Nucleotides as nucleophiles: Reactions of nucleotides with phosphoimidazolide activated guanosine

An earlier study of the reaction of phosphoimidazolide activated nucleosides (ImpN) in aqueous phosphate buffers indicated two modes of reaction of the phosphate monoanion and dianion. The first mode is catalysis of the hydrolysis of the P-N bond in ImpN's which leads to imidazole and nucleoside 5′-monophosphate. The second represents a nucleophilic substitution of the imidazole to yield the nucleoside 5′-diphosphate. This earlier study thus served as a model for the reaction of ImpN with nucleoside monophosphates (pN) because the latter can be regarded as phosphate derivatives. In the present study we investigated the reaction of guanosine 5′-phosphate-2-methylimidazolide, 2-MeImpG, in the presence of pN (N=guanosine, adenosine and uridine) in the range 6.9 ≤ pH ≤ 7.7. We observed that pN's do act as nucleophiles to form NppG, and as general base to enhance the hydrolysis of the P-N bond in 2-MeImpG, i.e. pN show the same behavior as inorganic phosphate. The kinetic analysis yields the following rate constants for the dianion pN²⁻:k _n ^pN =0.17±0.02 M⁻¹ h⁻¹ for nucleophilic attack andk _h ^pN =0.11±0.07 M⁻¹ h⁻¹ for general base catalysis of the hydrolysis. These rate constants which are independent of the nucleobase compare withk _{p 2}=0.415 M⁻¹ h⁻¹ and $k_h^{p^2 } $ =0.217 M⁻¹ h⁻¹ for the reactions of HPO ₄ ²⁻ . In addition, this study shows that under conditions where pN presumably form stacks, the reaction mechanism remains unchanged although in quantitative terms stacked pN are somewhat less reactive. Attack by the 2′-OH and 3′-OH groups of the ribose moiety in amounts ≥1% is not observed; this is attributed to the large difference in nucleophilicity in the neutral pH range between the phosphate group and the ribose hydroxyls. This nucleophilicity rank is not altered by stacking.