Essential Functional Interplay of the Catalytic Groups in Acid Phosphatase.

The cooperative interplay between the functional devices of a preorganized active site is fundamental to enzyme catalysis. An in-depth understanding of this phenomenon is central to elucidating the remarkable efficiency of natural enzymes and provides an essential benchmark for enzyme design and engineering. Here, we study the functional interconnectedness of the catalytic nucleophile (His18) in an acid phosphatase by analyzing the consequences of its replacement with aspartate. We present crystallographic, biochemical, and computational evidence for a conserved mechanistic pathway via a phospho-enzyme intermediate on Asp18. Linear free-energy relationships for phosphoryl transfer from phosphomonoester substrates to His18/Asp18 provide evidence for the cooperative interplay between the nucleophilic and general-acid catalytic groups in the wild-type enzyme, and its substantial loss in the H18D variant. As an isolated factor of phosphatase efficiency, the advantage of a histidine compared to an aspartate nucleophile is ∼104-fold. Cooperativity with the catalytic acid adds ≥102-fold to that advantage. Empirical valence bond simulations of phosphoryl transfer from glucose 1-phosphate to His and Asp in the enzyme explain the loss of activity of the Asp18 enzyme through a combination of impaired substrate positioning in the Michaelis complex, as well as a shift from early to late protonation of the leaving group in the H18D variant. The evidence presented furthermore suggests that the cooperative nature of catalysis distinguishes the enzymatic reaction from the corresponding reaction in solution and is enabled by the electrostatic preorganization of the active site. Our results reveal sophisticated discrimination in multifunctional catalysis of a highly proficient phosphatase active site.

Biosynthesis of the Fungal Organophosphonate Fosfonochlorin Involves an Iron(II) and 2-(Oxo)glutarate Dependent Oxacyclase.

The fungal metabolite Fosfonochlorin features a chloroacetyl moiety that is unusual within known phosphonate natural product biochemistry. Putative biosynthetic genes encoding Fosfonochlorin in Fusarium and Talaromyces spp. were investigated through reactions of encoded enzymes with synthetic substrates and isotope labelling studies. We show that the early biosynthetic steps for Fosfonochlorin involve the reduction of phosphonoacetaldehyde to form 2-hydroxyethylphosphonic acid, followed by oxidative intramolecular cyclization of the resulting alcohol to form (S)-epoxyethylphosphonic acid. The latter reaction is catalyzed by FfnD, a rare example of a non-heme iron/2-(oxo)glutarate dependent oxacyclase. In contrast, FfnD behaves as a more typical oxygenase with ethylphosphonic acid, producing (S)-1-hydroxyethylphosphonic acid. FfnD thus represents a new example of a ferryl generating enzyme that can suppress the typical oxygen rebound reaction that follows abstraction of a substrate hydrogen by a ferryl oxygen, thereby directing the substrate radical towards a fate other than hydroxylation.

Overall Retention of Methyl Stereochemistry during B12-Dependent Radical SAM Methyl Transfer in Fosfomycin Biosynthesis.

Methylcobalamin-dependent radical S-adenosylmethionine (SAM) enzymes methylate non-nucleophilic atoms in a range of substrates. The mechanism of the methyl transfer from cobalt to the receiving atom is still mostly unresolved. Here we determine the stereochemical course of this process at the methyl group during the biosynthesis of the clinically used antibiotic fosfomycin. In vitro reaction of the methyltransferase Fom3 using SAM labeled with 1H, 2H, and 3H in a stereochemically defined manner, followed by chemoenzymatic conversion of the Fom3 product to acetate and subsequent stereochemical analysis, shows that the overall reaction occurs with retention of configuration. This outcome is consistent with a double-inversion process, first in the SN2 reaction of cob(I)alamin with SAM to form methylcobalamin and again in a radical transfer of the methyl group from methylcobalamin to the substrate. The methods developed during this study allow high-yield in situ generation of labeled SAM and recombinant expression and purification of the malate synthase needed for chiral methyl analysis. These methods facilitate the broader use of in vitro chiral methyl analysis techniques to investigate the mechanisms of other novel enzymes.

