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

STRUCTURES, ABSOLUTE CONFIGURATIONS, AND SYNTHESES OF VOLATILE SIGNALS FROM THREE SYMPATRIC ANT-LION SPECIES, Euroleon nostras, Grocus bore, and Myrmeleon formicarius (NEUROPTERA: MYRMELEONTIDAE).

PETER B A E C K S T R O M , I G U N N A R B E R G S T R O M , 2 FREDRIK BJORKLING, I HE H U I - Z H U , t'5 H A N S - E R I K H O G B E R G , 3 ULLA

J A C O B S S O N , 1'6 L I N G U O - Q I A N G , 1'5 J A N L O F Q V I S T , 4 WASSGREN 2

TORBJORN N O R I N , 1 a n d A N N - B R I T T

~Department of Organic Chemistry, Royal Institute of Technology S-100 44 Stockholm, Sweden 2Department of Chemical Ecology, GOteborg University S-400 33 GOteborg, Sweden 3University College of Sundsvall/H?irnOsand S-851 24 Sundsvall, Sweden 4Department of Anirnal Ecology, Ecology Building, Lund University S-223 62 Lund, Sweden (Received April 19, 1987; accepted October 23, 1987) Abstract--The thoracic gland of the ant-lion Euroleon nostras was found to contain nerol oxide (la) and (Z)-6-undecen-2-ol (nostrenol, 3) while the species Grocus bore contained 10-homonerol oxide (lb) and nostrenol (3). Nerol (2a) and 10-homonerol (2b) were found in a third species, Myrmeleon formicarius. 10-Homonerol, racemic 10-homonerol oxide, and racemic as well as (R)- and (S)-nostrenol were synthesized. The nerol oxide of E. nostras and the 10-homonerol oxide of G. bore were found to be racemic, while both species contained optically pure (R)-nostrenol (28). Key Words--Euroleon nostras, Grocus bore, Myrmeleon formicarius, Neuroptera, Myrmeleontidae, nerol oxide, nostrenol, (R)- and (S)-(Z)-6-undecen-2-ol, 10-homonerol oxide, nerol, 10-homonerol, chiral shift reagent, chiral GC phase. SVisiting research scientists from Institute of Photographic Chemistry, Academia Sinica, Beijing, China (H. H.-Z.) and Shanghai Institute of Organic Chemistry, Academia Sinica, 345 Ling Lu, Shanghai, China (L. G.-Q.). 6To whom correspondence should be addressed. 61 0098-0331/89/0100-0061 $06.00/0 © 1989 Plenum Publishing Coq~oratlon

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INTRODUCTION

The three ant-lion species, Euroleon nostras (Fourc.), Grocus bore Tjed., and Myrmeleon formicarius L., are sympatric both in time and space in high populations in limited areas on the northern part of Oland, an island in the Baltic in southern Sweden. Their biology indicates pheromone communication as species-specific isolating mechanisms. A morphological study revealed an unusually big, paired, thoracic gland in the males (Elofsson and L6fqvist, 1974). The gland was present in females only as a tiny remnant, which supports the hypothesis of the gland secretion as a species separating pheromone. Two small brushes, which are present only in males at the basal hind margin of the back wings, fit into small pits on the thorax when the wings are folded. The gland secretion is emptied into the pits and is dispersed in the air by the brushes when the males flutter their wings. The volatile secretions of the glands in males of E. nostras, G. bore, and M. formicarius were analyzed with capillary gas chromatography and gas chromatography-mass spectrometry (L6fqvist and Bergstr6m, 1980). In each species the secretion consisted of only two major components present in about equal proportions. Only traces of minor components were found. One of the components (MS, role = 170) was present in E. nostras and G. bore, but it was not identified. In addition, nerol oxide was identified from males of E. nostras. The other component in G. bore had a mass spectrum very similar to nerol oxide, and it was suggested to be a homonerol oxide. M. formicarius contained nerol and an unknown compound (MS, m/e = 168). The intention with this study was to determine the chemical structures, including the stereochemistry, of significant compounds in E. nostras, G. bore, and M. formicarius as a basis for studies of their biological role. Methods for synthesis of the compounds and their isomers have also been developed.

METHODS AND MATERIALS

Insect Material Larvae of E. nostras and G. bore were collected in southern Sweden at Byerum in the northern part of (91and, an island in the Baltic. The larvae were bred in the laboratory up to the time of pupation and emergence of imagines according to the procedure of L6fqvist and Bergstr6m (1980).

Preparation of Extracts The thoracic glands of 3 to 5-day-old males were dissected and immersed into ca. 200 ~1 distilled pentane. After concentration to about 100/~1 the extracts

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were run on a preparative capillary gas chromatograph (injected volume: 2 14, recovery: 80%) equipped with a revolving fraction collector (Wassgren and Bergstr6m, 1984). The analysis was performed using a fused silica column with stationary phase OV-351, 25 m × 0.35 mm ID, df 1/~m. Thus, the solvent was removed from the volatile compounds, and a separation of the compounds was simultaneously achieved. The neat compounds were then dissolved in chloroform-d for analysis by [1H]NMR.

