COMPOSITION OF CHEMICALS FOR MANIPULATING THE

BEHAVIOUR OF THE PISTACHIO TWIG BORER, KERMANIA PISTACIELLA (LEPIDOPTERA: OINOPHILIDAE)

FIELD OF THE INVENTION

The invention relates to a composition and procedure for manipulating the behavior of the pistachio twig borer, Kermania pistaciella (K. pistaciella) . More specifically, this invention relates to the preparation and use of racemic (12Z)-2-acetoxy-12-heptadecene, or optical isomers thereof, and of racemic (12Z)-2-hydroxy-12-heptadecene, or optical isomers thereof, for manipulating the behavior of K. pistaciella.

BACKGROUND OF THE INVENTION

The pistachio twig borer, K. pistaciella, is one of the most significant and harmful insect pests in plantations of pistachio, Pistacia vera, in Iran and Turkey. K. pistaciella larvae feed on terminal buds of twigs, and in twigs and shoots, causing abscission of fruit buds and die-back of twigs. Moreover, K. pistaciella may directly damage fruit clusters (1). Many pistachio plantations in Turkey and all plantations in Iran are treated annually with insecticides to reduce damage caused by K. pistaciella larvae. Insecticides are harmful to humans and the environment.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.

The invention is directed to a composition for manipulating the behavior of K. pistaciella, said composition comprising: a chemical selected from one or more of the group consisting of (25',12Z)-2-acetoxy-12-heρtadecene, (2R, 12Z)-2-acetoxy-12-heρtadecene, (2S, 12Z)-2-hydroxy-12-heptadecene, and (2i?,12Z)-2-hydroxy-12-heptadecene. The chemicals can be formulated in a viscous matrix, or can be microencapsulated.

The invention can include a trap that captures attracted male K. pistaciella, or a release device, containing a composition according to the invention.

The invention is also directed to a method of preventing male K. pistaciella from locating, and mating with, female K. pistaciella, comprising using a pheromone-containing release device or matrix, a trap containing a pheromone release device, or microencapsulated pheromone, said pheromone being a chemical selected from one or more of the group consisting of

(2S, 12Z)-2-acetoxy- 12-heptadecene, (2R,12Z)-2-acetoxy-12-heptadecene, (2S, 12Z)-2-hydroxy-12-heptadecene, and (2R, 12Z)-2-hydroxy-12-heptadecene.

The invention also pertains to a process for preparing racemic (12Z)-2-hydroxy-12-heptadecene which comprises adding (llZ)-hexadecenal to an ether solution of methyl magnesium halogenide.

The invention also pertains to a process for preparing racemic (12Z)-2-acetoxy-12-heptadecene, which comprises acetylating (12Z)-2-hydroxy- 12-heptadecene.

The invention also pertains to a process for preparing (ZS, 12Z)-2-hydroxy- 12-heptadecene, (2R, 12Z)-2-acetoxy-12-heptadecene, and (2S, 12Z)-2-acetoxy-2-heptadecene, which comprises adding immobilized lipase Novozym 435 to a solution of (12Z)-2-hydroxy-heptadecene and vinylacetate, separating (25',12Z)-2-hydroxy-12-heptadecene and (2R,12Z)-2-acetoxy-12-heptadecene, and acetylating (25,122)-2-hydroxy-12-heptadecene.

The invention also pertains to a three-step synthetic process for obtaining additional quantities of (25',12Z)-2-acetoxy-12-heptadecene [based on inversion of the absolute configuration of (2R, 122)-2-acetoxy-heptadecene] which comprises stirring (2R,12Z)-2-acetoxy-12-heptadecene and sodium or potassium acetate in alcohol to afford (2R,12Z)-2-hydroxy-12-heptadecene, sulfonylating (2R,12Z)-2-hydroxy-12-heptadecene, and stirring the sulfonate of

(2/?,12Z)-2-hydroxy-12-heptadecene in polar solvent with sodium or potassium acetate to obtain (2S',12Z)-2-acetoxy-12-heptadecene.

