Enzymatic RNA Production from NTPs Synthesized from Nucleosides and Trimetaphosphate*.

A mechanism of nucleoside triphosphorylation would have been critical in an evolving "RNA world" to provide high-energy substrates for reactions such as RNA polymerization. However, synthetic approaches to produce ribonucleoside triphosphates (rNTPs) have suffered from conditions such as high temperatures or high pH that lead to increased RNA degradation, as well as substrate production that cannot sustain replication. Previous reports have demonstrated that cyclic trimetaphosphate (cTmp) can react with nucleosides to form rNTPs under prebiotically-relevant conditions, but their reaction rates were unknown and the influence of reaction conditions not well-characterized. Here we established a sensitive assay that allowed for the determination of second-order rate constants for all four rNTPs, ranging from 1.7×10-6 to 6.5×10-6  M-1 s-1 . The ATP reaction shows a linear dependence on pH and Mg2+ , and an enthalpy of activation of 88±4 kJ/mol. At millimolar nucleoside and cTmp concentrations, the rNTP production rate is sufficient to facilitate RNA synthesis by both T7 RNA polymerase and a polymerase ribozyme. We suggest that the optimized reaction of cTmp with nucleosides may provide a viable connection between prebiotic nucleotide synthesis and RNA replication.

Fosmidomycin transport through the phosphate-specific porins OprO and OprP of Pseudomonas aeruginosa.

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen, responsible for many hospital-acquired infections. The bacterium is quite resistant toward many antibiotics, in particular because of the fine-tuned permeability of its outer membrane (OM). General diffusion outer membrane pores are quite rare in this organism. Instead, its OM contains many substrate-specific porins. Their expression is varying according to growth conditions and virulence. Phosphate limitations, as well as pathogenicity factors, result in the induction of the two mono- and polyphosphate-specific porins, OprP and OprO, respectively, together with an inner membrane uptake mechanism and a periplasmic binding protein. These outer membrane channels could serve as outer membrane pathways for the uptake of phosphonates. Among them are not only herbicides, but also potent antibiotics, such as fosfomycin and fosmidomycin. In this study, we investigated the interaction between OprP and OprO and fosmidomycin in detail. We could demonstrate that fosmidomycin is able to bind to the phosphate-specific binding site inside the two porins. The inhibition of chloride conductance of OprP and OprO by fosmidomycin is considerably less than that of phosphate or diphosphate, but it can be measured in titration experiments of chloride conductance and also in single-channel experiments. The results suggest that fosmidomycin transport across the OM of P. aeruginosa occurs through OprP and OprO. Our data with the ones already known in the literature show that phosphonic acid-containing antibiotics are in general good candidates to treat the infections of P. aeruginosa at the very beginning through a favorable OM transport system.

Discovery of Potent and Selective Methylenephosphonic Acid CD73 Inhibitors.

Solid tumors are often associated with high levels of extracellular ATP. Ectonucleotidases catalyze the sequential hydrolysis of ATP to adenosine, which potently suppresses T-cell and NK-cell functions via the adenosine receptors (A2a and A2b). The ectonucleotidase CD73 catalyzes the conversion of AMP to adenosine. Thus, increased CD73 enzymatic activity in the tumor microenvironment is a potential mechanism for tumor immune evasion and has been associated with poor prognosis in the clinic. CD73 inhibition is anticipated to restore immune function by skirting this major mechanism of adenosine generation. We have developed a series of potent and selective methylenephosphonic acid CD73 inhibitors via a structure-based design. Key binding interactions of the known inhibitor adenosine-5'-(α,ß-methylene)diphosphate (AMPCP) with hCD73 provided the foundation for our early designs. The structure-activity relationship study guided by this structure-based design led to the discovery of 4a, which exhibits excellent potency against CD73, exquisite selectivity against related ectonucleotidases, and a favorable pharmacokinetic profile.

C-H Bond Cleavage Is Rate-Limiting for Oxidative C-P Bond Cleavage by the Mixed Valence Diiron-Dependent Oxygenase PhnZ.

