Phosphorothioate nucleotides

Andreola, M.L., Calmels, C., Michel, I, Toulme, J.J. and Litvak, S. (2000) Towards the selection of phosphorothioate aptamers Optimizing in vitro selection steps with phosphorothioate nucleotides. Eur.J. Biochem., 267, 5032-5040. 

Vitravene (fomivirsen sodium, ISIS Pharmaceuticals see Table 9) remains the only antisense-based biopharmaceutical approved for general medical use (see also Part III, Chapter 3). The product is a 21-base phosphorothioate nucleotide that displays a base sequence complementary to certain human cytomegaloviral mRNA transcripts. Its administration inhibits viral replication through an antisense mechanism. Approved in the US in 1998 and in the EU in 1999, the product is indicated for the treatment of cytomegalovirus retinitis by intraocular injection in AIDS patients. It was withdrawn from the EU market in May 2002 for commercial reasons. 

Aside from the 5 and 3 ends, some internal modihcations of the mRNA such as a phosphorothiate backbone or T modifications of residues can be brought into the mRNA as long as the modified residues are substrate for the RNA polymerases routinely used for in vitro transcription (T7, T3, or SP6 RNA polymerases) and do not interfere with the translation process. It seems that only a few modifications such as phosphorothioate nucleotides and 2 amino residues can be used [14]. However, they lower the efficacy of transcription and translation while they do not noticeably increase the stability of the mRNA toward RNases. Some more work may allow the identifications of modifications that may allow the efficacious production of mRNA, which resists extracellular RNases and remains well translatable by the ribosome. Such mRNA would be optimized compared with native mRNA for therapeutic uses. 

DNAs containing a-phosphorothioate nucleotide analogs are not effectively hydrolyzed by the 3 — 5 -exonuclease activity. With Pol Ik, the thio linkage... 

The principle of cadmium(II) rescue experiments is as follows (see also refs [8,10]) Phosphorothioate nucleotides are inserted into a RNA at single, defined sites. In cases of significantly decreased or abolished enzymatic activity of the RNA and subsequent restoration of activity upon addition of soft metal ions like Cd ", one assumes that the respective phosphate moiety coordinates a metal ion which is relevant for the catalytic activity of the ribozymes. 

In August 1998, Vitravene (tradename) became the first (and thus far the only) antisense product to be approved for general medical use by the FDA. It gained approval within the EU the following year, although it has since been withdrawn from the EU market due to commercial rather than technical reasons. The product is a 21-nucleotide phosphorothioate based product of the following base sequence ... 

Figure 3.9 Mode of action for antisense drags. An example is Fomivirsen (Vitravene, Isis Pharmaceuticals), which is a 21-nucleotide phosphorothioate that binds to the complementary mRNA of cytomegalovirus and blocks the translation process. Cytomegalovirus is a virus that belongs to the herpes group. (Source Chang YT. Keyword of the Post Genomic Era—Library, New York University, New York, 2002. http //www. nyu.edu/classes/ytchang/book/e003.html [accessed September 10, 2002].)...

There are many examples of each of these possibilities (see figure 8.8). In the case of the 2 sugar position, most of the 2 -substititutions have been achieved with 2 -0-alkyl, 2 -amino, and 2 -fluoro replacements. Purine base substitutions have been performed with 2-aminopurine, xanthosine, and isoguanosine. Phosphodiester replacements have been effected using phosphorothioate substitutions. As for the non-nucleotide linkers, propanediol linkers have been employed. Using these various bioisosteric substitutions,... 

Nucleoside 5 -phosphorothioates have also been employed as activated nucleotide derivatives for synthesis of pyrophosphates.321 The interaction of tributylammonium 2, 3 -di-0-benzoyluridine 5 -phosphorothioate (73) with silver a-D-glucopyranosyl and a-D-galac-topyranosyl phosphates in pyridine solution, with subsequent de-benzoylation, gave the corresponding glycosyl esters in 60-70% yield. This procedure can probably be classified as a variant of the mixed-anhydride method, the driving force of the reaction being the formation of insoluble silver sulfide. 

A. General description Fomivirsen sodium (molecular weight about 7kDa) is a phosphorothioate oligonucleotide, 21 nucleotides in length, with the following sequence 5 -GCG TTT GCT CTT CTT CTT GCG-3. ... 

A supported sulfide source has been demonstrated by Zhang et al.16 to synthesize oligonucleotide phosphorothioates (entry 12). Aminodithiazole-thione attached to a methacrylate-ethyleneglycol copolymer is used to efficiently convert a nucleotide phosphite to a phosphorothioate. The product nucleotide possesses protecting groups suitable for solid-phase oligonucleotide synthesis and thus is a valuable building block for nucleic acid therapeutics. 

The term (XG + Xc) is the sum of the mole fractions of guanine and cytidine in the antisense strand. The mole fraction of any nucleobase is equal to the number of nucleotides containing that base divided by the total number of nucleotides in the oligonucleotide strand. [M+] is the molar concentration of monovalent cations. In a typical mammalian cell, [K + ] is 140 mM, and [Na+ ] is 10 mM. L is the length of the duplex in base pairs. Based on Equation 6.2 and the assumption that phosphorothioate oligonucleotides behave as regular DNA, fomivirsen (6.17) would have a predicted Tm of 59 °C. Equation 6.1 predicts 64 °C. More complex forms of Equation 6.2 with increased accuracy appear regularly in the literature.8... 

T. Burckin, L. Truong, and A.A. Levin. 1997. Pharmacokinetic properties of phosphorothioate oligonucleotides. Nucleosides Nucleotides 16 1689-1693. 

E. Zylber-Katz, N.M. Dean, S.D. Rabkin, R.L. Martuza, and R.I. Glazer. 1996.Treatment of glioblastoma U-87 by systemic administration of an antisense protein kinase C-a phosphorothioate oligodeoxy-nucleotide. Mol. Pharmacol. 50 ... 

Butler M, Stecker K, Bennett CF. Flistological localization of phosphorothioate oligodeoxynucleotides in normal rodent tissue. Nucleosides Nucleotides 1997 16(7-9) 1761-4. 

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