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Assembling and Unraveling Carbohydrates Structures: Conformational analysis of synthesized branched oligosaccharides
Stockholm University, Faculty of Science, Department of Organic Chemistry.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Advances in the elaboration of vaccines and enzyme inhibitors rely on acquiring more knowledge about protein-carbohydrate binding events. Furthermore, the relationships between biological function and the three-dimensional properties of large glycans can be studied by focusing on the structural components they contained, namely, by scaling down the system under analysis. Chemical methods are useful assets as they allow the isolation and determination of epitopes; these small and recognizable fragments that lead to very specific interactions. In this thesis, biologically relevant saccharides were obtained using recently developed concepts in carbohydrate synthesis and NMR spectroscopy was used to unravel their conformational preferences.

In paper I, the convergent synthesis of the tetrasaccharide found in the natural product solaradixine is described. Reactivity enhanced disaccharide glycosyl donors were coupled to a disaccharide acceptor in a 2 + 2 fashion. The computer program CASPER was subsequently used to verify the synthesized structure.

The conformation arming concept employed in paper I was further investigated in paper II. An NMR-based methodology enabled the determination of the ring conformations of a set of donors. Subsequently, glycosylation reactions were performed and yields were correlated to donors ring shapes. Perturbations in the rings shape caused by bulky silyl ether protective groups were sufficient to boost the potency of several donors. As a matter of fact, complex branched oligosaccharides could be obtained in good to excellent yields.

In paper III, NMR spectroscopy observables were measured to elucidate the ring shape, the mutual orientation of the rings across the glycosidic bond and the positions of the side chains of 5 trisaccharides found in larger structures. With the aid of molecular dynamics simulations, their overall conformational propensities were revealed.

Finally, the software CASPER prediction skills were improved by adding, inter alia, NMR information of synthesized mono- and disaccharides to its database. Unassigned chemical shifts from polysaccharides served as input to challenge its ability to solve large carbohydrate structures.

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University , 2016. , 85 p.
Keyword [en]
Convergent carbohydrate synthesis, Super-armed donors, Glycosylation, Conformational analysis, NMR, Chemical shift prediction
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-133106ISBN: 978-91-7649-473-8 (print)OAI: oai:DiVA.org:su-133106DiVA: diva2:957006
Public defence
2016-10-21, 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 2: Manuscript. Paper 4: Manuscript.

Available from: 2016-09-28 Created: 2016-08-31 Last updated: 2017-10-11Bibliographically approved
List of papers
1. Synthesis of the tetrasaccharide glycoside moiety of Solaradixine and rapid NMR-based structure verification using the program CASPER
Open this publication in new window or tab >>Synthesis of the tetrasaccharide glycoside moiety of Solaradixine and rapid NMR-based structure verification using the program CASPER
2016 (English)In: Tetrahedron, ISSN 0040-4020, E-ISSN 1464-5416, Vol. 72, no 7, 912-927 p.Article in journal (Refereed) Published
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.

Keyword
Glycosylation, Oligosaccharide, Saponin, CASPER, Chemical shift prediction
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-127344 (URN)10.1016/j.tet.2015.12.042 (DOI)000369461900004 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2016-03-11 Created: 2016-03-02 Last updated: 2017-11-30Bibliographically approved
2. Structure-Reactivity Relationships of Conformationally Armed Disaccharide Donors and Their Use in the Synthesis of a Hexasaccharide Related to the Capsular Polysaccharide from Streptococcus pneumoniae Type 37
Open this publication in new window or tab >>Structure-Reactivity Relationships of Conformationally Armed Disaccharide Donors and Their Use in the Synthesis of a Hexasaccharide Related to the Capsular Polysaccharide from Streptococcus pneumoniae Type 37
2017 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 82, no 15, 8123-8140 p.Article in journal (Refereed) Published
Abstract [en]

