Isolation and purification of deoxyribonucleosides from 90% ¹³C-enriched DNA of algal cells and their characterization by ¹H and ¹³C NMR

¹³C-enriched deoxyribonucleosides have been isolated from the DNA of Algal cells grown in an atmosphere of 90% ¹³C-labelled carbon dioxide. The ¹³ enriched DNA was quantitatively hydrolysed with DNase I, snake venom phosphodiesterase I and alkaline phosphatase of intestinal mucosa. The resulting deoxyribonucleosides were separated by preparative reversed-phase high pressure liquid chromatography in 60 minutes with detection by ultraviolet absorption at 254 nm. The final products were obtained in milligram quantities in high purity and in high yield. The ¹H resonances of the base and sugar protons of these deoxyribonucleosides appear as well resolved multiplets in the 600 MHz NMR spectrum, due to the extensive ¹H-¹³C couplings. Similarly, the ¹³C resonances of these deoxyribonucleosides appear as multiplets in the 75.5 MHz ¹³C NMR spectrum, due to¹³C-¹³C couplings. The ¹H-¹³C and ¹³C-¹³C coupling constants were also measured and tabulated. The isotopic enrichment of ¹³C these deoxyribonucleosides was obtained by integration of the ¹H and/or ¹³C NMR spectra. It was found that the enrichment varied from carbon to carbon and species to species in the range of 70-89%, suggesting differential uptake and assimilation of 90% ¹³CO₂ during metabolism pathways. This protocol provides experimentally useful quantities of ¹³C-enriched deoxyribonucleosides, which may be incorporated into site-specifically labeled oligonucleotides by chemical synthesis.