Enzymatic Synthesis of 6 R-[U- 14C]Tetrahydrobiopterin from [U- 14C]GTP

Abstract

An enzymatic method for preparing 6 R-[U- 14C]tetrahydrobiopterin from [U- 14C]GTP is presented. This method utilizes purified preparations of three biosynthetic enzymes for 6 R-tetrahydrobiopterin, i.e., Escherichia coli GTP cyclohydrolase I, rat 6-pyruvoyltetrahydropterin synthase, and rat sepiapterin reductase. Based on the catalytic properties of these enzymes, the reaction conditions were optimized to complete the entire conversion reaction from GTP to 6 R-tetrahydrobiopterin in a single reaction solution without the need to isolate unstable intermediates after each enzymatic reaction. The reaction conditions thereby established yielded [U- 14C]biopterin in an amount equivalent to 75%, on a molar basis, of the initial amount of [U- 14C]GTP. The product was subsequently isolated by high-performance liquid chromatography. The method produced labeled 6 R-tetrahydrobiopterin with a specific activity of 450 Ci/mol and an overall yield of more than 60%.