Preparation of adenosine nucleotide derivatives suitable for affinity chromatography

Abstract

Methods of synthesizing a series of chemically-defined AMP, ADP, ATP, adenylyl imidodiphosphate and pyrophosphate derivatives suitable for affinity chromatography are extensively described. Each derivative has a single primary amino group at the end of a hexamethylene ;spacer' chain for attachment to CNBr-activated agarose. The synthesis of the derivative where the ;spacer' arm is attached directly to the 8 position of the adenine ring to produce 8-(6-aminohexyl)amino-AMP involves the direct bromination of AMP in the 8 position followed by displacement of the halogen by 1,6-diaminohexane. This monophosphate derivative can then be converted into the corresponding di- or triphosphate forms by direct phosphate condensation with carbonyl di-imidazole. A second series of adenosine phosphate derivatives with the phosphate moieties unsubstituted has been similarly prepared from N(6)-(6-aminohexyl)-AMP (Guilford et al., 1972). A third type of ligand has been synthesized by condensing the phosphoryl imidazolide of AMP with 6-aminohex-1-yl phosphate. This compound, P(1)-(6-aminohex-1-yl) P(2)-(5'-adenosyl) pyrophosphate, has an unsubstituted adenine ring. The synthesis of a fourth type of ligand, 6-aminohex-1-yl pyrophosphate, was done by heating 6-aminohexan-1-ol with crystalline pyrophosphoric acid under reduced pressure. The structures of the synthesized compounds were confirmed by chemical, electrophoretic and chromatographic methods and by u.v. spectrometry. The general applicability of the synthetic methods used is discussed in relation to the preparation of other affinity adsorbents. Examples are given where these derivatives have been successful in reversibly binding dehydrogenases, kinases and myosin and its proteolytic subfragments. The partial purification of rat liver glucokinase on an ADP derivative is shown.

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

“…1. In principle, Ado-5'-P is brominated in the %position, followed by nucleophilic displacement with suitable diaminoalkanes [22,23,27] and subsequent coupling of an N-protected amino acid to the terminal amino group by a carbodiimide-promoted reaction. The method is facile and, in general, proceeds with an estimated overall yield of about 40% based on the parent nucleotide,…”

“…1) and the properties of the intermediates and products all support the structure assigned. The shift in ultraviolet absorption maximum from 262.5 nm for brsAdo-5'-P to 279 nm on reaction with diaminoalkanes [22,23,27] The compounds showed the expected behaviour on thin-layer chromatography in several systems and gave the anticipated reaction to ultraviolet and ninhydrin.…”

“…The synthesis of br8Ado-5'-P was based on the methods of Ikehara and Uesugi [22] and Trayer et a f . [23]. NazAdo-5'-P(1) (4.5 g, 10 mmol) was dissolved in 1 M sodium acetate buffer pH 4.0 (420 ml) and bromine/water (0.87 ml bromine, 17 mmol, dissolved in 87 ml water with vigorous shaking) added.…”

Binding of ATP to the progesterone receptor.

“…In most cases, the immobilized nucleotides have been used as a step in enzyme purification (8,(20)(21)(22)(23)(24)(25). They have also been used as characterization tools for studies such as the phosphorylation of succinyl CoA synthetase (24) and the interaction between myosin species and ATP (25).…”

A new and rapid method for the isolation of myosin from small amounts of muscle and non‐muscle tissue by affinity chromatography

“…Preliminary experiments in which rabbit muscle myosin, purified by conventional techniques, was applied directly to Sepharose derivatives of either C8-ADP or N6 -ADP indicated: (a) that this enzyme could be bound quite tightly to these columns in the presence of either Mg", Ca2+ or in the absence of divalent cations (in the presence of EDTA) at the high ionic strength required for its solubility; and (b) that buffers containing high concentrations of Cl-inhibited this binding [7]. The final buffer chosen for the chromatography work therefore contained 0.6 M potassium or ammonium acetate in order to solubilize the myosin.…”