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 @,coo-diaminoalkanes gave the corresponding 8-(co-aminoalkyl)-Ado-5'-P derivatives. Condensation of these analogues with N-trifluoroacetyl-glycine or /I-alanine in the presence of a water-soluble carbodiimide generated several 8-substituted derivatives. These analogues : I OH comprised an %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 CNBractivated 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, (Ado-5'-P)-8-NH CH2CH2CH2CH2CH2CH2NH2 and (Ado-5'-P)-S-NH CH2-CH CH2NH CO CHzNH2 were competitive with NADH for rabbit muscle lactate dehydrogenase, OHwith 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-(w-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.

Affinity Chromatography on Immobilised Nucleotides

“…The column was washed with water (2 1) to remove unreacted 1,6-diaminohexane until the effluent was negative to the trinitrobenzene sulphonate test [14,18]. No ultraviolet-absorbing material was found in the washings.…”

“…cm-' at 250 nm and Base-Substituted AMP Ligands (Fig. 1 C, D ) The synthesis and characterisation of N6-(6-aminohexy1)-adenosine 5'-monophosphate (Ahx6AMP) and 8-(6-aminohexyl)-adenosine 5'-monophosphate (Ahx*-AMP) was performed as described previously [14,18]. In each case, the diaminoalkane 1,6-diaminohexane, was purified by distillation under reduced pressure prior to use.…”

“…Nucleotide derivatives were localised by ultraviolet light (254 nm) and free amino groups visualised with a ninhydrin spray (BDH Chemicals Ltd) [18]. Ultraviolet spectra were recorded on a Unicam SP1800 spectrophotometer.…”

“…Ultraviolet spectra were recorded on a Unicam SP1800 spectrophotometer. Phosphate and amino group determinations were performed as described previously [14,18]. 'H-NMR spectra were obtained with a Perkin-Elmer R-12B NMR spectrometer operating at 60 MHz or a Varian XL100-12 operating at 100 MHz.…”

“…The gels were tumbled gently overnight at 0 -4 "C and washed with water (50 ml), water (500 ml), 1 M NaCl (500 ml), 2 M NaCl (500 ml) and finally water (800 ml). Ligand concentrations were determined as described by Lowe [18]. The immobilised nucleotide stock gels were diluted with appropriate amounts of unmodified Sepharose 4B in order to equalise final ligand concentrations.…”