The synthesis of dithymidine boranophosphate by the oxathiaphospholane approach.

The application of the oxathiaphospholane approach for the synthesis of dithymidine boranphospate was evaluated. It was shown, that although the nucleoside-3'-O-oxathiaphospholane-borane complexes 2 or 6 could not be chromatographically separated into diastereomerically pure species due to their apparent instability to moisture, they can be successfully applied to the non-stereocontrolled formation of internucleotide boranophosphate bond by reaction with 5'-OH-nucleoside in the presence of DBU. Attempts to apply the related dithiaphospholane approach for the preparation of dithymidine boranophosphorothioate were unsuccessful.

Stereochemistry of cleavage of internucleotide bonds by Serratia marcescens endonuclease.

Endonuclease from Serratia marcescens hydrolyzes internucleotide phosphorothioate linkages of R(P) configuration with inversion of configuration at P-atom. This observation supports a reported architecture of the active site, with 3'-bridging and pro-S(P) non-bridging oxygen atoms of the scissile phosphate group involved in direct contact with hydrated magnesium cation, while His-89 activates a water molecule which attacks the phosphorus atom according to a one-step in-line mechanism. The presence of a phosphorothioate bond of S(P) configuration downstream to that one being cleaved reduces the rate of hydrolysis. This suggests participation of the pro-S(P) oxygen atom of that phosphate bond in the mechanism of action of the enzyme, which was not detected in published crystallographic analyses.

The mononucleotide-dependent, nonantisense mechanism of action of phosphodiester and phosphorothioate oligonucleotides depends upon the activity of an ecto-5'-nucleotidase.

Many reports indicate different nonantisense yet sequence-specific effects of antisense phosphorothioate oligonucleotides. Products of enzymatic degradation of the oligonucleotides can also influence cell proliferation. The cytotoxic effects of deoxyribonucleoside-5'-phosphates (dNMPs) and their 5'-phosphorothioate analogs, deoxyribonucleoside-5'-monophosphorothioates (dNMPSs) on 4 human cell types (HeLa, HL-60, K-562, and endothelial cells) were examined, and the effects were correlated with the catabolism of these compounds. The results indicate that differences in cytotoxicity of dNMPs or dNMPSs in these cells depend upon different activity of an ecto-5'-nucleotidase. It has also been found that dNMPSs stimulate proliferation of human umbilical vein endothelial cells and HL-60 cells in a concentration-dependent manner. This stimulation might be caused by the binding of deoxynucleoside-5'-phosphorothioates to as-yet unidentified nucleotide receptor(s) at the cell surface. (Blood. 2001;98:995-1002)

Regulation of PAI-1 concentration in platelets by systemic administration of antisense oligonucleotides to rats.

In this report we tested the effect of oligodeoxyribonucleotides antisense to PAI-1 mRNA administered into rats on PAI-1 concentration in platelets. Low doses of the antisense oligonucleotide (MPO-16R) reduced PAI-1 activity, both in rat blood plasma and platelet lysates by 20.5% and 28.7%, respectively. There was no change in platelet count after treatment with MPO-16R but treated platelets showed lower aggregability as compared with controls (37 +/- 13% and 54 +/- 12%, respectively). In an experimental model of rat arterial thrombosis, low doses of MPO-16R caused a significant delay in the occlusion time (31.8%). These data further support for the role of PAI-1 as a major determinant of arterial thrombolysis resistance and for the first time demonstrate the possibility of reduction of platelet PAI-1 concentration by antisense approach.

(S)-(-)-Bromofosfamide (CBM-11): synthesis and antitumor activity and toxicity in mice.

(S)-(-)-Bromofosfamide (CBM-11), an enantiomerically pure bromo analog of ifosfamide, was found to be potent against several model tumors in mice. Therapeutic indices of CBM-11 were more favorable as compared to those received for ifosfamide.

Recent advances in the elucidation of the mechanisms of action of ribozymes.

