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The objective of this study was to investigate the changes of the fatty acid in breast (BM) and leg (LM) muscles from 17-wk-old female White Kołuda geese packaged in a vacuum and stored in freezing conditions at -20°C. During 17 weeks, the geese were fed ad libitum on the same complete feed. The samples (18 LM and 18 BM) from the right part of the carcasses were stored for 30, 90, 80, 270, and 365 d. The changes in the fatty acid profile were established by gas chromatography. In this work, there were also calculated lipid profile indicators such as Σ PUFA n-6/Σ PUFA n-3, Σ UFA/Σ SFA, and Σ PUFA/Σ SFA. Time of frozen storage affected the decrease in Σ SFA, Σ MUFA, and Σ PUFA of BM and LM. The statistical analysis of the obtained data shows that the type of muscle also generally affected the fatty acid profile. The BM are characterized higher proportion of Σ SFA, and the LM are defined as containing more Σ MUFA and Σ PUFA. Extending frozen storage time caused only the deterioration of Σ PUFA n-6/Σ PUFA n-3. The Σ PUFA n-6/Σ PUFA n-3 were the highest in BM and LM on the 365th day of storage. Although the Σ PUFA n-6/Σ PUFA n-3 ratio in muscles stored for 180, 270, and 365 d was higher than the recommended values. The lipid profile indicators (Σ UFA/Σ SFA, and Σ PUFA/Σ SFA) were similar in raw meat and in all frozen storage samples. It means that frozen storage didn't affect this index and the BM and LM have the same quality from the dietary point of view. Leg muscles during frozen storage are characterized by higher Σ UFA/Σ SFA and Σ PUFA/Σ SFA than the breast muscles.

The study aimed to determine the technological and sensory properties of the breast and thigh muscles of geese from the Polish native varieties: Kartuska (Ka) and Suwalska (Su) (from northern Poland) as well as Lubelska (Lu) and Kielecka (Ki) (from southern Poland). The color parameters: L*, a*, b*, ΔE, C, h°, total heme pigments (THPs), and share of myoglobin (Mb), metmyoglobin (MMb), and oxymyoglobin (MbO

GPR119 receptor has been proposed as a metabolic regulator playing a pivotal role in the modulation of glucose homeostasis in type 2 diabetes. GPR119 was identified on pancreatic β cells and its ligands have the ability to enhance glucose-stimulated insulin secretion (GSIS). Lysophosphatidylcholine (LPC) was shown to potentiate GSIS and our present studies indicate that 2-methoxy-lysophosphatidylcholine (2-OMe-LPC) analogues, unable to undergo 1 → 2 acyl migration, stimulate GSIS from murine βTC-3 pancreatic cells even more efficiently. Moreover, biological assays in engineered Tango™ GPR119-bla U2OS cells were carried out to ascertain the agonist activity of 2-OMe-LPC at GPR119. 2-OMe-LPC possessing in sn-1 position the residues of myristic, palmitic, stearic and oleic acid were also evaluated as factors regulating [Ca2 +]i mobilization and cAMP levels. Extension of these studies to R- and S-enantiomers of 14:0 2-OMe-LPC revealed that the overall impact on GSIS does not depend on chirality, however, the intracellular calcium mobilization data show that the R enantiomer is significantly more active than S one. Taking into account differences in chemical structure between various native LPCs and their 2-methoxy counterparts the possible binding mode of 2-OMe-LPC to the GPR119 receptor was determined using molecular modeling approach.

