Enzymatic synthesis of ATP analogs and their purification by reverse-phase high-performance liquid chromatography.
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.
Schobert
B
B
Department of Physiology and Biophysics, University of California, Irvine 92717, USA.
D000227:Adenine Nucleotides / Q000138:chemical synthesis / Q000302:isolation & purification; D000244:Adenosine Diphosphate / Q000032:analysis; D000249:Adenosine Monophosphate / Q000032:analysis; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000032:analysis / Q000138:chemical synthesis* / Q000302:isolation & purification; D000263:Adenylate Kinase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004985:Ethenoadenosine Triphosphate / Q000138:chemical synthesis / Q000302:isolation & purification; D005573:Formycins / Q000138:chemical synthesis / Q000302:isolation & purification; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism; D011770:Pyruvate Kinase / Q000378:metabolism; D012265:Ribonucleotides / Q000138:chemical synthesis / Q000302:isolation & purification; D014372:Tubercidin / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000302:isolation & purification
D000227:Adenine Nucleotides; D004985:Ethenoadenosine Triphosphate; D005573:Formycins; D012265:Ribonucleotides; C009609:tubercidin 5'-triphosphate; C032552:formycin triphosphate; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; D000263:Adenylate Kinase; D009703:Nucleoside-Phosphate Kinase; D014372:Tubercidin