ABSTRACT
PURPOSE: To predict potential inhibitors of alpha-enolase to reduce plasminogen binding of Streptococcus pneumoniae (S. pneumoniae) that may lead as an orally active drug. S. pneumoniae remains dominant in causing invasive diseases. Fibrinolytic pathway is a critical factor of S. pneumoniae to invade and progression of disease in the host body. Besides the low mass on the cell surface, alpha-enolase possesses significant plasminogen binding among all exposed proteins. METHODS: In-silico based drug designing approach was implemented for evaluating potential inhibitors against alpha-enolase based on their binding affinities, energy score and pharmacokinetics. Lipinski's rule of five (LRo5) and Egan's (Brain Or IntestinaL EstimateD) BOILED-Egg methods were executed to predict the best ligand for biological systems. RESULTS: Molecular docking analysis revealed, Sodium (1,5-dihydroxy-2-oxopyrrolidin-3-yl)-hydroxy-dioxidophosphanium (SF-2312) as a promising inhibitor that fabricates finest attractive charges and conventional hydrogen bonds with S. pneumoniae alpha-enolase. Moreover, the pharmacokinetics of SF-2312 predict it as a therapeutic inhibitor for clinical trials. Like SF-2312, phosphono-acetohydroxamate (PhAH) also constructed adequate interactions at the active site of alpha-enolase, but it predicted less favourable than SF-2312 based on binding affinity. CONCLUSION: Briefly, SF-2312 and PhAH ligands could inhibit the role of alpha-enolase to restrain plasminogen binding, invasion and progression of S. pneumoniae. As per our investigation and analysis, SF-2312 is the most potent naturally existing inhibitor of S. pneumoniae alpha-enolase in current time.
Subject(s)
Phosphopyruvate Hydratase/chemistry , Streptococcus pneumoniae/enzymology , Administration, Oral , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacokinetics , Molecular Docking Simulation , Organophosphonates/chemistry , Organophosphonates/pharmacokinetics , Phosphonoacetic Acid/analogs & derivatives , Phosphonoacetic Acid/chemistry , Phosphonoacetic Acid/pharmacokinetics , Phosphopyruvate Hydratase/antagonists & inhibitors , Phosphopyruvate Hydratase/metabolism , Pneumococcal Infections/drug therapy , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacokineticsABSTRACT
Chemically modified oligonucleotides are being developed as a new class of medicines for curing conditions that previously remained untreatable. Three primary classes of therapeutic oligonucleotides are single-stranded antisense oligonucleotides (ASOs), double stranded small interfering RNAs (siRNAs), and oligonucleotides that induce exon skipping. Recently, ASOs, siRNAs, and exon skipping oligonucleotides have been approved for patients with unmet medical needs, and many other candidates are being tested in late stage clinical trials. In coming years, therapeutic oligonucleotides may match the promise of small molecules and antibodies. Interestingly, in the 1980s when we developed chemical methods for synthesizing oligonucleotides, no one would have imagined that these highly charged macromolecules could become future medicines. Indeed, the anionic nature and poor metabolic stability of the natural phosphodiester backbone provided a major challenge for the use of oligonucleotides as therapeutic drugs. Thus, chemical modifications of oligonucleotides were essential in order to improve their pharmacokinetic properties. Keeping this view in mind, my laboratory has developed a series of novel oligonucleotides where one or both nonbridging oxygens in the phosphodiester backbone are replaced with an atom or molecule that introduces molecular properties that enhance biological activity. We followed two complementary approaches. One was the use of phosphoramidites that could act directly as synthons for the solid phase synthesis of oligonucleotide analogues. This approach sometimes was not feasible due to instability of various synthons toward the reagents used during synthesis of oligonucleotides. Therefore, using a complementary approach, we developed phosphoramidite synthons that can be incorporated into oligonucleotides with minimum changes in the solid phase DNA synthesis protocols but contain a handle for generating appropriate analogues postsynthetically.This Account summarizes our efforts toward preparing these types of analogues over the past three decades and discusses synthesis and properties of backbone modified oligonucleotides that originated from the Caruthers' laboratory. For example, by replacing one of the internucleotide oxygens with an acetate group, we obtained so-called phosphonoacetate oligonucleotides that were stable to nucleases and, when delivered as esters, entered into cells unaided. Alternatively oligonucleotides bearing borane phosphonate linkages were found to be RNase H active and compatible with the endogenous RNA induced silencing complex (RISC). Oligonucleotides containing an alkyne group directly linked to phosphorus in the backbone were prepared as well and used to attach molecules such as amino acids and peptides.
