The Synthesis of Three AMP‐Analogues: N 6 ‐(6‐Aminohexyl)‐adenosine 5′‐Monophosphate, N 6 ‐(6‐Aminohexyl)‐adenosine 2′,5′‐Bisphosphate, and N 6 ‐(6‐Aminohexyl)‐adenosine 3′,5′‐Bisphosphate and Their Application as General Ligands in Biospecific Affinity Chromatography

Phosphorylation of 6‐chloropurine riboside with phosphorus oxychloride and phosphorus trichloride gave a mixture of the two isomers, 6‐chloropurine‐riboside 2′,5′‐bisphosphate and 6‐chloropurine‐riboside 3′,5′‐bisphosphate. Reaction with 1,6‐diaminohexane followed by resolution of the isomeric mixture on a Dowex 1‐X2 column yielded N 6 ‐(6‐aminohexyl)‐adenosine 2′,5′‐bisphosphate and N 6 ‐(6‐aminohexyl)‐adenosine 3′,5′‐bisphosphate. The inhibition of several NADP + ‐dependent and NAD + ‐dependent dehydrogenases by N 6 ‐(6‐aminohexyl)‐adenosine 2′,5′‐bisphosphate, N 6 ‐(6‐aminohexyl)‐adenosine 3′,5′‐bisphosphate and N 6 ‐(6‐aminohexyl)‐adenosine 5′‐monophosphate was examined. These three AMP‐analogues were attached to Sepharose 4B by the cyanogen bromide method and the binding of several NAD(P) + ‐dependent enzymes were investigated. NADP + ‐dependent enzymes were bound to Sepharose substituted with N 6 ‐(6‐aminohexyl)‐adenosine 2′,5′‐bisphosphate, whereas NAD + ‐dependent enzymes were not bound under the same conditions. Conversely, when N 6 ‐(6‐aminohexyl)‐adenosine 5′‐monophosphate was used as the immobilised ligand only the NAD + ‐dependent enzymes were bound, as well as glucose‐6‐phosphate dehydrogenase showing weak affinity. These observations strongly suggest that these two immobilised analogues represent true biospecific and group‐specific adsorbents. The enzymes were eluted with their complementary nucleotides, NAD(H) and NADP(H). These techniques were utilised to purify several NADP + ‐dependent enzymes from a crude Candida utilis extract by chromatography on the new biospecific adsorbent.