PhnZ utilizes a mixed valence diiron(II/III) cofactor and O2 to oxidatively cleave the carbon-phosphorus bond of (R)-2-amino-1-hydroxyethylphosphonic acid to form glycine and orthophosphate. The active site residues Y24 and E27 are proposed to mediate induced-fit recognition of the substrate and access of O2 to one of the active site Fe ions. H62 is proposed to deprotonate the C1-hydroxyl of the substrate during catalysis. Kinetic isotope effects (KIEs), pH-rate dependence, and site-directed mutagenesis were used to probe the rate-determining transition state and the roles of these three active site residues. Primary deuterium KIE values of 5.5 ± 0.3 for D(V) and 2.2 ± 0.4 for D(V/K) were measured with (R)-2-amino[1-2H1]-1-hydroxyethylphosphonic acid, indicating that cleavage of the C1-H bond of the substrate is rate-limiting. This step is also rate-limiting for PhnZ Y24F, as shown by a significant deuterium KIE value of 2.3 ± 0.1 for D(V). In contrast, a different reaction step appears to be rate-limiting for the PhnZ E27A and H62A variants, which exhibited D(V) values near unity. A solvent KIE of 2.2 ± 0.3 for D2O(V) is observed for PhnZ. Significant solvent KIE values are also observed for the PhnZ Y24F and E27A variants. In contrast, the PhnZ H62A variant does not show a significant solvent KIE, suggesting that H62 is mediating proton transfer in the transition state. A proton inventory study with PhnZ indicates that 1.5 ± 0.6 protons are in flight in the rate-determining step. Overall, the rate-determining transition state for oxidative C-P bond cleavage by PhnZ is proposed to involve C-H bond cleavage that is coupled to deprotonation of the substrate C1-hydroxyl by H62.

Whole-Cell Detection of C-P Bonds in Bacteria.

Naturally produced molecules possessing a C-P bond, such as phosphonates and phosphinates, remain vastly underexplored. Although success stories like fosfomycin have reinvigorated small molecule phosphonate discovery efforts, bioinformatic analyses predict an enormous unexplored biological reservoir of C-P bond-containing molecules, including those attached to complex macromolecules. However, high polarity, a lack of chromophores, and complex macromolecular association impede phosphonate discovery and characterization. Here we detect widespread transcriptional activation of phosphonate biosynthetic machinery across diverse bacterial phyla and describe the use of solid-state nuclear magnetic resonance to detect C-P bonds in whole cells of representative Gram-negative and Gram-positive bacterial species. These results suggest that phosphonate tailoring is more prevalent than previously recognized and set the stage for elucidating the fascinating chemistry and biology of these modifications.

Discovery of a Phosphonoacetic Acid Derived Natural Product by Pathway Refactoring.

The activation of silent natural product gene clusters is a synthetic biology problem of great interest. As the rate at which gene clusters are identified outpaces the discovery rate of new molecules, this unknown chemical space is rapidly growing, as too are the rewards for developing technologies to exploit it. One class of natural products that has been underrepresented is phosphonic acids, which have important medical and agricultural uses. Hundreds of phosphonic acid biosynthetic gene clusters have been identified encoding for unknown molecules. Although methods exist to elicit secondary metabolite gene clusters in native hosts, they require the strain to be amenable to genetic manipulation. One method to circumvent this is pathway refactoring, which we implemented in an effort to discover new phosphonic acids from a gene cluster from Streptomyces sp. strain NRRL F-525. By reengineering this cluster for expression in the production host Streptomyces lividans, utility of refactoring is demonstrated with the isolation of a novel phosphonic acid, O-phosphonoacetic acid serine, and the characterization of its biosynthesis. In addition, a new biosynthetic branch point is identified with a phosphonoacetaldehyde dehydrogenase, which was used to identify additional phosphonic acid gene clusters that share phosphonoacetic acid as an intermediate.

Screening of nerve agent markers with hollow fiber-chemosorption of phosphonic acids.