Subject(s)
DNA/chemistry , Phosphorus/chemistry , Boranes/chemistry , Foscarnet/chemistry , Oligonucleotides/chemistry , Organophosphonates/chemistry , Organophosphorus Compounds/chemistry , Phosphines/chemistry , Phosphonoacetic Acid/chemistryABSTRACT
The mild acetylating agent, methyl acetyl phosphate, is used to estimate the p Ka values of some of the amine groups in peptides with sequences corresponding to a segment of the N-terminal tail of histone H4. When Ser-1 is not phosphorylated, the Lys ε amines have p Ka values in the range of 7.8-8.3, which are much lower than the currently assumed values. When Ser-1 is phosphorylated, the p Ka values of these Lys amines are elevated to the range of 8.8-10.3, thus providing the rationale for reports that they are then better substrates for acetyltransferases. Thus, reversal of suppressed p Ka values of Lys ε amines by Ser phosphorylation represents the basis for signaling in histone N-terminal tails to promote hyperacetylation, which is a hallmark of transcriptionally active euchromatin. In contrast, a state of hypoacetylation is present in the absence of phosphorylation as in transcriptionally inactive heterochromatin. A novel approach for estimating p Ka values based on a linkage between the Henderson-Hasselbalch and Michaelis-Menten equations indicates that the p Ka values of the Lys ε amines in H3 and H4 N-terminal tails have a highly variable charge gradient dependent on the location and proximity to the phosphorylation site.
Subject(s)
Histones/chemistry , Histones/metabolism , Acetylation , Amino Acid Sequence , Animals , Binding Sites , Histone Code , Histones/genetics , Humans , Kinetics , Lysine/chemistry , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphonoacetic Acid/analogs & derivatives , Phosphonoacetic Acid/chemistry , Phosphonoacetic Acid/metabolism , Serine/chemistry , Signal Transduction , Transcription, GeneticABSTRACT
Besides chemically interacting with hard tooth tissue, acidic functional monomers of self-etch adhesives should etch the prepared tooth surface to dissolve the smear layer and to provide surface micro-retention. Although the etching efficacy of functional monomers is commonly determined in terms of pH, the pH of adhesives cannot accurately be measured. Better is to measure the hydroxyapatite (HAp)-dissolving capacity, also considering that functional monomers may form monomer-Ca salts. Here, the etching efficacy of 6 functional monomers (GPDM, phenyl-P, MTEGP, 4-META, 6-MHP and 10-MDP) was investigated. Solutions containing 15 wt% monomer, 45 wt% ethanol, and 40 wt% water were prepared. Initially, we observed enamel surfaces exposed to monomer solution by scanning electron microscopy (SEM). X-ray diffraction (XRD) was employed to detect monomer-Ca salt formation. Phenyl-P exhibited a strong etching effect, while 10-MDP-treated enamel showed substance deposition, which was identified by XRD as 10-MDP-Ca salt. To confirm these SEM/XRD findings, we determined the etching efficacy of functional monomers by measuring both the concentration of Ca released from HAp using inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and the amount of monomer-Ca salt formation using 31P magic-angle spinning (MAS) nuclear magnetic resonance (NMR). ICP-AES revealed that the highest Ca concentration was produced by phenyl-P and the lowest Ca concentration, almost equally, by 4-META and 10-MDP. Only 10-MDP formed 10-MDP-Ca salts, indicating that 10-MDP released more Ca from HAp than was measured by ICP-AES. Part of the released Ca was consumed to form 10-MDP-Ca salts. It is concluded that the repeatedly reported higher bonding effectiveness of 10-MDP-based adhesives must not only be attributed to the more intense chemical bonding of 10-MDP but also to its higher etching potential, a combination the other functional monomers investigated lack.