C-H Bond Cleavage Is Rate-Limiting for Oxidative C-P Bond Cleavage by the Mixed Valence Diiron-Dependent Oxygenase PhnZ.

PhnZ utilizes a mixed valence diiron(II/III) cofactor and O2 to oxidatively cleave the carbon-phosphorus bond of (R)-2-amino-1-hydroxyethylphosphonic acid to form glycine and orthophosphate. The active site residues Y24 and E27 are proposed to mediate induced-fit recognition of the substrate and access of O2 to one of the active site Fe ions. H62 is proposed to deprotonate the C1-hydroxyl of the substrate during catalysis. Kinetic isotope effects (KIEs), pH-rate dependence, and site-directed mutagenesis were used to probe the rate-determining transition state and the roles of these three active site residues. Primary deuterium KIE values of 5.5 ± 0.3 for D(V) and 2.2 ± 0.4 for D(V/K) were measured with (R)-2-amino[1-2H1]-1-hydroxyethylphosphonic acid, indicating that cleavage of the C1-H bond of the substrate is rate-limiting. This step is also rate-limiting for PhnZ Y24F, as shown by a significant deuterium KIE value of 2.3 ± 0.1 for D(V). In contrast, a different reaction step appears to be rate-limiting for the PhnZ E27A and H62A variants, which exhibited D(V) values near unity. A solvent KIE of 2.2 ± 0.3 for D2O(V) is observed for PhnZ. Significant solvent KIE values are also observed for the PhnZ Y24F and E27A variants. In contrast, the PhnZ H62A variant does not show a significant solvent KIE, suggesting that H62 is mediating proton transfer in the transition state. A proton inventory study with PhnZ indicates that 1.5 ± 0.6 protons are in flight in the rate-determining step. Overall, the rate-determining transition state for oxidative C-P bond cleavage by PhnZ is proposed to involve C-H bond cleavage that is coupled to deprotonation of the substrate C1-hydroxyl by H62.

An Oxidative Pathway for Microbial Utilization of Methylphosphonic Acid as a Phosphate Source.

Methylphosphonic acid is synthesized by marine bacteria and is a prominent component of dissolved organic phosphorus. Consequently, methylphosphonic acid also serves as a source of inorganic phosphate (Pi) for marine bacteria that are starved of this nutrient. Conversion of methylphosphonic acid into Pi is currently only known to occur through the carbon-phosphorus lyase pathway, yielding methane as a byproduct. In this work, we describe an oxidative pathway for the catabolism of methylphosphonic acid in Gimesia maris DSM8797. G. maris can use methylphosphonic acid as Pi sources despite lacking a phn operon encoding a carbon-phosphorus lyase pathway. Instead, the genome contains a locus encoding homologues of the non-heme Fe(II) dependent oxygenases HF130PhnY* and HF130PhnZ, which were previously shown to convert 2-aminoethylphosphonic acid into glycine and Pi. GmPhnY* and GmPhnZ1 were produced in E. coli and purified for characterization in vitro. The substrate specificities of the enzymes were evaluated with a panel of synthetic phosphonates. Via 31P NMR spectroscopy, it is demonstrated that the GmPhnY* converts methylphosphonic acid to hydroxymethylphosphonic acid, which in turn is oxidized by GmPhnZ1 to produce formic acid and Pi. In contrast, 2-aminoethylphosphonic acid is not a substrate for GmPhnY* and is therefore not a substrate for this pathway. These results thus reveal a new metabolic fate for methylphosphonic acid.

2-Nitroimidazole-Furanoside Derivatives for Hypoxia Imaging-Investigation of Nucleoside Transporter Interaction, 18F-Labeling and Preclinical PET Imaging.

