiments, and the OS was sequenced using a combination of tandem mass spectrometry (MS/MS) data and NMR (CNMR)-C-13 glycosylation shifts. The structure of the native PS was determined using NMR spectroscopy, and it consists of branched pentasaccharide repeating units joined by phosphodiester linkages: -> 4)[alpha-L-QuipNAc-(1 -> 3)]-alpha-D-GalpNAc6Ac-(1 -> 6)-alpha-D-Glcp-(1 -> P-4)-alpha-L-QuipNAc-(1 -> 3)-beta-D-GlcpNAc-(1 ->; the O-acetyl groups represent 0.4 equivalents per repeating unit. Both the OS and PSs exhibit rare conformational behavior since two of the five anomeric proton resonances could only be observed at an elevated temperature.

  • 13.
    Fontana, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Weintraub, Andrej
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Structural studies and biosynthetic aspects of the O-antigen polysaccharide from Escherichia coli O422015In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 403, p. 174-181Article in journal (Refereed)
    Abstract [en]

    The structure of the O-antigen polysaccharide (PS) from Escherichia coli O42 has been investigated by NMR spectroscopy as the main method, which was complemented with sugar analysis, mass spectrometry, and analysis of biosynthetic information. The O-specific chain of the O-deacylated lipopolysaccharide (LPS-OH) consists of branched tetrasaccharide-glycerol repeating units joined by phosphodiester linkages. The lipid-free polysaccharide contains 0.8 equiv of O-acetyl groups per repeating unit and has the following teichoic acid-like structure: Based on biosynthetic aspects, this should also be the biological repeating unit. This O-antigen structure is remarkably similar to that of E. coli O28ac, differing only in the presence or absence, respectively, of a glucose residue at the branching point. The structural similarity explains the serological cross-reactivity observed between strains of these two serogroups, and also their almost identical O-antigen gene cluster sequences. -> 2)-(R)-Gro-(1-P-4)-beta-D-GlcpNAc-(1 -> 3)-beta-D-Galf2Ac-(1 -> 3)-alpha-D-GlcpNAc-(1 -> vertical bar a-D-Glcp-(1 -> 3)

  • 14.
    Hamark, Christoffer
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Landström, Jens
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    SEAL by NMR: Glyco-Based Selenium-Labeled Affinity Ligands Detected by NMR Spectroscopy2014In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 20, no 43, p. 13905-13908Article in journal (Refereed)
    Abstract [en]

    We report a method for the screening of interactions between proteins and selenium-labeled carbohydrate ligands. SEAL by NMR is demonstrated with selenoglycosides binding to lectins where the selenium nucleus serves as an NMR-active handle and reports on binding through Se-77 NMR spectroscopy. In terms of overall sensitivity, this nucleus is comparable to C-13 NMR, while the NMR spectral width is ten times larger, yielding little overlap in Se-77 NMR spectroscopy, even for similar compounds. The studied ligands are singly selenated bioisosteres of methyl glycosides for which straightforward preparation methods are at hand and libraries can readily be generated. The strength of the approach lies in its simplicity, sensitivity to binding events, the tolerance to additives and the possibility of having several ligands in the assay. This study extends the increasing potential of selenium in structure biology and medicinal chemistry. We anticipate that SEAL by NMR will be a beneficial tool for the development of selenium-based bioactive compounds, such as glycomimetic drug candidates.

  • 15. Harper, James K.
    et al.
    Tishler, Derek
    Richardson, David
    Lokvam, John
    Pendrill, Robert
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Solid-State NMR Characterization of the Molecular Conformation in Disordered Methyl alpha-L-Rhamnofuranoside2013In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 117, no 26, p. 5534-5541Article in journal (Refereed)
    Abstract [en]

    A combination of solid-state C-13 NMR tensor data and DFT computational methods is utilized to predict the conformation in disordered methyl alpha-L-rhamnofuranoside. This previously uncharacterized solid is found to be crystalline and consists of at least six distinct conformations that exchange on the kHz time scale. A total of 66 model structures were evaluated, and six were identified as being consistent with experimental C-13 NMR data. All feasible structures have very similar carbon and oxygen positions and differ most significantly in OH hydrogen orientations. A concerted rearrangement of OH hydrogens is proposed to account for the observed dynamic disorder. This rearrangement is accompanied by smaller changes in ring conformation and is slow enough to be observed on the NMR time scale due to severe steric crowding among ring substituents. The relatively minor differences in non-hydrogen atom positions in the final structures suggest that characterization of a complete crystal structure by X-ray powder diffraction may be feasible.

