ω-Azido fatty acids as probes to detect fatty acid biosynthesis, degradation, and modification

Abstract

while decreasing lipophilicity, which in turn increases signal resolution on reversed phase HPLC systems ( 7 ).The method we developed for HPLC/MS-based detection of FA intermediates involves three steps : i ) simple preparation of FAs with a terminal azido group (AFAs) that allows most FA modifi cations to occur; ii ) use of these AFAs as metabolic probes and labeling of the in vivo formed derivatives in the corresponding organism with tetramethoxydibenzoazacyclooctyne (TDAC) ( 1 , Fig. 1A ), a cyclooctyne synthesized originally by Starke, Walther, and Pietzsch ( 8 ,9 ); and iii ) the detection of the clicked compounds by HPLC/MS. Although usually a reporter function such as a fl uorophore is linked to the alkyne reagent, no such modifi cations were needed here, because the formation of an electron-rich triazole ring make it quite susceptible to protonation and thus detection by MS, especially as the molecular mass range of the clicked FAs differs strongly from other lipophilic compounds found in most cells. AFAs are bioorthogonal, do not react with other functional groups other than alkynes, are easily taken up by the cells like other FAs when externally added, and can be used to follow the fate of AFAs in a given organism in real time. Thus AFAs will add to the overall toolbox of lipid analysis in general. Similarily, alkyne-modifi ed cholesterol has been used recently to trace cellular cholesterol metabolism and localization ( 10 ). MATERIALS AND METHODS General experimental proceduresSolvents and reagents were obtained from Sigma-Aldrich (München, Germany). TDAC was synthesized using the procedure described by Starke, Walther, and Pietzsch ( 9 ) FAs are found in all known living organisms, playing a vital role in cell compartmentalization, energy storage, and secondary metabolite production. In bacteria, most FAs are found in the cell membrane as part of the lipid bilayer ( 1 ). The actual FA profi le of a cell can strongly vary depending on environmental and developmental conditions requiring de novo biosynthesis, degradation, and modifi cation of the FAs involved ( 2-5 ). The monitoring of these metabolic pathways is usually conducted by GC-MS of FA methyl esters or other volatile FA derivatives ( 6 ). In spite of its merits, such as high sensibility and direct observability of especially volatile natural products, it is desirable to also have simple and effective methods of FA analysis via HPLC/MS, which today is a widespread tool in analytical chemistry as well. However, HPLC/MS-based FA analysis is diffi cult, mainly because of little ionizability and hence little signal strength. Small modifi cations introduced to certain FAs, however, can greatly increase ionizability

Solid‐Phase Enrichment and Analysis of Azide‐Labeled Natural Products: Fishing Downstream of Biochemical Pathways

“…The use of a bioorthogonal label offers high selectivity in the process, as opposed to enrichment methods that target biologically ubiquitous functional groups such as amines and thiols 36. As was previously shown for FA analysis,10,13,37 derivatized azides show an excellent propensity for protonation. This makes LC/MS analysis especially simple, since otherwise neutral or negatively charged compounds become readily discernible within a single analytical run (Figure S14).…”

Interrogating Plant-Microbe Interactions with Chemical Tools: Click Chemistry Reagents for Metabolic Labeling and Activity-Based Probes

“…Metabolic labeling of plants and microbes using bioorthogonal click chemistry reagents has led to the development of a broad range of tools, including synthetic analogs of monolignols [ 20 , 21 , 22 , 23 ], glycans [ 24 , 25 , 26 ], amino acids [ 27 , 28 , 29 ], and fatty acids [ 30 , 31 , 32 ] for investigating the production or modification of various biomolecular species. These bioorthogonal reagents have been designed to mimic the structure of native compounds and contain an alkyne or azide functionality for downstream attachment of reporter tags using click chemistry ( Figure 2 ).…”

Synthesis and Characterization of Fluorescent Probes for the Development of a Ceramide Synthase FRET-Based Assay

“…Even though many azido-functionalized lipids have already been described in the literature, [26][27][28][29][30][31] it is still unclear how the azido group emulates a carbon chain, and authors often differ in whether the nitrogen atoms of the azide should or should not be counted when designing an ω-azido fatty acid analogue of a particular chain length. Therefore, both RBM5-065 and RBM5-068 were synthesized as potential palmitic acid surrogates, as shown in Scheme 5.…”