To advance the field of glycobiology, efficient synthesis methods of oligosaccharides and glycoconjugates are a requisite. In glycosylation reactions using superarmed donors, both selectivity and reactivity issues must be considered, and we herein investigate these aspects for differently protected beta-linked 2-O-glycosylated glucosyl donors carrying bulky tert-butyldimethylsilyl groups to different extents. The acceptors in reactions being secondary alcohols presents a challenging situation with respect to steric crowding. Conformational pyranose ring equilibria of the superarmed disaccharide donors with axial-rich substituents contained skew and boat conformations, and three-state models were generally assumed. With NIS/TfOH as the promotor, 2,6-di-tert-butyl-4-methylpyridine as the base, and a dichloromethane/toluene solvent mixture, ethyl 1-thio-beta-d-glucosyl disaccharide donors having 6-O-benzyl group(s) besides tert-butyldimethylsilyl groups were efficiently coupled at -40 degrees C to the hydroxyl group at position 3 of glucopyranosyl acceptors to form beta-(1 -> 2),beta-(1 -> 3)-linked trisaccharides, isolated in excellent 95% yield. The more axial-rich donors in skew and boat conformations are thus preorganized closer to the assumed transition state in these glycosylation reactions. The developed methodology was subsequently applied in the synthesis of a multibranched hexasaccharide related to the capsular polysaccharide from Streptococcus pneumoniae type 37, which consists of a beta-(1 -> 3)-linked backbone and a beta-(1 -> 2)-linked side chain of D-glucosyl residues in disaccharide repeating units.

National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-147106 (URN)10.1021/acs.joc.7b01264 (DOI)000407307700041 ()28682619 (PubMedID)
Available from: 2017-10-11 Created: 2017-10-11 Last updated: 2017-10-11Bibliographically approved
3. Delineating the conformational flexibility of trisaccharides from NMR spectroscopy experiments and computer simulations
Open this publication in new window or tab >>Delineating the conformational flexibility of trisaccharides from NMR spectroscopy experiments and computer simulations
Show others...
2016 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 18, no 28, 18776-18794 p.Article in journal (Refereed) Published
Abstract [en]

The conformation of saccharides in solution is challenging to characterize in the context of a single well-defined three-dimensional structure. Instead, they are better represented by an ensemble of conformations associated with their structural diversity and flexibility. In this study, we delineate the conformational heterogeneity of five trisaccharides via a combination of experimental and computational techniques. Experimental NMR measurements target conformationally sensitive parameters, including J couplings and effective distances around the glycosidic linkages, while the computational simulations apply the well-calibrated additive CHARMM carbohydrate force field in combination with efficient enhanced sampling molecular dynamics simulation methods. Analysis of conformational heterogeneity is performed based on sampling of discreet states as defined by dihedral angles, on root-mean-square differences of Cartesian coordinates and on the extent of volume sampled. Conformational clustering, based on the glycosidic linkage dihedral angles, shows that accounting for the full range of sampled conformations is required to reproduce the experimental data, emphasizing the utility of the molecular simulations in obtaining an atomic detailed description of the conformational properties of the saccharides. Results show the presence of differential conformational preferences as a function of primary sequence and glycosidic linkage types. Significant differences in conformational ensembles associated with the anomeric configuration of a single glycosidic linkage reinforce the impact of such changes on the conformational properties of carbohydrates. The present structural insights of the studied trisaccharides represent a foundation for understanding the range of conformations adopted in larger oligosaccharides and how these molecules encode their conformational heterogeneity into the monosaccharide sequence.

National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-133040 (URN)10.1039/c6cp02970a (DOI)000379939100017 ()
External cooperation:
Available from: 2016-08-29 Created: 2016-08-29 Last updated: 2017-11-21Bibliographically approved
4. 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 CASPER
Open this publication in new window or tab >>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 CASPER
Show others...
2013 (English)In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 380, 156-166 p.Article in journal (Refereed) Published
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.

Keyword
Oligosaccharide, Glycan, Chemical shift prediction, Automation
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-95416 (URN)10.1016/j.carres.2013.06.026 (DOI)000325167400024 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

AuthorCount:12;

Available from: 2013-10-31 Created: 2013-10-28 Last updated: 2017-09-07Bibliographically approved

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