The benefits of PET imaging of tumor hypoxia in patient management has been demonstrated in many examples and with various tracers over the last years. Although, the optimal hypoxia imaging agent has yet to be found, 2-nitroimidazole (azomycin) sugar derivatives-mimicking nucleosides-have proven their potential with [18F]FAZA ([18F]fluoro-azomycin-α-arabinoside) as a prominent representative in clinical use. Still, for all of these tracers, cellular uptake by passive diffusion is postulated with the disadvantage of slow kinetics and low tumor-to-background ratios. We recently evaluated [18F]fluoro-azomycin-ß-deoxyriboside (ß-[18F]FAZDR), with a structure more similar to nucleosides than [18F]FAZA and possible interaction with nucleoside transporters. For a deeper insight, we comparatively studied the interaction of FAZA, ß-FAZA, α-FAZDR and ß-FAZDR with nucleoside transporters (SLC29A1/2 and SLC28A1/2/3) in vitro, showing variable interactions of the compounds. The highest interactions being for ß-FAZDR (IC50 124 ± 33 µM for SLC28A3), but also for FAZA with the non-nucleosidic α-configuration, the interactions were remarkable (290 ± 44 µM {SLC28A1}; 640 ± 10 µM {SLC28A2}). An improved synthesis was developed for ß-FAZA. For a PET study in tumor-bearing mice, α-[18F]FAZDR was synthesized (radiochemical yield: 15.9 ± 9.0% (n = 3), max. 10.3 GBq, molar activity > 50 GBq/µmol) and compared to ß-[18F]FAZDR and [18F]FMISO, the hypoxia imaging gold standard. We observed highest tumor-to-muscle ratios (TMR) for ß-[18F]FAZDR already at 1 h p.i. (2.52 ± 0.94, n = 4) in comparison to [18F]FMISO (1.37 ± 0.11, n = 5) and α-[18F]FAZDR (1.93 ± 0.39, n = 4), with possible mediation by the involvement of nucleoside transporters. After 3 h p.i., TMR were not significantly different for all 3 tracers (2.5⁻3.0). Highest clearance from tumor tissue was observed for ß-[18F]FAZDR (56.6 ± 6.8%, 2 h p.i.), followed by α-[18F]FAZDR (34.2 ± 7.5%) and [18F]FMISO (11.8 ± 6.5%). In conclusion, both isomers of [18F]FAZDR showed their potential as PET hypoxia tracers. Differences in uptake behavior may be attributed to a potential variable involvement of transport mechanisms.

C-Methylation Catalyzed by Fom3, a Cobalamin-Dependent Radical S-adenosyl-l-methionine Enzyme in Fosfomycin Biosynthesis, Proceeds with Inversion of Configuration.

Fom3, a cobalamin-dependent radical S-adenosyl-l-methionine (SAM) methyltransferase, catalyzes C-methylation at the C2 position of cytidylylated 2-hydroxyethylphosphonate (HEP-CMP) to afford cytidylylated 2-hydroxypropylphosphonate (HPP-CMP) in fosfomycin biosynthesis. In this study, the Fom3 reaction product HPP-CMP was reanalyzed by chiral ligand exchange chromatography to confirm its stereochemistry. The Fom3 methylation product was found to be ( S)-HPP-CMP only, indicating that the stereochemistry of the C-methylation catalyzed by Fom3 is ( S)-selective. In addition, Fom3 reaction was performed with ( S)-[2-2H1]HEP-CMP and ( R)-[2-2H1]HEP-CMP to elucidate the stereoselectivity during the abstraction of the hydrogen atom from C2 of HEP-CMP. Liquid chromatography-electrospray ionization mass spectrometry analysis of the 5'-deoxyadenosine produced showed that the 2H atom of ( R)-[2-2H1]HEP-CMP was incorporated into 5'-deoxyadenosine but that from ( S)-[2-2H1]HEP-CMP was not. Retention of the 2H atom of ( S)-[2-2H1]HEP-CMP in HPP-CMP was also observed. These results indicate that the 5'-deoxyadenosyl radical stereoselectively abstracts the pro-R hydrogen atom at the C2 position of HEP-CMP and the substrate radical intermediate reacts with the methyl group on cobalamin that is located on the opposite side of the substrate from SAM. Consequently, it was clarified that the C-methylation catalyzed by Fom3 proceeds with inversion of configuration.

The α-hydroxyphosphonate-phosphate rearrangement of a noncyclic substrate - some new observations.

