Schobert B
Department of Physiology and Biophysics, University of California, Irvine 92717, USA.
Anal Biochem. 1995 Apr 10;226(2):288-92. doi: 10.1006/abio.1995.1227.
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.
描述了从各自的单磷酸酯和二磷酸酯酶促合成ATP类似物，如结核菌素5'-三磷酸、间型霉素A 5'-三磷酸和乙烯基-ATP的方法。反应产物通过反相高效液相色谱法进行纯化，使用C-18基质和pH值为7的挥发性流动相，以三丁胺作为离子对试剂。每种类似物都需要一种组成略有不同的缓冲液来实现反应产物和反应物的基线分离。洗脱采用等度洗脱，允许进行几次连续运行，而无需对色谱柱进行任何中间平衡。合并馏分冷冻干燥后，合成的核苷三磷酸的产率约为70%。所述方法适用于分析研究或半制备目的。