The cleavage of RNA can be accelerated by a number of factors. These factors include an acidic group (Lewis acid) or a basic group that aids in the deprotonation of the attacking nucleophile, in effect enhancing the nucleophilicity of the nucleophile; an acidic group that can neutralize and stabilize the leaving group; and any environment that can stabilize the pentavalent species that is either a transition state or a short-lived intermediate. The catalytic properties of ribozymes are due to factors that are derived from the complicated and specific structure of the ribozyme-substrate complex. It was postulated initially that nature had adopted a rather narrowly defined mechanism for the cleavage of RNA. However, recent findings have clearly demonstrated the diversity of the mechanisms of ribozyme-catalyzed reactions. Such mechanisms include the metal-independent cleavage that occurs in reactions catalyzed by hairpin ribozymes and the general double-metal-ion mechanism of catalysis in reactions catalyzed by the Tetrahymena group I ribozyme. Furthermore, the architecture of the complex between the substrate and the hepatitis delta virus ribozyme allows perturbation of the pK(a) of ring nitrogens of cytosine and adenine. The resultant perturbed ring nitrogens appear to be directly involved in acid/base catalysis. Moreover, while high concentrations of monovalent metal ions or polyamines can facilitate cleavage by hammerhead ribozymes, divalent metal ions are the most effective acid/base catalysts under physiological conditions.

Stereocontrolled synthesis of LNA dinucleoside phosphorothioate by the oxathiaphospholane approach.

The application of the oxathiaphospholane approach for the stereocontrolled synthesis of LNA dinucleoside phosphorothioate is described. The reaction of ring opening condensation proceeds in CH3CN solution in high yield and with over 96% stereoselectivity. One of diastereomers of LNA dinucleoside phosphorothioate (presumably R(P)) was found to be readily digested by svPDE.

Differences among mechanisms of ribozyme-catalyzed reactions.

The catalytic properties of ribozymes depend on the sophisticated structures of the respective ribozyme-substrate complexes. Although it has been suggested that ribozyme-mediated cleavage of RNA occurs via a rather strictly defined mechanism, recent findings have clearly demonstrated the diversity of reaction mechanisms.

Stereochemical outcome and kinetic effects of Rp- and Sp-phosphorothioate substitutions at the cleavage site of vaccinia type I DNA topoisomerase.

To probe the mechanism of the reversible DNA phosphodiester bond cleavage and religation mechanism of the type I topoisomerase from vaccinia virus, we have synthesized DNA substrates carrying a single nonbridging Rp- or Sp-phosphorothioate (Ps) modification at the scissile phosphodiester (Pd) bond. Analysis of the stereochemical outcome of the net cleavage and rejoining reaction established that the reaction proceeds with retention of configuration, as expected for a double-displacement mechanism. Single-turnover kinetic studies on irreversible strand cleavage using 18/24 mer suicide substrates showed thio effects (k(Pd)/k(Ps)) of 340- and 30-fold for the Rp-Ps and Sp-Ps stereoisomers, respectively, but approximately 10-fold smaller thio effects for the reverse single-turnover religation reaction (Rp-Ps = 30 and Sp-Ps = 3). As compared to the smaller suicide cleavage substrates, approach-to-equilibrium cleavage studies using 32/32 mer substrates showed 7-9-fold smaller thio effects on cleavage, similar effects on religation, and the same ratio of the Rp to Sp thio effect as the suicide cleavage reaction ( approximately 10). In general, thio effects of 2.4-7.2-fold on the cleavage equilibrium are observed for the wild-type and H265A enzymes, suggesting differences in the interactions of the enzyme with the nonbridging sulfur in the noncovalent and covalent complexes. Studies of the cleavage, religation, and approach-to-equilibrium reactions catalyzed by the H265A active site mutant revealed a stereoselective, 11-fold decrease in the Rp-thio effect on cleavage and religation as compared to the wild-type enzyme. This result suggests that His-265 interacts with the nonbridging pro-Rp oxygen in the transition state for cleavage and religation, consistent with the arrangement of this conserved residue in the crystal structure of the human topoisomerase-DNA complex. In general, the greatest effect of thio substitution and the H265A mutation is to destabilize the transition state, with smaller effects on substrate binding. The interaction of His-265 with the pro-Rp nonbridging oxygen is inconsistent with the proposal that this conserved residue acts as a general acid in the strand cleavage reaction.