Analytical Techniques Gas Chromatography. Gas chromatographic analyses were performed on a Hewlett Packard (HP) 5880 gas chromatograph equipped with an N/P-detector and a flame ionization detector. Fused silica columns were used with stationary phases as follows: Superox FA, 27 m x 0.5 mm ID, df 0.4/~m; OV351, 25 m x 0.35 mm ID, df 1.0/~m; XE-60-(S)-valine-(S)-2-phenylethylamide, 50 m x 0.23 mm ID, df0.12 /zm; Ni(II)-bis[3-heptafluorobutyryl-l(R)camphorate], 25 m x 0.25 mm ID; Mn(II)-bis[3-heptafluorobutyryl-l(R)-camphorate], 35 m x 0.25 mm ID. Mass Spectrometry. Finnigan 4021 GC-MS (quadropole) and LKB 2091 GC-MS instruments (magnetic type) were used. [IH]NMR Spectrometry. IH (200.3 MHz) NMR spectra were measured in CDC13 with tetramethylsilane as an internal standard on a Bruker WP200 spectrometer. Determination of Absolute Configuration. Lanthanide-Induced Shift (LIS) Studies on Nerol Oxide (la). Several chiral shift reagents were investigated in order to resolve nerol oxide. Tris[3-(trifluoromethylhydroxymethylene)-d-camphor]europium [Eu(TFC)3 Stohler Isotope Chemicals] gave no observable shift changes. Tris[d,d-dicampholylmethanato)europium [Eu(dcm)3, Alfa] gave small shift changes but no shift separation of the two enantiomers. However, the third shift reagent tris[3-(heptafluoropropylhydroxymethylene)-d-camphor]europium [Eu(HFC)3] proved to be successful. The quality of the commercial shift reagents varied a lot. Initially Stohler I.C. delivered a sample that gave shift differences of the enantiomers. However, later deliveries proved inactive (and insoluble in chloroform-d). Then samples from Ega, Lancaster Synthesis Ltd., and Aldrich were tried with varying success. Sublimation of shift reagent did not improve the result. The conditions for the LIS studies had to be worked out carefully. In a typical test run Eu(HFC)3 dissolved in chloroform-d was added in portions (0.1 equiv.) to the nerol oxide (7 mg) in chloroform-d. The result of each addition was checked by [IH]NMR, and a lanthanide reagent to substrate ratio (L/S) of 0.5 : 1 was found to be optimal. Pentane extracts of thoracic glands from 20

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males of E. nostras were fractionated by preparative capillary gas chromatography to give ca. 200/zg of nerol oxide. A microtube (5/2.5 mm, Wilmad Cat. No 520-1) was used with a sample volume of 100 ~1. Chiral GC Separations. GC separations of the enantiomers of nerol oxide and 10-homonerol oxide were carded out on a fused silica capillary column (35 m × 0.25 mm ID) coated with Chirametal Mn(II)-bis[3-heptafluorobutyryll(R)-camphorate] in OV-101 (Schurig and Weber, 1981); conditions: isothermal at 65°C, carrier gas N2 (9 psi), ~ = 16 cm/sec. Retention times: (S)neroloxide, 29.6 min; (R)-nerol oxide, 30.6 min; 10-homonerol oxide, 59 min and 61.3 min (tentatively assigned to S and R, respectively). It is interesting to note that only the enantiomers of nerol oxide could be separated on the closely related phase Ni(II)-bis[3-heptafluorobutyryI-I (R )-camphorate] in SE-54, 25 m × 0.23 mm ID (Schurig and Weber, 1984); conditions: isothermal at 93°C, carrier gas Nz (7 psi), ~ = 15 cm/sec, split 40/1. Retention times: (S)-neroloxide, 12.6 min; (R)-neroloxide, 12.4 min. Separations of the E/Z isomers as well as the enantiomers of synthetic (Z)and (E)-6-undecen-2-ol were carded out by tandem GC of the isopropyl carbamate derivatives. The first fused silica capillary column (12 m x 0.22 mm ID, df 0. I #m) was coated with chemically bonded SE-54. The second fused silica capillary column (50 m × 0.23 mm ID, dyO. 12 ~m) was coated with XE60-(S)-valine-(S)-2-phenylethylamide (Krnig, 1982; Krnig et al., 1982). Running conditions were as follows: isothermal at 120°C for 60 min and then programmed to 165°C at a rate of l°C/min, carder gas N2 (22 psi), ~ = 12 cm/ sec, N/P-detector. The isopropyl carbamate derivatives of synthetic and natural materials were prepared according to the procedure described by Krnig et al. (1982). After evaporation of the solvent, the residue was dissolved in ca. 50/zl methylene chloride. Then 20/zl isopropyl isocyanate was added followed by heating in an aluminium block at 100°C for 20 min. The solvent was then evaporated and the residue was dissolved in 20/zl methylene chloride; 2/zl of the solution was injected for analysis on a gas chromatograph as described above.

Synthetic Compounds Column chromatography separations were made on a column that had been dry packed with Merck 60 silica gel, 230-400 mesh. Light petroleum, bp 4060°C, with increased amounts of ethyl acetate (0, 1.25, 2.5, 5, 10, 20, 40, and 80%) was delivered by a metering pump at a rate of 30 ml/min for 12.5 mm ID columns. Analytical GLC of the synthetic compounds was performed on a Pye Unicam 204 instrument with an FID detector connected to a computing integrator; 25-m columns were used coated either with Carbowax 20 M or with SP-1000. A Finnigan model 4021 spectrometer connected to an INCOS data

ANT LION VOLATILES

65

system was used to record GC-MS spectra. The [~H]NMR spectra were recorded in CDC13 with tetramethylsilane as internal standard using Varian EM360, Jeol PMX60Si, and Bruker WP200 spectrometers. The coupling constants (J) are in Hz. The following abbrevations are used: DMSO = dimethyl sulfoxide, THF = tetrahydrofuran, DIBAL = diisobutylaluminium hydride, PCC = pyridinium chlorochromate.

