Synthesis and Reactions of 5-Alkyltetrahydrofolates

Abstract

5-Methyltetrahydrofolic acid, 5-n-butyltetrahydrofolicacid and tetrahydromethotrexate have been successfully prepared. The solubility of 5-methyltetrahydrofolic acid was determined in the pH range 1 to 7; also, the pK value for the ionisation of the 3,4-amide function was measured by a spectrophotometric method. An attempt was made to separate the diastereoisomers of 5-methyltetrahydrofolic acid on a DEAE cellulose column and also by fractional crystallisation. A detailed kinetic study of the autoxidation of 5-methyltetrahydrofolic was carried out using a simple manometric method and the rate of oxidation was found to havef irst order dependence on both 5-methyltetrahydrofolic acid concentration and oxygen partial pressure. The effects of pH and various catalysts and inhibitors were studied. The rate was found to increase ongoing from pH 9 to pH 13 but decreased markedly from pH 5 to pH 1. Copper ions were found to increase the reaction rate by a factor of 10, whereas phenol (a free-radical scavenger) halved the reaction rate. Product analysis was undertaken using n.m.r., t.l.c.u.v., i.r., e.s.r., and microbiological assay techniques and the major products of oxidation were found to be 5-methyl-5 ,6-dihydrofolic acid and 8-dehydro-4a-hydroxy-5-methyltetrahydrofolic acid. A search for observable intermediates proved fruitless. A mechanism in keeping with the above observations is proposed,