SYNTHESIS AND PROPERTIES OF DIURIDINE PHOSPHATE ANALOGS CONTAINING THIO AND AMINO MODIFICATIONS

Several analogues of diuridine phosphate (UpU) were synthesized in ord er 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 Up U 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 (U psU) 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 atta ck, on the phosphorus by the 2'-oxo anion is rate-limiting in the hydr olysis 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 constan t up to pH 11.5, thus indicating an acid-catalyzed reaction. The analo gue 2'-amino-2'-deoxyuridylyl (3'-5') 5'-thio-5'-deoxyuridine (amUpsU) was readily hydrolyzed above pH 7, in contrast to the hydrolytic stab ility 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, w here protonation of the 5'-amine is rate limiting. The cleavage rates of UpsU, UpnU, and amUpsU were accelerated in the presence of Mg2+, Zn 2+, and Cd2+ ions, but a correlation with interaction between metal io n and leaving group could only be demonstrated for amUpsU. UpsU and Up nU are also substrates for RNase A with UpsU having similar Michaelis- Menten parameters to UpU. In contrast, UpnU is more rapidly degraded w ith an approximate 35-fold increase in catalytic efficiency, which is reflected purely in an increase in the value of k(cat).