Dextran-Linked Purine Nucleosides as Substrates and Inhibitors of Adenosine Deaminase

Abstract

Dextran-bound adenosine, inosine, and nebularine have been prepared by carbodiimide coupling of their 2',3'-O-(4-carboxyethyl-l-methylbutylidene) cyclic acetal derivatives to 6-aminohexyldextran or 12-aminododecanyldextran. The latter polymers were prepared by cyanogen-bromide activation of dextran T80 followed by reaction with 1,6-diaminohexane or 1 ,12-diaminododecane. A high CNBr concentration leads to high-molecular-weight material, probably due to cross-linking, accompanied by a decrease in the digestion velocity using endo-dextranase from Penicillium species (EC 3.2.1.1 1).The dextran-bound nucleosides, as well as the nucleoside 2', 3'-O-(4-ethoxycarbonyI-l -methylbutylidene) acetal derivatives, were tested as substrates and inhibitors for adenosine deaminase. The K, of the adenosine acetal ester is identical to that of adenosine which shows that acetalation does not hinder complex formation. Since the maximum velocity of deamination is decreased fourfold, the modified substrate does not fit as well as the nucleoside. The polymer-bound acetals show a 3 -8-fold increase of K, or Ki and unchanged V compared to the corresponding acetals while dextranase digestion of the support does not alter the kinetic data. This indicates that the length of the polysaccharide chain does not interfere either with the complex formation or with the catalytic activity of the modified substrate. Since the activation energies of the deamination reactions of adenosine, its acetal ester, and dextran-linked adenosine are all similar (29.8 -32.3 kJ mol-') it is concluded that no diffusion control of the enzymatic reaction results from the binding of the nucleoside acetals to dextran T80.Heterogeneous enzyme-catalysed reactions can be divided into two categories: (a) reactions between soluble substrates and enzymes embedded in biological membranes or other native complex structures ; (b) reactions between a soluble enzyme and a polymer-bound or insoluble substrate [l -61.This prompted us to immobilise substrates and inhibitors of adenosine deaminase via cyclic acetal derivatives onto soluble dextran T80. Dextrans are a group of structurally related polysaccharides (mr, lo5 -10') elaborated by various microorganisms, especially by strains of Leuconostoc. All dextrans contain linear chains of 1 .+ 6-linked a-D-glucopyranose residues as the dominant structural feature [7]. Since we have already immobilised adenosine deaminase on dextran T80 [8], we now have a complete kinetic model system in which either the enzyme or its binding partners are covalently bound to a polymer matrix.The polymer attachment of nucleosides and nucleoside antimetabolites has several implications. (a) From the pharmacological point of view, the brief plasma persistance of small substrates and inhibitors can be overcome by linking the biomolecule to a polymer such as dextran T80, which is used as a blood volume expander. High activity of the modified substrate can only be expected if the position of attachment is carefully chosen and, if possible, a stereochemica...

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