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W J Stec

Publications and source records attributed to W J Stec.

At least 37 records · Page 2Linked to original sources
PubMed1997

Novel cost-effective methanephosphonoanilidothioate approach to the stereoselective synthesis of dinucleoside (3',5')-methanephosphonates.

A new method of stereoselective preparation of di(2'-deoxy or 2'-OMe)ribonucleoside (3',5')-methanephosphonate 5 is presented. The DBU/LiCl-assisted reaction of 5'-O-DMT-(2'-deoxy or 2'-OMe)ribonucleoside 3'-O-(S-alkyl methanephosphonothioate) 9 with 5'-OH nucleosides proceeds with full stereospecificity, giving 5 in moderate to good yield. The conversion of 5'-O-DMT-(2'-deoxy or 2'-OMe) ribonucleoside 3'-methanephosphonoanilidothioates 8 and 3'-O-methanephosphonoanilidates 10 by means of NaH/CX2 (X = O,S) followed by S-alkylation leads to monomers 9, with the possibility of use of both separated diastereomers of 8 for the preparation of one selected diastereomer of 5. The relative configuration at the P atom in 2'-OMe and deoxynucleoside derivatives of compounds 9 was established by means of stereoselective degradation of nucleoside 3'-O-methanephosphonothioates 11 (precursors of 9) with nuclease P1.

Chromatography, High Pressure Liquid↗
PubMed1997

Stability of stereoregular oligo(nucleoside phosphorothioate)s in human plasma: diastereoselectivity of plasma 3'-exonuclease.

The stability of stereoregular oligo(nucleoside phosphorothioate)s (PS-oligos) in human plasma has been studied. 3'-Exonuclease present in human plasma appeared to be RP specific, that is, it cleaves internucleotide phosphorothioate linkages of [RP]-configuration and not those of [SP]-configuration. Therefore, PS-oligos containing all phosphorothioate internucleotide linkages of [RP]-configuration [RP-PS-oligos]) are more effectively degraded by the enzyme than PS-oligos prepared via nonstereo-controlled methods (so-called random mixture of diastereomers [Mix-PS-oligos]), whereas oligo(nucleoside phosphorothioate)s of [S(P)]-configuration remain intact. The enzyme activity depends on the sequence of nucleobases. The presence of deoxycytidine units (three or more residues) at the 3'-end of PS-oligo substrate significantly inhibits the enzyme activity.

Drug Stability↗
PubMed1997

Stereodependent inhibition of plasminogen activator inhibitor type 1 by phosphorothioate oligonucleotides: proof of sequence specificity in cell culture and in vivo rat experiments.

Hexadecadeoxyribonucleotides complementary to a fragment of human PAI-1 mRNA located upstream of the start codon and their phosphorothioate analogs were studied in cultured HUVECs as sequence-dependent inhibitors of PAI-1 expression. The activity of the random mixture of diastereomers of phosphorothioate hexadecanucleotide PS-16H has been compared with that of isosequential, stereoregular [All-Sp] and [All-Rp] isomers. The highest inhibitory effect on PAI-1 synthesis was observed with the [All-Sp] diastereomer. Stereorandom phosphorothioate oligonucleotide PS-16R complementary to the same region of rat PAI-1 mRNA, when injected into tail vein of rats, substantially decreased the level of PAI-1 in blood plasma.

Animals↗
PubMed1996

Chiral phosphorothioates as probes of protein interactions with individual DNA phosphoryl oxygens: essential interactions of EcoRI endonuclease with the phosphate at pGAATTC.

The contact between EcoRI endonuclease and the "primary clamp" phosphate of its recognition site pGAATTC is absolutely required for recognition of the canonical and all variant DNA sites. We have probed this contact using oligonucleotides containing single stereospecific (Rp)- or (Sp)- phosphorothioates (Ps). At the GAApTTC position, where the endonuclease interacts with only one phosphoryl oxygen at the central DNA kink, Rp-Ps inhibits and Sp-Ps stimulates binding and cleavage [Lesser et al. (1992) J. Biol. Chem. 267, 24810-24818]: in contrast, at the pGAATTC position both diastereomers inhibit binding. For single-strand substitution, the penalty in binding free energy (delta delta G0bind) is slightly greater for Sp-Ps (+ 0.9 kcal/mol) than for Rp-Ps (+ 0.7 kcal/mol). Binding penalties are approximately additive for double-strand substitution (Rp,Rp-Ps or Sp,Sp-Ps). Neither Ps diastereomer in one DNA strand affects the first-order rate constants for cleavage in the unmodified DNA strand, and only Sp-Ps inhibits the cleavage rate constant (3-fold) in the modified DNA strand. Thus, the second-order cleavage rate (including binding and catalysis) is inhibited 14-fold by Sp-Ps and 45-fold by Sp,Sp-Ps. In the canonical complex, the phosphate at pGAATTC is completely surrounded by protein and each nonbridging phosphoryl oxygen receives two hydrogen bonds from the endonuclease, such that in either orientation the increased bond length of P-S- inhibits binding. However, the pro-Sp oxygen interacts with residues that are connected (by proximity or inter-side-chain hydrogen bonding) to side chains with essential roles in catalysis, so cleavage is preferentially inhibited when these side chains are slightly displaced by the Sp-Ps diastereomer.

