Synthesis and Characterization of 8-Nitroguanosine 3',5'-Cyclic Monophosphorothioate Rp-Isomer as a Potent Inhibitor of Protein Kinase G1α.

Guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinases (PKG) are kinases regulating diverse physiological functions including vascular smooth muscle relaxation, neuronal synaptic plasticity, and platelet activities. Certain PKG inhibitors, such as Rp-diastereomers of derivatives of guanosine 3',5'-cyclic monophosphorothioate (Rp-cGMPS), have been designed and used to study PKG-regulated cell signaling. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is an endogenous cGMP derivative formed as a result of excess production of reactive oxygen species and nitric oxide. 8-Nitro-cGMP causes persistent activation of PKG1α through covalent attachment of cGMP moieties to cysteine residues of the enzyme (i.e., the process called protein S-guanylation). In this study, we synthesized a nitrated analogue of Rp-cGMPS, 8-nitroguanosine 3',5'-cyclic monophosphorothioate Rp-isomer (Rp-8-nitro-cGMPS), and investigated its effects on PKG1α activity. We synthesized Rp-8-nitro-cGMPS by reacting Rp-8-bromoguanosine 3',5'-cyclic monophosphorothioate (Rp-8-bromo-cGMPS) with sodium nitrite. Rp-8-Nitro-cGMPS reacted with the thiol compounds cysteine and glutathione to form Rp-8-thioalkoxy-cGMPS adducts to a similar extent as did 8-nitro-cGMP. As an important finding, a protein S-guanylation-like modification was clearly observed, by using Western blotting, in the reaction between recombinant PKG1α and Rp-8-nitro-cGMPS. Rp-8-Nitro-cGMPS inhibited PKG1α activity with an inhibitory constant of 22 µM in a competitive manner. An organ bath assay with mouse aorta demonstrated that Rp-8-nitro-cGMPS inhibited vascular relaxation induced by acetylcholine or 8-bromo-cGMP more than Rp-8-bromo-cGMPS did. These findings suggest that Rp-8-nitro-cGMPS inhibits PKG through induction of an S-guanylation-like modification by attaching the Rp-cGMPS moiety to the enzyme. Additional study is warranted to explore the potential application of Rp-8-nitro-cGMPS to biochemical and therapeutic research involving PKG1α activation.

Phosphorothioate anti-sense oligonucleotides: the kinetics and mechanism of the generation of the sulfurising agent from phenylacetyl disulfide (PADS).

The synthesis of phosphorothioate oligonucleotides is often accomplished in the pharmaceutical industry by the sulfurisation of the nucleotide-phosphite using phenylacetyl disulfide (PADS) which has an optimal combination of properties. This is best achieved by an initial 'ageing' of PADS for 48 h in acetonitrile with 3-picoline to generate polysulfides. The initial base-catalysed degradation of PADS occurs by an E1cB-type elimination to generate a ketene and acyldisulfide anion. Proton abstraction to reversibly generate a carbanion is demonstrated by H/D exchange, the rate of which is greatly increased by electron-withdrawing substituents in the aromatic ring of PADS. The ketene can be trapped intramolecularly by an o-allyl group. The disulfide anion generated subsequently attacks unreacted PADS on sulfur to give polysulfides, the active sulfurising agent. The rate of degradation of PADS is decreased by less basic substituted pyridines and is only first order in PADS indicating that the rate-limiting step is formation of the disulfide anion from the carbanion.

A general and efficient approach for the construction of RNA oligonucleotides containing a 5'-phosphorothiolate linkage.

Oligoribonucleotides containing a 5'-phosphorothiolate linkage have provided effective tools to study the mechanisms of RNA catalysis, allowing resolution of kinetic ambiguity associated with mechanistic dissection and providing a strategy to establish linkage between catalysis and specific functional groups. However, challenges associated with their synthesis have limited wider application of these modified nucleic acids. Here, we describe a general semisynthetic strategy to obtain these oligoribonucleotides reliably and relatively efficiently. The approach begins with the chemical synthesis of an RNA dinucleotide containing the 5'-phosphorothiolate linkage, with the adjacent 2'-hydroxyl group protected as the photolabile 2'-O-o-nitrobenzyl or 2'-O-α-methyl-o-nitrobenzyl derivative. Enzymatic ligation of the 2'-protected dinucleotide to transcribed or chemically synthesized 5' and 3' flanking RNAs yields the full-length oligoribonucleotide. The photolabile protecting group increases the chemical stability of these highly activated oligoribonucleotides during synthesis and long-term storage but is easily removed with UV irradiation under neutral conditions, allowing immediate use of the modified RNA in biochemical experiments.

