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Synthesis and analysis of conformationally restricted ceramide analogs
(Synthese und Untersuchung konformativ fixierter Ceramidanaloga)

dc.contributor.advisorKolter, Thomas
dc.contributor.authorWirtz, Jennifer

Ceramide (e.g. (2S,3R,4E)-2-Octadecanoylamino-octadec-4-en-1,3-diol) is a structural component of membrane glycosphingolipids and sphingomyelin, and occurs in free form as well as bound to proteins in the human skin (Kolter and Sandhoff, 1999). Certain conformationally rigid analogs of this lipid, such as 5b and 24, showed an unexpected interference with the biosynthesis of complex glycosphingolipids such as that of ganglioside GM2 (GalNAcβ1,4-(NeuAcα2,3)Galβ1,4Glcβ1Cer; Figure 1.2).
To get insight into the molecular mechanism of this apparent inhibition, a series of experiments have been carried out in this work:
1) New structural analogs of the halogenated oxazinanone (5a) and oxazolidinone (5b) have been prepared, and analysed in cultured cells.
1a) The preparative route that gave access to these compounds was used for the preparation of the brominated compounds 7.2a and 7.2b. At least in cultured fibroblasts, the biosynthetic incorporation of 3-[14C]L-serine into cellular glycosphingolipids in the presence of 5b and 7.2b were similar.
1b) To further address the role of the halogen atom, oxazinanones bearing a hydroxyl group instead of a halogen (18a,b) were prepared from phytosphingosine. As has been shown before, these substances were not accessible on the route used otherwise due to a competing rearrangement reaction. 18a and 18b showed entirely different lipid labelling patterns, when glycosphingolipid biosynthesis is analysed in cultured neurons. These data indicate a vital role of the halogen atom for the observed effects.
1c) Other structural analogs of 5a and 5b were prepared and analyzed: Title compounds bearing an additional methyl group in the head group were prepared from L-threonine. 23.2a behaved similar to 5a when analysed in cultured fibroblasts, indicating that the substituent does not hinder the molecular interaction leading to the observed effects.
2) An appropriate way to investigate metabolism of 5a or 5b, was well as to address the question, if these substances are covalently bound to certain cellular proteins, is the incorporation of a suitable radioisotope into these substances. In model experiments, conditions for two reactions were found, under which incorporation of a tritium atom into newly synthesized suitable precursor substances should be possible, at least in low yields.
3) Oxazinanone 12 with an azide group within the alkyl chain was prepared. This substance should allow the identification of potential metabolites via Staudinger ligation, together with the identification of putative protein targets after affinity purification.
4) Initially, 5a and 5b, as well as potential glycosidated metabolites, have been investigated on their effect of ganglioside GM2 synthase, a membrane bound N-acetylgalactosaminyltransferase of the Golgi-apparatus. Baculovirus-infected insect cells overexpressing this enzyme were used as enzyme-source to investigate 5b in a liposomal assay system, since alteration of membrane properties caused by the compound has to be excluded as reason for the observed effects. Due to the instability of the enzyme in the proteoliposome preparation, no conditions could be found to analyse this possibility. Also a micellar GalNAc transferase assay in the presence of lipid extracts derived from fibroblast cells incubated with 5b showed no inhibition.
5) Metabolites of 5b were searched for by thin layer chromatography and mass spectrometry. For this purpose, fragmentation pattern of 5a, 5b, and a glucosylated potential metabolite were recorded and used for parent ion- and neutral loss-scans in the lipid extract of cultured fibroblasts. No metabolites could be found by this method. For glycolipid quantification and analysis of molecular species distribution in the lipid extract of cells by electrospray (ESI) and atmospheric pressure chemical ionisation (APCI) mass spectrometry, a ganglioside GM3 and a sulfatide derivative were prepared as glycolipid standards with appropriate alkyl chain lengths for further investigation.
6) It could be demonstrated by fluorescence microscopy, that the Golgi apparatus is fragmented in response to 5b in cultured human fibroblasts. Although it cannot be distinguished at the moment, if this is cause or effect of altered sphingolipid metabolism induced by 5b, this observation points to a protein target of 5a and 5b that is required for vesicular transport through the Golgi apparatus.
7) To investigate the role of the benzene substituent in another conformationally rigid ceramid analogue, the benzazepinone 24, phenylacetylsphingosine was prepared as an open-chain analog and analyzed in cultured murine granule cells and human fibroblasts. Since the ganglioside patterns in the presence of both compounds are similar, glycosyltransferase inhibition by this substance is not caused by conformational restriction, but by the presence of the benzene moiety. Analysis of the substances in fibroblasts indicated a higher toxicity of the open chain compound, which is in agreement with the current view on cytotoxicity of cell-permeable ceramide analogs.
8.) In chapter 4.9, a direct influence of 5b and 24 on protein kinase C as another potential mediator of the indirect effect on glycosphingolipid metabolism has been ruled out experimentally.
Although the molecular target of 5a,b could not been identified, novel tools to address this question have been prepared in this work, several possibilities have been ruled out, and the integrity of the Golgi apparatus has been shown to be affected by these substances.

dc.rightsIn Copyright
dc.subjectceramide analogs
dc.subjectconformational restriction
dc.subjectganglioside biosynthesis
dc.subjectGolgi apparatus
dc.subject.ddc500 Naturwissenschaften
dc.subject.ddc540 Chemie
dc.subject.ddc570 Biowissenschaften, Biologie
dc.titleSynthesis and analysis of conformationally restricted ceramide analogs
dc.title.alternative(Synthese und Untersuchung konformativ fixierter Ceramidanaloga)
dc.typeDissertation oder Habilitation
dc.publisher.nameUniversitäts- und Landesbibliothek Bonn
ulbbnediss.affiliation.nameRheinische Friedrich-Wilhelms-Universität Bonn
ulbbnediss.fakultaetMathematisch-Naturwissenschaftliche Fakultät
dc.contributor.coRefereeSandhoff, Konrad

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