The Synthesis of Several 8‐Substituted Derivatives of Adenosine 5′‐Monophosphate to Study the Effect of the Nature of the Spacer Arm in Affinity Chromatography

Abstract

Reaction of adenosine 5′‐monophosphate (Ado‐5′‐P) with bromine at pH 4.0 yielded 8‐bromoadenosine 5′‐monophosphate and following reaction with several α,ω‐diaminoalkanes gave the corresponding 8‐(ω‐aminoalkyl)‐Ado‐5′‐P derivatives. Condensation of these analogues with N‐trifluoroacetyl‐glycine or β‐alanine in the presence of a water‐soluble carbodiimide generated several 8‐substituted derivatives. These analogues: (Formula Presented.) comprised an 8‐substituted Ado‐5′‐P ligand to which a spacer molecule of similar length but differing hydrophobicity was attached. The derivatives were purified, characterised and attached to CNBr‐activated Sepharose. The chromatographic behaviour of the resulting adsorbents was investigated in terms of their ability to bind both lactate and alanine dehydrogenase. The enzymes bound tighter to the more hydrophobic derivatives with the strength of the interaction decreasing with increasing hydrophilicity. The effect of the introduction of a single hydroxyl group in the spacer arm was also studied with several enzymes known to exhibit anomalous behaviour on affinity chromatography. In each case binding was weaker to the derivative bearing the more hydrophilic spacer arm. In free solution, were competitive with NADH for rabbit muscle lactate dehydrogenase, with Ki values of 2.0 and 1.8 mM respectively. These data suggest a difference in accessibility of the hydrophobic and hydrophilic derivatives when attached to Sepharose rather than a fundamental difference in affinity. This suggestion is supported by the fact that there is no obvious correlation between the Ki values in free solution of a series of 8‐(ω‐aminoalkyl)‐Ado‐5′‐P derivatives of increasing chain length, and hence hydrophobicity, and their chromatographic behaviour when immobilised. The data suggest that the hydrophobicity/hydrophilicity of the spacer arm determine the accessibility of the immobilised ligand to interaction with the complementary enzyme. Copyright © 1977, Wiley Blackwell. All rights reserved