Preparation of Nucleoside H-Phosphonoselenoate Monoesters Via the Phosphinate Approach

An efficient entry to nucleoside 3′-H-phosphonoselenoate monoesters via phosphinate intermediates was developed. It involves a reaction of suitably protected nucleosides with triethylammonium phosphinate in the presence of pivaloyl chloride, followed by selenization of the intermediate nucleoside phosphinates with triphenylphosphine selenide, to produce the corresponding nucleoside H-phosphonoselenoates in 86–92% yields.     

Synthesis and evaluation of some novel phosphate and phosphinate derivatives of araA. Studies on the mechanism of action of phosphate triesters.

A number of novel phosphinate and phosphate triester derivatives of the anti-viral nucleoside analogue araA have been prepared. Spectroscopic and analytical data have been collected on both the reagents and the nucleotides. An in vitro assay indicated inhibition of DNA synthesis by mammalian cells, by each of the nucleotide derivatives, in the range 3-30 microM. Inhibition was reduced, but not abolished, for the phosphinates relative to the phosphates. These results are consistent with a mode of action involving release of the free nucleoside araA and the nucleotide araAMP.     

Synthesis of 1,2-dipalmitoyloxypropyl-3-(2-trimethylammoniumethyl)phosphinate

The total synthesis of 1,2-dipalmitoyloxypropyl-3-(2-trimethylammoniumethyl)phosphinate, the phosphinate analog of phosphatidylcholine, is described. The phosphinate analog has been essentially prepared by an Arbusov type reaction between 1,2-dipalmitoyl-sn-glycerolbromohydrin and 2-bromoethyl phosphonic acid dimethyl ester for 48 h at 170°C, followed by removal of the methyl ester with sodium iodide and reaction with aqueous trimethylamine to yield the final product. The phosphinate analog of phosphatidylcholine was characterized by elemental analysis, thin-layer chromatography (TLC), IR spectroscopy and phosphonophosphorus determinations.     

ATP-dependent ligases in trypanothione biosynthesis--kinetics of catalysis and inhibition by phosphinic acid pseudopeptides

Glutathionylspermidine is an intermediate formed in the biosynthesis of trypanothione, an essential metabolite in defence against chemical and oxidative stress in the Kinetoplastida. The kinetic mechanism for glutathionylspermidine synthetase (EC 6.3.1.8) from Crithidia fasciculata (CfGspS) obeys a rapid equilibrium random ter-ter model with kinetic constants K(GSH) = 609 microM, K(Spd) = 157 microM and K(ATP) = 215 microM. Phosphonate and phosphinate analogues of glutathionylspermidine, previously shown to be potent inhibitors of GspS from Escherichia coli, are equally potent against CfGspS. The tetrahedral phosphonate acts as a simple ground state analogue of glutathione (GSH) (K(i) approximately 156 microM), whereas the phosphinate behaves as a stable mimic of the postulated unstable tetrahedral intermediate. Kinetic studies showed that the phosphinate behaves as a slow-binding bisubstrate inhibitor [competitive with respect to GSH and spermidine (Spd)] with rate constants k(3) (on rate) = 6.98 x 10(4) M(-1) x s(-1) and k(4) (off rate) = 1.3 x 10(-3) s(-1), providing a dissociation constant K(i) = 18.6 nM. The phosphinate analogue also inhibited recombinant trypanothione synthetase (EC 6.3.1.9) from C. fasciculata, Leishmania major, Trypanosoma cruzi and Trypanosoma brucei with K(i)(app) values 20-40-fold greater than that of CfGspS. This phosphinate analogue remains the most potent enzyme inhibitor identified to date, and represents a good starting point for drug discovery for trypanosomiasis and leishmaniasis.     

Silanediol-based inhibitor of thermolysin

The first silanediol inhibitor of thermolysin is reported, prepared by analogy with the Grobelny/Bartlett phosphinate inhibitor. A Cbz group on nitrogen proved to be unstable to the triflic acid mediated silanediol deprotection and was replaced with a dihydrocinnamoyl group. The silanediol was prepared in high purity by hydrolysis of a difluorosilane intermediate and proved to be an effective inhibitor, differing from the phosphinate by a factor of 4 (K_i=41 nM).     

Spontaneous reactivation of phosphinylated human erythrocyte acetylcholinesterase and human serum butyrylcholinesterase

This report documents studies on the spontaneous reactivation of human erythrocyte acetylcholinesterase and human serum butyrylcholinesterase following inhibition by organophosphinate esters. The spontaneous reactivation reactions were carried out at 26.0 degrees C in 0.10 M phosphate buffer of pH 7.6. Based upon results at 24 h, human serum butyrylcholinesterase inhibited with 4-nitrophenyl methyl (4-methoxyphenyl) phosphinate was the most responsive (92.5% recovery) of the nine esters studied. Using the same criteria, the most active compound in the human erythrocyte acetylcholinesterase studies was 4-nitrophenyl methyl(phenyl)phosphinate (74.2% recovery). With seven of the nine compounds examined the response was greater from the serum enzyme than from the erythrocyte enzyme.     

