Mass spectrometry-directed structure elucidation and total synthesis of ultra-long chain (O-acyl)-ω-hydroxy fatty acids

The (O-acyl)-ω-hydroxy fatty acids (OAHFA) are an unusual lipid subclass present in the skin, vernix caseosa and meibomian gland secretions. While they are structurally related to the general class of fatty acid esters of hydroxy fatty acids (FAHFA), the ultra-long chain (30-34 carbons) and putative ω-substitution of the backbone hydroxy fatty acid suggest OAHFA have unique biochemistry. Complete structural elucidation of OAHFA has been challenging due to their low abundance within complex lipid matrices. Furthermore, the occurrence of these compounds as a mixture of closely related isomers has meant that no spectroscopic data have been obtained to guide structure confirmation by total synthesis. Exploiting the gas-phase purification of lipids through multi-stage mass spectrometry and novel, multi-dimensional ion activation methods, we describe the full molecular structure of ultra-long chain OAHFAs extracted from human meibum; assigning sites of unsaturation, stereochemical configuration of carbon-carbon double bonds, and ester linkage regiochemistry. Such isomer-resolved mass spectrometry has guided the first total synthesis of (O-acyl)-ω-hydroxy fatty acids which, in turn, confirm the structure of the most abundant OAHFA found in human meibum, OAHFA 50:2. The availability of a synthetic OAHFA opens new territory for future investigations into the unique biophysical and biochemical properties of these lipids.