Naturally occurring phosphonates such as phosphinothricin (Glufosinate a commercially used herbicide) and fosfomycin (Monurol a clinically used antibiotic) have proved to be potent and useful biocides. method for the selective detection of natural products containing phosphonate and phosphinate functional groups. We have used this method to identify a new phosphonate metabolite phosacetamycin whose PFI-2 structure biological activity and biosynthetic gene cluster are reported. gene (1). The commonality of to many phosphonate biosynthetic pathways offers a way to prescreen organisms for the genetic capacity to produce phosphonates but so far a robust and sensitive method for screening cultures for the presence and identity of phosphonate compounds has PFI-2 not been developed. Phosphorus nuclear magnetic resonance spectroscopy (31P NMR) is a robust and extremely selective technique that allows for detection of molecules with a C-P bond due to the characteristic chemical shift range (14) in complex matrices however the relatively low sensitivity throughput (15) and limited structural information 31P NMR provides prompted development of a complementary method that would address these limitations. The flexibility of liquid chromatography (LC) and the level of detailed information that can be obtained from complex samples using mass spectrometry and tandem mass spectrometry (MS and MS/MS) make LC-MS and LC-MS/MS attractive tools for the screening of microbial PFI-2 extracts for the presence of high value or high interest compounds; however detection and identification of small hydrophilic organic acids such as phosphinates phosphonates phosphorylated compounds and carboxylic acids presents an analytical challenge when employing this approach. The reason analysis of these types of compounds can be problematic using LC-MS is that they are found in matrices that have a high concentration of nonvolatile salts which are a major source of interference. Selective removal of these nonvolatile salts is required to make the sample suitable for mass spectrometric analysis and presents a challenge due to their high concentration in biological samples and co-migration with small organic acids through most chromatographic media. An additional challenge that arises when conducting the analysis of secondary metabolites such as phosphonates is that they are present in much lower concentrations than phosphorylated metabolites and phosphate salts. For example phosphate and phosphorylated metabolites can be in the range of 1-10 mM (16) whereas phosphonate metabolites such as fosfomycin are typically present PFI-2 in much lower concentrations (17). To solve this problem we developed a phosphonate enrichment protocol based on iron-immobilized metal affinity chromatography (IMAC) patterned after phosphopeptide enrichment strategies (18) that includes steps to reduce the background of contaminating phosphorylated compounds and phosphate salts. We PFI-2 couple this enrichment to RTKN hydrophilic interaction chromatography (HILIC) (19) for the separation and precursor ion scanning mass spectrometry for the selective detection of phosphonate metabolites. The detection method can be scaled up for preparative scale purification for full structure elucidation and biological activity determination. Application of the method enabled discovery of a novel phosphonate antibiotic that we designated phosacetamycin whose structure bioactivity and biosynthetic gene PFI-2 cluster is reported here. We also propose the biosynthetic pathway of phosacetamycin based upon sequencing of the biosynthetic gene cluster. RESULTS AND DISCUSSION The current lack of methods to quickly detect and identify phosphonate and phosphinate metabolites first prompted the development of a high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) based screening platform that would allow rapid evaluation of microbes whose genomes contain and therefore the genetic capacity for the production of phosphonate compounds (79 and 63 corresponding to the elimination of PO3? and PO2? respectively (20). Unlike phosphorylated compounds however we found phosphonates preferentially fragment to give the 63 ion (Figure 1). The differential fragmentation patterns of phosphonates relative to phosphates provides a potential way to discriminate between highly abundant phosphorylated compounds and phosphate from the cell extracts and culture media of microorganisms and phosphonate metabolites of interest. Unfortunately not all phosphate derivatives were observed to obey this trend; phosphoenoyl pyruvate (PEP) phosphoethanolamine (PEA).