Stereocontrolled solid-phase synthesis of phosphorothioate oligoribonucleotides using 2'-O-(2-cyanoethoxymethyl)-nucleoside 3'-O-oxazaphospholidine monomers.

A method for the synthesis of P-stereodefined phosphorothioate oligoribonucleotides (PS-ORNs) was developed. PS-ORNs of mixed sequence (up to 12mers) were successfully synthesized by this method with sufficient coupling efficiency (94-99%) and diastereoselectivity (≥98:2). The coupling efficiency was greatly improved by the use of 2-cyanoethoxymethyl (CEM) groups in place of the conventional TBS groups for the 2'-O-protection of nucleoside 3'-O-oxazaphospholidine monomers. The resultant diastereopure PS-ORNs allowed us to clearly demonstrate that an ORN containing an all-(Rp)-PS-backbone stabilizes its duplex with the complementary ORN, whereas its all-(Sp)-counterpart has a destabilizing effect.

Optimization of pyrosequencing reads by superior successive incorporation efficiency of improved 2'-deoxyadenosine-5'-triphosphate analogs.

Conventional pyrosequencing using 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) (dATPαS) is problematic due to the high cost of the substrate (dATPαS) and deterioration in the accuracy of incorporation to read through poly(T) regions. One reason for these problems is that dATPαS has a sulfur on the α-phosphate and also has isomers (Sp and Rp). To solve these problems, 11 nucleotide substrates, which could replace dATPαS in pyrosequencing, were newly synthesized. All substrates were modified on the seventh or eighth position of the adenine base from normal dATP. We found that the substrate that had an ethenyl-linked modified group on the seventh position of the adenine base had low activity in the luciferase reaction and high incorporation efficiency with the thymine base. One substrate in particular had 10-fold better incorporation efficiency than dATPαS. The new nucleotide substrate satisfied all conditions as a replacement of dATPαS.

Synthesis and characterization of 2'-modified-4'-thioRNA: a comprehensive comparison of nuclease stability.

We report herein the synthesis and physical and physiological characterization of fully modified 2'-modified-4'-thioRNAs, i.e. 2'-fluoro-4'-thioRNA (F-SRNA) and 2'-O-Me-4'-thioRNA (Me-SRNA), which can be considered as a hybrid chemical modification based on 2'-modified oligonucleotides (ONs) and 4'-thioRNA (SRNA). In its hybridization with a complementary RNA, F-SRNA (15mer) showed the highest T(m) value (+16 degrees C relative to the natural RNA duplex). In addition, both F-SRNA and Me-SRNA preferred RNA as a complementary partner rather than DNA in duplex formation. The results of a comprehensive comparison of nuclease stability of single-stranded F-SRNA and Me-SRNA along with 2'-fluoroRNA (FRNA), 2'-O-MeRNA (MeRNA), SRNA, and natural RNA and DNA, revealed that Me-SRNA had the highest stability with t(1/2) values of > 24 h against S1 nuclease (an endonuclease) and 79.2 min against SVPD (a 3'-exonuclease). Moreover, the stability of Me-SRNA was significantly improved in 50% human plasma (t(1/2) = 1631 min) compared with FRNA (t(1/2) = 53.2 min) and MeRNA (t(1/2) = 187 min), whose modifications are currently used as components of therapeutic aptamers. The results presented in this article will, it is hoped, contribute to the development of 2'-modified-4'-thioRNAs, especially Me-SRNA, as a new RNA molecule for therapeutic applications.

Phosphorothioate analogs of m7GTP are enzymatically stable inhibitors of cap-dependent translation.

We report synthesis and properties of a pair of new potent inhibitors of translation, namely two diastereomers of 7-methylguanosine 5'-(1-thiotriphosphate). These new analogs of mRNA 5'cap (referred to as m(7)GTPalphaS (D1) and (D2)) are recognized by translational factor eIF4E with high affinity and are not susceptible to hydrolysis by Decapping Scavenger pyrophosphatase (DcpS). The more potent of diastereomers, m(7)GTPalphaS (D1), inhibited cap-dependent translation in rabbit reticulocyte lysate approximately 8-fold and approximately 15-fold more efficiently than m(7)GTP and m(7)GpppG, respectively. Both analogs were also significantly more stable in RRL than unmodified ones.

