Preparation of Cocarboxylase

Abstract

From the reaction mixture which I have recently described 1 the synthetic cocarboxylase (thiaminpyrophosphate) may be obtained in crystalline form by the following procedure: 500 mg sodium pyrophosphate are placed in a Pyrex test-tube and heated until all of the water of crystallization is removed. One cc of orthophosphoric acid (cp 85%) is placed in another large Pyrex tube and heated until a slight amount of solid deposit forms on the side of the tube. Then the pyrophosphate is added and the mixture gently heated until solution takes place. After cooling 500 mg vitamin B₁.HCl are added. The tube is placed in an oil bath of 155°, kept there for 3 min and constantly stirred. Then the tube is removed and after cooling the solid mass is dissolved in 10 cc of cold water. Cold saturated Ba(OH)₂ solution is added until no more precipitate forms and the solution is just commencing to turn yellow. The precipitate is centrifuged off and the supernatant fluid is decanted. The precipitate is extracted 4 times for 5 minutes with 40 cc of cold water. All 5 supernatant fluids are united and after cooling 3% H₂SO₄ is added to slight blue reaction of congo red paper. The BaSO₄ is centrifuged off and discarded. The Ba-free solution is concentrated to 30 cc in vacuum at 25°. It is cooled in ice water and 15 volumes of a mixture of 1 part of absolute alcohol and 2 parts of ether are added which precipitates the cocarboxylase in the form of microscopic needles. Sometimes a gummy mass will form which turns into long macroscopic needles on short standing in the cold. The preparation obtained on 6 recrystallizations from the alcohol-ether mixture, redissolved each time in 10 cc of N/S HCl, and dried in vacuum is readily soluble in water and is free of inorganic salts.

This synthetic preparation is practically as active as natural cocarboxylase 2 (very kindly furnished by Professor Lohmann). Phosphorus and thiamin content, however, indicate that my cocarboxylase still contains a small amount of impurities. The synthetic cocarboxylase gives a yellow color with the formaldehyde-azo-test of Kinnersley and Peters. Thiamin gives a red color.

I wish to thank Merck and Company, and Winthrop Chemical Company, for generous gifts of synthetic vitamin B₁.