Dinucleoside Phosphorothioates Research Articles

Electronic Structures of LNA Phosphorothioate Oligonucleotides.

Important oligonucleotides in anti-sense research have been investigated in silico and experimentally. This involves quantum mechanical (QM) calculations and chromatography experiments on locked nucleic acid (LNA) phosphorothioate (PS) oligonucleotides. iso-potential electrostatic surfaces are essential in this study and have been calculated from the wave functions derived from the QM calculations that provide binding information and other properties of these molecules. The QM calculations give details of the electronic structures in terms of e.g., energy and bonding, which make them distinguish or differentiate between the individual PS diastereoisomers determined by the position of sulfur atoms. Rules are derived from the electronic calculations of these molecules and include the effects of the phosphorothioate chirality and formation of electrostatic potential surfaces. Physical and electrochemical descriptors of the PS oligonucleotides are compared to the experiments in which chiral states on these molecules can be distinguished. The calculations demonstrate that electronic structure, electrostatic potential, and topology are highly sensitive to single PS configuration changes and can give a lead to understanding the activity of the molecules.

Stereocontrolled synthesis of dinucleoside phosphorothioates using a fluorous tag

Dinucleoside phosphorothioates were synthesized in a stereocontrolled manner (diastereoselectivity>99:1) by using thymidine derivatives bearing a simple perfluoroalkyl tag (C6F13 or C8F17) at the 3′-end and diastereopure nucleoside 3′-O-oxazaphospholidine monomers. Fluorous-tagged intermediates in the synthesis were easily purified by a simple fluorous solid-phase extraction process.

Synthesis of a 35S‐labeled dinucleoside phosphorothioate prodrug, an orally bioavailable anti‐HBV agent

The 35S‐labeled, dinucleoside phosphorothioate 1, an orally available agent against hepatitis B virus, was prepared in eight steps with high specific activity and radiochemical purity. Radiolabeled 3H‐benzodithiole‐3‐one‐1,1‐dioxide was synthesized in four steps from 35S8 and was used as the sulfurizing reagent. Copyright © 2012 John Wiley & Sons, Ltd.

Metabolism, Pharmacokinetics, Tissue Distribution, and Stability Studies of the Prodrug Analog of an Anti-Hepatitis B Virus Dinucleoside Phosphorothioate

The alkoxycarbonyloxy dinucleotide prodrug R(p), S(p)-2 is an orally bioavailable anti-hepatitis B virus agent. The compound is efficiently metabolized to the active dinucleoside phosphorothioate R(p), S(p)-1 by human liver microsomes and S9 fraction without cytochrome P450-mediated oxidation or conjugation. The conversion of R(p), S(p)-2 to R(p), S(p)-1 appears to be mediated by liver esterases, occurs in a stereospecific manner, and is consistent with our earlier reported studies of serum-mediated hydrolytic conversion of R(p), S(p)-2 to R(p), S(p)-1. However, further metabolism of R(p), S(p)-1 does not occur. The presence of a minor metabolite, the desulfurized product 10 was noted. The prodrug R(p), S(p)-2 was quite stable in simulated gastric fluid, whereas the active R(p), S(p)-1 had a half-life of <15 min. In simulated intestinal fluid, the prodrug 2 was fully converted to 1 in approximately 3 h, whereas 1 remained stable. To ascertain the tissue distribution of the prodrug 2 in rats, the synthesis of (35)S-labeled R(p), S(p)-2 was undertaken. Tissue distribution studies of orally and intravenously administered radiolabeled [(35)S]2 demonstrated that the radioactivity concentrates in the liver, with the highest liver/plasma ratio in the intravenous group at 1 h being 3.89 (females) and in the oral group at 1 h being 2.86 (males). The preferential distribution of the dinucleotide 1 and its prodrug 2 into liver may be attributed to the presence of nucleoside phosphorothioate backbone because phosphorothioate oligonucleotides also reveal a similar tissue distribution profile upon intravenous administration.

