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A simple method for the synthesis of tetrahydrofolic acid
SHORT
A Simple
459
CO;Mh1UNICATIONS
Method for Tetrahydrofolic
the
Synthesis Acid
of
Procedures presently employed for the preparation of tetrahydrofolic acid such as catalytic hydrogenation (1) and Na,S,O, reduction (2) are time consuming, require apparatus, and oftentimes entail column chromatography to obtain purified material. This communication describes a rapid and simple method to give a dl-tetrahydrofolic acid preparation suitable for biochemical studies. It involves the use of Na&04 and folic acid in a solid state reaction, absorption of the tetrahydrofolic acid into glacial acetic acid, and precipitation with ether containing mercaptoethanol. The synthesis was carried out as follows: 350 mg of commercial folic acid (97% purity) was suspended in 90 ml of glacial acetic acid which contained 2% 2-mercaptoethanol, 2 gm of Na&O, was then added, and the suspension was mixed with a magnetic stirrer at room temperature for 20-30 min. The mixture was filtered under helium into 200 ml of ether containing 2% 2-mercaptoethanol. The precipitate was collected by centrifugation and washed five times with 100 ml portions of ether containing 2% 2-mercaptoethanol. The product was dried under vacuum in test tubes covered with aluminum foil to keep out light. The yield on a weight basis was 95% of theoretical. The product, a near-white powder, showed an absorption maximum of 297 rnp in 0.1% mercaptoethanol. Reaction of the product with formic acid resulted in the formation of a yellow solution which when examined spectrophotometrically gave a spectrum identical to that of W,,V”methenyltetrahydrofolic acid. The purity of the compound was found to be 90% based on the quantitative conversion of tetrahydrofolic acid to X”,A”“-mcthenyltetrahydrofolate by the method of Zakrzewski (3). To calculate the concentration of N”,N*“-methenyltetrahydrofolatc the molar extinction coefficient at 350 mp of 25.1 X lo3 (4) was used. Methcnyltetrahydrofolate formed in situ was functional in the Nj,N’“-methylenetetrahydrofolate dehydrogenase system without further purification, which would indicate that no by-products have been formed during this preparation that are of an inhibitory nature to the enzyme. As is to be expected of tetrahydrofolic acid, aeration of the compound gave a spectrum characteristic of dihydrofolic acid and prolonged exposure of the solid material resulted in the characteristic change in color to a reddish brown.
460
SHORT
COMMUNICATIONS
ACKNOWLEDGMENTS The author problem. The
wishes author
to express thanks to Dr. Ii. is the recipient of a Lederle
0. Donaldson for suggesting Medical Faculty Award.
this
REFERENCES 1. O’DELL,
B. L., VANDERBILT,
J. M.,
BLOOM,
E. S., AND PFIFFNER,
J. J., 1. Am.
Chem.
Sot. 69, 250 (1947). 2. SILVERMAN, (1961). 3. ZAKRZEWSRI, 4. RAM.ASASTRI,
M.,
AND SORONHA,
H.
S. F., J. Biol. Chem. B. V., AND BLAKELY,
M.,
Biochem.
Biophys.
CommlLn. 4,
Res.
241, 2962 (1966). R. L., J. Biol.
Chem.
239,
106 (1964). LEODIS
Department of Biochemistry Howard University Medical Washington, D, C. 20001 Received April 15. 1968
Application
180.
DAVIS
School
of
Glass
for Separation
Fiber of Cl9
Chromatography Steroids1
In the conversion of androst-5-en-3/3-ol-17-one (DHA) (l-3) and its sulfate ester (DHA-sulfate) (4) to androst-4-en-3-one-17/3-ol (Testosterone), androst-5-ene-3P,17&diol (A5-androstenediol) has been implicated as an intermediate. It is not known if this pathway also involves anclro&-4-ene-3,8,17P-diol (Aa-androstenediol). This question is not easily answered because the A’- and A”-androstenediols were not readily separated either by paper chromatography (3, 5) or by thin-layer chromatography 16). This communication describes a method for rapid resolution of A”-anclrostenediol and A*-androstenediol utilizing AgNO, treated silica gel impregnated glass fiber sheets (ITLC-SG, Gelman Instrument Company). In addition the chromatographic behavior on ITLC-SG and AgNO, impregnated ITLC-SG (Ag-ITLC-SG) of other C,, steroids is presented. This procedure was developed for studies of the metabolism of isotopically labeled DHA and DHA-sulfate in tissue fractions. The method of extraction of the steroids has been described (7). The extracts were first chromatographed on paper (Whatman 2) using the Zaffaroni system (8). The formamide impregnated paper was developed with descending cyclohexane/benzene l/l. Allowing the solvent front to ’ This investigation States Public Health
was supported Service.