Racemic ethyl hydrogen (1-hydroxy-2-methylsulfanyl-1-phenylethyl)phosphonate was resolved with (R)-1-phenylethylamine. The (R)-configuration of the (-)-enantiomer was determined by chemical correlation. Esterification of the (-)-enantiomer with a substituted diazomethane derived from 3-hydroxy-1,3,5(10)-estratrien-17-one delivered two epimeric phosphonates separated by HPLC. Methylation with methyl fluorosulfate at the sulfur atom and treatment with a strong base induced an α-hydroxyphosphonate-phosphate rearrangement with formation of dimethyl sulphide and two enantiomerically pure enol phosphates. Their oily nature interfered with a single crystal X-ray structure analysis to determine the stereochemistry at the phosphorus atom.

Stereochemical Course of Methyl Transfer by Cobalamin-Dependent Radical SAM Methyltransferase in Fosfomycin Biosynthesis.

The methyl groups of [ methyl-( S)]- and [ methyl-( R)]-[ methyl-D,T]-l-methionine fed to Streptomyces fradiae were incorporated into fosfomycin, which was chemically degraded to chiral AcONa. The enzymatic test gave the ( S)-configuration for the chiral AcONa derived from methionine with the ( S)-[D,T]methyl group ( F = 31.7) and ( R) for the one derived from methionine with the ( R)-[D,T]methyl group ( F = 83.0). The radical SAM methyltransferase transfers the methyl group of MeCbl to HEP-CMP with inversion of configuration.

On the rearrangement of N-aryl-N-Boc-phosphoramidates to N-Boc-protected o-aminoarylphosphonates.

ABSTRACT: Various arylamines were converted in two steps to N-Boc-N-arylphosphoramidates. LiTMP and LDA induced directed ortho-metalation at temperatures from -78 to 0 °C. The ensuing [1,3]-migration of the phosphorus atom with its substituents from the nitrogen to the ortho-carbanionic carbon atom gave N-Boc-protected o-aminoarylphosphonates. The nature of the substituent of 3-substituted phenylphosphoramidates strongly influenced the regioselectivity of phosphonate formation. A crossover experiment with a deuterated phosphoramidate proved the intramolecular course of the rearrangement. Three representative N-Boc-o-aminoarylphosphonates were deprotected to access the corresponding o-aminoarylphosphonic acids.

Preparation of Phosphonic Acid Analogues of Proline and Proline Analogues and Their Biological Evaluation as δ1-Pyrroline-5-carboxylate Reductase Inhibitors.

Racemic 1-hydroxy-3-butenyl-, 3-chloro-1-hydroxypropyl-, and 3-bromo-1-hydroxypropylphosphonate and the corresponding (S)-enantiomers obtained by lipase-catalyzed resolution of the respective racemic chloroacetates were subjected to functional group manipulations. These comprised ozonolysis, Mitsunobu reactions with hydrazoic acid and N-hydroxyphthalimide, alkylation of hydrazine derivative, removal of phthaloyl group followed by intramolecular substitution, and global deprotection to deliver the racemates and (R)-enantiomers (ee 92-99% by chiral high-performance liquid chromatography) of pyrrolidin-2-yl-, oxazolidin-3-yl-, oxazolidin-5-yl-, pyrazolidin-3-yl-, and 1,2-oxazinan-3-ylphosphonic acids. These phosphonic acids were evaluated as analogues of proline and proline analogues for the ability to inhibit γ-glutamyl kinase, δ1-pyrroline-5-carboxylate synthetase, and δ1-pyrroline-5-carboxylate reductase. Only the latter enzyme was inhibited by two of them at concentrations exceeding 1 mM.

A Methylidene Group in the Phosphonic Acid Analogue of Phenylalanine Reverses the Enantiopreference of Binding to Phenylalanine Ammonia-Lyases.