Time‐Dependent Thermocontrol of the Hydrophilic and Lipophilic Properties of DNA Oligonucleotide Prodrugs

This unit describes the preparation of alkylthioalkylated and formamidoalkylated alcohols, an amidoalkylated alcohol, a hydroxylalkylated phosphoramidate, and their phosphoramidothioate derivatives, all of which have been identified as heat-sensitive thiophosphate-protecting groups in the development of thermolytic immunostimulatory DNA prodrugs. The alcohols are converted to their deoxyribonucleoside phosphoramidite derivatives, which are then used in the preparation of thermosensitive dinucleoside phosphorothioates. The thiophosphate-protecting groups of these dinucleoside phosphorothioates presumably undergo thermolytic cyclodeesterification at elevated temperature under essentially neutral conditions to release the desired phosphorothioate diester function. On the basis of their thermolytic deprotection kinetics, one can identify those thiophosphate-protecting groups that (i) may be useful for thiophosphate protection of CpG motifs of immunostimulatory DNA oligonucleotides (CpG ODNs); (ii) are suitable for protection of phosphodiester functions flanking the CpG motifs; and (iii) offer adequate protection of terminal phosphodiester functions against ubiquitous extracellular and intracellular exonucleases that may be found in biological environments.

Hydroxyalkylated phosphoramidate, phosphoramidothioate and phosphorodiamidothioate derivatives as thiophosphate protecting groups in the development of thermolytic DNA prodrugs

The hydroxyalkylated phosphoramidate 4a, phosphoramidothioates 4b, 4e–j, and phosphorodiamidothioates 4c and 4d have been identified as a new class of heat-sensitive thiophosphate protecting groups in the development of thermolytic immunomodulatory DNA prodrugs. These alcohols are converted to their deoxyribonucleoside phosphoramidite derivatives 6a–j, which are then used in the preparation of the thermosensitive dinucleoside phosphorothioates 7a–j. The negatively charged thiophosphate protecting groups of 7a–b and 7e–j presumably undergo thermolytic cyclodeesterification at elevated temperature under essentially neutral conditions. The thiophosphate protecting groups of 7e and 7f show relatively rapid deprotection kinetics at 37 °C (t½ = 20 and 42 h, respectively) and are therefore suitable for the protection of phosphodiester functions flanking the CpG motifs of immunomodulatory DNA sequences, whereas the thiophosphate protecting groups of 7g–j with thermolytic deprotection half-lives in the range of 94–265 h at 37 °C are more appropriate for the thiophosphate protection of CpG motifs. Furthermore, the thermostability of the group protecting the thiophosphate function of 7a (t½ = 82 min at 90 °C) should offer adequate protection of the 5′- and/or 3′-terminal phosphodiester functions of DNA prodrugs against ubiquitous extracellular and intracellular exonucleases.

Studies of Asymmetric Induction in the Synthesis of Dinucleoside Phosphorothioates from 2-Oxo-1,3,2-dithiaphospholane Nucleoside Derivatives

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.

DNA Oligonucleotides Containing Stereodefined Phosphorothioate Linkages in Selected Positions

This unit describes a method for the synthesis of DNA chimeric PO/PS-oligonucleotides with a stereodefined phosphorothioate bond in the selected position. Diastereomerically pure 5'-O-DMTr-N-protected-deoxyribonucleoside-3'-O-(2-thio-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane)s obtained according to the previously described protocol (UNIT 4.17) are transformed via a stereospecific 1,3,2-oxathiaphospholane-ring opening condensation into the corresponding dinucleoside phosphorothioates. Such dimers cannot be introduced into an oligonucleotide chain via the phosphoramidite approach since the unprotected P-S(-) bond is easily oxidized during routine I(2)/Py/water oxidation of the phosphite function. In the methodology described here, the reversible alkylation of the PS function is applied. Subsequently, the 3'-phosphoramidites of such PS-protected dimers prepared in situ are used for routine synthesis of chimeric PO/PS-oligonucleotides according to the phosphoramidite method. The presence of the alkylated PS-function requires modified conditions for oligonucleotide deprotection and cleavage from the solid support. Detailed procedures for the synthesis of PS-dimers and their incorporation into an oligonucleotide chain, as well as deprotection/purification steps are presented.

