Utilisation of structurally diverse organophosphonates by Streptomycetes.

A group of streptomycete strains was found able to utilise a wide range of structurally diverse phosphonates as a sole phosphorus source. No relation could be observed between ability to synthesise compounds containing a direct carbon-to-phosphorus (C-P) bond and biodegradative potential towards phosphonates in the strains studied. Streptomyces morookaensis DSM 40565 could degrade 2-amino-4-phosphonobutyrate as a sole nitrogen and phosphorus source in a stereoselective-like manner. This result suggests the existence of a new metabolic pathway for C-P bond breakage.

Metabolism of the phosphonate herbicide glyphosate by a non-nitrate-utilizing strain of Penicillium chrysogenum.

A Penicillium chrysogenum strain was isolated for its ability to grow in minimal medium containing the herbicide glyphosate as the only nitrogen source. The presence of concentrations up to 25 mM progressively stimulated the fungal growth rate, which was negligible in media lacking reduced nitrogen. However, glyphosate utilization never exceeded 1 mmol g-1 mycelial dry mass, and below a threshold concentration both herbicide uptake and fungal growth were subject to a lag phase, suggesting that the herbicide may enter the cell by either simple passive diffusion or inducible carriers. Amino acids, possible products of glyphosate breakdown, as well as ammonia, were found to replace the herbicide in restoring mycelial growth. Cells were devoid of detectable nitrate reductase activity, thus the isolate seems to be impaired in its ability to convert nitrate to ammonium. In vitro activity of 5-enol-pyruvyl-shikimate-3-phosphate synthase, the target site of glyphosate action, was highly sensitive to the herbicide. Fungal growth rate was considerably lower when the herbicide was also the only phosphorus source, whereas glyphosate utilization was substantially unaffected, suggesting an unusual route for its degradation. Herbicide metabolism was strongly reduced when other sources of organic nitrogen were made available.

Aminophosphonic acids of potential medical importance.

Aminophosphonic acids were almost unknown in 1959 but today they are the subject of more than 6000 papers. Their negligible mammalian toxicity, and the fact that they very efficiently mimic aminocarboxylic acids makes them extremely important antimetabolites, which compete with their carboxylic counterparts for the active sites of enzymes and other cell receptors. Although biological importance of these compounds was recognized over 50 years ago they still represent promising and somewhat undiscovered class of potential drugs.

Reductive biotransformation of diethyl beta-, gamma- and delta-oxoalkylphosphonates by cells of baker's yeast.

Enantiomerically pure hydroxyalkylphosphonates (over 95% of enantiomeric excess) were obtained by asymmetric reductive biotransformation of a variety of oxoalkylphosphonates catalyzed by baker's yeast. In the most cases the biotransformations were carried out in water under aerobic conditions using whole cell system. In the case of compounds unreactive under these conditions the anaerobic reduction was applied.

The Herbicidally Active Compound N-2-(6-Methyl-Pyridyl)-Aminomethylene Bisphosphonic Acid Inhibits In Vivo Aromatic Biosynthesis.

The effect of N-2-(6-methyl-pyridyl)-aminomethylene bisphosphonic acid (M-pyr-AMBPA), a compound previously shown to exhibit herbicidal properties on whole plants and to inhibit in vitro activity of the first enzyme in the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase, was investigated on Nicotiana plumbaginifolia suspension cultured cells and compared to that of the herbicide glyphosate. The addition of M-pyr-AMBPA from 10(-4) to 10(-3) M was found to cause a severe cell growth reduction. Kinetic analysis of partially purified DAHP synthase accounted for non-competitive inhibition type with respect to both phospho-enol-pyruvate and erythrose-4-phosphate, with K(I) values of 0.43 and 0.62 mM, respectively. Amino acid pool measurements of cells grown in the presence of sublethal doses of M-pyr-AMBPA pointed to an actual reduction of free aromatic amino acids, showing that DAHP synthase inhibition takes place in vivo, and suggesting that the interference of this aminophosphonate with plant aromatic biosynthesis may account for a large part of its phytotoxicity. However, exogenous supply of a mixture of phenylalanine, tyrosine and tryptophan failed to achieve full reversal of cell growth inhibition, yet the occurrence of other target(s) cannot be ruled out.

Degradation of phosphonates by streptomycete isolates.

Wild-type Streptomyces sp. strains, able to utilise both naturally occurring and synthetic organophosphonates, were isolated. High levels of inorganic phosphate were necessary for their growth in complete medium as well as in medium, supplemented with phosphonates as the sole carbon or nitrogen source. Isolate StA expressed detectable enzymatic activity against 2-aminoethylphosphonate in vivo. Streptomycete StC had a surprising ability to degrade N-phosphonomethylglycine (glyphosate) in a phosphate-independent manner via C-P bond cleavage accompanied by sarcosine formation.

The ability of soil-borne fungi to degrade organophosphonate carbon-to-phosphorus bonds.