Subject(s)
Dental Cements/chemistry , Dental Etching/methods , Methacrylates/chemistry , Phosphonoacetic Acid/analogs & derivatives , Glycerolphosphate Dehydrogenase/chemistry , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Materials Testing , Microscopy, Electron, Scanning , Organophosphorus Compounds/chemistry , Phosphonoacetic Acid/chemistry , Spectrophotometry, Atomic , Surface Properties , X-Ray DiffractionABSTRACT
The surface modification of Fe3O4-based magnetic nanoparticles (MNPs) with N-(phosphonomethyl)iminodiacetic acid (PMIDA) was studied, and the possibility of their use as magnetic resonance imaging contrast agents was shown. The effect of the added PMIDA amount, the reaction temperature and time on the degree of immobilization of this reagent on MNPs, and the hydrodynamic characteristics of their aqueous colloidal solutions have been systematically investigated for the first time. It has been shown that the optimum condition for the modification of MNPs is the reaction at 40 °C with an equimolar amount of PMIDA for 3.5 h. The modified MNPs were characterized by X-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric, and CHN elemental analyses. The dependence of the hydrodynamic characteristics of the MNP colloidal solutions on the concentration and pH of the medium was studied by the dynamic light scattering method. On the basis of the obtained data, we can assume that the PMIDA molecules are fixed on the surface of the MNPs as a monomolecular layer. The modified MNPs had good colloidal stability and high magnetic properties. The calculated relaxivities r2 and r1 were 341 and 102 mmol-1 s-1, respectively. The possibility of using colloidal solutions of PMIDA-modified MNPs as a T2 contrast agent for liver studies in vivo (at a dose of 0.6 mg kg-1) was demonstrated for the first time.
Subject(s)
Contrast Media/pharmacology , Liver/metabolism , Magnetite Nanoparticles/chemistry , Phosphonoacetic Acid/analogs & derivatives , Animals , CHO Cells , Cell Line, Tumor , Contrast Media/chemistry , Contrast Media/toxicity , Cricetulus , Humans , Magnetic Resonance Imaging/methods , Magnetite Nanoparticles/toxicity , Male , Mesocricetus , Phosphonoacetic Acid/chemistry , Phosphonoacetic Acid/pharmacology , Phosphonoacetic Acid/toxicity , TemperatureABSTRACT
Small molecule inhibitors have a powerful blocking action on viral polymerases. The bioavailability of the inhibitor, nevertheless, often raise a significant selectivity constraint and may substantially limit the efficacy of therapy. Phosphonoacetic acid has long been known to possess a restricted potential to block DNA biosynthesis. In order to achieve a better affinity, this compound has been linked with natural nucleotide at different positions. The structural context of the resulted conjugates has been found to be crucial for the acquisition by DNA polymerases. We show that nucleobase-conjugated phosphonoacetic acid is being accepted, but this alters the processivity of DNA polymerases. The data presented here not only provide a mechanistic rationale for a switch in the mode of DNA synthesis, but also highlight the nucleobase-targeted nucleotide functionalization as a route for enhancing the specificity of small molecule inhibitors.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , Enzyme Inhibitors/pharmacology , Nucleotides/pharmacology , Phosphonoacetic Acid/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , HIV-1/enzymology , Molecular Structure , Moloney murine leukemia virus/enzymology , Nucleotides/chemistry , Phosphonoacetic Acid/chemical synthesis , Phosphonoacetic Acid/chemistryABSTRACT
CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2'-O-methyl-3'-phosphonoacetate, or 'MP') incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications.