Base Sequence↗
PubMed1996

Anti-sense oligodeoxynucleoside phosphorothioates nonspecifically inhibit invasion of red blood cells by malaria parasites.

Anti-sense and sense oligodeoxynucleoside phosphorothioates, based on analysis of the secondary structure of Plasmodium falciparumdihydrofolate reductase-thymidylate synthase mRNA, were synthesized. Their effects on P. falciparum growth in vitro were examined by microscopy and [3H] hypoxanthine incorporation. Both anti-sense and sense oligodeoxynucleoside phosphorothioates inhibit invasion of red blood cells by merozoites and this is interpreted as being caused by their polyanionic nature. Specific anti-sense effects of the oligonucleoside phosphorothioates could not however be demonstrated.

Animals↗
PubMed1996

Internalization of oligodeoxynucleotide antisense to type-1 plasminogen activator inhibitor mRNA in endothelial cells: a three-dimensional reconstruction by confocal microscopy.

A three-dimensional reconstruction analysis of localization of phosphodiester and phosphorothioate oligonucleotide antisense to type-1 plasminogen activator inhibitor (PAI-1) mRNA within endothelial cells is described. When EA.hy 926 cells were incubated with fluorescently labelled phosphodiester (PO-16) or phosphorothioate (PS-16) oligonucleotides at low, not cytotoxical concentrations, the relative brightness composition of the images of the particular samples was much higher for PS-16 than PO-16 and dependent upon the extracellular concentration and the incubation time. The 3-D reconstructions based on the series of optical sections of the samples, spaced every 1.5 microns, showed the punctuate accumulation of the oligonucleotides and a striking difference in a spatial distribution between PO-16 and PS-16 within the cytoplasm. Even after 24 h incubation of endothelial cells with 2.5 microM of PO-16 and PS-16 oligonucleotides, there was a predominant oligonucleotide localization within the cytoplasm and only traces of oligonucleotides could be seen in the cell nucleus and/or perinuclear organelles.

Antisense Elements (Genetics)↗
PubMed1995

Stereodifferentiation--the effect of P chirality of oligo(nucleoside phosphorothioates) on the activity of bacterial RNase H.

P stereoregular phosphorothioate analogs of pentadecamer 5'-d(AGATGTTTGAGCTCT)-3' were synthesized by the oxathiaphospholane method. Their diastereomeric purity was assigned by means of enzymatic degradation with nuclease P1 and, independently, with snake venom phosphodiesterase. DNA-RNA hybrids formed by phosphorothioate oligonucleotides (PS-oligos) with the corresponding complementary pentadecaribonucleotide were treated with bacterial RNase H. The DNA-RNA complex containing the PS-oligo of [all-RP] configuration was found to be more susceptible to RNase H-dependent degradation of the pentadecaribonucleotide compared with hybrids containing either the [all-SP] counterpart or the so called 'random mixture of diastereomers' of the pentadeca(nucleoside phosphorothioate). This stereodependence of RNase H action was also observed for a polyribonucleotide (475 nt) hybridized with these phosphorothioate oligonucleotides. The results of melting studies of PS-oligo-RNA hybrids allowed a rationalization of the observed stereodifferentiation in terms of the higher stability of heterodimers formed between oligoribonucleotides and [all-RP]-oligo(nucleoside phosphorothioates), compared with the less stable heterodimers formed with [all-SP]-oligo(nucleoside phosphorothioates) or the random mixture of diastereomers.

Bacteria↗
PubMed1995

Analysis of oligo(deoxynucleoside phosphorothioate)s and their diastereomeric composition.

Short oligo(deoxynucleoside phosphorothioate)s were analyzed as a pool of individual diastereomeric species. The composition of such mixtures, determined by means of HPLC, indicates that consecutive couplings in commonly used phosphoramidite chemistry lead to increasing contents of the Rp isomer. Methods of analysis and mathematical basis for diastereomeric composition are discussed. Data presented include all 16 possible combinations of nucleosides in dinucleotide phosphorothioates, as well as examples of trimers and tetramers.