New NTP analogs: the synthesis of 4'-thioUTP and 4'-thioCTP and their utility for SELEX.

The synthesis of the triphosphates of 4'-thiouridine and 4'-thiocytidine, 4'-thioUTP (7; thioUTP) and 4'-thioCTP (8; thioCTP), and their utility for SELEX (systematic evolution of ligands by exponential enrichment) is described. The new nucleoside triphosphate (NTP) analogs 7 and 8 were prepared from appropriately protected 4'-thiouridine and -cytidine derivatives using the one-pot method reported by J. Ludwig and F. Eckstein [(1989) J. Org. Chem., 54, 631-635]. Because SELEX requires both in vitro transcription and reverse transcription, we examined the ability of 7 and 8 for SELEX by focusing on the two steps. Incorporation of 7 and 8 by T7 RNA polymerase to give 4'-thioRNA (thioRNA) proceeded well and was superior to those of the two sets of frequently used modified NTP analogs for SELEX (2'-NH2dUTP and 2'-NH2dCTP; 2'-FdUTP and 2'-FdCTP), when an adequate leader sequence of DNA template was selected. We revealed that a leader sequence of about +15 of DNA template is important for the effective incorporation of modified NTP analogs by T7 RNA polymerase. In addition, reverse transcription of the resulting thioRNA into the complementary DNA in the presence of 2'-deoxynucleoside triphosphates (dNTPs) also proceeded smoothly and precisely. The stability of the thioRNA toward RNase A was 50 times greater than that of the corresponding natural RNA. With these successful results in hand, we attempted the selection of thioRNA aptamers to human alpha-thrombin using thioUTP and thioCTP, and found a thioRNA aptamer with high binding affinity (K(d) = 4.7 nM).

Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides.

Oligonucleotides composed of 2'-O-methyl and locked nucleic acid residues complementary to HIV-1 trans-activation responsive element TAR block Tat-dependent trans-activation in a HeLa cell assay when delivered by cationic lipids. We describe an improved procedure for synthesis and purification under highly denaturing conditions of 5'-disulphide-linked conjugates of 3'-fluorescein labelled oligonucleotides with a range of cell-penetrating peptides and investigate their abilities to enter HeLa cells and block trans-activation. Free uptake of 12mer OMe/LNA oligonucleotide conjugates to Tat (48-58), Penetratin and R9F2 was observed in cytosolic compartments of HeLa cells. Uptake of the Tat conjugate was enhanced by N-terminal addition of four Lys or Arg residues or a second Tat peptide. None of the conjugates entered the nucleus or inhibited trans-activation when freely delivered, but inhibition was obtained in the presence of cationic lipids. Nuclear exclusion was seen for free delivery of Tat (48-58), Penetratin and R9 conjugates of 16mer phosphorothioate OMe oligonucleotide. Uptake into human fibroblast cytosolic compartments was seen for Tat, Penetratin, R9F2 and Transportan conjugates. Large enhancements of HeLa cell uptake into cytosolic compartments were seen when free Tat peptide was added to Tat conjugate of 12mer OMe/LNA oligonucleotide or Penetratin peptide to Penetratin conjugate of the same oligonucleotide.

A novel universal linker for efficient synthesis of phosphorothioate oligonucleotides.

A versatile and conformationally preorganized universal linker molecule is reported here for efficient synthesis of phosphorothioate oligonucleotides. With respect to nucleoside loaded support, comparable yield and quality based on ion-pair LC-MS are obtained for both deoxy and 2'-O-methoxyethyl modified phosphorothioate oligonucleotides. No 3'-phosphate or phosphorothioate monoester or any modification of universal molecule still attached to oligonucleotide was observed. [structure: see text]

Recent advances in the stereocontrolled synthesis of antisense phosphorothioates.

Antisense technology has advanced substantially in the past few decades and now is a well-established therapeutic approach in medicinal chemistry, and it may prove to be a valuable tool in the treatment of a wide range of diseases. Phosphorothioate oligonucleotides are among the most important and promising antisense agents. However, the key drawback lies in their polydiastereomerism, which manifests itself in the different chemical and biological properties of the diastereomeric species. Methodologies towards the stereocontrolled synthesis of antisense phosphorothioate oligonucleotides have been well investigated in recent years. In this review, the progress in this field is summarized.

Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5'-protected thiol function and a 3'-amino group.

