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Glycoconjugates: Solid-phase synthesis and biological applications
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glycoconjugates are biologically important molecules with diverse functions. They consist of carbohydrates of varying size and complexity, attached to a non-sugar moiety as a lipid or a protein. Glycoconjugate structures are often very complex and their intricate biosynthetic pathways makes overexpression difficult. This renders the isolation of pure, structurally defined compounds from natural sources cumbersome. Therefore, to better address questions in glycobiology, synthetic glycoconjugates are an appealing alternative. In addition, synthetic methods allow for the preparation of non-natural glycoconjugates that can enhance the understanding of the influence of structural features on the biological responses.

In this thesis, synthetic methods for the preparation of glycoconjugates, especially glycolipid analogues, have been developed. These methods make use of solid-phase chemistry and are amenable to library synthesis of series of similar compounds. Solid-phase synthesis is a technique where the starting material of the reaction is attached to small plastic beads through a linker. This allows large excess of reagents to speed up the reactions and the sometimes difficult purifications of intermediate products are reduced to simple washings of the beads.

One problem with solid-phase synthesis is the difficulties to monitor the reactions and characterize the intermediate products. Gel-phase 19 F-NMR spectroscopy, using fluorinated linkers and protecting groups, is an excellent tool to overcome this problem and to monitor solid-phase synthesis of e.g. glycoconjugates. Two novel fluorinated linkers for the attachment of carboxylic acids have been developed and are presented in the thesis. These linkers can be cleaved with both acids of varying strengths and nucleophiles like hydroxide ions, and they are stable to glycosylation conditions. In addition, a novel filter reactor for solid-phase synthesis was designed. The reactor fits into an ordinary NMR spectrometer to facilitate the reaction monitoring with gel-phase 19 F-NMR spectroscopy.

The biological applications of the synthesized glycolipids were demonstrated in two different settings. The CD1d restricted binding of glycolipids carrying the monosaccharide α-GalNAc as carbohydrate could be detected on viable cells of mouse origin. CD1d is one of several antigen presenting molecules (the CD1 proteins) that presents lipids and glycolipids to circulating T-cells that in turn can initiate an immune response. The CD1 molecules are relatively sparsely investigated, and the method to measure glycolipid binding on viable cells, as described in the thesis, has the possibility to greatly enhance the knowledge of the structural requirements for CD1-binding.

Serine-based neoglycolipids with terminal carboxylic acids were used to prepare glycoconjugate arrays with covalent bonds to secondary amines on microtiter plates. Carbohydrate arrays have great possibilities to simplify the study of interactions between carbohydrates and e.g. proteins and microbes. The usefulness of the glycolipid arrays constructed in the thesis was illustrated with two lectins, RCA120 from Ricinus communis and BS-1 from Bandeiraea simplicifolia. Both lectins bound to the array of neoglycolipids in agreement with their respective specificity for galactosides.

Glycobiology is a large area of great interest and the methods described in this thesis can be used to answer a variety of glycoconjugaterelated biological questions.

Place, publisher, year, edition, pages
Umeå: Kemi , 2005. , 68 p.
Keyword [en]
Glycoconjugate, Glycolipid, Solid-phase synthesis, Glycosylation, Gel-phase 19F-NMR spectroscopy, Fluorinated linker, Carbohydrat array, CD1
National Category
Organic Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-630ISBN: 91-7305-984-6 (print)OAI: oai:DiVA.org:umu-630DiVA: diva2:144063
Public defence
2005-12-10, 10:00
Supervisors
Available from: 2005-11-16 Created: 2005-11-16 Last updated: 2011-03-10Bibliographically approved
List of papers
1. Loading of the antigen-presenting protein CD1d with synthetic glycolipids
Open this publication in new window or tab >>Loading of the antigen-presenting protein CD1d with synthetic glycolipids
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2004 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 5, no 4, 437-444 p.Article in journal (Refereed) Published
Abstract [en]

