Uridine dihydrouridine

If polyribonucleotides are treated simultaneously with methoxylamine and bisulphite, cytidine residues are converted into 5,6-dihydro-7V4-methoxycytidine-6-sulphonate,154 and uridine into 5,6-dihydrouridine-6-sulphonate.155 Treatment with dilute ammonia regenerates the uridine residues, leaving the dihydrocytidine derivatives unaffected. When only the cytidine residues have been derivatized, pancreatic ribonuclease becomes uridyl ribonuclease, since it is unable to cleave the chain on the 3 -side of the modified cytidine.154 This allows the isolation of blocks of modified cytidine residues. T2 ribonuclease may also be used. Alternatively, a ribonuclease from Physarum polycephalum has been found to hydrolyse CpX links very slowly, allowing the isolation of cytidine blocks.156 If both uridine and cytidine residues are modified, T2 ribonuclease acts as puryl ribonuclease, allowing the isolation of cumulative blocks of pyrimidines.155 This ability to alter the specificity of nuclease cleavage is a useful tool in sequence analysis. 

Pulse radiolysis experiments have shown that "OH radical adds preferentially at C5 of the uracil moiety, giving rise to the reducing 5-hydroxy-5,6-uracil-6-yl radical. Interestingly, the two cis diastereomers of 6-hydroperoxy-5-hydroxy-5,6-dihydrouridine, two of the expected final products of the latter radicals in aerated aqueous solutions, have been prepared by trifluoroacetic acid treatment of uridine (3, R = H, = ribose) in the presence of H202 (equation 14). The mechanism of the reaction that involves transient formation of an epoxide-type intermediate followed by nucleophilic attack by a perhy-droxyl group at C6 presents similarities with the substitution of thymine bromohydrin by... 

Hydrogenation of uridine 5 -(a-D-glucopyranosyl pyrophosphate) over rhodium on alumina resulted in the 5,6-dihydrouridine derivative229,324 (75). Several modifications in the heterocyclic base of adenosine 5 -(a-D-glucopyranosyl pyrophosphate) have been described. 

The effect, on substrate properties, of structural changes in the nucleoside residue, as studied with uridine 5 -(a-D-glucopyranosyl pyrophosphate) 4"-epimerases from liver339,364,394 and mung bean,364,377 is qualitatively similar to that just discussed for uridine 5 -(a-D-glu-copyranosyl pyrophosphate) dehydrogenase. The enzymes tolerate various substitutions at C-6 and C-5 (such as those resulting in derivatives of 5,6-dihydrouridine, 6-azauridine, and 5-methyluridine)... 

Transfer RNA molecules are notable for containing unusual nucleotides (Fig. 5) such as 1-methylguanosine (m G), pseudouridine OF), dihydrouridine (D), inosine (I) and 4-thiouridine (S4U). These are created by modification of guano-sine and uridine after tRNA synthesis. For example, inosine is generated by deamination of guanosine. 

Levene and LaForge,1 in 1912, made the interesting observation that the nucleoside uridine I, which is obtained by hydrolysis of nucleic acid, is reduced practically quantitatively to dihydrouridine II by PaaTs2 method of catalytic reduction, namely, by means of colloidal palladium and hydrogen. This transformation involves an addition of hydrogen at the double bond joining positions 4 and 5 in the uracil nucleus and the change is represented as follows ... 

As far as the writers are aware, this is the first and only case described in the literature of the application of a catalytic process of reduction in the pyrimidine series. A striking fact revealed by the work of these investigators is the remarkable ease with which the sugar can be detached from this dihydrouridine molecule II by hydrolysis with acids. The corresponding uridine combination I is very stable and resistant to hydrolysis. 

The ring structure of the ribose residue was ascertained" in the same general manner as for adenosine and guanosine. Triacetyl-dihydrouridine was prepared by the hydrogenation of triacetyl-uridine. On simultaneous deacetylation and methylation this was transformed to the fully methylated dihydro-uridine. By simultaneous hydrolysis and oxidation of this product, with hydrobromic acid and bromine, trimethyl -D-ribonolactone was formed, its identity being confirmed by oxidation to meso-dimethoxy-succinic acid. It follows that the ribose component has the furanose ring structure, and that uridine is 3 -D-ribofuranosyl-uracil. 

Posttranscription modification of tRNA The synthesis of tRNA involves modification of some uridine nucleotides to unusual nucleotides, such as pseudouridine, ribothymidine, and dihydrouridine. 

Most tRNAs are 7495 nucleotides in length, and differ by the number of nucleotides in the variable loop. The TyC loop is named for the presence of an invariant pseudo-uridine base modification (y) and the D loop contains several dihydrouridine residues. 

Dihydrouridine is an abundant modified nucleoside found in tRNA predominantly on the D-loop. DUSs, flavin-containing proteins that catalyze the conversion of uridine to dihydrouridine on tRNA, have been... 

Little mechanistic work has been published on these enzymes to date. The four DUS enzymes in S. cerevisiae each show distinct site specificity on tRNA, with each enzyme reducing either one or two specific uridines in tRNA. An in vivo DUS-complementation assay has been developed to screen for residues important in catalysis in which an E. coli strain that has all the DUS genes knocked out is complemented with a plasmid-borne DUS and the dihydrouridine content of the tRNA is then determined. This approach was used to identity a cysteine residue that is essential for DUS activity. Mapping this cysteine onto the crystal structure of a DUS from Thermatoga maritimc shows that it is positioned in the active site to act as a catalytic acid. Presumably, the mechanism of uridine reduction involves hydride transfer from N5 of the flavin to C6 of uridine and protonation of C5 of uridine by an active site acid. The actual substrate of DUSs has not been identified. The enzymes reduce in transcribed tRNAs several orders of magnitude slower than naturally modified tRNA. ° The critical modification for DUS activity has not yet been identified. 

Figure 29-2. Sequence and projection of the conformation of the phenyl alanine specific f-RNAP " of yeast. D, Dihydrouridine DiMeG, 2,2-dimethylguanosine 2 OMeC, 2 methyl cytidine ij/, pseudo-uridine (base bonded to the sugar group via C ).

Nucleosides, Nucleotides, Derivatives, and Related Compounds. — 5-Substituted uracils were examined for conformational dependence on the substituents, uridine 5 -phosphate disodium salt, 2 -deoxyuridine 5 -phosphate disodium salt, " 6-methyl-2 -deoxyuridine, 5-C-acetyl-2 -deoxyuridine, dihydrouridine 3 -phosphate potassium salt, 5-hydroxymethyl-2 -deoxyuridine, 4-thio--uridine, 2,5 -anhydro-2, 3 -0-isopropylidene-2-thiouracil, 2,2 -an-hydro-1 - 3-D-arabinofuranosyl-2-thiouracil. ... 

A model for the major uridine intermediate generated by attack of hydroxyl radical, the 5,6-dihydrouridin-6-yl radical, has been selectively generated from a ketone precursor (5) via Norrish type I photocleavage in a dinucleotide, single-stranded, and double-stranded RNA. ... 

SYNTHESIS OF 5-SUBSTITUTED URIDINES via THE AMIDE a-ANION OF 5,6-DIHYDROURIDINE... 

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