Subject(s)
CRISPR-Cas Systems , DNA Cleavage , Gene Editing/methods , RNA, Guide, Kinetoplastida/genetics , Base Sequence , Binding Sites/genetics , Humans , K562 Cells , Phosphonoacetic Acid/chemistry , RNA, Guide, Kinetoplastida/chemistry , RNA, Guide, Kinetoplastida/metabolismABSTRACT
The enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) is a key enzyme in the methylerythritol 4-phosphate pathway and is a target for the development of antibiotics, herbicides, and antimalarial drugs. DXPS catalyzes the formation of 1-deoxy-d-xylulose 5-phosphate (DXP), a branch point metabolite in isoprenoid biosynthesis, and is also used in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6). Previously, we found that DXPS is unique among the superfamily of thiamin diphosphate (ThDP)-dependent enzymes in stabilizing the predecarboxylation intermediate, C2-alpha-lactyl-thiamin diphosphate (LThDP), which has subsequent decarboxylation that is triggered by d-glyceraldehyde 3-phosphate (GAP). Herein, we applied hydrogen-deuterium (H/D) exchange MS (HDX-MS) of full-length Escherichia coli DXPS to provide a snapshot of the conformational dynamics of this enzyme, leading to the following conclusions. (i) The high sequence coverage of DXPS allowed us to monitor structural changes throughout the entire enzyme, including two segments (spanning residues 183-238 and 292-317) not observed by X-ray crystallography. (ii) Three regions of DXPS (spanning residues 42-58, 183-199, and 278-298) near the active center displayed both EX1 (monomolecular) and EX2 (bimolecuar) H/D exchange (HDX) kinetic behavior in both ligand-free and ligand-bound states. All other peptides behaved according to the common EX2 kinetic mechanism. (iii) The observation of conformational changes on DXPS provides support for the role of conformational dynamics in the DXPS mechanism: The closed conformation of DXPS is critical for stabilization of LThDP, whereas addition of GAP converts DXPS to the open conformation that coincides with decarboxylation of LThDP and DXP release.
Subject(s)
Mass Spectrometry/methods , Transferases/metabolism , Glyceraldehyde 3-Phosphate/chemistry , Glyceraldehyde 3-Phosphate/metabolism , Models, Molecular , Pentosephosphates/chemistry , Pentosephosphates/metabolism , Phosphonoacetic Acid/analogs & derivatives , Phosphonoacetic Acid/chemistry , Phosphonoacetic Acid/metabolism , Protein Binding , Protein ConformationABSTRACT
A versatile and general catalytic strategy has been developed for the α-arylation of phosphonoacetates utilizing parallel microscale experimentation. These α-substituted phosphonoacetates are widely useful, notably as substrates in the Horner-Wadsworth-Emmons-type olefinations. However, the current routes to these products involve harsh conditions, limiting the variety of functionality. The reported method can be used with a variety of aryl chlorides and aryl bromides, including several heterocyclic examples.
Subject(s)
Alkenes/chemistry , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/chemical synthesis , Phosphonoacetic Acid/chemical synthesis , Catalysis , Molecular Structure , Phosphonoacetic Acid/chemistry , StereoisomerismABSTRACT
BACKGROUND: A deeper knowledge on the effects of the degradation of magnetic nanoparticles on their magnetic properties is required to develop tools for the identification and quantification of magnetic nanoparticles in biological media by magnetic means. METHODS: Citric acid and phosphonoacetic acid-coated magnetic nanoparticles have been degraded in a medium that mimics lysosomal conditions. Magnetic measurements and transmission electron microscopy have been used to follow up the degradation process. RESULTS: Particle size is reduced significantly in 24 h at pH 4.5 and body temperature. These transformations affect the magnetic properties of the compounds. A reduction of the interparticle interactions is observed just 4 h after the beginning of the degradation process. A strong paramagnetic contribution coming from the degradation products appears with time. CONCLUSIONS: A model for the in vivo degradation of magnetic nanoparticles has been followed to gain insight on the changes of the magnetic properties of iron oxides during their degradation. The degradation kinetics is affected by the particle coating, in our case being the phosphonoacetic acid-coated particles degraded faster than the citric acid-coated ones.
Subject(s)
Citric Acid/chemistry , Lysosomes/chemistry , Lysosomes/ultrastructure , Magnetite Nanoparticles/chemistry , Magnetite Nanoparticles/ultrastructure , Phosphonoacetic Acid/chemistry , Biomimetic Materials/chemistry , Biomimetic Materials/radiation effects , Body Fluids/chemistry , Coated Materials, Biocompatible/chemistry , Coated Materials, Biocompatible/radiation effects , Electric Impedance , Kinetics , Magnetite Nanoparticles/radiation effects , Materials Testing , Particle Size , TemperatureABSTRACT
A novel route for the synthesis of 2',3'-difluorinated 5'-deoxythreosyl phosphonic acid nucleosides from glyceraldehyde using the Horner-Emmons reaction in the presence of triethyl α-fluorophosphonoacetate is described. The second fluorination at the 2'-position was an electrophilic reaction performed using N-fluorodibenzenesulfonimide. Glycosylation reactions between the nucleosidic bases and glycosyl donor 9 generated nucleosides that were further phosphonated and hydrolyzed to produce the desired nucleoside analogues. The synthesized nucleoside analogues 13, 16, 20, and 23 were tested for anti- human immunodeficiency virus (HIV) activity as well as cytotoxicity. Adenine derivative 16 showed significant anti-HIV activity up to 100 µM.
