Synthesis and evaluation of the inhibitory activity of the four stereoisomers of the potent and selective human γ-glutamyl transpeptidase inhibitor GGsTop

2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (R_P/S_P). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (k_on = 174 M^?1 s^?1) was ca. 8-fold more potent than the d-isomer (k_on = 21.5 M^?1 s^?1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be S_P. The S_P-isomers inhibited human GGT (k_on = 21.5–174 M^?1 s^?1), while the R_P-isomers were inactive even at concentrations of 0.1 mM.     

Mononucleoside SATE Glucosyl Phosphorothiolates as a New Series of Pronucleotides

Abstract

The synthesis and the study of two phosphorothiolate derivatives of 3′-azido-2′,3′-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and glucosyl residues associated to the phosphorus atom by a 2-oxyethyl link, are reported. These derivatives could be considered as prototypes of a new series of nucleotide prodrugs (pronucleotides).     

Uranium complexes of cyclic O,O-bidentate ligands with the P-N-P backbone

The formation of uranium complexes of novel ligands belonging to phosphorylated 2-oxo-1,2-azaphospholane series, namely 2-ethoxy-1-diethoxyphosphoryl-2-oxo-1,2λ⁵-azaphospholane (1a) and both individual R^∗,R^∗- and R^∗,S^∗-diastereomers of the related 2-oxo-2-phenyl-1,2λ⁵-azaphospholanes 1b,c with different surrounding at the exocyclic phosphorus atom, has been studied. The structures of the complexes of ML composition obtained in the reaction with uranyl nitrate in 1:1 ratio were found to depend on the difference in donor properties of the oxygen atom of endo- and exocyclic phosphoryl groups. The ligand 1a possessing the greater difference, serves as O-monodentate one with metal-oxygen bonding via the endocyclic PO function while both isomers of 1b,c coordinate to uranyl cation in a O,O-bidentate fashion. In solutions the ML complexes reacted with air oxygen to afford (μ₂-peroxo)-bridged uranium complexes [{UO₂(L)NO₃}₂(μ₂-O₂)] which structures were confirmed by X-ray crystallography data.     

Synthesis and characterization of CpMCl(PR3)(S or W-η-butadienesulfonyl) compounds of rhodium and iridium

A metathesis reaction of [Cp^∗MCl₂(PR₃)] [M = Rh, R = Ph (1), Me (3); M = Ir, R = Ph (2), Me (4)] takes place in the presence of potassium butadienesulfinate (SO₂CHCHCHCH₂)K (9) to afford the mononuclear compounds [Cp^∗M(Cl)(PR₃)(η¹-SO₂CHCHCHCH₂)] [M = Rh, R = Ph (11S), (11W); M = Rh, R = Me (13S), (13W)] and [M = Ir, R = Ph (12S); M = Ir, R = Me (14S), (14W)] under different reaction conditions. The addition of PR₃ (R = Ph, Me) to Cp^∗Ir(Cl)[(1,2,5-η)-SO₂CHCHCHCH₂] (7) affords the corresponding iridium isomers 12S, 12W and 14S, in a non-selective reaction, along with the corresponding dichloride compounds 2 or 4. The ¹H and ¹³C{¹H} NMR data are consistent with the butadienesulfonyl ligands coordinated exclusively through the sulfur atom, and they show the presence of two isomers, described as the S and W conformers, which can be isolated separately. There is clear evidence that these isomers correspond to the kinetic and thermodynamic derivatives, respectively.     

Conformation of Diastereomers of Adenosine Cyclic 3′, 5′-Phosphorothioate

Abstract

¹H and ³¹P NMR spectra of cAMP (1) and both diastereomers of cAMPS (2 and 3) were compared with these of structurally related bicyclic phosphate 4 and phosphorothioates 5 and 6. Conformational analysis was also performed by NMR techniques for bicyclic phosphoranilidates 7 and 8 and (Rp)-cdAMP anilidate (9). Chair conformation is predominant for all investigated compounds 1–8, while the phosphoranilidate 9 exists in solution in chair-twist equilibrium. Thus, antagonistic properties of (Rp)-cAMPS with respect to cAMP are inferred by the change in the overall molecular shape caused by the presence of the bulky sulfur atom in the equatorial position of the cAMPS molecule.     