  • 16. He, Xibing
    et al.
    Hatcher, Elizabeth
    Eriksson, Lars
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    MacKerell, Alexander D., Jr.
    Bifurcated Hydrogen Bonding and Asymmetric Fluctuations in a Carbohydrate Crystal Studied via X-ray Crystallography and Computational Analysis2013In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, no 25, p. 7546-7553Article in journal (Refereed)
    Abstract [en]

    The structure of the O-methyl glycoside of the naturally occurring 6-O-[(R)-1-carboxyethyl]-alpha-D-galactopyranose, C10H18O8, has been determined by X-ray crystallography at 100 K, supplementing the previously determined structure obtained at 293 K (Acta Crystallogr. 1996, C52, 2285-2287). Molecular dynamics simulations of this glycoside were Performed in the crystal environment with different numbers of units cells included in the primary simulation system at both 100 and 293 K. The Calculated unit cell Parameters and the intramolecular geometries (bonds, angles, and dihedrals) agree well with experimental results. Atomic fluctuations, including B-factors and anisotropies, are in good agreement with respect to the relative values on an atom-by-atom basis. In addition, the fluctuations increase with increasing simulation system size, with the simulated values converging to values lower than those observed experimentally indicating that the simulation model is not accounting for all possible contributions to the experimentally observed B-factors, which may be related to either the simulation time scale or size. In the simulation's, the hydroxyl group of O7 is found to from bifurcated hydrogen bonds with O6 and O8 of an adjacent molecule, with the interactions dominated by the interaction HO7-O6 interaction. Quantum mechanical calculations support this observation.

  • 17.
    Kapla, Jon
    et al.
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Engström, Olof
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Stevensson, Baltzar
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Wohlert, Jakob
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Maliniak, Arnold
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Molecular dynamics simulations and NMR spectroscopy studies of trehalose-lipid bilayer systems2015In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 17, no 34, p. 22438-22447Article in journal (Refereed)
    Abstract [en]

    The disaccharide trehalose (TRH) strongly affects the physical properties of lipid bilayers. We investigate interactions between lipid membranes formed by 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and TRH using NMR spectroscopy and molecular dynamics (MD) computer simulations. We compare dipolar couplings derived from DMPC/TRH trajectories with those determined (i) experimentally in TRH using conventional high-resolution NMR in a weakly ordered solvent (bicelles), and (ii) by solid-state NMR in multilamellar vesicles (MLV) formed by DMPC. Analysis of the experimental and MD-derived couplings in DMPC indicated that the force field used in the simulations reasonably well describes the experimental results with the exception for the glycerol fragment that exhibits significant deviations. The signs of dipolar couplings, not available from the experiments on highly ordered systems, were determined from the trajectory analysis. The crucial step in the analysis of residual dipolar couplings (RDCs) in TRH determined in a bicelle-environment was access to the conformational distributions derived from the MD trajectory. Furthermore, the conformational behavior of TRH, investigated by J-couplings, in the ordered and isotropic phases is essentially identical, indicating that the general assumptions in the analyses of RDCs are well founded.

  • 18.
    Lihammar, Richard
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Rönnols, Jerk
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Bäckvall, Jan-Erling
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Epimerization of Glycal Derivatives by a Cyclopentadienylruthenium Catalyst: Application to Metalloenzymatic DYKAT2014In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 20, no 45, p. 14756-14762Article in journal (Refereed)
    Abstract [en]

    Epimerization of a non-anomeric stereogenic center in carbohydrates is an important transformation in the synthesis of natural products. In this study an epimerization procedure of the allylic alcohols of glycals by cyclopentadienylruthenium catalyst 1 is presented. The epimerization of 4,6-O-benzylidene-D-glucal 4 in toluene is rapid, and an equlibrium with its D-allal epimer 5 is established within 5min at room temperature. Exchange rates for allal and glucal formation were determined by 1D H-1 EXSY NMR experiments to be 0.055s(-1) and 0.075s(-1), respectively. For 4-O-benzyl-L-rhamnal 8 the epimerization was less rapid and four days of epimerization was required to achieve equilibration of the epimers at room temperature. The epimerization methodology was subsequently combined with acylating enzymes in a dynamic kinetic asymmetric transformation (DYKAT), giving stereoselective acylation to the desired stereoisomers 12, 13, and 15. The net effect of this process is an inversion of a stereogenic center on the glycal, and yields ranging from 71% to 83% of the epimer were obtained.

