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.

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.

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.

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.

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.

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.

On the configurational stability of chiral, nonracemic fluoro- and iodo-[D(1)]methyllithiums.

Enantiomerically pure fluoro-[D1]methyllithium and iodo-[D1]methyllithiums of up to 92% ee were generated by transmetalation of the corresponding stannanes with MeLi in THF at various temperatures. The intermediate halo-[D1]methyllithiums were trapped with benzaldehyde or acetophenone already present in excess in the reaction mixture to either give halohydrins or to disintegrate to carbene. The fluoro-[D1]methyllithiums were found to be microscopically configurationally stable within the tested range of -95 to 0 °C, but chemically only stable at temperatures below -95 °C due to a rapidly increasing portion disintegrating to carbene. The iodo-[D1]methyllithiums were configurationally labile relative to the rate of addition to PhCHO at all temperatures tested (-95 to -30 °C). Disintegration to carbene interfered as well.

[(3)H]metyrapol and 4-[(131)i]iodometomidate label overlapping, but not identical, binding sites on rat adrenal membranes.

Metyrapone, metyrapol, and etomidate are competitive inhibitors of 11-deoxycorticosterone hydroxylation by 11ß-hydroxylase. [(3)H]Metyrapol and 4-[(131)I]iodometomidate bind with high affinity to membranes prepared from bovine and rat adrenals. Here we report inhibitory potencies of several compounds structurally related to one or both of these adrenostatic drugs, against the binding of both radioligands to rat adrenal membranes. While derivatives of etomidate inhibited the binding of both radioligands with similar potencies, derivatives of metyrapone inhibited the binding of 4-[(131)I]iodometomidate about 10 times weaker than the binding of [(3)H]metyrapol. By X-ray structure analysis the absolute configuration of (+)-1-(2-fluorophenyl)-2-methyl-2-(pyridin-3-yl)-1-propanol [(+)-11, a derivative of metyrapol] was established as (R). We introduce 1-(2-fluorophenyl)-2-methyl-2-(pyridin-3-yl)-1-propanone (9; Ki = 6 nM), 2-(1-imidazolyl)-2-methyl-1-phenyl-1-propanone (13; 2 nM), and (R)-(+)-[1-(4-iodophenyl)ethyl]-1H-imidazole (34; 4 nM) as new high affinity ligands for the metyrapol binding site on 11ß-hydroxylase and discuss our results in relation to a proposed active site model of 11ß-hydroxylase.

Determination of absolute configuration of the phosphonic acid moiety of fosfazinomycins.

Fosfazinomycins A and B produced by Streptomyces lavendofoliae share the same phosphonate moiety with one chiral centre of unknown configuration which was determined by synthesising both enantiomers of 2-hydroxy-2-phosphonoacetic acid methyl ester. A chiral cyclic phosphite was reacted with methyl glyoxylate in a Pudovik reaction to give a pair of diastereomeric α-hydroxyphosphonates, which were separated by HPLC. The configurations at C-2 were assigned on the basis of single crystal X-ray structure analysis. Deprotection of these diastereomers furnished the enantiomeric α-hydroxyphosphonic acids, of which the (S)-configured had the same sign of optical rotation as the phosphonic acid moiety of the two fosfazinomycins.

On the Configurational Stability of Chiral Heteroatom-Substituted [D1]Methylpalladium Complexes as Intermediates of Stille and Suzuki-Miyaura Cross-Coupling Reactions.

Enantiomerically pure (S)-tributylstannyl[D1]methanol and (R)- and (S)-tributylstannyl[D1]methyl benzoates were Stille-coupled with bromobenzene and benzoyl chloride in 1,4-dioxane and toluene using [(Ph3P)4Pd] or [(Ph3P)2PdCl2] either alone or in combination with CuCN as cocatalyst at temperatures up to 80 °C. The products were found to be enantiomerically pure. (R)- and (S)-N-(tributylstannyl[D1]methyl)phthalimides gave enantiomerically pure products with benzoyl chloride, but with bromobenzene protected phenyl[D1]methylamines gave products of only 52-69 % ee depending on the solvent used. Tributyl(thio[D1]methyl)stannanes could not be Stille-coupled with benzoyl chloride or with bromobenzene. Similarly, dimethyl phenyl[D1]methylboronate underwent a Suzuki-Miyaura coupling with bromobenzene to give phenyl[D1]methylsilane with 99 % ee. All couplings followed a retentive course and, except in one case, the intermediate [XCHDPdL n ] complexes were found to be microscopically configurationally stable.

On the preparation and determination of configurational stability of chiral thio- and bromo[D1]methyllithiums.

Thio- and bromo[D(1)]methyllithiums (ee 99%) were generated from the respective stannanes by tin-lithium exchange at temperatures ranging from 0 to -95 °C. Thio[D(1)]methyllithiums 6 were found to be microscopically configurationally labile on the time scale of the thiophosphate-α-mercaptophosphonate rearrangement even at -95 °C. Thio[D(1)]methyllithiums 13a and 13b underwent a thia-[2,3]-Wittig rearrangement down to -95 °C and 13b only down to -50 °C. The former were microscopically configurationally stable below -95 °C, and the latter racemized completely at -50 °C. Chiral bromo[D(1)]methyllithiums are chemically unstable at -78 °C but microscopically configurationally stable at the time scale of their addition to benzaldehyde and acetophenone.

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.

Mechanism and substrate recognition of 2-hydroxyethylphosphonate dioxygenase.

HEPD belongs to the superfamily of 2-His-1-carboxylate non-heme iron-dependent dioxygenases. It converts 2-hydroxyethylphosphonate (2-HEP) to hydroxymethylphosphonate (HMP) and formate. Previously postulated mechanisms for the reaction catalyzed by HEPD cannot explain its conversion of 1-HEP to acetylphosphate. Alternative mechanisms that involve either phosphite or methylphosphonate as intermediates, which potentially explain all experimental studies including isotope labeling experiments and use of substrate analogues, were investigated. The results of these studies reveal that these alternative mechanisms are not correct. Site-directed mutagenesis studies of Lys16, Arg90, and Tyr98 support roles of these residues in binding of 2-HEP. Mutation of Lys16 to Ala resulted in an inactive enzyme, whereas mutation of Arg90 to Ala or Tyr98 to Phe greatly decreased k(cat)/K(m,2-HEP). Furthermore, the latter mutants could not be saturated in O(2). These results suggest that proper binding of 2-HEP is important for O(2) activation and that the enzyme uses a compulsory binding order with 2-HEP binding before O(2). The Y98F mutant produces methylphosphonate as a minor side product providing indirect support for the proposal that the last step during catalysis involves a ferric hydroxide reacting with a methylphosphonate radical.