P-stereocontrolled synthesis of oligo(nucleoside N3'→O5' phosphoramidothioate)s - opportunities and limitations.

3'--(2-Thio-1,3,2-oxathiaphospholane) derivatives of 5'--DMT-3'-amino-2',3'-dideoxy-ribonucleosides (OTP-N), that bear a 4,4-unsubstituted, 4,4-dimethyl, or 4,4-pentamethylene substituted oxathiaphospholane ring, were synthesized. Within these three series, OTP-N differed by canonical nucleobases (, Ade, Cyt, Gua, or Thy). The monomers were chromatographically separated into P-diastereomers, which were further used to prepare NN' dinucleotides (3), as well as short P-stereodefined oligo(deoxyribonucleoside N3'→O5' phosphoramidothioate)s (NPS-) and chimeric NPS/PO- and NPS/PS-oligomers.

Phosphorothioate analogs of P1,P3-di(nucleosid-5'-yl) triphosphates: Synthesis, assignment of the absolute configuration at P-atoms and P-stereodependent recognition by Fhit hydrolase.

Di(nucleosid-5'-yl) polyphosphates (NN) are involved in various biological processes, and constitute signaling molecules in the intermolecular purinergic systems. They exert tumor suppression function and are substrates for specific hydrolases (e.g.

Crystallographic studies of the complex of human HINT1 protein with a non-hydrolyzable analog of Ap4A.

Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch in the histidine triad proteins superfamily. HINT1 plays an important role in various biological processes, and it has been found in many species. Here, we report the first structure (at a 2.

Synthesis of PS/PO-chimeric oligonucleotides using mixed oxathiaphospholane and phosphoramidite chemistry.

Chimeric oligonucleotides containing phosphodiester and phosphorothioate linkages have been obtained using the solid phase synthesis. The oligonucleotide parts possessing natural internucleotide phosphate bonds were assembled using commercially available nucleoside 3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites 7 whereas the phosphorothioate segment was built using nucleoside 3'-O-(2-thio-1,3,2-oxathiaphospholanes) 3. The oxidation steps, crucial for the conversion of phosphite linkages into the phosphate moieties, were conducted using tert-butylperoxy-trimethylsilane, and this reagent was not harmful to the diester phosphorothioate linkages.

N-Acyl-phosphoramidates as potential novel form of gemcitabine prodrugs.

Gemcitabine (dFdC) is a cytidine analog remarkably active against a wide range of solid tumors. Inside a cell, gemcitabine is phosphorylated by deoxycytidine kinase to yield gemcitabine monophosphate, further converted to gemcitabine di- and triphosphate. The most frequent form of acquired resistance to gemcitabine in vitro is the deoxycytidine kinase deficiency.

Hint2, the mitochondrial nucleoside 5'-phosphoramidate hydrolase; properties of the homogeneous protein from sheep (Ovis aries) liver.

Adenosine 5'-phosphoramidate (NH2-pA) is a rare natural nucleotide and its biochemistry and biological functions are poorly recognized. All organisms have proteins that may be involved in the catabolism of NH2-pA. They are members of the HIT protein family and catalyze hydrolytic splitting of NH2-pA to 5'-AMP and ammonia.

Synthesis and an antiviral activity evaluation of nucleoside 5´-O-(N-acyl) phosphoramidates.

Background: pyrimidine nucleoside analogues represent an established class of clinically useful antiviral agents. Once inside the cell, they are activated by a series of intracellular phosphorylation steps to produce 5´-triphosphate derivatives. In many cases, nucleoside analogues are poor substrates for the cellular kinases needed for their activation.

Mutations that increase both Hsp90 ATPase activity in vitro and Hsp90 drug resistance in vivo.

Hsp90 inhibitors are currently tested in clinical trials as anticancer agents. We investigated whether inhibitor resistance may arise as a result of a point mutation in Hsp90. We used yeast cells that expressed human Hsp90beta to select inhibitor-resistant mutants from the randomly mutagenized library.

Stereochemistry of rHint1 hydrolase assisted cleavage of P-N bond in nucleoside 5'-O-phosphoramidothioates.

The Hint-1 hydrolase assisted cleavage of the P-N bond in adenosine-5'-O-[N-(tryptophanylamide)]phosphoramidothioate proceeds with retention of configuration at the phosphorus atom which is consistent with the formation of a covalent enzyme-substrate complex.

Limited usefulness of the PFA-100 for the monitoring of ADP receptor antagonists--in vitro experience.

