- Email: [email protected]
Thin-layer chromatography of N,N-dimethyl-p-amino-benzolazophenacyl esters of volatile fatty acids and hydroxy acids
ANALYTIC.AL
BIOCHEMISTKY
Thin-Layer
46, 62-66 (1972)
Chromatography
of
benzolazophenacyl Acids
and
Esters
of Volatile
Hydroxy
Acids
I. M. SELIGMANl Division
N,N-Dimethyl-p-amino-
F. A. DOY
AND
of Entomology, Commonwealth Scientific Organization, Canberra,A.C.T.,
Received April
Fatty
and
Industrial
Research
Austrnlia
2, 1971
During an investigation of the biosynthesis of volatile branched-chain fatty acids in various papilionid species, it was necessary to develop a sensitive procedure for the detection of these acids as products of selected radioactive precursors. Colored nonvolatile derivatives were required for this purpose. In 1964, Churacek et al. (1) described a procedure for the synthesis of various azophenacyl esters of fatty acids and the separation of these esters in a reverse-phase paper chromatographic system. During the course of this work, Churacek and Pechova (2) demonstrated that these azophenacyl esters are conveniently separated on silica gel thin-layer plates. In this paper the thin-layer chromatographic separation of hydroxy acid azo esters is described, and a new solvent system for the separation of the esters of volatile fatty acids is also considered. Purification of radioactive azophenacyl esters to constant specific activity can be achieved with minimal loss of material through successive purification steps, and t,he amounts of material recovered can be monitored photometrically. This technique was applied to the determination of the radiochemical purity of a volatile fatty acid isolated from a biological source. MATERIALS
AND
METHODS
Preparation of Reagents N,N-Dimethyl-p-aminobenzolphenacyl chloride was prepared by a Friedel-Craft synthesis from p-aminophenacyl chloride according to the technique of Churacek et al. (1). l Present address: Department ence, Rehovot, Israel.
of Biodynamics,
62 @ 1972 by Academic
Press, Inc.
The Weizmann
Institute
of Sci-
TLC
OF
LOWER
FATTY
AKD
HYDROXY
ACIDS
63
,!3-Hydroxyisobutyric acid was prepared from formaldehyde and ethyl a-bromopropionate by a Reformat’sky reaction, according to the technique of Blaise and Herman as described by Robinson and Coon (3). ,&Hydroxypropionic acid was synthesized from ethylene cyanohydrin according to Robinson and Coon. Methylbutyric acid was prepared by hydrogenation of tiglic acid. Uniformly labeled l*C-L-valine (0.26 &i/mmole) was obtained from Amersham. All other reagents are obtainable from commercial sources. Collection and Processing of Biological Materials Papilio larvae store copious quantities of defensive secretion in evertible glands (osmcteria) located behind the head of t’he caterpillar (4,5). Microliter quantities of this secretion can be obtained by applying a capillary tube to the everted glands of animals made to assume a defensive posture. The combined concentrations of isobutyric and methylbutyric acids in the secretion are high (approximately 0.5 M to a phenolphthalein end-point titrated with dilute NaOH) . Synthesis of Azophenacyl
Esters
Free acids (approximately 1 ~1) or oFmeteria1 secretions were neutralized to phenolphthalein with dilute alcoholic NaOH (0.01 X) and evaporated to dryness. Considering the low concentration of reagents used it was unnecessary to back-titrate as Churacek et al. recommend (1). Equimolar quantities of the sodium salts and axo-dye reagent were mixed in a total volume of 0.2 to 1.0 ml dimethylformamide/water (15: 1) and heated on a boiling water bath in a stoppered round-bottom flask for 1 hr. The mixture was then diluted with water and the esters were extracted into eit’her chloroform or ethyl acetate. The colored organic phase was evaporated to dryness in vacua and the red dye was redissolved in a minimal volume of acetone. Thin-Layer
Chromatography
250 p silica gel G thin-layer plates were prepared according to standard procedures. The acetone solution of the dye was applied as either a spot or a streak to the origin on the plate. The best mobile phase for the separation of C&C, fatty acids is t)enzene/ethyl acetate (20: 1). Hydroxy acids are separated with benzene/ethyl acetate (3: 1). Adequate separation for most compounds was achieved in 30-60 min. The minimum quantity of acid that1 yielded a visible spot was 0.5 x 10e9mole (i.e., approximately O.OTi11.g).Heat act8ivat’ion did not affect migration of the azo esters.