Pt(II) coordination, like of cis -(NH 3 ) 2 Pt(II), affects the acid-base properties of guanosine derivatives. The acidity constants of such complexes are calculated by curve-fitting procedures using published 1 H NMR shift data measured in aqueous solution (D 2 O) in dependence on pH (pD). Comparison of the p K a values of the ligands with those of the Pt(II) complexes reveals the expected behavior for (N7)-platination, i.e., (N1)H sites are acidified due to charge repulsion. These effects of Pt(II) are compared with those of Ni 2+ and Cd 2+ , allowing predictions for the acidification of Zn 2+ . Studied are the cis -(NH 3 ) 2 Pt(II) complexes of inosylyl(3′ → 5′)inosine, guanylyl(3′ → 5′)guanine (GpG) − , its 2′-deoxy relative d(GpG) − , and its phosphorylated derivative d(pGpG) 3− . The Pt(II) effect is mostly rather symmetrical, like with d(CpGpG) 2− , but it can also be quite asymmetrical, like with d(CpCpGpG) 3− or in the cyclohexylamine-substituted Cisplatin complex of d(GpG) − . Potential consequences for base pairing between the platinated guanine and the complementary cytosine are discussed. It is proposed that partial deprotonation of dG under the influence of Pt(II) at physiological pH leads in water to a marked reduction in pairs with intact Watson–Crick hydrogen bonds, thereby adding to the well established thermal destabilization of double-stranded DNA by G,G intrastrand cross-links of Cisplatin.

The chemical synthesis of phosphorothioate/phosphodiester analogues of 2-methoxy-lysophosphatidylethanolamine has been described. For the preparation of phosphorothioate derivatives oxathiaphospholane approach has been employed. The phosphodiester compounds were prepared by OXONE® oxidation of corresponding phosphorothioates. Each lysophospholipid analogue was synthesized as a series of four compounds, bearing different fatty acid residues both saturated (14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1→2 acyl migration. The cytotoxicity of newly synthesized 2-methoxy-lysophosphatidylethanolamine derivatives was evaluated with resazurin-based method in prostate cancer PC3 cell line. The highest reduction of cell viability was noted for LPE analogues containing myristoyl acyl chain.

The chemical synthesis of phosphorothioate/phosphorodithioate analogues of 2-methoxy-lysophosphatidylcholine has been described. For the preparation of new sulfur derivatives of lysophosphatidylcholine both oxathiaphospholane and dithiaphospholane approaches have been employed. Each lysophospholipid analogue was synthesized as a series of five compounds, bearing different fatty acid residues both saturated (12:0, 14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1→2 acyl migration. The cellular toxicity of newly synthesized 2-methoxy-lysophosphatidylcholine derivatives was measured using MTT viability assay and lactate dehydrogenase release method.

The intrinsic acid-base properties of the hexa-2'-deoxynucleoside pentaphosphate, d(ApGpGpCpCpT) [=(A1∙G2∙G3∙C4∙C5∙T6)=(HNPP)⁵⁻] have been determined by ¹H NMR shift experiments. The pKa values of the individual sites of the adenosine (A), guanosine (G), cytidine (C), and thymidine (T) residues were measured in water under single-strand conditions (i.e., 10% D₂O, 47 °C, I=0.1 M, NaClO₄). These results quantify the release of H⁺ from the two (N7)H⁺ (G∙G), the two (N3)H⁺ (C∙C), and the (N1)H⁺ (A) units, as well as from the two (N1)H (G∙G) and the (N3)H (T) sites. Based on measurements with 2'-deoxynucleosides at 25 °C and 47 °C, they were transferred to pKa values valid in water at 25 °C and I=0.1 M. Intramolecular stacks between the nucleobases A1 and G2 as well as most likely also between G2 and G3 are formed. For HNPP three pKa clusters occur, that is those encompassing the pKa values of 2.44, 2.97, and 3.71 of G2(N7)H⁺, G3(N7)H⁺, and A1(N1)H⁺, respectively, with overlapping buffer regions. The tautomer populations were estimated, giving for the release of a single proton from five-fold protonated H₅(HNPP)(±) , the tautomers (G2)N7, (G3)N7, and (A1)N1 with formation degrees of about 74, 22, and 4%, respectively. Tautomer distributions reveal pathways for proton-donating as well as for proton-accepting reactions both being expected to be fast and to occur practically at no "cost". The eight pKa values for H₅(HNPP)(±) are compared with data for nucleosides and nucleotides, revealing that the nucleoside residues are in part affected very differently by their neighbors. In addition, the intrinsic acidity constants for the RNA derivative r(A1∙G2∙G3∙C4∙C5∙U6), where U=uridine, were calculated. Finally, the effect of metal ions on the pKa values of nucleobase sites is briefly discussed because in this way deprotonation reactions can easily be shifted to the physiological pH range.