Syntheses of lO-Homonerol (2b) and lO-Homonerol Oxide (lb) (Z)-2-Ethyl-6-methyl-l-iodo-l ,5-heptadiene (6). 1-Bromo-4-methyl-3pentene (4, 2.64 g, 16.2 mmol) in dry ether (30 ml) was slowly added to Mg metal (480 mg) and a small crystal of iodine. After 1.5 hr at reflux, the solution was carefully transferred into a solution of CuBr/MezS (3 g, 0.9 equiv) in 10 ml of ether and 20 ml dimethylsulfide at - 5 5 ° C . This temperature was kept for 2.5 hr. Then 1-butyne (5, 1.26 ml, 870 rag) was added via a cooled syringe during 1 rain at - 4 5 ° C . The mixture was warmed to - 2 3 ° C for 2 hr and then recooled to - 4 0 ° C . Iodine was added in small portions and the mixture was allowed to warm to - 10°C for 0.5 hr. Ammonium chloride (20 ml, 10% aq.) was added and the organic layer was diluted with pentane, separated, filtered and washed (Na2S203, NH3 aq., NaC1 10% aq.). This procedure yielded 2.6 g (61%) of the desired product 6; [~H]NMR (200 MHz) 6 1.0 (t, 3H), 1.6 (s, 3 H), 1.7 (s, 3H), 2.0-2.4 (m, 6H), 5.1 (t, 1H), and 5.9 (bs, 1H). (Z)-3-Ethyl-7-methyl-2,6-octadienoic Acid (7). An ether solution of n-butyllithium (1.2 M, 1.14 mmol) was added under stirring at - 7 0 ° C to the iodoalkene 6 (1 mmol) in ether (10 ml). The mixture was stirred at - 6 0 ° C for ca. 30 min. The reaction was complete when the Gilman test was negative (Fieser and Fieser, 1967). Then excess carbon dioxide was bubbled through the mixture at - 7 8 ° C . The reaction mixture was allowed to warm to - 2 0 ° C . Water (5 ml) was added followed by an aqueous sodium bicarbonate solution until the mixture was alkaline. The aqueous layer was then acidified with hydrochloric acid (1 M) followed by extraction with ethyl ether (3 × 10 ml). The organic solution was dried (MgSO4) and the solvent evaporated to give the acid 7, 62 mg (34%); [IH]NMR (200 MHz) 6 1.08 (t, 3H, J = 7.4), 1.62 (s 3H), 1.69 (s, 3H), 2.10-2.26) (m, 4H), 2.63 (t, 2H, J = 7.4), 5.15 (bt, 1H), 5.66 (bs, 1H), and 10.7 (broad, 1H). (Z)-3-Ethyl- 7-methyl-2, 6-octadien-l-ol, l O-Homonerol (2b). A solution of the acid 7 (62 mg, 0.34 mmol) in anhydrous ethyl ether (5 ml) was added dropwise to lithium aluminium hydride (25.8 rag, 0.68 mmol) in anhydrous ethyl ether (5 ml) to create a gentle reflux. After 2 hr, sodium sulfate decahydrate-celite was added (Baeckstrrm, 1978). After another 1.5 hr, the solid was filtered off and washed with ethyl ether. After evaporation of the solvent and flash-chromatography, the combined ether filtrates gave the 10-homonerol (2b),

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30 mg (45%); MS (re~e) 168 (M), 150 (M-18), 135 (M-18-15), 121, 107, 79, 69 and 41; [IH]NMR (200 MHz) ~ 1.03 (t, 3H, J = 7.4), 1.60 (s, 4H), 1.69 (s, 3H), 1.9-2.1 (m, 6H), 4.13 (d, 2H, J = 7.0), 5.11 (bt, 1H), and 5.42 (t, 1H). Photooxidation of lO-Homonerol (2b). 10-Homonerol (2b, 30 mg, 0.18 mmol) was dissolved in a chloroform solution of Rose Bengal (3 ml). The solution was irradiated in a Rayonet reactor equipped with 16 RPR 350 nm lamps. Oxygen was bubbled through the solution. Tetrabutylammonium borohydride (46 mg, 0.18 mmol) was added in three portions (23, 12, and 11 rag, respectively) after 0, 60, and 80 min of irradiation (Baeckstr6m et al., 1982). After 1.5 hr, the chloroform was removed by evaporation. Potassium iodide (60 mg, 0.36 mmol) in water (1 ml) and ethyl ether (6 ml) was then added to the residue. After stirring for 10 min, the resulting crystals were removed and washed by filtration. The combined red ethereal layer containing the alcohols 8 and 9 was separated from the aqueous phase and dried (MgSO4). lO-Homonerol Oxide (lb). The obtained ethereal solution of the alcohols 8 and 9 was mixed with 2 drops of perchloric acid in ether (4 ml) and stirred for 30 rain. After addition of a small amount of sodium carbonate, the resulting crystals were filtered off and washed with ethyl ether. Silica gel (2 g) was added to the ethyl ether solution and the solvent was evaporated in vacuo. The red powder with the adsorbed reaction products was added to the top of a column for flash-chromatography on basic alumina. 10-Homonerol oxide was obtained in a pure state. The GC-MS and [IH]NMR spectra were identical with those of the natural compound (cf. Figures 2 and 3). [IH]NMR (200 MHz) 6 1.01 (t, 3H, J = 7.5), 1.69 (d, 3H, J = 1.2), 1.73 (d, 3H, J = 1.2), 1.85-2.16 (m, 4H), 4.19 (m, 3H), 5.22 (bd, 1H), and 5.39 (bs, 1H).

Syntheses of Nostrenol (3) and Related Alcohols 10 and 11 6-Methyl-6-hexanolide (18). m-Chloroperbenzoic acid (11.9 g) in methylene chloride (100 ml) was added to a stirred solution of 2-methylcyclohexanone (5.6 g) in methylene chloride (20 ml). The resulting mixture was refluxed for 2 hr, cooled to 0°C, filtered and the filtrate was concentrated in vacuo. Bulbto-bulb distillation (10 mm Hg/air bath at 100-120°C) gave an oil (4.70 g, 73%). NMR (60 MHz) 6 1.4 (d, 3H), 1.4-2.1 (m, 6H), 2.5-2.8 (m, 2H), and 4.1-4.8 (m, IH). Cyclic Lactols 12, 13, and 14. One of the lactones 18, 19, or 20 (0.1 mol) was stirred in dry THF (100 ml) at - 5 0 ° C under argon. DIBAL (25 ml, 0.21 mol) in THF (I00 ml) was added (30 min) and stirring at - 5 0 ° C was continued for an additional 30 min. A powdered mixture of celite (1 vol), sodium sulfate decahydrate (70 g, 1 voI), and ethyl ether (300 ml, saturated with water) was added at - 5 0 ° C . The mixture was allowed to warm slowly to room tempera-