Nucleophilic N1-->N3 rearrangement of 5'-O-trityl-O2,3'-cycloanhydrothymidine.

5'-O-Trityl-O2,3'-cycloanhydrothymidine (1) heated at 150 degrees C in the presence of O,O-diethyl phosphate or O,O-diethyl phosphorothioate anions undergoes rearrangement into N3-isomer (2); its structure was established by both advanced NMR methods and X-ray crystallographic studies. The most probable mechanism of 1-->2 rearrangement relies upon reversibility of glycosidic bond cleavage process.

Oligomeric building block approach to the synthesis of diastereomerically pure pentathymidine 3',5'-methanephosphonates.

[formula: see text] A method for a large-scale synthesis of stereodefined oligo(nucleoside 3',5'-methanephosphonates) has been developed, based on transient 3'-O protection, which allows for the conversion of the protecting chirally defined methanephosphonanilidate group, located at the 3' end of a stereoregular oligomer, into diastereomerically pure "oligomeric building blocks" for stereospecific coupling with the 5'-OH group of another oligonucleotide.

The cytotoxicity of anti-PAI-1 oligonucleotides and their conjugates.

The cytotoxicity of anti-PAI-5 hexadecanucleotides (phosphodiesters and phosphorothioates) and their conjugates with lipophilic alcohols was tested in EA.hy 926 hybrid endothelial cells. Some cytotoxicity was found for cholesteryl and bornyl conjugates at concentrations higher than those used for antisense inhibition experiments.

Inhibition of plasminogen activator inhibitor release in endothelial cell cultures by antisense oligodeoxyribonucleotides with a 5'-end lipophilic modification.

A series of conjugates containing residues of lipophilic alcohols covalently bound to 5' end of oligodeoxyribonucleotides targeted against human plasminogen activator inhibitor (PAI-1) mRNA was synthesized via the oxathiaphospholane approach. The highest anti-PAI-1 activity in EA.hy 926 endothelial cell cultures was found for conjugates containing menthyl or heptadecanyl groups linked with an oligonucleotide complementary to a segment of human PAI-1 mRNA. The phosphodiester antisense oligonucleotides, which otherwise exhibit only limited anti-PAI-1 activity, were found to be more active than phosphorothioate oligonucleotides when conjugated to lipophilic alcohol residues. For menthyl conjugates an evidence of antisense mechanism of inhibition was found.

Antisense oligodeoxyribonucleotide against the MLL-LTG19 chimeric transcript inhibits cell growth and induces apoptosis in cells of an infantile leukemia cell line carrying the t(11;19) chromosomal translocation.

To clarify the role of the multiple lineage leukemia gene-leukemia translocation gene of chromosome 19 (MLL-LTG19) protein in leukemogenesis, we synthesized antisense oligodeoxyribonucleotide (ODN) against the fused region of the MLL-LTG19 chimeric transcript and treated KOCL33 cells carrying the t(11;19) translocation with antisense ODN. The antisense ODN inhibited cell growth and induced apoptosis in KOCL33 cells but not in Daudi cells, which have no t(11;19). The levels of MLL-LTG19 mRNA and MLL-LTG19 protein in KOCL33 cells treated with antisense ODN were shown to decrease with time by reverse transcription-PCR and Western blot analysis. These results suggest that the MLL-LTG19 fusion protein contributes to cell proliferation and malignant transformation in infantile acute leukemia cells carrying the t(11;19) translocation.

Effect of P-chirality of oligo(deoxyribonucleoside phosphorothioate)s on the activity of terminal deoxyribonucleotidyl transferase.

