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.

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.

Sulfurization of dinucleoside phosphite triesters with chiral disulfides.

Sixteen chiral analogues of phenylacetyl disulfide (PADS) and 5-methyl-3H-1,2,4-dithiazol-3-one (MEDITH) were used to sulfurize five dithymidine phosphite triesters, each incorporating a ß-cyanoethoxy or siloxy group. Each mixture of S(P):R(P) phosphite triester diastereomers was combined with approximately one fourth of an equivalent of each of the sulfurizing reagents, and the R(PS):S(PS) diastereomer ratios of the resulting phosphite sulfides or phosphorothioates were determined by reverse-phase HPLC. Diastereoselectivities and corresponding diastereomeric excess (de) values were calculated by correcting for the starting triester diastereomer ratios. The highest de values for R(PS) and S(PS) phosphorothioates were 14.7% and 7.9%, respectively, both using MEDITH analogues.

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.

Microfluidic assembly of lipid-based oligonucleotide nanoparticles.

BACKGROUND: Oligonucleotides (ONs) have shown great promise as therapeutic agents for various diseases. It is necessary to provide a protocol for preparation of ON-loaded lipid nanoparticles (LNPs) in a reproducible manner on a laboratory scale. MATERIALS AND METHODS: A 3-inlet microfluidic (MF) chip-based device was used to synthesize LNPs at the lipid/ON ratio of 10/1 (w/w) and at flow rates ranging from 50 to 1100 µl/min. A series of LNPs containing either antisense oligodeoxyribonucleotide (AS-ODN) or small-interfering RNA (siRNA) were synthesized. Bulk mixing was used as control. RESULTS: The MF method was shown to be particularly useful for synthesis of LNPs loaded with AS-ODN. The optimal range of flow rates for AS-ODN LNPs was found to be 100 to 200 µl/min. MF synthesis produced LNPs with lower polydispersity values. However, the MF was less effective in preparing LNPs loaded with siRNA, which may have been due to greater rigidity of double-stranded siRNA comparing to single-stranded AS-ODN. CONCLUSION: MF technology is a simple, affordable and reproducible method for production of ON-LNPs.

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.

Solid-supported reagents for synthesis of nucleoside monothiophosphates, dithiodiphosphates, and trithiotriphosphates.

This unit describes procedures for the selective synthesis of nucleoside monothiophosphates, dithiodiphosphates, and trithiotriphosphates from solid-supported phosphitylating reagents. Rigid and sterically hindered polymer-bound 1,3,2-oxathiaphospholane is reacted selectively with the 5'-hydroxyl group of nucleosides in the presence of 1H-tetrazole. Sulfurization in the presence of Beaucage's reagent (3H-1,2-benzodithiole-3-one 1,1-dioxide) followed by ring-opening with 3-hydroxypropionitrile and basic cleavage in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) through the elimination of polymer-bound ethylene episulfide afford nucleoside monothiophosphates. Furthermore, reaction of polymer-bound diphosphitylating and triphosphitylating reagents, prepared from polymer-bound benzyl alcohol, with unprotected nucleosides, sulfurization with Beaucage's reagent, and acidic cleavage using trifluoroacetic acid cocktail produce nucleoside dithiodiphosphates and trithiotriphosphates in moderate yields.

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.

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.

Incorporation of 3'-S-phosphorothiolates into RNA: potential applications in RNAi.

Using solid-phase synthesis, oligoribonucleotides containing multiple 3'-S-phosphorothiolate modifications have been successfully synthesized, purified and characterized, utilizing a 2'-deoxyuridine phosphorothioamidite monomer.

Synthesis and properties of 2'-modified-4'-thioRNA.

As a part of our ongoing research projects, we envisioned the synthesis of 2'-modified-4'-thioRNA, such as 2'-fluoro-4'-thioRNA (2'-F-4'-thioRNA) and 2'-O-methyl-4'-thioRNA (2'-OMe-4'-thioRNA) to enhance the potential of 4'-thioRNA. Appropriately protected 2'-deoxy-2'-fluoro-4'-thiouridine (6), -cytidine (10), and - adenosine (18), substrates for the synthesis of novel modified RNAs were successfully synthesized. The fullymodified RNA consisting of 2'-deoxy-2'-fluoro-4'-thionucleosides was synthesised, and we examined its abilities of hybridization and stability against nucleases. It was found that 2'-F-4'-thioRNA shows high hybridization ability to complementary RNA. Furthermore, 2'-F-4'-thioRNA has strong resistance toward degradation in 50% human plasma.

