Synthesis and hybridizing properties of P-stereodefined chimeric [PS]-{DNA:RNA} and [PS]-{DNA:(2'-OMe)-RNA} oligomers

Abstract

Oxathiaphospholane derivatives of 2'-OMe-ribonucleosides and 2'-O-TBDMS-ribonucleosides (^MN-OTP and ^TN-OTP, respectively; nucleobase protected) were synthesized and separated into pure P-diastereomers. X-ray analysis showed the R _P absolute configuration of the phosphorus atom in the fast-eluting diastereomer of ^TA-OTP. The fast- and slow-eluting P-diastereomers of ^MN-OTP and ^TN-OTP were used in the solid-phase synthesis of phosphorothioate dinucleotides (^MN_PST and N_PST, respectively), which were subsequently hydrolyzed with R _P-selective phosphodiesterase svPDE and S _P-selective nuclease P1 to determine the absolute configuration of the phosphorus atoms. P-Stereodefined phosphorothioate ([PS]) 10-mer chimeric oligomers [PS]-{DNA:(2'-OMe)-RNA} and isosequential [PS]-{DNA:RNA} containing two ^MN_PS or N_PS units were synthesized. Melting experiments performed for their complexes with Watson-Crick paired DNA matrix showed that ^MN_PS or N_PS units decrease the thermal stability of the duplexes (ΔT _m = -0.5 ÷ -5.5 °C per modification) regardless of the absolute configuration of the P-atoms. When the (2'-OMe)-RNA matrix was used an increase in T _m was noted in all cases (ΔT _m = +1 ÷ +7 °C per modification). The changes in thermal stability of the duplexes formed by [PS]-chimeras with DNA and (2'-OMe)-RNA matrices do not correlate with the absolute configuration of the phosphorus atoms.

Chart 1. Classes of the OTP derivatives of 2′-deoxyribo- and ribonucleosides. B′ = Ade^Bz, Cyt^Bz, Gua^iBu, or Ura. For those with R = H or Me, see ref. and , respectively.

Scheme 1. Synthesis of OTP monomers 2 and 3 (R,R = –(CH₂)₅–) and the mechanistic principle of the coupling step.

Fig. 1. X-ray ORTEP diagram of the detritylated fast-eluting P-diastereomer of 3a showing the R_P absolute configuration. The displacement ellipsoids are shown at the 50% probability level. Colors: red – oxygen, blue – nitrogen, pink – phosphorus, yellow – sulfur, orange – silicon, black – carbon, white – hydrogen.

Fig. 2. Parts of the X-ray ORTEP diagram show: left image–a DNA-like C2′-endo conformation of detritylated fast-eluting 3a; right image – spatial orientation of substituents on the phosphorus atom. Colors as in Fig. 1.

Fig. 3. Decay of absorption (at 504 nm) of the DMT-cation released in consecutive detritylation steps.

Fig. 4. HPLC profiles recorded for ^MGS during DMT-ON (the upper panel) and DMT-OFF (the lower panel) purification steps.

Fig. 5. Normalized melting curves for selected duplexes (see legend) dissolved in pH 7.2 buffer (10 mM Tris–HCl, 100 mM NaCl, and 10 mM MgCl₂). The curves designations mA and mAS (in the legend) correspond to ^MA and ^MAS, respectively.

Fig. 6. Changes in melting temperature of duplexes (ΔT_m; °C) formed by [PO]-{DNA:^MRNA} chimeras with Md (green bars) or Mm (black bars), and PSCh oligomers with Md (blue bars) and Mm (red bars). The ΔT_m values were calculated using the T_m values for the corresponding duplexes Bd/Md, Bd/Mm, B(R,S)/Md and B(R,S)/Mm. Designations along the horizontal axis such as mAR or mGS correspond to ^MAR and ^MGS, respectively.