The invention also pertains to a synthetic process for scale-up production of (2»S,12Z)-2-acetoxy-12-heptadecene, which comprises converting

(Z)-l-bromo-9-tetradecene to a Grignard reagent, and then adding (5)-propylene

oxide and a catalytic amount of copper (I) iodide, and acetylating the resulting (2S, 12Z)-2-hydroxy-12-heptadecene to (2S, 12Z)-2-acetoxy-12-heρtadecene.

The invention also pertains to a method of alleviating damage in a pistachio planta- tion caused by K. pistaciella which comprises deploying one or more field release devices or micro-encapsulations which contain a chemical selected from one or more of the group consisting of (25',12Z)-2-acetoxy-12-heptadecene, (2R, 12Z)-2-acetoxy- 12-heptadecene, (25',12Z)-2-hydroxy-12-heptadecene, and (2R,12Z)-2-hydroxy-12-heptadecene.

The invention also pertains to a method of diagnosing the population density of K. pistaciella in pistachio plantations which comprises deploying in plantations one or more traps baited with a release device containing a chemical selected from one or more of the group consisting of (25',12Z)-2-acetoxy-12-heptadecene and (2R, 12Z)-2-acetoxy-12-heptadecene.

The invention also pertains to a method of determining the onset of an annual K. pistaciella flight period in a pistachio plantation which comprises deploying in a pistachio plantation one or more traps baited with a release device containing a chemical selected from one or more of the group consisting of

(2S, 12Z)-2-acetoxy-12-heptadecene and (2R, 12Z)-2-acetoxy-12-heptadecene.

The invention also pertains to the use of a composition comprising a chemical selected from one or more traps baited with a release device containing a chemical selected from one or more of the group consisting of

(25',12Z)-2-acetoxy-12-heptadecene and (2i?,12Z)-2-acetoxy-12-heptadecene in manipulating the behaviour of the pistachio twig borer, K. pistaciella.

In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following detailed descriptions.

DRAWINGS

Drawings illustrate specific embodiments of the invention, but should not be construed as restricting the spirit or scope of the invention in any way:

- A -

FIG. 1 illustrates flame ionization detector (FID) and electroantennographic detector (EAD: male K. pistaciella antenna) responses to one female equivalent of female K. pistadella pheromone extract.

FIG. 2 illustrates a mass spectrum of candidate pheromone component A in pheromone gland extracts that elicited the strongest response from male K. pistaciella antenna.

FIG. 3 illustrates a scheme for the syntheses of racemic (12Z)-2-hydroxy-12-heptadecene and (12Z)-2-acetoxy-12-heptadecene.

FIG. 4 illustrates a scheme for the enantioselective syntheses of (25, 12Z)-2-hydroxy-12-heptadecene, (2R, 12Z)-2-acetoxy-12-heptadecene, and (25',12Z)-2-acetoxy-12-heptadecene.

FIG. 5 illustrates a scheme for the syntheses of enantiospecific (2/?,12Z)-2-hydroxy-12-heptadecene, and additional quantities of (25',12Z)-2-acetoxy-12-heptadecene.

FIG. 6 illustrates a scheme for alternative syntheses of

(2S, 12Z)-2-hydroxy-12-heptadecene and (2S, 12Z)-2-acetoxy-12-heptadecene.

FIG. 7 illustrates a scheme for large-scale synthesis of (2S, 12Z)-2-acetoxy- 12-heptadecene .

FIG. 8 illustrates graphical data of captures of male K. pistaciella in field experiment 1 (Turkey) in traps baited with synthetic candidate pheromone components.

FIG. 9 illustrates graphical data of captures of male K. pistaciella in field experi- ment 2 (Turkey) in traps baited with synthetic candidate pheromone components.

FIG. 10 illustrates graphical data of captures of male K. pistaciella in field experiment 3 (Turkey) in traps baited with synthetic candidate pheromone components.

FIG. 11 illustrates graphical data of captures of male K. pistaciella in field experiment 4 (Turkey) in traps baited with synthetic candidate pheromone components.

FIG. 12 illustrates graphical data of captures of male K. pistaciella in field experiment 5 (Iran) in traps baited with synthetic candidate pheromone components.

