Journal of Chemical Ecology, Vol. 15, No. 1, 1989

PRESENCE OF LONG-CHAIN DIALKYL ETHERS IN CUTICULAR WAX OF THE AUSTRALIAN CHRYSOMELID BEETLE Monolepta australis I

I A N A. S O U T H W E L L and IAN A. S T I F F North Coast Agricultural Institute Wollongbar, N.S. W. 2480, Australia

(Received August 24, 1987; accepted November 13, 1987) Abstract--lnvestigation of the lipid extract of the Australian chrysomelid beetle, Monolepta australis, has revealed a novel homologous series of longchain, unsaturated-saturated dialkyl ethers in the cuticular wax. Gas chromatography-mass spectrometry, proton magnetic resonance, infrared spectroscopy, and chemical degradation have shown that ethers of formula CH3(CH2)gCH=CH(CH2)60(CH2)I_, 16CH3 predominate. Key Words--Monolepta beetle, red-shouldered leaf beetle, Monolepta austrails, lipid extract, unsaturated aliphatic ethers, 7-octadecenyl alkyl ethers, gas chromatography-mass spectometry, Coleoptera, Chrysomelidae.

INTRODUCTION Long-chain, saturated aliphatic ethers have recently been reported from the cuticular waxes o f the locust Locusta migratoria cinerascens (Genin et al., 1987). Concurrently we isolated and orally reported a homologous series of long-chain, unsaturated-saturated dialkyl ethers from the indigenous monolepta or redshouldered leaf beetle, M o n o l e p t a australis (Jacoby). M. australis is a chrysomelid beetle approximately 6 mm in length endemic to the subtropical central coast of eastern Australia. Swarms of the beetle now attack the foliage, flowers, and fruit o f avocado, macadamia, lychee, mango, stone fruit, citrus, and eucalyptus windbreak species (Murray, 1982). Present methods o f monitoring and control could be greatly improved by the use of t Presented at the 9th National Conference of the Organic Chemistry Division of the Royal Australian Chemical Institute in Adelaide, South Australia, May 11-15, 1986. 255 0098-033118910100-0255506.0010 ~_1 1989 Plcnum Publishing Corporation

256

SOUTHWELL AND STIFF

pheromone-based traps. Consequently, the chemical constituents of M. australis were extracted and fractionated and are currently being purified and evaluated for pheromone-type activity. This first paper reports the isolation and identification of a novel series of long-chain ethers from the lipid fraction of the beetle.

METHODS AND MATERIALS

Swarms of M. australis (1.5 kg, approx. 93,000) were collected from ornamental trees at Wollongbar, New South Wales (28°50'S, 153°25'E) in summer and immediately soaked in doubly distilled petroleum spirit (bp 50-70°C May and Baker). After 20 days, the beetles were filtered, washed (2 × 1 liter, 5 min), soaked (2 liters, 14 hr), filtered, washed (2 x 1 liter) with petroleum spirit and the combined solutions concentrated to give a yellow wax (21.5 g, 1.4%). The washed beetles were then extracted at 25°C with dichloromethane and acetone for the investigation of more polar constituents. The yellow wax (21.5 g) was preabsorbed onto alumina (40 g) and subject to flash liquid chromatography on alumina (160 g). Elution with aliquots (100 ml) of petroleum spirit (50-70°C) gave first a colorless wax fraction (IR 720 cm -t) (0.62 g) followed by an orange-colored oil (3.10 g) showing some carbonyl (1740 cm -I) and ether (1100-1200 cm -j) infrared absorption. This second fraction was also preabsorbed on alumina (20 g) and subjected to flash chromatography on alumina (100 g). Elution with aliquots (50 ml) of light petroleum gave further wax (0.86 g) from fractions 1 and 2 and then an ether mixture (0.45 g) (IR 1118 cm -~) from fractions 3-5. Thin-layer chromatography on silica gel when run in 4 % ethyl acetate in petroleum spirit gave a single spot (Ry0.47). This mixture distilled at 220°C at 0.5 mm. [tH]NMR indicated an olefinic triplet at (55.35 (J 5.5 Hz, 2H), an oxymethylene triplet at (53.39 (J = 8.2 Hz, 4H), a methyl triplet at (50.88 (J = 7.0 Hz, 6H), and a prominent methylene signal at 61.26 (46-50 H). Signals for/3oxy and allylic methylenes were evident at ~51.56 (4H) and (52.01 (4H), respectively. Analytical gas chromatography was conducted on a Perkin Elmer Sigma 2B chromatograph using a 10-m x 0.22-mm ID, BPl-coated, fused-silica, opentubular (FSOT) capillary column using N2 as carder gas. Individual runs were temperature programmed from 100°C to 300°C with no initial holding period. Percentage compositions were computed with a Perkin Elmer Sigma 10B data station, and Kovats indices were calculated with respect to straight-chain hydrocarbon standards. Preliminary mass spectral data were obtained on an AEI MS 12 mass spectrometer at 70 eV ionizing voltage, 8000 V accelerating voltage with ion source at 200°C.

