Practical synthesis of N-(di-n-butylamino)methylene-protected 2-aminopurine riboside phosphoramidite for RNA solid-phase synthesis

Abstract

2-Aminopurine (Ap) is a fluorescent nucleobase analog that is frequently used as structure-sensitive reporter to study the chemical and biophysical properties of nucleic acids. In particular, thermodynamics and kinetics of RNA folding and RNAligand binding, as well as RNA catalytic activity are addressable by pursuing the Ap fluorescence signal in response to external stimuli. Site-specific incorporation of Ap into RNA is usually achieved by RNA solid-phase synthesis and requires appropriately functionalized Ap riboside building blocks. Here, we introduce a robust synthetic path toward a 2-aminopurine riboside phosphoramidite whose N 2 functionality is masked with the N-(di-n-butylamino)methylene group. This protection is considered advantageous over previously described N-(dimethylamino)methylene or acyl protection patterns needed for the fine-tuned deprotection conditions to achieve large synthetic RNAs.

Synthesis of N4-acetylated 3-methylcytidine phosphoramidites for RNA solid-phase synthesis

“…This problem can usually be circumvented if N 4 -acetyl instead of benzoyl protection is applied [ 22 , 23 ]. Moreover, for 2′-O protection we envisaged both 2′- O -TBDMS (to satisfy the most widespread concept of commercially available nucleoside building blocks) [ 24 – 27 ] and 2′- O -TOM protection [ 28 – 31 ]. The latter is conducive for the solid-phase synthesis of long RNAs (> 50–60 nt) due to reduced steric hindrance during the coupling step and hence very high coupling yields [ 28 , 29 ].…”