Intramolecular Participation of Amino Groups in the Cleavage and Isomerization of Ribonucleoside 3′‐Phosphodiesters: The Role in Stabilization of the Phosphorane Intermediate

Abstract

A dinucleoside-3',5'-phosphodiester model, 5'-amino-4'-aminomethyl-5'-deoxyuridylyl-3',5'-thymidine, incorporating two aminomethyl functions in the 4'-position of the 3'-linked nucleoside has been prepared and its hydrolytic reactions studied over a wide pH range. The amino functions were found to accelerate the cleavage and isomerization of the phosphodiester linkage in both protonated and neutral form. When present in protonated form, the cleavage of the 3',5'-phosphodiester linkage and its isomerization to a 2',5'-linkage are pH-independent and 50-80 times as fast as the corresponding reactions of uridylyl-3',5'-uridine (3',5'-UpU). The cleavage of the resulting 2',5'-isomer is also accelerated, albeit less than with the 3',5'-isomer, whereas isomerization back to the 3',5'-diester is not enhanced. When the amino groups are deprotonated, the cleavage reactions of both isomers are again pH-independent and up to 1000-fold faster than the pH-independent cleavage of UpU. Interestingly, the 2'- to 3'-isomerization is now much faster than its reverse reaction. The mechanisms of these reactions are discussed. The rate accelerations are largely accounted for by electrostatic and hydrogen-bonding interactions of the protonated amino groups with the phosphorane intermediate.

Phosphodiester models for cleavage of nucleic acids

“…The possible role of the lysine ε-amino group in the catalytic center of RNase A has been elucidated by incorporating an amino group covalently in the vicinity of the scissile phosphodiester linkage of the model compound. For this purpose, compound 12a bearing two aminomethyl groups at C4´ was prepared and its reactions were compared to the reactions of UpU [ 86 ] and 4´-hydoxymethyl-UpT ( 12b ) [ 87 ]. The p K a values for the mono- and diammonium ions of 12a were determined to be 7.2 and 5.8, respectively.…”

“…At pH > 9, the cleavage of 12a is hydroxide-ion-catalyzed and as fast as the respective reaction of UpU and 12b . Over a narrow pH range 7.5–8.5, where both amino groups still are deprotonated, the behavior of 12a , however, differs from that of UpU or 12b ; another pH-independent cleavage occurs [ 86 ]. This reaction is one order of magnitude faster than the pH-independent cleavage of 12a at pH 3–5, i.e., when both amino groups are protonated.…”

“… Mechanism for the pH-independent cleavage and isomerization of model compound 12a in the pH-range 7.5–8.5 [ 86 ]. …”

Participation of an additional 4′-hydroxymethyl group in the cleavage and isomerization of ribonucleoside 3′-phosphodiesters

“…20 The free hydroxy functions of 5 were first protected as 4,4′dimethoxytrityl ethers, giving 6. Transformation of uridine into 4′hydroxymethyl-2′,3′-bis(tert-butyldimethylsilyl)uridine (5), used as the starting material, has been previously described.…”

“…20 In those cases where significant buffer catalysis was observed (viz. For the first 8 min, isocratic elution with 100% buffer was used, followed by a linear acetonitrile gradient from 0 to 30% during 22 min.…”