Quantitative High-Field NMR- and Mass Spectrometry-Based Fatty Acid Sequencing Reveals Internal Structure in Ru-Catalyzed Deuteration of Docosahexaenoic Acid

Ru-based catalysis results in highly unsaturated fatty
acid (HUFA)
ethyl esters (EE) deuterated to various extents. The products carry 2H (D) mainly at their bis-allylic positions,
where they are resistant to autoxidation compared to natural HUFA
and are promising as neurological and retinal drugs. We characterized
the extent of deuteration at each allylic position of docosa-4,7,10,13,16,19-hexaenoic
acid deuterated to completion at bis-allylic and
allylic positions (D-DHA) by two-dimensional (2D) and high-field (600
and 950 MHz) NMR. In separate experiments, the kinetics of docosahexaenoic
acid (DHA) EE deuteration was evaluated using Paternò–Büchi
(PB) reaction tandem mass spectrometry (MS/MS) analysis, enabling
deuteration to be quantitatively characterized for isotopologues (D0–D14
DHA) at each internal allylic position. NMR analysis shows that the
net deuteration of the isotopologue mixture is about 94% at the bis-allylic positions, and less than 1% remained as the
protiated −CH2–. MS analysis shows that deuteration
kinetics follow an increasing curve at bis-allylic
positions with higher rate for internal bis-allylic
positions. Percent D of bis-allylic positions increases
linearly from D1 to D9 in which all internal bis-allylic
positions (C9, C12, C15) deuterate uniformly and more rapidly than
external bis-allylic positions (C6, C18). The mono-allylic positions near the methyl end (C21) show a
steep increase of D only after the D10 isotopologue has been deuterated
to >90%, while the mono-allylic position near
the
carboxyl position, C3, deuterates last and least. These data establish
detailed methods for the characterization of Ru-catalyzed deuteration
of HUFA as well as the phenomenological reaction kinetics as net product
is formed.