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ANT LION VOLATILES

ture, after which it was filtered and the solid was thoroughly washed with ethyl ether. The filtrate was dried (MgSO4) and concentrated to give an oil (9.5-10,1 g, 93-99%), which was used in the next step without further purification. Wittig Reaction of Cyclic Lactols 12, 13, and 14. Sodium hydride (300 mg, 80% in oil, 20 mmol) was washed free of oil with dry THF in portions under argon. The residual THF was evaporated under vacuum and the flask filled with argon. DMSO (40 ml, freshly distilled from Call2) was added, and the mixture was stirred and heated slowly to 60°C until all the hydride had reacted. After cooling to 20°C an alkyltriphenylphosphonium bromide (20 mmol) was added to the stirred solution. The ylide (15, 16, or 17) was formed rapidly, and the resulting intensely red solution was cooled to 15°C and a solution of the lactol (12, 13, or 14, 20 mmol) in DMSO (10 ml) was added dropwise. After stirring at 15°C for 1.5 hr, the reaction mixture was poured into water and extracted with pentane. The pentane solution was concentrated to 10 ml and filtered. The solid was washed with a few milliliters of pentane, and the solution was concentrated to give an oil, which was subjected to flash-chromatography to give the (Z)-alkenol (10, 11, or 21) after bulb-to-bulb distillation (air bath at 130°C/7 mm Hg, yield 14-16 mmol, 70-80%). The products contained less than 8% of the E-isomers (GC). The NMR spectra of 10 and 11 are shown in Figure 5. (Z)-5-Decen-l-ol (21). Bp 58-60°C/1 mm Hg. GC showed the ratio (Z/ E) to be 93/7; n°5 1.4504; IR (film) um~x 3320 cm -1 (very broad, H-bonded OH); NMR (60 MHz) 6 0.9 (t, 3H), 1.1-1.8 (m, 8H), 1.8-2.3 (m, 4H), 2.80 (s, 1H disappears on addition of D20), 3.60 (t, 2H), and 5.37 (m, 2H). (Z)-5-Decenal (22). (Z)-5-Decen-l-ol (21, 5 g, 32 mmol) was stirred in methylene chloride (20 ml). PCC (16 mg, 50 mmol) in methylene chloride (100 ml) was added. The mixture was stirred at room temperature for 2 hr, poured into ethyl ether (1.5 1), and the organic phase was washed with aqueous HC1 (2 M) followed by saturated sodium bicarbonate solution and water. The organic phase was dried (MgSO4) and concentrated to give an oil, which, after flashchromatography and distillation (bp 80-85°C/7 mm Hg), furnished the pure aldehyde (4.3 g, yield 87%, 99% pure, Z/E = 93/7 by GC); n°5 1.4491; IR (film) ~co 1725 cm - l (strong); NMR (60 MHz) 6 0.93 (t, 3H), 1.1-2.2 (m, 10H), 2.43 (dt, 2H, J 6.8 and 1.6), 5.40 (m, 2H), and 9.82 (t, 1H, J = 1.6). Nostrenol, rac-(Z)-6-Undecen-2-ol (3). Methyllithium (1.6 M in ethyl ether, 50 ml) was stirred in dry ethyl ether (100 ml) at - 8 0 ° C under argon. (Z)-5-Decenal (22, 4.1 g) in ethyl ether (25 ml) was added dropwise. After stirring for 2 hr, the temperature was raised to - 5 0 ° C and 2 M aqueous HCI was added. After warming to room temperature the reaction mixture was poured into water. The organic phase was washed with water, dried (MgSO4), and the solvent evaporated to give an oil, which was flash-chromatographed and dis=

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tilled (3.2 g, yield 72%, bp 108-109°C, 12 mm Hg, 99% pure, Z/E = 95/5 by GC). This compound had identical IR, [mH]NMR, and MS spectra and GC behavior (achiral columns) as those of natural nostrenol.

Syntheses of (S)- and (R)-Nostrenol (26 and 28, respectively) (Z)-l-Chloro-3-octene (25). (Z)-3-Octen-l-ol (23, 2.62 g, 20.5 mmol; Zhong et al., 1982), triphenylphosphine (6 g, 23 mmol) and carbon tetrachloride (4 ml) were mixed and heated to 120°C (of. Hooz and Gilani, 1968). A vigorous reaction occurred. Heating was continued for 15 min. Ethanol (0.5 ml) was added and heating was continued for an additional 15 min. After cooling, pentane (25 ml) was added. Precipitated triphenylphosphine oxide was illtered off and the filtrate was carefully concentrated to give an oil, which, on addition of pentane (10 ml), precipitated further oxide that was removed, and the pentane was evaporated to give an oil. This was chromatographed on silica gel. Pentane eluted the desired product, which was distilled to give 2.92 g (88%). Bp 60-61 °C/5 mm Hg; n°5 1.4486; [IH]NMR (60 MHz) ~50.93 (t, 3H), 1.2-1.7 (m, 4H), 1.9-2.3 (m, 2H), 2.54 (quint., 2H, J = 7.5), and 5.48 (t, 2H, J = 7.5). (S)-Nostrenol, (S)-(Z)-6-Undecen-2-ol (26). (Z)-l-Chloro-3-octene (25, 2.94 g, 20 mmol) in dry THF (15 ml) was added to magnesium turnings (0.72 g, 30 mmol), which had been activated by a few drops of 1,2-dibromoethane under argon. The resulting mixture was heated to reflux for 1 hr. After cooling, the solution of the Grignard reagent was decanted from the excess magnesium and added via a syringe to a solution of CuBr/MezS (1 mmol) in THF (20 ml) at - 3 0 ° C under argon (cf. Huynh et al., 1979). (S)-Epoxypropane (24, 1.2 g, 21 mmol) was added. After stirring at - 3 0 ° C for 1 hr, the temperature was raised to 20°C. The reaction mixture was poured into an aqueous solution of ammonium chloride. The resulting mixture was diluted with pentane and the aqueous layer extracted twice with pentane. The combined organic phases were washed with water, dried (MgSO4), and the solvent was carefully evaporated to give 2.72 g (80 %) after flash-chromatography and distillation, bp 108-109 ° C/ 12 mm Hg; [c~]o = +6.02 ° (neat); ZIE ratio > 98.5/1.5 by GC (Carbowax 20 M, 135°C). The [~H]NMR spectrum 200 MHz) was identical with that of natural nostrenol. (R)-Nostrenyl benzoate, (R)-(Z)-6-Undecen-2-yl benzoate (27). (S)-Nostrenol (26, 1.70 g, 10 mmol), triphenylphosphine (6.55 g, 25 mmol), and benzoic acid (3.05 g, 25 mmol) was stirred in dry THF (100 ml) under argon at 0°C. Diethyl azodicarboxylate (4.52 g, 26 mmol) in THF (10 ml) was added during 5 min. After stirring overnight at room temperature, methanol (0.6 ml) was added to destroy excess reagents. After concentration to 25 ml, the mixture was poured into pentane. The precipitate was removed by filtration and the

