Chromatographic Separation of the Diastereoisomers

Reduction of folic acid to the tetrahydro level introduces a second asymmetrical center into the molecule at carbon atom 6.1 The resulting tetrahydrofolate, and subsequent derivatives, prepared chemically consist of dl mixtures that are only 50% active in biological reactions. The need for fully reactive isomers of reduced folates led to the enzymatic synthesis of tetrahydrofolate from 7, %dihydrofolate via folic reductase and reduced triphosphopyridine nucleotide (1). An inactive isomer of 5-methyltetrahydrofolate has been isolated after enzymatic oxidation of the active component of a mixture of diastereoisomers of 5-methyltetrahydrofolate to 5, lo-methylenetetrahydrofolate (2). However, since folic acid has an optically active carbon in the L-glutamic acid moiety, the reduced folates consist of a mixture of diastereoisomers that differ in physical properties. Thus differential solubility has served as a basis for the separation of optical isomers of chemically synthesized calcium salt of N5-formyltetrahydrofolic acid (3). During the purification of chemically prepared 5, lo-methylenetetrahydrofolate, resolution of the two diastereoisomers on a cellulose ion exchange column was observed. The evidence for the separation of the two isomers of 5, lo-methylenetetrahydrofolate is presented in this communication.