1,3,2-Oxathiaphospholane approach to the synthesis of P-chiral stereodefined analogs of oligonucleotides and biologically relevant nucleoside polyphosphates

Abstract Among the various classes of modified nucleotides and oligonucleotides, phosphorothioate analogs, in which the sugar-phosphate backbone is modified by the substitution of a sulfur atom for one of the nonbridging oxygen atoms, have been most extensively studied in both in vitro and in vivo experiments. However, this substitution induces P-chirality of the dinucleoside phosphorothioate moiety. Consequently, even short phosphorothioate oligonucleotides synthesized using standard chemical methods exist as mixtures of many diastereoisomers. In our laboratory, the oxathiaphospholane (OTP) method has been developed for a stereocontrolled synthesis of oligo(deoxyribonucleoside phosphorothioate)s. Recently, this approach has been extended to ribonucleoside derivatives, and stereodefined phosphorothioate diribonucleotides were incorporated into oligomers suitable for mechanistic studies on deoxyribozymes. Next, it was found that the OTP ring can be opened with nucleophiles as weak as the phosphate or pyrophosphate anion, giving rise to nucleoside α-thiopolyphosphates. Surprisingly, the reaction between nucleoside OTP and O,O-dialkyl H-phosphonate or O,O-dialkyl H-phosphonothioate led to nucleoside 5'-O-(α-thio-β-O,O-dialkyl-hypophosphate) or 5'-O-(α,β-dithio-β-O,O-dialkyl-hypophosphate), respectively, i.e., derivatives containing a direct P-P bond.

Enhanced P-stereodependent stability of complexes formed by phosphorothioate oligonucleotides due to involvement of sulfur as strong hydrogen bond acceptor

Abstract The antisense or antigene properties are exerted not only by natural oligonucleotides, but also by their different analogs. Among them, phosphorothioate oligonucleotides (PS-DNA), in which the sugar-phosphate backbone is modified due to substitution of the sulfur atom for one of the nonbridging oxygens, are much more resistant toward nucleases and, simultaneously, maintain good hybridization properties. However, the substitution induces the P-chirality of dinucleoside phosphorothioate moiety and even short PS-DNA synthesized using standard chemical methods exist as a mixture of hundreds or thousands of diastereoisomers. Diastereomerically pure oligomers of [PS]-d(CG)4 and [PS]-d(GC)4 series, obtained using the oxathiaphospholane method, were investigated with respect to their ability to adopt the left-handed conformation at high sodium chloride concentration. Obtained data allow us to postulate the formation of a strong intramolecular hydrogen bond with anionic sulfur atom as an acceptor. Homopurine [All-RP-PS]-oligos, but not [All-SP-PS]-oligos, form with the RNA templates extremely stable triplex structures, so far not described in the literature. Most likely, the triplexes are stabilized by hydrogen bonds or water bridges with the participation of sulfur atoms of internucleotide linkage. Notably, target RNA molecules are "arrested" by properly designed [All-RP-PS]-DNA probes at submicromolar concentration, and as the result, they are not recognized by reverse transcriptase.

An Efficient Reagent for the Phosphorylation of Deoxyribonucleosides, DNA Oligonucleotides, and Their Thermolytic Analogues

[reaction: see text] The phosphoramidite 11 was prepared in three steps from methyl 2-mercaptoacetate and demonstrated efficiency in the synthesis of conventional 5'-/3'-phosphate/thiophosphate monoester derivatives of 2'-deoxyribonucleosides and DNA oligonucleotides. Moreover, the use of 11 has enabled the preparation of the dinucleoside phosphorothioate analogue 26 in high yields (>95%) with minimal cleavage (<2%) of the thermolytic thiophosphate protecting group.

New approach to the synthesis of oligodeoxyribonucleotides modified with phosphorothioates of predetermined sense of P-chirality

Appropriately protected, diastereomerically pure dinucleoside phosphorothioates, obtained by the stereocontrolled oxathiaphospholane method, were S-alkylated with 2-nitrobenzyl bromide and then converted into their 3′- O-phosphoramidites. The corresponding S-protected dinucleotide building blocks were successfully used for the synthesis of oligonucleotides containing P-stereodefined phosphorothioate bonds at preselected positions.

