Diesters of Diols in Wheat Leaf Wax1 A.P. TULLOCH, National Research Council of Canada, Prairie, Regional Laboratory, Saskatoon, Saskatchewan, Canada

described by Allebone et al. (3) and gave quantitative recovery.

ABSTRACT

Diesters have been isolated from the leaf wax of spring wheat, T r i t i c u m aestir u m , L. (Selkirk variety) by chromatography. The diesters, which form 3% of the wax and which were shown by gas liquid chromatography to be a mixture of C51-C60 esters, consist largely of trans 2-docosenoic and trans 2-tetracosenoic acid esters of 1,9-nonanediol, 1,10-decanediol, 1,11-undecanediol and 1,12dodecanediol. The structures of the components were confirmed by synthesis.

Gas Liquid Chromatography

I N T R O D U C T I ON

Small amounts of esters of trans 2-docosenoic and trans 2 - t e t r a c o s e n o i c acids were detected in leaf wax of Little Club wheat (1) but their complete structure was not determined. Similar esters have now been isolated from leaf wax of Selkirk variety of spring wheat, T r i t i c u m aestir u m L. and shown to consist mainly of diesters of C9-C12 a,co-diols and the above two acids. E X P E R I M E N T A L PROCEDURES

NMR spectra were obtained with a Varian HA-100 spectrometer; chemical shifts are in part per million from internal tetramethylsilane and the solvent was carbon tetrachloride. Thin Layer Chromatography

Analytical thin layer chromatography (TLC) carried out using Silica Gel G plates, prepared with spreader setting 0.025 in (0.675 ram), the solvent was chloroform containing 1% ethanol v/v (2), and Rf values were as follows: tetracosyl tetracosanoate, 0.72; diol diester and methyl tetracosanoate, 0.55 ; tetracosanol, 0.15 ; spots were detected by spraying with 50% H2SO 4 and heating with an IR lamp. Ethanolysis products were examined in benzene containing 30% ethyl acetate by volume and Rf values were: ethyl trans 2-tetracosenoate, 0.72; tetracosanol, 0.43; 1,10-decanediol 0.04. Preparative TLC was carried out on 20x20 cm plates of Silica Gel G, 1.3 mm thick, using chloroform containing 1% ethanol by volume as solvent; the rest of the procedure was as was

lIssued as NRCC No. 12064

Analytical gas liquid chromatography (GLC) was performed using an F and M model 402 gas chromatograph with flame ionization detectors, fitted with a 5 ft x 1/8 in. stainless steel column packed with 80-100 mesh, acid washed and silanized, Chromosorb W coated with 2% silicone SE 30. The temperature was programmed, at 2 C/min, from 300 to 380 C for analysis of diol diesters, from 100 to 160 C for diol diacetates and from 125 to 225 C for ethyl esters; the flow rate was 40 ml helium/min. Preparative GLC was carried out with a unit of conventional design with thermal conductivity detectors fitted with a 3 ft x lJ4 in. stainless steel column packed with 10% silicone SE-30 on 60-80 mesh Anachrom ABS; the flow rate w a s 20 ml helium/min. isolation of Diol Diesters

Leaf wax (10.5 g), isolated as previously described (1), was chromatographed on Biosil A (Bio Rad Laboratories) (200 g). When most of the free alcohols had been eluted by hexanechloroform (17:3), a mixture (3.5 g ) o f free acids, hydroxy 3-diketones, diol diesters, alcohols and unidentified material was eluted by hexane-chloroform, 1:1. After treatment with diazomethane, this mixture was rechromatographed on Biosil A ( t 0 0 g). Methyl esters (from free acids) and alcohols were eluted first and crude diol diesters were then eluted by hexane-chloroform (17:3). Pure diesters (0.3 g) were obtained by preparative TLC. NMR: CH 3, 0.88; CH2, 1.27; H-4 of a f t - u n s a t u r a t e d ester, 2.18 (multiplet); CH 2 of -CH2-O-COR, 4.03 (triplet); H-2, 5.70 (doublet, J=16 cps); H-3, 6.82 (two triplets, J=16 cps). The ratio of the intensities of the last four signals was 2:2:1:1 in agreement with the foregoing assignments. A portion of the diesters was hydrogenated in ethyl acetate over 5% palladium charcoal and the product completely recovered by extraction of the catalyst with boiling chloroform. Ethanolysis of Diol Diesters

Diol diesters (0.21 g) were refluxed overnight in ethanol containing 5% HC1 by weight (25 ml), chloroform (25 ml) was added, the mixture neutralized with Ag2CO3, filtered, Ag

641

A.P. TULLOCH

642 T

70-

mixed mp 80.0-81.5 C. The C 9 diol was not isolated but was identified by the retention times of the free diol and the diacetate.

|

60

Synthesis of the Unsaturated Acids

50 54

55

50 -

w co z

20-

a..