Aromatic amino acid ammonia-lyases and aromatic amino acid 2,3-aminomutases contain the post-translationally formed prosthetic 3,5-dihydro-4-methylidene-5H-imidazol-5-one (MIO) group. MIO enzymes catalyze the stereoselective synthesis of α- or ß-amino acid enantiomers, making these chemical processes environmentally friendly and affordable. Characterization of novel inhibitors enables structural understanding of enzyme mechanism and recognizes promising herbicide candidates as well. The present study found that both enantiomers of the aminophosphonic acid analogue of the natural substrate phenylalanine and a novel derivative bearing a methylidene at the ß-position inhibited phenylalanine ammonia-lyases (PAL), representing MIO enzymes. X-ray methods unambiguously determined the absolute configuration of all tested enantiomers during their synthesis. Enzyme kinetic measurements revealed the enantiomer of the methylidene-substituted substrate analogue as being a mirror image relation to the natural l-phenylalanine as the strongest inhibitor. Isothermal titration calorimetry (ITC) confirmed the binding constants and provided a detailed analysis of the thermodynamic driving forces of ligand binding. Molecular docking suggested that binding of the (R)- and (S)-enantiomers is possible by a mirror image packing.

Chemical Synthesis of (RP)- and (SP)-[16O,17O,18O]Phosphoenol Pyruvate.

Enzymes and chirality are intimately associated. For their mechanisms to be studied, chiral substrates are needed as probes. Here, we report a concise synthesis of (RP)- and (SP)-[16O,17O,18O]phosphoenol pyruvate starting from enantiomerically pure (R)-2-chloro-1-phenylethanol, which was transformed into 18O-labeled 3-methyl-1-phenylbutane-1,3-diol. The diol was reacted with tris(dimethylamino)phosphane and consecutively with H217O to yield a mixture of cyclic H-phosphonates labeled with 17O and 18O. They were silylated and subjected to a Perkow reaction with ethyl 3-chloropyruvate. Two protected-[16O,17O,18O]phosphoenol pyruvates were formed and finally globally deprotected. Their configuration was reassessed by a known enzymatic test in combination with conversion of the formed d-glucose-6-phosphate into mixtures of labeled methyl d-glucose-4,6-phosphates, which were analyzed by 31P NMR spectroscopy. The enzymatic test supported the configuration assigned to labeled stereogenic phosphorus atoms on the basis of synthesis.

Phosphonodifluoropyruvate is a mechanism-based inhibitor of phosphonopyruvate decarboxylase from Bacteroides fragilis.

Bacteroides fragilis, a human pathogen, helps in the formation of intra-abdominal abscesses and is involved in 90% of anaerobic peritoneal infections. Phosphonopyruvate decarboxylase (PnPDC), a thiamin diphosphate (ThDP)-dependent enzyme, plays a key role in the formation of 2-aminoethylphosphonate, a component of the cell wall of B. fragilis. As such PnPDC is a possible target for therapeutic intervention in this, and other phosphonate producing organisms. However, the enzyme is of more general interest as it appears to be an evolutionary forerunner to the decarboxylase family of ThDP-dependent enzymes. To date, PnPDC has proved difficult to crystallize and no X-ray structures are available. In the past we have shown that ThDP-dependent enzymes will often crystallize if the cofactor has been irreversibly inactivated. To explore this possibility, and the utility of inhibitors of phosphonate biosynthesis as potential antibiotics, we synthesized phosphonodifluoropyruvate (PnDFP) as a prospective mechanism-based inhibitor of PnPDC. Here we provide evidence that PnDFP indeed inactivates the enzyme, that the inactivation is irreversible, and is accompanied by release of fluoride ion, i.e., PnDFP bears all the hallmarks of a mechanism-based inhibitor. Unfortunately, the enzyme remains refractive to crystallization.

Efficient preparation of 2-nitroimidazole nucleosides as precursors for hypoxia PET tracers.

ABSTRACT: 2-Deoxy-D-ribose was converted to α/ß-mixtures of methyl 3-O-acetyl- and methyl 3-O-benzoyl-2-deoxy-5-(p-toluenesulfonyl)-D-ribofuranosides. These were reacted with boron trichloride to generate ribofuranosyl chlorides, which afforded precursors for tracers to image tumor hypoxia on substitution with salts of 2-nitroimidazole. The anomeric ratio of the nucleosides was delicately influenced by the reaction conditions.

[18F]Fluoro-azomycin-2´-deoxy-ß-d-ribofuranoside - A new imaging agent for tumor hypoxia in comparison with [18F]FAZA.

