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Preparation of C14-cholesterol from C14-cholestenone
456
LETTERS
TO
THE
EDITORS
cnte that the chick livers contained sufficient enzymes to liberate by autolysis all pantothenic acid contained therein. No attempt was made to determine whether the pantothenic acid that accumulated in the livers in vitamin B12 deficiency w&s free or conjugated. REFERENCES GROSCHKE, A. C., AND EVANS, R. J., Poultry Sci. 29, 616 (1950). 2. MILLER, D. C., AND GROSCHKE, A. C., Mich. Agr. Expt. &a. Quart. Bull. 32, 279 (1950). 3. SKEQQS, H. R., AND WRIQHT, L. D., J. Biol. Chem. 156, 21 (1944). 4. BUSKIRK, H. H., BERODAHL, A. M., AND DELOR, R. A., J. Biol. Chem. 172, 671 (1948). 5. NEILANDS, J. B., AND STRONG, F. M., Arch. Biochem. 19, 287 (1948). 6. LIPMANN, F., KAPLAN, N. O., NOVELLI, G. D., TUTTLE, L. C., AND GUIRARD, B. M., J. Biol. Chem. 167, 869 (1947). 7. NISHI, H., KING, T. E., AND CHELDELIN, V. H., J. Nutrition 41, 279 (1950).
.l.
Department of Agricultural Chemistry and Poultry Husbandry, Michigan State College, East Lansing, Michigan Received January %?, 1961
Preparation
of CWCholesterol
ROBERT
JOHN
EVANS
A. C. GROSCHKE HELEN A. BUTTS
from C14-Cholestenone 1
The best method for the preparation of C14-cholesterol hitherto published consists in the reduction of the enol acetate of A%holesten&one with lithium aluminum hydride (1). However, a yield of only about 3501, can be secured following the published directions. We have found that a considerable increase in yield can be obtained when sodium borohydride is used as a hydrogen source in this reduction in the place of lithium ahuninum hydride. Pure cholesterol can be prepared by this modification in yields of 60% of the theory (2). EXPERIMENTAL
Three grams of C1~A%holesten-3-one enol acetates labeled in the C-3 position with Cl4 and having a count of 1162 counts/min./mg. was ,dissolved without heating in 2000 ml. absolute ethanol containing 1 g. NaBHd; 2 mg. more of NaBH4 was added 1 Supported in part by grants from the American Cancer Society (Massachusetts Division, Inc.); the Public Health Service, Grant No. C321; and the Damon Runyon Memorial Fund for Cancer Research, Inc. 2 This substance was made from C14-Aecholesten-3-one prepared according to TUIXNER, R. B., J. Am. Chem. Sot. 72, 579 (1950), by Dr. Lillian Levy in this laboratory. A small amount of this substance was diluted with nonradioactive A4-cholesten3-one and then converted into the enol acetate by Westphal’s method, Chem. B. Sot. 70, 2128 (1937). It was found advantageous to add triethylamine to the crude enol acetate after removal of acetyl chloride and acetic anhydride in UUCUO.
LETTERS
TO
THE
457
EDITORS
and the mixture was left at room temperature for 47 hr. with occasional shaking. The NaBH, dissolved completely in about 5 hr. and only a slight turbidity remained in the mixture. The alcohol was distilled in vacuum and the residue taken up in ether. This solution was washed with sodium hydroxide solution and water, dried, and evaporated. The white residue weighed 2.8 g. It was dissolved in 100 ml. of 95% ethanol, 3 ml. of cont. HCl was added and the mixture was refluxed for 2 hr. This solution was again worked up by evaporation, extraction with ether, and washing of the ether solution with water, sodium hydroxide solution, and water. After drying and evaporation the weight was 2.7 g. This residue was dissolved in 450 ml. of 95% ethanol, and was precipitated while hot with a hot solution of 8 g. digitonin in 800 ml. of 90% ethanol. The digitonide which precipitated slowly was filtered by suction after standing for 2 days at room temperature, washed with 90% ethanol and ether, and finally dried. The digitonide which weighed 8.6 g. was decomposed into crude cholesterol with pyridine and ether. Two grams yield (co. 74%) was obtained. The cholesterol was dissolved in 200 ml. hexane and put on a column of 60 g. silica gel. Seven eluates of 200 ml. hexane and then of benzene gave only traces of material. The next eluates with 200 ml. ether-benzene (1:9) gave 1.93 g. (71’%) of cholesterol, m.p. 146148”, which was recrystallized from methanol with the addition of ethyl acetate using charcoal and Celite. The following fractions were obtained: Fraction
1 1.30 g.; sint. 146”; m.p. 147-148”. 2 0.37 g.; sint. 147”; m.p. 148-148.6”. 3 0.0972 g.; sint. 139”; m.p. 142.5-145”.