Identification of the stereochemical requirements in the 4-aryl-2-cycloalkylidenhydrazinylthiazole scaffold for the design of selective human monoamine oxidase B inhibitors

Exploring the effect that substituents on the cycloaliphatic ring had on the inhibitory activity against human monoamine oxidase B of a series of 4-aryl-2-cycloalkylidenhydrazinylthiazoles led to the synthesis of a new series of 2-methylcyclopentyl and 3-methylcyclopentyl derivatives which were tested in vitro as mixtures of diastereoisomers. In fact, due to the presence of a chiral center on the cycloaliphatic ring and a trisubstituted CN bond, they exist as four diastereoisomers ((E)-(R), (E)-(S), (Z)-(R), (Z)-(S)). 4-(2,4-Difluorophenyl)-2-(2-(3-methylcyclopentylidene)hydrazinyl)thiazole was chosen as a model to investigate the influence of stereochemical requirements on the inhibitory activity against hMAO-B of these derivatives after a stereoconservative synthesis and semi-preparative HPLC diastereoseparation. (R)-(Z) isomer of this compound was endowed with a potent and selective hMAO-B inhibition higher than that of reference drugs as also corroborated by molecular modeling studies.     

Evaluation of enantioselectivity in lipase-catalyzed acylation of hydroxyalkylphosphine oxides

The lipase-catalyzed optical resolution of 2-, 3-, and 5-hydroxyalkyl phosphorus compounds 1 provided the corresponding optically pure diastereomers in good yields. (S_P, R)- and (R_P, S)-1 were acylated faster than (S_P, S)- and (R_P, R)-1. The stereoselectivity at the phosphorus atom changed with the flexibility of the active sites in the lipases. The stereoselectivity at the phosphorus atom was higher in the reaction of 1a than in the reaction of 1b,c. The reaction rate of -hydroxyalkylphosphine oxide 1c was faster than that of 1a, although less enantioselectivity was observed at the phosphorus atom.     

Stable spiro-endoperoxides by sunlight-mediated photooxygenation of 1,2-O-alkylidene-5(E)-eno-5,6,8-trideoxy-alpha-D-xylo-oct-1,4-furano-7-uloses

Sunlight-mediated photooxygenation of 3-O-acetyl and 3-O-methyl derivatives of 1,2-O-alkylidene-5(E)-eno-5,6,8-trideoxy-α-d-xylo-oct-1,4-furano-7-uloses (1a-e) in carbon tetrachloride solution gave stable 4,7-epidioxy derivatives in 4R (2a-e) and 4S (3a-e) configurations. The presence of an endo alkyl, on the 1,2-O-alkylidene group and its size, resulted in an increase of the yield of the 4S isomers. 3-O-Acetyl derivatives yielded products as a mixture of C-7 anomers, whereas 3-O-methyl derivatives gave pure single stereoisomers.     

Regioselectivity in the glycosylation of 5-(3-chlorobenzo[b]thien-2-yl)-4H-1,2,4-triazole-3-thiol

The glycosylation of 5-(3-chlorobenzo[b]thien-2-yl)-4H-1,2,4-triazole-3-thiol (1) and its 3-benzylsulfanyl and 3-methylsulfanyl derivatives with different glycosyl halides 2-4 has been studied in presence of base. The S-glycosides 5-7 were obtained in the presence of triethylamine, whereas the respective S,N⁴-bis(glycosyl) derivatives 8-10 were synthesized in the presence of potassium carbonate; the S,N²-bis(glycosyl) isomer 11 could also be isolated in the case of the galactosyl analog. Similarly, after protecting 1 as 3-benzyl(methyl)sulfanyl derivatives 12 or 13, the N⁴-glycosyl analogs 14-19 as well as minor amounts of S,N²-bis(galactosyl) isomers 20 and 21 were formed. The theoretical calculations using AM1 semiempirical methods agreed with the experimental results. Microwave irradiation (MWI) led to higher yields in much less time than the conventional methods, and no change in regioselectivity has been noticed.     