The nucleoside derivatives, containing at 3′-O or 5′-O-position 2-oxo-1,3,2-dithiaphospholane function were found to react in the presence of DBU with a hydroxyl group of appropriately protected nucleosides to yield, after deprotection, dinucleoside phosphorothioates in moderate yields. An asymmetric induction connected with the formation of new chiral phosphorothioate center has been examined.

Two coordination compounds of palladium(II) with N -allylimidazole ( l ) of the general formula [PdL 4 ]Cl 2 · 3H 2 O ( 1 ) and trans -[PdL 2 Cl 2 ] ( 2 ) have been synthesized. The crystal and molecular structure of complexes 1 and 2 was established by single-crystal X-ray diffraction analysis. The X-ray structural data were supplemented by solid-state 13 C NMR measurements (CP MAS and PASS 2D). The 1D and 2D NMR studies in solution reveal that complex 1 is unstable at room temperature and undergoes reversible decomposition to 2 . The method for how to preserve a complex with four allyl-imidazole ligands in solution is shown.

The crystal and molecular structure of the title compound ( 1 ) was established by an X-ray diffraction analysis. Some geometrical parameters including a slightly pyramidal shape around its ylidic Cβ-atom were determined (trans-bent type conformation), providing evidence for a strong electron delocalisation in the PαCβPγOδ backbone. The charge density redistribution within this molecular unit and its other fully optimised geometries was evaluated in ab initio MO calculations using both the HF and DFT (B3LYP) formalism, which supported such a concept. As a result, the double zwitterionic form (structure C ) was suggested as the best description of 1 . The absence of an experimentally NMR observable 2JPα–Cβ–Hβ coupling was tentatively rationalised in terms of fast pyramidalisation of the Cβ-anionic site. Crystallographic and solution NMR data for ylide 1 were compared with those reported for the other mesomerically stabilised Wittig-type reagents and structurally related anionic species. It was concluded that all aforementioned systems have almost identical P-ylidic bonding most likely governed mainly by very strong electrostatic interactions, with a small contribution of negative hyperconjugation.

The stability constants of the 1:1 complexes formed in aqueous solution between Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Zn2+, or Cd2+ (M2+) and methyl thiophosphate (MeOPS(2-)) or uridine 5'-O-thiomonophosphate (UMPS(2-)) (PS(2-)=MeOPS(2-) or UMPS(2-)) have been determined (potentiometric pH titrations; 25 degrees C; I = 0.1 M, NaNO(3)). Comparison of these results for M(PS) complexes with those known for the parent M(PO) phosphate species, where PO(2-)=CH(3)OPO(2-)(3) or UMP(2-) (uridine 5'-monophosphate), shows that the alkaline earth metal ions, as well as Mn2+, Co2+, and Ni2+ have a higher affinity for phosphate groups than for their thio analogues. However, based on the linear log K(M)(M(R-PO3)) versus pK(H)(H(R-PO3)) relationships (R-PO(2-)(3) simple phosphate monoester or phosphonate ligands with a non-interacting residue R) it becomes clear that the indicated observation is only the result of the lower basicity of the thiophosphate residue. In contrast, the thio complexes of Zn2+ and Cd2+ are more stable than their parent phosphate ones, and this despite the lower basicity of the PS(2-) ligands. This stability increase is identical for M(MeOPS) and M(UMPS) species and amounts to about 0.6 and 2.4 log units for Zn(PS) and Cd(PS), respectively. Since no other binding site is available in MeOPS(2-), this enhanced stability has to be attributed to the S atom. Indeed, from the mentioned stability differences it follows that Cd2+ in Cd(PS) is coordinated by more than 99% to the thiophosphate S atom; the same value holds for Pb(PS), which was studied earlier. The formation degree of the Sbonded isomer amounts to 76+/-6 % for Zn(PS) and is close to zero for the corresponding Mg2+, Ca2+, and Mn2+ species. It is further shown that Zn(MeOPS)(aq)(2+) releases a proton from a coordinated water molecule with pK(a) approximately 6.9; i.e., this deprotonation occurs at a lower pH value than that for the same reaction in Zn(aq)(2+). Since Mg2+, Ca2+, Mn2+, and Cd2+ have a relatively low tendency for hydroxo complex formation, it was possible, for these M2+, to also quantify the stability of the binuclear complexes, M(2)(UMPS-H)+, where one M2+ is thiophosphate-coordinated and the other is coordinated at (N3)(-) of the uracil residue. The impact of the results presented herein regarding M2+/nucleic acid interactions, including those of ribozymes (rescue experiments), is briefly discussed.

