Natural products containing phosphorus-carbon bonds have found widespread use in medicine and agriculture1. One such compound, phosphinothricin tripeptide (PTT), contains the unusual amino acid phosphinothricin (PT) attached to two alanine residues (Fig. 1). Synthetic PT (glufosinate) is a component of two top-selling herbicides (Basta® and Liberty®), and is widely used with resistant transgenic crops including corn, cotton and canola. Recent genetic and biochemical studies showed that during PTT biosynthesis 2-hydroxyethylphosphonate (HEP) is converted to hydroxymethylphosphonate (HMP) (Fig. 1)2. Reported here are the in vitro reconstitution of this unprecedented C(sp3)-C(sp3) bond cleavage reaction and X-ray crystal structures of the enzyme. The protein is a mononuclear non-heme iron(II)-dependent dioxygenase that converts HEP to HMP and formate. In contrast to most other members of this family, the oxidative consumption of HEP does not require additional cofactors or the input of exogenous electrons. The current study expands the scope of reactions catalyzed by the 2-His-1-carboxylate mononuclear non-heme iron family of enzymes.

Phosphinothricin-tripeptide (PTT, phosphinothricyl-alanyl-alanine) is a natural product antibiotic and potent herbicide that is produced by Streptomyces hygroscopicus ATCC 21705 1 and Streptomyces viridochromogenes DSM 40736 2 . PTT has attracted widespread interest due to its commercial applications and unique phosphinic acid functional group. Despite intensive study since its discovery in 1972 (see 3 for a comprehensive review), a number of steps early in the PTT biosynthetic pathway remain uncharacterized. Here we report a series of interdisciplinary experiments involving the construction of defined S. viridochromogenes mutants, chemical characterization of accumulated intermediates, and in vitro assay of selected enzymes to examine these critical steps in PTT biosynthesis. Our results indicate that early PTT biosynthesis involves a series of heretofore undescribed catalyses, including a highly unusual reaction for carbon bond cleavage. In sum, we define a more complex pathway for early PTT biosynthesis that includes biochemically unprecedented and chemically interesting steps. KeywordsStreptomyces viridochromogenes; phosphinothricin; biosynthesis; bialaphos; phosphonate metabolism * Corresponding Author: Department of Microbiology, University of Illinois at Urbana-Champaign, 601 South Goodwin Avenue, Urbana, IL 61801, Phone: 217-244-1943, Fax: 217-244-6697, metcalf@uiuc.edu. # Present Address: Shanghai Chemspec Corp., No. 3 Lan 1273. Accession codesSequence data from the PTT biosynthetic gene cluster that was used in designing the experiments reported here was derived solely from previously available sequence (GenBank accession number AY632421). Sequence data generated in this study that corrects apparent sequencing errors in S. hygroscopicus PTT biosynthetic genes (found in GenBank accessions AB029917.1 and D37878.1) were deposited in GenBank under accession numbers EF486265 and EF486266 respectively. The bioactive portion of the PTT (also known as bialaphos, 1) tripeptide is phosphinothricin (PT, 2). PT is the only known naturally occurring compound to incorporate a direct carbon to phosphorus to carbon (C-P-C) bond motif. PTT has become a model for the biosynthesis of phosphonic acid antibiotics based on numerous studies 3 , leading to a pathway that is regarded as mostly solved. It should be noted, however, that many of the PTT non-producing mutants analyzed to define the pathway were generated by random chemical mutagenesis and some mutations were not rigorously mapped to individual genes. Accordingly, the resulting biosynthetic model is based upon a patchwork of chemical, enzymatic, and genetic data. Further, though many PTT biosynthetic genes were previously cloned and sequenced, the full gene cluster was not completely sequenced from either producer until very recently, when the cluster from S. viridochromogenes was isolated and characterized 7,8 . Sequence analysis of the cluster revealed that only about half of the genes identified had rigorous genetic or biochemical data establishing the...

Hydroxyethylphosphonate is a required intermediate in fosfomycin biosynthesis.