Phosphorothioate analogues of oligonucleotides (PS-oligos) of predetermined chirality at the phosphorus atom at each internucleotide linkage have been used as primers for terminal deoxyribonucleotidyl transferase (TdT, EC 2.7.7.31). The enzyme catalyzes efficient elongation of PS primers in which all phosphorothioate internucleotide linkages are uniformly of the [R(P)] configuration, while the presence of the linkage(s) of the [S(P)] configuration significantly decreases or completely inhibits the primer extension. Our results indicate that for the elongation of phosphorothioate oligomers the most important is the internucleotide bond located between the second and the third nucleoside from the 3'-end. The presence of [S(P)] linkage at this position strongly reduces the enzyme activity while the [R(P)] bond allows for effective elongation of the primer. The activity of the enzyme is also influenced by base composition and sequence of phosphorothioate primer as well as the dNTP used for elongation process.

X-ray and high resolution selenium-77 solid state NMR spectroscopy as complementary probes to structural studies of organophosphorus diselenides.

77Se high resolution solid state NMR spectroscopy was employed to study structural properties of bis(diisopropoxyphosphorothioyl) diselenide 1 and bis(dineopentoxyphosphorothioyl) diselenide 2. The principal elements Tii of 77Se effective dipolar/chemical shift tensor were calculated from spinning sideband intensities employing the WIN-MAS program. The values of anisotropy and asymmetry parameters reflect the distortion of the selenium environment. It was found that the T33 component mostly contributes to changes in the isotropic chemical shifts. 77Se CP/MAS experiments were used to decide the assignment of space group by counting the number of crystallographically unique selenium centers in the unit cell. Crystals of diselenide 1 are triclinic, space group P1 with a = 8.485(3) A, b = 8.508(1) A, c = 8.511(2) A, alpha = 98.835(15) degrees, beta = 111.653(24) degrees, gamma = 93.524(21) degrees, V = 559.5(3) A3, Dc = 1.544(2) g/cm3 and Z = 1. Refinement using 2222 reflections for 157 variables gives R = 0.037. Crystals of diselenide 2 are triclinic, space group P1 with a = 9.1418(8) A, b = 9.1465(8) A, c = 9.9200(9) A, alpha = 74.751(8) degrees, beta = 74.629(7) degrees, gamma = 82.216(7) degrees, V = 769.7(1) A3, Dc = 1.365(2) g/cm3 and Z = 1. Refinement using 3316 reflections for 297 variables gives R = 0.0272.

Synthesis, biochemical and biological studies on oligonucleotides bearing a lipophilic dimethoxytrityl group.

Dimethoxytritylphosphono-oligonucleotide conjugates have been prepared. They are totally resistant to nucleases present in human serum and do not affect cleavage of a complementary oligoribonucleotide by RNase H. Conjugates possessing a phosphate backbone gave better antisense inhibition of expression of plasminogen activator inhibitor type-1 within endothelial cells as compared with unconjugated oligonucleotides.

Synthesis and interaction with thymidylate synthase of 5'-dithiophosphate and 5'-fluorothiophosphate of thymidine.

Thymidine-5'-fluorothiophosphate, dTMP(S)-F, was synthesized by the oxathiaphospholane, and thymidine 5'-dithiophosphate, dTMPS2, by the dithiaphospholane, method. To estimate the role of 5'-phosphate group ionization in binding of pyrimidine nucleotides by thymidylate synthase, dTMP(S)-F was studied as an inhibitor of mouse tumour (L1210) enzyme, and its inhibitory properties were compared with those of dTMPS2, a close dTMP analogue. While dTMPS2 proved to be an inhibitor, competitive vs dUMP, with K(i)app = 94 microM, the 5'-fluorothiophosphate congener displayed no activity, indicating that the enzyme requires for binding the presence of a dianionic 5'-phosphate group in a nucleotide.