Alkali metal salts of diethyl phosphites act as nucleophiles or electron donors in reactions with diethyl phosphorochloridate. Evidence is provided that formation of the direct P(IV)P(IV) bond proceeds via a single electron transfer process (SET) from phosphite anion to phosphorochloridate.

Novel analogues of acyclic nucleosides based on a bis(hydroxymethyl)phosphinic acid (BHPA) backbone were obtained by condensation of the bis(4,4′-dimethoxytrityl) derivative of BHPA with N -1- or N -3-(2-hydroxyethyl)thymine in the presence of MSNT, or by an Appel reaction with N -1- or N -3-(2-aminoethyl)thymine. After selective deprotection and phosphitylation they were used as monomers for automated solid support synthesis of short oligomers by the phosphoramidite approach.

The acidity constants of twofold protonated methyl thiophosphate (MeOPS(2-)) and of monoprotonated uridine 5'- O-thiomonophosphate (UMPS(2-)) have been determined in aqueous solution (25 degrees C; I= 0.1 M, NaNO(3)) by potentiometric pH titration. The stability constants of their 1:1 complexes formed with Pb(2+), i.e. Pb(MeOPS) and Pb(UMPS), have also been measured. The results show that replacement of a phosphate oxygen by a sulfur atom increases the acidity by about 1.4 p K units. On the basis of recently established log versus plots ( = simple phosphate or phosphonate ligands where R is a non-coordinating residue), it is shown that the stability of the Pb(thiophosphate) complexes is by log Delta= 2.43+/-0.09 larger than expected for a Pb(2+)-phosphate interaction. The identity of the stability increase (log Delta) observed for Pb(MeOPS) and Pb(UMPS) shows that the nucleobase residue in the Pb(UMPS) complex has no influence on complex formation. To be able to carry out the mentioned comparisons, we have also determined the stability constant of the complex formed between Pb(2+) and methyl phosphate; the corresponding data for Pb(UMP) were already known from our earlier studies. The present results allow an evaluation of other Pb(2+) complexes formed with thiophosphate derivatives and they are applied now to the Pb(2+) complexes of adenosine 5'- O-thiomonophosphate (AMPS(2-)). The stability constants of the Pb(H;AMPS)(+) and Pb(AMPS) complexes were measured and it is shown that, within the error limits, the stability of the Pb(AMPS) complex is determined by the basicity of the thiophosphate group of AMPS(2-); in other words, no hint for macrochelate formation involving N7 was observed. More important, with the aid of micro-stability-constant considerations it is concluded that the structure of the dominating isomer of the Pb(H;AMPS)(+) species is the one where the proton is located at the N1 site of the adenine residue and Pb(2+) is coordinated to the deprotonated thiophosphate group. The insights gained from this study with regard to thiophosphate-altered single-stranded nucleic acids and their affinity towards Pb(2+) are discussed.

