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Asymmetric triglycerides from Impatiens edgeworthii seed oil
475
BIOCHIMICA ET BIOPHYSICA ACTA BBA 55320
ASYMMETRIC
TRIGLYCERIDES
FROM IMPATIENS
EDGEWORTHII
SEED OIL M. 0. BAGBU
AND
C. R. SMITH,
JR.
NortAern Regional Research Laboratory*, (Received
Peoria, Ill. (U.S.A.)
September rgth, 1966)
SUMMARY
Impatiens edgeworthii Hook F. seed oil, which contains acetate, parinarate, and the more common fatty acylgroups, was hydrogenated to give mainly cr-acetodistearin. The hydrogenated oil produced a plain negative optical rotatory dispersion curve. Comparison of the dispersion curve with those of cr-acetodiacyl triglycerides synthesized by stereoselective methods indicates that the triglycerides of Impatiens edgeworthii seed oil have the (SJ-configuration.
INTRODUCTION
During an investigation1 of the stereochemistry of cl-parinaric acid from Impatiens edgeworthii Hook F. seed oil, we identified acetic acid as a constituent of the oil. KAUFMANN AND KELLER~ isolated a large quantity of acetodiparinarin from other Tmpatiens species. The possible presence of acetodiparinarin, together with the reports+6 that measurable optical rotation can be observed for asymmetric triglycerides containing greatly different fatty acyl groups, prompted our further study of the glyceryl diacylacetates ** from I. edgeworthii seed oil. Three reviewss+f@ in 1965 summarize the many attempts to determine whether triglycerides are ~ymmet~cally biosynthesized and whether they exist as optically active enantiomers or as racemic mixtures. Such studies have been greatly facilitated by the availability of sensitive ultraviolet spectropolarimeters. MORRIS' developed a procedure that enabled him to detect optical rotation of a modified triglyceride and thus to demonstrate that some natural triglycerides occur predominantly as a single enantiomer. KLEIMAN et al.8 reported that the acetic acid in Euonymus verrZtcos@s seed oil occurs on the cc-position of glycerol. Those Euonymus triglycerides had measurable optical rotation, and they were of the (S)-configuration. BROCKERHOFF etaZ.a~lo have concluded that some natural triglycerides are asymmetric but they did not measure rotations. * This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture. ** Awls as used in this paper refer to long-chain fatty groups, such as palmitoyl and stearoyl, with the exclusion of acetyl. Biochim. Biophys.
Acta, 137 (1967) 475-477
476
M. 0.
EAGBY, C. R. SMITH
EXPERIMENTALPROCEDURESANDRESULTS The I. e~ge~o~~~~~ seed oil (stored as a pentane-hexane solution after isolation as reported previouslyl) was freed of solvent under a stream of N, with care being taken to exclude air and light. To eliminate the reactivity of the polyunsaturated acids in the oil and to provide a material amenable to handling, the oil was hydrogenated in absolute ethanol with a platinum catalyst. The saturated oil had an infrared spectrum (Ccl, solution) like that of ol-acetodistearinll. (Found: C, 73.2; H, 11.6; N, 0.0; P, 0.0; S, 0.0, Acetodisteari~, C41H,80B, requires C, 73.8; H, x1.8). Thin-layer chromatography on silica gel G* (solvent system : 10% diethyl ether in light petroleum, b.p. 33-57”, spots visualized by charring with H,SO,) revealed mainly diacyl acetoglyceride with very weak spots corresponding to conventional triglycerides and unsaponifiables. Gas-liquid chromatography essentially as described by LITCHFIELD, HARLOW AND REISER I2 on 3% JXR Gaschrom Q, 0.5 mx3 mm stainless-steel column, programmed from 120' at 4” per min with on-column injection 320°, flame