  • 19.
    Lundborg, Magnus
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Ali, Eunus
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    An in silico virtual screening study for the design of norovirus inhibitors: fragment-based molecular docking and binding free energy calculations2013In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 378, p. 133-138Article in journal (Refereed)
    Abstract [en]

    Gastrointestinal infections caused by noroviruses may be prevented by the inhibition of their binding to histo-blood group carbohydrate antigens. A fragment-based virtual screening approach was used, employing docking followed by molecular dynamics simulations in order to enable binding free energy calculations using the linear interaction energy method. The resulting structures, composed of high-affinity fragments, can be a good starting point for lead optimizations and four molecules that pass both REOS and SYLVIA filters, which can remove known toxic features and assess the synthetic accessibility, respectively, are proposed as inhibitors.

  • 20. Mally, Manuela
    et al.
    Fontana, Carolina
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    LeibundGut-Landmann, Salome
    Laacisse, Lamia
    Fan, Yao-Yun
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Aebi, Markus
    Glycoengineering of host mimicking type-2 LacNAc polymersand Lewis X antigens on bacterial cell surfaces2013In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 87, no 1, p. 112-131Article in journal (Refereed)
    Abstract [en]

    Bacterial carbohydrate structures play a central role in mediating a variety of host-pathogen interactions. Glycans can either elicit protective immune response or lead to escape of immune surveillance by mimicking host structures. Lipopolysaccharide (LPS), a major component on the surface of Gram-negative bacteria, is composed of a lipid A-core and the O-antigen polysaccharide. Pathogens like Neisseria meningitidis expose a lipooligosaccharide (LOS), which outermost glycans mimick mammalian epitopes to avoid immune recognition. Lewis X (Gal beta 1-4(Fuc alpha 1-3)GlcNAc) antigens of Helicobacter pylori or of the helminth Schistosoma mansoni modulate the immune response by interacting with receptors on human dendritic cells. In a glycoengineering approach we generate human carbohydrate structures on the surface of recombinant Gram-negative bacteria, such as Escherichia coli and Salmonella enterica sv. Typhimurium that lack O-antigen. A ubiquitous building block in mammalian N-linked protein glycans is Gal beta 1-4GlcNAc, referred to as a type-2 N-acetyllactosamine, LacNAc, sequence. Strains displaying polymeric LacNAc were generated by introducing a combination of glycosyltransferases that act on modified lipid A-cores, resulting in efficient expression of the carbohydrate epitope on bacterial cell surfaces. The poly-LacNAc scaffold was used as an acceptor for fucosylation leading to polymers of Lewis X antigens. We analysed the distribution of the carbohydrate epitopes by FACS, microscopy and ELISA and confirmed engineered LOS containing LacNAc and Lewis X repeats by MALDI-TOF and NMR analysis. Glycoengineered LOS induced pro-inflammatory response in murine dendritic cells. These bacterial strains can thus serve as tools to analyse the role of defined carbohydrate structures in different biological processes.

  • 21. Mensch, Carl
    et al.
    Pendrill, Robert
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Johannessen, Christian
    Studying the Glycan Moiety of RNase B by Means of Raman and Raman Optical Activity2014In: ChemPhysChem, ISSN 1439-4235, E-ISSN 1439-7641, Vol. 15, no 11, p. 2252-2254Article in journal (Refereed)
    Abstract [en]

    Raman and Raman optical activity (ROA) spectroscopy are used to study the solution-phase structure of the glycan moiety of the protein ribonuclease B (RNase B). Spectral data of the intact glycan moiety of RNase B is obtained by subtracting high-quality spectral data of RNase A, the non-glycosylated form of the RNase, from the spectra of the glycoprotein. The remaining difference spectra are compared to spectra generated from Raman and ROA data of the constituent disaccharides of the RNase glycan, achieving convincing spectral overlap. The results show that ROA spectroscopy is able to extract detailed spectral data of the glycan moieties of proteins, provided that the non-glycosylated isoform is available. Furthermore, good comparison between the full glycan spectrum and the regenerated spectra based on the disaccharide data lends great promise to ROA as a tool for the solution-phase structural analysis of this structurally elusive class of biomolecules.

