De novo structure determination of 3-((3-aminopropyl)amino)-4-hydroxybenzoic acid, a novel and abundant metabolite in Acinetobacter baylyi ADP1

IntroductionMetabolite identification remains a major bottleneck in the understanding of metabolism. Many metabolomics studies end up with unknown compounds, leaving a landscape of metabolites and metabolic pathways to be unraveled. Therefore, identifying novel compounds within a metabolome is an entry point into the dark side' of metabolism.ObjectivesThis work aimed at elucidating the structure of a novel metabolite that was first detected in the soil bacterium Acinetobacter baylyi ADP1 (ADP1).MethodsWe used high resolution multi-stage tandem mass spectrometry for characterizing the metabolite within the metabolome. We purified the molecule for 1D- and 2D-NMR (H-1, C-13, H-1-H-1-COSY, H-1-C-13-HSQC, H-1-C-13-HMBC and H-1-N-15-HMBC) analyses. Synthetic standards were chemically prepared from MS and NMR data interpretation.ResultsWe determined the de novo structure of a previously unreported metabolite: 3-((3-aminopropyl)amino)-4-hydroxybenzoic acid. The proposed structure was validated by comparison to a synthetic standard. With a concentration in the millimolar range, this compound appears as a major metabolite in ADP1, which we anticipate to participate to an unsuspected metabolic pathway. This novel metabolite was also detected in another -proteobacterium.ConclusionStructure elucidation of this abundant and novel metabolite in ADP1 urges to decipher its biosynthetic pathway and cellular function.