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(11Z,13E)-HEXADECADIEN-1-YL ACETATE: SEX PHEROMONE OF THE GRASS WEBWORM Herpetogramma licarsisalis - IDENTIFICATION, SYNTHESIS AND FIELD BIOASSAYS

ANDREW R. GIBB,1,* D. MAX SUCKLING,1 ASHRAF M. EL-SAYED,1 BJÖRN BOHMAN,2 C. RIKARD UNELIUS,2 JENNIFER J. DYMOCK,3 MICHELLE L. LARSEN,4 and BRUCE E. WILLOUGHBY5

1Horticulture and Food Research Institute of New Zealand Ltd., PO Box 51, Lincoln, New Zealand 2Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden 329A Seaview Road, Cable Bay, Mangonui 0557, Northland, New Zealand 4Bugs Ed., P.O. Box 446, The Gap, Queensland 4061, Australia 5AgResearch Ltd., Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand

* To whom correspondence should be addressed. E-mail: [email protected]

1 METHODS AND MATERIALS

Syntheses and Purification of Pheromone Compounds. (11Z,13E)- Hexadecadien-1-yl acetate, (11E,13Z)-hexadecadien-1-yl acetate, and (11Z,13E)- hexadecadien-1-ol were synthesized according to Schemes 1 and 2. Preparative medium-pressure liquid chromatography (MPLC) was performed on silica gel (Merck 60, 0.040-0.063 mm) in 20 or 25 mm i.d. glass columns with gradient elution, using hexane and increasing proportions of ethyl acetate (Baeckström et al., 1987). 1H-NMR spectra of CDCl3 solutions were recorded at 250 MHz, using a Bruker AM spectrometer. The starting materials employed were obtained from commercial suppliers, and used without further purification. The reactions involving anhydrous solvents were carried out under an atmosphere of nitrogen. General Synthetic Strategies. The synthesis of (11Z,13E)-hexadecadien-1-ol (5; Scheme 1) and the corresponding acetate, (11Z,13E)-hexadecadien-1-yl acetate (6; Scheme 1), started with 11-bromoundecanol 1, which was protected using dihydropyran with Amberlyst 15 as catalyst (Santangelo et al., 2002). The THP- protected bromide 2 was transformed to the phosphonium salt 3 with PPh3 (Legrand et al., 2004). A Wittig reaction between 3 and (2E)-pentenal in the presence of sodium ethoxide gave, after deprotection, the (Z,E)-dien-1-ol 5 as the major product. Acetylation of 5 using acetic anhydride in pyridine gave product 6 (Santangelo et al., 2002).

i HO Br THPO Br 1 2 O ii H

THPO THPO PPh3Br 4 iii 3

iv

v HO AcO 5 6

Scheme 1. Syntheses of (11Z,13E)-hexadecadien-1-ol (5) and (11Z,13E)-hexadecadien-1-yl acetate (6). Reaction conditions: i) Amberlyst 15, t-butylated hydroxyanisole (BHA), 3,4-dihydropyran, diethyl ether, 0C to room temperature (RT); ii) PPh3, MeCN, reflux; iii) sodium ethoxide, THF, reflux then -100°C, addition of (2E)-pentenal, then to RT; iv) Amberlyst 15, BHA, MeOH; 45C; v) Ac2O/pyridine, RT.

The synthesis of (11E,13Z)-hexadecadien-1-yl acetate (11; Scheme 2) started with the protection of 11-bromoundecanol 1, using dihydropyran with Amberlyst 15 as catalyst (Santangelo et al., 2002) to give 2. Oxidation by N-methylmorpholine N- oxide monohydrate in DMSO yielded the aldehyde 7 (Ando et al., 1988; Albini, 1993; Mozuraitis et al., 2002). A Wittig reaction starting from formylmethyl- triphenylphosphonium chloride and 7 resulted in the alkenal 8. A subsequent Wittig reaction between 8 and the ylide formed from propyltriphenylphosphonium bromide and sodium ethoxide, yielded the product 9, which after deprotection (Santangelo et al., 2002) yielded the diene 10. However, because of difficulties in removing the intermediate 7 from the product 8, the Wittig reaction also yielded a monoene that