  • 22. Patel, Dhilon S.
    et al.
    Pendrill, Robert
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Mallajosyula, Sairam S.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    MacKerell, Alexander D., Jr.
    Conformational Properties of alpha- or beta-(1 -> 6)-Linked Oligosaccharides: Hamiltonian Replica Exchange MD Simulations and NMR Experiments2014In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, no 11, p. 2851-2871Article in journal (Refereed)
    Abstract [en]

    Conformational sampling for a set of 10 alpha- or beta-(1 -> 6)-linked oligosaccharides has been studied using explicit solvent Hamiltonian replica exchange (HREX) simulations and NMR spectroscopy techniques. Validation of the force field and simulation methodology is done by comparing calculated transglycosidic J coupling constants and proton-proton distances with the corresponding NMR data. Initial calculations showed poor agreement, for example, with >3 Hz deviation of the calculated (3)J(H5,H6R) values from the experimental data, prompting optimization of the omega torsion angle parameters associated with (1 -> 6)-linkages. The resulting force field is in overall good agreement (i.e., within similar to 0.5 Hz deviation) from experimental (3)J(H5,H6R) values, although some small limitations are evident. Detailed hydrogen bonding analysis indicates that most of the compounds lack direct intramolecular H-bonds between the two monosaccharides; however, minor sampling of the O6 center dot center dot center dot HO2' hydrogen bond is present in three compounds. The results verify the role of the gauche effect between O5 and O6 atoms in gluco- and manno-configured pyranosides causing the omega torsion angle to sample an equilibrium between the gt and gg rotamers. Conversely, galacto-configured pyranosides sample a population distribution in equilibrium between gt and tg rotamers, while the gg rotamer populations are minor. Water radial distribution functions suggest decreased accessibility to the O6 atom in the (1 -> 6)-linkage as compared to the O6' atom in the nonreducing sugar. The role of bridging water molecules between two sugar moieties on the distributions of omega torsion angles in oligosaccharides is also explored.

  • 23.
    Pendrill, Robert
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Eriksson, Lars
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Methyl 4-O-benzyl-alpha-l-rhamno-pyrano-side2014In: Acta Crystallographica Section E: Structure Reports Online, ISSN 1600-5368, E-ISSN 1600-5368, Vol. 70, p. o561-o562Article in journal (Refereed)
  • 24.
    Pendrill, Robert
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Jonsson, K. Hanna M.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Glycan synthesis, structure, and dynamics: A selection2013In: Pure and Applied Chemistry, ISSN 0033-4545, E-ISSN 1365-3075, Vol. 85, no 9, p. 1759-1770Article in journal (Refereed)
    Abstract [en]

    Glycan structural information is a prerequisite for elucidation of carbohydrate function in biological systems. To this end we employ a tripod approach for investigation of carbo hydrate 3D structure and dynamics based on organic synthesis; different experimental spectroscopy techniques, NMR being of prime importance; and molecular simulations using, in particular, molecular dynamics (MD) simulations. The synthesis of oligosaccharides in the form of glucosyl fluorides is described, and their use as substrates for the Lam16A E115S glucosyl synthase is exemplified as well as a conformational analysis of a cyclic beta-(1 -> 3)-heptaglucan based on molecular simulations. The flexibility of the N-acetyl group of aminosugars is by MD simulations indicated to function as a gatekeeper for transitions of glycosidic torsion angles to other regions of conformational space. A novel approach to visualize glycoprotein (GP) structures is presented in which the protein is shown by, for example, ribbons, but instead of stick or space-filling models for the carbohydrate portion it is visualized by the colored geometrical figures known as CFG representation in a 3D way, which we denote 3D-CFG, thereby effectively highlighting the sugar residues of the glycan part of the GP and the position(s) on the protein.

  • 25.
    Pendrill, Robert
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Säwén, Elin
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Conformation and Dynamics at a Flexible Glycosidic Linkage Revealed by NMR Spectroscopy and Molecular Dynamics Simulations: Analysis of β-ʟ-Fucp-(1→6)-α-ᴅ-Glcp-OMe in Water Solution2013In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, no 47, p. 14709-14722Article in journal (Refereed)
    Abstract [en]

    The intrinsic flexibility of carbohydrates facilitates different 3D structures in response to altered environments. At glycosidic (1 -> 46)-linkages, three torsion angles are variable, and herein the conformation and dynamics of beta-1.-Fucp-(1 -> 6)-alpha-D-Glcp-OMe are investigated using a combination of NMR spectroscopy and molecular dynamics (MD) simulations. The disaccharide shows evidence of conformational averaging for the psi and co torsion angles, best explained by a four-state conformational distribution. Notably, there is a significant population of conformations having psi = 85 degrees (clinal) in addition to those having psi = 180 degrees (anfiperiplanar). Moderate differences in C-13 R-1 relaxation rates are found to be best explained by axially symmetric tumbling in combination with minor differences in librational motion for the two residues, whereas the isomerization motions are occurring too slowly to significantly to the observed relaxation rates. The MD simulation was found to give a reasonably good agreement with experiment, especially with respect to diffusive properties, among which the rotational anisotropy, D parallel to/D parallel to, is found to be 2.35. The force field employed showed too narrow omega torsion angles in the gauche trans and gauche gauche states as well as overestimating the population of the gauche trans conformer. This information can subsequently be used in directing parameter developments and emphasizes the need for refinement of force fields for (1 -> 6)-linked carbohydrates.