This report describes a method developed for extracting nerve gas markers such as phosphonic acids from urine and other aqueous samples. It involves single-step microextraction with chemosorption to hollow fibers that have been pre-soaked in a solution containing a derivatization reagent (3,5 triflouro methyl benzene diazomethane). The derivatives it forms with phosphonic acids can be sensitively detected by mass spectrometric detectors operating in negative chemical ionization (NCI) mode. Limits of quantification obtained in analyses of water and urine extracts by GC/MS in negative chemical ionization and selected ion monitoring mode were 0.1-10 and 0.5-10ng/mL, respectively. Pentaflourophenyl diazomethane can also be used as a derivatization reagent, and the micro-extracts (which generate low background signals) can be sensitively analyzed by GC-MS/MS in NCI selected reaction monitoring (SRM) mode, using two specific transitions for both reagents. Thus, this sensitive approach can be flexibly modified to obtain confirmatory information, or address potential problems caused by interferences in some samples.

Discovery of potent and selective inhibitors of human aminopeptidases ERAP1 and ERAP2 by screening libraries of phosphorus-containing amino acid and dipeptide analogues.

A collection of fifty phosphonic and phosphinic acids was screened for inhibition of ERAP1 and ERAP2, the human endoplasmic reticulum aminopeptidases. The cooperative action of these enzymes is manifested by trimming a variety of antigenic precursors to be presented on the cell surface by major histocompatibility class I. The SAR studies revealed several potent compounds, particularly among the phosphinic dipeptide analogues, that were strong inhibitors of ERAP2 (Ki=100-350nM). A wide structural diversity of the applied organophosphorus compounds, predominantly non-proteinogenic analogues, allowed identification of representatives selective toward only one form of ERAP. For example, N'-substituted α,ß-diaminophosphonates and phosphinates exhibited potency only toward ERAP2, which is in agreement with the P1 basic substrate-oriented specificity. Such discriminating ligands are invaluable tools for elucidating the precise role of a particular aminopeptidase in the concerted function of antigen processing and in human diseases.

Bioavailability and bioconcentration potential of perfluoroalkyl-phosphinic and -phosphonic acids in zebrafish (Danio rerio): Comparison to perfluorocarboxylates and perfluorosulfonates.

Currently, information regarding bioavailability and bioconcentration potential of perfluoroalkyl phosphinic acids (PFPiAs) in aquatic organisms does not exist. The main objective of the present study was to assess uptake and elimination kinetics of PFPiAs in zebrafish (Danio rerio) following aqueous exposure. The results showed that PFPiA exposure can result in very high steady-state bioconentration factors (BCFss), compared to perfluorocarboxylates and perfluorosulfonates.C6/C10 PFPiA exhibited the highest BCFss, ranging between 10(7) and 10(10), orders of magnitude higher than those for long-chain perfluorocarboxylates. Strong positive relationships were observed between BCFss versus the membrane-water distribution coefficient (Dmw) and the protein-water partition coefficient (Kpw) of the studied perfluoroalkyl substances. However, BCFss exhibited a substantial drop for the very hydrophobic PFPiAs (C8/C10 and C6/C12 PFPiAs). The reduced BCFss of these long-chain PFPiAs (perfluoroalkyl chain length=18; Dmw=10(9)) is likely the result of reduced bioavailability due to interaction with solute molecules/organic matter present in the water phase and/or reduced gill membrane permeability. While PFPiAs can be metabolized to perfluoroalkyl phosphonic acids, the metabolic transformation rate seems insufficient to counteract the high degree of uptake across gill membranes. These findings help to better understand exposure pathways and bioaccumulation behavior of these important perfluorinated acids in aquatic systems.

Kinetic, thermodynamic and structural analysis of tamiphosphor binding to neuraminidase of H1N1 (2009) pandemic influenza.