257

LONG-CHAIN D1ALKYL ESTERS

Combined gas chromatography-mass spectrometry was conducted on a Hewlett Packard MSD instrument using on-column injection into a 12-m x 0.2mm ID BP1 FSOT capillary column. Temperature was programmed to rise from 30°C to 220°C at 30°C/min and then to 300°C at 4°C/min. Proton magnetic resonance spectra were recorded at 300 MHz in CDC13 on a Bruker CXP 300 spectrometer. Chemical Degradation. The M. australis lipid ether fraction (1) (65.5 mg) in dry diethyl ether (2.0 ml) was treated with 3-chloroperbenzoic acid (46.8 rag, approx. 2 equiv.) at 25°C in the dark. After 64 hr, the solution was diluted with diethyl ether (20 ml), washed with: (1) 5% sodium bisulfite solution (5 ml), (2) 5 % sodium bicarbonate solution (2 x 5 ml), and (3) saturated sodium chloride solution (5 ml). The epoxide (5) solution was dried (Na2SO4) and without concentration stirred at 25°C with freshly prepared periodic acid (178 mg) (Willard, 1939). After 1 hr the mixture was diluted with diethyl ether (10 ml), washed with: (1) 10% sodium thiosulfate solution (5 ml), (2) saturated sodium chloride solution (5 ml), and dried (Na2SO4). Without concentration, the aldehyde mixture [6 + 7 (see Scheme 2 below)] was treated with lithium aluminium hydride (50.0 mg) and stirred. After 16 hr, excess reagent was decomposed by the dropwise addition of 10% ammonium chloride solution, dried (Na2SO4), filtered, and concentrated to give a colorless mixture of alcohols 8 + 9 (45.7 mg). This alcohol mixture was dissolved in dry chloroform (2.0 ml) and refluxed with excess trimethylsilyliodide (0.2 ml, 281 mg). After 1 hr, the mixture was cooled and decolorized by the dropwise addition of 10% sodium thiosulfate solution, diluted with diethyl ether, dried (Na2SO4), and concentrated. The resulting iodide mixture, 10 + 11 + 12, in dry diethyl ether (2.0 ml) was added to lithium aluminium hydride (103.6 mg) suspended in dry diethyl ether (1.0 ml) and stirred at 25°C. After 16 hr excess reagent was destroyed by the cautious dropwise addition of saturated ammonium chloride solution. The solution was dried (Na2SO4) and concentrated at 25 °C to give the hydrocarbon mixture 13 + 14 + 15. All reactions were followed to completion using TLC, GLC, and IR spectroscopy. Hydrocarbon products 13, 14, and 15 were also confirmed by GC coinjection of authentic standards.

RESULTS AND DISCUSSION

The aliphatic ethers in M. australis comprise in excess of 2 % of the total lipid fraction and in excess of 300 ppm of the entire fresh insect. Their presence in the cuticular wax layer was established by isolating the ether fraction from a brief (10 min), cold (0°C) extraction. Identification was based on IR and NMR spectroscopy, gas chromatography (GC), and gas chromatography-mass spectrometry (GC-MS). IR absorption at 1118 cm -~, when coupled with retention data from thin-layer, liquid,