Synthesis,characterization, and biological assessment of the four stereoisomers of the H3 receptor antagonist 5-fluoro-2-methyl-N-[2-methyl-4-(2-methyl[1,3′]bipyrrolidinyl-1′-yl)phenyl]benzamide

This Letter describes the asymmetric synthesis of the four stereoisomers (8a–8d) of a potent and highly selective histamine H₃ receptor (H₃R) antagonist, 5-fluoro-2-methyl-N-[2-methyl-4-(2-methyl[1,3′]bipyrrolidinyl-1′-yl) phenyl]benzamide (1). The physico-chemical properties, in vitro H₃R affinities and ADME of 8a–8d were determined. Stereoisomer 8c (2S,3′S) displayed superior in vitro H₃R affinity over other three stereoisomers and was selected for further profiling in in vivo PK and drug safety. Compound 8c exhibited excellent PK properties with high exposure, desired brain to plasma ratio and reasonable brain half life. However, all stereoisomers showed similar unwanted hERG affinities.     

Glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of γ-glutamyl transpeptidase for probing cysteinyl-glycine binding site

γ-Glutamyl transpeptidase (GGT) catalyzing the cleavage of γ-glutamyl bond of glutathione and its S-conjugates is involved in a number of physiological and pathological processes through glutathione homeostasis. Defining its Cys-Gly binding site is extremely important not only in defining the physiological function of GGT, but also in designing specific and effective inhibitors for pharmaceutical purposes. Here we report the synthesis and evaluation of a series of glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of human and Escherichia coli GGTs to probe the structural and stereochemical preferences in the Cys-Gly binding site. Both enzymes were inhibited strongly and irreversibly by the peptidyl phosphorus esters with a good leaving group (phenoxide). Human GGT was highly selective for l-aliphatic amino acid such as l-2-aminobutyrate (l-Cys mimic) at the Cys binding site, whereas E. coli GGT significantly preferred l-Phe mimic at this site. The C-terminal Gly and a l-amino acid analogue at the Cys binding site were necessary for inhibition, suggesting that human GGT was highly selective for glutathione (γ-Glu-l-Cys-Gly), whereas E. coli GGT are not selective for glutathione, but still retained the dipeptide (l-AA-Gly) binding site. The diastereoisomers with respect to the chiral phosphorus were separated. Both GGTs were inactivated by only one of the stereoisomers with the same stereochemistry at phosphorus. The strict recognition of phosphorus stereochemistry gave insights into the stereochemical course of the catalyzed reaction. Ion-spray mass analysis of the inhibited E. coli GGT confirmed the formation of a 1:1 covalent adduct with the catalytic subunit (small subunit) with concomitant loss of phenoxide, leaving the peptidyl moiety that presumably occupies the Cys-Gly binding site. The peptidyl phosphonate inhibitors are highly useful as a ligand for X-ray structural analysis of GGT for defining hitherto unidentified Cys-Gly binding site to design specific inhibitors.     

Prodrugs of fumarate esters for the treatment of psoriasis and multiple sclerosis—a computational approach

Density functional theory (DFT) calculations at B3LYP/6-31 G (d,p) and B3LYP/6-311?+?G(d,p) levels for the substituted pyridine-catalyzed isomerization of monomethyl maleate revealed that isomerization proceeds via four steps, with the rate-limiting step being proton transfer from the substituted pyridinium ion to the C=C double bond in INT1. In addition, it was found that the isomerization rate (maleate to fumarate) is solvent dependent. Polar solvents, such as water, tend to accelerate the isomerization rate, whereas apolar solvents, such as chloroform, act to slow down the reaction. A linear correlation was obtained between the isomerization activation energy and the dielectric constant of the solvent. Furthermore, linearity was achieved when the activation energy was plotted against the pK _a value of the catalyst. Substituted-pyridine derivatives with high pK _a values were able to catalyze isomerization more efficiently than those with low pK _a values. The calculated relative rates for prodrugs 1–6 were: 1 (406.7), 2 (7.6?×?10⁶), 3 (1.0), 4 (20.7), 5 (13.5) and 6 (2.2?×?10³). This result indicates that isomerizations of prodrugs 1 and 3–5 are expected to be slow and that of prodrugs 2 and 6 are expected to be relatively fast. Hence, prodrugs 2 and 3–5 have the potential to be utilized as prodrugs for the slow release of monomethylfumarate in the treatment of psoriasis and multiple sclerosis.