Subject(s)
Anti-HIV Agents/chemical synthesis , Nucleosides/chemistry , Phosphorous Acids/chemical synthesis , Acetates/chemistry , Adenine/chemistry , Anti-HIV Agents/chemistry , Benzophenones/chemistry , Chromatography, Thin Layer , Drug Design , Glyceraldehyde/chemistry , Guanine/chemistry , HIV Infections/drug therapy , Humans , Hydrogen/chemistry , Leukocytes, Mononuclear/drug effects , Magnetic Resonance Spectroscopy , Phosphonoacetic Acid/analogs & derivatives , Phosphonoacetic Acid/chemistry , Phosphorous Acids/chemistry , Spectrophotometry , Spectroscopy, Fourier Transform Infrared , Sulfonamides/chemistryABSTRACT
CoCr alloy is commonly used in various cardiovascular medical devices for its excellent physical and mechanical properties. However, the formation of blood clots on the alloy surfaces is a serious concern. This research is focused on the surface modification of CoCr alloy using varying concentrations (1, 25, 50, 75, and 100 mM) of phosphoric acid (PA) and phosphonoacetic acid (PAA) to generate various surfaces with different wettability, chemistry, and roughness. Then, the adsorption of blood plasma proteins such as albumin and fibrinogen and the adhesion, activation, and aggregation of platelets with the various surfaces generated were investigated. Contact angle analysis showed PA and PAA coatings on CoCr provided a gradient of hydrophilic surfaces. FTIR showed PA and PAA were covalently bound to CoCr surface and formed different bonding configurations depending on the concentrations of coating solutions used. AFM showed the formation of homogeneous PA and PAA coatings on CoCr. The single and dual protein adsorption studies showed that the amount of albumin and fibrinogen adsorbed on the alloy surfaces strongly depend on the type of PA and PAA coatings prepared by different concentrations of coating solutions. All PA coated CoCr showed reduced platelet adhesion and activation when compared to control CoCr. Also, 75 and 100 mM PA-CoCr showed reduced platelet aggregation. For PAA coated CoCr, no significant difference in platelet adhesion and activation was observed between PAA coated CoCr and control CoCr. Thus, this study demonstrated that CoCr can be surface modified using PA for potentially reducing the formation of blood clots and improving the blood compatibility of the alloy.
Subject(s)
Albumins/chemistry , Chromium/chemistry , Cobalt/chemistry , Fibrinogen/chemistry , Phosphonoacetic Acid/chemistry , Phosphoric Acids/chemistry , Platelet Adhesiveness , Adsorption , Albumins/metabolism , Alloys/chemistry , Blood Platelets/metabolism , Fibrinogen/metabolism , Spectroscopy, Fourier Transform Infrared , Surface PropertiesABSTRACT
A new, general method for the synthesis of phosphono-γ-butyrolactones has been achieved through Mn(OAc)3-mediated radical oxidative phosphonation and lactonization of alkenoic acids with H-phosphonates and H-phosphine oxide. Mn(OAc)3 can be readily prepared from Mn(OAc)2 in the laboratory. This transformation allows the direct formation of a P-C bond and the construction of a lactone ring in one reaction.
