Toward Quantitative Sequencing of Deuteration of Unsaturated Hydrocarbon Chains in Fatty Acids

No general method currently is available for the quantitative determination of deuterium (D) at C positions along a hydrocarbon chain. Bis-allylic deuterated highly unsaturated fatty acids (D-HUFA) are a novel class of drugs stabilized against H-abstraction-mediated oxidation by deuteration at the most labile positions. Ru-based catalytic deuteration overcomes the limited scale of bis-allylic D-HUFA production by total organic synthesis; however, it produces a complex mixture of bis-allylic D isotopologues and isotopomers, requiring detailed sequencing for characterization. We report here adaptation and application of the Paternó-Büchi (PB) reaction of 2-acetylpyridine to a series of D-HUFA with analysis by shotgun lipidomics to determine position-specific quantitative D abundances. Sodiated ^PBD-HUFA result in diagnostic ions of high abundance upon collision-induced dissociation (CID) activation, enabling sensitive differentiation and quantification of D fraction at each bis- and mono-allylic position for each isotopologue. Catalytically deuterated isotopologues D5-7 linolenic acid (D5-7 LnA), D6-8 arachidonic acid (D6-8 ARA), D7-9 eicosapentaenoic acid (D7-9 EPA), and D9-11 docosahexaenoic acid (D9-11 DHA) incorporate 80-98, 95-100, 81-100, and 83-100% D at their bis-allylic positions, respectively. D-HUFA isotopologues having D number greater than or equal to bis-allylic sites (e.g., D10-DHA or D11-DHA) deuterated >95% at bis-allylic positions, except for D-LnA. The mono-allylic position near the methyl end deuterates to a much greater extent than the mono-allylic position near the carboxyl end, and both positions deuterate only when bis-allylic D is near-saturated. This method enables rapid, accurate characterization of position and isotopomer-specific D composition and enables sequencing along the chain.