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69

solvent evaporated. The remaining oil was flash-chromatographed. Ethyl acetate (2-3 %) in light petroleum eluted 2.15 g (78 %), pure by GC; [c~]o --- - 26.4 ° (neat); [1HI NMR (60 MHz) 6 0.9 (t, 3H), 1.0-2.2 (m, 12 H), 1.37 (d, 3H), 5.2 (sextet, 1H), 5.3 (m, 2H), and 7.2-8.2 (m, 5H). (R)-Nostrenol, (R)-(Z)-6-Undecen-2-ol (28). (R)-Nostrenyl benzoate (27, 1.67 g, 0.61 mmol) was dissolved in dry ether (25 ml) under argon. A suspension of lithium aluminium hydride in ether (1 M, 20 ml) was added. The mixture was stirred overnight, when aqueous HC1 (2 M) was carefully added to pH 1. After extraction twice with pentane, the organic phase was washed with water, dried (MgSO4), and concentrated to give an oil, which was flash-chromatographed. Ethyl acetate (3-4%) in light petroleum eluted a colorless oil, which was distilled (Kugelrohr 10 mm Hg/air bath 130°C), 0.9 g (87%); [~] = - 6 . 0 8 ° (neat). rac-(E)-6-Undecen-2-ol. This compound was prepared via the reaction sequence described for (S)-(Z)-nostrenol (26) except that the starting materials were rac-(E)-3-octen-2-ol (Zhong et al., 1982) and racemic epoxypropane; colorless oil, [IH]NMR (60 MHz) 8 0.85 (t, 3H), 1.10 (d, 3H) 1.1-2.2 (m, 9H), 3.8 (m, 1H), and 5.3 (m, 2H); IR (film) 970 cm -I [(E)-CH = CH-]. RESULTS

Identification Typical capillary gas chromatograms of the volatile secretions from E. nostras and G. bore are given in Figure 1. The two major components in each species were nerol oxide (la) and (Z)-6-undecen-2-ol (nostrenol, 3) in E. nostras, and 10-homonerol oxide (lb) and nostrenol (3) in G. bore (Scheme 1). In both species these compounds are found in the proportions 3:2 (in the order mentioned above). The small peaks present in the gas chromatograms emanate from the solvent used for extraction (hydrocarbon isomer and phthalates). Similarly, the major components of M. formicarius were found to be nerol (2a) and 10-homonerol (2b). R

1 o R=Me b R=EI

2 a R=Me b R=Et SCHEME1.

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ET AL.

lcl A

d

lb

!

60 t-'~ 5 °/rain

i

150

3

u

200 isothermal

FIG. 1. Capillary gas chromatographic separation of the volatile compounds from the thoracic glands of E. nostras (A) and G. bore (B). Column: Superox FA 27 m × 0.25 mm. Temperature program: isothermal at 60°C for2 min and then programmed to 200°C at a rate of 5°C/min.

Structure Determinations lO-Homonerol Oxide (lb). This compound, present in the secretion from males of G. bore, has a mass spectrum (m/e = 166) very similar to that of nerol oxide (la). L6fqvist and Bergstr6m (1980) suggested that it was a homonerol oxide. This was confirmed by a methyl triplet present in the [~H]NMR spectrum, which indicates that the structure can be 10-homonerol oxide (lb) with an ethyl substituent instead o f the methyl group in the 3-position (Figures 2 and 3). The other possible isomers, 8- and 9-homonerol oxide (Rohjahn and Bruhn,

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67

100

82

Ib

50 ¸

41

M

55

219 ,,h..,

I].l,..,.,,t,,J. 137 i

M/E

40

60

,

i

, .,t

i

166 ,'

I.

80 100 120 140 160 180

FIG. 2. Mass spectrum of natural 10-homonerol oxide (lb) from G. bore. 1978), were not identical with the natural product as shown by comparisons (GC-MS; N M R ) with an authentic sample o f a mixture of the two isomers. In order to confirm the structure o f 10-homonerol oxide ( l b ) a synthesis was designed according to Scheme 2. The alkyl cuprate of 4, prepared from the

lb

FIG. 3. [tH]NMR of natural 10-homonerol oxide (lb); approx. 50 #g (in CDCI3) derived from an extract of 45 males of G. bore. The large peak at (5 1.54 belongs to water.

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corresponding magnesium reagent, was added to 1-butyne (5) followed by treatment with iodine to form the vinyl iodide 6. Lithiation of 6 followed by alkylation with carbon dioxide yielded the acid 7 (Cahiez et al., 1976). Reduction with lithium aluminium hydride (LAH) afforded the 10-homonerol (2b). Photooxidation with singlet oxygen of 2b and simultaneous reduction with tetramethylammonium borohydride (QBH4; Baeckstrrm et al., 1982) gave the two isomeric alcohols 8 and 9. Acidic cyclization of the alcohol mixture (Ohloff et al., 1980) gave the 10-homonerol oxide (lb), which was separated from the unreacted alcohol by column chromatography. The GC-MS data and the [~H]NMR spectrum synthetic l b were identical with those of the isolated natural product of G. bore.

