Oligo phosphorothioate

Data from [8]. Labeled methylphosphonate (MP-oligo), phosphorothioate (S-oligo), alternating methyl-phosphonate-phosphodiester (Alt-MP-oligo), and unmodified phosphodiester (D-oligo) oligodeoxynu-cleotides. 

One of the advantages of H-phosphonate chemistry is the ease with which phosphate analogs are prepared (Fig. 3). Phosphorothioates are readily prepared by oxidation with Sg in pyridine/CS2. This is a very clean reaction leading to virtually 100% substitution by sulfur on phosphate. This reaction has been extended to the synthesis of oligo-phosphorothioates (7,12,13), as well as uniformly labeled phosphorothioates (29,30). The synthesis of phosphorothioates has been discussed in greater detail in Chapter 8. 

The precise mechanism(s) by which oligos enter cells is not fully understood. Most are charged molecules, sometimes displaying a molecular mass of up to 10-12 kDa. Receptor-mediated endocytosis appears to be the most common mechanism by which charged oligos, such as phosphorothioates, enter most cells. One putative phosphorothioate receptor appears to consist of an 80 kDa surface protein, associated with a smaller 34 kDa membrane protein. However, this in itself seems to be an inefficient process, with only a small proportion of the administered drug eventually being transferred across the plasma membrane. 

Two nucleosides at the 5 -end and four nucleosides at the 3 -end are 2 -0-methyl ribonucleosides (bold letters) the remaining are deoxynucleosides. The underlined nucleosides of Oligo ASM are those of mismatched controls compared to Oligo AS. For both modified oligonucleotides, all intemucleotide linkages are phosphorothioate. 

Rifai, A.,W. Byrsch, K. Fadden, J. Clark, and K.-H. Schlingensipen. 1996. Clearance kinetics, biodistribution, and organ saturability of phosphorothioate oligo-deoxynucleotides in mice. Am. J. Pathol. 149 717-725. 

Bigelow, J.C., L.R. Chirin, L.A. Mathews, and J.J. McCormack. 1990. High-performance liquid chromatographic analysis of phosphorothioate analogues of oligo-deoxynudeotides in biological fluids. 

Phosphorothioate oligo An oligo (usually a probe) containing S in place of P (Fig. 1C). 

The early attempts to use relatively easily available diastereomerically pure nucleoside 3 -0-(2-cyanoethyl-AT,AT-diisopropylphosphoramidite) monomers for the stereospecific synthesis of PS-Oligos failed because of inevitable racemiza-tion of Pm intermediate caused by an excess of lff-tetrazole necessary for efficient elongation of oligonucleotide chain [13]. An idea to use for that purpose appropriately protected nucleosides functionalized at 3 -0 position with 2-thio-1,3,2-oxathiaphospholane moiety arose from the studies on the reactions of di-substituted phosphorothioates with oxiranes [14,15], and in particular from the observation that PS-PO exchange in 0,0-diphenyl phosphorothioate (8) upon treatment with ethylene oxide in methanol solution resulted in formation of... 

Detailed studies on the stereocontrolled synthesis of oligo(nucleoside phosphorothioate by the oxathiaphospholane approach were performed with monomers 15 [20].The optimization ofthe coupling step was performed [20,23] involving the amount of the monomer (20 mg for 1 pmol synthesis), the concentration of DBU (1 mol/1) and the coupling time (450 s). To complete the pro-... 

Stereocontrolled Synthesis of Isotopomeric Oligo(nudeoside PS180-Phosphorothioates and -P180-Phosphates)... 

Monomers [180]-15a-d were successfully used for the synthesis of several stereodefined oligo(nucleoside [180]phosphorothioate)s. Their stereochemistry and isotopic enrichment were confirmed by a combined enzymatic-mass spectrometry method employing snake venom phosphodiesterase or calf spleen nuclease, and MALDI TOF mass spectrometry. 

The Application of 1,3,2-Oxathiaphospholane Derivatives for the Synthesis of Stereodefined Oligo(ribonudeoside Phosphorothioate)s and their 2, 5 -Analogues... 

Stereodefined oligo(ribonucleoside phosphorothioate)s were available only by the enzymatic method [51 -54] or by partially stereocontrolled H-phosphonate procedure [55,56]. However, both procedures could provide only products with RP configuration. Alternatively, compounds containing the single phosphorothioate modification were synthesized by nonstereospecific phosphoramidite or H-phosphonate methods and then separated into diastereomers by tedious HPLC procedure with various degree of success [57-59]. Such constructs became important tools for investigation of the mechanism of action of ri-bozymes. 

In order to check the applicability of the oxathiaphospholane method for the stereocontrolled synthesis of oligo(ribonucleoside 3, 5 -phosphorothioate)s, appropriate oxathiaphospholane monomers possessing 2 -hydroxyl function protected with ferf-butyldimethylsilyl group (40 a-d) were synthesized and sepa-... 

Stec extended his work on P-chiral phosphorothioates and described the synthesis of P-chiral, isotopomeric deoxyribonucleoside-phosphorothioates and -phosphates (76a-p) labelled with an oxygen isotope. To obtain stereodefined PS 0-oligos, the 5 -0-DMT-nucleoside-3 -0-(2-thio- spiro -4,4-pentamethy-lene-l,3,2-[0 ]oxathiaphospholanes were synthesised by phosphitylation of the correct protected nucleoside with chirally pure [O ]oxathiaphospholane, with subsequent sulfurisation. The resulting oxadithiaphospholanes were separated by chromatography into diastereomerically pure forms and individually treated with Se02 to yield the oxathiaphospholanes. ... 

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