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Antigens derived from the mucin MUC1: Solution and solid-phase synthesis of saccharides, peptides and glycopeptides
Umeå University, Faculty of Science and Technology, Chemistry.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mucin is a term used to describe a large family of heavily glycosylated proteins which are present on the surfaces of secretory epithelial cells and are overexpressed by many carcinomas. Membrane-bound mucin MUC1 is of special interest. Its backbone consists of repeating units of twenty amino acids with five potential glycosylation sites. These sites are expanded to structures like the T (Galβ(1->3)GalNA-Ser/Thr) and Tn (GalNA-Ser/Thr) antigens by the action of various glycosyltransferases. In different types of carcinomas these epitopes are being terminated by sialic acid residues to form among others: 2,3-sialyl-T and sialyl-Tn structures due to the elevated levels of different sialyltransferases.

Solid-phase synthesis of the selected antigens derived from the mucin MUC1 has been developed and optimized. A chemoenzymatic approach has been used to effectively prepare 2,3-sialyl-T and 2,6-sialyl-Tn glycopeptides. The formation of intramolecular sialic acid lactones in presence of acetic acid was investigated. The stability of lactones formed from 2,3-sialyl-T towards water was studied using NMR spectroscopy and it appeared that 1''->2' lactone displayed remarkable strength to hydrolysis and it was suggested as a candidate for cancer vaccine.

Gel-phase 19F NMR spectroscopy is known to be a very good tool to characterize resin-bound products using fluorinated protecting groups and linker molecules. The hydrophobic peptide LLLLTVLTV, which is a fragment from the MUC1 signal sequence, was prepared using solid-phase synthesis according to a modified Fmoc protocol with more active coupling reagent, stronger base, and the isopropylidene dipeptide Fmoc-Leu-Thr-(ΨMe,Mepro)-OH. Gel-phase 19F NMR spectroscopy was used to evaluate peptide chain aggregation and coupling and deprotection efficiency.

A carbamate linker strategy proved to be effective in solid-phase synthesis of serine-based neoglycolipids with terminal amino functionality. Neoglycolipids were covalently bound to secondary amines in microtiter plates using squaric acid ester methodology. These arrays have potential to study the interactions between carbohydrates and e.g. proteins and microbes.

The new fluorinated α-amino protective group [1-(4-(4-fluorophenyl)-2,6-dioxocyclohexylidene)ethyl] Fde was developed. This group is cleaved with hydrazine in DMF solution. By using amino acids protected with this group, it was possible to quantify the efficiency of peptide coupling using gel-phase 19F NMR spectroscopy.

Place, publisher, year, edition, pages
Umeå: Kemi , 2008. , 76 p.
Keyword [en]
Lactones, glycopeptides, signal peptide, neoglycolipid arrays, solid-phase synthesis, MUC1, gel-phase 19F NMR spectroscopy, fluorinated alpha-amino protective group
National Category
Organic Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-1630ISBN: 978-91-7264-557-8 (print)OAI: oai:DiVA.org:umu-1630DiVA: diva2:141611
Public defence
2008-05-24, KB3B1, Stora Hörsalen, KBC, Linneaus v., Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2010-01-21Bibliographically approved
List of papers
1. Formation of lactones from sialylated MUC1 glycopeptides
Open this publication in new window or tab >>Formation of lactones from sialylated MUC1 glycopeptides
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2006 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 4, no 4, 713-20 p.Article in journal (Refereed) Published
Abstract [en]

The tumor-associated carbohydrate antigens TN, T, sialyl TN and sialyl T are expressed on mucins in several epithelial cancers. This has stimulated studies directed towards development of glycopeptide-based anticancer vaccines. Formation of intramolecular lactones involving sialic acid residues and suitably positioned hydroxyl groups in neighboring saccharide moieties is known to occur for glycolipids such as gangliosides. It has been suggested that these lactones are more immunogenic and tumor-specific than their native counterparts and that they might find use as cancer vaccines. We have now investigated if lactonization also occurs for the sialyl TN and T antigens of mucins. It was found that the model compound sialyl T benzyl glycoside , and the glycopeptide Ala-Pro-Asp-Thr-Arg-Pro-Ala from the tandem repeat of the mucin MUC1, in which Thr stands for the 2,3-sialyl-T antigen, lactonized during treatment with glacial acetic acid. Compound gave the 1''--> 2' lactone as the major product and the corresponding 1''--> 4' lactone as the minor product. For glycopeptide the 1''--> 4' lactone constitued the major product, whereas the 1''--> 2' lactone was the minor one. When lactonized was dissolved in water the 1''--> 4' lactone underwent slow hydrolysis, whereas the 1''--> 2' remained stable even after a 30 days incubation. In contrast the corresponding 2,6-sialyl-TN glycopeptide did not lactonize in glacial acetic acid.

