The Synthesis of Three AMP‐Analogues: N⁶‐(6‐Aminohexyl)‐adenosine 5′‐Monophosphate, N⁶‐(6‐Aminohexyl)‐adenosine 2′,5′‐Bisphosphate, and N⁶‐(6‐Aminohexyl)‐adenosine 3′,5′‐Bisphosphate and Their Application as General Ligands in Biospecific Affinity Chromatography

Abstract

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 Iy6-diaminohexane followed by resolution of the isomeric mixture on a Dowex 1-X2 column yielded N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate and N6-(6-aminohexyl)-adenosine 3',5'-bisphosphate.The inhibition of several NADP+-dependent and NAD+-dependent dehydrogenases by N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate, N6-(6-aminohexyl)-adenosine 3',5'-bisphosphate and N6-(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 N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate, whereas NAD+-dependent enzymes were not bound under the same conditions. Conversely, when N6-(6-aminohexy1)-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 NADPf-dependent enzymes from a crude Candida utilis extract by chromatography on the new biospecific adsorbent.The development during the last three years of biospecific adsorbents of the "general ligand" type to be used in affinity chromatography has proven most valuable [I-51. Ligands of this nature have, as the name implies, affinity for large groups of enzymes and therefore great versatility. The separation or purification of one particular enzyme can subsequently be achieved by application of biospecific elution.Enzymes.

Purification of glutathione reductase from erythrocytes by the use of affinity chromatography on 2′,5′‐ADP‐sepharose 4‐B

“…[9] ). However, the more well-defined matrix obtained by coupling N 6-(aminohexyl)-adenosine 2',5'-bisphosphate to Sepharose [3] (marketed under the name of 2',5'-ADP-Sepharose 4-B) seemed to be the most promising adsorbent, especially since glutathione reductase from a yeast extract had been purified 56-fold on this material [3]. The present investigation confirms this conclusion.…”

“…1858) was purchased from Pharmacia Fine Chemicals. The ligand was linked to agarose via the N ~ amino group in the adenine ring as described by Brodelius et al [3]. Fresh pig blood was obtained from a local slaughterhouse.…”

“…The present paper shows that affinity chromatography on 2',5'-ADP-Sepharose 4-B, which has been designed for purification of NADP÷-linked enzymes [3], is a very efficient step in the purification of glutathione reductase from human or porcine erythrocytes. Glucose 6-phosphate dehydrogenase (EC 1.1.1.49), which has previously been purified simultaneously with glutathione reductase [1 ], can also be purified by the same technique.…”

Isolation and characterization of the glucose-6-phosphate dehydrogenase gene of Drosophila melanogaster

“…The homogenate was centrifuged at 16 000 x g for 30 min, and the supernatant was applied to a 2', 5'-ADP agarose (PL Biochemicals) affinity column (110 x 10 mm) equilibrated with TEMG. After washing with 45 ml of TEMG, G6PD was eluted with 9.0 ml of TEMG containing 0.1 mM NADP + (Brodelius et al, 1974). The enzyme was placed on a DE-52 cellulose column (50 x 5 mM) equilibrated with TEMG and the column was washed with 10 ml of TEMG, containing 50 mM NaCl.…”

Immobilized Coenzymes in General Ligand Affinity Chromatography and Their Use as Active Coenzymes

“…The first general ligand thus tailored for immobilization and of unambiguous, defined structure was N6-(6-aminohexyl)-AMP (5,8,10,11) (compound 1 in Table 11). In subsequent studies the corresponding X6-(6-aminohexyl)-2',5'-ADP (compound 6) and N 6 -(6-aminohexyl)-3',5'-ADP (compound 8) were prepared ( 12). All these analogs are modified at the same site (i.e., position N 6 ) , the exocyclic nitrogen on the adenine moiety, and by the same spacer molecule.…”