64
SELIGMAN
Determination
AND
of Specific Activity
DOY
of Radioactive Isobutyric
Acid
Several Pap&o aegeus last instars larvae were immobilized on ice and injected with 1 ~1 14C-valine (6 m&i). The defensive secretion was milked from the osmeterium 15-20 hr later, and azophenacyl esters were prepared as described above. The esters were chromatographed on silica gel with benzene/ethylacetate (2O:l). The azophenacyl ester of isobutyric acid was scraped off the glass plate and eluted from the silica gel with acetone through a nonabsorbent cotten plug jammed into the neck of a glass funnel. Radioactivity was assayed in a Nuclear-Chicago gasflow counter on stainless-steel planchets coated with a monolayer of silicone grease to facilitate controlled spreading of the dye. Sufficient counts were allowed to accumulate t,o yield an error of 5% per sample. As yields of secretion were variable, estimates of total incorporation are not very meaningful. Recovery of label ranged from 0.01% to 0.1%. Estimates of amounts of material used for specific activity measurements are based on the optical density at 460 mp of the azo ester diluted in acetone. The molar extinction coefficient of the isobutyric acid ester at this wave length is 2 X lo4 M-l cm-l. RESULTS
AND
DISCUSSION
The R, values for the esters of twelve acids are listed in Table 1. The mean Rf values and standard deviations for the esters of propionic acid and isobutyric acid are 0.301 -+ 0.010 and 0.348 * 0.006, respecRI
Values Silica
of Azophenacyl Gel G Thin-Layer Ester
Formic acid Acetic acid Propionic acid Butyric acid Valerie acid Caproic acid Caprylic acid Pelargonic acid Crotonic acid Tiglic acid Isobutyric acid Methylbutyric acid N,N-Dimethyl-p-aminobenzolazophenacyl ‘6 R&l = migration relative C = number of carbon atoms
TABLE 1 Esters of Lower Fatty Acids Plates with Benzene/Ethyl
Chromatographed Acetate (20: 1)
on
of
chloride
1 2 3 4 5 6 8 9 4 5 4 5 -
to N,N-dimethyl-p-aminobenzolazophenacyl in acid.
0.21 0.19 0.25 0.32 0.40 0.43 0.45 0.45 0.25 0.32 0.32 0.40 0.47
0.46 0.40 0.53 0.67 0.85 0.92 0.97 0.97 0.53 0.67 0.67 0.85 1.00
chloride.
TLC
Rf Values
OF
LOWER
FATTY
relative
65
ACIDS
Chromatographed on Silica Acetate (3: 1)
of
or-Hydroxypropionic acid (lactic cu-Hydroxyisobutyric acid cY-Hydroxyisocaproic acid @-Hydroxypropionic acid P-Hydroxy-n-butyric acid /%Hydroxyisobutyric acid N,N-Dimethyl-p-aminobenzolazophenacyl = migration
HYDROXY
TABLE 2 Esters of Hydroxy ilcids Plates wit.h Benzene/Ethyl
of Azophenacyl Gel G Thin-Layer Ester
a Rx
AND
acid)
0.29 0.35 0.50 0.18 0.25 0.72 0.77
chloride
0.38 0.45 0.65 0.23 0.32 0.93 1.00
to N,N-dimethyl-p-aminobenzolazophenacyl
chloride.
tively. Each of these estimates is based on eight measurements on one plate. Isomers cannot be separated, but migration of esters in a homologous series is correlated with the molecular weight of the acid within the range C, to Cs. The ester of formic acid, the first member of the series, behaves anomalously-its Rf is higher than that of the ester of acetic acid. A double bond decreases the Rf of the azo ester to that of the lower homolog, e.g., the crotonic ester and the propionic ester cannot be separated, and the tiglic ester has the same Rf as a saturated C, ester. The Rf values of the esters of some (Y- and ,8-hydroxy acids are in Table 2. In this case isomers are clearly distinguishable: (Y- and /3-hydroxypropionic esters separate quite readily, as do the esters of the three hydroxybutyric acids tested. The Rf of esters of hydroxy acids within a homologous series is also related to the chain length of the acid. The esters of two biologically important hydroxy acids, lactic acid and /3-hydroxy-n-butyric acid, migrate close to each other. However, they can be easily distinguished either by cochromatography or by overrunning the chromatogram with the benzene/ethyl acetate (3:l) solvent. In Table 3, the specific activity of the azo ester of W-isobutyric
Specific
Activity WkValine
Chromatographic run
TABLE 3 of Radioactive Isobutyricazophenacyl Injected into Pup&o ageus Last Total counts recovered
2 3 5 Specific
154 117 activity
= total
counts
Ester,
Est,er Derived Instar Larvaea Specific x
mrmole 0.22
G mrmoles
activitya 10-Z 7.0 7.8
0.l.i
recovered
from
ester.