CD1 proteins present mammalian and microbial lipid and glycolipid antigens to different subsets of T cells. Few such antigens have been identified and the binding of these to CD1 molecules has mainly been studied by using responding T cells in cellular assays or recombinant solid-phase CD1 proteins. In the present study we use four different glycolipids, some of which contain tumor-associated carbohydrate antigens, to develop a procedure to easily detect binding of glycolipids to CD1 proteins on viable cells. Two of these glycolipids are novel glycoconjugates containing -D-N-acetylgalactosamine (-GalNAc) that were prepared by a combined solution and solid-phase approach. The key step, a Fischer glycosylation of 9-fluorenylmethoxycarbonylaminoethanol with GalNAc, furnished the -glycoside 4 in 34 % yield. Cells were incubated with glycolipids and stained with monoclonal antibodies specific for the carbohydrate part. The level of glycolipid bound to cells was then determined by flow cytometry with a secondary antibody labeled with fluorescein isothiocyanate. All four glycolipids were found to bind to CD1d but with different selectivity. The loading was dose dependent and could be inhibited by an established CD1d ligand, -galactosylceramide. Through use of this procedure, glycolipids were selectively loaded onto CD1d expressed on professional antigen-presenting cells for future use as cellular vaccines. Moreover, the glycolipids described in this study represent novel CD1d-binding ligands that will be useful derivatives in the study of CD1d-dependent immune responses, for example, against tumors.

Place, publisher, year, edition, pages
Weinheim: Wiley-VCH, 2004
Keyword
antigens, glycolipids, glycosylation, immunochemistry, proteins
Identifiers
urn:nbn:se:umu:diva-14087 (URN)doi:10.1002/cbic.200300655 (DOI)
Available from: 2007-05-22 Created: 2007-05-22 Last updated: 2017-12-14Bibliographically approved
2. Solid-phase synthesis of serine-based glycosphingolipid analogues for preparation of glycoconjugate arrays
Open this publication in new window or tab >>Solid-phase synthesis of serine-based glycosphingolipid analogues for preparation of glycoconjugate arrays
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2005 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 3, no 2, 309-315 p.Article in journal (Refereed) Published
Abstract [en]

Synthetic glycolipids with defined structures are important tools in the study of glycolipid biology. In this paper we describe a solid-phase synthesis of three galactosylated serine-based glycosphingolipid analogues using the novel linker 2-fluoro-4-(hydroxymethyl)-phenoxyacetic acid. Gel-phase 19F-NMR spectroscopy was used to measure the yield and stereochemical outcome of the solid-phase glycosylations. Under NIS–TfOH promotion, α- and β-selective glycosylations were performed at room temperature with thioglycoside donors carrying fluorine labelled protective groups. Finally, the glycolipids were covalently linked to microtiter plates and labelled lectins with different selectivity for α- and β-galactosides could bind to the glycolipid arrays.

Place, publisher, year, edition, pages
London: Royal Society of Chemistry, 2005
Identifiers
urn:nbn:se:umu:diva-4815 (URN)10.1039/b415436c (DOI)
Available from: 2005-11-16 Created: 2005-11-16 Last updated: 2017-12-14Bibliographically approved
3. A Novel, Acid-Labile, Fluorinated Linker for Solid-Phase Organic Chemistry
Open this publication in new window or tab >>A Novel, Acid-Labile, Fluorinated Linker for Solid-Phase Organic Chemistry
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4816 (URN)
Available from: 2005-11-16 Created: 2005-11-16 Last updated: 2010-01-13Bibliographically approved
4. An NMR Tube Filter Reactor for Solid-Phase Synthesis and Gel-Phase 19F-NMR Spectroscopy
Open this publication in new window or tab >>An NMR Tube Filter Reactor for Solid-Phase Synthesis and Gel-Phase 19F-NMR Spectroscopy
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4817 (URN)
Available from: 2005-11-16 Created: 2005-11-16 Last updated: 2010-01-13Bibliographically approved

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