detector 350”, indicated only triglycerides with one acetyl group. The aceto-palmito-stearin represented about 3.3% of the total area of the gas-liquid chromatographic peaks. The presence of acetic acid was confirmed as the only low molecular weight acid, as described earlier 1. To remove the small amount of contaminants from the diacyl acetoglyceride, the hydrogenated oil (0.19 g) was chromatographed on IO g of silica gel (Adsorbosil, 3060 mesh) with diethyl ether-light petroleum, b.p. 33-57O, as a discontinuous gradient using gradually increasing amounts of diethyl ether. The bulk of the material was eluted with 7% diethyl ether (v/v). Homogeneity of various fractions was determined by thin-layer chromatography, and fractions consisting of only a-acetotriglyceride were combined to give 0.14 g of a white solid. The aggregate was slurried with Darco G6o charcoal in acetone, filtered, and freed of solvent to give 0.~3 g of product. It was then recrystallized from I5 ml of ethanol at 8” to give 0.11 g of a solid which had m-p. 51-52.5’; [cc]g-0.6”, [cr]&--I.r”, [cc]~&--2.0°, [cc]&%-2.9”, [a&&%-9.1' in hexane (c, 2.28). Optical rotatory dispersion was obtained with a Cary recording spectropolarimeter. Thin-layer chromatography showed only one spot ; it corresponded to a-acetyl diacyl triglyceride13. Saponification of the or-acetyl diacyl triglyceride yielded acetic, palmitic, and stearic acids as the only acid components determined by gas-liquid chromatography. Gas-liquid chromatographic analysis of methyl esters of the water-insoluble acids showed 4.5% of methyl palmitate and 95.5% of methyl stearate. This composition compares to that of the original hydrogenated oil’. DISCUSSION The results from thin-layer chromatography, gas-liquid chromatography, and elemental analyses indicated that hydrogenated I. edgeworthii seed oil consists mainly of cr-acetodistearin. Comparison of its optical rotatory dispersion with that of a-acetotriglycerides, synthesized by R. KLEIMAN of this laboratory** by the stereoselective * The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar productsnot mentioned. ** R. KLEIMAN, personal communication.
477
ASYMMETRIC TRIGLYCERIDES FROM A SEED OIL methods
of SOWDEN AND FISHER”
hydrogenated
acetotriglyceride
Application convention
provides
asymmetry
seed
that the main acetotriglyceride
must be the result of a selective
evidence
suggesting
mating the KENNEDY’*
for plants, one might speculate acid intermediate necessary
postulated
natural
when applying
by KENNEDY
process
some of the prevalent
in
resulting
The
I. edgeworthii
seed, HIRAYAMA
was acetylated
biosynthes~ed
the major
AND HUJII~*
in a manner
approxi-
of triglycerides
holds
from the L-phosphatidic as a final step.
triglycerides
emphasizes
theories of fatty
the caution
acid distribution
in
fats.
The specificity might
soybean
that the diglyceride
that
is (S)-3-aceto-r,z-distearin.
biosynthetic
If this scheme for biosynthesis
Our finding asymm~t~ca~y
that the
The HIRSCHMANN”
that seed oil might be synthesized
pathway.
revealed
rulel691’I indicates
oil have the (S)-configuration.
seed. In their study of lipid classes in maturing found
(enzymatic~,
all one enantiomer.
of the CAHN-INGOLD-PRELOG
of I. edgeworthG
triglycerides
and RENKONEN~~
was essentially
provide
of triglyceride
a useful system
synthesis
for studying
in I. edgezworthii the biosynthesis
seed suggests
that it
of triglycerides
in the
plant kingdom. ACKNOWLEDGEMENTS Optical analyses
rotatory
dispersion
by R. KLEIMAN,
by
R.
G.
and microanalyses
POWELL,
gas-liquid
chromatographic
by i%rs. C. I&GREW.