Phosphonic acids encompass a common yet chemically diverse class of natural products that often possess potent biological activities. Here we report that, despite the significant structural differences among many of these compounds, their biosynthetic routes contain an unexpected common intermediate, 2-hydroxyethyl-phosphonate, which is synthesized from phosphonoacetaldehyde by a distinct family of metal-dependent alcohol dehydrogenases (ADHs). Although the sequence identity of the ADH family members is relatively low (34 -37%), in vitro biochemical characterization of the homologs involved in biosynthesis of the antibiotics fosfomycin, phosphinothricin tripeptide, and dehydrophos (formerly A53868) unequivocally confirms their enzymatic activities. These unique ADHs have exquisite substrate specificity, unusual metal requirements, and an unprecedented monomeric quaternary structure. Further, sequence analysis shows that these ADHs form a monophyletic group along with additional family members encoded by putative phosphonate biosynthetic gene clusters. Thus, the reduction of phosphonoacetaldehyde to hydroxyethyl-phosphonate may represent a common step in the biosynthesis of many phosphonate natural products, a finding that lends insight into the evolution of phosphonate biosynthetic pathways and the chemical structures of new C-P containing secondary metabolites.

The late stages of biosynthesis of phosphinothricin tripeptide (PTT) involve peptide formation and methylation on phosphorus. The exact timing of these transformations is not known. To provide insight into this question, we developed a heterologous expression system for PhsA, one of three NRPS proteins in PTT biosynthesis. The apparent k cat /K m value for ATP-pyrophosphate exchange activity for D,L-N-acetylphosphinothricin was 3.5 μM -1 min -1 , whereas the k cat /K m,app for L-N-acetyldemethylphosphinothricin was 0.5 μM -1 min -1 , suggesting the former might be the physiological substrate. Each substrate could be loaded onto the phosphopantetheine arm of the thiolation domain as observed by Fourier transform mass spectrometry (FTMS).

Fluorinated amino acids are useful building blocks for the preparation of biologically active peptides and peptidomimetics with increased metabolic stability. We report here the synthesis of two fluorinated amino acids, β-difluoroalanine and γ-difluorothreonine as analogs of Ser and Thr, respectively. These compounds were suitably protected for Fmoc-based solid phase peptide synthesis. Once incorporated into peptides, they may serve as alternative substrates or inhibitors of lantibiotic synthetases that post-translationally dehydrate Ser and Thr residues to dehydroalanine and dehydrobutyrine, respectively.Michael acceptors have been popular functionalities for the design of enzyme inhibitors and active site affinity labels. 1 Dehydroalanines (Dha) and dehydrobutyrines (Dhb) are potential Michael acceptors and are present in a large number of natural products, including the microcystins, 2 nodularin, 3 thiostrepton and other thiopeptides, 4 and the lantibiotics. 5 The electrophilicity of dehydroalanine (Dha) is significantly moderated compared to acrylamides due to the inherent enamine functionality in dehydro amino acids. This may explain why natural products containing dehydroalanines often interact with their targets by non-covalent mechanisms 6 despite the presence of the Michael acceptor. Increasing the electrophilicity of dehydroalanines in natural products or designed inhibitors may lead to the development of powerful tools for mechanistic enzymology or for use in cell biology and signal transduction. Introduction of an electron withdrawing group on the terminal vinyl carbon would provide for the desired increased reactivity. If this functionality also consisted of a good leaving group, the Michael addition could be rendered irreversible by way of an addition-elimination pathway (Scheme 1). 7 Fluorine-substituted dehydroalanines would be particularly attractive because of the small steric requirements of the fluorine substituent. Moreover, fluorinated amino acids have received much attention due to their potential applications in medicine. 8,9,10 In previous studies we have developed synthetic methodology to prepare fluorinated dehydroalanines. 11 In this contribution we describe the synthesis of precursor amino acids that are envisioned to be potential substrates for lantibiotic synthetases. This class of enzymes catalyzes the dehydration of Ser and Thr residues in their substrate peptides resulting in formation of Dha and Dhb structures. 5 With the recent successful in vitro reconstitution of these proteins, 12 they can now be evaluated as potential tools for the construction of more reactive Dha and Dhb analogs. One possibility would be replacement of a Ser in the substrates for dehydration by a difluoroalanine. Enzymatic elimination of one of the fluorines of difluoroalanine 11b would then produce a fluoro-Dha (Scheme 2). Alternatively, replacement of the methyl group of Thr with a fluorinated methyl group and subsequent enzymatic dehydration would result in a Dhb analog with increased electrop...

The trifluoromethanide anion is the postulated key intermediate in nucleophilic trifluoromethylation reactions. It is believed to be incompatible with Na+ cation due to the strong Na–F bond. However, herein we demonstrate that it could be prepared for the first time. Trialkylamines can be used as cation chelating agents to stabilize the isolated –CF3 ion to realize trifluoromethylation reaction. With this strategy, trifluoromethyl aromatic ketones could be effectively synthesized from fluoroform and aromatic esters with diisopropyl aminosodium (NaDA) and trialkylamines.