The ability of a wide variety of soil-borne fungal strains to degrade four structurally different compounds containing P-C bonds, namely the naturally occurring amino acid ciliatine, the popular herbicide glyphosate, phosphonoacetic acid and 2-amino-3-phosphonopropionic acid, was studied in order to show that soil fungi may play an important role in the biodegradation of organophosphonates. Most of the strains appeared to utilize ciliatine as the sole source of phosphorus for growth. Only a limited number of strains were able to grow on the other phosphonates used in this work. The strains of Trichoderma harzianum, Scopulariopsis sp. and Aspergillus niger chosen for more detailed study show the ability to degrade ciliatine, glyphosate and also amino(3-methoxyphenyl)methylphosphonic acid effectively.

Organophosphonate Utilization by the Wild-Type Strain of Penicillium notatum.

We studied the biodegradation of compounds containing phosphorus-to-carbon bonds by using a wild-type strain of Penicillium notatum. The substrate specificity of this strain was studied, and we found that it is able to utilize structurally diverse organophosphonates as sole sources of phosphorus. This ability seems to be inducible, as indicated by the presence of a lag phase during growth. A popular herbicide, glyphosate, inhibited fungal growth, but it was also degraded by the fungus if it was applied in sublethal doses. This indicates that P. notatum may play an important role in biodegradation of organophosphonates. The strain which we used did not metabolize any of the phosphonates which we tested when they were used as sole carbon or nitrogen sources.

Antibacterial activity of phosphono peptides based on 4-amino-4-phosphonobutyric acid.

Phosphono dipeptides based on 4-amino-4-phosphonobutyric acid (phosphonic acid analogue of glutamic acid, GluP) were synthesized and evaluated for their antibacterial activity. Dipeptides containing N-terminal alanine, leucine, isoleucine, phenylalanine or lysine showed marked antibacterial activity against Escherichia coli, whilst those containing alanine, leucine, valine or proline were active against Serratia marcescens. AlaGluP and LeuGluP were nearly equipotent with the respective dipeptides based on 1-aminoethylphosphonic acid (phosphonic acid analogue of alanine). The structure-activity relationship, i.e. dependence of the activity of phosphono dipeptides on the nature of their N-terminal component, indicated that transport of the peptide through the bacterial cytoplasmic membrane constitutes a crucial step in its antibacterial activity.

Inhibition of aminopeptidases by phosphonic acid and phosphinic acid analogues of aspartic and glutamic acids.

More than 30 phosphonic and phosphinic acid analogues of aspartic and glutamic acids were synthesized in order to probe how the structural differences of these molecules were reflected in their ability to inhibit cytosolic (LAP) and microsomal (APM) aminopeptidases. Although most of the compounds studied were found to exert only a modest inhibitory effect, the studies provide some information on the structural requirements of the binding subsites and catalytic centers of both enzymes.

Organophosphonate utilization by the wild-type strain of Pseudomonas fluorescens.

The wild-type strain of Pseudomonas fluorescens was found to utilize a range of structurally diverse organophosphonates as its sole carbon or nitrogen sources. Representative compounds included aminoalkylphosphonates, hydroxyalkylphosphonates, oxoalkylphosphonates, and phosphono dipeptides. Among them, amino(phenyl)methylphosphonate,2-aminoethylphosphonate, aminomethylphosphonate, diisopropyl 9-aminofluoren-9-ylphosphonate, and 2-oxoalkylphosphonates were used by P. fluorescens as its sole sources of phosphorus. Only slight growth was observed on the herbicide glyphosate (N-phosphonomethylglycine), which was metabolized to aminomethylphosphonate. Neither phosphinothricin nor its dialanyl tripeptide, bialaphos, supported growth of P. fluorescens. The possible mechanisms of organophosphonate degradation by this strain are discussed.

Phosphonic and phosphinic acid analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) as potential antimelanotic agents.

Phosphonic acid and phosphinic acid analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) were found to influence tyrosinase activity, thus being potential regulators of melanization in mammalian cells. The analogues were tested for their cytostatic activity in vitro, by means of total cell protein determination, on mouse B16 melanoma and, for comparison, on human KB carcinoma cell lines. Four compounds out of 22 revealed promising cytostatic activity for both cell lines, being nearly equipotent with dopa. Those compounds which most effectively suppressed the growth of both tumour cell lines were the same as those which had a significant influence on tyrosinase activity regardless of whether they acted as inhibitors or substrates.

Antibacterial activity of phosphono dipeptides based on 1-amino-1-methylethanephosphonic acid.

Phosphono dipeptides containing 1-amino-1-methylethanephosphonic acid (phosphonic acid analogue of alpha-methylalanine, MeAlaP) and glycine, alanine, valine, leucine phenylalanine, proline, methionine or lysine as N- terminal component were synthesized in order to determine their antibacterial properties. Peptides containing alanine, leucine, valine phenylalanine and methionine showed marked in vitro activity, especially against Escherichia coli and Serratia marcescens strains. There were, however, generally less potent than the respective phosphono dipeptides based on 1-aminoethanephosphonic acid (phosphonic acid analogue of alanine, AlaP). The possible mechanism of action of the peptides of MeAlaP involves their active transport into the bacterial cell, followed by intracellular release of MeAlaP, which most likely inhibits alanine racemase, a key enzyme in peptidoglycan biosynthesis. Studies on the uptake of AlaMeAlaP and LeuMeAlaP by Escherichia coli mutants defective in the oligopeptide permease suggest that these peptides are not transported by the oligopeptide transport system.

