P-Stereodefined phosphorothioate analogs of glycol nucleic acids-synthesis and structural properties

Abstract

Enantiomerically pure, protected acyclic nucleosides of the GNA type (glycol nucleic acids) (^GN'), obtained from (R)-(+)- and (S)-(-)-glycidols and the four canonical DNA nucleobases (Ade, Cyt, Gua and Thy), were transformed into 3'-O-DMT-protected 2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane derivatives (OTP-^GN') containing a second stereogenic center at the phosphorus atom. These monomers were chromatographically separated into P-diastereoisomers, which were further used for the synthesis of P-stereodefined "dinucleoside" phosphorothioates ^GN_PST (^GN = ^GA, ^GC, ^GG, ^GT). The absolute configuration at the phosphorus atom for all eight ^GN_PST was established enzymatically and verified chemically, and correlated with chromatographic mobility of the OTP-^GN' monomers on silica gel. The ^GN_PS units (derived from (R)-(+)-glycidol) were introduced into self-complementary PS-(DNA/GNA) octamers of preselected, uniform absolute configuration at P-atoms. Thermal dissociation experiments showed that the thermodynamic stability of the duplexes depends on the stereochemistry of the phosphorus centers and relative arrangement of the ^GN units in the oligonucleotide strands. These results correlate with the changes of conformation assessed from circular dichroism spectra.

Fig. 1. Structures of (A) P-stereodefined PS-DNA, (B) C-stereodefined GNA, and (C) P,C-stereodefined PS-(DNA/GNA) oligomers.

Scheme 1. Synthesis of ^DMT-GN′ (4a–c) and their conversion into the oxathiaphospholane monomers 5a–d. In the inset, the numbers in parentheses indicate the priority of substituents (in terms of the Cahn–Ingold–Prelog's rules) around the stereogenic centers. The synthesis of ^DMT-GG^iBu (4d) is shown in Scheme 2.

Scheme 2. Synthesis of ^DMT-GG^iBu (4d).

Scheme 3. A possible mechanism of spontaneous oxathiaphospholane ring-opening/cyclization during detritylation of OTP-^GT (5a).

Scheme 4. Synthesis of “dinucleotides” ^GN_PST (11a–d) using OTP-^GN′ (5a–d) and products of their hydrolysis (if successful) with svPDE.

Scheme 5. The “inverted” synthesis of ^GA_PST (11*b) using ^DMT-GA^Bz and 5′-OTP-T_Camph (12).

Fig. 2. HPLC profiles for the mixture of standards 11*b (an inset in panel A) and co-injections of 11*b and ^GA_PST (11b) obtained from fast- and slow-eluting (R_C)-OTP-^GA^Bz5b (panels A and B, respectively). HPLC conditions: a Kinetex column, 250 × 4.6 mm; flow rate 1 mL min⁻¹, buffer A: 0.1 M TEAB, buffer B: 40% CH₃CN in 0.1 TEAB, gradient: 0–38% of buffer B over 20 min.

Fig. 3. CD spectra for selfcomplementary oligomers 15–19 (dissolved in pH 7.2 buffer containing 10 mM Tris–HCl, 100 mM NaCl, and 10 mM MgCl₂) recorded after 15 minutes incubation at 15 °C.

Fig. 4. CD spectra for selfcomplementary oligomers 14, 16, 14/16 and 15/17 (dissolved in pH 7.2 buffer containing 10 mM Tris–HCl, 100 mM NaCl, and 10 mM MgCl₂) recorded after 15 minutes incubation at 15 °C.