Phosphonate Monoester Research Articles

Effect of Structural Variation within Lipophilic Lariat Ether Phosphonic Acid Monoesters on the Selectivity and Efficiency of Competitive Alkali Metal Cation Extraction into Chloroform

Lipophilic lariat ether phosphonic acid monoethyl esters with systematic crown ether ring size variation from 12-crown-4 to 24-crown-8 are utilized for competitive alkali metal cation extractions from aqueous solutions into chloroform. Effective alkali metal cation extraction from weakly acidic, neutral, and basic aqueous solutions is achieved. With 4, 5, and 6 oxygens in the crown ether rings, selectivities for Li(+), Na(+), and K(+), respectively, are observed. An 18-crown-6 phosphonic acid monoester exhibits excellent extraction selectivity for K(+) with K(+)/Li(+) and K(+)/Na(+) > 100. The lipophilic group attachment site, as well as the crown ether ring size, is shown to influence the extraction selectivity for the lariat ether phosphonic monoesters.

Anti-Cocaine Catalytic Antibodies

Over 50,000,000 Americans have used cocaine (1) since 1980 and approximately 2,000,000 are presently addicted with disastrous medical and social consequences.1 At present, no efficient drug therapy is available for the management of cocaine abuse and dependence. Cocaine is hypothesized to exert its addictive effect by blocking a transport protein for the neurotransmitter dopamine, and the difficulties inherent in blocking a blocker may in part explain the failure to develop a useful therapeutic agent. One alternative to the classical receptor antagonist approach would be a circulating catalytic antibody that could degrade cocaine and interfere with the delivery of the drug to the central nervous system. To obtain antibodies able to catalyze the hydrolysis of cocaine to the inactive products, ecgonine methyl ester (2) and benzoic acid (3), we synthesized a series of stable phosphonate monoester transition state analogs for benzoyl esterolysis (4a-c). With these immunogens we elicited the first artificial enzymes able to degrade cocaine in vitro.2.3

Anti-Cocaine Catalytic Antibodies

Over 50,000,000 Americans have used cocaine (1) since 1980 and approximately 2,000,000 are presently addicted with disastrous medical and social consequences.1 At present, no efficient drug therapy is available for the management of cocaine abuse and dependence. Cocaine is hypothesized to exert its addictive effect by blocking a transport protein for the neurotransmitter dopamine, and the difficulties inherent in blocking a blocker may in part explain the failure to develop a useful therapeutic agent. One alternative to the classical receptor antagonist approach would be a circulating catalytic antibody that could degrade cocaine and interfere with the delivery of the drug to the central nervous system. To obtain antibodies able to catalyze the hydrolysis of cocaine to the inactive products, ecgonine methyl ester (2) and benzoic acid (3), we synthesized a series of stable phosphonate monoester transition state analogs for benzoyl esterolysis (4a-c). With these immunogens we elicited the first artificial enzymes able to degrade cocaine in vitro.2.3

Reaction of soluble penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus with beta-lactams and acyclic substrates: kinetics in homogeneous solution.

The kinetics of reaction of solubilized penicillin-binding protein 2a (sPBP2a) of methicillin-resistant Staphylococcus aureus with a variety of beta-lactams and acyclic species was studied in homogeneous aqueous solution at 37 degreesC in 25 mM Hepes buffer, pH7.0, containing 1 M NaCl. Under these conditions, but not at lower salt concentrations, protein precipitation did not occur either during or after the reaction. The reactions of beta-lactams in general could be monitored by competition with a chromophoric beta-lactam, nitrocefin, or directly in certain cases by protein fluorescence. Rate constants for reaction of a wide variety of beta-lactams are reported. The interactions are characterized by a slow second-order acylation reaction followed by a slower deacylation. For example, the rate constants for benzylpenicillin were 12 M-1.s-1 and 3x10(-5) s-1 respectively. The acylation is slow in comparison with those of normal non-resistant high-molecular-mass penicillin-binding proteins. sPBP2a also seemed to catalyse the slow hydrolysis of a variety of acyclic depsipeptides but not that of a d-Ala-d-Ala peptide. The reactions with certain depsipeptides also led to protein precipitation. These reactions were, however, not affected by prior blockage of the beta-lactam-binding site by benzylpenicillin and thus might take place elsewhere on the enzyme. Two classes of potential transition- state analogue inhibitors, phosphonate monoesters and boronates, seemed to have little effect on the rate of reaction of sPBP2a with nitrocefin and therefore seem to have little affinity for the beta-lactam-binding/D,D-peptidase site.