Subject(s)
4-Butyrolactone/chemical synthesis , Acetates/chemistry , Manganese/chemistry , Organometallic Compounds/chemistry , Phosphonoacetic Acid/chemistry , 4-Butyrolactone/chemistry , Lactones/chemistry , Molecular StructureABSTRACT
Phosphonates (C-PO3²â») have applications as antibiotics, herbicides, and detergents. In some environments, these molecules represent the predominant source of phosphorus, and several microbes have evolved dedicated enzymatic machineries for phosphonate degradation. For example, most common naturally occurring phosphonates can be catabolized to either phosphonoacetaldehyde or phosphonoacetate, which can then be hydrolyzed to generate inorganic phosphate and acetaldehyde or acetate, respectively. The phosphonoacetaldehyde oxidase gene (phnY) links these two hydrolytic processes and provides a previously unknown catabolic mechanism for phosphonoacetate production in the microbial metabolome. Here, we present biochemical characterization of PhnY and high-resolution crystal structures of the apo state, as well as complexes with substrate, cofactor, and product. Kinetic analysis of active site mutants demonstrates how a highly conserved aldehyde dehydrogenase active site has been modified in nature to generate activity with a phosphonate substrate.
Subject(s)
Acetaldehyde/analogs & derivatives , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Phosphonoacetic Acid/metabolism , Acetaldehyde/chemistry , Acetaldehyde/metabolism , Apoproteins/chemistry , Apoproteins/metabolism , Catalytic Domain/genetics , Crystallography, X-Ray , Kinetics , Models, Molecular , Molecular Structure , NAD/chemistry , NAD/metabolism , Oxidoreductases/genetics , Phosphonoacetic Acid/chemistry , Sinorhizobium meliloti/enzymologyABSTRACT
Aspartate transcarbamoylase (ATCase) catalyzes the synthesis of N-carbamoyl-L-aspartate from carbamoyl phosphate and aspartate in the second step of the de novo biosynthesis of pyrimidines. In prokaryotes, the first three activities of the pathway, namely carbamoyl phosphate synthetase (CPSase), ATCase and dihydroorotase (DHOase), are encoded as distinct proteins that function independently or in noncovalent association. In animals, CPSase, ATCase and DHOase are part of a 243 kDa multifunctional polypeptide named CAD. Up-regulation of CAD is essential for normal and tumour cell proliferation. Although the structures of numerous prokaryotic ATCases have been determined, there is no structural information about any eukaryotic ATCase. In fact, the only detailed structural information about CAD is that it self-assembles into hexamers and trimers through interactions of the ATCase domains. Here, the expression, purification and crystallization of the ATCase domain of human CAD is reported. The recombinant protein, which was expressed in bacteria and purified with good yield, formed homotrimers in solution. Crystallization experiments both in the absence and in the presence of the inhibitor PALA yielded small crystals that diffracted X-rays to 2.1 Å resolution using synchrotron radiation. The crystals appeared to belong to the hexagonal space group P6(3)22, and Matthews coefficient calculation indicated the presence of one ATCase subunit per asymmetric unit, with a solvent content of 48%. However, analysis of the intensity statistics suggests a special case of the P21 lattice with pseudo-symmetry and possibly twinning.
Subject(s)
Aspartate Carbamoyltransferase/chemistry , Aspartic Acid/analogs & derivatives , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/chemistry , Dihydroorotase/chemistry , Enzyme Inhibitors/chemistry , Phosphonoacetic Acid/analogs & derivatives , Aspartate Carbamoyltransferase/genetics , Aspartate Carbamoyltransferase/metabolism , Aspartic Acid/chemistry , Aspartic Acid/metabolism , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/metabolism , Crystallization , Crystallography, X-Ray , Dihydroorotase/genetics , Dihydroorotase/metabolism , Enzyme Inhibitors/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Models, Molecular , Phosphonoacetic Acid/chemistry , Phosphonoacetic Acid/metabolism , Protein Multimerization , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SynchrotronsABSTRACT
The degradation of phosphonates in the natural environment constitutes a major route by which orthophosphate (Pi) is regenerated from organic phosphorus and recently implicated in marine methane production, with ramifications to environmental pollution issues and global climate change concerns. This work explores the application of stable oxygen isotope analysis in elucidating the CP bond cleavage mechanism(s) of phosphonates by UV photo-oxidation and for tracing their sources in the environment. The two model phosphonates used, glyphosate and phosphonoacetic acid were effectively degraded after exposure to UV irradiation. The isotope results indicate the involvement of both ambient water and atmospheric oxygen in the CP bond cleavage and generally consistent with previously posited mechanisms of UV-photon excitation reactions. A model developed to calculate the oxygen isotopic composition of the original phosphonate P-moiety, shows both synthetic phosphonates having distinctly lower values compared to naturally derived organophosphorus compounds. Such mechanistic models, based on O-isotope probing, are useful for tracing the sources and reactions of phosphonates in the environment.