Phosphate-limited growth of Chromatium vinosum in continuous culture

Chromatium vinosum DSM 185 was grown in continuous culture at a constant dilution rate of 0.071 h^-1 with sulfide as the only electron donor. The organism was subjected to conditions ranging from phosphate limitation (S _R-phosphate=2.7 M and S _R-sulfide=1.8 mM) to sulfide limitation (S _R-phosphate=86 M and S _R-sulfide=1.8 mM). At values of S _R-phosphate below 7.5 M the culture was washed out, whereas S _R-phosphate above this value resulted in steady states. The saturation constant (K ) for growth on phosphate was estimated to be between 2.6 and 4.1 M. The specific phosphorus content of the cells increased from 0.30 to 0.85 mol P mg^-1 protein with increasing S _R-phosphate. The specific rate of phosphate uptake increased with increasing S _R-phosphate, and displayed a non-hyperbolic saturation relationship with respect to the concentration of phosphate in the inflowing medium. Approximation of a hyperbolic saturation function yielded a maximum uptake rate (V _max) of 85 nmol P mg^-1 protein h^-1, and a saturation constant for uptake (K _t) of 0.7 M. When phosphate was supplied in excess 8.5% of the phosphate taken up by the cells was excreted as organic phosphorus at a specific rate of 8 nmol P mg^-1 protein h^-1.Non-standard abbreviations BChla bacteriochlorophyll a - D dilution rate; _max, maximum specific growth rate - maximum specific growth rate if the substrate were not inhibitory - K saturation constant for growth on phosphate - V _max maximum rate of phosphate uptake - K _i saturation constant for phosphate uptake - K _i inhibition constant for growth in the presence of sulfide - S _R concentration of substrate in the inflowing medium     

Asymmetric total synthesis and antiproliferative activity of goniothalamin oxide isomers

Goniothalamin oxide (1) is a styryl lactone which was isolated from bark and leaves of several Goniothalamus species. This natural product has some interesting biological properties such as larvicidal and tripanocidal activities. However, no studies on the antiproliferative profile of goniothalamin oxide (1) and its stereoisomers have been reported yet. Here, goniothalamin epoxide (1), isogoniothalamin epoxide (2) and their enantiomers were prepared via epoxidation of (R)-and (S)-goniothalamin (4). A 3:2 molar ratio in favor of goniothalamin oxide (1) and ent-1 was observed from (R)- and (S)-4, respectively, when 3-chloroperbenzoic acid (mCPBA) was employed while an increase to 6:1 molar ratio was achieved with (S,S)-Jacobsen’s catalyst. Antiproliferative activity of these epoxides revealed that ent-isogoniothalamin oxide (ent-2) was the most active against the eight cancer cell lines studied. These results indicate that 6S, 7R and 8R absolute configurations are beneficial for the activity of these epoxides.     

Evaluation of enantioselectivity in lipase-catalyzed acylation of hydroxyalkylphosphine oxides

The lipase-catalyzed optical resolution of 2-, 3-, and 5-hydroxyalkyl phosphorus compounds 1 provided the corresponding optically pure diastereomers in good yields. (S_P, R)- and (R_P, S)-1 were acylated faster than (S_P, S)- and (R_P, R)-1. The stereoselectivity at the phosphorus atom changed with the flexibility of the active sites in the lipases. The stereoselectivity at the phosphorus atom was higher in the reaction of 1a than in the reaction of 1b,c. The reaction rate of ɛ-hydroxyalkylphosphine oxide 1c was faster than that of 1a, although less enantioselectivity was observed at the phosphorus atom.     