Co-ordination chemistry and molecular mechanics study of the magnesium(II) and calcium(II) complexes of trisubstituted 1,4,7-triazacyclononane derivatives

The affinities of 1,4,7-tris(2-hydroxyalkyl)-1,4,7-triazacyclononane derivatives for MgII and CaII were found to differ greatly. Whereas 1,4,7-tris(2-hydroxyethyl)- (L1), 1,4,7-tris(2-hydroxy-2-methylpropyl)- (L2) and the unsymmetrical 1,4,7-tris(2-hydroxypropyl)-1,4,7-triazacyclononane derivative L3b barely discriminated between MgII and CaII, the symmetrical isomer L3a was more than 500 times more selective for MgII than for CaII. Similar selectivity differences were observed between the diastereomers of 1,4,7-tris(2-hydroxy-2-phenylethyl)- (L4) and 1,4,7-tris(2-hydroxydodecyl)-1,4,7-triazacyclononane (L5). These selectivities were related to the structure of the magnesium complexes as shown by molecular mechanics (MMX) calculations. Only those ligands favoring the formation of a magnesium complex with a large twist angle between the planes of co-ordinating oxygens and ring nitrogens resulting in a small, tight cavity showed a large preference for MgII over CaII. The MMX calculations predicted large twist angle structures for phosphinate derivatives, and for a phosphonate monoester derivative, and these ligands were found to have a high selectivity for MgII. Similarly, the calculated preference for the smaller twist angle correctly predicted the lack of MgII/CaII selectivity for acetate and amide derivatives and for a phosphonate diester derivative. Equilibration of the complexes of the ligands in the presence of both MgII and CaII was slow, as shown for example by kd,0 = 2.1 × 10–4 s–1 for CaL3a. These dissociation rates were a factor of 100 times larger at the boundary of a two-phase (water–chloroform) system.

Structure-activity studies of the inhibition of serine β-lactamases by phosphonate monoesters

A new series of phosphonyl derivatives has been prepared and tested for inhibition of serine (class A and C) β-lactamases. Variations of the leaving group in a series of methyl phosphonates showed that leaving groups better than the previously employed p-nitrophenoxide could give more effective inhibitors. Inclusion of a negative charge in the leaving group did not, per se, lead to better inhibitors. Aryl phosphonates appeared more effective than those with electronically comparable but smaller leaving groups. The combination of a good leaving group, 2,4-dinitrophenoxide, with an amido side-chain, phenylmethylsulfonamido—the latter rather than phenylacetamido in order to increase the stability of the compound with respect to intramolecular nucleophilic catalysis of hydrolysis by the amide group—did not yield overall a better inhibitor than previously employed p-nitrophenyl phosphonates. These results give the first indication of specific interactions between a β-lactamase and the leaving group of a phosphonate inhibitor. Only one enantiomer of a chiral thiophosphonate, presumably the R p isomer, was an effective inhibitor. Addition of either a d- or a l-methyl group to the methylene group of a p-nitrophenyl amidomethylphosphonate did not enhance the inhibitory ability of the phosphonate. Class A β-lactamases remain refractory to phosphonates.

Phosphonate Diester and Phosphonamide Synthesis. Reaction Coordinate Analysis by 31P NMR Spectroscopy: Identification of Pyrophosphonate Anhydrides and Highly Reactive Phosphonylammonium Salts1

A series of phosphonochloridates was prepared from the corresponding phosphonate monoesters, and their reactions with alcohols, amines, and the bisnucleophile 4-aminobutan-1-ol have been investigated using 31P NMR spectroscopy. In the conversion of phosphonate monoesters to phosphonochloridates via the addition of thionyl chloride or oxalyl chloride, pyrophosphonate anhydrides were found to be formed readily as byproducts. The anhydrides reacted readily with alcohols, but more slowly than the corresponding phosphonochloridates, and only sluggishly, if at all, with amines. Therefore, when phosphonamides are prepared, anhydride formation must be suppressed. This is accomplished when the monoester is added to the chloridating agent. Unhindered phosphonochloridates reacted predominantly with the amino function of 4-aminobutan-1-ol to furnish the phosphonamidates, whereas a sterically hindered phosphonochloridate demonstrated a preference for O-coupling. This result indictes that the energy gained during P−O bond formation surmounts the kinetic barrier resulting from steric hindrance more effectively than formation of the weaker P−N bond. Importantly, treatment of the phosphonochloridates with tertiary amines prior to addition of the nucleophile resulted in the formation of hitherto unrecognized phosphonylating agents, which we formulated as phosphonyltrialkylammonium salts. The latter, unlike the anhydrides, are more reactive than the phosphonochloridates toward both alcohols and amines, affording improved yields of phosphonate esters and amides. These improved yields are not obtained when triethylamine is added simultaneously with the nucleophile merely to neutralize acid rather than as a deliberate step to generate the phosphonyltrialkylammonium salts. Use of these novel phosphonylating agents proceeded without concomitant racemization at stereogenic centers α to phosphorous. Interestingly, reaction of even an unhindered phosphonyltriethylammonium salt with 4-aminobutan-1-ol favored O-phosphonylation over N-phosphonylation by a factor of 8, demonstrating that both the charge transfer in the transition state and steric hindrance affect the propensity for P−O vis a vis P−N bond formation. In marked contrast, simultaneous addition of this bisnucleophile and triethylamine, like coupling in the absence of tertiary amine, afforded the phosphonate and phosphonamide in nearly equal amounts.