Keyword
Glycopeptides/*chemistry/metabolism, Hydrolysis, Lactones/*chemical synthesis/chemistry, Magnetic Resonance Spectroscopy, Molecular Structure, Mucins/*chemistry, Sialic Acids/*chemistry
Identifiers
urn:nbn:se:umu:diva-11794 (URN)10.1039/b514918e (DOI)16467946 (PubMedID)
Available from: 2007-08-28 Created: 2007-08-28 Last updated: 2017-12-14Bibliographically approved
2. Application of gel-phase 19F NMR spectroscopy for optimization of solid-phase synthesis of a hydrophobic peptide from the signal sequence of the mucin MUC1
Open this publication in new window or tab >>Application of gel-phase 19F NMR spectroscopy for optimization of solid-phase synthesis of a hydrophobic peptide from the signal sequence of the mucin MUC1
2007 (English)In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 13, no 5, 354-361 p.Article in journal (Refereed) Published
Abstract [en]

This paper describes the manual Fmoc/t-Bu solid-phase synthesis of a difficult nine-residue hydrophobic peptide LLLLTVLTV from one of the signal sequences that flank the tandem repeat of the mucin MUC1. Gel-phase 19F NMR spectroscopy was used as a straightforward method for optimization of the solid-phase synthesis. Different approaches were applied for comparative studies. The strategy based on modified solid-phase conditions using DIC/HOAt for coupling, DBU for Fmoc deprotection, and the incorporation of the pseudo proline dipeptide Fmoc-Leu-Thr(Me, Me pro)-OH as a backbone-protecting group was found to be superior according to gel-phase 19F NMR spectroscopy. Implementation of the optimized Fmoc protocol enabled an effective synthesis of signal peptide LLLLTVLTV.

Keyword
solid-phase peptide synthesis, difficult sequence, gel-phase 19F NMR, pseudo proline
Identifiers
urn:nbn:se:umu:diva-12721 (URN)doi:10.1002/psc.850 (DOI)
Available from: 2008-05-19 Created: 2008-05-19 Last updated: 2017-12-14Bibliographically approved
3. Arrays for investigations of carbohydrate protein interactions: Solid-phase synthesis of amino-functionalized glycoconjugates using a carbamate linker strategy
Open this publication in new window or tab >>Arrays for investigations of carbohydrate protein interactions: Solid-phase synthesis of amino-functionalized glycoconjugates using a carbamate linker strategy
(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Identifiers
urn:nbn:se:umu:diva-3131 (URN)
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2010-01-14Bibliographically approved
4. Synthesis and application of N-[1-(4-(4-fluorophenyl)-2,6-dioxocyclohexylidene)ethyl] (Fde)-protected amino acids for optimization of solid-phase peptide synthesis using gel-phase (19)F NMR spectroscopy.
Open this publication in new window or tab >>Synthesis and application of N-[1-(4-(4-fluorophenyl)-2,6-dioxocyclohexylidene)ethyl] (Fde)-protected amino acids for optimization of solid-phase peptide synthesis using gel-phase (19)F NMR spectroscopy.
2009 (English)In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 15, no 4, 264-71 p.Article in journal (Refereed) Published
Abstract [en]

N-[1-(4-(4-fluorophenyl)-2,6-dioxocyclohexylidene)ethyl] (Fde) protected amino acids have been prepared and applied in solid-phase peptide synthesis monitored by gel-phase (19)F NMR spectroscopy. The Fde protective group could be cleaved with 2% hydrazine or 5% hydroxylamine solution in DMF as determined with gel-phase (19)F NMR spectroscopy. The dipeptide Ac-L-Val-L-Val-NH(2) 12 was constructed using Fde-L-Val-OH and no noticeable racemization took place during the amino acid coupling with N,N'-diisopropylcarbodiimide and 1-hydroxy-7-azabenzotriazole or Fde deblocking. To extend the scope of Fde protection, the hydrophobic nonapeptide LLLLTVLTV from the signal sequence of mucin MUC1 was successfully prepared using Fde-L-Leu-OH at diagnostic positions. Copyright (c) 2009 European Peptide Society and John Wiley & Sons, Ltd.

Place, publisher, year, edition, pages
European Peptide Society and John Wiley & Sons, Ltd., 2009
Keyword
solid-phase peptide synthesis, gel-phase 19F NMR spectroscopy, fluorinated -amino protective group, difficult peptide
Identifiers
urn:nbn:se:umu:diva-18745 (URN)10.1002/psc.1110 (DOI)19235188 (PubMedID)
Available from: 2009-02-24 Created: 2009-02-24 Last updated: 2017-12-13

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