DETAILED DESCRIPTION OF THE INVENTION Throughout the following description specific details are set forth in order to provide a more thorough understanding to persons skilled in the art. However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense.

Female K. pistaciella have been observed by the inventors to attract male K. pistaciella for mating. This invention relates to the identification, synthesis and field testing of the female K. pistaciella pheromone that attracts male K. pistaciella. This pheromone is new and has not been known previously as a pheromone of K. pistaciella, or as a pheromone in any other insect species.

Gas chromatographic-electroantennographic detection (GC-EAD) analysis of pheromone gland extract of female K. pistaciella both from Turkey and Iran, revealed one compound that elicited strong responses from male K. pistaciella antennae. This compound was identified as (122)-2-acetoxy-12-heptadecene. In field experiments in Turkey, traps baited with racemic (12Z)-2-acetoxy-12-heptadecene captured significant numbers of male K. pistaciella.

(12Z)-2~Acetoxy-12-heptadecene together with the corresponding alcohol (12Z)-2-hydroxy-12-heptadecene was significantly less attractive to male K. pistaciella than was (122)-2-acetoxy-12-heptadecene by itself. Enantiomerically enriched (25',12Z)-2-acetoxy-12-heptadecene strongly attracted male K. pistaciella, whereas (2R,12Z)-2-acetoxy-12-heptadecene was as unattractive as unbaited control traps. Moreover, (2R,122)-2-acetoxy-12-heptadecene when added to (2S, 12Z)-2-acetoxy-12-heptadecene significantly reduced attractiveness of the latter. Either (25 ^r ,12Z)-2-hydroxy-12-heptadecene, (2R,12Z)-2-hydroxy-12-heptadecene, of both, when added to (2.S,12Z)-2-acetoxy-12-heptadecene, strongly reduced attraction of male K. pistaciella to the pheromone (25',12Z)-2-acetoxy-12-heptadecene. In a field experiment in Iran, traps baited with (25',12Z)-2-acetoxy-12-heptadecene strongly attracted male K. pistaciella, whereas traps baited with

(2R,12Z)-2-acetoxy-12-heptadecene, or with (25',12Z)-2-acetoxy-12-heptadecene

plus (2R,12Z)-2-acetoxy-12-heptadecene, were as unattractive to male K. pistaciella as unbaited control traps.

The essence of the invention is the preparation and use of racemic (12Z)-2-acetoxy-12-heptadecene, or optical isomers thereof, and of

(12Z)-2-hydroxy-12-heptadecene, or optical isomers thereof, for manipulating the behavior of K. pistaciella. The invention is directed to the preparation and use of chemicals for manipulating the behavior of K. pistaciella, said chemicals comprising (25, 12Z)-2-acetoxy-12-heptadecene, (2R, 12Z)-2-acetoxy-12-heptadecene, (25,12Z)-2-hydroxy-12-heρtadecene, and (2R,12Z)-2-hydroxy-12-heptadecene.

Chemical proportions can cover all possible combinations and ratios. The composition can be contained in, and released from, slow release devices. Pheromone release devices can consist of a viscous matrix that is impregnated with pheromone and can be laced with insecticide. Such devices can be placed on trees to attract and kill male K. pistaciella, or can be held in traps to capture attracted male K. pistaciella. Pheromone release devices, or microencapsulated pheromone, can also be distributed in pistachio plantations to prevent male K. pistaciella from locating, and mating with, female K. pistaciella.

Analysis of pheromone extract of female K. pistaciella

K. pistaciella from both Turkey and Iran were analysed, and the following descriptions of analytical procedures and results pertain to K. pistaciella both from Iran and Turkey.

Abdominal tips with pheromone glands of female K. pistaciella were removed and extracted in hexane for 5 minutes. Aliquots of one female equivalent of such extracts were analysed by coupled gas chromatographic-electroantennographic detection (GC-EAD) (2). These analyses revealed 2 components (A and B) that elicited a strong response from male K. pistaciella antennae (FIG. 1).

