Next-Generation Paternò-Büchi Reagents for Lipid Analysis by Mass Spectrometry.

The Paternò-Büchi (PB) reaction is a photochemical reaction involving [2 + 2] cycloaddition between electronically excited carbonyl and carbon-carbon double bond (C═C). It has been established as a lipid derivatization strategy, leading to confident assignment of C═C locations in lipids when coupled with tandem mass spectrometry (MS/MS). Although acetone and several aryl-containing ketones or aldehydes have been explored as PB reagents, the chemical properties critical to achieving efficient conversion and minimum side reactions remain unclear. Herein, we investigated a set of acetophenone (AP) derivatives, aiming to provide insights into the development of new PB reagents with enhanced performance for lipid analysis. For AP derivatives, we found that electron-withdrawing groups (e.g., -F and -CF) on the benzene ring improved the overall conversion, while a bulky group at the position decreased the conversion. Norrish Type I cleavage was largely diminished; however, the Norrish Type II side reaction was more competitive, producing products isomeric to the PB reaction products. Among all AP derivatives tested, 2',4',6'-trifluoroacetophenone (triFAP) showed the best performance. It offered a relatively high PB yield (20-30%) for different types of C═C, high sensitivity (sub-nM) for C═C identification, and accurate isomer quantitation. Due to the significantly reduced chemical interferences in shotgun analysis, triFAP provided better performance than that from acetone PB-MS/MS. An offline triFAP PB reaction was implemented in a liquid chromatography analysis workflow, which enabled the large-scale identification of phospholipids including C═C location isomers from a complex lipid extract.