Synthesis-driven reverse metabolomics reveals 3-hydroxy N-acyl amides as gut microbial molecules.

UNLABELLED: 3-Hydroxy N-acyl amides are bioactive lipids with reported anti-obesity and glucose-regulating effects, yet they are rarely detected in untargeted metabolomics studies because they are largely absent from existing spectral reference libraries. To address this gap, we synthesized an MS/MS spectral resource comprising 436 structurally diverse 3-hydroxy N-acyl amides, spanning 3- to 18-carbon chains with a wide range of amine headgroups, such as ornithine, valine, and dopamine. Using a synthesis-driven reverse metabolomics approach, we found 161,626 spectral matches across 54,744 publicly available files in untargeted metabolomics data sets, revealing widespread occurrences in biological samples, including human-derived specimens. Of these molecules detected through MS/MS spectral matching, 334 represent newly reported biological entities. We further confirmed their presence in human saliva, stool, and skin using retention time and ion mobility measurements. Frequent detection in microbial data sets and validation in communities of human-derived gut bacteria support microbial production. Several metabolites also showed altered abundance in individuals with diabetes mellitus, showing that this lipid class is modulated in human metabolic disease. Together, these findings establish 3-hydroxy N-acyl amides as a distinct and biologically relevant lipid class, and the accompanying MS/MS spectral resource will enable their broader recognition and study in untargeted metabolomics data. IMPORTANCE: Microbe-host interactions lead to the production of metabolites that play an important role in human health and disease, though the annotation of such metabolites remains scarce. We created a re-usable MS/MS spectral library of an underrepresented class of compounds-3-hydroxy N-acyl amides. Our study characterized the presence of over 400 3-hydroxy N-acyl amides among thousands of public untargeted metabolomics datasets. We found over 100,000 instances of these metabolites within microbial, plant, and animal data, and used orthogonal validation methods to confirm nine previously unreported molecules within human feces, saliva, and skin samples. We cultured communities of human-derived gut bacteria and found evidence of bacterial production of four 3-hydroxy N-acyl amides. Our results suggest that these metabolites play important roles in human metabolism and disease, and sharing this curated resource enables the detection of these metabolites in future studies.