Crystal Structures, Reactivity and Inferred Acylation Transition States for 2‘-Amine Substituted RNA

Ribose 2‘-amine substitutions are broadly useful as structural probes in nucleic acids. In addition,
structure-selective chemical reaction at 2‘-amine groups is a robust technology for interrogating local
nucleotide flexibility and conformational changes in RNA and DNA. We analyzed crystal structures for
several RNA duplexes containing 2‘-amino cytidine (CN) residues that form either CN−G base pairs or
CN−A mismatches. The 2‘-amine substitution is readily accommodated in an A-form RNA helix and thus
differs from the C2‘-endo conformation observed for free nucleosides. The 2‘-amide product structure was
visualized directly by acylating a CN−A mismatch in intact crystals and is also compatible with A-form
geometry. To visualize conformations able to facilitate formation of the amide-forming transition state, in
which the amine nucleophile carries a positive partial charge, we analyzed crystals of the CN−A duplex at
pH 5, where the 2‘-amine is protonated. The protonated amine moves to form a strong electrostatic
interaction with the 3‘-phosphodiester. Taken together with solution-phase experiments, 2‘-amine acylation
is likely facilitated by either of two transition states, both involving precise positioning of the adjacent 3‘-phosphodiester group.