Determination of dissociation constant of phosphinate group in phosphinic pseudopeptides by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was used for determination of dissociation constant of phosphinate group in phosphinic pseudopeptides, i.e. peptides where one peptide bond is substituted by phosphinic acid moiety -PO2--CH2-. The dissociation constants were determined for a set of newly synthesized pseudopeptides derived from a structure N-Ac-Val-Ala(psi)(PO2--CH2)Leu-His-NH2 by nonlinear regression of experimentally measured pH dependence of their effective electrophoretic mobilities. CZE experiments were carried out in Tris-phosphate background electrolytes in the pH range 1.4-3.2. The pseudopeptides were synthesized as a mixture of four diastereomers, the separation of which was achieved in most cases. Moreover, differences of the effective mobilities of the pseudopeptide diastereomers enabled simultaneous determination of the dissociation constant of their phosphinate group without necessity of previous isolation of individual isomers.     

Structure-based design and synthesis of phosphinate isosteres of phosphotyrosine for incorporation in Grb2-SH2 domain inhibitors. Part 2

A series of novel phosphinates, derived from 4-phosphonomethylphenylalanine, are described as isosteres of phosphotyrosine. Benzyl (or alkyl) phosphinomethylphenylalanine derivatives were prepared by alkylation of an amino acid P-H phosphinate.     

Inhibitors of the bacterial cell wall biosynthesis enzyme MurC

A series of phosphinate transition-state analogues of the L-alanine adding enzyme (MurC) of bacterial peptidoglycan biosynthesis was prepared and tested as inhibitors of the Escherichia coli enzyme. Compound 4 was identified as a potent inhibitor of MurC from Escherichia coli with an IC(50) of 49nM.     

Synthesis of new pyrimidine 5'-H-thiophosphonates]

Thymidine and 3'-deoxythymidine 5'-H-thiophosphonates were synthesized by interaction of 3'-O-acetylthymidine or 3'-deoxythymidine and ammonium phosphinate in the presence of pivaloyl chloride and following oxidation in situ by sulfur of the intermediate phosphinates. The compounds seemed to be 1:1 mixture of diastereomers with chiral phosphorus. The prepared compounds were tested as potential inhibitors of HIV production.     

Novel hydroxamic acid-related phosphinates: inhibition of neutral aminopeptidase N (APN)

Here we describe the inhibitory activity toward neutral aminopeptidase of three new families of phosphinate inhibitors related in structure to hydroxamic acids. These compounds, even as racemic mixtures, are good inhibitors of APN and show strong structure activity relationship (SAR) depending on the substituents in P1 and P1' positions.     

Crystal structures of active LytM

Lysostaphin-type enzymes are metalloendopeptidases that are present in bacteriophages and in bacteria. They share the catalytic domain, but normally contain other domains as well. The well-characterized enzymes in this group are all specific for the pentaglycine crosslinks in the cell walls of some Gram-positive bacterial species. Lysostaphin-type enzymes are synthesized as secreted preproenzymes and require proteolytic activation for maturation. Although lysostaphin, the prototypical peptidase in the group, is widely used as a tool in biotechnology and developed as an antistaphylococcal agent, the detailed structure of this enzyme is unknown. So far, only one lysostaphin-type enzyme, the Staphylococcus aureus autolysin LytM, has been crystallized in its full-length, inactive form. Here, we describe the synthesis of a convenient reporter substrate, characterize the metal and pH-dependence of an active LytM fragment, and present its crystal structure in three crystal forms at different pH values that either support or do not support activity. In all structures, we find an extended, long and narrow groove that has the active site at its bottom and is delineated on the sides by the most flexible regions of the molecule. In two cases, the groove is partially filled by a loop of a neighbouring molecule in the crystal. As the loop contains three consecutive glycine residues, this crystal packing effect supports the interpretation that the groove is the substrate-binding cleft. To characterize the substrate-binding mode more closely, a phosphinate analogue of tetraglycine was synthesized. Although tetraglycine is a substrate of the active LytM fragment, the phosphinate analogue turned out to be a very poor inhibitor. Crystals that were grown in its presence contained an L+-tartrate molecule from the crystallization buffer and not the phosphinate in the active site.     

Structure-based design and synthesis of phosphinate isosteres of phosphotyrosine for incorporation in Grb2-SH2 domain inhibitors. Part 1

Based on X-ray crystal structure information, mono charged phosphinate isosteres of phosphotyrosine have been designed and incorporated in a short inhibitory peptide sequence of the Grb2-SH2 domain. The resulting compounds, by exploiting additional interactions, inhibit binding to the Grb2-SH2 domain as potently as the corresponding doubly charged (phosphonomethyl)phenylalanine analogue.     