Oxygenated verticillene derivatives from Bursera suntui

Medium polarity fractions of the hexane extracts of the stems of Bursera suntui afforded six previously known (1-6) and four hitherto unknown verticillane derivatives: (1S,3Z,7S,8S,11S,12S)-(+)-7,8-epoxyverticill-3-en-12,20-diol (7), (1S,3Z,7S,8S,11S,12S)-(+)-7,8-epoxyverticill-3-en-12,20-diol 20-acetate (8), (1S,3Z,7S,11S,12S)-(+)-verticilla-3,8(19)-dien-7,12,20-triol (9), and (1S,3Z,7S,11S,12S)-(+)-verticilla-3,8(19)-dien-7,12,20-triol 20-acetate (10). Acetylation of 9 and 10 yielded (1S,3Z,7S,11S,12S)-(+)-verticilla-3,8(19)-dien-7,12,20-triol 7,20-diacetate (11), while hydrolysis of 8 gave 7. The structures and stereochemistry of 7-11 were established by spectroscopic analyses, particularly by 1D and 2D NMR spectra and HRESIMS. The conformational preferences of 7-11 were studied by molecular mechanics modelling employing the Monte Carlo protocol followed by B3LYP/DGDZVP DFT calculation, thus supporting the observed ¹H NMR NOESY cross peaks.     

Structure of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway for Phosphonate Degradation

Carbon-phosphorus lyase is a multienzyme system encoded by the phn operon that enables bacteria to metabolize organophosphonates when the preferred nutrient, inorganic phosphate, is scarce. One of the enzymes encoded by this operon, PhnP, is predicted by sequence homology to be a metal-dependent hydrolase of the β-lactamase superfamily. Screening with a wide array of hydrolytically sensitive substrates indicated that PhnP is an enzyme with phosphodiesterase activity, having the greatest specificity toward bis(p-nitrophenyl)phosphate and 2′,3′-cyclic nucleotides. No activity was observed toward RNA. The metal ion dependence of PhnP with bis(p-nitrophenyl)phosphate as substrate revealed a distinct preference for Mn²⁺ and Ni²⁺ for catalysis, whereas Zn²⁺ afforded poor activity. The three-dimensional structure of PhnP was solved by x-ray crystallography to 1.4 resolution. The overall fold of PhnP is very similar to that of the tRNase Z endonucleases but lacks the long exosite module used by these enzymes to bind their tRNA substrates. The active site of PhnP contains what are probably two Mn²⁺ ions surrounded by an array of active site residues that are identical to those observed in the tRNase Z enzymes. A second, remote Zn²⁺ binding site is also observed, composed of a set of cysteine and histidine residues that are strictly conserved in the PhnP family. This second metal ion site appears to stabilize a structural motif.In many environments inorganic phosphate, an essential nutrient, can fall to extremely low concentrations, forcing microorganisms to utilize other forms of phosphorus to survive. In such cases, organophosphonates can comprise a major fraction of the total phosphorus available to biological systems (e.g. 2-aminoethylphosphonate is a widespread natural product). However, cleavage of the highly stable carbon-phosphorus (CP)⁵ bond to release inorganic phosphate requires specialized enzymes. One such enzyme activity found widely in bacteria is CP-lyase (1). Cleavage of the CP bond of organophosphonates by CP-lyase yields inorganic phosphate and, remarkably, a hydrocarbon. CP-lyase is actually a multienzyme system, encoded by the phn operon (phnCDEFGHIJKLMNOP), which is induced by low concentrations of phosphate as part of the pho regulon. Gene deletion studies in Escherichia coli have shown that phnGHIJKLM are essential for catalysis of CP bond cleavage, whereas the remaining genes probably encode transport, regulatory, or accessory functions (2). Only a handful of the proteins encoded by the phn operon have been characterized to date. PhnD was shown to be a periplasmic binding protein with high affinity for organophosphonates (3); the three-dimensional structure of PhnH, one of the proteins essential for CP-lyase catalysis, was recently solved, but a function has yet to be determined (4); PhnN was shown to be an ATP-dependent kinase that provides a redundant pathway to 5-phospho-d-ribofuranosyl-α-1-diphosphate (5); and PhnO was demonstrated to be an acetyl-CoA-dependent N-acyltransferase with activity toward a wide range of aminoalkylphosphonates (6).Although the phnP gene is not essential for CP bond cleavage by cells in liquid culture (2), cell growth on solid media supplemented with methylphosphonate or phosphite as the sole phosphorus source is prevented by phnP mutations (7), suggesting a critical regulatory or accessory role for PhnP. Accordingly, phnP appears frequently in the phn operon in various species of bacteria, typically following the phnN gene (8). PhnP is predicted based on its sequence to be a member of the β-lactamase family of metal-dependent hydrolases with greatest homology to enzymes from the tRNase Z (ProDom family PD352433) and ElaC families (9), the latter erroneously annotated as composed of arylsulfatases but later determined to also belong to the tRNase Z family (10, 11). The tRNase Z enzymes are endonucleases used by prokaryotes and eukaryotes to cleave a specific phosphodiester bond near the 3′-end of pre-tRNA, yielding a 3′-end that can be coupled to an amino acid. These enzymes typically use two active site bound Zn²⁺ ions to simultaneously lower the pK_a of a nucleophilic water molecule and stabilize negative charge development on the phosphodiester linkage undergoing nucleophilic attack (12). Since it is not clear how a tRNase activity would support cell growth with an organophosphonate as a sole phosphorus source, we set out to characterize the substrate specificity and three-dimensional structure of PhnP to learn more about this critical CP-lyase enzyme.     

