Compounds with C-P bonds represent an understudied class of natural products with proven and attractiveproperties as drugs for humans or herbicides. With stable carbon-phosphorus bonds, phosphonates andphosphinates are useful scaffolds that mimic phosphates and display desirable pharmacological properties.This major class of bioactive compounds will be addressed by an intensely collaborative team at theUniversity of Illinois.The Kelleher Laboratory will leverage its core expertise in ultra-high performance Fourier-Transform MassSpectrometry (FTMS) to implement instrumentation and tailored software for phosphonate compounds andbiosynthetic intermediates in both targeted and discovery modes. For known phosphonates, both smallmolecule MS (metabolomics) and large molecule MS (FTMS-based assays), will be used to interrogatephosphonate intermediates both free and bound as thioesters to non-ribosomal peptide synthetases.Further, the compound K-26 will be linked to its biosynthetic gene cluster using a general fosmid-screeningapproach employing a highly automated FTMS-based screen. The enzymology underlying the biosynthesisof phosphinothricin tripeptide (PTT) will be elucidated. With emphasis on the PTT system, the KelleherLaboratory will use its extensive experience in non-ribosomal peptide synthesis to dissect the timing of Pmethylationand the role of a curious tandem thiolation domain in the biosynthetic assembly line. Armed withmechanistic understanding of the thiotemplate portion of this gene cluster, a series of phs mutants will bescreened using large molecule FTMS to engineer the NRPS portion of the PTT cluster from S.vidriochromogenes, with production of unnatural PTT analogues in a heterologous producer, S. lividans, tofollow.For both targeted analysis and discovery, the negative mass defect of phosphorus along with selectiveMS/MS detection approaches will require tailored software for 'phosphonate-directed' metabolomics to filterlarge datasets emanating from ion trap/Fourier-Transform hybrid mass spectrometers operating at 7, 12,and 14.5 Tesla. A particular strength of a MS approach to phosphonate discovery is that new compoundsare identified based on structural features and not modes of biosynthesis or spectrum of activity in abioassay. The FTMS approaches to engineered production and discovery of new phosphonatescomplements the many other strategies presented in this integrated P01.
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