Phosphinate, sulfonate, and sulfonamidate dipeptides as potential inhibitors of Escherichia coli aminopeptidase N

In an effort to prepare novel inhibitors of bacterial aminopeptidase N (PepN), the phosphinate, propenylphosphinate, decylphosphinate, sulfonate, and sulfonamidate analogs of Ala-Ala were synthesized and tested as inhibitors. Phosphinate 1 was shown to inhibit PepN with a K(i) of 10microM, and propenylphosphinate 2 and decylphosphinate 3 inhibited PepN with a K(i) of ca. 1microM. Sulfonate and sulfonamidate analogs did not inhibit PepN.     

Synthetic studies towards a novel, chemical stable, abasic site analogue of DNA

We synthesized the phosphinate 7 via photoaddition of methanol to the alpha, beta unsaturated deoxyribono lactone as the key step, followed by an Arbusov reaction for the introduction of phosphorous. Precursor 7 serves for the synthesis and incorporation into DNA of a novel chemically stable abasic site analogue that might act as an inhibitor for DNA glycosylases.     

Design, synthesis and structure-activity relationships of new phosphinate inhibitors of MurD

A series of new phosphinate compounds were designed and synthesized as inhibitors of the d-glutamic acid-adding enzyme (MurD) involved in peptidoglycan biosynthesis. They were tested against the MurD enzyme from Escherichia coli, allowing initial structure-activity relationships to be deduced. Two compounds had IC(50) values near 100 microM and constitute a promising starting point for further development.     

Design, modelling, synthesis and biological evaluation of peptidomimetic phosphinates as inhibitors of matrix metalloproteinases MMP-2 and MMP-8

Three novel peptidomimetic phosphinate inhibitors have been synthesized and evaluated as inhibitors of matrix metalloproteinases MMP-2 and MMP-8. Their IC50 values are in the micromolar range, and one of them showed to be the most effective inhibitor of MMP-2. The differences in binding affinities for MMP-2 and MMP-8 of the three phosphinates have been rationalized by means of modelling studies and molecular dynamics simulations.     

Submicromolar phosphinic inhibitors of Escherichia coli aspartate transcarbamoylase

The design, syntheses, and enzymatic activity of two submicromolar competitive inhibitors of aspartate transcarbamoylase (ATCase) are described. The phosphinate inhibitors are analogs of N-phosphonacetyl-l-aspartate (PALA) but have a reduced charge at the phosphorus moiety. The mechanistic implications are discussed in terms of a possible cyclic transition-state during enzymatic catalysis.     

Specific soman-hydrolyzing enzyme activity in a clonal neuronal cell culture

An enzymatic activity that specifically hydrolyzes the highly toxic organophosphorus anticholinesterase compound soman (pinacolyl methylphosphonofluoridate) has been identified and partially characterized in the clonal neuronal neuroblastoma-glioma hybrid NG108-15 cell line. Using the whole cell homogenate as the enzyme source and 1 mM substrate, the relative rate of hydrolysis of two other toxic anticholinesterase compounds sarin (isopropyl methylphosphonofluoridate) and tabun (ethyl-N-dimethyl phosphoramidocyanidate) is approximately one-tenth the rate of hydrolysis of soman, while DFP (diisopropyl phosphorofluoridate), paraoxon (p-nitrophenyl diethylphosphate), and a phosphinate PNMPP (p-nitrophenyl methyl (phenyl) phosphinate) are not hydrolyzed. Analysis of the kinetics of soman hydrolysis reveals two components of the enzyme activity with different affinities and reaction rates. Unlike previously reported enzymes of this type, this enzyme lacks chiral specificity and thus hydrolyzes both toxic and non-toxic soman stereoisomers at equal rates. The enzyme activity is stable at low temperature, found almost exclusively in the soluble fraction of these cells, and enhanced significantly by Mn2+ and by chemical differentiation of these cells in culture. The results suggest possible application of this enzyme for soman detection and/or detoxication, and use of the NG108-15 cell line to study the natural function(s) of enzymes of this type.     

Kinetic properties of a nucleoside phosphotransferase of chick embryo

1. A nonspecific nucleoside phosphotransferase (nucleotide : 3'-deoxynucleotide 5'-phosphotransferase, EC 2.7.1.77), purified from chick embryos, catalyzes the transfer of phosphate ester from a nucleotide donor to a nucleoside acceptor. 2. The enzyme exhibits sigmoidal kinetics with respect to nucleoside monophosphate donors, but with respect to nucleoside di- or triphosphate donors and nucleoside acceptors hyperbolic kinetics were obtained. 3. The nucleoside phosphotransferase of chick embryo is unstable to heat and is protected from inactivation by a large number of nucleosides. 4. Nucleoside di- and triphosphates lower both the concentration of nucleoside monophosphates required for half-maximal velocity and the kinetic order of reaction measured with these phosphate donors. On the contrary, nucleoside di- or triphosphate do not modify the kinetic parameters evaluated for nucleoside acceptors. 5. We suggest that the nucleoside phosphotransferase contains both substrate and regulatory sites. It seems that the free apoenzyme is converted, by means of cooperative interactions between regulatory sites, into an enzyme-nucleotide complex, which is particularly stable at 37 degrees C.