Remote Control of Diastereoselectivity of Dn-Ase Ii and Endonuclease Eco Ri Towards Phosphorothioate Analogues of Oligonucleotides

(Rp)-Thymidyl 3′-(4-nitrophenyl phosphorothioate) (TP(S)NP) is converted in the presence of DN-ase II into (Sp,Rp)-thymidyl (3′–5′)-thymidyl phosphorothioate 3′-(4-nitrophenyl phosphorothioate) (TP(S)TP(S)NP), while (Sp)-TP(S)NP under analogous conditions gives (Rp,Sp)-TP(S)TP(S)NP, but as a minor product only. The preponderant product was recognized as (Sp,Rp)-thymidyl (3′–5′) thymidyl phosphorothioate 5′–(4-nitrophenyl phosphorothioate) (NPP(S)TP(S)T). All phosphorothioyl transfer reactions occur with retention of configuration at phosphorus atoms, which speaks for a double-displacement process and involvement of phosphorothioylated enzymes as reactive intermediates. Nucleolytic activity of DN-ase II, characteristic for this enzyme with respect to natural oligonucleotides, is not observed if TP(S)NP are used as the substrates. However, the transferase activity is even extended for the (4-nitrophenyl phosphorothioate)-shift from 3′ to 5′-position of dinucleotide, but for diastereoisomer (Rp,Sp)-TP(S)TP(S)NP only. Such “remote” control of diastereoselectivity of DN-ase II is not unique, because Endonuclease Eco RI exerts similar selectivity towards diastereoisomers of octanucleotide GP(S)GAATTCC. (Rp)-isomer, but not (Sp)-counterpart is the substrate for the enzyme causing its degradation to (Rp)-GP(S)G and PAATTCC. The influence of absolute configuration at phosphorothioate in 5′-“adjacent” position to the “scissile” internucleotide phosphate bond is discussed in terms of conformational changes of oligonucleotide or diastereofacial character of DNA-protein interaction and importance of protein-adjacent phosphate charge-charge interactions.