Novel internucleotide 3'-NH-P(CH3)(O)-O-5' linkage. Oligo(deoxyribonucleoside methanephosphonamidates); synthesis, structure and hybridization properties.

Diastereomeric dithymidine methanephosphonamidates (TnpmT) were synthesized by reaction of 3'-amino-3'-deoxythymidine with 3'- O -acetylthymidin-5-yl-methanephosphonochloridate. Separated dinucleotide TnpmT(fast) and TnpmT(slow) diastereomers were used as building blocks to prepare chimeric dodecathy-midylates, possessing one to four modified linkages, by means of phosphoramidite automated solid phase synthesis. As expected, the methanephosphonamidate internucleotide linkage is resistant to nuclease P1, snake venom PDE and 3'-exonuclease from human plasma. Degradation of dodecathymidylates possessing modified internucleotide linkages in alternate positions proved the 'hopping' properties of 3'-exonuclease. Oligo(deoxyribonucleotide methanephosphonamidates) were tested for their binding affinity to complementary oligomers in thermal denaturation experiments. All the oligomers showed lower binding affinity to DNA and RNA targets, however, oligomers originating from the TnpmT(fast) dimeric unit exhibited better hybridization properties than their diastereomeric TnpmT(slow) counterparts. A lowering of T m of approximately 2.4 degrees C (1.0-1.8 degrees C) was observed for each introduced TnpmT(fast) modification and 6.0 degrees C (4.2-5.0 degrees C) for each TnpmT(slow) modification in duplexes of modified dodecathymidylates with dA12(A12) oligomers. The oligo(deoxyribonucleoside methanephosphonamidate) designated F4, possessing four modified methanephosphonate linkages originating from the TnpmT(fast) diastereomeric unit, exhibits a tendency for triplex formation, as was demonstrated in thermal denaturation experiments with the d(A21C4T21) hairpin oligomer.

Phosphorothioate oligodeoxyribonucleotides antisense to PAI-1 mRNA increase fibrinolysis and modify experimental thrombosis in rats.

The effect of systemic inhibition of PAI-1 expression in rats by PS-16R, a phosphorothioate analogue of hexadecadeoxyribonucleotide complementary to a signal peptide coding sequence of rat PAI-1 mRNA, on PAI-1 activity in blood plasma and thrombus formation was studied in rat models for experimental thrombosis. In previous in vitro studies, oligonucleotides of PS-16R family have been shown to inhibit efficiently PAI-1 synthesis in endothelial cells by antisense mechanism. When PS-16R was administered intravenously as a single bolus injection (1 to 5 mg per rat), it produced a significant reduction in PAI-1 activity of blood plasma. This effect was both time- and concentration-dependent. Under the same conditions, three groups of rats were treated with control oligodeoxynucleotides such as PS-16R with double mismatches, with scrambled sequence, and an oligodeoxynucleotide with sense sequence (complementary to PS-16R), respectively. Based on these preliminary experiments, a low dose of 1.5 mg per rat was selected to produce approximately 20-30% reduction of PAI-1 activity in blood plasma and the effect of such a decrease in PAI-1 expression was tested on thrombus formation in two rat models for experimentally induced thrombosis. Such a limited decrease in PAI-1 activity produced a significant antithrombotic effect in the arterial thrombosis model. There was a profound delay in the occlusion time in rats treated with PS-16R when compared to control animals (80 +/- 3 and 55 +/- 3 h, respectively), although blood plasma activity of PAI-1 in the same groups of rats differed only by 20%. There was also a tendency to reduce both an incidence of venous thrombosis (58.33 and 68.11%, respectively) and thrombus weight (2.1 +/- 0.4 and 2.9 +/- 0.9 mg, respectively) in the animals treated with PS-16R. However, this effect was not significant. Thus, low dose of PS-16R through inhibition of PAI-1 synthesis in targeted cells in rats reduced PAI-1 activity in blood plasma and protected against arterial thrombus formation in the rat.