ABSTRACT
Normal phase HPLC and OPTLC methods are described for the analysis of Hevizos (5-isopropyl-2'-beta-deoxyuridine) and it's impurities. The eluent is 50% water saturated ethyl-acetate to which 0.5-1.5% methanol is added depending on the silica used. The water content is important as it practically eliminates tailing. The methods can also be used for the determination of 5-isopropyl-2'-beta-deoxyuridine and it's impurities in Hevizos ointment after a simple extraction procedure. The OPTLC method uses a CHROMPRES 10 apparatus, aluminum backed HPTLC plates and the same eluent-system as the HPLC method. The reproducibility of the HPLC method (rel. st. dev.) is 0.8%, the correlation coefficient is > 0.999 for the main component, while the impurities can be determined at the 0.1% level with a reproducibility of 20%, and a correlation coefficient of > 0.997. The reproducibility of the OPTLC method (rel. st. dev.) is 4% for the main component, while for the impurities at the 0.5% level the reproducibility is 20%.
Subject(s)
Antiviral Agents/analysis , Deoxyuridine/analogs & derivatives , Antiviral Agents/isolation & purification , Chromatography, High Pressure Liquid/methods , Deoxyuridine/analysis , Deoxyuridine/isolation & purification , Indicators and ReagentsABSTRACT
The carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine, C-BVDU, is a very potent and selective anti-herpes-virus compound. In order to synthesize and study the properties of a DNA that contains C-BVDU, the 5'-triphosphate, C-BVDUTP was prepared and evaluated as a potential substrate of the E. coli Klenow DNA polymerase enzyme. Although C-BVDUTP proved to be a very poor substrate also of this enzyme, it could be incorporated up to 3.6% into the synthetic DNA, poly(dA-dT, C-BVDU). This level of substitution decreased significantly the template activity for DNA and RNA polymerases, as compared to that of poly(dA-dT).
Subject(s)
Bromodeoxyuridine/analogs & derivatives , DNA Polymerase I/metabolism , DNA/biosynthesis , Deoxyuracil Nucleotides/metabolism , Chromatography, High Pressure Liquid , Nucleic Acid Conformation , Nucleic Acid Denaturation , Peptide Fragments , Spectrum Analysis , Substrate Specificity , Templates, Genetic , Thymine Nucleotides/metabolismABSTRACT
Reversed-phase and reversed-phase ion-pair chromatographic methods were used to determine 5-alkyluracils in the presence of purine bases (mainly adenine) in the hydrolysates of enzymatically synthesized DNA. The effect of ionic strength, pH, concentration of the ion-pairing agent and methanol on the selectivity between alkyluracils and purine bases was examined in order to simplify the routine work and to reduce the time necessary for analysis. Optimal conditions could be developed for the isocratic separation of the various mixtures obtained by hydrolysis of the products of enzymatic synthesis.