  • 26.
    Pendrill, Robert
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Sørensen, Ole W.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Suppressing one-bond homonuclear 13C,13C scalar couplings in the J-HMBC NMR experiment: application to 13C site-specifically labeled oligosaccharides2014In: Magnetic Resonance in Chemistry, ISSN 0749-1581, E-ISSN 1097-458X, Vol. 52, no 3, p. 82-86Article in journal (Refereed)
    Abstract [en]

    Site-specific C-13 isotope labeling is a useful approach that allows for the measurement of homonuclear C-13,C-13 coupling constants. For three site-specifically labeled oligosaccharides, it is demonstrated that using the J-HMBC experiment for measuring heteronuclear long-range coupling constants is problematical for the carbons adjacent to the spin label. By incorporating either a selective inversion pulse or a constant-time element in the pulse sequence, the interference from one-bond C-13,C-13 scalar couplings is suppressed, allowing the coupling constants of interest to be measured without complications. Experimental spectra are compared with spectra of a nonlabeled compound as well as with simulated spectra. The work extends the use of the J-HMBC experiments to site-specifically labeled molecules, thereby increasing the number of coupling constants that can be obtained from a single preparation of a molecule.

  • 27. Perepelov, Andrei V.
    et al.
    Shashkov, Alexander S.
    Guo, Xi
    Filatov, Andrei V.
    Weintraub, Andrej
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Knirel, Yuriy A.
    Structure and genetics of the O-antigen of Escherichia coli O169 related to the O-antigen of Shigella boydii type 62015In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 414, p. 46-50Article in journal (Refereed)
    Abstract [en]

    The O-polysaccharide (O-antigen) of Escherichia coli O169 was studied by sugar analysis along with 1D and 2D H-1 and C-13 NMR spectroscopy. The following structure of the branched hexasaccharide repeating unit was established: [GRAPHICS] The O-polysaccharide of E. coli O169 differs from that of Shigella boydii type 6 only in the presence of a side-chain glucose residue. A comparison of the O-antigen biosynthesis gene clusters between the galF to gnd genes in the genomes of the two bacteria revealed their close relationship. The glycosyltransferase gene responsible for the formation of the beta-D-Glcp-(1 -> 6)-alpha-D-Galp linkage in the O-antigen was identified in the gene cluster.

  • 28. Perepelov, Andrei V.
    et al.
    Wang, Quan
    Filatov, Andrei V.
    Xia, Xianghong
    Shashkov, Alexander S.
    Weintraub, Andrej
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Wang, Lei
    Knirel, Yuriy A.
    Structures and gene clusters of the closely related O-antigens of Escherichia coli O46 and O134, both containing D-glucuronoyl-D-allothreonine2015In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 409, p. 20-24Article in journal (Refereed)
    Abstract [en]

    The O-polysaccharides (O-antigens) were isolated by mild acid degradation of the lipopolysaccharide (LPS) of Escherichia coli O46 and O134. The structures of their linear tetrasaccharide repeating units were established by sugar analysis along with 1D and 2D H-1 and C-13 NMR spectroscopy: [GRAPHICS] where D-aThr indicates D-allothreonine and R indicates O-acetyl substitution (similar to 70% on aThr and similar to 15% on GalNAc) in E. coli O46 whereas the O-acetylation is absent in E. coli O134. Functions of genes in the essentially identical O-antigen gene clusters of E. coli O46 and O134 were tentatively assigned by a comparison with sequences in available databases and found to be in agreement with the O-polysaccharide structures established.

  • 29. Rojas-Macias, Miguel A.
    et al.
    Ståhle, Jonas
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Luetteke, Thomas
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Development of the ECODAB into a relational database for Escherichia coli O-antigens and other bacterial polysaccharides2015In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 25, no 3, p. 341-347Article in journal (Refereed)
    Abstract [en]

    Escherichia coli O-antigen database (ECODAB) is aweb-based application to support the collection of E. coli O-antigen structures, polymerase and flippase amino acid sequences, NMR chemical shift data of O-antigens as well as information on glycosyltransferases (GTs) involved in the assembly of O-antigen polysaccharides. The database content has been compiled from scientific literature. Furthermore, the system has evolved from being a repository to one that can be used for generating novel data on its own. GT specificity is suggested through sequence comparison with GTs whose function is known. The migration of ECODAB to a relational database has allowed the automation of all processes to update, retrieve and present information, thereby, endowing the system with greater flexibility and improved overall performance. ECODAB is freely available at http://www.casper.organ.su.se/ECODAB/. Currently, data on 169 E. coli unique O-antigen entries and 338 GTs is covered. Moreover, the scope of the database has been extended so that polysaccharide structure and related information from other bacteria subsequently can be added, for example, from Streptococcus pneumoniae.

