The H-phosphonate approach to the solution phase synthesis of linear and cyclic oligoribonucleotides

The solution phase synthesis of the tetraribonucleoside triphosphate r(ApCpGpU) 18 and the corresponding cyclic tetraribonucleotide 19 is described. The synthetic methodology is based on 5′- O -(DMTr)-2′- O -(Fpmp)-ribonucleoside-3′- H -phosphonate building blocks 10. Coupling, which is rapid and quantitative, is effected with di-(2-chlorophenyl) phosphorochloridate 5 at −40°C; it is followed by in situ treatment with 2-(4-methylphenyl) sulphanyl-1 H -isoindole-1,3(2 H )-dione 6b. The resulting sulphur transfer reaction also proceeds rapidly and quantitatively at −40°C. The same coupling and sulphur transfer steps are used in the cyclization reaction, but a 5′- H -phosphonate intermediate 24 is involved. The final three-step unblocking process involves treatment with (i) E-2-nitrobenzaldoxime 7 and N 1, N 1, N 3, N 3-tetramethylguanidine (TMG) 8 in acetonitrile, (ii) concentrated aqueous ammonia at 50°C and (iii) 0.5 mol/dm3 sodium acetate buffer (pH 4.0) at 40°C. The fully unblocked products 18 and 19 were characterized by NMR spectroscopy and by enzymatic digestion.