Anti-Cocaine Catalytic Antibodies—A Novel Approach to the Problem of Addiction

ABSTRACT Cocaine reinforces its self-administration in relation to the magnitude of and rate of rise to the peak serum concentration of the drug. Catalytic antibodies are artificial enzymes which could reduce serum cocaine concentrations, deprive the abuser of cocaine's reinforcing effect and thus favor extinction of the addiction. Catalytic antibodies are elicited by immunization with a stable analog of a transition-state for a chemical reaction. Through our new method for synthesizing phosphonate monoesters, we constructed several phos-phonate-based transition-state analogs of cocaine hydrolysis. Using these analogs, monoclonal antibodies were elicited and, thus far, nine anti-analog antibodies with hydrolytic activity against cocaine have been identified, cloned and studied. The activity of one of these antibodies, 15A10, is sufficient to commence preclinical studies.

Identification, Characterization, and Cloning of a Phosphonate Monoester Hydrolase from Burkholderia caryophilli PG2982

The glyphosate-degrading bacterium, Burkholderia caryophilli PG2982, was observed to utilize glyceryl glyphosate as a sole phosphorus source. The hydrolysis of glyceryl glyphosate to glyphosate by a phosphonate ester hydrolase (PEH) was identified as the first metabolic step in the mineralization pathway. This observation provides the first biological role for a phosphonate ester hydrolase activity. Purified PEH enzyme hydrolyzed several phosphonate esters including p-nitrophenyl phenylphosphonate, beta-naphthyl phenylphosphonate, and 5-bromo-4-chloro-3-indolyl phenylphosphonate. The purified PEH also hydrolyzed some phosphodiesters including p-nitrophenyl 5'-thymidine monophosphate and p-nitrophenyl phosphorylcholine. The most catalytically efficient substrate identified was bis-(p-nitrophenyl) phosphate with a Km of 0.9 mM and a kcat of 6.2 x 10(2) min-1, suggesting that the enzyme may also function in vivo as a phosphodiesterase. The native enzyme was a homotetramer of 58-kDa subunits and exhibited a pI of 4.2. The enzyme activity had a pH activity optimum of 9.0 and was stimulated 14-fold by Mn2+ ions, but a metal cofactor was not essential for activity. N-terminal and tryptic fragment amino acid sequences were obtained from the purified PEH protein and used to clone the B. caryophilli PG2982 gene, designated pehA. The unique substrate specificity of the enzyme and potential use as a novel conditional lethal gene in plants are discussed.

A phosphonate monoester hydrolase from Burkholderia caryophilli PG2982 is useful as a conditional lethal gene in plants.

A bacterial phosphonate monoester hydrolase was evaluated in plants as a conditional lethal gene useful for cell ablation and negative selection. Glyphosate is a potent herbicide whereas its phosphonate monoester derivative, glyceryl glyphosate, is approximately 50-fold less active. A phosphonate monoesterase gene (pehA) encoding an enzyme that hydrolyzes phosphonate esters including glyceryl glyphosate to glyphosate and glycerol was cloned from the glyphosate metabolizing bacterium, Burkholderia caryophilli PG2982. Constitutive expression of the pehA gene in Escherichia coli and Arabidopsis thaliana RLD had no observable phenotypic effects on growth and development. However, cells and plants expressing the pehA gene were killed when treated with glyceryl glyphosate. The phytotoxicity resulted from the hydrolysis of glyceryl glyphosate to glyphosate and subsequent inhibition of aromatic amino acid biosynthesis. As an example of tissue-specific cell ablation, floral sterility without vegetative toxicity was demonstrated by expressing the pehA gene using a tapetal-specific promoter and treating the mature plants with glyceryl glyphosate. A chromogenic phosphonate ester substrate, 5-bromo-4-chloro-indolyl phenylphosphonate, was used to monitor in situ expression of the pehA gene. The general utility of the pehA gene as a heterologous conditional lethal gene in plants is discussed.

