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Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems.

The enzymatic resolutions of two racemic ethyl hydroxyalkane(P-phenyl)phosphinates were performed by both esterification and hydrolysis approaches. The first reaction was performed in anhydrous diisopropyl ether with triethylamine or pyridine as additives by using lipases from three different sources (Candida cylindracea, Aspergillus niger, and Mucor javanicus). The increase in enantioselectivity was observed when NEt3 was applied. The second reaction—lipase-catalysed hydrolysis of ethyl butyryloxyalkane(P-phenyl)phosphinates—was carried out by Candida cylindracea lipase in diisopropyl ether saturated with water or in aqueous solutions containing MgCl2, LiCl, or Triton X-100. The usefulness of biphasic systems consisting of diisopropyl ether and water or aqueous solution of MgCl2, LiCl, or Triton X-100 also were tested. The use of biphasic system in the presence of Triton X-100 resulted in the higher conversion of the substrates.

Long range spin–spin couplings found between two phosphorus atoms are quite rare phenomena. A new example of such a coupling through seven bonds is described for 1-[(diethoxyphosphoryl)butyryloxymethyl]-4-[(diethoxyphosphoryl)hydroxymethyl]benzene and 1,4-bis[phosphorylhydroxymethyl]benzene and through six bonds for tetraethyl phenylene-1,3-(bishydroxymethylphosphonate). Coupling constants in this system are also well predicted by theoretical studies.

The use of quinine as a chiral solvating agent allows us to determine a tentative absolute configuration at the phosphorus atom of hydroxyphosphinates with two stereogenic centres (at the phosphorus and α-carbon atoms). Two ethyl butyryloxyalkane( P -phenyl)phosphinates were hydrolysed using various lipases. In all cases isomers possessing α-carbon atom with an ( S )-configuration were hydrolysed preferentially. The absolute configuration of both chiral centres of obtained α-hydroxyphosphinates was determined by using ( S )-(+)-MTPA-Cl and quinine. The mode of chiral discrimination of α-hydroxyphosphinates by quinine was studied by means of computational chemistry, which confirmed the experimental findings that the signals in 31 P NMR spectra of compounds with an ( R P )-configuration are situated upfield when compared with the respective ( S P ) isomers.

The changes in NMR spectra of O , O -dimethyl-1,4-oxoazetin-2-ylphosphonate (DMOAP) in polar and non-polar solvents were studied by means of experimental and theoretical approach. The precise chemical shift assignments of DMOAP were obtained by means of 1 H, 1 H{ 31 P}, 13 C{ 1 H}, 31 P{H} NMR and theoretical calculations (DTF, using the IGLO II and IGLO III basis set and solvent model PCM for CHCl 3 , MeOH, H 2 O). An optimized structure, calculated using B3LYP/6-31G( d , p ) solvent model PCM, gives three conformers in their energetic minima with various values of N C P O angle. Distribution of particular conformers, their structural differences and effect of internal or external hydrogen bonds in polar and non-polar solvents were considered as potential causes of the observed diversities in NMR spectra.

Biotransformation of diethyl of 2-oxo-2-phenylethylphosphonate using different fungal cells as biocatalysts yielded optically active O-phosphorylated derivative of 2-hydroxy-2-phenylethylphosphonate of satisfactory enantiomeric excess up to 99%.

An easy and efficient method for the microbial kinetic resolution of a substrate ethyl hydroxy(phenyl)methane(P-phenyl)phosphinate, which contains two stereogenic centers, is reported. To obtain the desired purpose biocatalyst, living fungal cells of Geotrichum candidum were cultivated with or without the supplementation of chemical additives influencing its activity. The biotransformation products were isolated with good yield and in enantiomerically pure forms.