2-Alkoxy-2-thiono-1,3,2-oxathiaphospholanes are readily transformed into phosphorothioate monoesters of the corresponding alcohols in a one-pot process, involving the reaction with 3-hydroxypropionitrile in the presence of DBU, followed by treatment with aqueous ammonia. In this way a series of nucleoside-3'-O- and 5'-O-phosphorothioates were prepared, as well as phosphorothioate derivatives of selected polyols.

2-Thiono-1,3,2-oxathiaphospholane derivatives of lipophilic alcohols including borneol, cholesterol, menthol and heptadecanol were synthesized and reacted with support-bound oligodeoxyribonucleotides containing free 5′-hydroxyl groups. The reaction is catalyzed by DBU and leads to oligodeoxyribonucleotide conjugates possessing a lipophilic alcohol residue bound at the 5′-end via a phosphorothioate linkage.

Oligodeoxynucleotide N3'-->P5' phosphoramidates are promising candidates for antisense therapeutics, as well as for diagnostic applications. We recently reported a new method for the synthesis of these oligonucleotide analogs which makes use of a phosphoramidite amine-exchange reaction in the key coupling step. We report herein an improved set of monomers that utilize a more reactive, hindered phosphoramidite to produce optimal yields in a single coupling step followed by oxidation, thereby eliminating the need for the previously reported couple-oxidize-couple-oxidize approach. On the 10 micromol scale, the synthesis is performed using only 3.6 equivalents (equiv.) of monomer. An improved oxidation reagent consisting of hydrogen peroxide, water, pyridine and THF is also introduced. Reported here for the first time is the use of a reverse-phase purification methodology employing a ribonucleotide purification handle that is removed under non-acidic conditions, in contrast to the conventional dimethoxytrityl group. The synthesis and purification of uniformly modified N3'-->P5' phosphoramidate oligodeoxy-nucleotides, as well as their chimera containing phosphodiester and/or phosphorothioate linkages at predefined positions, using these new methodologies are included herein. The results of31P NMR studies that led to this improved amine-exchange methodology are also described.

The correlation between induction factors (Fi) of phosphorothioates in the iodine–azide reaction and their detection limits (DLs) using the iodine–azide reagent has been established. The iodine–azide reagent has been used for the selective thin-layer chromatographic detection of several sugar phosphorothioates and related compounds. In several cases, hydrolytic treatment of phosphorothioates, prior to their detection on thin-layer chromatography (TLC) plates with the iodine–azide reagent, was accompanied by a dramatic increase in the detectability of the starting compounds. Comparison of TLC detection systems for phosphorothioates using iodine–azide procedures and other representative procedures are presented. The application of the iodine–azide procedure combined with the molybdate procedure for selective TLC detection of phosphates and phosphorothioates is illustrated.

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.

Nitric oxide (NO) plays an important role in cellular signaling and host defense, and it also contributes to the deleterious effects of immune response. Until recently, the lack of specific inhibitors of various forms of nitric oxide synthase (NOS) hampered a stringent evaluation of the role played by inducible NOS (iNOS) in cell damage. The present study investigated the use of antisense oligodeoxynucleotides (AS-ODNs) to selectively inhibit the expression of iNOS. AS-ODNs (1-10 microM) inhibited, in a time-dependent and dose-dependent manner, iNOS activity in RAW 264.7 murine macrophages. Maximal inhibitory effect was90%, and control ODNs had little or no effect on NO production. Treatment with AS-ODNs decreased iNOS protein and mRNA level in studied cell, and control ODNs again were ineffective. The decreased levels of the target mRNA in AS-ODN-treated samples suggest that the AS-ODNs used act as substrates for ribonuclease (RNase) H. Lipofection enhanced the effect of AS-ODNs on iNOS activity. However, this potentiation appears to be different from the antisense effect, in which the AS-ODNs studied were involved. Liposaccharide/interferon-gamma (LPS/IFN-gamma) induced iNOS, and increased NO production impaired the viability of macrophages. Treatment of RAW 264.7 cells with 10 microM AS-ODNs prevented the NO-induced lethal cell damage.