  • 30.
    Rönnols, Jerk
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Manner, Sophie
    Ellervik, Ulf
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Conformational effects due to stereochemistry and C3-substituents in xylopyranoside derivatives as studied by NMR spectroscopy2014In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 12, no 40, p. 8031-8035Article in journal (Refereed)
    Abstract [en]

    Glycosaminoglycans contain a beta-D-xylopyranose residue at its reducing end, which links the polysaccharide to the protein in proteoglycans. 2-Naphthyl beta-D-xylopyranosides have shown inhibition of tumor growth and we herein investigate conformation and dynamics of compounds structurally and stereochemically modified at the C3 position as well as the influence of solvent. The 3-deoxygenated compound, the 3-C-methyl-substituted beta-D-xylopyranoside, beta-D-ribopyranoside, the 3-C-methyl-substituted beta-D-ribopyranoside as well as 2-naphthyl beta-D-xylopyranoside were analyzed by NMR spectroscopy. Conformational equilibria were dependent on the solvent of choice, either methanol-d(4) or chloroform-d, with mainly C-4(1) and C-1(4) conformations present but also skew conformations to some extent. Intramolecular hydrogen bonding was concluded to be important for the 3-C-methyl-substituted beta-D-xylopyranosides in the non-polar solvent. Dynamic NMR (DNMR) spectroscopy was carried out for the 3-deoxygenated compound, which at 25 degrees C in methanol-d(4) exists with equally populated states of the C-4(1) and the C-1(4) conformations, but at -100 degrees C only a few percent is present of the latter. Using C-13 NMR detection for DNMR, resonance lines were shown to broaden at -40 degrees C and to sharpen again below -90 degrees C, without the emergence of a second set of NMR resonances, a typical behavior for an unequally populated equilibrium. The enthalpy and entropy activation barriers were calculated and resulted in Delta H-double dagger = 47.3 kJ mol(-1) and Delta S-double dagger = 54 J mol(-1) K-1.

  • 31.
    Rönnols, Jerk
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Pendrill, Robert
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Fontana, Carolina
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Hamark, Christoffer
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Angles d'Ortoli, Thibault
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Engström, Olof
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Ståhle, Jonas
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Zaccheus, Mona V.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Säwén, Elin
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Hahn, Liljan E.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Iqbal, Shahzad
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Complete H-1 and C-13 NMR chemical shift assignments of mono- to tetrasaccharides as basis for NMR chemical shift predictions of oligosaccharides using the computer program CASPER2013In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 380, p. 156-166Article in journal (Refereed)
    Abstract [en]

    H-1 and C-13 NMR chemical shift data are used by the computer program CASPER to predict chemical shifts of oligo- and polysaccharides. Three types of data are used, namely, those from monosaccharides, disaccharides, and trisaccharides. To improve the accuracy of these predictions we have assigned the H-1 and C-13 NMR chemical shifts of eleven monosaccharides, eleven disaccharides, twenty trisaccharides, and one tetrasaccharide; in total 43 compounds. Five of the oligosaccharides gave two distinct sets of NMR resonances due to the alpha- and beta-anomeric forms resulting in 48 H-1 and C-13 NMR chemical shift data sets. In addition, the pyranose ring forms of Neu5Ac were assigned at two temperatures, due to chemical shift displacements as a function of temperature. The H-1 NMR chemical shifts were refined using total line-shape analysis with the PERCH NMR software. H-1 and C-13 NMR chemical shift predictions were subsequently carried out by the CASPER program (http://www.casper.organ.su.se/casper/) for three branched oligosaccharides having different functional groups at their reducing ends, namely, a mannose-containing pentasaccharide, and two fucose-containing heptasaccharides having N-acetyllactosamine residues in the backbone of their structures. Good to excellent agreement was observed between predicted and experimental H-1 and C-13 NMR chemical shifts showing the utility of the method for structural determination or confirmation of synthesized oligosaccharides.