INTRODUCTION: Radiolabeled 2-nitroimidazoles (azomycins) are a prominent class of biomarkers for PET imaging of hypoxia. [18F]Fluoro-azomycin-α-arabinoside ([18F]FAZA) - already in clinical use - may be seen as α-configuration nucleoside, but enters cells only via diffusion and is not transported by cellular nucleoside transporters. To enhance image contrast in comparison to [18F]FAZA our objective was to 18F-radiolabel an azomycin-2´-deoxyriboside with ß-configuration ([18F]FAZDR, [18F]-ß-8) to mimic nucleosides more closely and comparatively evaluate it versus [18F]FAZA. METHODS: Precursor and cold standards for [18F]FAZDR were synthesized from methyl 2-deoxy-d-ribofuranosides α- and ß-1 in 6 steps yielding precursors α- and ß-5. ß-5 was radiolabeled in a GE TRACERlab FXF-N synthesizer in DMSO and deprotected with NH4OH to give [18F]FAZDR ([18F]-ß-8). [18F]FAZA or [18F]FAZDR was injected in BALB/c mice bearing CT26 colon carcinoma xenografts, PET scans (10min) were performed after 1, 2 and 3h post injection (p.i.). On a subset of mice injected with [18F]FAZDR, we analyzed biodistribution. RESULTS: [18F]FAZDR was obtained in non-corrected yields of 10.9±2.4% (9.1±2.2GBq, n=4) 60min EOB, with radiochemical purity >98% and specific activity >50GBq/µmol. Small animal PET imaging showed a decrease in uptake over time for both [18F]FAZDR (1h p.i.: 0.56±0.22% ID/cc, 3h: 0.17±0.08% ID/cc, n=9) and [18F]FAZA (1h: 1.95±0.59% ID/cc, 3h: 0.87±0.55% ID/cc), whereas T/M ratios were significantly higher for [18F]FAZDR at 1h (2.76) compared to [18F]FAZA (1.69, P<0.001), 3h p.i. ratios showed no significant difference. Moreover, [18F]FAZDR showed an inverse correlation between tracer uptake in carcinomas and oxygen breathing, while muscle tissue uptake was not affected by switching from air to oxygen. CONCLUSIONS: First PET imaging results with [18F]FAZDR showed advantages over [18F]FAZA regarding higher tumor contrast at earlier time points p.i. Availability of precursor and cold fluoro standard together with high output radiosynthesis will allow for a more detailed quantitative evaluation of [18F]FAZDR, especially with regard to mechanistic studies whether active transport processes are involved, compared to passive diffusion as observed for [18F]FAZA.

Synthesis and preclinical characterization of 1-(6'-deoxy-6'-[18F]fluoro-ß-d-allofuranosyl)-2-nitroimidazole (ß-6'-[18F]FAZAL) as a positron emission tomography radiotracer to assess tumor hypoxia.

Positron emission tomography (PET) using fluorine-18 (18F)-labeled 2-nitroimidazole radiotracers has proven useful for assessment of tumor oxygenation. However, the passive diffusion-driven cellular uptake of currently available radiotracers results in slow kinetics and low tumor-to-background ratios. With the aim to develop a compound that is actively transported into cells, 1-(6'-deoxy-6'-[18F]fluoro-ß-d-allofuranosyl)-2-nitroimidazole (ß-[18F]1), a putative nucleoside transporter substrate, was synthetized by nucleophilic [18F]fluoride substitution of an acetyl protected labeling precursor with a tosylate leaving group (ß-6) in a final radiochemical yield of 12±8% (n=10, based on [18F]fluoride starting activity) in a total synthesis time of 60min with a specific activity at end of synthesis of 218±58GBq/µmol (n=10). Both radiolabeling precursor ß-6 and unlabeled reference compound ß-1 were prepared in multistep syntheses starting from 1,2:5,6-di-O-isopropylidene-α-d-allofuranose. In vitro experiments demonstrated an interaction of ß-1 with SLC29A1 and SLC28A1/2/3 nucleoside transporter as well as hypoxia specific retention of ß-[18F]1 in tumor cell lines. In biodistribution studies in healthy mice ß-[18F]1 showed homogenous tissue distribution and excellent metabolic stability, which was unaffected by tissue oxygenation. PET studies in tumor bearing mice showed tumor-to-muscle ratios of 2.13±0.22 (n=4) at 2h after administration of ß-[18F]1. In ex vivo autoradiography experiments ß-[18F]1 distribution closely matched staining with the hypoxia marker pimonidazole. In conclusion, ß-[18F]1 shows potential as PET hypoxia radiotracer which merits further investigation.

