Effect of phosphorothioate chirality on i-motif structure and stability

Abstract

The P-chiral stereo-defined phosphorothioate groups have been introduced into all of the four internucleotide positions of d(T(PS1)C(PS2)C(PS3)C(PS4)C) (PSn = phosphorothioate group), and among the 16 possible diastereomers of PS-d(TC(4)), 10 stereomers have been synthesized to investigate the effects of the sense of the P-chirality upon the structure and stability of the i-motif structure. The temperature dependence of circular dichroism spectra showed that the melting temperature (T(m)) of the [all R(p)]-PS-d(TC(4)) i-motifs was 31 degrees C, identical to that of the parent oligomer, PO-d(TC(4)), while that of the [all S(p)]-PS-d(TC(4)) i-motif was largely decreased by 11 degrees C. Single substitution of R(p) with S(p) caused a decrease of T(m) by 3-4 degrees C at positions of PS1, PS2, and PS3 and by 1 degrees C at that of PS4, showing the additive property of the T(m) suppression. The comparison of the NOESY spectra between [all R(p)]-PS-, [all S(p)]-PS-, and PO-d(TC(4)) showed that intraresidual H6-H3' and H2' '-H4' NOE cross-peaks of the all S(p) isomer are weaker than those of the all R(p) isomer and PO-d(TC(4)), indicating the change in the C3'-endo conformation and glycosidic bond angle. The structural alternation for the i-motif formed by [all S(p)]-PS-d(TC(4)) is also suggested by the chemical shift differences of C2/C3/C4 H2''and H4' protons from those of [all R(p)]-PS-d(TC(4)) and PO-d(TC(4)). These results suggest that the S(p) configuration at phosphorus of the phosphorothioate linkage changes the sugar-phosphorothioate conformation and intermolecular interaction in the narrow groove, leading to the destabilization of the i-motif structure.