The P‐Stereocontrolled Synthesis of PO/PS‐Chimeric Oligonucleotides by Incorporation of Dinucleoside Phosphorothioates Bearing an O‐4‐Nitrophenyl Phosphorothioate Protecting Group

AbstractThe synthesis of protected model dinucleoside (3',5')‐O‐aryl phosphorothioates, their separation into pure diastereomers, and their successful incorporation into oligonucleotides followed by stereospecific deprotection of the O‐aryl phosphorothioate function with oximate ion (inversion) enables the preparation of chimeric PO/PS‐oligonucleotides with a predetermined sense of P‐chirality at each internucleotide phosphorothioate position. The absolute configuration at the phosphorus of the internucleotide O‐aryl phosphorothioate in “dimeric building blocks” has been assigned. The 3'‐terminal SP‐phosphorothioate linkages effectively protect such chimeric constructs from degradation by human plasma exonuclease (© Wiley‐VCH Verlag GmbH &amp; Co. KGaA, 69451 Weinheim, Germany, 2005)

Diastereocontrolled Synthesis of Phosphorothioate DNA via an Oxazaphospholidine Approach Using a Novel Class of Activators, Dialkyl(cyanomethyl)ammonium Salts

AbstractFor Abstract see ChemInform Abstract in Full Text.

Diastereocontrolled Synthesis of Phosphorothioate DNA via an Oxazaphospholidine Approach Using a Novel Class of Activators, Dialkyl(cyanomethyl)ammonium Salts

Dialkyl(cyanomethyl)ammonium salts 1 were synthesized and used as a novel class of activators for the stereospecific condensations of diastereopure nucleoside 3′-O-oxazaphospholidines with a nucleoside. This new oxazaphospholidine method could efficiently produce both (Rp)- and (Sp)-dinucleoside phosphorothioates.

An oxazaphospholidine approach for the stereocontrolled synthesis of oligonucleoside phosphorothioates.

The stereocontrolled synthesis of oligodeoxyribonucleoside phosphorothioates (PS-ODNs) using nucleoside 3'-O-oxazaphospholidine derivatives as monomer units is described. 2-Chloro-1,3,2-oxazaphospholidine derivatives were prepared from six kinds of enantiopure 1,2-amino alcohols and used for the phosphitylation reactions of 5'-O-protected nucleosides. A detailed study of these reactions revealed that the diastereoselectivity of the reaction depended on the structure of the enantiopure 1,2-amino alcohol, the reaction temperature, and the amine used as a scavenger of HCl. In addition, ab initio molecular orbital calculations for the 2-chlorooxazaphospholidine derivatives were carried out to elucidate the mechanism of these diastereoselective phosphitylation reactions. The LUMO of the 2-chloro-5-phenyloxazaphospholidine derivatives on the phosphorus atom was found to be almost orthogonal to the P-Cl bond. This LUMO may be involved in the phosphitylation reactions with predominant retention of the P-configuration. A series of dialkyl(cyanomethyl)ammonium salts were developed and used as activators for the condensation reactions of the diastereopure nucleoside 3'-O-oxazaphospholidines with 3'-O-protected nucleosides. In the presence of the new activators, the reactions proceeded rapidly to give the corresponding dinucleoside phosphite triesters. The diastereoselectivity of the condensation reaction did not depend on the counteranion but on the structure of the dialkyl(cyanomethyl)amine. In the presence of the activator, which consists of a relatively small dialkyl(cyanomethyl)amine, the condensation proceeded with excellent diastereoselectivity. After sulfurization and deprotection, diastereopure (R(p))- and (S(p))-dinucleoside phosphorothioates were obtained in excellent yields. The present methodology was also applied to the solid-phase synthesis of stereoregulated PS-ODNs. all-(R(p))-[T(PS)](3)T, all-(S(p))-[T(PS)](3)T, all-(R(p))-d[G(PS)A(PS)C(PS)]T, and all-(R(p))-[T(PS)](9)T were synthesized on a highly cross-linked polystyrene resin.

Diastereocontrolled synthesis of dinucleoside phosphorothioates using a novel class of activators, dialkyl(cyanomethyl)ammonium tetrafluoroborates.

A novel class of activators, dialkyl(cyanomethyl)ammonium tetrafluoroborates 1a-c, has been developed and applied to the condensations of diastereopure 5'-O-tert-butyldiphenylsilylthymidine 3'-cyclic phosphoramidites 3a-d with 3'-O-tert-butyldimethylsilylthymidine (4a). Among them, the condensation of 3a with 4a in the presence of 1a completed within 5 min and gave only one diastereoisomer of the corresponding phosphite 5a. After sulfurization and deprtection, almost diastereopure (Rp)-TpsT 7 was obtained (d.r. = 99:1). Next the 5'-O-(DMTr)nucleoside 3'-phosphoramidites 8a-d containing thymine, N6-benzoyladenine, N4-benzoylcytosine, and N2-phenylacetylguanine have been synthesized and allowed to condense with 3'-O-protected thymidine and 2'-deoxyadenosine. The 5'-O-DMTr group and the N-acyl groups of the nucleobases were compatible with the reaction conditions and the condensations completed quickly with excellent diastereoselectivity.

