Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis
- Here were report the combination of biocompatible click chemistry of omega-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that omega-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, omega-azidosphinganineHere were report the combination of biocompatible click chemistry of omega-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that omega-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, omega-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection.…
Verfasserangaben: | Julian FinkGND, Fabian SchumacherORCiDGND, Jan SchlegelORCiDGND, Philipp Stenzel, Dominik WiggerGND, Markus SauerORCiDGND, Burkhard KleuserORCiDGND, Jürgen SeibelORCiDGND |
---|---|
DOI: | https://doi.org/10.1039/d0ob02592e |
ISSN: | 1477-0520 |
ISSN: | 1477-0539 |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/33496698 |
Titel des übergeordneten Werks (Englisch): | Organic & biomolecular chemistry : an international journal of synthetic, physical and biomolecular organic chemistry |
Verlag: | Royal Society of Chemistry |
Verlagsort: | Cambridge |
Publikationstyp: | Wissenschaftlicher Artikel |
Sprache: | Englisch |
Datum der Erstveröffentlichung: | 20.01.2021 |
Erscheinungsjahr: | 2021 |
Datum der Freischaltung: | 01.03.2024 |
Band: | 19 |
Ausgabe: | 10 |
Seitenanzahl: | 11 |
Erste Seite: | 2203 |
Letzte Seite: | 2212 |
Fördernde Institution: | German Research Foundation (DFG)German Research Foundation (DFG) [RU 2123, RTG 2581] |
Organisationseinheiten: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Ernährungswissenschaft |
DDC-Klassifikation: | 5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften |
Peer Review: | Referiert |