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Exploring the structure of oligo- and polysaccharides: Synthesis and NMR spectroscopy studies
Stockholm University, Faculty of Science, Department of Organic Chemistry. (Göran Widmalm)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A deeper understanding of the diversity of carbohydrates and the many applications of oligo- and polysaccharides found in nature are of high interest. Many of the processes involving carbohydrates affect our everyday life. This thesis is based on six papers all contributing to an extended perspective of carbohydrate property and functionality. An introduction to carbohydrate chemistry together with a presentation of selected carbohydrate synthesis and analysis methods introduces the reader to the research field. The first paper is an NMR spectroscopy reinvestigation of the structures of the O-antigens from the lipopolysaccharides (LPS) of Shigella dysenteriae type 3 and Escherichia coli O124. The repeating units were concluded to be built of identical branched pentasaccharides now with the correct anomeric configurations. Paper II is a structural investigation of the O-antigen from the LPS of E. coli O74 which is built of branched tetrasaccharide repeating units including the uncommon monosaccharide d-Fuc3NAc. Paper III is a conformational study of a rhamnose derivative, using NMR spectroscopy and X-ray crystallography. The benzoyl ester group positioned at C4 prefers an “eclipsed” conformation in the crystal as well as in solution. The use of site-specifically 13C-labeled compounds in conformational studies is discussed in Papers IV and V. The disaccharide α-L-Rhap-(1→2)-α-L-Rhap-OMe was synthesized together with two 13C-isotopologues and studied with NMR spectroscopy to give seven J-couplings related to torsion angles φ and ψ. The trisaccharide α-L-Rhap-(1→2)[α-L-Rhap-(1→3)]-α-L-Rhap-OMe was synthesized with 13C-labeling at two positions which presented a solution to a problem of overlapping signals in the 1H NMR spectrum. The site-specific labeling also facilitated the measurement of two 3JCC and two 2JCH coupling constants. Finally, chapter 6 gives a short introduction to glycosynthase chemistry and discusses the synthesis of α-glycosyl fluorides. A novel cyclic heptasaccharide was synthesized from α-laminariheptaosyl fluoride using a mutant of the enzyme laminarase 16A and subsequently analyzed by NMR spectroscopy.

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University , 2010. , 61 p.
Keyword [en]
Carbohydrates, NMR spectroscopy, synthesis, lipopolysaccharides, conformational studies
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-37680ISBN: 978-91-7447-041-3 (print)OAI: oai:DiVA.org:su-37680DiVA: diva2:306294
Public defence
2010-05-07, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.Available from: 2010-04-15 Created: 2010-03-19 Last updated: 2010-04-08Bibliographically approved
List of papers
1. Structural studies of the O-antigenic polysaccharide from Shigella dysenteriae type 3 and Escherichia coli O124, a reinvestigation
Open this publication in new window or tab >>Structural studies of the O-antigenic polysaccharide from Shigella dysenteriae type 3 and Escherichia coli O124, a reinvestigation
2006 (English)In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 341, 2986-2989 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
Elesevier, 2006
Keyword
Cross-reactivity; Escherichia coli O164; Lactyl; Lipopolysaccharide; NMR
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-21529 (URN)10.1016/j.carres.2006.10.005 (DOI)
Available from: 2007-12-12 Created: 2007-12-12 Last updated: 2010-04-08Bibliographically approved
2. Structural determination of the O-antigenic polysaccharide from Escherichia coli O74
Open this publication in new window or tab >>Structural determination of the O-antigenic polysaccharide from Escherichia coli O74
2009 (English)In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 344, no 12, 1592-1595 p.Article in journal (Refereed) Published
Abstract [en]

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O74 has been determined. Component analysis, together with 1H and 13C NMR spectroscopy as well as 1H,15N-HSQC experiments were employed to elucidate the structure. Inter-residue correlations were determined by 1H,1H-NOESY and 1H,13C-heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure:

Full-size image (5K)

Cross-peaks of low intensity from an α-linked N-acetylglucosamine residue were present in the NMR spectra, and spectral analysis indicates that they originate from the penultimate residue in the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. The 1H, 13C and 15N NMR chemical shifts of the α- and β-anomeric forms of d-Fucp3NAc are also reported. The repeating unit of the E. coli O74 O-antigen is identical to that of the capsular polysaccharide from E. coli K45.