10

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)

52

oI :

o u.l

5O

3O 55 20

l0it300

5 ~ 320

340 360 TEMPERATURE,

3so

400

C

FIG. t. GLC separation of hydrogenated diol diesters; A, before, and B, after addition of 1,10-deeanediol didocosanoate. salts washed with chloroform and the combined filtrates evaporated. TLC (benzene containing 30% ethyl acetate) showed that complete ethanolysis had occurred giving ethyl esters and diols. The mixture was acetylated (acetic anhydride and pyridine) and chromatographed on a silicic acid column. Ethyl esters (0.15 g) were eluted with hexane-ether (97:3) and diol acetates (0.044 g) with hexane-ether (9:1). The ethyl esters were identified by GLC and NMR spectroscopy as previously described (1). Ethyl t r a n s 2-docosenoate and trans 2-tetracosenoate were separated by preparative GLC at 240 C, saponified and the acids crystallized from acetone. T r a n s 2-docosenoic acid had mp and mixed mp 71-72 C and trans 2-tetracosenoic acid had mp and mixed up 76.5-77.5 C. Diol acetates were separated by preparative GLC at 150 C and free diols, obtained by refluxing with methanolic hydrogen chloride, were crystallized from benzene. 1,10-Decanediol had mp and mixed mp 72.5-73.5 C, 1,11-undecanediol had mp and mixed mp 60.5-61.5 C and 1,12-dodecanediol had mp and LIPIDS, VOL. 6, NO. 9

T r a n s 2-docosenoic acid was synthesized as described by Artamonov (4) and had mp 70.5-71.5 C reference 4 gives 68.5-69 C). The methyl ester had mp 48-49C. C23H4402 calculated: C 78.34, H 12.58. Found: C 78.54, H 12.64. T r a n s 2-tetracosenoic acid was synthesized by elimination of acetic acid from methyl 3-acetoxytetracosanoate (5). 3-Hydroxytetracosanoic acid (6) was converted to methyl 3 - h y d r o x y t e t r a c o s a n o a t e which had mp 69.5-71.5 C. C25H5oO3 calculated: C 75.32, H 12.64. Found: C 75.42, H 12.51. The hydroxy ester was acetylated (acetic anhydride and pyridine) and distilled, bp/0.1 mm 200 C. A solution of acetoxy ester (2.18 g) in t-butanol (200 ml) was refluxed and a mixture of 0.1 N aqueous NaOH (99 ml) and t-butanol (70 ml) added slowly over 40 rain. The solution was refluxed for a further 30 rain, cooled, acidified and the products extracted with chloroform. The products were converted to methyl esters with methanol containing 5% HC1 by weight and separated on a silicic acid column. Methyl t r a n s 2-tetracosenoate (1.09 g, 58% yield) was eluted with hexane-ether 96:4 and methyl 3-hydroxytetracosanoate (0.81 g) was eluted with hexane-acetone 95:5. Crystallization from acetone gave methyl t r a n s 2-tetracosenoate with mp 53.5-54.5 C. C25H4802 calculated: C 78.88, H 12.7t. Found: C 79.06, H 12.77. Trans 2-tetracosenoic acid was obtained by saponification and after crystallization from acetone and had mp 76.5-77.5 C. C24H4602 calculated: C 78.62,. H 12.65. Found: C 78.50,

H 12.52.

Synthesis of Diols and C54 Diol Diester

Diols were obtained by reduction of the corresponding dicarboxylic acids with LiA1H4 in the usual way and crystallized from benzene. The C1o diol had mp 72-72.5 C (lit (7) 32 C), The C 11 diol nap 60.5-61.5 C (lit (8) 62-62.5 C) and the C12 diol mp 80-81 C (lit (9)80-81 C). 1,10-Decanediol didocosanoate. 1,10-Decanediol (0.61 g) was dissolved in methylene chloride (20 rot) and pyridine (2 ml) and a solution of docosanoyl chloride (2.50 g) in methylene chloride (5 ml) added and the mixture refluxed overnight. The product was extracted with chloroform, washed with 2 N CH1 and with water and crystallized from chloroform. The mp was 78.5-80 C. C 5 4 H l o 6 0 4 calculated: C 79.15, H 13,04.

643

DIESTERS OF WHEAT LEAF WAX TABLE I

TABLE II

Composition of Diol Diesters

Composition of Acids of Diol Diesters

Carbon atoms No. 51 52 53 54 55 56 57 58 59 60

Composition (by weight) 0.7 2.3 8.0 13.6 21.4 22.2 16.7 10.2 3.l 1.8

Composition calculated from random distribution ----3.9 9.3 16.6 21.7 20.1 16.8 "7.4 4.2

Acid

Percentage (by weight).

