Genomics-enabled discovery of phosphonate natural products and their biosynthetic pathways

Phosphonate natural products have proven to be a rich source of useful pharmaceutical, agricultural, and biotechnology products, whereas study of their biosynthetic pathways has revealed numerous intriguing enzymes that catalyze unprecedented biochemistry. Here we review the history of phosphonate natural product discovery, highlighting technological advances that have played a key role in the recent advances in their discovery. Central to these developments has been the application of genomics, which allowed discovery and development of a global phosphonate metabolic framework to guide research efforts. This framework suggests that the future of phosphonate natural products remains bright, with many new compounds and pathways yet to be discovered.     

Biochemical bases for a widespread tolerance of cyanobacteria to the phosphonate herbicide glyphosate

Possible non-target effects of the widely used, non-selective herbicide glyphosate were examined in six cyanobacterial strains, and the basis of their resistance was investigated. All cyanobacteria showed a remarkable tolerance to the herbicide up to millimolar levels. Two of them were found to possess an insensitive form of glyphosate target, the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase. Four strains were able to use the phosphonate as the only phosphorus source. Low uptake rates were measured only under phosphorus deprivation. Experimental evidence for glyphosate metabolism was also obtained in strains apparently unable to use the phosphonate. Results suggest that various mechanisms may concur in providing cyanobacterial strains with herbicide tolerance. The data also account for their widespread ability to metabolize the phosphonate. However, such a capability seems limited by low cell permeability to glyphosate, and is rapidly repressed when inorganic phosphate is available.     

Phosphonate ester probes for proteolytic antibodies

The reactivity of phosphonate ester probes with several available proteolytic antibody (Ab) fragments was characterized. Irreversible, active site-directed inhibition of the peptidase activity was evident. Stable phosphonate diester-Ab adducts were resolved by column chromatography and denaturing electrophoresis. Biotinylated phosphonate esters were applied for chemical capture of phage particles displaying Fv and light chain repertoires. Selected Ab fragments displayed enriched catalytic activity inhibitable by the selection reagent. Somewhat unexpectedly, a phosphonate monoester also formed stable adducts with the Abs. Improved catalytic activity of phage Abs selected by monoester binding was evident. Turnover values (kcat) for a selected Fv construct and a light chain against their preferred model peptide substrates were 0.5 and 0.2 min(-1), respectively, and the corresponding Michaelis-Menten constants (Km) were 10 and 8 microm. The covalent reactivity of Abs with phosphonate esters suggests their ability to recapitulate the catalytic mechanism utilized by classical serine proteases.     

Hydrolysis of phosphonate esters catalyzed by 5'-nucleotide phosphodiesterase.

4-Nitrophenyl and 2-napthyl monoesters of phenylphosphonic acid have been synthesized, and an enzyme catalyzing their hydrolysis was resolved from alkaline phosphatase of a commerical calf intestinal alkaline phosphatase preparation by extensive ion-exchange chromatography, chromatography on L-phenylalanyl-Sepharose with a decreasing gradient of (NH4) 2SO4, and gel filtration. Detergent-solubilized enzyme from fresh bovine intestine was purified after (NH4)2SO4 fractionation by the same technique. The purified enzyme is homogeneous by polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. It has a molecular weight of 108,000, contains approximately 21% carbohydrate, and has an amino acid composition considerably different from that reported from alkaline phosphatase from the same tissue. The homogeneous intestinal enzyme, an efficient catalyst of phosphonate ester hydoolysis but not of phosphate monoester hydrolysis, was identified as a 5'-nucleotide phosphodiesterase by its ability to hydrolyze 4-nitrophenyl esters of 5'-TMP but not of 3'-TMP. Also consistent with this identification was the ability of the enzyme to hydrolyze 5'-ATP to 5'-AMP and PPi, NAD+ to 5'-AMP and NMN, TpT to 5'-TMP and thymidine, pApApApA to 5'-AMP, and only the single-stranded portion of tRNA from the 3'-OH end. Snake venom 5'-nucleotide phosphodiesterase also hydrolyzes phosphonate esters, but 3'-nucleotide phosphodiesterase of spleen and cyclic 3',5'-AMP phosphodiesterase do not. Thus, types of phosphodiesterases can be conveniently distinguished by their ability to hydrolyze phosphonate esters. As substrates for 5'-nucleotide phosphodiesterases, phosphonate esters are preferable to the more conventional esters of nucleotides and bis(4-nitrophenyl) phosphate because of their superior stability and ease of synthesis. Furthermore, the rate of hydrolysis of phosphonate esters under saturating conditions is greater than that of the conventional substrates. At substrate concentrations of 1 mM the rates of hydrolysis of phosphonate esters and of nucleotide esters are comparable and both superior to that of bis(4-nitrophenyl) phosphate.     