66
SELIGMAW
AND
DOT
acid made in vivo from W-valine in Papilio aegeus is given. After three chromatographic runs, it is clear that the azo-dye ester has been purified to constant specific activity, indicating that this osmeterial secretion component is indeed derived from valine. The catabolic degradation of branched-chain amino acids in Pap&o aegeus will be considered at length in a subsequent publication (6). SUMMARY
A micro method has been developed for the thin-layer chromatography of lower fatty acids and hydroxy acids on silica gel. The basis of the procedure is conversion of the sodium salts of the acids to colored azophenacyl esters. The procedure was applied to the purification of a radioactive metabolite of YI2-valine, isobutyric acid, in Pap&o aegeus. ACKNOWLEDGMENTS We are grateful to Dr. B. P. Moore for a sample of methylbutyric to Mrs. M. Rozdarz for maintaining the Papilio culture.
acid and
REFERENCES 1. 2. 3. 4. 5.
6.
J., KOPECNY, F., KULHAVY, M., AND JURECEK, M., 2. Anal. Chem. 208, 102 (1965). CHURACEK, J., AND PECHOVA, H., J. Chromatogr. 48, 250 (1970). ROBINSON, W. G., AND COON, M. J., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. VI, p. 551. Academic Press, New York/London, 1963. CROSSLEY, A. C., AND WATERHOUSE, D. F., Tissue und Cell 1, 525 (1969). EISNER, T., AND MEINWALD, Y. C., Science 150, 1733 (1965). SELIGMAN, I. M., AND DOY, F. A., in preparation, 1971. CHUFLGEK,
BIOCHEMISTKY
Thin-Layer
46, 62-66 (1972)
Chromatography
of
benzolazophenacyl Acids
and
Esters
of Volatile
Hydroxy
Acids
I. M. SELIGMANl Division
N,N-Dimethyl-p-amino-
F. A. DOY
AND
of Entomology, Commonwealth Scientific Organization, Canberra,A.C.T.,
Received April
Fatty
and
Industrial
Research
Austrnlia
2, 1971
During an investigation of the biosynthesis of volatile branched-chain fatty acids in various papilionid species, it was necessary to develop a sensitive procedure for the detection of these acids as products of selected radioactive precursors. Colored nonvolatile derivatives were required for this purpose. In 1964, Churacek et al. (1) described a procedure for the synthesis of various azophenacyl esters of fatty acids and the separation of these esters in a reverse-phase paper chromatographic system. During the course of this work, Churacek and Pechova (2) demonstrated that these azophenacyl esters are conveniently separated on silica gel thin-layer plates. In this paper the thin-layer chromatographic separation of hydroxy acid azo esters is described, and a new solvent system for the separation of the esters of volatile fatty acids is also considered. Purification of radioactive azophenacyl esters to constant specific activity can be achieved with minimal loss of material through successive purification steps, and t,he amounts of material recovered can be monitored photometrically. This technique was applied to the determination of the radiochemical purity of a volatile fatty acid isolated from a biological source. MATERIALS
AND
METHODS
Preparation of Reagents N,N-Dimethyl-p-aminobenzolphenacyl chloride was prepared by a Friedel-Craft synthesis from p-aminophenacyl chloride according to the technique of Churacek et al. (1). l Present address: Department ence, Rehovot, Israel.
of Biodynamics,
62 @ 1972 by Academic
Press, Inc.