Subject(s)
Glycine/analogs & derivatives , Organophosphonates/chemistry , Oxygen Isotopes/chemistry , Phosphonoacetic Acid/chemistry , Biodegradation, Environmental , Environmental Pollutants/chemistry , Glycine/chemistry , Hydrolysis , Methane/chemistry , Models, Chemical , Oxidants/chemistry , Phosphates/chemistry , Photolysis , Time Factors , Ultraviolet Rays , GlyphosateABSTRACT
Thiamin diphosphate (ThDP)-dependent enzymes play vital roles in cellular metabolism in all kingdoms of life. In previous kinetic and structural studies, a communication between the active centers in terms of a negative cooperativity had been suggested for some but not all ThDP enzymes, which typically operate as functional dimers. To further underline this hypothesis and to test its universality, we investigated the binding of substrate analogue methyl acetylphosphonate (MAP) to three different ThDP-dependent enzymes acting on substrate pyruvate, namely, the Escherichia coli E1 component of the pyruvate dehydrogenase complex, E. coli acetohydroxyacid synthase isoenzyme I, and the Lactobacillus plantarum pyruvate oxidase using isothermal titration calorimetry. The results unambiguously show for all three enzymes studied that only one active center of the functional dimers accomplishes covalent binding of the substrate analogue, supporting the proposed alternating sites reactivity as a common feature of all ThDP enzymes and resolving the recent controversy in the field.
Subject(s)
Enzymes/chemistry , Enzymes/metabolism , Thiamine Pyrophosphate/metabolism , Acetolactate Synthase/chemistry , Acetolactate Synthase/metabolism , Binding Sites , Calorimetry/methods , Catalytic Domain , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Kinetics , Nuclear Magnetic Resonance, Biomolecular , Phosphonoacetic Acid/analogs & derivatives , Phosphonoacetic Acid/chemistry , Phosphonoacetic Acid/metabolism , Protein Binding , Pyruvate Dehydrogenase (Lipoamide)/chemistry , Pyruvate Dehydrogenase (Lipoamide)/metabolism , Pyruvate Oxidase/chemistry , Pyruvate Oxidase/metabolism , Thermodynamics , Thiamine Pyrophosphate/chemistryABSTRACT
We have established an efficient method for preparing methyl bis(2,2,2-trifluoroethoxy)bromophosphonoacetate, which we developed for the stereoselective synthesis of (E)-α-bromoacrylates. This improved method enables the reagent to be prepared reproducibly in one step from methyl bis(2,2,2-trifluoroethoxy)phosphonoacetate.
Subject(s)
Acrylates/chemical synthesis , Phosphonoacetic Acid/analogs & derivatives , Phosphonoacetic Acid/chemical synthesis , Acrylates/chemistry , Halogenation , Phosphonoacetic Acid/chemistry , StereoisomerismABSTRACT
This study aimed to examine the shear bond strength between cobalt chromium alloy and autopolymerizing acrylic resin using experimental primers containing 5, 10, and 15 wt% of 4-methacryloxyethyl trimellitic anhydride or 1, 2, and 3 wt% of 3-methacryloxypropyl-trimethoxysilane comparison to 5 commercial primers (ML primers, Alloy primer, Metal/Zirconia primer, Monobond S, and Monobond plus). Sixty alloy specimens were sandblasted and treated with each primer before bonded with an acrylic resin. The control group was not primed. The shear bond strengths were tested and statistically compared. Specimens treated with commercial primers significantly increased the shear bond strength of acrylic resin to cobalt chromium alloy (p<0.05). The highest shear bond strength was found in the Alloy primer group. Among experimental group, using 10 wt% of 4-methacryloxyethyl trimellitic anhydride -or 2 wt% of 3-methacryloxypropyltrimethoxysilane enhanced highest shear bond strength. The experimental and commercial primers in this study all improved bonding of acrylic resin to cobalt chromium alloy.