Preparation of (6S)-5-Formyltetrahydrofolate Labeled at High Specific Activity with ¹⁴C and ³H

This chapter describes preparation of (6S)-5-CHO-H₄PteGlu labeled either with ¹⁴C at position 5 or with ³H at positions 3′,5′,7, and 9 at specific activities equivalent to those of the labeled starting materials (l0–60 Ci/mmol and 50–60 mCi/mmol, respectively). This preparation method takes advantage of the reactivity of the 5 position of tetrahydrofolate towards attack by carboxylic acids in the presence of carbodiimides. The mild conditions used allow good yields of both labeled compounds. The formylation of H₄PteGlu promoted by carbodiimides is extremely sensitive to pH. This preparation can be scaled up by proportionately increasing [³H]PteGlu, reduced nicotinamide adenine diphosphate (NADPH), and dihydrofolate reductase while holding reaction times and volumes constant. [³H]PteGlu is incubated with dihydrofolate reductase and NADPH in 0.1 ml of 50 mM phosphate buffer, containing 50 mM 2-mercaptoethanol (2-ME). After 10 min at 37°, 900 μl of 100 mM formic acid containing 50 mM phosphate and 150 mM 2-ME is added.