2 was two carbons shorter than 10. This by-product, and trace amounts of other isomers were separated by repeated chromatography. The purified alcohol was treated with acetic anhydride in pyridine to give the corresponding acetate 11 (Björkling et al., 1985, 1987). A GC-MS comparative-analysis with the insect-produced C16 dien-1-yl acetate showed that E11,Z13-16:Ac was the wrong isomer, so no isomeric purification was carried out.

i ii HO Br THPO Br 2 1

O iii O THPO H O THPO H ClPPh 8 7 3 H

iv v THPO HO 9 10 Ph3P

vi AcO 11 Scheme 2. Synthesis of (11E,13Z)-hexadecadien-1-yl acetate (11). Reaction conditions: i) Amberlyst 15, t-butylated hydroxyanisole (BHA), 3,4-dihydropyran, diethyl ether, 0C to room temperature (RT); ii) N-methylmorpholine N-oxide monohydrate, dimethyl sulfoxide, reflux; iii) a - sodium ethoxide, toluene, reflux; b - tetrahydrofuran (THF), addition of 7, reflux; iv) sodium ethoxide, THF, reflux, addition of 8 at -15C then to RT; v) Amberlyst 15, BHA, MeOH, 45C; vi) Ac2O/pyridine, RT.

Syntheses of (11Z,13E)-hexadecadien-1-ol and (11Z,13E)-hexadecadien-1-yl acetate (Scheme 1). 11-(2-Tetrahydropyranyloxy)-1-undecyl-triphenylphosphonium bromide (3). A solution of 2 (6.00 g, 17.9 mmol) in dry acetonitrile (11 ml) was treated with triphenylphosphine (4.94 g, 18.8 mmol). The reaction mixture was refluxed under N2 for 22 hr. After cooling to room temperature (RT), some toluene was added and the acetonitrile was removed by azeotropic distillation. The crude product 3 (11.22 g), still containing some toluene, was used as such for the next step. (11Z,13E)-Hexadecadien-1-ol (5). The crude product 3 (5.00 g) was dissolved in THF. Toluene was added to a solution of 21% NaOEt (3.24 g, 10.1 mmol), and the solvents were removed by azeotropic distillation. The salt residue was dissolved in THF and added to the solution of 3. The mixture was refluxed for 90 min, while the ethanol formed was removed by distillation. The THF solution was cooled close to the freezing point of the mixture using liquid nitrogen, and then (2E)-pentenal (1.06 g, 12.6 mmol) was added. The reaction was stirred at RT overnight. The mixture was then poured into saturated NH4Cl, and the water phase was extracted three times with hexane. The combined organic phases were dried over MgSO4, filtered and the solvent was removed in vacuo. After MPLC the yield of THP-protected product 4 was 605 mg (22%). The product was dissolved in methanol (10 ml), and Amberlyst 15 (130 mg) and BHA (10 mg) were added to the stirred solution at 45C. After 3 hr of additional stirring, the solvents were removed in vacuo. MPLC yielded 348 mg (17%) of the alcohol 5 as a colorless oil. 1H NMR: : 6.30 (ddd, J = 15.0, 10.9, 1.1 Hz, 1H), 5.95 (t, J = 10.8 Hz, 1H), 5.70 (dt, J = 15.1, 6.6 Hz, 1H), 5.30 (dt, J = 10.8, 7.6 Hz, 1H), 3.64 (t, J = 6.3 Hz, 2H), 1.96-2.26 (m, 4H), 1.51-1.61 (m, 2H), 1.24-1.39 (m, 14H), 1.01 (t, J =7.4 Hz, 3H). To improve the isomeric purity the E,E-isomer was removed in a urea clathrate. A solution of urea (1.82 g, 31.0 mmol) in methanol (10 ml) was heated on a water bath. A 0.8-ml aliquot of this hot solution was added to 89 mg (0.37 mmol) of 5 (90%

