Acetal Levulinyl Ester (ALE) groups for 2′-hydroxyl protection of ribonucleosides in the synthesis of oligoribonucleotides on glass and microarrays

We describe a synthetic strategy that permits both the growth and deprotection of RNA chains that remain attached to a solid polymer support or chip surface. The key synthons for RNA synthesis are novel 5′-O-DMTr 2′-acetal levulinyl ester (2′-O-ALE) ribonucleoside 3′-phosphoramidite derivatives. In the presence of 4,5-dicyanoimidazole (DCI) as the activator, these monomers coupled to Q-CPG solid support with excellent coupling efficiency (∼98.7%). The method was extended to the light directed synthesis of poly rU and poly rA on a microarray through the use of a 5′-O-(2-(2-nitrophenyl)propoxycarbonyl)-2′-O-ALE-3′- phosphoramidite derivative. A two-stage deprotection strategy was employed to fully deblock the RNA directly on the Q-CPG or microarray support without releasing it from the support's surface: phosphate group deblocking with NEt₃ in acetonitrile (ACN) (2:3 v/v; 1 h, r.t.) followed by removal of the 2′-O-ALE groups under mild hydrazinolysis conditions (0.5-4 h, r.t.). This last treatment also removed the levulinyl (Lv) group on adenine (N⁶) and cytosine (N⁴) and the dimethylformamidine (dmf) group on guanine (N²). The chemistry and methods described here pave the way to the fabrication of microarrays of immobilized RNA probes for analyzing molecular interactions of biological interest.