RESUMEN
The enzymatic syntheses of ATP analogs, such as tubercidin 5'-triphosphate, formycin A 5'-triphosphate, and etheno-ATP, from their respective mono- and diphosphate are described. The reaction products were purified by reverse-phase HPLC using a C-18 matrix and a volatile mobile phase at pH 7, with tributylamine as the ion-pairing agent. Each of the analogs required a buffer of somewhat different composition for the baseline separation of reaction product and reactants. The elutions were isocratic and allowed several successive runs without any intermediate equilibration of the column. After freeze-drying of the pooled fractions, the yield of the synthesized nucleoside triphosphate was approximately 70%. The described procedures are applicable either for analytical investigations or for semi-preparative purposes.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/síntesis química , Nucleótidos de Adenina/síntesis química , Nucleótidos de Adenina/aislamiento & purificación , Adenosina Difosfato/análisis , Adenosina Monofosfato/análisis , Adenosina Trifosfato/análisis , Adenosina Trifosfato/aislamiento & purificación , Adenilato Quinasa/metabolismo , Cromatografía Líquida de Alta Presión , Etenoadenosina Trifosfato/síntesis química , Etenoadenosina Trifosfato/aislamiento & purificación , Formicinas/síntesis química , Formicinas/aislamiento & purificación , Nucleósido-Fosfato Quinasa/metabolismo , Piruvato Quinasa/metabolismo , Ribonucleótidos/síntesis química , Ribonucleótidos/aislamiento & purificación , Tubercidina/análogos & derivados , Tubercidina/síntesis química , Tubercidina/aislamiento & purificaciónRESUMEN
Cellular extraction and high-performance liquid chromatographic methods were developed for the isolation of etheno adducts from nucleotide pools formed in vivo following exposure to the chemical carcinogen ethyl carbamate. These techniques were employed to detect etheno adduct formation using BDF1 mice and rainbow trout (Salmo gairdneri) as test species following inter-peritoneal injection of the chemical. Ethenoadenine was detected in splenocyte nucleotide pools of mice after acute (24 h) exposure and chronic (two weeks) exposure. Several etheno adducts (i.e. ethenoadenine, etheno-AMP, etheno-ADP and etheno-ATP) were also detected in total spleen cell nucleotide pools of trout following acute ethyl carbamate exposure.
Asunto(s)
Adenosina Difosfato/análogos & derivados , Carcinógenos/farmacología , Nucleósidos/aislamiento & purificación , Uretano/farmacología , Adenina/análogos & derivados , Adenina/aislamiento & purificación , Adenosina Difosfato/aislamiento & purificación , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/aislamiento & purificación , Animales , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Etenoadenosina Trifosfato/aislamiento & purificación , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos , Bazo/efectos de los fármacos , Bazo/metabolismo , TruchaRESUMEN
This paper describes techniques and strategies for semi-preparative high-performance liquid chromatographic (HPLC) purification of 2'-deoxynucleoside 5'-triphosphates (dNTPs). The procedure yields dNTPs that are sufficiently pure for use in a sensitive electrophoretic assay of misincorporation during DNA synthesis. Anion-exchange HPLC was used to purify the four normal dNTPs (dATP, dGTP, dCTP and dTTP), plus the chemically modified analogues, 5-BrdUTP, 5-IodUTP and 1,N6-etheno-dATP (epsilon dATP). Baseline separations were achieved by isocratic elution of dNTPs with potassium dihydrogen phosphate mobile phase. In general, the resolution of dNTPs was highly dependent on pH, although the influence of mobile phase composition on separation of dNTPs was not the same for all three HPLC packing materials used. A Hewlett-Packard diode array detector was extremely valuable in the identification of contaminating peaks and in the development of optimal mobile phase conditions for dNTP purification. The pure dNTPs were used in the electrophoretic assay of misincorporation, yielding information about the mispairing potential of the modified dNTPs. BrdUMP and IodUMP were misincorporated in place of dCMP during chain elongation catalyzed by purified DNA polymerase I of Escherichia coli. epsilon dAMP was incorporated into DNA in place of dAMP, although at much lower efficiency than dAMP.