Synthesis of phosphonate derivatives of uridine, cytidine, and cytosine arabinoside

The vinyl phosphonate derivatives of uridine, cytidine, and cytosine arabinoside (ara-C) have been prepared through oxidation of appropriately protected nucleosides to the 5' aldehydes and Wittig condensation with [(diethoxyphosphinyl)methylidine]triphenylphosphorane. Dihydroxylation of these vinyl phosphonates with an AD-mix reagent generated the new 5',6'-dihydroxy-6'-phosphonates. After hydrolysis of the phosphonate esters and the various protecting groups, the six phosphonic acids were tested for their ability to serve as substrates for the enzyme nucleotide monophosphate kinase and for their toxicity to K562 cells.     

Synthesis and phosphodiesterase 5 inhibitory activity of new sildenafil analogues containing a phosphonate group in the 5(')-sulfonamide moiety of phenyl ring

Synthesis of new sildenafil analogues containing a phosphonate group in the 5(')-sulfonamide moiety of the phenyl ring, 12a-e, 13a-d, and 14a-d, and evaluation of their in vitro PDE5 inhibitory activity are disclosed. Enzyme assays revealed that maximum 10-fold increase in PDE5 inhibitory activity, compared with sildenafil, was achieved by introducing a phosphonate group in the 5(')-sulfonamide moiety. Docking model of (PDE5: 12d) complex shows that the PDE5-bound conformation of 12d matches completely with that of sildenafil, while 12d is partially overlapped with cGMP with ethyl phosphonate group of 12d superimposed onto the cyclic phosphate group of cGMP.     

Convergent and divergent biosynthetic strategies towards phosphonic acid natural products

The phosphonate class of natural products have received significant interests in the post-genomic era due to the relative ease with which their biosynthetic genes may be identified and the resultant final products be characterized. Recent large-scale studies of the elucidation and distributions of phosphonate pathways have provided a robust landscape for deciphering the underlying biosynthetic logic. A recurrent theme in phosphonate biosynthetic pathways is the interweaving of enzymatic reactions across different routes, which enables diversification to elaborate chemically novel scaffolds. Here, we provide a few vignettes of how Nature has utilized both convergent and divergent biosynthetic strategies to compile pathways for production of novel phosphonates. These examples illustrate how common intermediates may either be generated or intercepted to diversify chemical scaffolds and provides a starting point for both biotechnological and synthetic biological applications towards new phosphonates by similar combinatorial approaches.     

19-F NMR studies of the binding of a fluorine-labeled phosphonate ion to E. coli alkaline phosphatase.

The interaction of a fluorinated phosphonate with Zn-2+-and Mn-2+-alkaline phosphatase as studied by 19-F NMR revealed a stoichiometry of 1:1 for the binding of the phosphonate anion to the enzyme. In the presence of two metal ions, one fluorinated phosphonate ion was found to interact strongly with the enzyme, while a different interaction was observed when the number of metal ions per enzyme exceeded two. Phosphate replaced enzyme bound phosphonate, as is shown by the 19-F NMR spectra. No direct interaction between the fluorinated phosphonate and the metal ion responsible for enzyme activity was indicated by the 19-F NMR data. This observation supports the idea of a considerable distance between metal ion and substrate binding site in Escherichia coli alkaline phosphatase.     

Biosynthesis of 2-hydroxyethylphosphonate, an unexpected intermediate common to multiple phosphonate biosynthetic pathways

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.     