The Weizmann
Institute
of Sci-
TLC
OF
LOWER
FATTY
AKD
HYDROXY
ACIDS
63
,!3-Hydroxyisobutyric acid was prepared from formaldehyde and ethyl a-bromopropionate by a Reformat’sky reaction, according to the technique of Blaise and Herman as described by Robinson and Coon (3). ,&Hydroxypropionic acid was synthesized from ethylene cyanohydrin according to Robinson and Coon. Methylbutyric acid was prepared by hydrogenation of tiglic acid. Uniformly labeled l*C-L-valine (0.26 &i/mmole) was obtained from Amersham. All other reagents are obtainable from commercial sources. Collection and Processing of Biological Materials Papilio larvae store copious quantities of defensive secretion in evertible glands (osmcteria) located behind the head of t’he caterpillar (4,5). Microliter quantities of this secretion can be obtained by applying a capillary tube to the everted glands of animals made to assume a defensive posture. The combined concentrations of isobutyric and methylbutyric acids in the secretion are high (approximately 0.5 M to a phenolphthalein end-point titrated with dilute NaOH) . Synthesis of Azophenacyl
Esters
Free acids (approximately 1 ~1) or oFmeteria1 secretions were neutralized to phenolphthalein with dilute alcoholic NaOH (0.01 X) and evaporated to dryness. Considering the low concentration of reagents used it was unnecessary to back-titrate as Churacek et al. recommend (1). Equimolar quantities of the sodium salts and axo-dye reagent were mixed in a total volume of 0.2 to 1.0 ml dimethylformamide/water (15: 1) and heated on a boiling water bath in a stoppered round-bottom flask for 1 hr. The mixture was then diluted with water and the esters were extracted into eit’her chloroform or ethyl acetate. The colored organic phase was evaporated to dryness in vacua and the red dye was redissolved in a minimal volume of acetone. Thin-Layer
Chromatography
250 p silica gel G thin-layer plates were prepared according to standard procedures. The acetone solution of the dye was applied as either a spot or a streak to the origin on the plate. The best mobile phase for the separation of C&C, fatty acids is t)enzene/ethyl acetate (20: 1). Hydroxy acids are separated with benzene/ethyl acetate (3: 1). Adequate separation for most compounds was achieved in 30-60 min. The minimum quantity of acid that1 yielded a visible spot was 0.5 x 10e9mole (i.e., approximately O.OTi11.g).Heat act8ivat’ion did not affect migration of the azo esters.
64
SELIGMAN
Determination
AND
of Specific Activity
DOY
of Radioactive Isobutyric
Acid
Several Pap&o aegeus last instars larvae were immobilized on ice and injected with 1 ~1 14C-valine (6 m&i). The defensive secretion was milked from the osmeterium 15-20 hr later, and azophenacyl esters were prepared as described above. The esters were chromatographed on silica gel with benzene/ethylacetate (2O:l). The azophenacyl ester of isobutyric acid was scraped off the glass plate and eluted from the silica gel with acetone through a nonabsorbent cotten plug jammed into the neck of a glass funnel. Radioactivity was assayed in a Nuclear-Chicago gasflow counter on stainless-steel planchets coated with a monolayer of silicone grease to facilitate controlled spreading of the dye. Sufficient counts were allowed to accumulate t,o yield an error of 5% per sample. As yields of secretion were variable, estimates of total incorporation are not very meaningful. Recovery of label ranged from 0.01% to 0.1%. Estimates of amounts of material used for specific activity measurements are based on the optical density at 460 mp of the azo ester diluted in acetone. The molar extinction coefficient of the isobutyric acid ester at this wave length is 2 X lo4 M-l cm-l. RESULTS
AND
DISCUSSION
The R, values for the esters of twelve acids are listed in Table 1. The mean Rf values and standard deviations for the esters of propionic acid and isobutyric acid are 0.301 -+ 0.010 and 0.348 * 0.006, respecRI
Values Silica
of Azophenacyl Gel G Thin-Layer Ester
Formic acid Acetic acid Propionic acid Butyric acid Valerie acid Caproic acid Caprylic acid Pelargonic acid Crotonic acid Tiglic acid Isobutyric acid Methylbutyric acid N,N-Dimethyl-p-aminobenzolazophenacyl ‘6 R&l = migration relative C = number of carbon atoms
TABLE 1 Esters of Lower Fatty Acids Plates with Benzene/Ethyl
Chromatographed Acetate (20: 1)
on
of
chloride
1 2 3 4 5 6 8 9 4 5 4 5 -
to N,N-dimethyl-p-aminobenzolazophenacyl in acid.
