Synthesis of the bifuncttonal dinucleotide AMP-ATP and its application in general ligand affinity chromatography

A new AMP derivative substituted with spacer arms both at position N⁶ and C8 of the adenine moiety was synthesized and immobilized to Sepharose. To the immobilized ligand was subsequently coupled C8-substituted ATP in a solid-phase synthesis fashion yielding the bifunctional general ligand AMP-ATP. This affinity material was used in the separation of two major groups of enzymes, dehydrogenases and kinases. It was found that on passage of crude homogenates obtained from mouse kidney through the affinity column, several dehydrogenases and kinases were bound, which could be eluted separately using pulses of NADH and ATP, respectively. In the fractions obtained on NADH elution, lactate dehydrogenase, malate dehydrogenase, and α-glycerol phosphate dehydrogenase were found, whereas ATP eluted 3-phosphoglyceric acid kinase, pyruvate kinase, and aldolase.