A Semiempirical Transition State Structure for the First Step in the Alkaline Hydrolysis of Cocaine. Comparison between the Transition State Structure, the Phosphonate Monoester Transition State Analog, and a Newly Designed Thiophosphonate Transition State Analog

Semiempirical molecular orbital calculations have been performed for the first step in the alkaline hydrolysis of the neutral benzoylester of cocaine. Successes, failures, and limitations of these calculations are reviewed. A PM3 calculated transition state structure is compared with the PM3 calculated structure for the hapten used to induce catalytic antibodies for the hydrolysis of cocaine. Implications of these calculations for the computer–aided design of transition state analogs for the induction of catalytic antibodies are discussed.

Studies on kinetics of forward and backward extraction of neodymium by using phosphonic acid monoester as an acidic extractant

Studies on the kinetics of neodymium transport through a bulk liquid membrane, in apparatus consisting of a source, membrane and receiving phases, have been carried out. Different experimental variables, such as the pH, carrier, metal ion and stripping agent concentrations, have been explored. However, the kinetic and solvent extraction methods were compared to determine the equilibrium extraction constant. Complexation mechanism models have been developed. The kinetic behaviour of the ion transport based on forward and backward extraction processes with the consideration of controlled parameters was predicted. The ion transport of neodymium in the receiving or membrane phase envisages the determination of forward and backward extraction rate constants.

Crystallographic Structure of a Phosphonate Derivative of the Enterobacter cloacae P99 Cephalosporinase: Mechanistic Interpretation of a .beta.-Lactamase Transition-State Analog

The crystal structure of a complex formed on reaction of the Enterobacter cloacae P99 cephalosporinase (beta-lactamase) with a phosphonate monoester inhibitor, m-carboxyphenyl [[N-[(p-iodophenyl)acetyl]amino]methyl]phosphonate, has been obtained at 2.3-A resolution. The structure shows that the inhibitor has phosphonylated the active site serine (Ser64) with loss of the m-carboxyphenol leaving group. The inhibitor is positioned in the active site in a way that can be interpreted in terms of a transition-state analog. The arylacetamido side chain is placed as anticipated from analogous beta-lactamoyl complexes of penicillin-recognizing enzymes, with the amino group hydrogen-bonded to the backbone carbonyl of Ser318 (of the B3 beta-strand) and to the amides of Gln120 and Asn152. There is support in the asymmetry of the hydrogen bonding of this side chain to the protein and in the 2-fold disorder of the benzyl group for the considerable breadth in substrate specificity exhibited by class C beta-lactamases. One phosphonyl oxygen atom is in the oxyanion hole, hydrogen-bonded to main-chain NH groups of Ser318 and Ser64, while the other oxygen is solvated, not within hydrogen-bonding distance of any amino acid side chain. The closest active site functional group to the solvated oxygen atom is the Tyr150 hydroxyl group (3.4A); Lys67 and Lys315 are quite distant (4.3 and 5.7 A, respectively). Rather, Tyr150 and Lys67 are more closely associated with Ser64O gamma (2.9 and 3.3 A). This arrangement is interpreted in terms of the transition state for breakdown of the tetrahedral intermediate in the deacylation step of catalysis, where the Tyr150 phenol seems the most likely general acid. Thus, Tyr150, as the phenoxide anion, would be the general base catalyst in acylation, as proposed by Oefner et al. [Nature (1990) 343, 284-288]. The structure is compared with that of a similar phosphonate derivative of a class A beta-lactamase [Chen et al. (1993) J. Mol. Biol. 234, 165-178], and mechanistic comparisons are made. The sensitivity of serine beta-lactamases, as opposed to serine proteinases, toward inhibition by phosphonate monoanions is supported by electrostatic calculations showing a net positive potential only in the catalytic sites of the beta-lactamases.

First structural characterisation of an amino phosphonate monoester metal complex

A new type of tetradentate amino phosphonate monoester has been synthesized by alkaline hydrolysis of the parent phosphonate diester; the crystal structure of its copper complex reveals a monoester ligand-bridged centrosymmetric dimeric five-co-ordinate copper(II) complex.

Relaxometry, animal biodistribution, and magnetic resonance imaging studies of some new gadolinium (III) macrocyclic phosphinate and phosphonate monoester complexes.