in part,
by a grant
(AM-02294)
from
the United
A Simple
459
CO;Mh1UNICATIONS
Method for Tetrahydrofolic
the
Synthesis Acid
of
Procedures presently employed for the preparation of tetrahydrofolic acid such as catalytic hydrogenation (1) and Na,S,O, reduction (2) are time consuming, require apparatus, and oftentimes entail column chromatography to obtain purified material. This communication describes a rapid and simple method to give a dl-tetrahydrofolic acid preparation suitable for biochemical studies. It involves the use of Na&04 and folic acid in a solid state reaction, absorption of the tetrahydrofolic acid into glacial acetic acid, and precipitation with ether containing mercaptoethanol. The synthesis was carried out as follows: 350 mg of commercial folic acid (97% purity) was suspended in 90 ml of glacial acetic acid which contained 2% 2-mercaptoethanol, 2 gm of Na&O, was then added, and the suspension was mixed with a magnetic stirrer at room temperature for 20-30 min. The mixture was filtered under helium into 200 ml of ether containing 2% 2-mercaptoethanol. The precipitate was collected by centrifugation and washed five times with 100 ml portions of ether containing 2% 2-mercaptoethanol. The product was dried under vacuum in test tubes covered with aluminum foil to keep out light. The yield on a weight basis was 95% of theoretical. The product, a near-white powder, showed an absorption maximum of 297 rnp in 0.1% mercaptoethanol. Reaction of the product with formic acid resulted in the formation of a yellow solution which when examined spectrophotometrically gave a spectrum identical to that of W,,V”methenyltetrahydrofolic acid. The purity of the compound was found to be 90% based on the quantitative conversion of tetrahydrofolic acid to X”,A”“-mcthenyltetrahydrofolate by the method of Zakrzewski (3). To calculate the concentration of N”,N*“-methenyltetrahydrofolatc the molar extinction coefficient at 350 mp of 25.1 X lo3 (4) was used. Methcnyltetrahydrofolate formed in situ was functional in the Nj,N’“-methylenetetrahydrofolate dehydrogenase system without further purification, which would indicate that no by-products have been formed during this preparation that are of an inhibitory nature to the enzyme. As is to be expected of tetrahydrofolic acid, aeration of the compound gave a spectrum characteristic of dihydrofolic acid and prolonged exposure of the solid material resulted in the characteristic change in color to a reddish brown.
460
SHORT
COMMUNICATIONS
ACKNOWLEDGMENTS The author problem. The
wishes author
to express thanks to Dr. Ii. is the recipient of a Lederle
0. Donaldson for suggesting Medical Faculty Award.
this
REFERENCES 1. O’DELL,
B. L., VANDERBILT,
J. M.,
BLOOM,
E. S., AND PFIFFNER,
J. J., 1. Am.
Chem.
Sot. 69, 250 (1947). 2. SILVERMAN, (1961). 3. ZAKRZEWSRI, 4. RAM.ASASTRI,
M.,
AND SORONHA,
H.
S. F., J. Biol. Chem. B. V., AND BLAKELY,
M.,
Biochem.
Biophys.
CommlLn. 4,
Res.
241, 2962 (1966). R. L., J. Biol.
Chem.
239,
106 (1964). LEODIS
Department of Biochemistry Howard University Medical Washington, D, C. 20001 Received April 15. 1968
Application
180.
DAVIS
School
of
Glass
for Separation
Fiber of Cl9
Chromatography Steroids1
In the conversion of androst-5-en-3/3-ol-17-one (DHA) (l-3) and its sulfate ester (DHA-sulfate) (4) to androst-4-en-3-one-17/3-ol (Testosterone), androst-5-ene-3P,17&diol (A5-androstenediol) has been implicated as an intermediate. It is not known if this pathway also involves anclro&-4-ene-3,8,17P-diol (Aa-androstenediol). This question is not easily answered because the A’- and A”-androstenediols were not readily separated either by paper chromatography (3, 5) or by thin-layer chromatography 16). This communication describes a method for rapid resolution of A”-anclrostenediol and A*-androstenediol utilizing AgNO, treated silica gel impregnated glass fiber sheets (ITLC-SG, Gelman Instrument Company). In addition the chromatographic behavior on ITLC-SG and AgNO, impregnated ITLC-SG (Ag-ITLC-SG) of other C,, steroids is presented. This procedure was developed for studies of the metabolism of isotopically labeled DHA and DHA-sulfate in tissue fractions. The method of extraction of the steroids has been described (7). The extracts were first chromatographed on paper (Whatman 2) using the Zaffaroni system (8). The formamide impregnated paper was developed with descending cyclohexane/benzene l/l. Allowing the solvent front to ’ This investigation States Public Health
was supported Service.
in part,
by a grant
(AM-02294)
from
the United