The kinetic resolution of ethyl (1-hydroxyethyl)phenylphosphinate was carried by enzymatic esterification with vinyl butyrate or by hydrolysis of ethyl (1-butyryloxyethyl)phenylphosphinate either by the use of lipases or whole cells of microorganisms (bacteria and fungi). Since no stereodifferentiation of the phosphinate moiety was observed, the biotransformations gave diastereoisomers of ethyl (1-hydroxyethyl)phenylphosphinate with excellent enantiomeric excesses above 98%, while the percentage conversion ranged from 10% to 49% depending upon the procedure and conditions of the biocatalysis.

Ethyl butyryloxy(phenyl)methane(P-phenyl)phosphinate was hydrolyzed using four bacterial species as biocatalysts. In all cases the reaction was stereoselective and isomers bearing an α-carbon atom with an (S)-configuration were hydrolyzed preferentially. Also a lack of stereoselectivity toward the phosphorus atom was observed. Hydrolysis of one enantiomeric mixture, namely mixture of (SP,R) and (RP,S) configuration afforded enantiomerically pure ethyl (RP,S)-hydroxy(phenyl)methane(P-phenyl)phosphinate, configuration of which was established by X-ray crystallography. The observed 1H and 31P NMR chemical shifts of Mosher esters of ethyl hydroxy(phenyl)methane(P-phenyl)phosphinate were correlated with the configurations of both stereogenic centers of all four stereoisomers.

Air-born mixed fungal and bacterial culture capable of complete degradation of ciliatine was isolated. The utilization of the natural organophosphonate proceeded in the phosphate independent manner. Enzymatic activity involved in ciliatine degradation studied in the fungal cell-free extract proved to be distinct from bacterial pathway described before.

The enzyme responsible for the hydrolysis of phosphonoacetic acid, a non-biogenic C-P compound, was purified to electrophoretic homogeneity from a wild-type strain of Penicillium oxalicum. A 50-fold enrichment was obtained by a combination of anion exchange, hydrophobic interaction and MonoQ-fast protein liquid chromatography, with a yield of one-third of the initial activity. A characterization of the protein showed both similarities and differences with respect to the well-characterized bacterial counterpart. The fungal phosphonoacetate hydrolase is a 43-kDa monomeric protein showing low affinity toward its substrate and high sensitivity to even mildly acidic pH values. Enzyme activity neither required nor was stimulated by the presence of divalent cations. Polyclonal antibodies were raised in mouse against the purified protein, allowing the study of enzyme induction as a function of the phosphate status of the cell. Peptide mass mapping led to the determination of about 20% of the primary structure. Despite the biochemical differences, amino acid alignment showed a high degree of similarity of the fungal hydrolase with the few sequences available to date for the bacterial enzyme. The possible physiological role of a phosphonoacetate hydrolase is discussed.

Nonracemic diethyl 2-oxopropylphosphonate of R and S configuration and of high enantiomeric purity was obtained by stereocontrolled bioreduction using: Rhodotorula rubra, Rhodotorula glutinis, Rhodotorula gracilis and baker’s yeast. The effect of the addition of chemical compounds influencing the stereoselectivity of biotransformation have been investigated.

Review on the use of whole-cell biocatalysts for the preparation of optically active hydroxyalkanephosphonates is presented. There are three general processes applied so far, namely the use of lipolytic organisms either for enantioselective hydrolysis of acyloxyalkanephosphonates or enantioselective acylation of hydroxyalkanephosphonates, the use of baker’s yeast and other fungi for bioreduction of ketophosphonates and the use of bacteria and fungi for hydrolytic oxirane ring opening in substituted 1,2-epoxyethanephosphonates.

Nonracemic 1-phenylethyl alcohol of R and S configuration and of high enantiomeric purity was obtained by bioreduction of acetophenone in nonaqueous environment—anhydrous hexane. Immobilized cells of five different kinds of microorganisms were tested as biocatalysts, namely: Rhodotorula rubra , Rhodotorula glutinis , Cladosporium sp., Verticillium sp. and baker’s yeast. The effect of parameters such as the addition of chemical compounds influencing the stereoselectivity of bioreduction and the mode of biocatalyst pretreatment (permeabilization or cultivation under anaerobic conditions) has been investigated.

