Synthesis of 9-(3,4-dioxopentyl)hypoxanthine, the first arginine-directed purine derivative: an irreversible inactivator for purine nucleoside phosphorylase

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Abstract
The synthesis of 2 potential arginine-directed purine-based analogs, 6-chloro-9-(3,4-dioxypentyl)purine (6) and 9-(3,4-dioxopentyl)hypoxanthine (7), is reported. Compound 7 was extensively tested as a potential affinity label of purine nucleoside phosphorylase from human erythrocytes. Evidence that 7 reacted with the catalytic center of purine nucleoside phosphorylase includes the following: time-dependent inactivation of the enzyme by 7 was observed; a plot of the pseudo-first-order rate constant for inactivation of the enzyme vs. concentration of 7 was hyperbolic, characteristic of saturation phenomenon; substrates (Pi, arsenate, inosine) and a competitive inhibitor (formycin B) protected the enzyme from inactivation by 7. Compound 7 was 25 times more effective in inhibiting purine nucleoside phosphorylase than butanedione. Evidence that 7 modified arginine(s) includes the following: when the inactivation was performed in borate, both the rate and the extent of inactivation were enhanced compared to those of the controls run in tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer; dialysis of inactivator reversed the inactivation of Tris-HCl but not in borate buffer. All the above evidence combined with the previous demonstration that butanedione modified only arginines in purine nucleoside phosphorylases and the results presented here demonstrating the similarities in the behavior of butanedione and 7 imply that compound 7 can be called an arginine-directed affinity label for purine nucleoside phosphorylase.