  • 32. Shashkov, Alexander S.
    et al.
    Wang, Tianwei
    Perepelov, Andrei V.
    Weintraub, Andrej
    Liu, Bin
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Knirel, Yuriy A.
    Structure elucidation and biosynthesis gene cluster organization of the O-antigen of Escherichia coli O1702015In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 417, p. 11-14Article in journal (Refereed)
    Abstract [en]

    Enterotoxigenic Escherichia coli are causative agents of diarrhea in humans as well as animals, and E. coli O170 belongs to this virotype. Upon mild acid degradation of the lipopolysaccharide of E. coli O170, the branched O-polysaccharide chain was partially cleaved at beta-D-glactofuranosidic linkages to give multiple products, including a linear tetrasaccharide and oligomers thereof. Studies of the acid degradation products and O-deacylated lipopolysaccharide by 1D and 2D H-1 and C-13 NMR spectroscopy enabled elucidation of the following O-polysaccharide structure: -> 4)-beta-D-GlcpNAc-(1 -> 4)-beta-D-GlcpA-(1 -> 3)-beta-D-Galf-(1 -> 3)-beta-D-GlcNAc-(1 -> [GRAPHICS] beta-D-Galf Functions of genes in the O-antigen biosynthesis gene cluster were tentatively assigned and found to be in agreement with the O-polysaccharide structure.

  • 33. Siegbahn, Anna
    et al.
    Manner, Sophie
    Persson, Andrea
    Tykesson, Emil
    Holmqvist, Karin
    Ochocinska, Agata
    Rönnols, Jerk
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Sundin, Anders
    Mani, Katrin
    Westergren-Thorsson, Gunilla
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Ellervik, Ulf
    Rules for priming and inhibition of glycosaminoglycan biosynthesis; probing the beta 4GalT7 active site2014In: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 5, no 9, p. 3501-3508Article in journal (Refereed)
    Abstract [en]

    beta-1,4-Gatactosyltransferase 7 (beta 4GalT7) is an essential enzyme in the biosynthesis of glycosaminoglycan (GAG) chains of proteoglycans (PGs). Mammalian cells produce PGs, which are involved in biological processes such as cell growth and differentiation. The PGs consist of a core protein, with one or several GAG chains attached. Both the structure of the PGs and the GAG chains, and the expression of the enzymes involved in their biosynthesis and degradation, vary between normal cells and tumor cells. The biosynthesis of GAG chains is initiated by xylosylation of a serine residue of the core protein, followed by galactosylation by beta 4GalT7. The biosynthesis can also be initiated by exogenously added beta-D-xylopyranosides with hydrophobic aglycons, which thus can act as acceptor substrates for beta 4GalT7. To determine the structural requirements for beta 4GalT7 activity, we have cloned and expressed the enzyme and designed a focused library of 2-naphthyl beta-D-xylopyranosides with modifications of the xylose moiety. Based on enzymatic studies, that is galactosylation and its inhibition, conformational analysis and molecular modeling using the crystal structure, we propose that the binding pocket of beta 4GalT7 is very narrow, with a precise set of important hydrogen bonds. Xylose appears to be the optimal acceptor substrate for galactosylation by beta 4GalT7. However, we show that modifications of the xylose moiety of the beta-D-xylopyranosides can render inhibitors of galactosylation. Such compounds will be valuable tools for the exploration of GAG and PG biosynthesis and a starting point for development of anti-tumor agents.

  • 34. Siegbahn, Anna
    et al.
    Thorsheim, Karin
    Ståhle, Jonas
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Manner, Sophie
    Hamark, Christoffer
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Persson, Andrea
    Tykesson, Emil
    Mani, Katrin
    Westergren-Thorsson, Gunilla
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Ellervik, Ulf
    Exploration of the active site of beta 4GalT7: modifications of the aglycon of aromatic xylosides2015In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 13, no 11, p. 3351-3362Article in journal (Refereed)
    Abstract [en]

    Proteoglycans (PGs) are macromolecules that consist of long linear polysaccharides, glycosaminoglycan (GAG) chains, covalently attached to a core protein by the carbohydrate xylose. The biosynthesis of GAG chains is initiated by xylosylation of the core protein followed by galactosylation by the galactosyltransferase beta 4GalT7. Some beta-D-xylosides, such as 2-naphthyl beta-D-xylopyranoside, can induce GAG synthesis by serving as acceptor substrates for beta 4GalT7 and by that also compete with the GAG synthesis on core proteins. Here we present structure-activity relationships for beta 4GalT7 and xylosides with modifications of the aromatic aglycon, using enzymatic assays, cell studies, and molecular docking simulations. The results show that the aglycons reside on the outside of the active site of the enzyme and that quite bulky aglycons are accepted. By separating the aromatic aglycon from the xylose moiety by linkers, a trend towards increased galactosylation with increased linker length is observed. The galactosylation is influenced by the identity and position of substituents in the aromatic framework, and generally, only xylosides with beta-glycosidic linkages function as good substrates for beta 4GalT7. We also show that the galactosylation ability of a xyloside is increased by replacing the anomeric oxygen with sulfur, but decreased by replacing it with carbon. Finally, we propose that reaction kinetics of galactosylation by beta 4GalT7 is dependent on subtle differences in orientation of the xylose moiety.