Oligonucleotides with a nucleotide sequence complementary to various regions of human plasminogen activator inhibitor type-1 (PAI-1) mRNA have been studied as antisense inhibitors of expression of PAI-1 protein in cultured cells [human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells, human hybrid endothelial cells]. Hexadeca(deoxyribonucleoside phosphorothioate) 13 complementary to a fragment of a signal peptide PAI-1 mRNA was found to be most active, giving ca. 70% inhibition of PAI-1 release in a time- and dose-dependent way. The stereo-regular All-S(P) and All-R(P) diastereomers of 13 were studied and found to inhibit PAI-1 synthesis in HUVEC in a stereo-dependent manner, with the All-S(P) diastereomer considerably more active than the stereo-random construct and All-R(P) isomer. The observed stereo-dependent activity of oligonucleotide phosphorothioate constructs is presumably governed by their resistance to nucleases. The corresponding phosphodiester analogue of 13 was not active unless covalently bound at its 5'-end to a lipophilic alcohol residue (menthol, heptadecanol). The observed antisense activity of phosphodiester oligonucleotide bioconjugates in cultured human hybrid endothelial cells was paralleled by their increased stability in human plasma with respect to unconjugated oligonucleotide. The oligo(deoxyribonucleoside phosphorothioate) complementary to the same signal peptide region of rat PAI-1 mRNA was found to reduce the PAI-1 level in blood plasma of rats after intravenous administration into the tail vein. The effect was both time- and dose-dependent. The same oligonucleotide was found to protect against arterial thrombus formation in the rat (lower incidence of venous thrombosis, lower thrombus weight, and increased occlusion time in experimentally induced thrombosis). An anti-PAI-1 inhibitory activity has been independently reported for a 20-mer oligo(2'-O-methyl-ribonucleoside phosphorothioate) complementary to a 3'-untranslated region of human PAI-1 mRNA in cultured HUVEC and human aortic smooth muscle cells.

The TLC detection limits of the phosphorothioate analogs of nucleotides and related compounds by means of UV, iodine, HCl vapours, the iodine–azide reagent and the molybdate reagent have been determined. The iodine–azide reagent has been applied for the selective TLC detection of the phosphorothioate analogs of nucleotides.

In this paper, the geometry and conformation of oxathiaphospholane and dithiaphospholane rings in the solid state are described on the basis of our recent work and compared with literature data. For oxathiaphospholane rings in the solid state, both envelope and half-chair conformations, differently distorted, are observed. In oxathiaphospholane rings with the envelope conformation, the endocyclic carbon atom next to the ring oxygen atom is in the flap position. For dithiaphospholane compounds, the heterocyclic rings prefer a half-chair conformation or the conformation intermediate between half-chair and envelope forms; the envelope conformation is observed only in one case.

Two “thiophospholane” derivatives of cholesterol: 2-cholesteryl-2-thio-1,3,2-oxathiaphospholane (1) and 2-cholesteryl-2-thio-1,3,2-dithiaphospholane (2) were synthesized as new reagents for introducing a cholesteryl moiety at the 5′-end of oligonucleotidesvia the phosphorothioate or phosphorodithioate bond. Compounds1 and2 were subjected to structural studies by X-ray methods. Both compounds crystallized in the orthorhombic system, space group P212121,1 witha=6.283(1) A,b=12.067(1) A,c=38.983(3) A,2 witha=6.371(1) A,b=11.971(1) A andc=39.502(3) A. The five-membered heterocyclic rings of both compounds attain a half-chair conformation in the solid state. In structures of1 and2 a disorder of some atoms is observed. The absolute configuration at the phosphorus atom in1 of the components of diastereoisomeric mixture has been established.