Influenza virus causes severe respiratory infections that are responsible for up to half a million deaths worldwide each year. Two inhibitors targeting viral neuraminidase have been approved to date (oseltamivir, zanamivir). However, the rapid development of antiviral drug resistance and the efficient transmission of resistant viruses among humans represent serious threats to public health. The approved influenza neuraminidase inhibitors have (oxa)cyclohexene scaffolds designed to mimic the oxonium transition state during enzymatic cleavage of sialic acid. Their active forms contain a carboxylate that interacts with three arginine residues in the enzyme active site. Recently, the phosphonate group was successfully used as an isostere of the carboxylate in oseltamivir, and the resulting compound, tamiphosphor, was identified as a highly active neuraminidase inhibitor. However, the structure of the complex of this promising inhibitor with neuraminidase has not yet been reported. Here, we analyzed the interaction of a set of oseltamivir and tamiphosphor derivatives with neuraminidase from the A/California/07/2009 (H1N1) influenza virus. We thermodynamically characterized the binding of oseltamivir carboxylate or tamiphosphor to the neuraminidase catalytic domain by protein microcalorimetry, and we determined crystal structure of the catalytic domain in complex with tamiphosphor at 1.8 Å resolution. This structural information should aid rational design of the next generation of neuraminidase inhibitors.

Crystal structures of the apo form and a complex of human LMW-PTP with a phosphonic acid provide new evidence of a secondary site potentially related to the anchorage of natural substrates.

Low molecular weight protein tyrosine phosphatases (LMW-PTP, EC 3.1.3.48) are a family of single-domain enzymes with molecular weight up to 18 kDa, expressed in different tissues and considered attractive pharmacological targets for cancer chemotherapy. Despite this, few LMW-PTP inhibitors have been described to date, and the structural information on LMW-PTP druggable binding sites is scarce. In this study, a small series of phosphonic acids were designed based on a new crystallographic structure of LMW-PTP complexed with benzylsulfonic acid, determined at 2.1Å. In silico docking was used as a tool to interpret the structural and enzyme kinetics data, as well as to design new analogs. From the synthesized series, two compounds were found to act as competitive inhibitors, with inhibition constants of 0.124 and 0.047 mM. We also report the 2.4Å structure of another complex in which LMW-PTP is bound to benzylphosphonic acid, and a structure of apo LMW-PTP determined at 2.3Å resolution. Although no appreciable conformation changes were observed, in the latter structures, amino acid residues from an expression tag were found bound to a hydrophobic region at the protein surface. This regions is neighbored by positively charged residues, adjacent to the active site pocket, suggesting that this region might be not a mere artefact of crystal contacts but an indication of a possible anchoring region for the natural substrate-which is a phosphorylated protein.

Production of xylitol and tetrahydrofurfuryl alcohol from xylan in napier grass by a hydrothermal process with phosphorus oxoacids followed by aqueous phase hydrogenation.

The production of xylitol and tetrahydrofurfuryl alcohol (THFA) from napier grass was studied using two steps: a hydrothermal process with phosphorus oxoacids followed by aqueous phase hydrogenation with Pd/C. Xylose obtained from the napier grass by the hydrothermal treatment with 3.0 wt% phosphorous acid was subsequently converted into xylitol at 51.6% yield of the xylan in napier grass by hydrogenation with 5.0 wt% Pd/C. The furfural produced from napier grass with a 3.0 wt% phosphoric acid treatment was also directly subjected to the hydrogenation as a hydrolysate to yield 41.4% THFA based on the xylan in napier grass. The yields of xylitol and THFA obtained by hydrogenation using the napier grass hydrolysate containing xylose or furfural were almost the same as those of hydrogenation using commercial materials. To our knowledge, this is the first report on the production of THFA in high yield by hydrogenation directly from biomass hydrolysate.

Discovery of an acyclic nucleoside phosphonate that inhibits Mycobacterium tuberculosis ThyX based on the binding mode of a 5-alkynyl substrate analogue.