A new deprotection procedure enables a medium scale preparation of phosphodiester and phosphor-othioate oligonucleotides substituted with a protected thiol function at their 5'-ends and an amino group at their 3'-ends in good yield (up to 72 OD units/micromol for a 19mer phosphorothioate). Syntheses of 3'-amino-substituted oligonucleotides were carried out on a modified support. A linker containing the thioacetyl moiety was manually coupled in two steps by first adding its phosphor-amidite derivative in the presence of tetrazole followed by either oxidation or sulfurization to afford the bis-derivatized oligonucleotide bound to the support. Deprotection was achieved by treating the fully protected oligonucleotide with a mixture of 2,2'-dithiodipyridine and concentrated aqueous ammonia in the presence of phenol and methanol. This proced-ure enables (i) cleavage of the oligonucleotide from the support, releasing the oligonucleotide with a free amino group at its 3'-end, (ii) deprotection of the phosphate groups and the amino functions of the nucleic bases, as well as (iii) transformation of the 5'-terminal S -acetyl function into a dithiopyridyl group. The bis-derivatized phosphorothioate oligomer was further substituted through a two-step procedure: first, the 3'-amino group was reacted with fluorescein isothiocyanate to yield a fluoresceinylated oligo-nucleotide; the 5'-dithio-pyridyl group was then -quantitatively reduced to give a free thiol group which was then substituted by reaction with an N alpha-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide-oligonucleotide conjugate.

Enzymatic preparation of adenosine 5'-O-(3-[35S]thiotriphosphate) by thiophosphorylation of adenosine diphosphate.

The very useful radiolabeled ATP analog, adenosine 5-O-(3-[35S] thiotriphosphate) or [35S] ATPgammaS, has been prepared by a technique based on the thiophosphorylation of ADP that results in much higher yields of [35S] ATPgammaS than does a thiophosphate exchange method [1].

Phosphodiester and phosphorothioate oligonucleotide condensation and preparation of antisense nanoparticles.

Protamine is a cationic peptide with a molecular mass of approx. 4000 Da that is able to condense DNA. In the present study it was used to complex antisense oligonucleotides (ODNs) and to form solid particles with initial diameters of 90-150 nm. The reaction was very rapid and occurred by simple mixing of diluted solutions of the polycation with the oligonucleotide. The aggregation was dependent on the oligonucleotide chain length and the protamine/ODN mass ratio. Particle formation required a minimal chain length of nine nucleotides and a mass ratio of 0.5:1. The particle surface charge and the number of particles depended on the mass ratio. With increasing amounts of the peptide, the number of particles and the zeta potential increased. Both negatively and positively charged particles improved the stability of oligonucleotides against DNase I digestion. Above a mass ratio of 2.5:1 no degradation was found. The uptake of unbound rhodamine-labelled ODNs and its complexes with protamine was determined with Vero cells under in vitro cell culture conditions at 37 degrees C and 4 degrees C. At 37 degrees C the cellular uptake increased with increasing mass ratio. The internalized oligonucleotides were localized in the cytoplasm and in the nucleus of the cells. When Vero cells were treated with these samples at 4 degrees C for 4 h, no fluorescence could be detected inside the cells. Therefore, our data indicate an energy dependent endocytotic uptake mechanism. In contrast, spermine and spermidine, which are also known condensation agents, did not aggregate with oligonucleotides into nanoparticles under the same conditions.

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VII. Chemical synthesis by phosphorylation of 6-thioguanosine 5'-monophosphate, 5'-diphosphate and 5'-triphosphate, and their purification and identification by reversed-phase/ion-pair high-performance liquid chromatography and by various enzymatic assays.

A fast and reliable two-step method has been established for the chemical synthesis of 6-thioguanosine 5'-monophosphate, 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate starting from the ribonucleoside. In the first step, 6-thioguanosine dissolved in triethyl phosphate, at high yield reacts with phosphorus oxide trichloride to 6-thioguanosine 5'-monophosphate which is purified by anion-exchange chromatography on DEAE-Sephadex using a step gradient of hydrochloric acid. In the second step, 6-thioguanosine 5'-monophosphate dissolved in water, reacts with phosphoric acid in the presence of pyridine/dicyclohexyl carbodiimide and is converted to 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate which are separated from each other and from the 6-thioguanosine 5'-monophosphate by anion-exchange chromatography on DEAE-Sephadex using a gradient of ammonium bicarbonate. Material from each step of the preparation procedure is separated by reversed-phase HPLC chromatography and analyzed for its free ribonucleoside content, 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and small amounts of unidentified phosphorylated compounds. The purity of the final preparations and the identity of each 6-thioguanosine 5'-phosphate are proven by highly specific enzymatic peak-shifting/HPLC analyses using alkaline phosphatase, 5'-nucleotidase, pyruvate kinase, nucleoside diphosphate kinase and combined hexokinase/glucose 6-phosphate dehydrogenase.

