ω-Alkynyl Lipid Surrogates for Polyunsaturated Fatty Acids: Free Radical and Enzymatic Oxidations

Abstract

Lipid and lipid metabolite profiling are important parameters in understanding the pathogenesis of many diseases. Alkynylated polyunsaturated fatty acids are potentially useful probes for tracking the fate of fatty acid metabolites. The nonenzymatic and enzymatic oxidations of ω-alkynyl linoleic acid and ω-alkynyl arachidonic acid were compared to that of linoleic and arachidonic acid. There was no detectable difference in the primary products of nonenzymatic oxidation, which comprised cis,trans-hydroxy fatty acids. Similar hydroxy fatty acid products were formed when ω-alkynyl linoleic acid and ω-alkynyl arachidonic acid were reacted with lipoxygenase enzymes that introduce oxygen at different positions in the carbon chains. The rates of oxidation of ω-alkynylated fatty acids were reduced compared to those of the natural fatty acids. Cyclooxygenase-1 and -2 did not oxidize alkynyl linoleic but efficiently oxidized alkynyl arachidonic acid. The products were identified as alkynyl 11-hydroxy-eicosatetraenoic acid, alkynyl 11-hydroxy-8,9-epoxy-eicosatrienoic acid, and alkynyl prostaglandins. This deviation from the metabolic profile of arachidonic acid may limit the utility of alkynyl arachidonic acid in the tracking of cyclooxygenase-based lipid oxidation. The formation of alkynyl 11-hydroxy-8,9-epoxy-eicosatrienoic acid compared to alkynyl prostaglandins suggests that the ω-alkyne group causes a conformational change in the fatty acid bound to the enzyme, which reduces the efficiency of cyclization of dioxalanyl intermediates to endoperoxide intermediates. Overall, ω-alkynyl linoleic acid and ω-alkynyl arachidonic acid appear to be metabolically competent surrogates for tracking the fate of polyunsaturated fatty acids when looking at models involving autoxidation and oxidation by lipoxygenases.

On the cellular metabolism of the click chemistry probe 19-alkyne arachidonic acid

“…consistent with a previous report comparing the kinetics of AA and AA-alk transformation by crude human 12-LOX preparations where the transformation of AA was slightly more efficient than that of AA-alk (18). Similarly, purified murine COXs were reported to efficiently oxidize AA-alk but poorly catalyzed the cyclization of dioxalanyl intermediates to endoperoxides (18), which is consistent with the very limited biosynthesis of HHTrE-alk by stimulated platelets.…”

“…Importantly, corresponding ions with m/z 313, 295, 269, 251, and 199 were observed in the MS/MS product ion spectra of LTB 4 -alk that are analogous to fragments generated from LTB 4 but with m/z values shifted by 4 units, consistent with ions containing an omega-terminal alkyne (supplemental Figure S6B). Additionally, the peak identified as 5-HETE-alk showed the expected parent ion and fragmentation pattern that was previously reported for 5-LOX-catalyzed 5-HETE-alk synthesis (supplemental Figure S7) (18).…”

“…Similarly, purified murine COXs were reported to efficiently oxidize AA-alk but poorly catalyzed the cyclization of dioxalanyl intermediates to endoperoxides (18), which is consistent with the very limited biosynthesis of HHTrE-alk by stimulated platelets. However, the murine COX-catalyzed production of 11-HETE-alk that resulted from the poor cyclization was not detected in stimulated human platelets.…”

“…In cells incubated with AA-alk, a peak corresponding to AA-alk was observed on chromatograms, as well as second peak eluting dehydration/decarboxylation products with m/z of 297 and 253, respectively, were present, as were the expected fragmentation ions of a 12-hydroxylated product with m/z of 179 and 208 (supplemental Figure S3B) (18,42). Importantly, the expected fragmentation ion of 11-HETE-alk with an m/z of 167 (18,42) was absent. Platelets stimulated in the presence of exogenous AA-alk also produced 12-HETE and HHTrE generated from endogenous AA.…”

A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins

“…The tag has been synthetically introduced at the omega-terminal position of fatty acyl chains (23,25), in sphingoid bases (24), as part of phospholipid headgroup precursors (16), within sterol side chains (18,26,27), or at a cholesterol methyl group (19). Given its size and properties, the alkyne group does not alter the polarity and only minimally changes the structure and hence biochemical characteristics of the lipid (24,28).…”