258

SOUTHWELL AND STIFF

and gas chromatography clearly indicated aliphatic ethers. GC indicated a mixture containing nine components, each contributing more than 1% to the total ether fraction (Figure 1). From the retention index data, the major (60%) component (Figure 1, peak 3) seemed to represent the middle member of a fivemember homologous series. Traces of members of this series preceding peak 1 (Figure 1) were also evident from the GC and MS data. [~H]NMR indicated - - C H 2 - - C H = C H - - C H 2 (X 1), - - C H 2 - - C H 2 - - O - - C H 2 - - C H 2 (X 1), - - CH 2-- ( X 23-25) and - - C H 2 - - CH 3 ( x 2), suggesting a long-chain ether series with formula: CH3--(CH2)a--CH=CH--(CH2) b-O-(cH2) c-CH 3

(1)

GC-MS analysis defined each member of the series more specifically (Table 1). Molecular ion peaks were prominent for all except peak 6. All components gave strong fragments at m/z 250 and 281, representing fragment ions 2 and 4, resulting, respectively, from C - - O and ~ - - C - - C cleavage (Scheme 1). The alternative a - - C - - C fragmentation was evident from prominent mass spectral peaks at M-267 in all members of the series except chromatographic peak 2. The alternative fragmentation in the unsaturated chain was absent. Support for this assignment is seen in the typical C,,H2,,+~, C,,H2,,_I, and C,,H2~-2 decomposition fragments in the mass spectra. Thus an octadecenyl structure for the unsaturated half of the ether is established. The position of the double bond in the

3

Peak No. i

5

10

I

I

2'5

15 I I

30

20

I

I 35

25

I

30

I

40

RI

%

3114 3212 3322 3345 3411 3422 3487 3510 3560

18.4 2.8 60.0 1.4 1.6 1.6 1.4 4.2 4.0

R t (min) RI

FIG. 1. The total ion current gas chromatogram of the long-chain ether fraction of Monolepta australis extract showing retention time (R,) and retention index (R~) axes and percentagecontribution of major components.

259

LONG-CHAIN DIALKYL ESTERS

/m(CH2)cCH3 /z M3- 2 6 7 x i ,

i

~

~ i

•

CH3-(CH2)a-CH=CH-(CH2)b~O'CH2'- (CH2)c_I-CH 3 Y

1

/H

4

4" [CHa-ICH2)

a-cH mlz

=CH-lCH21

b]-H

250 2

SCHEME | .

TABLE 1. MASS SPECTRAL INTENSITIES OF SIGNIFICANT PEAKS (MOL WT IN PARENTHESES) FOR MAIN HOMOLOGOUS SERIES MEMBERS OF ALIPHATIC ETHER LIPIDS OF Monolepta australis

Peak No. m/z

M+ 281 250 M-267

1

2

3

6

8

10.7 (450) 5,3 40.6 9.9 (183)

4.4 (464) 5.2 10.5 < 1.0 (197)

6.5 (478) 3.3 16.2 6.6 (211)

< 1.0 (492) 10.8 19.5 6.3 (225)

5,4 (506) 6.8 25.4 5.4 (239)

