Oxathiaphospholane Approach to the Synthesis of P-Chiral, Isotopomeric Deoxy(ribonucleoside phosphorothioate)s and Phosphates Labeled with an Oxygen Isotope

Abstract

Introduced by Eckstein, phosphorothioate analogues of nucleotides have become an indispensable tool for studying the metabolism of nucleic acids. [1] Standard chemical methods for the synthesis of oligo(deoxyribonucleoside phosphorothioate)s (PS-Oligos) provide a mixture of 2 n diastereoisomers, where n is the number of phosphorothioate linkages. [2] The enzymatic synthesis of stereodefined PS-Oligos is limited to the preparation of (all-R P )-oligomers because of the stereoselectivity of available DNA and RNA polymerases. The first method for stereocontrolled chemical synthesis of PS-Oligos was elaborated in our group, [3] and several alternative methods were recently reported. [4,5] Stereodefined PS-Oligos were used for studying the mode of action of several bacterial and human enzymes [6±8] and the stereodependent avidity of PS-Oligos toward complementary DNA or RNA. [9] However, the presence of a sulfur atom affects the properties of internucleotide bonds, mostly due to the different steric requirements of sulfur atoms (PÀS vs PÀO bond length), different affinity towards metal ions, and changes in the distribution of the negative charge in the phosphorothioate anion.[10] Therefore, the hydration pattern of PS-Oligos is different from that of natural oligonucleotides, [11] and this obstructs the evaluation of kinetic data of ªrescue effectsº of thiophilic metal ions, and makes analysis of direct or watermediated contacts between metal ions and phosphate groups much more difficult. These inconveniences could be avoided by using P-chiral isotopomeric phosphates. [12] Here we describe the synthesis of stereodefined oligo(deoxyribonucleoside [ 18 O]phosphorothioate)s (PS 18 O-Oligos) and oligo-(deoxyribonucleoside [ 18 O]phosphate)s (P 18 O-Oligos), in which both of the nonbridging oxygen atoms of the internucleotide bond were replaced by S and 18 O, or one of them was replaced by 18 O, respectively. Oligonucleotides containing a single P-chiral [ 16 O, 18 O] internucleotide bond were first used by Eckstein [13] in studies on Eco RI endonuclease. Stereodefined P 18 O-Oligos can be used to investigate the interaction of particular oxygen atoms with other molecules or metal ions, given analytical methods that allow the isotopic effect to be measured with satisfactory accuracy. [14]

Thiophosphorylation of Biologically Relevant Alcohols by the Oxathiaphospholane Approach

“…21 Therefore, since compounds 1b were found to react with alcohols in a fully stereospecific manner, 16 the methodology presented in this paper has been also applied for the synthesis of diastereomerically pure isotopomeric nucleoside-3¢-[ 18 O]-phosphorothioates. 22 The successful preparation of nucleoside phosphorothioates prompted us to apply the aforementioned oxathiaphospholane procedure for the synthesis of phosphoromonothioate derivatives of selected polyols (3c, see Table 3). Thus, 2-thiono-1,3,2-oxathiaphospholane derivatives of 1,8-octanediol (1c: R i = R 1 , n = 2), 2-methyl-1,3-propanediol (1c: R i = R 2 , n = 2), glycerol (1c: R i = R 3 , n = 3), and pentaerythritol (1c: R i = R 4 , n = 4) were prepared according to the procedure described earlier, 23 and treated on a 2-4 mmol scale, for 2 hours at room temperature, in anhydrous acetonitrile solution, with two molar equivalent of 3-hydroxypropionitrile and one molar equivalent of DBU (calculated with respect to the number of oxathiaphospholane functions in the molecule 1c).…”

Synthesis of Phosphorothioate Oligonucleotides with Stereodefined Phosphorothioate Linkages

“…Longer oligonucleotides were obtained in poor yields and the syntheses were not reproducible. Oxathiaphospholanes that are 18 O‐labeled at the endocyclic position allowed for the synthesis of PS‐oligos with internucleotide PS[ 18 O]‐phosphorothioate moieties of predetermined chirality (Guga et al, ).…”