Sigmund F. Zakrzewski

Resistance to folic acid analogues in a strain of Streptococcus faecalis.

Summary Bioautographs of several 4-amino analogues of folic acid indicated the presence of contaminants which corresponded to the demethylated, deaminated, or pteroic acid analogues. All the antagonists studied contained sufficient pteroylglutamic acid or pteroic acid to account for their apparent utilization for growth by an antagonist-resistant strain of Streptococcus faecalis. This strain had a lower requirement for PGA and pteroic acid and a much greater capacity to convert PGA to CF than did the parent antagonist-sensitive strain. Significant inhibition of the formation of CF from PGA by the resistant cells was obtained only with very high concentrations of Aminopterin or A-methopterin.

[68] Preparation of tritiated dihydrofolic acid of high specific activity

Publisher Summary This chapter describes the preparation of tritiated dihydrofolic acid of high specific activity. In this method, dihydrofolic acid is formed, by the reduction of folic acid, with sodium dithionite at room temperature. The product is isolated from the reaction mixture, by precipitation, with hydrochloric acid. Tritiated dihydrofolic acid of high specific activity is also prepared by dithionite reduction of tritiated folic acid. However, due to the small quantities of starting material to be used in such preparations, the methodology is modified. In this case, the reaction is carried out in a capillary tube and the product is isolated, by chromatography on a small column of diethylaminoethyl cellulose (DEAE)-cellulose, with ammonium bicarbonate as the developing solvent. When dihydrofolate of specific radioactivity 170 mCi/mmol, recovered from such a column, is lyophilized to complete removal of the bicarbonate, partial decomposition to folate occurs. Because nonradioactive dihydrofolate prepared in an identical way could be recovered without decomposition, degradation appears to be due to the radiation effect. It has also been established that the radioactive dihydrofolate could be stored without decomposition in the presence of excess ammonium bicarbonate in evacuated ampules at –20°.

[187] Preparation of folinic acid (N5-formyltetrahydro folic acid)

Publisher Summary This chapter describes the preparation of folinic acid (N 5 -Formyltetrahydro Folic Acid). Folinic acid is formed upon treatment of N 5 , N l0 -methenyltetrahydrofolic acid with alkali at elevated temperature. The synthesis of N 5 , N l0 -methenyltetrahydrofolic acid by catalytic hydrogenation of folic acid in formic acid is also described. The synthesis of N 5 , N l0 -methenyltetrahydrofolic acid from tetrahydrofolic acid, its conversion to folinic acid, and purification on diethylaminoethyl (DEAE)-cellulose are highlighted. d l -L-folinic acid is commercially available and the method is useful only when either l- L-folinic acid is desired or when preparation of radioactive material is being considered. The reagents used are tetrahydrofolic acid, formic acid, 2-mercaptoethanol, or acetone. To ascertain the purity of the preparation some of its properties are compared with those of the calcium salt of leucovorin. On paper chromatography in 0.1 M phosphate buffer, pH 7, each compound appears as a single light-absorbing spot with an R f : 0.73-0.74. In both preparations, only traces of fluorescing materials are found.

[185] A new preparation of dihydrofolic acid

Publisher Summary Reduction of folic acid to dihydrofolic acid by sodium dithionite is described. The alternate method involving reduction of folic acid with zinc in alkaline solution is focused in the chapter. Use of the zinc reduction method is advantageous for preparation of tritium-labeled dihydrofolate. The reaction is carried out in the presence of tritiated water. Of the total tritium incorporated, about 30% is at C-9 and the rest at C-7. Starting with tritiated water of specific activity 90 mCi/ml, the yield of tritium incorporated is about 10%. Under comparable conditions the yield of tritium incorporation during the dithionite reduction is about 2.5%. In addition zinc reduction is applicable to derivatives of folate such as 7-methylfolic acid, which cannot be reduced by dithionite. The reagents used are folic acid, sodium hydroxide, zinc dust, or sodium ascorbate solution. To obtain pure compound the crude material is recrystallized or purified by ion-exchange chromatography on DEAE-cellulose (diethylaminoethyl-C).