Role of 2-oxoglutarate dehydrogenase in brain pathologies involving glutamate neurotoxicity

Decreased activity of the mitochondrial thiamin-dependent 2-oxoglutarate dehydrogenase complex (OGDHC) is associated with a number of inborn and acquired neuropathologies. We hypothesized that perturbation in flux through the complex influences brain development and function, in particular, because the OGDHC reaction is linked to the synthesis/degradation of neurotransmitters glutamate and GABA. Developmental impact of this metabolic knot was studied by characterizing the brain OGDHC activity in offspring of rats exposed to acute hypobaric hypoxia at a critical organogenesis period of pregnancy. In this model, we detected the hypoxia-induced changes in the brain OGDHC activity and in certain physiologic and morphometric parameters. The changes were mostly abrogated by application of specific effector of cellular OGDHC, the phosphonate analog of 2-oxoglutarate (succinyl phosphonate), shortly before hypoxia. The glutamate excitotoxicity known to greatly contribute to hypoxic damage was alleviated by succinyl phosphonate in situ. That is, the delayed calcium deregulation, mitochondrial depolarization and reactive oxygen species (ROS) production became less pronounced in cultivated neurons loaded with succinyl phosphonate. In vitro, succinyl phosphonate protected OGDHC from the catalysis-induced inactivation. Thus, the protective effects of the phosphonate upon hypoxic insult in vivo may result from the preservation of mitochondrial function and Ca²⁺ homeostasis due to the phosphonate inhibition of both the OGDHC-dependent ROS production and associated OGDHC inactivation. As a result, we showed for the first time that the hypoxia- and glutamate-induced cerebral damage is linked to the function of OGDHC, introducing the phosphonate analogs of 2-oxoglutarate as promising diagnostic tools to reveal the role of OGDHC in brain function and development.     

A novel biotinylated suicide inhibitor for directed molecular evolution of lipolytic enzymes

A bifunctional activity label (8) for directed molecular evolution of lipolytic enzymes has been designed and synthesized. The structure is composed of a 4-nitrophenyl activated phosphonate, that is, a suicide substrate of lipases/esterases, connected to a biotin moiety through a spacer containing a disulfide bridge. The phosphonate (3) was prepared by Michaelis-Arbuzov reaction of trimethylsilyl-protected 11-bromoundecanol (2) with triethyl phosphite. The deprotected omega-hydroxyalkylphosphonate (4) was transformed into an active N-hydroxysuccinimide carbonate (5) followed by 4-nitrophenyl activation of the phosphonate using standard procedures. The biotinylated phosphonate inhibitor (8) was then synthesised by coupling the phosphonate inhibitor (6) to the epsilon-amino-caproic acid and cystamine containing biotinyl spacer (7). The function of all relevant groups of the final activity label (8) (biotin-label, cleavable disulfide bridge, phosphonate-inhibitor) have been successfully tested with the commercial lipase Lipolase (Novo Nordisk). Hence, a tool for directed molecular evolution of lipolytic enzymes has been developed.     

Chemoenzymatic Synthesis, Inhibition Studies, and X-ray Crystallographic Analysis of the Phosphono Analog of UDP-Galp as an Inhibitor and Mechanistic Probe for UDP-Galactopyranose Mutase

UDP (uridine diphosphate) galactopyranose mutase (UGM) is involved in the cell wall biosynthesis of many pathogenic microorganisms. UGM catalyzes the reversible conversion of UDP-α-d-galactopyranose into UDP-α-d-galactofuranose, with the latter being the precursor of galactofuranose (Galf) residues in cell walls. Glycoconjugates of Galf are essential components in the cell wall of various pathogenic bacteria, including Mycobacterium tuberculosis, the causative agent of tuberculosis. The absence of Galf in humans and its bacterial requirement make UGM a potential target for developing novel antibacterial agents. In this article, we report the synthesis, inhibitory activity, and X-ray crystallographic studies of UDP-phosphono-galactopyranose, a nonhydrolyzable C-glycosidic phosphonate. This is the first report on the synthesis of a phosphonate analog of UDP-α-d-galactopyranose by a chemoenzymatic phosphoryl coupling method. The phosphonate was evaluated against three bacterial UGMs and showed only moderate inhibition. We determined the crystal structure of the phosphonate analog bound to Deinococcus radiodurans UGM at 2.6 Å resolution. The phosphonate analog is bound in a novel conformation not observed in UGM-substrate complex structures or in other enzyme-sugar nucleotide phosphonate complexes. This complex structure provides a structural basis for the observed micromolar inhibition towards UGM. Steric clashes, loss of electrostatic stabilization between an active-site arginine (Arg305) and the phosphonate analog, and a 180° flip of the hexose moiety account for the differences in the binding orientations of the isosteric phosphonate analog and the physiological substrate. This provides new insight into the ability of a sugar-nucleotide-binding enzyme to orient a substrate analog in an unexpected geometry and should be taken into consideration in designing such enzyme inhibitors.     