~... .~Br 4

+

k

=

5

--

1. Mg.elher ~"~r 2, CuBr/Me2S'3. 12 L"

1. n-BuLl -60°C,ether" 2, CO2

6

I•COOH ••oCHH LAH =

7

I~C1_120H

QBH4

2b

2OH + ~~HH2 OH

8

hv/02 _

- -H ..÷ 11~

9

lo

SCHEME 2.

lO-Homonerol (2b). From M. formicarius we have earlier identified nerol (2a) in the male volatile secretion. A second compound gave m/e = 168 (L6fqvist and Bergstrrm, 1980). It has now been identified as 10-homonerol (2b) by comparison of GC-MS data (magnetic and quadropole instrument) of the natural substance and the synthetic compound produced as an intermediate in the synthesis of 10-homonerol oxide.

73

A N T LION V O L A T I L E S

(Z)-6-Undecen-2-ol (Nostrenol, 3). This compound was found to be present in the secretions of E. nostras and G. bore, as shown by identical mass spectra (Figure 4) and GC retention times (Figure 1). It was identified by [tH]NMR as an undecen-2-ol, which was named nostrenol (3). The spectral data gathered for nostrenol (3) were inconclusive regarding the double bond position. However, preliminary inspection of the [IH]NMR spectrum suggests the most probable isomers to be 3, 10, and 11. The synthetic procedures of those isomers are outlined in Scheme 3. The key step is the Wittig reaction of the lactols 12, 13, and 14 with the appropriate alkylidene phosphoranes 15, 16, and 17, respectively. Wittig reactions in dimethylsulfoxide are known to give a high ratio of Z to E configuration (Goto et al., 1975; Hall et al., 1975), and this was also found in our case ( Z / E > 10 : 1 ). The lactols 12, 13, and 14 were prepared in excellent yields by reduction of the corresponding lactones 18, 19, and 20 with diisobutylaluminium hydride in tetrahydrofuran. The lactone 18 was obtained from 2-methylcyclohexanone on oxidation with m-chloroperbenzoic acid. Racemic compound 3 was prepared from (Z)-5-decenol (21) via oxidation to (Z)-5-decenal (22) by pyridinium chlorochromate. The aldehyde was then reacted with excess methyllithium in ethyl ether at low temperature which furnished (Z)-6-undecen-2-ol (3). The [IH]NMR spectra of the (Z)-undecen-2-ols 3, 10, and 11 as well as that of the natural product isolated from E. nostras are shown in Figure 5. From

54

100

6

81

Li 5o

OH 3

il 95 110 M II .,.:~

....

M/E 40

;i

.,.,

.......

60

'h h ".l .!,.,.r

80

, ""

122 .1

100

,,~,..J;.-.,

120

152, 170 ....

i .....

140

J . . . . . . . .

-

160

FIG. 4. Mass spectrum of natural nostrenol (3) from E. nostras, also present in G. bore.

0

MCPBA

21

2O

O

19

DIBAL THF

DIBAL THF '~

THF

DIBAL,,_

PCC ,,,.-

©

18 0

0

12 OH

~

~DMsoP(Ph)3'I'~5

- 80('C

~P(Ph)3.1L DMSO

H

NeLl

~P{Ph)3'IE DMSO

SCHEME 3.

1/-.,

OH

22

13

~_~

~

OH

~

O

11

3

21

10

H

OH

OH

OH

>. r*

t'n

©:

("3

>

75

A N T LION V O L A T I L E S

3. E, nostms

L_

3

L.__

10

OH

11

5

/~

3

2

1

FIG. 5. [IH]NMR spectra of extract from E. nostras (top), (Z)-6-undecen-2-ol (3), (Z)7-undecen-2-ol (10), and (Z)-5-undecen-2-ol (11) (bottom). For comparison the signals from the vinylic protons are expanded.

the comparison of these spectra it is evident that (Z)-6-undecen-2-ol (3) is identical with the natural nostrenol. Absolute Configurations Nerol oxide (la). It was of interest to investigate whether the identified compounds from the ant-lions were optically active and if there was any difference in chirality between the three species. Samples of nerol oxide with known absolute configurations (Ohloff et al., 1980) were investigated. Eu(HFC)3 was added to (S)-nerol oxide (L/S = 0.5), and the [1H]NMR showed that the lowfield doublet at ~ 5.6-5.8 belonged to H-7 of (S)-nerol oxide. When a solution of racemic nerol oxide and Eu(I-IFC)3 (L/S = 0.5) was added to the sample of (S)-nerol oxide and Eu(HFC)3, the ratio between the two doublets changed according to the ratio between the S-enantiomer and the racemate. Thus (R)-

