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

• Published in: Journal of organic chemistry Vol. 61; no. 18; pp. 6273 - 6281
• Main Authors: Thomson, James B, Patel, Bhisma K, Jiménez, Victor, Eckart, Klaus, Eckstein, Fritz
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 06.09.1996; Amer Chemical Soc
• Subjects: Chemistry; Chemistry, Organic; Physical Sciences; Science & Technology; CATALYSIS; RNA; MECHANISM; 3-DIMENSIONAL MODEL; STABILITY; HAMMERHEAD RIBOZYME; ESTERS; HYDROLYSIS; TERTIARY STRUCTURE; METAL-IONS
• ISSN: 0022-3263; 1520-6904
• DOI: 10.1021/jo960795l

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 × 10-6 s-1 at pH 6 to 3200 × 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 × 10-6 s-1 at pH 5 to 140 × 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 × 10-6 s-1 and 138 × 10-6 s-1. The hydrolysis of 2‘-amino-2‘-deoxyuridylyl (3‘−5‘) 5‘-amino-5‘-deoxythymidine (amUpnT) rose from 17 × 10-6 s-1 at pH 11.5 to 11 685 × 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 Mg2+, Zn2+, and Cd2+ 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.