Inhibition of aminopeptidases by aminophosphonates.

More than 30 aminophosphonates were synthesized to probe how the structural changes introduced into the phosphonic acid analogue of leucine, a potent inhibitor of cytosolic leucine aminopeptidase (Giannousis & Bartlett, 1987), affect their ability to inhibit cytosolic (EC 3.4.11.1) and microsomal (EC 3.4.11.2) aminopeptidases. Although most of the compounds studied were found to exert only a modest competitive inhibitory effect, nearly every modification of the structure of the phosphonic acid analogue of leucine was reflected in a marked difference in the affinities of these compounds for the two enzymes. [1-Amino-2-(N-alkylamino)ethyl]phosphonic acids are effective inhibitors of the microsomal enzyme, acting in a time-dependent manner. Kinetic data obtained for these inhibitors correspond to the mechanism for a biphasic slow-binding inhibition process: E + I in equilibrium E* in equilibrium E*I, in which the slow initial isomerization of the enzyme is followed by the fast formation of enzyme-inhibitor complex. The most effective inhibitor of this type was [1-amino-2-(N-cyclohexylamino)ethyl]phosphonic acid, which has a Ki value of 0.87 microM toward the microsomal aminopeptidase--a value that can be considered as equipotent with bestatin and with leucinal and hydroxamic acids, the strongest known nonpeptide inhibitors of this enzyme.

Phosphonic analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) influence mushroom tyrosinase activity.

A series of phosphonic analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) were synthesized in order to study their interaction with mushroom tyrosinase. 1-Amino-2-(3,4-dihydroxyphenyl)ethylphosphonic acid and 1-amino-2-(3,4-dimethoxyphenyl)ethylphosphonic acid turned out to be substrates for mushroom tyrosinase with Km values of 3.3 mM and 9.3 mM respectively. Shortening of the alkyl chain by one methylene group gave amino-(3,4-dihydroxyphenyl)methylphosphonic acid, one of the most powerful known inhibitors of this enzyme. This compound, racemic as well as in its optically active forms, exerts a mixed type of inhibition with an affinity for the enzyme one order of magnitude greater than that of the natural substrate.

Antibacterial activity of phosphono dipeptides related to alafosfalin.

A series of dipeptides containing N-terminal alanine or leucine and a wide range of P-terminal racemic 1-amino-alkanephosphonates were prepared and tested in vitro for their ability to inhibit the growth of various bacterial species. The results demonstrate that peptides containing 4-amino-4-phosphonobutyric acid and 1-amino-1-methylethanephosphonic acid exhibit antibacterial activity comparable with that observed in the case of peptides containing P-terminal racemic 1-aminoethanephosphonic acid (analogue of alanine) used as a positive control.

Technetium-99m-labeled phosphonic acid analog of serine: bone uptake.

A phosphonic acid analog of serine (PAAS), its P-methyl derivative, and an allied compound were labeled with 99mTc, and evaluated in experimental animals. These molecules were able to bind 99mTc in the presence of stannous ions, but the biologic behavior of the later two labeled compounds was quite different from that of the first one. Technetium-99m-labeled PAAS behaved like 99mTc-labeled methylene diphosphonate, but its P-methyl derivative and the third agent showed little bone accumulation. It appears that both hydroxyl groups attached to the phosphorus atom are essential for uptake in bone, but that only one is required for binding 99mTc.

N-(Phosphonoacetyl)amino phosphonates. Phosphonate analogues of N-(phosphonoacetyl)-L-aspartic acid (PALA).

Michaelis-Arbuzov reaction of N-(chloroacetyl)amino phosphonic acids or their esters, followed by acidolysis, gives moderate yields of N-(phosphonoacetyl) derivatives of a variety of (aminoalkyl)phosphonic acids, including analogues of the cytostatic agent PALA, in which the alpha- or beta-carboxylic groups in the aspartate moiety are replaced by a PO3H2 function. Assay of cytostatic activity with human KB cell lines indicates that the substitution of any of the COOH groups in PALA with PO3H2 results in total loss of cytostatic activity. No activity was observed also in the case of other [N-(phosphonoacetyl)amino]alkylphosphonic acids described in this report.

Enzymatic activity of activated sludge in biological treatment of wastewater containing dimethylamine, dimethylformamide and methylethylketone.

Activated sludge treating synthetic wastewater carrying dimethylamine (DMA), dimethylformamide (DMF) and methylethylketone (MEK) was examined. The compounds in question were found to affect the enzymatic activity of the activated sludge. Dehydrogenase activity was higher than in control activated sludge and stabilization of this activity was achieved on the twelfth day of the run. Alkaline phosphatase activity was lower than in the control sludge. No correlation between activity of the studied enzymes and biodegradation of DMA, DMF and MEK was observed.