REFERENCES I 2 3 4 5 6 7 8 g 10
II 12 13 14 15 16 17 18 19
M. 0. BAGBY, C. R. SMITH, JR. AND I. A. WOLFF,L@&, I (1966) 263. H.P. KAUFMANN AND M.KELLER,C~~~. Bev., 81 (1948) 152. W. SCHLENK, JR.,J. Am. Oil Chemists’ Sot., 42 (1965) 945. W. SCHLENK, JR.,Angew. Chem. Intern.Ed. En&, 4 (1965) 139. L. J. MORRIS, Biochem.Biophys. Res. Commun., 18 (1905) 495. M. H. COLEMAN, J. Am. Oil Chemists’ Sot., 42 (1965) 1040. L. J. MORRIS, Biochem.Bio$hys. Res. Commun., 20 (1965) 340. R. KLEIMAN, R. W.MILLER, F. R. EARLE AND I. A. WOLFF,Lipids, I (1966) 286. H. BROCKERHOFF, R. J. HOYLEANDN. WOLMARK, Biochim.BioPhys. Acta. 116(1966) .I 67. , H. BROCKERHOFF AND M. YURKOWSKI, J. Lipid Res., 7(x966)62: M. A. HOEFNAGEL, A.VANVEEN AND P. E. VERKADE, Rec. Trav. China.,81 (x962)461. C. LITCHFIELD, R. D. HARLOW AND R. REISER, J. Am. O~~Chem~sts* Sot., 42 (1965) 849. 0. RENKONEN, J. Am. Oil Chemists'Sot.,42 (1965) 298. J.C. SOWDEN AND H.O. L.FIsHER,J. Am. Chem. Soc.,63 (1941) 3244. R.S.CAHN,C. K.INGOLDANDV.PRELOG, Eqberientia, 12(1956) 81. R.S. CAHN, 1.Chem. Educ.. 41 11964) 116. H. HIRSCH&N, J.Biol.Chem.,‘235'ir960)2762. 0. HIRAYAMA AND K. HUJII, Agr. Biol. Chem. Tokyo, 29 (1965) I. E. P. KENNEDY, Federation PYOC., 20 (1961) 934. Biochim. Bioehys.
Acta, 137 (1967) 475-477
BIOCHIMICA ET BIOPHYSICA ACTA BBA 55320
ASYMMETRIC
TRIGLYCERIDES
FROM IMPATIENS
EDGEWORTHII
SEED OIL M. 0. BAGBU
AND
C. R. SMITH,
JR.
NortAern Regional Research Laboratory*, (Received
Peoria, Ill. (U.S.A.)
September rgth, 1966)
SUMMARY
Impatiens edgeworthii Hook F. seed oil, which contains acetate, parinarate, and the more common fatty acylgroups, was hydrogenated to give mainly cr-acetodistearin. The hydrogenated oil produced a plain negative optical rotatory dispersion curve. Comparison of the dispersion curve with those of cr-acetodiacyl triglycerides synthesized by stereoselective methods indicates that the triglycerides of Impatiens edgeworthii seed oil have the (SJ-configuration.
INTRODUCTION
During an investigation1 of the stereochemistry of cl-parinaric acid from Impatiens edgeworthii Hook F. seed oil, we identified acetic acid as a constituent of the oil. KAUFMANN AND KELLER~ isolated a large quantity of acetodiparinarin from other Tmpatiens species. The possible presence of acetodiparinarin, together with the reports+6 that measurable optical rotation can be observed for asymmetric triglycerides containing greatly different fatty acyl groups, prompted our further study of the glyceryl diacylacetates ** from I. edgeworthii seed oil. Three reviewss+f@ in 1965 summarize the many attempts to determine whether triglycerides are ~ymmet~cally biosynthesized and whether they exist as optically active enantiomers or as racemic mixtures. Such studies have been greatly facilitated by the availability of sensitive ultraviolet spectropolarimeters. MORRIS' developed a procedure that enabled him to detect optical rotation of a modified triglyceride and thus to demonstrate that some natural triglycerides occur predominantly as a single enantiomer. KLEIMAN et al.8 reported that the acetic acid in Euonymus verrZtcos@s seed oil occurs on the cc-position of glycerol. Those Euonymus triglycerides had measurable optical rotation, and they were of the (S)-configuration. BROCKERHOFF etaZ.a~lo have concluded that some natural triglycerides are asymmetric but they did not measure rotations. * This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture. ** Awls as used in this paper refer to long-chain fatty groups, such as palmitoyl and stearoyl, with the exclusion of acetyl. Biochim. Biophys.