Effects of Phosphonic Acid Analogues of Glycerol-3-Phosphate on the Growth of Escherichia coli

The four-carbon phosphonate, 3,4-dihydroxybutyl-1-phosphonate, is similar to glycerol-3-phosphate in its ability to inhibit cell growth of Escherichia coli strain 8 cultured in low-phosphate synthetic medium supplemented with either succinate or casein hydrolysate as the sole carbon source. The three-carbon phosphonate, 2,3-dihydroxypropyl-1-phosphonate, does not appear to exhibit a similar effect. The inhibition caused by the four-carbon phosphonate differs from that caused by glycerol-3-phosphate in at least three ways. (i) Its inhibitory effect is not offset by the presence of glucose in the culture medium. (ii) It is capable of exerting its inhibitory effect on cells containing an active aerobic glycerol-3-phosphate dehydrogenase. (iii) Its inhibitory effect is maintained in synthetic medium containing high concentrations of inorganic phosphate. The four-carbon phosphonate appears to be bacteriostatic and inhibits the uptake of labeled glycerol-3-phosphate by E. coli strain 8.     

Synthesis and in vitro evaluation of S-acyl-3-thiopropyl prodrugs of Foscarnet

A new enzyme-labile group called S-acyl-3-thiopropyl group (SATP) has been synthesized from allylic esters of phosphonate. After demonstration of the enzyme-labile character of the SATP in cellular extracts, it has been introduced onto the phosphonate moiety of PFA (Foscarnet) to obtain potential lipophilic prodrugs. To ponder the lipophilicity of the triesters of PFA, esters of monomethylether of polyethyleneglycols and of thioglycerol were introduced on the PFA carboxylate moiety. The SATP groups were introduced in an attempt to deliver PFA after bioactivation inside the cells. The PFA prodrugs were evaluated in vitro for their activity against human immunodeficiency viruses (HIV-1 and HIV-2).     

Global and seasonal variation of marine phosphonate metabolism

Marine microbial communities rely on dissolved organic phosphorus (DOP) remineralisation to meet phosphorus (P) requirements. We extensively surveyed the genomic and metagenomic distribution of genes directing phosphonate biosynthesis, substrate-specific catabolism of 2-aminoethylphosphonate (2-AEP, the most abundant phosphonate in the marine environment), and broad-specificity catabolism of phosphonates by the C-P lyase (including methylphosphonate, a major source of methane). We developed comprehensive enzyme databases by curating publicly available sequences and then screened metagenomes from TARA Oceans and Munida Microbial Observatory Time Series (MOTS) to assess spatial and seasonal variation in phosphonate metabolism pathways. Phosphonate cycling genes were encoded in diverse gene clusters by 35 marine bacterial and archaeal classes. More than 65% of marine phosphonate cycling genes mapped to Proteobacteria with production demonstrating wider taxonomic diversity than catabolism. Hydrolysis of 2-AEP was the dominant phosphonate catabolism strategy, enabling microbes to assimilate carbon and nitrogen alongside P. Genes for broad-specificity catabolism by the C-P lyase were far less widespread, though enriched in the extremely P-deplete environment of the Mediterranean Sea. Phosphonate cycling genes were abundant in marine metagenomes, particularly from the mesopelagic zone and winter sampling dates. Disparity between prevalence of substrate-specific and broad-specificity catabolism may be due to higher resource expenditure from the cell to build and retain the C-P lyase. This study is the most comprehensive metagenomic survey of marine microbial phosphonate cycling to date and provides curated databases for 14 genes involved in phosphonate cycling.Subject terms: Water microbiology, Microbial ecology, Microbial biooceanography, Metagenomics     

Broadly distributed chemical reactivity of natural antibodies expressed in coordination with specific antigen binding activity