Chromatography, High Pressure Liquid↗
PubMed1995

Inhibition by modified oligodeoxynucleotides of the expression of type-1 plasminogen activator inhibitor in human endothelial cells.

Antisense phosphodiester and phosphorothioate oligodeoxynucleotides (23-residue or 24-residue oligodeoxynucleotides) were constructed for sequences of type-1 plasminogen-activator-inhibitor mRNA to assess their capability to modulate type-1 plasminogen-activator-inhibitor-mediated fibrinolysis. Antisense oligodeoxynucleotides were targeted at the mRNA sequence coding a signal peptide, at a part of the reactive center Ile342-Pro349, and at an internally translated segment Asn265-Leu272. The effect of antisense oligonucleotides on the concentration of type-1 plasminogen activator inhibitor in conditioned media and human endothelial cells was determined by the activity test with fibrin as a substrate, and by immunoprecipitation after metabolic labelling of cells with [35S]methionine. Three phosphorothioate oligodeoxynucleotides were specifically inhibitory while phosphodiester oligodeoxynucleotides with the same sequence did not show any activity. Phosphorothioate oligodeoxynucleotides 2, 4 and 6 inhibited the synthesis of type-1 plasminogen activator inhibitor in endothelial cells in a time-dependent and concentration-dependent manner. These data suggest that antisense oligodeoxynucleotides may be useful in the design of antithrombolytic therapeutics.

Base Sequence↗
PubMed1994

The epidermal growth factor-like domain from tissue plasminogen activator. Cloning in E. coli, purification and ESR studies of its interaction with human blood platelets.

To examine whether the epidermal growth factor (EGF)-like domain Pro47-Asp87 is involved in the interaction of tissue plasminogen activator (t-PA) with platelets, we have expressed this domain in E. coli. The peptide fragment was produced from a plasmid expression vector as a fusion protein with beta-galactosidase Met1-Val444 at high yield in eight clones of E. coli. The fusion protein was purified and subjected to mild acid hydrolysis with formic acid, then the peptide Pro47-Asp87, identified by immunoblotting using specific antibodies to t-PA, was isolated by HPLC. After incubation with blood platelets spin labelled with 16-doxylstearic acid or 5-doxylstearic acid, the Pro47-Asp87 peptide fragment reduced fluidity of the membrane lipid bilayer to the same extent as did intact t-PA as indicated by ESR measurements. Our data suggest that the EGF-like domain of t-PA can directly interact with blood platelets and thus it seems to contain those sites of the t-PA molecule that bind the platelet membrane components.

Base Sequence↗
PubMed1992

Stereoselective interaction with chiral phosphorothioates at the central DNA kink of the EcoRI endonuclease-GAATTC complex.

We have probed the contacts between EcoRI endonuclease and the central phosphate of its recognition site GAApTTC, using synthetic oligonucleotides containing single stereospecific Rp- or Sp-phosphorothioates (Ps). These substitutions produce subtle stereospecific effects on EcoRI endonuclease binding and cleavage. An Sp-Ps substitution in one strand of the DNA duplex improves binding free energy by -1.5 kcal/mol, whereas the Rp-Ps substitution has an unfavorable effect (+0.3 kcal/mol) on binding free energy. These effects derive principally from changes in the first order rate constants for dissociation of the enzyme-DNA complexes. The first order rate constants for strand scission are also affected, in that a strand containing Sp-Ps substitution is cleaved 2 to 3 times more rapidly than a strand containing a normal prochiral phosphate, whereas a strand containing Rp-Ps substitution is cleaved about 3 times slower than normal. As a result, single-strand substitutions produce pronounced asymmetry in the rates of cleavage of the two DNA strands, and this effect is exaggerated in an Rp,Sp-heteroduplex. Ethylation-interference footprinting indicates that none of the Ps substitutions cause any major change in contacts between endonuclease and DNA phosphates. When an Sp-Ps localizes P = O in the DNA major groove, a hydrogen-bonding interaction with the backbone amide-NH of Gly116 of the endonuclease is improved relative to that with a prochiral phosphate having intermediate P-O bond order and delocalized charge.

Base Sequence↗
PubMed1992

Application of phosphate-backbone-modified oligonucleotides in the studies on EcoRI endonuclease mechanism of action.

