114] Preparation and properties of “active formaldehyde” and “active formate”

Abstract

This chapter discusses the preparation and properties of “active formaldehyde” and “active formate.” “Active formaldehyde,” an adduct between formaldehyde and tetrahydrofolic acid, has been identified as N5,N10-methylenetetra-hydrofolic acid (I). The compound is synthesized most easily by allowing tetrahydrofolic acid to interact with an excess of formaldehyde at a slightly acidic pH and purifying the product by column chromatography. There are three known forms of “active formate”—namely, N5-formyl-tetrahydrofolic acid (folinic acid), N10-formyltetrahydrofolic acid, and N5,N10-methenyltetrahydrofolic acid, which are encountered in enzymatic systems. Folinic acid is commercially available. N5,N10-Methenyltetrahydrofolic acid (II) is readily prepared as a stable, crystalline material by the procedure discussed in the chapter, and N10-formyltetrahydrofolate can be generated in situ by adjusting solutions of the methenyl derivative to pH 7 or higher. When the prepared “active formate” product is examined by descending paper chromatography on Whatman No. 1 paper with 1.0 M formic acid–0.01 M mercaptoethanol as the solvent system, a single white fluorescent spot is observed under ultraviolet light. The failure of this spot to respond to the spray reagent, which reductively cleaves many folic acid compounds at the C9–N10 linkage, is referable to the stabilizing effect of the methenyl bridge between the N5- and N10-positions.