The count of this cholesterol was 1088 counts,/min./mg. counts/min./mg. for fraction 2.
for fraction
1 and 1020
REFERENCES 1. DAUBEN, W. G., AND EASTHAM, J. F., J. Am. Chem. Sot. 72, 2305 (1950). 2. WENDLER, N. L., GRABER, R. P., JONES, R. E., AND TISHLER, M., J. Am. Chem. Sot. 72,5793 (1950), in their work on the synthesis of 17-hydroxycorticosterone have also found that LiBH4 or NaBHI gave better results than LiAlH,. Cf. Chem. Worcester Shrewsbury, Received
Eng.
News
Foundation for Massachusetts February
29, 194 (1951). Experimental
Biology,
14, I.951
Acetoin Synthesis by Highly Purified
ERWIN SCHWENK MARCEL GUT JOSEPHBELIS~
a-Carboxylase
1
Although the enzymatic formation of acetoin (acetyl methyl carbinol) by yeast (l), animal tissues, and bacteria has been studied for many years, its mechanism of formation from pyruvate or pyruvate and acetaldehyde still remains unsettled. In certain bacteria a-acetolactate has been shown to be an intermediate (2,3), but in yeast, animal tissues, and plants another mechanism appears to be involved (3). i This work was aided by grants from the National Vitamin Foundation, the Williams-Waterman Fund of the Research Corporation, and the Division of Research Grants and Fellowships, National Institutes of Health.
LETTERS
TO
THE
EDITORS
cnte that the chick livers contained sufficient enzymes to liberate by autolysis all pantothenic acid contained therein. No attempt was made to determine whether the pantothenic acid that accumulated in the livers in vitamin B12 deficiency w&s free or conjugated. REFERENCES GROSCHKE, A. C., AND EVANS, R. J., Poultry Sci. 29, 616 (1950). 2. MILLER, D. C., AND GROSCHKE, A. C., Mich. Agr. Expt. &a. Quart. Bull. 32, 279 (1950). 3. SKEQQS, H. R., AND WRIQHT, L. D., J. Biol. Chem. 156, 21 (1944). 4. BUSKIRK, H. H., BERODAHL, A. M., AND DELOR, R. A., J. Biol. Chem. 172, 671 (1948). 5. NEILANDS, J. B., AND STRONG, F. M., Arch. Biochem. 19, 287 (1948). 6. LIPMANN, F., KAPLAN, N. O., NOVELLI, G. D., TUTTLE, L. C., AND GUIRARD, B. M., J. Biol. Chem. 167, 869 (1947). 7. NISHI, H., KING, T. E., AND CHELDELIN, V. H., J. Nutrition 41, 279 (1950).
.l.
