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 RNA–ligand 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 N2 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.Graphic abstract
Highlights
The nucleobase analog 2-aminopurine (Ap) is widely used as minimally invasive fluorescent reporter in RNA for the investigation of folding and ligand–RNA interactions [1,2,3,4,5]
The exocyclic 2-amino function was selectively protected by treatment with N,N-dibutylformamide dimethyl acetal (DBFDMA) [25,26,27,28,29] producing nucleoside derivative 2
The 5′ and 3′ hydroxyl groups were simultaneously protected by reaction with di-tert-butylsilyl bis(trifluoromethanesulfonate) ((tBu)2Si(OTf)2) [30, 31], followed by silylation of the 2′-hydroxyl group with tert-butyldimethylsilyl chloride (TBDMSCl) to give compound 3
Summary
The nucleobase analog 2-aminopurine (Ap) is widely used as minimally invasive fluorescent reporter in RNA for the investigation of folding and ligand–RNA interactions [1,2,3,4,5]. Several reports on the synthesis of 2-aminopurine 2′-deoxyriboside phosphoramidite and its incorporation into DNA oligomers followed.
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