Quinine was used as a 31 P NMR shift reagent for the determination of the enantiomeric excess of diethyl 1,2-dihydroxyethanephosphonates obtained either by chemical or biocatalytic synthesis. Sharpless asymmetric dihydroxylation of diethyl trans -vinylphosphonates enabled differentiation of threo and erythro isomers of 1,2-dihydroxyphosphonates, and provided standards for the determination of the absolute configuration of each of enantiomeric pair of threo isomers.

Five different species of microorganisms, namely, Rhodotorula rubra, Rhodotorula glutinis, Cladosporium sp., Verticillium sp., and baker's yeast, turned out to be useful biocatalysts for enantioselective reduction of a variety of diethyl 1-oxoalkylphosphonates. To suppress substrate decomposition, bioreductions were carried out under anhydrous conditions, using lyophilized cells immobilized on Celite R 630. The influence of reaction conditions such as biotransformation time and chemical additives on the yield of the reaction is discussed.

A strain of Geobacillus caldoxylosilyticus from central heating system water could utilize a number of organophosphonates as the sole phosphorus source for growth at 60°C. During growth on glyphosate, aminomethylphosphonate release to the medium was observed, and in cell extracts, a glyphosate oxidoreductase-type activity, producing stoichiometric amounts of aminomethylphosphonate and glyoxylate from glyphosate, was detectable.

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.

Herbicidal derivatives of aminomethylenebisphosphonic acids discovered over 20 years ago were considered to be some kind of curiosity until 1995. Renewed interest in these compounds brought controversies about their modes and cellular targets of action. © 2000 John Wiley & Sons, Inc. Heteroatom Chem 11:449–453, 2000

Reaction of commercially available trialkyl phosphites with N-phthalylamino acids gave mixtures of seven products, whereas the same reaction carried out with pure triethyl phosphite yielded only the desired 2-(N-phthalytamino)-1-oxoalkanephosphonates. These compounds underwent rearrangement to the same types of products that were obtained with the commercial phosphites. This latter series of reactions was promoted by the presence of dialkyl phosphites.

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.

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.

A simple and efficient method was developed for the preparation of optically active 1-hydroxyalkylphosphonates. Diethyl 1-butyryloxyalkylphosphonates were hydrolysed by whole cell cultures of Pseudomonas fluorescens or Penicillium citrinum to the afford corresponding hydroxyphosphonates with moderate to good yields and satisfactory optical purity. The substrate specificity of these two microorganisms is discussed in detail. Chirality 11:109–114, 1999. © 1999 Wiley-Liss, Inc.

An analogue of aspartic acid obtained by the replacement of the α-carboxylic group by a phosphonic acid group underwent chemical transformation upon the action of zinc(II) ions under neutral and slightly alkaline conditions yielding, most probably, a cyclic phosphonamidate composed of two ligand molecules. In contrast, an analogue obtained by the replacement of the β-carboxylic group by a phosphonic acid moiety did not undergo such a transformation. In order to determine which structural features are responsible for the observed cyclization a series of aminophosphonic acids was synthesized and their behaviour in zinc(II) solutions studied by means of NMR spectroscopy. Cyclization of 1-aminoalkylphosphonic acids upon the action of zinc(II) ions in neutral and slightly alkaline media seems to be a general property of these acids. The crystal structures of two of the studied compounds were also determined.

Both R- and S-phenylethyl alcohol of high enantiomeric purity (98%) and with a satisfactory yield (40-80%) were obtained by bioreduction of acetophenone, catalyzed by whole cells of baker's yeast.

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.