260

Sou'rHWELLANDS'nFF

chain was, as anticipated (Hallgren et al., 1959; Ryhage and Stenhagen, 1963; McCloskey and McClelland, 1965), not available from the mass spectral data. The M-267 and subsequent C,,H2,,+~ fragments indicated that chromatographic peaks 1, 2, 3, 6, and 8 were, respectively, tridecyl (c = 12), tetradecyl (c = 13), pentadecyl (c = 14), hexadecyl (c = 15), and heptadecyl (c = 16) octadecenyl ether. Of the numerous methods available for the location of double bonds in unsaturated fatty acid or other alkene derivatives (e.g., McCloskey and McClelland, 1965; Capella and Zorzut, 1968; Niehus and Ryhage, 1968; Abley et al., 1970; Dommes et al., 1976; Blomquist et al., 1980; Suzuki et al., 1981), a chemical degradation method best suited to monoenic series (Privett, 1966) was used. In our method (Scheme 2), the mixture of ethers (1) was epoxidized with 3-chloroperbenzoic acid, and the resulting epoxide series (5) was cleaved with periodic acid to give aldehydes 6 and 7. Without separation, this mixture was reduced to the alcohols 8 and 9 and treated with the ether-cleaving agent trimethylsilyliodide (Jung and Lyster, 1977; Olah, 1979) to give iodides 10, 11, and 12. These were reduced to their respective alkanes undecane 13, heptane 14, and a tridecane-heptadecane mixture (15)'fixing the double bond in the 7-position of the octadecenyl chain in contrast with the 9-position as would be expected from an ether derived from the more usual oleyl derivatives. The retention times and indices of the major starting material and products are shown in Table 2. The saturated side of the ether linkage of the homologous series was confirmed by using GC to follow the presence of the five-membered homologous series through the entire reaction sequence (Scheme 2). In each reaction mixture, the presence of C~3 to C~7 alkyl chains in an approximate ratio of 13:3:60.: 2:3 was cleady observed, giving finally tridecane, tetradecane, pentadecane, hexadecane, and heptadecane (15). This then confirmed the structure of the homologous series as 1 where a = 9, b = 6, and c = 12-16, and GC peaks 1, 2, 3, 6, and 8 (Figure 1) were assigned, respectively, to tridecyl, tetradecyl, pentadecyl, hexadecyl, and heptadecyl 7-octadecenyl ether. Peaks 4, 5, 7, and 9 and their mass spectra are less definitive and await further identification.

CONCLUSION This occurrence of long-chain, alkenyl-alkyl ethers in the lipid extract of

Monolepta australis is, to our knowledge, the first reported occurrence of such compounds in either their natural or synthetic state. Recent reviews of insect lipids have not included any such ethers (Blomquist and Jackson, 1979; Hadley, 1981; Lockey, 1985; Lockey, 1986, personal communication). As far as we know, this then becomes the second formally reported occurrence of long-chain

261

LONG-CHAIN DIALKYL ESTERS

CHa(CH2) O - C H = C H - ( C H z )

6-O-(cH2)cCH3

1

CH 3(CH2) 0 - C H - C H - ( C Hz) 6 - O - ( C H 2 ) c C H a

"o"

CHa(CH2) 9 ~ c H O

5

-I-

C H O - ( C H 2 ) 6 - O - (CH2)cCH3

6

7 iii

CH3(CH2)o- CH2OH -F

CHzOH-(CH2)|-O-(CH2)cCH3 9

8

CHa(CHi)e-CH21

-I-CH21-(CH2)6-1

10

11

CH3(CHz)eCH3 13

i

mcpba,

12

"k CH3(CHz,)6 H 14

Et20, 2 5 ° 6 4 h r

Jii LiA lt4, Et20 ' 2 5 ° 1 6 h r

ii

-I- I - ( C H 2 ) c C H 3

-4- H(CH2)cC Ha 15

o

HsIO 6 , E t 2 0 , 2 5 , I h r

iv TMSI. CHCI3, 62° I h r

o v L i A f H 4 . E t 2 0 , 25. 16hr SCHEME 2.

ethers in a biological environment. Although the isomeric 11-octadecenoxy radical is known in insects (vaccenyl acetate: Brieger and Butterworth, 1970; Meinwald et al., 1966; Jackson et al; 1981), the occurrence of 7-octadecenoxy derivatives in any biological environment is not known to us. On the other hand, the alkyl chain with predominantly odd numbers of carbon atoms is common and similar to half the Locusta migratoria lipid ethers, with carbon numbers Ct3, Cis, and Ci7 predominating (Genin et al., 1987). Speculation about the taxonomic significance of the occurrence of these

262

SOUTHWELL AND STIFF

TABLE 2. RETENTION TIMES (Rt) AND INDICES (R/) FOR MAJOR STARTING MATERIAL AND PRODUCTS OF REACTION SEQUENCE SHOWN IN SCHEME 2 (C = 14) Compound

R, Rt

1

5

6

7

8

9

10

11

12

13

14

1:

20.13 3322

21.47 3516

2.63 1290

14.09 2460

3.27 1364

14.61 2528

5.08 1526

5.55 1572

16.15 2735

1.20 1100

4.95" 700

4.' 151

"BP1 40-m column: 1 rain at 40°C, 10°C/min to 250°C.