FIG. 1 illustrates flame ionization detector (FID) and electroantennographic detector (EAD: male K. pistaciella antenna) responses to one female equivalent of female K. pistaciella pheromone extract. Chromatography: Hewlett Packard (HP) gas chromatograph (GC) 5890 equipped with a GC column (30 m x 0.25 mm ID) coated with DB-5 (J&W, Folsom, California); splitless injection, temperature of injection port and FID: 24O ⁰ C; temperature program: 5O ⁰ C (1 min), 2O ⁰ C per min to 28O ⁰ C. Retention indices (3) of compound A on columns coated with DB-23, DB-5

and DB-210 were 2373, 2017 and 2309, respectively. Retention indices (RIs) (3) of compound B on columns coated with DB-23, DB-5 and DB-210 were 2395, 1896 and 2112, respectively.

The mass spectrum of compound A (FIG. 2) with diagnostic fragment ion m/z 61 indicated an acetate functionality, and fragment ion m/z 236 [MW (296)-60] suggested that compound A had one double bond.

FIG. 2 illustrates the mass spectrum of compound A obtained from a Varian 2000 GC-tandem mass spectrometer (MS/MS).

Hydrogenation of pheromone gland extract, followed by renewed GC-EAD and GC-MS analyses, revealed a new EAD-active compound (Al), suggesting that hydrogenation of A had resulted in a saturated volatile (Al) with different chromato- graphic characteristics [Retention indices: 2440 (DB-23); 2108 (DB-5); and 2395 (DB-210)]. Compound Al was determined to be 2-acetoxy-heptadecane through comparative GC and GC-MS analyses of Al and synthetic acetoxy-heptadecanes with the acetoxy group in Cl, C2, C3, C4, C5, C6, and C7, respectively. Retention indices of female K. pistaciella-iprodaced A further suggested the double bond location was either in C 10, C 11 , C 12 or C 13.

Synthetic (12Z)-2-acetoxy-heptadecene, but not any of (10Z)-2-acetoxy-10-heptadecene, (1 lZ)-2-acetoxy-l 1-heptadecene, (13Z)- 2-acetoxy-13-heptadecene, (10E)- 2-acetoxy-lO-heptadecene, (HE)- 2-acetoxy-l 1-heptadecene, or (12E)-2-acetoxy-12-heptadecene, co-chromatographed with female-produced A and elicited comparable responses from male K. pistaciella antennae. With the identification of component A as

(12Z)-2-acetoxy-12-heptadecene, component B was hypothesized and shown to be (12Z)-2-hydroxy-12~heptadecene through comparative GC, GC-MS and GC-εAD with an authentic standard. Female K. pistaciella were also shown to produce the S-enantiomer of A, as determined by comparative GC analysis (chiral Cyclodex-B column; J&W Scientific, Folsom, California) of insect-produced A and synthetic enantiomers of A.

Racemic (12Z)-2-hydroxy-12-heptadecene (Compound 2 in Figure 3) was synthesized starting with commercially available (112)-hexadecenal (1 in Figure 3, Bedoukian Research Inc., Danbury, CT). Compound 1 (225 g, 0.915 mol) in 400

ml of ether was added dropwise under argon at 0-10 ⁰ C during 3 hours to a stirred solution of methyl magnesium chloride (3M in ether; 333 ml, 1 mol). The mixture was allowed to warm-up to room temperature, quenched with 200 ml of saturated aqueous ammonium chloride (aq. NH ₄ Cl), and extracted three times with ether (600 ml). Extracts were combined, washed with brine, dried (anh. MgSO ₄ ), filtered and evaporated at 12-15 mm Hg, affording 233 g of (Z)-12-heptadecen-2-ol (2, 99% pure by GC, 0.906 mol, yield 99%) as a slightly yellowish oil. Racemic (12Z)-2-acetoxy-12-heptadecene (3 in Figure 1) was synthesized by adding dropwise at room temperature 94 ml (0.99 mol) of acetic anhydride to a stirred solution of 233 g (0.906 mol) of alcohol 2 in 100 ml (1.23 mol) of pyridine. After stirring 6 hours, the reaction product was extracted with ether/hexane (1:1) (3 x 250 ml). Combined extracts were washed successively with saturated aq. sodium bicarbonate (aq. NaHCO ₃ ), 10% HCl, water and brine, and dried (anh. MgSO ₄ ). Filtration and removal of solvents afforded 270 g of (12Z)-2-acetoxy-12-heptadecene (3, 99.5% pure by GC, quantitative yield).