The urgent need for new antibiotics poses a challenge to target un(der)exploited vital cellular processes. Thymidylate biosynthesis is one such process due to its crucial role in DNA replication and repair. Thymidylate synthases (TS) catalyze a crucial step in the biosynthesis of thymidine 5-triphosphate (TTP), an elementary building block required for DNA synthesis and repair. To date, TS inhibitors have only been successfully applied in anticancer therapy due to their lack of specificity for antimicrobial versus human enzymes. However, the discovery of a new family of TS enzymes (ThyX) in a range of pathogenic bacteria that is structurally and biochemically different from the "classic" TS (ThyA) has opened the possibility to develop selective ThyX inhibitors as potent antimicrobial drugs. Here, the interaction of the known inhibitor 5-(3-octanamidoprop-1yn-1yl)-2'-deoxyuridine-5'-monophosphate (1) with Mycobacterium tuberculosis ThyX enzyme is explored using molecular modeling starting from published crystal structures, with further confirmation through NMR experiments. While the deoxyuridylate (dUMP) moiety of compound 1 occupies the cavity of the natural substrate in ThyX, the rest of the ligand (the "5-alkynyl tail") extends to the outside of the enzyme between two of its four subunits. The hydrophobic pocket that accommodates the alkyl part of the tail is formed by displacement of Tyr 44.C, Tyr 108.A and Lys 165.A. Changes to the resonance of the Lys 165 NH3 group upon ligand binding were monitored in a titration experiment by 2D HISQC NMR. Guided by the results of the modeling and NMR studies, and inspired by the success of acyclic antiviral nucleosides, compounds where a 5-alkynyl uracyl moiety is coupled to an acyclic nucleoside phosphonate (ANP) were synthesized and evaluated. Of the compounds evaluated, sodium (6-(5-(3-octanamidoprop-1-yn-1-yl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hexyl)phosphonate (3 e) exhibited 43 % of inhibitory effect on ThyX at 50 µM. While only modest activity was achieved, this is the first example of an ANP inhibiting ThyX, and these results can be used to further guide structural modifications to this class to develop more potent compounds with potential application as antibacterial agents acting through a novel mechanism of action.

Carboxylic acid (bio)isosteres in drug design.

The carboxylic acid functional group can be an important constituent of a pharmacophore, however, the presence of this moiety can also be responsible for significant drawbacks, including metabolic instability, toxicity, as well as limited passive diffusion across biological membranes. To avoid some of these shortcomings while retaining the desired attributes of the carboxylic acid moiety, medicinal chemists often investigate the use of carboxylic acid (bio)isosteres. The same type of strategy can also be effective for a variety other purposes, for example, to increase the selectivity of a biologically active compound or to create new intellectual property. Several carboxylic acid isosteres have been reported, however, the outcome of any isosteric replacement cannot be readily predicted as this strategy is generally found to be dependent upon the particular context (i.e., the characteristic properties of the drug and the drug-target). As a result, screening of a panel of isosteres is typically required. In this context, the discovery and development of novel carboxylic acid surrogates that could complement the existing palette of isosteres remains an important area of research. The goal of this Minireview is to provide an overview of the most commonly employed carboxylic acid (bio)isosteres and to present representative examples demonstrating the use and utility of each isostere in drug design.

Catabolism and detoxification of 1-aminoalkylphosphonic acids: N-acetylation by the phnO gene product.

In Escherichia coli uptake and catabolism of organophosphonates are governed by the phnCDEFGHIJKLMNOP operon. The phnO cistron is shown to encode aminoalkylphosphonate N-acetyltransferase, which utilizes acetylcoenzyme A as acetyl donor and aminomethylphosphonate, (S)- and (R)-1-aminoethylphosphonate, 2-aminoethyl- and 3-aminopropylphosphonate as acetyl acceptors. Aminomethylphosphonate, (S)-1-aminoethylphosphonate, 2-aminoethyl- and 3-aminopropylphosphonate are used as phosphate source by E. coli phn(+) strains. 2-Aminoethyl- or 3-aminopropylphosphonate but not aminomethylphosphonate or (S)-1-aminoethylphosphonate is used as phosphate source by phnO strains. Neither phn(+) nor phnO strains can use (R)-1-aminoethylphosphonate as phosphate source. Utilization of aminomethylphosphonate or (S)-1-aminoethylphosphonate requires the expression of phnO. In the absence of phnO-expression (S)-1-aminoethylphosphonate is bacteriocidal and rescue of phnO strains requires the simultaneous addition of d-alanine and phosphate. An intermediate of the carbon-phosphorus lyase pathway, 5'-phospho-α-d-ribosyl 1'-(2-N-acetamidoethylphosphonate), a substrate for carbon-phosphorus lyase, was found to accumulate in cultures of a phnP mutant strain. The data show that the physiological role of N-acetylation by phnO-specified aminoalkylphosphonate N-acetyltransferase is to detoxify (S)-1-aminoethylphosphonate, an analog of d-alanine, and to prepare (S)-1-aminoethylphosphonate and aminomethylphosphonate for utilization of the phosphorus-containing moiety.