Phosphoenolpyruvate Phosphomutase Activity in an l-Phosphonoalanine-Mineralizing Strain of Burkholderia cepacia

A strain of Burkholderia cepacia isolated by enrichment culture utilized l-2-amino-3-phosphonopropionic acid (phosphonoalanine) at concentrations up to 20 mM as a carbon, nitrogen, and phosphorus source in a phosphate-insensitive manner. Cells contained phosphoenolpyruvate phosphomutase activity, presumed to be responsible for cleavage of the C—P bond of phosphonopyruvate, the transamination product of l-phosphonoalanine; this was inducible in the presence of phosphonoalanine.Organophosphonates are characterized by the presence of a stable, covalent carbon-to-phosphorus (C—P) bond. In the majority of previous studies they have been utilized only under phosphate-limited conditions and only as sole sources of phosphorus for microbial growth (3, 4, 21, 22). The C—P bond may be cleaved by at least three distinct bacterial enzymes: the C—P lyase enzyme complex(es) (17, 24, 25, 27, 28), phosphonoacetaldehyde hydrolase (5, 6, 9, 12), and phosphonoacetate hydrolase (14–16). The latter enzyme is unique in that its expression is independent of the phosphate status of the cell and is inducible solely by phosphonoacetate. It is likely that organophosphonate biodegradation in the environment is mediated largely by a C—P lyase(s), with organisms capable of mineralizing organophosphonates as sources of carbon and energy being rare (2, 13).Phosphonoalanine (2-amino-3-phosphonopropionic acid) is one of the naturally occurring C—P compounds synthesized by lower organisms, such as the sea anemone Zoanthus sociatus (10) and the protozoan Tetrahymena pyriformis (8, 23, 29). In this paper, we report the isolation of a bacterium capable of mineralizing l-phosphonoalanine as a carbon, energy, nitrogen, and phosphorus source independently of the phosphate status of the cell.Enrichment was carried out with a basal mineral salts medium which contained the following (per liter): KCl, 0.2 g; MgSO₄ · 7H₂O, 0.2 g; CaCl₂ · 2H₂O, 0.01 g; ferric ammonium citrate, 1.0 mg; trace element solution (11), 1 ml; and vitamin solution (14), 1 ml. Filter-sterilized (0.22-μm pore size) dl-phosphonoalanine (8 mM) was routinely added as a carbon, energy, nitrogen, and phosphorus source. The pH of the medium was initially adjusted to 7.2, and where required, filter-sterilized solutions of sodium pyruvate as a carbon source (final concentration, 10 g liter⁻¹), NH₄Cl as an inorganic nitrogen source (final concentration, 5 g liter⁻¹), and/or phosphate buffer (final concentration, 1 mM) were added to the medium. Enrichment cultures (25 ml in 250-ml Erlenmeyer flasks) were inoculated with a 0.5% (vol/vol) composite inoculum from an activated sludge plant (Dunmurry, Northern Ireland), a laundry waste disposal lagoon (Summit Lake, Wis.), and a sheep dip disposal site (County Antrim, Northern Ireland). All sites were known to have a history of exposure to organophosphonates. Cultures were incubated at 28°C on an orbital shaker at 100 rpm. Microbial growth was measured by the increase in optical density at 650 nm (OD₆₅₀) using a Pye-Unicam 8265 UV-visible light spectrophotometer (Pye-Unicam Ltd., Cambridge, United Kingdom). Release of inorganic phosphate and ammonium into culture supernatants was monitored by the methods of Fiske and SubbaRow (7) and Weatherburn (30), respectively.Three gram-negative isolates, each capable of growth on 8 mM dl-phosphonoalanine as a carbon, nitrogen, and phosphorus source were obtained following five rounds of serial enrichment. Of these, isolate Pal6 grew