Enantiomeric R-(−)- and (S)-(+)-2-anilino-2-oxo-1,3,2-oxazaphosphorinanes ( 1 ) have been synthesized by catalytic debenzylation of separated diastereomers of 3-(α-methylbenzyl)-2-anilino-2-oxo-1,3,2-oxazaphosphorinanes. Self-induced diastereomeric anisochronicity in non-racemic mixtures of these enantiomers was shown to vary with enantiomeric composition, overall specimen concentration, and temperature. The equilibrium constants of monomer-dimer and homo-cross-associate diastereomeric equilibria are estimated for the first time.

Appropriately protected nucleoside 5'-O-(2-thio-1,3,2-dithiaphospholanes) react with inorganic pyrophosphate in the presence of a strong base catalyst (DBU) to give nucleoside 5'-O-(1,1-dithiotriphosphates) 1a-g. The latter compounds, including an AZT analogue, show modest antivirial activity against HIV-1 and HIV-2 replication in CEM cells. The AZT and deoxyadenosine derivatives were found to be inhibitors of HIV reverse transcriptase.

Three compounds (1, 2, and 3) containing 1,3,2-oxathiaphospholane and 1,3,2-dithiaphospholane rings were subjected to structural studies by the X-ray method. Compounds 1 and 3 attain a half-chair conformation, whereas, for compound 2, an envelope conformation of the five-membered heterocyclic ring was found in the solid state. For all three compounds, a site occupation of some structural elements in a unit cell was observed.

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.

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 RP) was found to be readily digested by svPDE.

In this study we evaluated efficiency of DNAzymes to modulate motility of cancer cells, an important factor in the progression and metastasis of cancers. For this purpose we targeted β1 integrins that are predominant adhesive receptors in various carcinoma cell lines (CX1.1, HT29, LOVO, LS180, PC-3). To evaluate invasiveness of cancer cells, we used a transwell migration assay that allowed analyzing chemotactic migration of colon carcinoma cell lines across an ECM-coated membrane. Their adhesive properties were also characterized by the analysis of adhesion to fibronectin, laminin and collagen. In addition, the expression of major integrin subunits, selected intact β1 integrins, and other adhesive receptors (ICAM, E-selectin, uPAR) was analyzed by flow cytometry. Inhibition of β1 integrin expression by DNAzyme to β1 mRNA almost abolished the invasiveness of the CX1.1, HT29, LS180, LOVO and PC-3 cells in vitro. These data show that DNAzymes to β1 integrin subunit can be used to inhibit invasiveness of carcinoma cells.

Enantiomeric R-(−)- and (S)-(+)-2-anilino-2-oxo-1,3,2-oxazaphosphorinanes ( 1 ) have been synthesized by catalytic debenzylation of separated diastereomers of 3-(α-methylbenzyl)-2-anilino-2-oxo-1,3,2-oxazaphosphorinanes. Self-induced diastereomeric anisochronicity in non-racemic mixtures of these enantiomers was shown to vary with enantiomeric composition, overall specimen concentration, and temperature. The equilibrium constants of monomer-dimer and homo-cross-associate diastereomeric equilibria are estimated for the first time.

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 RP) was found to be readily digested by svPDE.

2-Alkoxy-2-thiono-1,3,2-oxathiaphospholanes are readily transformed into phosphorothioate monoesters of the corresponding alcohols in a one-pot process, involving the reaction with 3-hydroxypropionitrile in the presence of DBU, followed by treatment with aqueous ammonia. In this way a series of nucleoside-3'-O- and 5'-O-phosphorothioates were prepared, as well as phosphorothioate derivatives of selected polyols.

Oligonucleotides and their analogues have recently found broad application in biochemistry and molecular biology as new tools for studying interactions of nucleic acids with other biomolecules, and as potential candidates for therapeutics. In this chapter the synthetic methods leading to P-chiral oligonucleotide analogues are reviewed, including synthesis of isotopomeric phosphates and various nucleotide and oligonucleotide conjugates. In this respect special emphasis has been put on the application of oxathiaphospholane methodology, recently developed in the authors’ laboratory, for the stereocontrolled synthesis of P-chiral biophosphate analogues.