Phosphonic acid analogue of forfenicine, amino (p-formylbenzyl)-phosphonic acid, was synthesized and evaluated as antibacterial agent. As indicated by disc diffusion test this compound was found to inhibit significantly the growth ofBacillus subtilis andStaphylococcus aureus and moderately the growth ofEscherichia coli. Resistance ofPseudomonas fluorescens to the action of the aminophosphonate may result from the ability of the strain to degrade this compound.

The herbicidal action of N-pyridylaminomethylenebisphosphonic acids is accompanied by an impairment of anthocyanin biosynthesis. This suggests that they might act as inhibitors of some steps in aromatic amino acid biosynthesis. Herbicidal effects were reversed by aromatic amino acids using both bacterial and plant models, a finding that strongly supports this hypothesis. Structural features of these compounds suggest the sixth enzyme in the shikimate pathway 5-enol-pyruvoylshikimate-3-phosphate (EPSP) synthase as a possible target, since a strong structural similarity exists between aminomethylenebisphosphonic acid and an inhibitor of EPSP synthase, the herbicide glyphosate. This is, however, not the case since they did not act as inhibitors of this enzyme.

N-Pyridylaminomethylenebisphosphonic acids constitute a class of promising herbicides. Since their mode of action at the cellular level is still poorly understood, we studied the influence of N-pyridylaminomethylenebisphosphonic acids on plant growth, at the whole plant and undifferentiated tissue levels, using seedlings and cell suspension cultures of mono- and dicotyledonous species. These compounds exhibited strong herbicidal properties, being equipotent with the popular herbicide glyphosate. Since they also depressed buckweed anthocyanin biosynthesis, the shikimate pathway could represent a site of action of N-pyridylaminomethylenebisphosphonic acids.

A series of seven structurally diverse N -substituted aminomethylene bisphosphonic acids exhibiting remarkable herbicidal activity was found to inhibit the activity of the first enzyme in the prechorismate pathway, 3-deoxy- D - arabino -heptulosonate-7-phosphate synthase, partially purified from Nicotiana plumbaginifolia suspension cultured cells. At millimolar concentrations all compounds inhibited the Co 2+ -dependent, cytosol-localized enzyme form. However, the addition of excess divalent cations to the reaction mixture was able to completely relieve their effect, suggesting that the chelating properties of these compounds could account for their inhibitory effect. In contrast, only five compounds of seven reduced significantly the activity of the plastidial and Mn 2+ -stimulated isoform. The inhibition brought about by N -2-(6-methyl-pyridyl)- and N -2-(5-chloro-pyridyl)-aminomethylene bisphosphonic acid could not be relieved by raising the manganese concentration in the assay mixture. A kinetic analysis showed that the latter compound inhibits enzyme activity uncompetitively with respect to phospho enol pyruvate, competitively with respect to the other substrate, erythrose-4-phosphate. As manganese is a V max stimulator of the plastidial enzyme, this ruled out the possibility of an inhibition simply based upon metal chelation.

Reaction of C-ethyl esters of phosphonic- and phosphinic acid analogues of glutamic and aspartic acids with ethyl orthoformate provides the mixtures of N-formylamino- and N-ethoxymethyleneimino-derivatives with nearly quantitative yields. Scope and limitations of this procedure were studied by means of GC/MS technique.

(N-pyridylamino)methylenebisphosphonates exhibit strong herbicidal activity which may be reversed by supplementation of the growth media with aromatic amino acids. They appeare to be the inhibitors of aromatic amino acids biosynthesis acting as inhibitors of DAHP synthase the first enzyme of shikimate pathway. Over 40 analogues of these acids were synthesized in order to determine the structure-activity relationship.

A highly stereoselective synthesis of 2-amino-1-hydroxy-3-phenylpropylphosphonic acid was achieved by simple addition of diethyl phosphite to enantiomeric N-blocked phenylalaninals. These compouds exhibit significant herbicidal activity.