The Gd3+ complexes of three new phosphorus containing tetraaza macrocycles (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis (methylene ethylphosphonic acid), H4DOTEP; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis (methylene phosphonic acid monoethylester), H4DOTPME; and the corresponding n-butyl ester, H4DOTPMB) were prepared and examined for possible use as MRI contrast agents. Although thermodynamically and kinetically less stable than Gd(DOTA)- in saline and HSA solution, the stability of these new macrocyclic complexes appears to be sufficiently high for in vivo applications. NMRD relaxivity profiles of the three complexes indicate that the number of inner sphere water molecules for these chelates is < or = 1 and that the more hydrophobic chelate, Gd(DOTPMB), binds to human serum albumin (HSA). Biodistribution studies of the radioactive 153Sm or 159Gd chelates in rats, gamma imaging of the 153Sm chelates in rats, and proton MRI studies of the nonradioactive Gd3+ chelates in rabbits all indicate that the DOTPMB complexes accumulate preferentially in the liver, spleen, and small intestines while the more hydrophilic DOTEP and DOTPME complexes appear to display renal clearances similar to other low molecular weight contrast agents.

Kinetics and mechanism of .beta.-lactamase inhibition by phosphonamidates: The quest for a proton

Four phosphonamidates were synthesized as potential beta-lactamase inhibitors. Three were methanephosphonamidates [CH3PO2-NHR/Ar, where R/Ar = 4-methoxybenzyl (3a), phenyl (3b), and m-nitrophenyl (3c)], while the fourth, PhCH2OCONHCH2PO2-NHPh (2a), also contained a beta-lactamase active site-directed amido side chain. The pH-rate profiles for the hydrolyses of these compounds in the absence of enzyme demonstrated the necessity of nitrogen protonation in the transition state; the reactive neutral form was the zwitterion, IH. The four phosphonamidates irreversibly inhibited the class C beta-lactamase of Enterobacter cloacae P99 by phosphonylation of the active-site serine hydroxyl group, but they displayed strikingly different inhibition pH-rate profiles. The pH profile and inhibition rates of the N-alkyl derivative 3a could be understood in terms of a direct reaction between IH and EH, the form of the enzyme reactive with substrates and phosphonate monoester inhibitors. The pH profile for 2a also indicated that EH was the reactive enzyme form, but its direct reaction with IH is unlikely because of the low concentration of the latter, stemming from its low nitrogen pKa. In this case, proton uptake from solution subsequent to phosphonamidate anion binding probably accounts for the observed rates. The anilides 3b and 3c were weak inhibitors with respect to 2a and 3a. Their major inhibitory activity, observed at above neutral pH in contrast to that of 2a and 3a, probably involves modes of binding not typical of substrate analogs but which allow access to protons. Inhibition by 3c was interpreted to involve rate-determining protonation at high pH. At and above neutral pH, phosphonamidates will generally be less effective inhibitors than phosphonate p-nitrophenyl monoesters. Below pH 7, enzyme-specific phosphonamidates, especially N-alkyl derivatives, will become more effective than the esters. The results are consistent with the view that, because of the specific geometry of the phosphonyl-transfer transition state, the effectiveness of phosphonic acid derivatives as beta-lactamase inhibitors is limited by the absence of a suitably positioned general acid catalyst at the active site.

Synthesis of mixed phosphonate diester analogues of dipeptides using BOP or PyBOP reagents

Mixed phosphonate diesters of a N-protected α-amino phosphonic acid are prepared from phosphonate monoesters using BOP or PyBOP as activating agent. This reaction proceeds without racemisation.

Antibody-Catalyzed Degradation of Cocaine

Immunization with a phosphonate monoester transition-state analog of cocaine provided monoclonal antibodies capable of catalyzing the hydrolysis of the cocaine benzoyl ester group. An assay for the degradation of radiolabeled cocaine identified active enzymes. Benzoyl esterolysis yields ecgonine methyl ester and benzoic acid, fragments devoid of cocaine's stimulant activity. Passive immunization with such an artificial enzyme could provide a treatment for dependence by blunting reinforcement.

Phosphonate and Phosphate Monoester Hydrolysis

Abstract The reaction of phosphonate and phosphate monoesters with an epoxide yields β-hydroxyalkyl derivatives which upon treatment with base quantitatively release the original alcohol moiety of the ester in a relatively rapid simple procedure.

Mild Preparation of 1-Benzyloxyiminoalkylphosphonic Dichlorides: Application to the Synthesis of Cyclic Phosphonic Diesters and Cyclic Monoester Amides

1-Benzyloxyiminophosphonates (3) were converted under very mild conditions to the corresponding phosphonyl dichlorides (5). The application toward the synthesis of diastereomeric 1,3,2,-oxazaphospholidines (6/7), and diastereomeric 1,3,2-dioxaphosphorinanes (8/9) is reported. The structure of 9a was confirmed by X-ray analysis