Stereocontrolled synthesis of oligonucleoside phosphorothioates and PO/PS-chimeric oligonucleotides by using oxazaphospholidine derivatives.

Solid-phase synthesis of oligonucleotides having a fully-modified stereoregular phosphorothioate backbone or a stereoregular phosphate/phosphorothioate chimeric backbone was achieved by using diastereopure nucleoside 3'-bicyclic oxazaphospholidine derivatives and beta-cyanoethyl phosphoramidites as monomer units and N-(cyanomethyl)pyrrolidinium trifluoromethanesulfonate (CMPT) as an acidic activator. The trans-isomers of the oxazaphospholidines were obtained exclusively by the reaction of the 3'-OH of appropriately protected nucleosides and the corresponding 2-chloro-1,3,2-oxazaphospholidine derivative. The trans-isomers were configurationally stable and did not epimerize almost at all even in the presence of CMPT. As a result, the formation of phosphorothioate internucleotide linkages using the oxazaphospholidine derivatives and CMPT proceeded without any loss of diastereopurity. In addition to the synthesis of stereoregular phosphorothioate linkages, the synthesis of phosphate internucleotide linkages through the same method was studied. As a result of the study, stereoregular phosphate/phosphorothioate chimeric oligonucleotides as well as stereoregular oligonucleoside phosphorothioates were efficiently synthesized by using the same method.

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]

Solid-phase synthesis of stereoregulated oligodeoxyribonucleoside phosphorothioates.

An efficient stereocontrolled synthesis of oligodeoxyribonucleoside phosphorothioates on a solid-support by an oxazaphospholidine method was achieved. A detailed investigation on the reagents and conditions for the solid-phase synthesis revealed that the use of the capping and sulfurizing reagents which are regularly used for the conventional synthesis of phosphorothioate DNA resulted in a sluggish removal of the chiral auxiliaries on the phosphorothioate internucleotidic linkages. After optimization of the capping and sulfurizing steps, the deprotection of the internucleotidic linkages were successfully conducted under the regular basic conditions, and P-stereoregulated phosphorothioate DNA oligomers were successfully obtained with excellent diastereopurity.

Effect of the extent of thiolation and introduction of phosphorothioate internucleotide linkages on the anti-HIV activity of Suligovir [(s4dU)35].

Suligovir is a 35-mer homo-oligonucleotide, containing exclusively 4-thio deoxyuridylate, proved to be a potent inhibitor of HIV entry. In this paper, we described the effect of extent of thiolation and the introduction of nuclease-resistant phosphorothioate linkages on the anti-HIV activity of Suligovir. We found that the decreased thiolated nucleotide content decreases the anti-HIV potency of the compound and the introduction of phosphorothioate linkages does not improve its antiviral activity.

Antisense oligonucleotides: from design to therapeutic application.

1. An antisense oligonucleotide (ASO) is a short strand of deoxyribonucleotide analogue that hybridizes with the complementary mRNA in a sequence-specific manner via Watson-Crick base pairing. Formation of the ASO-mRNA heteroduplex either triggers RNase H activity, leading to mRNA degradation, induces translational arrest by steric hindrance of ribosomal activity, interferes with mRNA maturation by inhibiting splicing or destabilizes pre-mRNA in the nucleus, resulting in downregulation of target protein expression. 2. The ASO is not only a useful experimental tool in protein target identification and validation, but also a highly selective therapeutic strategy for diseases with dysregulated protein expression. 3. In the present review, we discuss various theoretical approaches to rational design of ASO, chemical modifications of ASO, ASO delivery systems and ASO-related toxicology. Finally, we survey ASO drugs in various current clinical studies.

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.

Development of siRNA for therapeutics: efficient synthesis of phosphorothioate RNA utilizing phenylacetyl disulfide (PADS).

Efficient synthesis of phosphorothioate RNA (PS-RNA) is demonstrated by using phenylacetyl disulfide (PADS) in a mixture of pyridine and acetonitrile (1:1, v/v) for 3 min. Sulfurization is achieved with >99.8% stepwise efficiency. This reagent also performs efficiently during synthesis of RNA containing PS:PO mixed backbone.