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The sweet side of molecular structure: NMR spectroscopic studies of glycans and their interactions with proteins
Stockholm University, Faculty of Science, Department of Organic Chemistry.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, within the topic of bioorganic chemistry, the molecular structure of carbohydrates has been studied. Carbohydrates, or glycans, are ubiquitous biomolecules exhibiting a wide range of biological roles. The specific functions of these molecules are largely determined by their interactions with proteins and molecular structure ultimately governs such specialized recognition events.

Glycan-binding proteins, such as lectins or enzymes, often interact with their sweet ligands in a transient fashion and nuclear magnetic resonance spectroscopy (NMR) is a viable technique to probe these complexes. In particular, ligand-based NMR techniques have been employed, typically in combination with other biophysical as well as biochemical and computational methods. The aim of this work has been to gain new insights about specific biological systems, to develop methods and to devise protocols for their studies.

The first two papers cover NMR-interaction studies of native ligands as well as inhibitor glycans with the enzyme hen egg-white lysozyme and the lectin botulinum neurotoxin type A. Screening experiments were performed to investigate ligand affinities and selectivities. Solution models in combination with X-ray crystal structures were compared in order to evaluate their agreement and the details of interactions.

A method for application in carbohydrate ligand NMR-screening was developed in paper three. The heteronucleus selenium was exploited as a reporter of selenoglycosides binding to lectins. 77Se NMR spectroscopy proved sensitive to binding events and the presented approach should be useful in large screenings of glycomimetic inhibitors.  In order to obtain sufficient amounts of glycans for bioorganic studies their production often relies on chemical synthesis. In the last paper, the structure of some conformationally highly activated glycosyl donors was thoroughly investigated and related to their reactivity in synthetic glycosylation reactions.  

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University , 2015. , 77 p.
Keyword [en]
bioorganic chemistry, glycans, ligand-based NMR, molecular simulations, protein-carbohydrate interactions, lectins, carbohydrate conformations, 77Se NMR, structure-reactivity relationships, super-armed donors
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-112350ISBN: 978-91-7649-083-9 (print)OAI: oai:DiVA.org:su-112350DiVA: diva2:779030
Public defence
2015-02-20, 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 3: Manuscript. Paper 4: Manuscript.

 

Available from: 2015-01-29 Created: 2015-01-12 Last updated: 2017-10-11Bibliographically approved
List of papers
1. Combining weak affinity chromatography, NMR spectroscopy and molecular simulations in carbohydrate-lysozyme interaction studies
Open this publication in new window or tab >>Combining weak affinity chromatography, NMR spectroscopy and molecular simulations in carbohydrate-lysozyme interaction studies
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2012 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 10, no 15, 3019-3032 p.Article in journal (Refereed) Published
Abstract [en]

By examining the interactions between the protein hen egg-white lysozyme (HEWL) and commercially available and chemically synthesized carbohydrate ligands using a combination of weak affinity chromatography (WAC), NMR spectroscopy and molecular simulations, we report on new affinity data as well as a detailed binding model for the HEWL protein. The equilibrium dissociation constants of the ligands were obtained by WAC but also by NMR spectroscopy, which agreed well. The structures of two HEWL-disaccharide complexes in solution were deduced by NMR spectroscopy using H-1 saturation transfer difference (STD) effects and transferred H-1,H-1-NOESY experiments, relaxation-matrix calculations, molecular docking and molecular dynamics simulations. In solution the two disaccharides beta-D-Galp-(1 -> 4)-beta-D-GlcpNAc-OMe and beta-D-GlcpNAc-(1 -> 4)-beta-D-GlcpNAc-OMe bind to the B and C sites of HEWL in a syn-conformation at the glycosidic linkage between the two sugar residues. Intermolecular hydrogen bonding and CH/pi-interactions form the basis of the protein-ligand complexes in a way characteristic of carbohydrate-protein interactions. Molecular dynamics simulations with explicit water molecules of both the apo-form of the protein and a ligand-protein complex showed structural change compared to a crystal structure of the protein. The flexibility of HEWL as indicated by a residue-based root-mean-square deviation analysis indicated similarities overall, with some residue specific differences, inter alia, for Arg61 that is situated prior to a flexible loop. The Arg61 flexibility was notably larger in the ligand-complexed form of HEWL. N,N'-Diacetylchitobiose has previously been observed to bind to HEWL at the B and C sites in water solution based on H-1 NMR chemical shift changes in the protein whereas the disaccharide binds at either the B and C sites or the C and D sites in different crystal complexes. The present study thus highlights that protein-ligand complexes may vary notably between the solution and solid states, underscoring the importance of targeting the pertinent binding site(s) for inhibition of protein activity and the advantages of combining different techniques in a screening process.

National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-85915 (URN)10.1039/c2ob07066a (DOI)000301958100017 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2013-01-10 Created: 2013-01-10 Last updated: 2017-12-06Bibliographically approved
2. SEAL by NMR: Glyco-Based Selenium-Labeled Affinity Ligands Detected by NMR Spectroscopy
Open this publication in new window or tab >>SEAL by NMR: Glyco-Based Selenium-Labeled Affinity Ligands Detected by NMR Spectroscopy
2014 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 20, no 43, 13905-13908 p.Article in journal (Refereed) Published
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.

Keyword
Se-77 NMR spectroscopy, lectins, protein-ligand interactions, SEAL, selenoglycosides
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-109971 (URN)10.1002/chem.201404933 (DOI)000343800700012 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

AuthorCount:3;

Available from: 2014-12-04 Created: 2014-12-02 Last updated: 2017-12-05Bibliographically approved
3. Recognition of Ganglioside Receptors by Botulinum Neurotoxin A
Open this publication in new window or tab >>Recognition of Ganglioside Receptors by Botulinum Neurotoxin A
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(English)Manuscript (preprint) (Other academic)
National Category
Chemical Sciences
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-112342 (URN)
Available from: 2015-01-12 Created: 2015-01-12 Last updated: 2016-01-29Bibliographically approved
4. 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

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