Synthesis and interactions with thymidylate synthase of 2,4-dithio analogues of dUMP and 5-fluoro-dUMP.

The 2,4-dithio analogues of 2'-deoxyuridine and 2'-deoxy-5-fluorouridine have been synthesized by thiation of the previously described 2-thio analogues, and then phosphorylated enzymatically or chemically to yield 2,4-dithio-dUMP and 2,4-dithio-5-fluoro-dUMP. In striking contrast to the 2-thio and 4-thio analogues of dUMP, which are good substrates of thymidylate synthase, 2,4-dithio-dUMP is not a substrate. But, surprisingly, it is a competitive inhibitor, relative to dUMP, of the purified enzymes from both parental and FdUrd-resistant L1210 cells, with K(i) values of 32 microM and 55 microM, respectively. Although 2,4-dithio-5-fluoro-dUMP behaved as a typical slow-binding inhibitor of the enzyme, its K(i) value was 10(3)-10(4)-fold higher than those for the corresponding 2-thio and 4-thio congeners. Similarly, 2,4-dithio-FdUrd was a much weaker inhibitor of tumour cell growth (IC50 approximately 10(-5)M) than FdUrd (IC50 approximately 10(-9)M), 2-thio-FdUrd(IC50 approximately 10(-7)M) or 4-thio-FdUrd (IC50 approximately 5x10(-8)M), while with 2,4-dithio-dUrd no influence on cell growth could be observed. Theoretical considerations, based on calculated aromaticities of the uracil and thiouracil rings, suggest that lack of substrate activity of 2,4-dithio-dUMP may result from increased pyrimidine ring aromaticity of the latter, leading to resistance of C(6) to nucleophilic attack by the enzyme active center cysteine.

Synthesis of nucleoside alpha-thiotriphosphates via an oxathiaphospholane approach.

[reaction: see text]. Nucleoside 5'-O-(alpha-thiotriphosphates) were obtained in reactions of the appropriate nucleoside 5'-O-(2-thio-1,3,2-oxathiaphospholanes) with pyrophosphate in the presence of DBU. The presented method allows also for preparation of alpha-seleno congeners and corresponding alpha-modified diphosphates.

Dimethylthiarum disulfide: new sulfur transfer reagent in phosphorothioates oligonucleotide synthesis.

Dimethylthiarum disulfide (DTD) has been developed as a new and efficient sulfur-transfer reagent for automated synthesis of phosphorothioate oligonucleotides using phosphoramidite chemistry. The traditional four-step automated oligonucleotide synthesis has been compressed to three-step protocol using DTD. This improvement allowed an overall 20% reduction in the solvent consumption and reduced the total synthesis time by 25%. The large-scale application of DTD has been successfully demonstrated by synthesis of therapeutically useful 20-mer phosphorothioate antisense oligonucleotides with excellent yield and purity.

Inhibition of murine dendritic cell activation by synthetic phosphorothioate oligodeoxynucleotides.

Depending on sequence and backbone structure, DNA can inhibit as well as stimulate immune responses. As previously shown, single-base phosphorothioate (Ps) oligodeoxynucleotides (ODN) can inhibit murine macrophage activation. To determine whether these compounds can also affect dendritic cells (DC), the effects of 30-mer Ps ODN (SdA, SdT, SdG, and SdC) on DC activation were assessed in an in vitro system. With DC preparations obtained from murine bone marrow cultured in granulocyte macrophage-colony stimulating factor, the Ps ODN blocked the production of interleukin-12 and nitric oxide induced by bacterial DNA, an immunostimulatory cytosine phosphate guanosine dinucleotide (CpG) ODN and lipopolysaccharide (LPS). Furthermore, these compounds inhibited up-regulation of costimulatory molecules CD40 and CD86 as well as major histocompatibility complex-II molecules, indicating an effect on DC maturation. Although the Ps ODN limited uptake of CpG ODN as assessed by flow cytometry, the Ps ODN did not affect LPS uptake, suggesting that these compounds inhibit DC responses by effects on downstream signaling pathways. Together, these observations extend the range of action of inhibitory ODN to DC and suggest a role of these compounds as immunomodulatory agents.