Baker's yeast catalyses the asymmetric reductive biotransformation of diethyl 2-oxoalkylphosphonates yielding diethyl 2-hydroxyalkylphosphonates in good yields and with high enantiometric excess (>97%). As indicated by spectral analyses these hydroxyphosphonates exist in conformation which is “frozen” by intramolecular hydrogen bonding between phosphonate oxygen and hydroxyl hydrogen

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.

A series of phosphonic, phosphinic, and phosphonous acid analogues of phenylglycine and phenylalanine was synthesized and tested as herbicides against Lepidium sativum and Cucumis sativus. Aminobenzylphosphonic acids exhibited notable herbicidal activity and thus represent a group of the most active herbicides found among aminophosphonic acids.

Isoxazole‐substituted 1‐aminoethyl‐ and 1‐hydroxyethyiphosphonates were synthesized by a multi‐step procedure and were screened for herbicidal activity against Lepidium sativum L. and Cucumis sativus L. All the synthesized compounds exhibited notable herbicidal activity.

A series of N‐(phosphonacetyl)amine derivatives were synthesized and screened for plant‐growth regulating activity on Lepidium sativum L. and Cucumis sativus L. Aromatic N‐(phosphonoacetyl)amines. which may be considered as possible analogues of N‐acylaniline herbicides obtained by replacement of their acyl group by the phosphonacetyl moiety, exhibited significant or moderate herbicidal activity. In contrast, N‐(phosphonoacetyl)amino acids and N‐(phosphonoacetyl)aminophosphonic acids promoted the growth of L. sativum and C. sativus roots.

Dipeptide and phosphono dipeptide hydrochlorides permeated well through a 1-decanol membrane supported in a porous polyethylene hollow fiber matrix. This transfer is easily accomplished either by passive or by carrier-facilitated (with Kryptofixes 222 or 5 present in the membrane phase) transport. The transport is stereoselective with l-l dipeptides being transported faster than their l-d isomers.

Aminophosphonates are analogues of amino acids in which a carboxylic moiety is replaced by phosphonic acid or related groups. Acting as antagonists of amino acids, they inhibit enzymes involved in amino acid metabolism and thus affect the physiological activity of the cell. These effects may be exerted as antibacterial, plant growth regulatory or neuromodulatory. Chosen representative examples of biologically active aminophosphonates are presented in some detail.

A highly stereoselective synthesis of 2-amino-1-hydroxy-3-phenylpropylphosphonic acid was achieved by simple addition of diethyl phosphite to enantiomeric N-blocked phenylalaninals. These compouds exhibit significant herbicidal activity.

Utilization of organophosphonates as the sole source of phosphorus, carbon or nitrogen by a soil isolate of Penicillium citrinum was studied. Penicillium citrinum was found to utilize 2-aminoethylphosphonic and 2-oxoalkylphosphonic acids as the sole phosphorus source whereas 1-hydroxyalkylphosphonates as well as 1-aminoalkylphosphonates and their dipeptides did not support the growth of the fungus. The mould did not metabolize any of the phosphonates tested, when they served as the sole carbon or nitrogen source. Penicillium citrinum is perhaps the first mould strain isolated from soil, shown to be capable of organophosphonate degradation.

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 glutamic and aspartic acids were synthesized and screened for herbicidal activity on Lepidium sativum L. Depending on the chemical structure, they exhibited significant or moderate herbicidal activity against L. sativum roots (with some representatives being equipotent with phosphinothricin), while their influence on shoot growth was negligible. Cucumis sativus L. appeared to be more tolerant to these analogues. The origin of this selectivity remains to be determined.

The biological activity and natural occurrence of the aminophosphonic acids were described half a century ago. Since then the chemistry and biology of this class of compounds have developed into the separate field of phosphorus chemistry. Today it is well acknowledged that these compounds possess a wide variety of promising, and in some cases commercially useful, physiological activities. Thus, they have found applications ranging from agrochemical (with the herbicides glyphosate and bialaphos being the most prominent examples) to medicinal (with the potent antihypertensive fosinopril and antiosteoporetic bisphosphonates being examples).