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.