Acta, 137 (1967) 475-477
476
M. 0.
EAGBY, C. R. SMITH
EXPERIMENTALPROCEDURESANDRESULTS The I. e~ge~o~~~~~ seed oil (stored as a pentane-hexane solution after isolation as reported previouslyl) was freed of solvent under a stream of N, with care being taken to exclude air and light. To eliminate the reactivity of the polyunsaturated acids in the oil and to provide a material amenable to handling, the oil was hydrogenated in absolute ethanol with a platinum catalyst. The saturated oil had an infrared spectrum (Ccl, solution) like that of ol-acetodistearinll. (Found: C, 73.2; H, 11.6; N, 0.0; P, 0.0; S, 0.0, Acetodisteari~, C41H,80B, requires C, 73.8; H, x1.8). Thin-layer chromatography on silica gel G* (solvent system : 10% diethyl ether in light petroleum, b.p. 33-57”, spots visualized by charring with H,SO,) revealed mainly diacyl acetoglyceride with very weak spots corresponding to conventional triglycerides and unsaponifiables. Gas-liquid chromatography essentially as described by LITCHFIELD, HARLOW AND REISER I2 on 3% JXR Gaschrom Q, 0.5 mx3 mm stainless-steel column, programmed from 120' at 4” per min with on-column injection 320°, flame detector 350”, indicated only triglycerides with one acetyl group. The aceto-palmito-stearin represented about 3.3% of the total area of the gas-liquid chromatographic peaks. The presence of acetic acid was confirmed as the only low molecular weight acid, as described earlier 1. To remove the small amount of contaminants from the diacyl acetoglyceride, the hydrogenated oil (0.19 g) was chromatographed on IO g of silica gel (Adsorbosil, 3060 mesh) with diethyl ether-light petroleum, b.p. 33-57O, as a discontinuous gradient using gradually increasing amounts of diethyl ether. The bulk of the material was eluted with 7% diethyl ether (v/v). Homogeneity of various fractions was determined by thin-layer chromatography, and fractions consisting of only a-acetotriglyceride were combined to give 0.14 g of a white solid. The aggregate was slurried with Darco G6o charcoal in acetone, filtered, and freed of solvent to give 0.~3 g of product. It was then recrystallized from I5 ml of ethanol at 8” to give 0.11 g of a solid which had m-p. 51-52.5’; [cc]g-0.6”, [cr]&--I.r”, [cc]~&--2.0°, [cc]&%-2.9”, [a&&%-9.1' in hexane (c, 2.28). Optical rotatory dispersion was obtained with a Cary recording spectropolarimeter. Thin-layer chromatography showed only one spot ; it corresponded to a-acetyl diacyl triglyceride13. Saponification of the or-acetyl diacyl triglyceride yielded acetic, palmitic, and stearic acids as the only acid components determined by gas-liquid chromatography. Gas-liquid chromatographic analysis of methyl esters of the water-insoluble acids showed 4.5% of methyl palmitate and 95.5% of methyl stearate. This composition compares to that of the original hydrogenated oil’. DISCUSSION The results from thin-layer chromatography, gas-liquid chromatography, and elemental analyses indicated that hydrogenated I. edgeworthii seed oil consists mainly of cr-acetodistearin. Comparison of its optical rotatory dispersion with that of a-acetotriglycerides, synthesized by R. KLEIMAN of this laboratory** by the stereoselective * The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar productsnot mentioned. ** R. KLEIMAN, personal communication.
