Synthesis and characterization of dodecanucleotides containing the XhoI recognition sequence with a phosphorothioate group at the cleavage site

The synthesis and characterization of diastreomeric dodecanucleotides, d[GATC_p(S)TCGAGATC], containing recognition sequence of the XhoI restriction endonuclease with a phosphorothioate internucleotidic linkage the cleavage site are described. R_p and S_p form of diastereomerically pure dinucleoside phosphorothioates d[C_p(S)T] were presynthesized and used for the addition to the growing oligonucleotide chain as a block. The stereochemistry of dinucleoside phosphorothioate was assigned by ³¹P NMR spectroscopy, enzyme digestion, and reverse-phase HPLC. XhoI restriction endonuclease cut only R_p diastereomer d[GATC_p(S)TCGAGATC]. The rate of hydrolysis is slower than that of the unmodified dodecamer d[GATCTCGAGATC]. The phosphorothioate nucleotide is using for determination of the stereochemical course of the XhoI catalyzed reaction.