Place, publisher, year, edition, pages
Elsevier Ltd., 2009
Keyword
Escherichia coli; Lipopolysaccharide; Capsular polysaccharide; NMR; Biological repeating unit
National Category
Other Basic Medicine
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-29260 (URN)10.1016/j.carres.2009.03.023 (DOI)000269734500028 ()
Available from: 2009-08-19 Created: 2009-08-19 Last updated: 2017-12-13Bibliographically approved
3. Methyl 4-O-benzoyl-2,3-O-isopropylidene-a-L-rhamnopyranoside
Open this publication in new window or tab >>Methyl 4-O-benzoyl-2,3-O-isopropylidene-a-L-rhamnopyranoside
2006 (English)In: Acta Crystallographica Section C: Crystal Structure Communications, ISSN 0108-2701, E-ISSN 1600-5759, Vol. 62, no 8, o447-o449 p.Article in journal (Refereed) Published
Abstract [en]

The title compound, C17H22O6, having an ester group at O4 of the hexopyranosyl sugar residue shows for the exo-cyclic C=O bond a conformation that is eclipsed to the C4-H4 bond. The two related torsion angles are denoted by syn and cis conformations. The q1 torsion angle (H4-C4-O4-C10) is indicated to have a similar conformation in solution as analyzed by NMR spectroscopy and a Karplus-type relationship.

Place, publisher, year, edition, pages
Wiley Interscience, 2006
National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-37681 (URN)10.1107/S0108270106021603 (DOI)
Available from: 2010-03-19 Created: 2010-03-19 Last updated: 2017-12-12Bibliographically approved
4. Studies on the conformational flexibility of α-L-Rhap-(1→2)-α-L-Rhap-OMe using molecular simulation and 13C-site-specific labeling: a model for a commonly occurring disaccharide in bacterial polysaccharides
Open this publication in new window or tab >>Studies on the conformational flexibility of α-L-Rhap-(1→2)-α-L-Rhap-OMe using molecular simulation and 13C-site-specific labeling: a model for a commonly occurring disaccharide in bacterial polysaccharides
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Bacterial polysaccharides are comprised of a variety of monosaccharides, L-rhamnose (6-deoxy-Lmannose) being one of them. This sugar is often part of α-(1→2)- and/or α-(1→3)-linkages and wehave therefore studied the disaccharide α-L-Rhap-(1→2)-α-L-Rhap-OMe to obtain information onconformational preferences at this glycosidic linkage. The target disaccharide was synthesized with 13C site-specific labeling at C1' and at C2', i.e., in the terminal group. 2D 1H,13C-HSQC-HECADE and 1H,13C-J-HMBC NMR experiments, 1D 13C and 1H NMR spectra together with total line-shape analysis were used to extract conformationally dependent hetero- and homonuclear spin-spincoupling constants. This resulted in the determination of 2JC2',H1' , 3JC1',C1 , 3JC1',C3 , 3JC2',C2 , 2JC1',C2 ,1JC1',C2' , and 1JC1',H1' . These data together with previously determined JC,H and 1H,1H NOEs result in fourteen conformationally dependent NMR parameters that are available for analysis of glycosidiclinkage flexibility and conformational preferences. A molecular dynamics simulation of the disaccharide with explicit water molecules as solvent showed a major conformational state at ΦH =40° and ψH = –35°, consistent with experimental NMR data.