477
ASYMMETRIC TRIGLYCERIDES FROM A SEED OIL methods
of SOWDEN AND FISHER”
hydrogenated
acetotriglyceride
Application convention
provides
asymmetry
seed
that the main acetotriglyceride
must be the result of a selective
evidence
suggesting
mating the KENNEDY’*
for plants, one might speculate acid intermediate necessary
postulated
natural
when applying
by KENNEDY
process
some of the prevalent
in
resulting
The
I. edgeworthii
seed, HIRAYAMA
was acetylated
biosynthes~ed
the major
AND HUJII~*
in a manner
approxi-
of triglycerides
holds
from the L-phosphatidic as a final step.
triglycerides
emphasizes
theories of fatty
the caution
acid distribution
in
fats.
The specificity might
soybean
that the diglyceride
that
is (S)-3-aceto-r,z-distearin.
biosynthetic
If this scheme for biosynthesis
Our finding asymm~t~ca~y
that the
The HIRSCHMANN”
that seed oil might be synthesized
pathway.
revealed
rulel691’I indicates
oil have the (S)-configuration.
seed. In their study of lipid classes in maturing found
(enzymatic~,
all one enantiomer.
of the CAHN-INGOLD-PRELOG
of I. edgeworthG
triglycerides
and RENKONEN~~
was essentially
provide
of triglyceride
a useful system
synthesis
for studying
in I. edgezworthii the biosynthesis
seed suggests
that it
of triglycerides
in the
plant kingdom. ACKNOWLEDGEMENTS Optical analyses
rotatory
dispersion
by R. KLEIMAN,
by
R.
G.
and microanalyses
POWELL,
gas-liquid
chromatographic
by i%rs. C. I&GREW.
REFERENCES I 2 3 4 5 6 7 8 g 10
II 12 13 14 15 16 17 18 19
M. 0. BAGBY, C. R. SMITH, JR. AND I. A. WOLFF,L@&, I (1966) 263. H.P. KAUFMANN AND M.KELLER,C~~~. Bev., 81 (1948) 152. W. SCHLENK, JR.,J. Am. Oil Chemists’ Sot., 42 (1965) 945. W. SCHLENK, JR.,Angew. Chem. Intern.Ed. En&, 4 (1965) 139. L. J. MORRIS, Biochem.Biophys. Res. Commun., 18 (1905) 495. M. H. COLEMAN, J. Am. Oil Chemists’ Sot., 42 (1965) 1040. L. J. MORRIS, Biochem.Bio$hys. Res. Commun., 20 (1965) 340. R. KLEIMAN, R. W.MILLER, F. R. EARLE AND I. A. WOLFF,Lipids, I (1966) 286. H. BROCKERHOFF, R. J. HOYLEANDN. WOLMARK, Biochim.BioPhys. Acta. 116(1966) .I 67. , H. BROCKERHOFF AND M. YURKOWSKI, J. Lipid Res., 7(x966)62: M. A. HOEFNAGEL, A.VANVEEN AND P. E. VERKADE, Rec. Trav. China.,81 (x962)461. C. LITCHFIELD, R. D. HARLOW AND R. REISER, J. Am. O~~Chem~sts* Sot., 42 (1965) 849. 0. RENKONEN, J. Am. Oil Chemists'Sot.,42 (1965) 298. J.C. SOWDEN AND H.O. L.FIsHER,J. Am. Chem. Soc.,63 (1941) 3244. R.S.CAHN,C. K.INGOLDANDV.PRELOG, Eqberientia, 12(1956) 81. R.S. CAHN, 1.Chem. Educ.. 41 11964) 116. H. HIRSCH&N, J.Biol.Chem.,‘235'ir960)2762. 0. HIRAYAMA AND K. HUJII, Agr. Biol. Chem. Tokyo, 29 (1965) I. E. P. KENNEDY, Federation PYOC., 20 (1961) 934. Biochim. Bioehys.
Acta, 137 (1967) 475-477