%0 Journal Article %1 fink2021azidosphinganine %A Fink, Julian %A Schumacher, Fabian %A Schlegel, Jan %A Stenzel, Philipp %A Wigger, Dominik %A Sauer, Markus %A Kleuser, Burkhard %A Seibel, Jürgen %D 2021 %I The Royal Society of Chemistry %J Org. Biomol. Chem. %K sauer %N 10 %P 2203--2212 %R 10.1039/D0OB02592E %T Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis %U http://dx.doi.org/10.1039/D0OB02592E %V 19 %X Here were report the combination of biocompatible click chemistry of ω-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 ω-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, ω-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.

@article{fink2021azidosphinganine, abstract = {Here were report the combination of biocompatible click chemistry of ω-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 ω-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, ω-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.}, added-at = {2021-04-12T10:14:36.000+0200}, author = {Fink, Julian and Schumacher, Fabian and Schlegel, Jan and Stenzel, Philipp and Wigger, Dominik and Sauer, Markus and Kleuser, Burkhard and Seibel, Jürgen}, biburl = {https://www.bibsonomy.org/bibtex/283e8360ae66e73a63119f441d0092a8a/reichert}, doi = {10.1039/D0OB02592E}, interhash = {dae3d46a9de40db06051f0307ae0a423}, intrahash = {83e8360ae66e73a63119f441d0092a8a}, issn = {14770520}, journal = {Org. Biomol. Chem.}, keywords = {sauer}, number = 10, pages = {2203--2212}, publisher = {The Royal Society of Chemistry}, timestamp = {2021-04-12T10:14:36.000+0200}, title = {Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis}, url = {http://dx.doi.org/10.1039/D0OB02592E}, volume = 19, year = 2021 }