Keyword
carbohydrates, torsion angle, NMR spectroscopy, molecular dynamics, heteronuclear long-range coupling constants
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:su:diva-38144 (URN)
Available from: 2010-03-29 Created: 2010-03-29 Last updated: 2010-04-08Bibliographically approved
5. NMR analysis of conformationally dependent nJCH and nJCC in the trisaccharide α-L-Rhap-(1→2)[α-L-Rhap-(1→3)]-α-L-Rhap-OMe and a site-specifically labeled isotopologue thereof
Open this publication in new window or tab >>NMR analysis of conformationally dependent nJCH and nJCC in the trisaccharide α-L-Rhap-(1→2)[α-L-Rhap-(1→3)]-α-L-Rhap-OMe and a site-specifically labeled isotopologue thereof
(English)Manuscript (preprint) (Other academic)
Abstract [en]

An array of NMR spectroscopy experiments have been carried out to obtain conformationallydependent 1H,13C- and 13C,13C-spin-spin coupling constants in the trisaccharide a-L-Rhap-(1®2)[a-LRhap-(1®3)]-a-L-Rhap-OMe. The trisaccharide was synthesized with 13C site-specific labeling at C2'and C2'', i.e., in the rhamnosyl groups in order to alleviate 1H spectral overlap. Using both the naturalabundance compound and the 13C-labeled sample 2D 1H,13C-J-HMBC and 1H,13C-HSQC-HECADENMR experiments, total line-shape analysis of 1H NMR spectra and 1D 13C NMR experiments wereemployed to extract 3C,H J , 2C,H J , 3C,C J , and1C,C J . The 13C site-specific labeling facilitates straightforward determination of nC,C J as the splitting of the 13C natural abundance resonances. This studyresulted in seven conformationally dependent coupling constants for the trisaccharide and illustrates theuse of 13C site-specific labeling as a valuable tool that extends the 1D and 2D NMR methods in currentuse to attain both hetero- and homonuclear spin-spin coupling constants.

Keyword
J-HMBC, HSQC-HECADE, HETCOR, oligosaccharide, heteronuclear long-range coupling constants
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:su:diva-38132 (URN)
Available from: 2010-03-29 Created: 2010-03-29 Last updated: 2010-04-08Bibliographically approved
6. Synthesis of cyclic β-glucan using Laminarinase 16A glycosynthase mutant from the basidiomycete Phanerochaete chrysosporium
Open this publication in new window or tab >>Synthesis of cyclic β-glucan using Laminarinase 16A glycosynthase mutant from the basidiomycete Phanerochaete chrysosporium
Show others...
2010 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 132, no 5, 1724-1730 p.Article in journal (Refereed) Published
Abstract [en]

Glycosynthases are precise molecular instruments for making specifically linked oligosaccharides. X-ray crystallography screening of ligands bound to the 1,3(4)-β-d-glucanase nucleophile mutant E115S of Phanerochaete chrysosporium Laminarinase 16A (Lam16A) showed that laminariheptaose (L7) bound in an arch with the reducing and nonreducing ends occupying either side of the catalytic cleft of the enzyme. The X-ray structure of Lam16A E115S in complex with α-laminariheptaosyl fluoride (αL7F) revealed how αL7F could make a nucleophilic attack upon itself. Indeed, when Lam16A E115S was allowed to react with αL7F the major product was a cyclic β-1,3-heptaglucan, as shown by mass spectrometry. NMR confirmed uniquely β-1,3-linkages and no reducing end. Molecular dynamics simulations indicate that the cyclic laminariheptaose molecule is not completely planar and that torsion angles at the glycosidic linkages fluctuate between two energy minima. This is the first report of a glycosynthase that joins the reducing and nonreducing ends of a single oligosaccharide and the first reported synthesis of cyclic β-glucan.

Place, publisher, year, edition, pages
American Chemical Society, 2010
National Category
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-37223 (URN)10.1021/ja909129b (DOI)000275084900063 ()
Available from: 2010-02-17 Created: 2010-02-17 Last updated: 2017-12-12Bibliographically approved

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