Department of Agricultural Chemistry and Poultry Husbandry, Michigan State College, East Lansing, Michigan Received January %?, 1961
Preparation
of CWCholesterol
ROBERT
JOHN
EVANS
A. C. GROSCHKE HELEN A. BUTTS
from C14-Cholestenone 1
The best method for the preparation of C14-cholesterol hitherto published consists in the reduction of the enol acetate of A%holesten&one with lithium aluminum hydride (1). However, a yield of only about 3501, can be secured following the published directions. We have found that a considerable increase in yield can be obtained when sodium borohydride is used as a hydrogen source in this reduction in the place of lithium ahuninum hydride. Pure cholesterol can be prepared by this modification in yields of 60% of the theory (2). EXPERIMENTAL
Three grams of C1~A%holesten-3-one enol acetates labeled in the C-3 position with Cl4 and having a count of 1162 counts/min./mg. was ,dissolved without heating in 2000 ml. absolute ethanol containing 1 g. NaBHd; 2 mg. more of NaBH4 was added 1 Supported in part by grants from the American Cancer Society (Massachusetts Division, Inc.); the Public Health Service, Grant No. C321; and the Damon Runyon Memorial Fund for Cancer Research, Inc. 2 This substance was made from C14-Aecholesten-3-one prepared according to TUIXNER, R. B., J. Am. Chem. Sot. 72, 579 (1950), by Dr. Lillian Levy in this laboratory. A small amount of this substance was diluted with nonradioactive A4-cholesten3-one and then converted into the enol acetate by Westphal’s method, Chem. B. Sot. 70, 2128 (1937). It was found advantageous to add triethylamine to the crude enol acetate after removal of acetyl chloride and acetic anhydride in UUCUO.
LETTERS
TO
THE
457
EDITORS
and the mixture was left at room temperature for 47 hr. with occasional shaking. The NaBH, dissolved completely in about 5 hr. and only a slight turbidity remained in the mixture. The alcohol was distilled in vacuum and the residue taken up in ether. This solution was washed with sodium hydroxide solution and water, dried, and evaporated. The white residue weighed 2.8 g. It was dissolved in 100 ml. of 95% ethanol, 3 ml. of cont. HCl was added and the mixture was refluxed for 2 hr. This solution was again worked up by evaporation, extraction with ether, and washing of the ether solution with water, sodium hydroxide solution, and water. After drying and evaporation the weight was 2.7 g. This residue was dissolved in 450 ml. of 95% ethanol, and was precipitated while hot with a hot solution of 8 g. digitonin in 800 ml. of 90% ethanol. The digitonide which precipitated slowly was filtered by suction after standing for 2 days at room temperature, washed with 90% ethanol and ether, and finally dried. The digitonide which weighed 8.6 g. was decomposed into crude cholesterol with pyridine and ether. Two grams yield (co. 74%) was obtained. The cholesterol was dissolved in 200 ml. hexane and put on a column of 60 g. silica gel. Seven eluates of 200 ml. hexane and then of benzene gave only traces of material. The next eluates with 200 ml. ether-benzene (1:9) gave 1.93 g. (71’%) of cholesterol, m.p. 146148”, which was recrystallized from methanol with the addition of ethyl acetate using charcoal and Celite. The following fractions were obtained: Fraction
1 1.30 g.; sint. 146”; m.p. 147-148”. 2 0.37 g.; sint. 147”; m.p. 148-148.6”. 3 0.0972 g.; sint. 139”; m.p. 142.5-145”.
The count of this cholesterol was 1088 counts,/min./mg. counts/min./mg. for fraction 2.
for fraction
1 and 1020
REFERENCES 1. DAUBEN, W. G., AND EASTHAM, J. F., J. Am. Chem. Sot. 72, 2305 (1950). 2. WENDLER, N. L., GRABER, R. P., JONES, R. E., AND TISHLER, M., J. Am. Chem. Sot. 72,5793 (1950), in their work on the synthesis of 17-hydroxycorticosterone have also found that LiBH4 or NaBHI gave better results than LiAlH,. Cf. Chem. Worcester Shrewsbury, Received
Eng.
News
Foundation for Massachusetts February
29, 194 (1951). Experimental
Biology,
14, I.951
Acetoin Synthesis by Highly Purified
ERWIN SCHWENK MARCEL GUT JOSEPHBELIS~
a-Carboxylase
1
Although the enzymatic formation of acetoin (acetyl methyl carbinol) by yeast (l), animal tissues, and bacteria has been studied for many years, its mechanism of formation from pyruvate or pyruvate and acetaldehyde still remains unsettled. In certain bacteria a-acetolactate has been shown to be an intermediate (2,3), but in yeast, animal tissues, and plants another mechanism appears to be involved (3). i This work was aided by grants from the National Vitamin Foundation, the Williams-Waterman Fund of the Research Corporation, and the Division of Research Grants and Fellowships, National Institutes of Health.