3 isomeric purity) in a heated Eppendorf tube. The mixture was slowly cooled to RT and then stored at 4C overnight. The tube was quickly centrifuged and the supernatant was poured into water and extracted three times with diethyl ether, dried over MgSO4, and the solvent was removed in vacuo. This yielded 50 mg of >98% isomerically pure product. (11Z,13E)-Hexadecadien-1-yl acetate (6) A mixture of 1:3 acetic anhydride/pyridine (6.5 ml) was added to the alcohol 5 (65 mg, 0.27 mmol) while stirring. The reaction was continued at 4C overnight, the mixture was poured over ice-water and the water phase was extracted four times with hexane. The combined organic phases were washed with 1 M H2SO4 (aq) and twice with CuSO4 (aq), dried over MgSO4, and the solvent was removed in vacuo. After MPLC the yield of acetate 6 as a colorless liquid was 75 mg (93%). No loss of isomeric purity was observed. 1H NMR: : 6.30 (ddd, J = 15.0, 10.9, 1.1 Hz, 1H), 5.95 (t, J = 10.8 Hz, 1H), 5.70 (dt, J = 15.1, 6.6 Hz, 1H), 5.30 (dt, J = 10.7, 7.6 Hz, 1H), 4.05 (t, J = 6.7 Hz, 2H), 2.07-2.18 (m, 4H), 2.05 (s, 3H), 1.24-1.40 (m, 16H), 1.01 (t, J = 7.4 Hz, 3H). The spectroscopic data are in agreement with those found in the literature for close analogs, e.g. Björkling et al. (1987). Synthesis of (11E,13Z)-hexadecadien-1-yl acetate (Scheme 2). 11-Bromo-1-(2- tetrahydropyranyloxy)-undecane (2) Amberlyst 15 (2.01 g) and t-butylated hydroxyanisole (BHA, 10 mg), followed by 3,4-dihydropyran (5.12 ml, 56.1 mmol) were added to a stirred solution of 11-bromo-1-undecanol 1 (11.57 g, 46.2 mmol) in diethyl ether at 0C. The reaction was left at RT for 3 hr. The catalyst was filtered off, and the ether was removed in vacuo. The yield of 2 as a colorless liquid after MPLC was 13.10 g (85%). 1H NMR: : 4.57 (t, 1H), 3.80-3.93 (m, 1H), 3.73 (dt, J = 9.5, 6.9 Hz, 1H), 3.32-3.54 (m, 2H), 3.40 (t, J = 6.9 Hz, 2H), 1.28-1.90 (m, 24H). 11-(2-Tetrahydroxypyranyloxy)-undecanal (7) 11-Bromo-1-(2- tetrahydropyranyloxy)-undecane (2) (2.67 g, 8.0 mmol) was added at RT to a stirred solution of N-methylmorpholine N-oxide monohydrate (2.31 g, 17.1 mmol) in dimethyl sulfoxide (DMSO, 15 ml). Molecular sieves (2 g, 4Å) were added to the solution. After stirring at 80°C for 80 min, the reaction mixture was poured into ice- cold saturated brine (100 ml), and extracted with 50% ethyl acetate in cyclohexane (6 x 60 ml). The organic phase was dried over MgSO4, and the solvent removed in vacuo. MPLC yielded 1.23 g (47%) of 11-(2-tetrahydropyranyloxy)-undecanal (7) as a colorless liquid. 1H NMR: : 9.76 (t, J = 1.8 Hz, 1H), 4.57 (t, 1H), 3.80-3.93 (m, 1H), 3.73 (dt, J = 9.5, 6.9 Hz, 1H), 3.44-3.55 (m, 1H), 3.37 (dt, J = 9.6, 6.7 Hz, 1H), 2.42 (dt, J = 7.3, 1.8 Hz, 2H), 1.28-1.90 (m, 22H). 13-(2-Tetrahydropyranyloxy)-(2E)-tridecenal (8). Formylmethyl- triphenylphosphonium chloride (4.8 g, 14.0 mmol), dissolved in toluene, was added to a 21% solution of sodium ethoxide in ethanol (4.7 g, 14.6 mmol). The mixture was refluxed for 1 hr, concentrated, dissolved in tetrahydrofuran (THF) and then 2.19 g (8.11 mmol) of 11-(2-tetrahydropyranyloxy)-undecanal was added. The reaction mixture was refluxed overnight, and silica gel (35 g) and some toluene were added. The solvents were removed by azeotropic distillation. MPLC gave 1.43 g (60%) of product 8 as a slightly yellow oil contaminated by the starting material. This impure product was used as such in the next step. 1H NMR: : 9.59 (d, J = 7.9 Hz, 1H), 6.85 (dt, J = 15.6, 6.8 Hz, 1H), 6.11 (ddt, J = 15.6, 7.9, 1.4 Hz, 1H), 4.57 (t, 1H), 3.81-3.92 (m, 1H), 3.73 (dt, J = 9.5, 6.9 Hz, 1H), 3.44-3.55 (m, 1H), 3.38 (dt, J = 9.5, 6.7 Hz, 1H), 2.33 (app. dd, J = 7.0, 1.4 Hz, 2H), 1.22-1.88 (m, 22H). (11E,13Z)-Hexadecadien-1-ol (10). Toluene was added to a solution of 21 % NaOEt (1.36 g, 4.25 mmol) in ethanol, and the solvents were removed by azeotropic