Stereoselective synthesis of dinucleoside phosphorothioate using enantiopure 1,2-amino alcohols as chiral auxiliaries.

Diastereopure nucleoside 3'-cyclic phosphoramidites were synthesized stereoselectively from enantiopure 1,2-amino alcohols. In the presence of a novel activator, these phosphoramidites underwent the condensation with 3'-O-tert-butyldimethylsilylthymidine to give the corresponding phosphite intermediates. Upon sulfurization, followed by deprotection, dithymidine phosphorothioate was obtained in good yield with good to excellent diastereoselectivity.

Stereocontrolled synthesis of LNA Dinucleoside phosphorothioate by the oxathiaphospholane approach

The application of the oxathiaphospholane approach for the stereocontrolled synthesis of LNA dinucleoside phosphorothioate is described. The reaction of ring opening condensation proceeds in CH 3CN solution in high yield and with over 96% stereoselectivity. One of diastereomers of LNA dinucleoside phosphorothioate (presumably R P) was found to be readily digested by svPDE.

Deoxyribonucleoside Cyclic N-Acylphosphoramidites as a New Class of Monomers for the Stereocontrolled Synthesis of Oligothymidylyl- and Oligodeoxycytidylyl- Phosphorothioates

A simple and straightforward synthesis of the pyrimidine 2‘-deoxyribonucleoside cyclic N-acylphosphoramidites RP-1 and SP-1 is described. Specifically, (±)-2-amino-1-phenylethanol 2 was chemoselectively N-acylated to 4 by treatment with ethyl fluoroacetate 3 followed by reaction with hexaethylphosphorus triamide to afford the cyclic N-acylphosphoramidite 5 as a mixture of diastereomeric rotamers (5a and 5b). Condensation of N4-benzoyl-5‘-O-(4,4‘-dimethoxytrityl)-2‘-deoxycytidine 8 with 5 in the presence of 1H-tetrazole gave, after silica gel chromatography, pure RP-1 and SP-1. 31P NMR studies indicated that when RP-1 or SP-1 is reacted with 3‘-O-acetylthymidine and N,N,N‘,N‘-tetramethylguanidine in CD3CN, the dinucleoside phosphotriester SP-9 or RP-9 is formed in near quantitative yield with total P-stereospecificity (δP 144.2 or 143.9 ppm, respectively). Sulfurization of SP-9 or RP-9 generated the P-stereodefined dinucleoside phosphorothioate RP-11 or SP-11 (δP 71.0 or 71.2 ppm, respectively). The 2‘-deoxycytidine cyclic N-acylphosphoramidite derivatives RP-1 and SP-1 were subsequently applied to the solid-phase synthesis of [RP,RP]- and [SP,SP]-trideoxycytidilyl diphosphorothioate d(CPSCPSC), and [RP,SP,RP]-tetradeoxycytidilyl triphosphorothioate d(CPSCPSCPSC). Following deprotection, reversed-phase (RP) HPLC analysis of these oligonucleotide analogues showed a single peak for each oligomer. By comparison, RP-HPLC analysis of purified P-diastereomeric d(CPSCPSC) and d(CPSCPSCPSC) prepared from standard 2-cyanoethyl deoxyribonucleoside phosphoramidites exhibited 4 and 8 peaks, respectively, each peak corresponding to a specific P-diastereomer (see Figure 3A). The thymidine cyclic N-acylphosphoramidite derivatives RP-14 and SP-14 were also prepared, purified, and used successfully in the solid-phase synthesis of [RP]11-d[(TPS)11T]. Thus, the application of deoxyribonucleoside cyclic N-acyl phosphoramidites to P-stereocontrolled synthesis of oligodeoxyribonucleoside phosphorothioates may offer a compelling alternative to the methods currently used for such syntheses.