Synthesis and Properties of Diuridine Phosphate Analogues Containing Thio and Amino Modifications

Publisher Website
Google Scholar
Abstract
Several analogues of diuridine phosphate (UpU) were synthesized in order to investigate why replacing the 2'-hydroxyl with a 2'-amino group prevents hydrolysis. These analogues were designed to investigate what influence the 2'-substituent and 5'-leaving group have upon the rate of hydrolysis. All the analogues were considerably more labile than UpU toward acid-base-catalyzed hydrolysis. In the pH region from 6 to 9, the rate of hydrolysis of uridylyl (3'-5') 5'-thio-5'-deoxyuridine (UpsU) hydrolysis rose, in a log linear fashion, from a value of 5 x 10(-)(6) s(-)(1) at pH 6 to 3200 x 10(-)(6) s(-)(1) at pH 9, indicating that attack on the phosphorus by the 2'-oxo anion is rate-limiting in the hydrolysis mechanism. In contrast, the rate of uridylyl (3'-5') 5'-amino-5'-deoxyuridine (UpnU) hydrolysis fell from a value of 1802 x 10(-)(6) s(-)(1) at pH 5 to 140 x 10(-)(6) s(-)(1) at pH 7.5, where it remained constant up to pH 11.5, thus indicating an acid-catalyzed reaction. The analogue 2'-amino-2'-deoxyuridylyl (3'-5') 5'-thio-5'-deoxyuridine (amUpsU) was readily hydrolyzed above pH 7, in contrast to the hydrolytic stability of amUpT, with rates between 85 x 10(-)(6) s(-)(1) and 138 x 10(-)(6) s(-)(1). The hydrolysis of 2'-amino-2'-deoxyuridylyl (3'-5') 5'-amino-5'-deoxythymidine (amUpnT) rose from 17 x 10(-)(6) s(-)(1) at pH 11.5 to 11 685 x 10(-)(6) s(-)(1) at pH 7.0, indicating an acid-catalyzed reaction, where protonation of the 5'-amine is rate limiting. The cleavage rates of UpsU, UpnU, and amUpsU were accelerated in the presence of Mg(2+), Zn(2+), and Cd(2+) ions, but a correlation with interaction between metal ion and leaving group could only be demonstrated for amUpsU. UpsU and UpnU are also substrates for RNase A with UpsU having similar Michaelis-Menten parameters to UpU. In contrast, UpnU is more rapidly degraded with an approximate 35-fold increase in catalytic efficiency, which is reflected purely in an increase in the value of k(cat).