4 distillation. The NaOEt residue was dissolved in THF. Propyltriphenylphosphonium bromide (1.45 g, 3.76 mmol) was added to the solution and the mixture refluxed for 90 min. The ethanol formed was removed by distillation. After cooling to -15°C, 840 mg of the impure enal 8 from the previous step was added. The mixture was then stirred overnight at RT. Silica gel (9 g) and some toluene were added before the solvents were removed by azeotropic distillation. MPLC gave 609 mg (67%) of the THP-protected product 9, which was then dissolved in methanol (4 ml). Amberlyst 15 (110 mg) and BHA (10 mg) were added to the stirred solution at 45C. After 2 hr of additional stirring the solvent was removed in vacuo. After MPLC the yield of the alcohol 10 (92% isomeric purity) as a colorless oil was 282 mg (47%). 1H NMR: : 6.30 (ddd, J = 15.0, 11.0, 1.2 Hz, 1H), 5.91 (t, J = 10.9 Hz, 1H), 5.66 (dt, J = 15.1, 7.0 Hz, 1H), 5.33 (m, 1H), 3.64 (t, J = 6.6 Hz, 2H), 2.18 (dt, J = 7.5, 1.4 Hz, 2H), 2.05 (m, 2H), 1.25-1.65 (m, 16H), 0.95 (t, J = 7.5 Hz, 3H). (11E,13Z)-Hexadecadien-1-yl acetate (11). The alcohol 10 (23 mg, 0.097 mmol) was added to acetic anhydride (0.5 ml) and pyridine (1.5 ml) at RT and left overnight. The mixture was then poured into ice-water and extracted with hexane (4 x 20 ml). The combined organic phases were washed with 1 M H2SO4 (aq) and 10% CuSO4 (aq), and dried over MgSO4. After MPLC the yield of the acetate 11 (92% isomeric purity) as a colorless liquid was 27 mg (99%). 1H NMR: : 6.30 (ddd, J = 15.0, 11.0, 1.2 Hz, 1H), 5.91 (t, J = 11.0 Hz, 1H), 5.66 (dt, J = 15.0, 7.0 Hz, 1H), 5.33 (m, 1H), 4.05 (t, J = 6.8 Hz, 2H), 2.18 (dt, J = 7.5, 1.5 Hz, 2H), 2.05 (m, 2H), 2.05 (s, 3H), 1.22-1.70 (m, 16H), 0.95 (t, J = 7.5 Hz, 3H). The spectroscopic data are in agreement with those found in the literature for close analogs, e.g. Björkling et al. (1987).

REFERENCES

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5 SANTANGELO, E. M., CORACINI, M., WITZGALL, P., CORREA, A. G., and UNELIUS, C. R. 2002. Identification, syntheses and characterization of the geometric isomers of 9,11-hexadecadienal from female pheromone glands of the sugar cane borer Diatraea saccharalis. J. Nat. Prod. 65:909-915.

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