76

BAECKSTROM ET AL,

nerol oxide gives rise to the upfield doublet in this region. Addition of 0.45 equivalents of Eu(HFC)3 to naturally occurring nerol oxide (200 /~g) from E. nostras resulted in two signals from H-7 in a 1 : 1 ratio, showing that this natural nerol oxide is racemic. The nerol oxide from E. nostras was also analyzed using fused silica capillary GC columns coated with either Ni(II)- or Mn(II)-bis[3-heptafluorobutyryl- 1(R)-camphorate] (Schurig and Weber, 1981, 1984). The results confirmed that the nerol oxide is racemic. (S)-Nerol oxide was used as reference. The elution order of the S-enantiomer compared to that of the R-enantiomer was reversed on the two columns (see Methods and Materials). lO-Homonerol Oxide (lb). The homonerol oxide isolated from G. bore was similarly tested and found to be racemic. However, only the Mn(II)-derived chiral phase separated the enantiomers of l b . The close relationship between l a and l b suggests also that (S)-10-homonerol oxide has the shorter retention time (see Methods and Materials). Nostrenol (3). In order to establish the chirality of natural nostrenol an asymmetric synthesis was outlined as shown in Scheme 4. (Z)-3-Octen-l-ol (23) (Zhong et al., 1982) and commercially available (S)-epoxypropane (24) were used as starting materials. The alcohol 23 was transformed into the chloride 25 on treatment with triphenylphosphine and tetrachloromethane (Hooz and Gilani, 1968). The chloride was converted to the alkyl cuprate via the corresponding Grignard reagent followed by reaction with (S)-epoxypropane (24) to furnish (S)-nostrenol (26). The R-enantiomer was prepared from (S)-nostrenol (26) via a Mitsunobu reaction (Mitsunobu, 1981). Thus, (S)-nostrenol (26) was reacted with triphenylphosphine, diethyl azodicarboxylate, and benzoic acid, which gave (R)-nostrenyl benzoate (27). Reduction with lithium aluminium hydride furnished (R)-nostrenol (28). Gas chromatography of the isopropyl carbamates of the two alcohols on a tandem GC column [SE-54/XE-60-(S)-valine(S)-2-phenylethylamide] showed that the synthetic enantiomers were almost optically pure ( = 9 3 % ee) and contained less than 2% of the E-isomer. The latter was prepared as the racemate as described above but starting from (E)-3octen-l-ol (Zhong et al., 1982) and racemic epoxypropane. Retention times were 118.9 min [(Z)-(R)-6-undecen-2-ol], 119.2 rain [(Z)-(S)], 119.4 rain [(E)(R)], and 119.7 min [(E)-(S)] on the tandem GC column. Nostrenol from E. nostras was analyzed by capillary GC as the isopropyl carbamate derivative. The naturally occurring sample was found to be enantiomerically pure ( > 99.9 %) and of R-configuration. The nostrenol isolated from G. bore was also found to be of R-configuration. The retention times were 66.4 min and 66.9 min for the (R)- and (S)-nostrenols, respectively, on the column coated with XE-60-(S)-valine-(S)-2-phenylethylamide.

PhCOOH P(Ph)3

23

~ _ _ ~ O H

_

_

27

oco~

Me

.

CCI4/PIPh)3_=

LAH

SCHEME4.

25

28

3, /_~,,,H 2z'Me

1. Mg CI 2-CuBr/Me2S_

Me

26

Me

78

BAECKSTROM ET AL.

DISCUSSION

The ant-lion species are old from an evolutionary point of view. Therefore, it is of interest to investigate the pheromone systems of these insects in order to gain knowledge of possible isolating mechanisms between closely related species. The male ant-lion species have large thoracic glands, producing characteristic volatile chemicals which exhibit close structural relationships between the different species. The adults live only for a few days during which they must mate. These observations suggest that the substances may form part of a communication system associated with mating and contributing towards isolating mechanisms between the sympatric species. Lrfqvist and Bergstr6m (1980) reported earlier that the thoracic glands of each of the three ant-lion species contained two major volatile constituents. One of the compounds in E. nostras was found to be nerol oxide (la), while nero! (2a) was identified from M. formicarius. In the present investigation we have used [IH]NMR and/or MS to identify the three remaining components as 10homonerol oxide (lb) in G. bore, 10-homonerol (2b) in M. formicarius, and (Z)-6-undecen-2-ol (nostrenol, 3) in E. nostras and G. bore. Four of these compounds are biosynthetically related and of isoprenoid origin, whereas the fifth compound, nostrenol (3), is of another biogenetic origin. It seems that the three sympatric ant-lion species make use of a combination of a minimum of two substances for reproductive isolation. Nerol (2a) and nerol oxide (la) are well-known plant constituents while their homologs 10-homonerol (2b) and 10-homonerol oxide (lb) have not been identified previously. Nostrenol (3) has been reported as a minor constituent of cognac (ter Heide et al., 1978), but its synthesis and spectral data have not, to our knowledge, been reported in the literature. Syntheses were designed for 10-homonerol, 10-homonerol oxide, and nostrenol in order to verify the structural assignments and for EAG measurements and bioassays. The biological role of the compounds is currently under investigation. Three of the isolated compounds possess an asymmetric center, and we have found that nostrenol from the two ant-lion species E. nostras and G. bore is enantiomerically pure and of R-configuration. The nerol oxide of E. nostras as well as the closely related 10-homonerol oxide of G. bore are racemic. Nerol oxide has not yet been found to occur in nature in a chiral form (Ohloff et al., 1980). A GC separation of a mixture of Z- and E-isomers of racemic 6-undecen2-ols into their chiral entities was successfully performed by connecting one column for the separation of geometrical isomers to a chiral column for subsequent separation into the two pairs of enantiomers. The corresponding isopropyl carbamates were employed for this separation, and use of a nitrogen-

79

ANT LION VOLATILES

sensitive flame photometric detector gave a sensitivity 10-20 times greater than that with a flame ionization detector. The amount o f the compounds isolated from E. n o s t r a s was relatively large compared with amounts o f pheromone components typically found in insects. Thus, it was possible to obtain a large enough amount of material to determine the chirality o f nerol oxide (200/zg) by [ t H ] N M R after addition o f a chiral shift reagent. The chirality o f nerol oxide was also determined in the nanogram range by GC on a column coated with Ni(II)-bis[3-heptafluorobutyryl-1 (R )-camphorate]. This chiral phase could not, however, separate the enantiomers of 10homonerol oxide. On the other hand the corresponding Mn(II)-derived GC phase separated the enantiomers o f both nerol oxide and 10-homonerol oxide. Furthermore, the order of elution of the two enantiomers of nerol oxide was reversed on these two chiral GC phases. The N M R technique for structural and configurational studies has rarely been used on insect pheromones, which are generally produced only in nanogram quantities. Silverstein and coworkers (Plummer et al., 1976; Stewart et al., 1977) have reported the enantiomeric composition of a few pheromones (alcohols and ketals) determined by the addition o f shift reagents. The quantity (150-200 ~g) of compound used in these and the present investigation is at present the minimum amount for determination of chirality by addition o f a chiral shift reagent. Acknowledgments--The authors thank Dr. G. Ohloff, Firmenich SA, for kindly supplying samples of (R)- and (S)-nerol oxide. Special thanks are also due to Dr. E. Klein, Dragoco, for showing interest in this work and for the sample of the 8- and 9-homonerol oxides, and to Dr. T. Ohlsson, Department of Organic Chemistry, Chalmers University of Technology, GSteborg, for the synthesis of manganese(lI)-bis[3-heptafluorobutyryl-l(R)-camphorate]. We also warmly thank Elisabeth Marling for valuable help with collecting and rearing the insect.