  • 35.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    A perspective on the primary and three-dimensional structures of carbohydrates2013In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 378, p. 123-132Article in journal (Refereed)
    Abstract [en]

    Carbohydrates, in more biologically oriented areas referred to as glycans, constitute one of the four groups of biomolecules. The glycans, often present as glycoproteins or glycolipids, form highly complex structures. In mammals ten monosaccharides are utilized in building glycoconjugates in the form of oligo-(up to about a dozen monomers) and polysaccharides. Subsequent modifications and additions create a large number of different compounds. In bacteria, more than a hundred monosaccharides have been reported to be constituents of lipopolysaccharides, capsular polysaccharides, and exopolysaccharides. Thus, the number of polysaccharide structures possible to create is huge. NMR spectroscopy plays an essential part in elucidating the primary structure, that is, monosaccharide identity and ring size, anomeric configuration, linkage position, and sequence, of the sugar residues. The structural studies may also employ computational approaches for NMR chemical shift predictions (CASPER program). Once the components and sequence of sugar residues have been unraveled, the three-dimensional arrangement of the sugar residues relative to each other (conformation), their flexibility (transitions between and populations of conformational states), together with the dynamics (timescales) should be addressed. To shed light on these aspects we have utilized a combination of experimental liquid state NMR techniques together with molecular dynamics simulations. For the latter a molecular mechanics force field such as our CHARMM-based PARM22/SU01 has been used. The experimental NMR parameters acquired are typically H-1, H-1 cross-relaxation rates (related to NOEs), (3)JCH and (3)JCC trans-glycosidic coupling constants and H-1, C-13-and H-1, H-1-residual dipolar couplings. At a glycosidic linkage two torsion angles phi and psi are defined and for 6-substituted residues also the omega torsion angle is required. Major conformers can be identified for which highly populated states are present. Thus, in many cases a well-defined albeit not rigid structure can be identified. However, on longer timescales, oligosaccharides must be considered as highly flexible molecules since also anti-conformations have been shown to exist with H-C-O-C torsion angles of similar to 180 degrees, compared to syn-conformations in which the protons at the carbon atoms forming the glycosidic linkage are in close proximity. The accessible conformational space governs possible interactions with proteins and both minor changes and significant alterations occur for the oligosaccharides in these interaction processes. Transferred NOE NMR experiments give information on the conformation of the glycan ligand when bound to the proteins whereas saturation transfer difference NMR experiments report on the carbohydrate part in contact with the protein. It is anticipated that the subtle differences in conformational preferences for glycan structures facilitate a means to regulate biochemical processes in different environments. Further developments in the analysis of glycan structure and in particular its role in interactions with other molecules, will lead to clarifications of the importance of structure in biochemical regulation processes essential to health and disease.

  • 36. Wu, Emilia L.
    et al.
    Fleming, Patrick J.
    Yeom, Min Sun
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Klauda, Jeffery B.
    Fleming, Karen G.
    Im, Wonpil
    E. coil Outer Membrane and Interactions with OmpLA2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 11, p. 2493-2502Article in journal (Refereed)
    Abstract [en]

    The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is similar to 3 angstrom thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.

  • 37.
    Šoltésová, Mária
    et al.
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK). Charles University in Prague.
    Kowalewski, Jozef
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Dynamics of exocyclic groups in the Escherichia coli O91 O-antigen polysaccharide in solution studied by carbon-13 NMR relaxation2013In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 57, no 1, p. 37-45Article in journal (Refereed)
    Abstract [en]

    Carbon-13 relaxation data are reported for exocyclic groups of hexopyranosyl sugar residues in the repeating unit within the Escherichia coli O91 O-antigen polysaccharide in a dilute D2O solution. The measurements of T 1, T 2 and heteronuclear nuclear Overhauser enhancements were carried out at 310 K at two magnetic fields (16.4 T, 21.1 T). The data were analyzed using the standard and extended Lipari–Szabo models, as well as a conformational jump model. The extended version of the Lipari–Szabo and the two-site jump models were most successful for the hydroxymethyl groups of Gal and GlcNAc sugar residues. Different dynamics was found for the hydroxymethyl groups associated with different configurations (d-gluco, d-galacto) of the sugar residues, the latter being faster than the former.

1 - 37 of 37
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