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

Abstract

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.

Paternò–Büchi Reaction Mass Spectrometry Enables Positional Assignment of Polymethylene-Interrupted Double Bonds in Food-Derived Lipids

“…For the various FAME analyzed, we also found that Δ5 F A at m / z 153 is much more abundant than Δ5 F O at m / z 242, suggesting a potential regioselective process during the PB adduction. Since 2-ACP can also add a proton to become charged, protonated precursors appear which could also be used to pinpoint the double bond positions. , However, the sodiated precursors are most intense in our hands and thus offer a more sensitive workflow for untargeted analyses. While [M + Na] + and [M + H] + ions do not generate significant isobars in our specific analyses, the addition of chromatography separation can prevent any isobar issues as well as provide clues as to double bond stereochemistry (Z/E).…”

“…Here, m / z 207 is not a companion ion to the strong ion m / z 209, which is a common phenomenon for other double bond characterization methods such as CACI-MS/MS and DMOX-electron ionization (EI)-MS. The observation that PB diagnostic ions appear as a single m / z value without any companion ion simplifies the detection of isomers with one or more overlapping double bond positions compared to other approaches; it was important for quantifying levels of intramolecular deuterium previously. , Therefore, m / z 207 is a fragment from sources other than 18:2­(9 Z ,12 Z ). Having a m / z value 2 unit smaller than Δ9 diagnostic ion from (18:2­(9 Z ,12 Z )), m / z 207 indicates a Δ9 double bond position in isomeric 18:2 with the other double bond proximal to the Δ9 double bond (where in (18:2­(9 Z ,12 Z ), the other double bond is terminal to the Δ9 double bond).…”

“…Paternò–Büchi (PB) reaction MS workflow was demonstrated to provide structural information for phospholipids, sphingomyelin, cholesteryl ester and fatty acids including conjugated linoleic acids (CLA) and isotopically labeled fatty acids. − The resolution of double-bond positional isomers has led to the discovery of biomarkers for various cancer tissues and diseases. ,− While promising, work specifically designed for the identification of unusual PMI-FA and glycerolipids containing PMI-FA by the PB workflow has yet to be carried out. In the current study, we tested the PB reaction MS workflow for de novo assignment of all the double bond positions in PMI-FA and triacylglycerol (TG) carrying PMI fatty acyl chain(s).…”

Fatty acid isomerism: analysis and selected biological functions

“…In this particular case, GC-CACI-MS/MS was not possible due to the presence of companion ions (±1 Da or ±2 Da) of the CACI diagnostic ions. 17,37 This special case of analysis of a unique pharmaceutical-like species illustrates the importance of detailed mass spectral interpretation and mechanism for new analytes.…”

DGet! An open source deuteration calculator for mass spectrometry data

“…Deuterated molecules can be synthesized using commercially available deuterated precursors (e.g., CD 3 I, CD 3 OD, DCO 2 D, CD 3 NH 2 , LiAlD 4 , NaBD 4 ) in standard chemistry techniques to produce site specific deuterated molecules [ 2 , 16 , 17 ]. Alternatively, deuterium can be incorporated using direct H/D exchange of protonated derivatives in D 2 O using metal catalysts [ 14 , 18 – 23 ], or by biosynthesis using biological organisms grown in D 2 O (Fig. 1 ) [ 13 , 24 – 26 ].…”