Antibody (Ab) nucleophilic reactivity was studied using hapten and polypeptide antigens containing biotinylated phosphonate diester groups (covalently reactive antigen analogs, CRAs). Polyclonal IgG from healthy donors formed covalent adducts with a positively charged hapten CRA at levels superior to trypsin. Each of the 16 single chain Fv clones studied expressed a similar reactivity, indicating the V domain location of the nucleophiles and their broad distribution in diverse Abs. The formation of hapten CRA-Fv adducts was correlated with Fv proteolytic activity determined by cleavage of a model peptide substrate. Despite excellent nucleophilicity, proteolysis by IgG proceeded at lower rates than trypsin, suggesting that events occurring after nucleophilic attack on the substrate limit the rate of Ab proteolysis. The extracellular domain of the epidermal growth factor receptor with phosphonate diester groups at Lys side chains and a synthetic peptide corresponding to residues 421- 431 of human immunodeficiency virus glycoprotein (gp) 120 with the phosphonate diester at the C terminus formed covalent adducts with specific polyclonal and monoclonal Abs raised by immunization with epidermal growth factor receptor and synthetic gp120-(421- 436) devoid of phosphonate diester groups, respectively. Adduct formation was inhibited by extracellular domain of the epidermal growth factor receptor (exEGFB) and synthetic gp120-(421- 436) devoid of phosphonate groups, suggesting that the nucleophiles are located within the antigen binding sites. These results suggest the innate character of the Ab nucleophilic reactivity, its functional coordination with non-covalent adaptive binding interactions developing over the course of B cell maturation, and novel routes toward permanent inhibition of Abs.     

Preparation and application of an affinity matrix for phosphatidylinositol-specific phospholipase C

A non-hydrolyzable phosphonate analogue of phosphatidyl inositol, racemic myo-inosityl-(1)-5-oxa-16-trifluoroacetamidohexadecyl phosphonate, was synthesized. This phosphonate inhibited the activity of phosphatidyl inositol-specific phospholipase C (PI-PLC) from Bacillus cereus with an IC50 of approximately 10 mM. Removal of the trifluoroacetyl blocking group followed by covalent binding of the phosphonate to cyanogen bromide activated Sepharose 4B via the amino group produced an affinity matrix specific for the PI-PLC from B. cereus. This affinity matrix was used to purify the phospholipase C from a complex mixture of proteins in a single step. Competition experiments with myo-inositol in the elution medium indicated that specific binding of the enzyme to the matrix most likely involves the enzyme active site. The inositol phosphonate derivatized matrix was stable over several months in neutral and alkaline media and was used repeatedly without loss of binding capacity. These results show that affinity matrices employing myo-inositol phosphonate ligands are useful for isolation and binding studies of PI-PLC and possibly of other enzymes interacting with phosphoinositides or myo-inositol phosphate derivatives.     

Preparation of 35S-labeled polyphosphorothioate oligodeoxyribonucleotides by use of hydrogen phosphonate chemistry.

The title compounds were chemically synthesized as their 5'-dimethoxytrityl derivatives by base-catalyzed reaction of 35S-enriched elemental sulfur with support-bound hydrogen phosphonate oligomer. This was derived from adamantane carbonyl chloride-activated coupling of nucleotide hydrogen phosphonate monomers, and similarly activated capping with isopropyl phosphite. A convenient, disposable, reversed-phase cartridge was utilized to purify and isolate the 5'-dimethoxytrityl derivative for subsequent in situ detritylation and elution of the final product. The specific activity obtained for the title compounds was ca. 10(7) cpm/mumols-eq P(O)S-. The procedure should be readily adaptable to appropriate syntheses of other P-S containing analogs of DNA and RNA.     

Chemistry and biochemistry of 1-desoxysphinganine 1-phosphonate (dihydrosphingosine-1-phosphonate)

The chemical synthesis of labelled 1-desoxy-D,L-sphinganine 1-phosphonate has been elaborated. This compound is an analog of sphinganine 1-phosphate, a naturally occurring intermediate in the biological degradation of long chain bases.The phosphonate is highly toxic when administered intravenously due to its hemolytic effect. The microsomal sphingosine 1-phosphate lyase(aldolase) cleaves [3-³H] 1-desoxysphinganine 1-phosphonate to [1-³H] hexadecanal and aminoethyl phosphonate like sphinganine 1-phosphate however at a reduced rate. The phosphonate is a competitive inhibitor of the lyase (aldolase). K_i has been determined. The molecular dimensions of the phosphonate have been discussed with reference to the aldolase mechanism and known properties of the enzyme.