Compounds with C-P bonds represent an understudied class of natural products with proven and attractive properties as drugs for humans or herbicides. With stable carbon-phosphorus bonds, phosphonates and phosphinates 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 the University of Illinois. The Kelleher Laboratory will leverage its core expertise in ultra-high performance Fourier-Transform Mass Spectrometry (FTMS) to implement instrumentation and tailored software for phosphonate compounds and biosynthetic intermediates in both targeted and discovery modes. For known phosphonates, both small molecule MS (metabolomics) and large molecule MS (FTMS-based assays), will be used to interrogate phosphonate 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-screening approach employing a highly automated FTMS-based screen. The enzymology underlying the biosynthesis of phosphinothricin tripeptide (PTT) will be elucidated. With emphasis on the PTT system, the Kelleher Laboratory will use its extensive experience in non-ribosomal peptide synthesis to dissect the timing of Pmethylation and the role of a curious tandem thiolation domain in the biosynthetic assembly line. Armed with mechanistic understanding of the thiotemplate portion of this gene cluster, a series of phs mutants will be screened 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, to follow. For both targeted analysis and discovery, the negative mass defect of phosphorus along with selective MS/MS detection approaches will require tailored software for """"""""phosphonate-directed"""""""" metabolomics to filter large 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 compounds are identified based on structural features and not modes of biosynthesis or spectrum of activity in a bioassay. The FTMS approaches to engineered production and discovery of new phosphonates complements the many other strategies presented in this integrated P01.
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