lipid ethers in insects as diverse as M. australis and L. migratoria would be premature until the lipid fractions of other insects have been investigated. Indeed, the advances in GC-MS technology that have facilitated the identification of these components may show that lipid ethers are widespread among insects. Speculation concerning biogenetic pathways is also awaiting the outcome of further investigation, although the presence of unsaturated chains with an even number of carbon atoms in M. australis and saturated chains with an even number of carbon atoms in L. migratoria indicates an extra step in the pathway of one species. Although our biological assays showed no pheromone activity for these ethers when tested on M. australis, their possible role in the physiology and/or behavior of M. australis also awaits the outcome of further investigation. Acknowledgments--We are indebted to Mr. Noel Davies, Central Science Laboratory, University of Tasmania, for GC-MS analysis, and Dr. J.J. Brophy, Department of Chemistry, Universily of NSW, for spectral determinations and useful discussion.

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LONG CHAIN DIALKYLESTERS

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DOMMES, V., WIRTZ-PEITZ, F., and KUNAO, W.-H., 1976. Structure determination of polyunsaturated fatty acids by gas chromatography-mass spectrometry--a comparison of fragmentation patterns of various derivatives. J. Chromatogr. Sci. 14:360-366. GENIN, E., JULLIEN, R., and RUZEAU-BRAESCH,S. 1987. New natural aliphatic ethers in cuticular waxes of gregarious and solitary locusts Locusta migratoria cinerascens. J. Chem. Ecol. 13:265-282. HADLEY, N.F. 1981. Cuticular lipids of terrestrial plants and arthropods: A comparison of their structure, composition and waterproofing function. Biol. Rev. 56:23-47. HALLGREN, B., RYHAGE, R., and STENHAGEN, E., 1959. Mass spectra of methyl oleate, methyl linoleate and methyl linolenate. Acta Chem. Scand. 13:845-847. JACKSON, L.L., ARNOLD, M.T., and BLOMQUIST,G.J. 1981. Surface lipids of Drosophila melanogaster: Comparison of the lipids from male and female wild type and sex-linked yellow mutant, bisect Biochem. 11:87-91. JUNG, M.E., and LYSTAR,M.A. 1977. Quantitative dealkylation of alkyl ethers via treatment with trimethylsilyl iodide. A new method for ether hydrolysis. J. Org. Chem. 42:3761-3764. LOCKEY, K.H. 1985. Insect cuticular lipids. Comp. Biochem. Physiol. 81B:263-273. McCLOSKEY, J.A., and MCCLELLAND,M.J. 1965. Mass spectra of O-isopropylidine derivatives of unsaturated fatty esters. J. Am. Chem. Soc. 87:5090-5093. MEINWALD, J., MEINWALD,Y.C., WHEELER, J.W., and EISNER, T. 1966. Major components in the exocrine secretions of a male butterfly (Lycorea). Science 151:583-585. MURRAY, D.A.H. 1982. Life history of Monolepta australis (Jacoby) (Coleoptera: Chrysomelidae). J. Aust. Entomol. Soc. 21:119-122. NIEHAUS, W.G., and RYHAGE, R., 1968. Determination of double bond positions in polyunsaturated fatty acids by combination gas chromatography-mass spectrometry. Anal. Chem. 40:1840-1847. OLAH, G.A. 1979. Synthetic methods and reactions. 62. Transformations with chlorotrimethylsilane/sodium iodide, a convenient in situ iodotrimethylsilane reagent. J. Org. Chem. 44:12471254. PRIVETT, O.S., 1966. Page 91, in R.T. Holman (ed.). Progress in the Chemistry of Fats and Other Lipids, Vol. IX, Part 1. Pergamon Press, Oxford. RYHAGE, R., and STENHAGEN,E. 1963. Chapter 9, in F.W. McLafferty (ed.). Mass Spectrometry of Organic Ions. Academic Press New York. SUZUKI, M., ARIGA,T., SEKINE,M., ARAKI,E., and MIYATAKE,T. 1981. Identification of double bond positions in polyunsaturated fatty acids by chemical ionisation mass spectrometry. Anal. Chem. 53:985-988. WILLARD,H.H. 1939. Pages 168-175, in H.S. Booth (ed.). Inorganic Syntheses, Vol. 1. McGrawHill, New York.