REFERENCES BAECKSTROM,P. 1978. Photochemical formation of chrysanthemic acid and cyclopropylacrylic acid derivatives. Tetrahedron 34:3331-3335. BAECKSTROM,P., OKECHA,S., DE SILVA,N., WIJEKOON,D., and NORIN,T., 1982. Photooxidation with simultaneous reduction of hydroperoxides with tetrabutylammonium borohydride. Synthesis of perillenal from myrcene. Acta Chem. Scand. B36:31-36. CAHIEZ, G., BERNARD,D, and NORMANT,J. F. 1976. Stereospecific syntheses of alkenyllithium reagents from alkenyl iodides. Synthesis 245-248. ELOFSSON, R., and LOFQVIST,J. 1974. The Eltringham organ and a new thoracic gland: Ultrastructure and presumed pheromone function (Insecta, Myrmeleontidae). Zool. Scripta 3:31-40. FIESER, L.F., and FIESER,M. 1967. Reagents for Organic Synthesis. John Wiley & Sons, New York, p. 417. GOTO, G., SHIMA,T., MASUYA,n., MASUOKA,Y., and HIRAGA,K. 1975. A stereoselective synthesis of (Z, E)-9,11-tetradecadienyl-l-acetate, a major component of the sex pheromone of Spodoptera litura. Chem. Lett. 103-106.

80

BAECKSTROM ET AL.

HALL, D.R., BEEVOR,P.S., LESTER, R., PoPPI, R.G., and NESBITT, B.F. 1975. Synthesis of the major sex pheromone of the Egyptian cotton leafworm Spodoptera littoralia" (Boisd). Chem. Ind. 216-217. TERHEIDE, R., DE VALOIS, P.J., VISSER,J., JAEGERS,P.P., and TIMMER, R., 1978. Concentration and identification of trace constituents in alcoholic beverages, pp. 249-281, in G. Charalambous (ed.). Analysis of Foods and Beverages. Symposium proceedings (1977), Academic Press, New York. Hooz, J., and GILANI, S.S.H. 1968. A rapid, mild procedure for the preparation of alkyl chlorides and bromides. Can. J. Chem. 46:86-87. HUYNH, C., DERGUINI-BOUMECHAL,F., and L1NSTRUMELLE,G. 1979. Copper-catalysed reactions of Grignard reagents with epoxides and oxetane. Tetrahedron Lett. 1503-1506. Kt3NIG, W.A. 1982. Separation of enantiomers by capillary gas chromatography with chiral stationary phases. HRC & CC 5:588-595. KtSNIG, W.A., FRANCKE,W., and BENECKE,I. 1982. Gas chromatographic enantiomer separation of chiral alcohols. J. Chromatogr. 239:227-231. LOFQVIST, J., and BERGSTROM,G. 1980. Nerol-derived volatile signals as a biochemical basis for reproductive isolation between sympatric populations of three species of ant-lions (Neuroptera: Myrmeleontidae). bisect Biochem. 10:1-10. MITSUNOBU, O., 1981. The use of diethyl azodicarboxylate and triphenylphosphine in synthesis and transformation of natural products. Synthesis 1-28. OHLOFF, G., GIERSCH, W., SCHULTE-ELTE,K.H., ENGGIST, P., and DEMOLE, D. 1980. Synthesis of (R)- and (S)-4-methyl-6,2'-methylprop-l'-enyl-5,6-dihydro-2H-pyran (nerol oxide) and natural occurrence of its racemate. Heir. Chim. Acta 63: 1582-1588. PLUMMER,E.L., STEWART,T.E., BYRNE, K., PEARCE,G.T., and SILVERSTEIN,R.M. 1976. Determination of the enantiomeric composition of several insect pheromone alcohols. J. Chem. Ecol. 2:307-331. ROJAHN,W., and BRUHN,W. 1978. The synthesis of certain homologous monocyclic terpene oxides. Dragoco Rep. 248-253. SCHURIG, V., and WEBER, R. 1981. Manganese(II)-bis(3-heptafluorobutyryl-l(R)-camphorate): A versatile agent for the resolution of racemic cyclic ethers by complexation gas chromatography. J. Chromatogr. 217:51-70. SCHURIG, V. and WEBER, R., I984. Use of glass and fused-silica open tubular columns for the separation of structural, configurational and optical isomers by selective complexation gas chromatography. J. Chromatogr. 289:321-332. SILVERSTEIN, R.M. 1979. Enantiomeric composition and bioactivity of chiral semiochemicals in insects, pp. 133-146, in F.J. Ritter (ed.). Chemical Ecology: Odour Communication in Animals. Elsevier, North Holland Biomedical Press, Amsterdam. STEWART,T.E., PLUMMER,E.L., MCCANDLESS,L.L., WEST,J.R., and SILVERSTEIN,R.M. 1977. Determination of enantiomer composition of several bicyclic ketal insect pheromone components. J. Chem. Ecol. 3:27-43. ZrtONG, T.-S., WANG, X.-Q., and LIN, G.-Q. 1982. Synthesis of the pink bollworm sex pheromone. Acta Chim. Sin. 40:856-860. WASSGREN,A.-B., and BERGSTRtSM,G. 1984. Revolving fraction collector for preparative capillary gas chromatography in the 100-ug to l-ng range. J. Chem. Ecol. 10:1543-1550.