Preparation of tritium-labeled tetrahydropteroylpolyglutamates of high specific radioactivity.

Tritium-labeled [6S]-tetrahydropteroylpolyglutamates of high radiospecific activity were prepared from the corresponding pteroylpolyglutamates. Malic enzyme and D,L-[2-3H]malate were used as a generating system to produce [4A-3H]NADPH which was coupled to the dihydrofolate reductase-catalyzed reduction of chemically prepared dihydropteroylpolyglutamate derivatives. Passage of the reaction mixtures through a column of immobilized boronate effectively removed NADPH, and the tetrahydropteroylpolyglutamates were subsequently purified by chromatography on DEAE-cellulose. Overall yields of the [6S]-tetrahydro derivatives were 18-48% and the radiospecific activities were 3-4.5 mCi X mumol-1.