0.21 0.19 0.25 0.32 0.40 0.43 0.45 0.45 0.25 0.32 0.32 0.40 0.47
0.46 0.40 0.53 0.67 0.85 0.92 0.97 0.97 0.53 0.67 0.67 0.85 1.00
chloride.
TLC
Rf Values
OF
LOWER
FATTY
relative
65
ACIDS
Chromatographed on Silica Acetate (3: 1)
of
or-Hydroxypropionic acid (lactic cu-Hydroxyisobutyric acid cY-Hydroxyisocaproic acid @-Hydroxypropionic acid P-Hydroxy-n-butyric acid /%Hydroxyisobutyric acid N,N-Dimethyl-p-aminobenzolazophenacyl = migration
HYDROXY
TABLE 2 Esters of Hydroxy ilcids Plates wit.h Benzene/Ethyl
of Azophenacyl Gel G Thin-Layer Ester
a Rx
AND
acid)
0.29 0.35 0.50 0.18 0.25 0.72 0.77
chloride
0.38 0.45 0.65 0.23 0.32 0.93 1.00
to N,N-dimethyl-p-aminobenzolazophenacyl
chloride.
tively. Each of these estimates is based on eight measurements on one plate. Isomers cannot be separated, but migration of esters in a homologous series is correlated with the molecular weight of the acid within the range C, to Cs. The ester of formic acid, the first member of the series, behaves anomalously-its Rf is higher than that of the ester of acetic acid. A double bond decreases the Rf of the azo ester to that of the lower homolog, e.g., the crotonic ester and the propionic ester cannot be separated, and the tiglic ester has the same Rf as a saturated C, ester. The Rf values of the esters of some (Y- and ,8-hydroxy acids are in Table 2. In this case isomers are clearly distinguishable: (Y- and /3-hydroxypropionic esters separate quite readily, as do the esters of the three hydroxybutyric acids tested. The Rf of esters of hydroxy acids within a homologous series is also related to the chain length of the acid. The esters of two biologically important hydroxy acids, lactic acid and /3-hydroxy-n-butyric acid, migrate close to each other. However, they can be easily distinguished either by cochromatography or by overrunning the chromatogram with the benzene/ethyl acetate (3:l) solvent. In Table 3, the specific activity of the azo ester of W-isobutyric
Specific
Activity WkValine
Chromatographic run
TABLE 3 of Radioactive Isobutyricazophenacyl Injected into Pup&o ageus Last Total counts recovered
2 3 5 Specific
154 117 activity
= total
counts
Ester,
Est,er Derived Instar Larvaea Specific x
mrmole 0.22
G mrmoles
activitya 10-Z 7.0 7.8
0.l.i
recovered
from
ester.
66
SELIGMAW
AND
DOT
acid made in vivo from W-valine in Papilio aegeus is given. After three chromatographic runs, it is clear that the azo-dye ester has been purified to constant specific activity, indicating that this osmeterial secretion component is indeed derived from valine. The catabolic degradation of branched-chain amino acids in Pap&o aegeus will be considered at length in a subsequent publication (6). SUMMARY
A micro method has been developed for the thin-layer chromatography of lower fatty acids and hydroxy acids on silica gel. The basis of the procedure is conversion of the sodium salts of the acids to colored azophenacyl esters. The procedure was applied to the purification of a radioactive metabolite of YI2-valine, isobutyric acid, in Pap&o aegeus. ACKNOWLEDGMENTS We are grateful to Dr. B. P. Moore for a sample of methylbutyric to Mrs. M. Rozdarz for maintaining the Papilio culture.
acid and
REFERENCES 1. 2. 3. 4. 5.
6.
J., KOPECNY, F., KULHAVY, M., AND JURECEK, M., 2. Anal. Chem. 208, 102 (1965). CHURACEK, J., AND PECHOVA, H., J. Chromatogr. 48, 250 (1970). ROBINSON, W. G., AND COON, M. J., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. VI, p. 551. Academic Press, New York/London, 1963. CROSSLEY, A. C., AND WATERHOUSE, D. F., Tissue und Cell 1, 525 (1969). EISNER, T., AND MEINWALD, Y. C., Science 150, 1733 (1965). SELIGMAN, I. M., AND DOY, F. A., in preparation, 1971. CHUFLGEK,