We have evaluated the usefulness of the PFA-100 system (collagen/ADP and collagen/epinephrine cartridges) to assess the in vitro effects of a few platelet function inhibitors: Aspisol (60 microg/ml), 4-[4-[4-(aminoiminomethyl]-1-piperazinyl]-1-piperidineactetic acid, hydrochloride trihydrate (GR144053F, fibrinogen receptor antagonist, 100 nM), adenosine-3',5'-diphosphate (A3P5P, P2Y1 ADP receptor antagonist, 500 microM) and Bis[(adenosine-5'-O-phosphorodithioyl)methylene]-phosphinic acid (APTMPA, P2Y12 ADP receptor antagonist, 500 microM) on platelet function, as compared with the other commonly used diagnostic technique, a whole blood electrical aggregometry (20 microM ADP or 0.5 mM arachidonic acid). The in vitro studies were carried out on a group of 38 subjects.

Biochemical, crystallographic, and mutagenic characterization of hint, the AMP-lysine hydrolase, with novel substrates and inhibitors.

Hint, histidine triad nucleotide-binding protein, is a universally conserved enzyme that hydrolyzes AMP linked to lysine and, in yeast, functions as a positive regulator of the RNA polymerase II C-terminal domain kinase, Kin28. To explore the biochemical and structural bases for the adenosine phosphoramidate hydrolase activity of rabbit Hint, we synthesized novel substrates linking a p-nitroaniline group to adenylate (AMP-pNA) and inhibitors that consist of an adenosine group and 5'-sulfamoyl (AdoOSO(2)NH(2)) or N-ethylsulfamoyl (AdoOSO(2)NHCH(2)CH(3)) group. AMP-pNA is a suitable substrate for Hint that allowed characterization of the inhibitors; titration of each inhibitor into AMP-pNA assays revealed their K(i) values.

Is platelet aggregation a more important contributor than platelet adhesion to the overall platelet-related primary haemostasis measured by PFA-100?

Background: Platelet-related primary haemostasis (PRPH), measured in PFA-100 as a closure time (CT), reflects platelets' combined ability to adhere and aggregate under higher shear stress. The inputs of platelet aggregation and platelet adhesion into the real values of CT remain unknown, and this poor discrimination results in the complexity of the PFA-100 measurement.

Objective: To estimate the particular contributions of two physiological phenomena, platelet aggregation and adhesion, and the importance of various membrane receptors underlying platelets' capability of the plug formation in PFA-100 cartridges.

Adenosine-5'-O-phosphorylated and adenosine-5'-O-phosphorothioylated polyols as strong inhibitors of (symmetrical) and (asymmetrical) dinucleoside tetraphosphatases.

Dinucleoside 5',5"'- P (1), P ( n )-polyphosphates, and particularly the diadenosine compounds, have been implicated in extracellular purinergic signalling and in various intracellular processes, including DNA metabolism, tumour suppression and stress responses. If permitted to accumulate, they may also be toxic. One approach to understanding their function is through the various specific degradative enzymes that regulate their levels.

Oxathiaphospholane approach to N- and O-phosphorothioylation of amino acids.

A method of highly efficient synthesis of N- and O-phosphorothioylated amino acids was developed. N- and O-(2-Thiono-1,3,2-oxathiaphospholanyl)amino acid methyl esters (3) were prepared in high yields in reaction of amino acid methyl esters with 2-chloro-1,3,2-oxathiaphospholane in pyridine in the presence of elemental sulfur. Compounds 3 were converted in high yield into the corresponding methyl or benzyl phosphorothioamides 6 and 7 by DBU-assisted treatment with methanol or benzyl alcohol.

Inhibition of ADP-triggered blood platelet aggregation by diadenosine polyphosphate analogues.

The synthesis and biological evaluation of new diadenosine polyphosphate analogues on blood platelet aggregation are reported. The most active are compounds with a sulfur atom replacing one or both non-bridging oxygens at phosphorus bound to adenosyl residues and hydroxymethyl groups of bis(hydroxymethyl)phosphinic acid.

Di-, tri- and tetra-5'-O-phosphorothioadenosyl substituted polyols as inhibitors of Fhit: Importance of the alpha-beta bridging oxygen and beta phosphorus replacement.

BACKGROUND: The human FHIT gene is inactivated early in the development of many human cancers and loss of Fhit in mouse predisposes to cancer while reintroduction of FHIT suppresses tumor formation via induction of apoptosis. Fhit protein, a diadenosine polyphosphate hydrolase, does not require hydrolase activity to function in tumor suppression and may signal for apoptosis as an enzyme-substrate complex. Thus, high affinity nonhydrolyzable substrate analogs may either promote or antagonize Fhit function, depending on their features, in Fhit + cells.