Chemical synthesis of oligodeoxyribonucleotides modified at a preselected internucleotide bond by the replacement of one of the two "nonbridging" oxygens by a sulfur atom or an ethoxy group yields model substrates for studies on DNA-protein interactions. Chromatographic (RP-HPLC) separation of the diastereomers of oligonucleotides containing EcoRI canonical sequence together with the assignment of the substituent orientation in the DNA molecule allowed study of the stereochemical aspects of DNA-EcoRI endonuclease interactions. The DNA segment involved in interactions between EcoRI protein and phosphate groups appeared to be larger than its canonical sequence, ...GAATTC..., and was extended to the nonamer. The modification of certain internucleotide bonds within this nonamer caused significant or complete protection against the nucleolytic action of EcoRI and, in some cases, manifested the diastereoselectivity of the enzyme. On the basis of the results of EcoRI-catalyzed hydrolysis of stereodefined phosphorothioate and phosphotriester substrates, we propose a model to explain this phenomenon at the molecular level.

Base Sequence↗
PubMed1992

Membrane-bound tumour necrosis factor alpha: immunocytochemical and ultrastructural studies of human monocytes and monocytic cell line and its induction by tumour cells in vitro.

Monoclonal antibody against recombinant human tumour necrosis factor alpha (rTNF) was used for the immunochemical detection of TNF in human blood monocytes and monocytic cell line U 937. Cells stimulated with phorbol myristate acetate (PMA) showed strong surface but not cytoplasmic staining. Unstimulated cells demonstrated weak or no staining. At early time after stimulation (1-2h) a spot reaction was seen in the Golgi area of the cytoplasm of stimulated cells. Coculture of tumour cells with monocytes also resulted in the induction of membrane TNF. Ultrastructural studies confirmed TNF localization within the cell membrane. These results indicate that TNF can be detected within the cells by immunocytochemistry which may make feasible studies on TNF appearance in cellular infiltrates in the tissues.

Antibodies, Monoclonal↗
PubMed1991

Novel route to oligo(deoxyribonucleoside phosphorothioates). Stereocontrolled synthesis of P-chiral oligo(deoxyribonucleoside phosphorothioates).

The synthesis and separation of diastereoisomerically pure 5'-O-DMT-nucleoside 3'-O-(2-thio-1,3,2-oxathiaphospholane) allows their use as synthons in DBU-catalyzed reaction with the 5'-hydroxyl function of solid-support-bound nucleoside moiety. Since this reaction is stereospecific (greater than 99%), this novel method allows preparation of oligo(nucleoside phosphorothioates) with predetermined chirality at each P-chiral internucleotide phosphorothioate centre.

Base Sequence↗
PubMed1991

Separation of [Sp]-3'-O-(5'-dimethoxytritylthymidyl)-5'-O-(3'camphanoyl thymidyl)-O-methyl phosphite.

First isolation of diastereoisomerically enriched (d.p. 95%) [Sp]-3'-O-(5'-dimethoxytrityl tjymidyl)-5'-O-(3'-camphanoyl thymidyl)-O-methyl phosphite (1) allowed to assign, via sulfuration and deprotection, its absolute configuration as Sp. Its reactions with methyl iodide, dimethoxytrityl chloride, or water, respectively, allowed to confirm correctness of assignment of absolute configuration in diastereisomers of TPMeT, and to assign absolute configuration in corresponding diastereoisomers of TPDMTT and TPHT.

Chromatography, High Pressure Liquid↗
PubMed1991

Novel method of synthesis of oligo(deoxyribonucleoside phosphorothioates).

The syntheses of 5'-O-DMT base-protected nucleoside 3'-O-[2-thio-1.3.2-oxathia-phospholanes] (1) and their subsequent reactions with nucleosides bound via 3'-oxygen to solid support, performed in the presence of DBU, formulate the basis to new methodology of preparation of title compounds--OligoS. Moreover separation of 1 into diastereoisomeric species allows the preparation of OligoS in stereocontrolled manner.

Chromatography↗
PubMed1990

Backbone-modified oligonucleotides containing a butanediol-1,3 moiety as a 'vicarious segment' for the deoxyribosyl moiety--synthesis and enzyme studies.

Sequential single replacement of nucleosides within the decanucleotide d[GGGAATTCCC] (7) by means of a butanediol-1,3 residue allowed us to obtain a set of ten decanucleotides containing 'vicarious' (V) carbon-phosphate fragments. These analogues were further used as substrates for svPDE, nuclease PI and EcoR1 endonuclease. Interestingly, replacement of any of the nucleosides within the canonical sequence ...GAATTC... by the butanediol-1,3 'vicarious' segment discriminates such constructs 8c-h as substrates for Eco-R1 enzyme.

Base Sequence↗