Docosanoic 7u 2-docosenoic Tetracosanoic Trans 2-tetracosenoic Unidentified a

5.0 46.7 5.2 40.4 2.7

.

afire unidentified components.

acids making a surprising constrast with the acids of the monoesters and free acids of the wax which contain about 70% of saturated C16-C30 acids in addition to the above two unsaturated acids (A.P. Tulloch and L.L. HoffFound: C 79.1 1, H 13.26. This diester had the man, unpublished work). Except for the previsame Rf on TLC as the natural compounds. ous finding in Little Club wax (1) these acids have not been found in nature before; trans RESULTS AND DISCUSSION 2-docosenoic acid has, however, been syntheThe diol diesters had unusual chromatog- sized (4). Of the diols only 1,12-dodecanediol has raphic properties in that they were eluted from a silicic acid column just after the major alcohol been reported previously in natural compounds; fraction but on TLC they had a much greater it occurs as an estolide in conifer waxes (10). Rf than the alcohols making it possible to The chain length distribution, approximately equal amounts of odd- and even-numbered purify them by preparative TLC. GLC analysis showed that the diesters had components, is quite unusual since long chain natural products are usually either almost all chain lengths ranging from CsI to C60. The NMR spectrum contained characteristic signals even-numbered or all odd-numbered. Very long chain diols have been isolated at 5.70 ppm and 6.82 ppm (with coupling constants of 16 cps) due to H-2 and H-3 from saponified carnauba wax (11) and apple wax (12) but whether they are present in the respectively of trans a f t - u n s a t u r a t e d esters (1). original wax as diol diesters or as hydroxy Ethanolysis of the diesters gave ethyl esters and monoesters is not clear. Diesters of long chain diols, GLC analysis indicated that the ethyl a,co-1 diols are present in beeswax (2) and esters were derivatives of trans 2-docosenoic and trans 2-tetracosenoic acids (1) and this was diesters of aft diols in animal skin lipids (13). confirmed by isolation of the individual acids The diesters of 2,3-diols in wax of turkey preen and comparison with authentic synthetic acids. gland (14) are of interest since the C19-C23 diols consist of approximately equal amounts The chain lengths of the diols were indicated by of even- and odd-numbered components. GLC analysis to be C9, CIO , C I I and C12 and Very small amounts of diesters of C2-C4 the structures were also confirmed by isolation diols may be present in plant lipids (15) but no and comparison with synthetic compounds. The diol diesters were not completely re- diesters of the medium chain length diols solved by GLC due to partial separation of reported here seem to have been described saturated and unsaturated components but a previously. fairly good separation (Fig. 1) was obtained ACKNOWLEDGMENTS after hydrogenation. The chain lengths of the components were established by reanalysis after Experimental assistance by L.L. Hoffman, NMR addition of synthetic Cs4 diester (Fig. 1B). The composition of the hydrogenated diesters is TABLE III given in Table I, the results being fairly similar Composition of Diols of Diol Diesters to those calculated assuming a random distribution and the presence of C22 and C24 acids

only. The composition of the acids is shown in Table II and that of the diols in Table llI. The acids of the diol diesters are thus limited almost entirely to the trans 2-unsaturated C22 and C24

Diol

1,9-Nonanediol 1,10-Decane diol 1,11 -Undecane diol 1,12-Dodecanediol

Percentage (by weight) 13.6 32.4 34.4 19.6

_

LIPIDS, VOL. 6, NO. 9

644

A.P. TULLOCH

spectra by M. Mazurek a n d elemental analyses by W.C. Haid. REFERENCES 1. T ulloch, A.P., and R.O. Weenink, Can. J. Chem. 4 7 : 3 1 1 9 (1969). 2. Tulloch, A.P., Chem. Phys. Lipids 6:235 (1971). 3. Allebone, J.E., R.J. Hamilton, B.A. Knights, B.S. Middleditch a n d D.M. Power, Ibid. 4:37 (1970). 4. A r t a m o n o v , P.A., Zh. Obseheh. Khim. 22:1992 (1952). 5. Tulloeh, A.P., and J.F.T. Speneer, Can. J. Chem. 4 2 : 8 3 0 (1964). 6. Skogh, M., Acta Chem. Scand. 6:809 (1952). 7. Hill, H.S., and H. Hibbert, J. Amer. Chem. Soe. 4 5 : 3 1 2 4 (1923).

LIPIDS, VOL. 6, NO. 9

8. Chuit, P., Helv. Chim. Aeta 9:264 (1926). 9. Lespieau, R., Compt. Rend. 187:605 (1928). 10. Kariyoni, T., H. Ageta a n d K. Isoi, Yakugaku Zasshi 79:54 (1959). 11. Downing, D.T., Z.H. Kranz a n d K.E. Murray, Aust. J. Chem. 14:619 (1961). 12. Mazliak, P. Phytoehem. 1:79 (1962). 13. Nicolaides, N., H.C. Fu a n d M.N.A. Ansari, Lipids 5:299 (1970). 14. Hansen, I.A., B.IC Tang a n d E. Edkins, J. Lipid Res. 10:267 (1969). 15. Bergelson, L.D., "Progress in the Chemistry of Fats a n d Other Lipids," Vol. 10, Pergamon Press, Oxford, 1969, p. 239.

[Received April 2, 1971]