Palatability of selected alpine plant litters for the decomposer Lumbricus rubellus (Lumbricidae).

On alpine pastureland the decline in large-bodied earthworm numbers and biomass after abandonment of management might be the result of a shift from highly palatable grass litter to poorly digestible leaf litter of dwarf shrubs. To test this hypothesis, we analysed nitrogen, phosphorous and total phenolic contents of fresh and aged litter of eight commonly occuring alpine plant species and compared consumption rates of these food sources in a controlled feeding experiment with Lumbricus rubellus (Lumbricidae). Furthermore, we analysed the microbial community structure of aged litter materials to check for a relationship between the microbial characteristics of the different plant litter types and the food choice of earthworms. Plant litters differed significantly in their chemical composition, earthworms, however, showed no preference for any litter species, but generally rejected fresh litter material. Microbial community structures of the litter types were significantly different, but we could find no evidence for selective feeding of L. rubellus. We conclude that L. rubellus is a widespread, adaptable ubiquist, which is able to feed on a variety of food sources differing in quality and palatability, as long as they have been exposed to wheathering.

Evidence for radical-mediated catalysis by HppE: a study using cyclopropyl and methylenecyclopropyl substrate analogues.

(S)-2-Hydroxypropylphosphonic acid epoxidase (HppE) is an unusual mononuclear iron enzyme that catalyzes the oxidative epoxidation of (S)-2-hydroxypropylphosphonic acid ((S)-HPP) in the biosynthesis of the antibiotic fosfomycin. HppE also recognizes (R)-2-hydroxypropylphosphonic acid ((R)-HPP) as a substrate and converts it to 2-oxo-propylphosphonic acid. To probe the mechanisms of these HppE-catalyzed oxidations, cyclopropyl- and methylenecyclopropyl-containing compounds were synthesized and studied as radical clock substrate analogues. Enzymatic assays indicated that the (S)- and (R)-isomers of the cyclopropyl-containing analogues were efficiently converted to epoxide and ketone products by HppE, respectively. In contrast, the ultrafast methylenecyclopropyl-containing probe inactivated HppE, consistent with a rapid radical-triggered ring-opening process that leads to enzyme inactivation. Taken together, these findings provide, for the first time, experimental evidence for the involvement of a C2-centered radical intermediate with a lifetime on the order of nanoseconds in the HppE-catalyzed oxidation of (R)-HPP.

The effect of Asp54 phosphorylation on the energetics and dynamics in the response regulator protein Spo0F studied by molecular dynamics.

The response regulator protein Spo0F acts as an intermediate phospho-messenger in the signal transduction pathway that controls initiation of the differentiation process of sporculation in the bacterium Bacillus subtilis. The regulatory domain of Spo0F contains a triad of three conserved aspartate residues, whereof one aspartate (Asp54) is phosphorylated. Using molecular dynamics simulations, we have studied the changes in flexibility induced by phosphorylation and estimated the free energy cost of introducing a phosphate group at this position using alchemical free energy calculations. The deduced conformational flexibility compares well with experimental NMR results. We find that the apo-conformation of the protein explores a rough energy landscape resulting in a broad population of conformational substates. Phosphorylation of Spo0F reduces protein flexibility, and in particular, the so-called anchor and recognition regions exhibit lower mobility relative to the apo-conformation. Phosphorylation of Asp54 (P-Asp54), in which the apo-structure coordinates to the magnesium ion, results in extension of the sidechain, and depending on which carboxylate oxygen is phosphorylated, distinct interactions between P-Asp54 and magnesium ion as well as residues in its proximity are established. However, phosphorylation does not affect the coordination number of the magnesium ion yielding, within the statistical uncertainties, the same free energy change.