Phosphorane intermediate vs. leaving group stabilization by intramolecular hydrogen bonding in the cleavage of trinucleoside monophosphates: implications for understanding catalysis by the large ribozymes

Abstract

Hydrolysis of 2′,3′-O-methyleneadenosin-5′-yl 5′-O-methyluridin-2′-yl 5′-O-methyl-2′-trifluoroacetamido-2′-deoxyuridin-3′-yl phosphate (1b) has been followed by HPLC over a wide pH range to study the effects of potential hydrogen bonding interactions of the 2′-trifluoroacetamido function on the rate and product distribution of the reaction. At pH < 2, decomposition of 1b (and its 3′,3′,5′-isomer 1a) is first-order in hydronium-ion concentration and cleavage of the P–O3′ bond of the 2′-trifluoroacetamido-modified nucleoside is slightly favored over cleavage of the P–O5′ bond. Between pH 2 and 4, the overall hydrolysis is pH-independent and the P–O3′ and P–O5′ bonds are cleaved at comparable rates. At pH 5, the reaction becomes first-order in hydroxide-ion concentration, with P–O3′ bond cleavage predominating. At 10 mmol L⁻¹ aqueous sodium hydroxide, no P–O5′ bond cleavage is observed. Compared to the 2′-OH counterpart 2, a modest rate enhancement is observed over the entire pH range studied. The absence of P–O5′ fission under alkaline conditions suggests hydrogen bond stabilization of the departing 3′-oxyanion by the neighboring 2′-trifluoroacetamido function.