The Stereochemical Course of the α-Hydroxyphosphonate-Phosphate Rearrangement.

The phosphonate-phosphate rearrangement is an isomerisation of α-hydroxyphosphonates bearing electron-withdrawing substituents at the α-carbon atom. We studied the stereochemical course of this rearrangement with respect to phosphorus. A set of four diastereomeric α-hydroxyphosphonates was prepared by a Pudovik reaction from two diastereomeric cyclic phosphites. The hydroxyphosphonates were separated and rearranged with Et3 N as base. In analogy to trichlorphon, which was the first reported compound undergoing this rearrangement. All four hydroxyphosphonates could be rearranged to 2,2-dichlorovinyl phosphates. Single-crystal X-ray structure analyses of the α-hydroxyphosphonates and the corresponding phosphates allowed us to show that the rearrangement proceeds with retention of configuration on the phosphorus atom.

Phosphonate­phosphinate rearrangement.

LiTMP metalated dimethyl N-Boc-phosphoramidates derived from 1-phenylethylamine and 1,2,3,4-tetrahydronaphthalen-1-ylamine highly selectively at the CH3O group to generate short-lived oxymethyllithiums. These isomerized to diastereomeric hydroxymethylphosphonamidates (phosphate­phosphonate rearrangement). However, s-BuLi converted the dimethyl N-Boc-phosphoramidate derived from 1-phenylethylamine to the N-Boc α-aminophosphonate preferentially. Only s-BuLi deprotonated dimethyl hydroxymethylphosphonamidates at the benzylic position and dimethyl N-Boc α-aminophosphonates at the CH3O group to induce phosphonate­phosphinate rearrangements. In the former case, the migration of the phosphorus substituent from the nitrogen to the carbon atom followed a retentive course with some racemization because of the involvement of a benzyllithium as an intermediate.

Crystal structure of PhnZ in complex with substrate reveals a di-iron oxygenase mechanism for catabolism of organophosphonates.

The enzymes PhnY and PhnZ comprise an oxidative catabolic pathway that enables marine bacteria to use 2-aminoethylphosphonic acid as a source of inorganic phosphate. PhnZ is notable for catalyzing the oxidative cleavage of a carbon-phosphorus bond using Fe(II) and dioxygen, despite belonging to a large family of hydrolytic enzymes, the HD-phosphohydrolase superfamily. We have determined high-resolution structures of PhnZ bound to its substrate, (R)-2-amino-1-hydroxyethylphosphonate (2.1 Å), and a buffer additive, l-tartrate (1.7 Å). The structures reveal PhnZ to have an active site containing two Fe ions coordinated by four histidines and two aspartates that is strikingly similar to the carbon-carbon bond cleaving enzyme, myo-inositol-oxygenase. The exception is Y24, which forms a transient ligand interaction at the dioxygen binding site of Fe2. Site-directed mutagenesis and kinetic analysis with substrate analogs revealed the roles of key active site residues. A fifth histidine that is conserved in the PhnZ subclade, H62, specifically interacts with the substrate 1-hydroxyl. The structures also revealed that Y24 and E27 mediate a unique induced-fit mechanism whereby E27 specifically recognizes the 2-amino group of the bound substrate and toggles the release of Y24 from the active site, thereby creating space for molecular oxygen to bind to Fe2. Structural comparisons of PhnZ reveal an evolutionary connection between Fe(II)-dependent hydrolysis of phosphate esters and oxidative carbon-phosphorus or carbon-carbon bond cleavage, thus uniting the diverse chemistries that are found in the HD superfamily.