most quickly on phosphonoalanine and was chosen for further investigation. It was identified by the National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, Scotland, as a strain of Burkholderia cepacia.When dl-phosphonoalanine (8 mM) was supplied as a carbon, nitrogen, and phosphorus source for growth of B. cepacia Pal6, some 47% of substrate phosphorus and 44% of substrate nitrogen was released concomitantly with growth as P_i and ammonium (results not shown). When the compound was supplied as the sole phosphorus source (Fig. ​(Fig.1),1), transient release of approximately 30% of substrate phosphorus to the medium as P_i was observed; this phenomenon has not previously been reported for the utilization of any organophosphorus compound as a phosphorus source. When B. cepacia Pal6 was grown on dl-phosphonoalanine as a nitrogen and phosphorus (Fig. ​(Fig.2)2) or nitrogen source, removal of 50% of phosphonoalanine from the medium was demonstrated by the method of Moore and Stein (18), along with release of just less than 50% of substrate phosphorus as P_i. A subsequent experiment in which the d- and l-enantiomers were separately supplied as sole sources of phosphorus indicated that only l-phosphonoalanine supported growth of B. cepacia Pal6. It is therefore clear that the catabolism of l-phosphonoalanine by this isolate is independent of the phosphate status of the cell, a marked departure from the many examples of classical pho regulon-controlled biodegradation of organophosphonates reported in the literature (26, 27). Open in a separate windowFIG. 1Growth of B. cepacia Pal6 on phosphonoalanine (1 mM) as the sole phosphorus source, with NH₄Cl as a nitrogen source (5 g liter⁻¹) and pyruvate as a carbon source (10 g liter⁻¹). Symbols: •, OD₆₅₀; ▴, phosphate release.Open in a separate windowFIG. 2Growth of B. cepacia Pal6 on phosphonoalanine (5 mM) as a nitrogen and phosphorus source, with pyruvate as a carbon source (10 g liter⁻¹). Symbols: •, OD₆₅₀; ▴, phosphate release (mM); □, phosphonoalanine remaining in medium (mM).B. cepacia Pal6 was grown on a range of dl-phosphonoalanine concentrations as carbon and nitrogen source in the presence of 1 mM inorganic phosphate. The cell yield was proportional to the concentration of phosphonoalanine supplied up to 20 mM, the highest concentration tested, again with release of less than 50% substrate phosphorus and nitrogen to the medium (results not shown), indicating no toxicity on the part of either the substrate or its breakdown products at these concentrations.In addition to phosphonoalanine, B. cepacia Pal6 was able to utilize 6 of 14 organophosphonate substrates supplied as the sole phosphorus source (Table ​(Table1);1); however, with the exception of 2-aminoethylphosphonic acid (2AEP), no phosphate release was observed during growth on these compounds, suggesting classical pho regulon control of their biodegradation and the involvement of a C—P lyase(s) or similar enzymes. B. cepacia Pal6 was also capable of growing on 2AEP as a carbon, energy, nitrogen, and phosphorus source, with concomitant release of excess phosphorus and nitrogen to the medium as inorganic phosphate and ammonium, respectively. It did not utilize any of the other phosphonates tested as the carbon and/or nitrogen and phosphorus source. The metabolism by B. cepacia Pal6 of 2AEP as a carbon, nitrogen and phosphorus source suggests that a phosphate-deregulated pathway is also responsible for the mineralization of this compound.

TABLE 1

Range of organophosphonate substrates utilized by B. cepacia Pal6 as the sole phosphorus source