FIG. 3 illustrates the scheme for the syntheses of racemic

( 12Z)-2-hydroxy- 12-heptadecene and ( 12Z)-2-acetoxy- 12-heptadecene .

Syntheses of (ZS, 12Z)-12-heptadecene (4 in FIG. 4), (2i?,12Z)-2-acetoxy-12-heptadecene (5 in Figure 4), and

(25 ^r ,12Z)-2-acetoxy-12-heptadecene (6 in FIG. 4) were initiated by adding 70 mg of immobilized lipase Novozym 435 (4) (10,000 units per gram, Sigma-Aldrich Company, St. Louis, MO.) to a stirred solution of 0.600 g of alcohol 2 (2.36 mmol) and 0.53 ml of vinylacetate (5.75 mmol, Aldrich Chemical Company, Milwaukee, WI) in 3 ml of hexane. After stirring 4 hours at 40 ⁰ C, the Novozym-containing resin was filtered, solvents were evaporated in vacuo, and alcohol 4 (0.280 g) and acetate 5 (0.340 g) were separated by flash chromatography [10 g of SiO ₂ , ether/hexane (1:20) as eluent]. Subsequent acetylation of alcohol 4 by acetic anhydride in pyridine, and usual work-up (see above) afforded 0.320 g (1.08 mmol) of (2S,12Z)-2-acetoxy-12-heptadecene [6, 99.5% pure, yield 45.8% (theory: 50%)]. Enantiomeric excess (ee) of (25 ^r ,12Z)-2-hydroxy-12-heptadecene (4), (2R,12Z)-2-hydroxy-12-heptadecene (5), and (ZS, 12Z)-2-acetoxy-l 2-heptadecene (6) was >99% ee, >95% ee, and >99% ee, respectively, as determined by chiral gas chromatography.

FIG. 4 illustrates the scheme for the enantioselective syntheses of (2-?,12Z)-2-hydroxy-12-heptadecene (4), (2R,12Z)-2-acetoxy-12-heptadecene (5), and (25 ^r ,12Z)-2-acetoxy-12-heptadecene (6).

Additional quantities of (2S, 12Z)~2-acetoxy-12-heptadecene (6) were obtained by a three-step-synthesis (FIG. 5), based on inversion of the absolute configuration of (2i?,12Z)-2-acetoxy-12-heptadecene (5). The mixture of 0.280 g of acetate 5 and 1.0 g of K ₂ CO ₃ in 10 ml of methanol was stirred 18 hours at room temperature. After removal of 9 ml of methanol in vacuo, 20 ml of ether and 5 ml of water were added to the reaction mixture. The organic layer was washed with water and brine, dried (anh. MgSO ₄ ), and solvents removed in vacuo to afford (2R,12Z)-2-hydroxy-12-heptadecene (7, 95% pure by GC). Without further purification alcohol 7 was mesylated at O ⁰ C with methanesulfonyl chloride (1.5 ml) in the presence of triethylamine (2 ml) in dichloromethane (10 ml). After 1 hour, the reaction was quenched with aq. NaHCO ₃ . Further extraction with ether (50 ml), washing of extract with 10% HCl, water, and brine, and drying and evaporation of solvent afforded the (R)-mesylate 8. Mesylate 8 without further purification was stirred 96 hours at 60-70°C in dry dimethylformamide (10 ml) with 3 g of sodium acetate. Usual work-up of the reaction mixture and purification of the product by flash column chromatography yielded 0.180 g of pure

(2)S,12Z)-2-acetoxy-12-heptadecene (6) with 90% ee. Overall yield based on acetate 5 was 64% .

FIG. 5 illustrates the scheme for the enantioselective syntheses of (2R,12Z)-2-hydroxy-12-heptadecene (7), and additional quantities of (2S, 12Z)-2-acetoxy-12-heptadecene (6).

The absolute configuration of acetates 5 and 6 was confirmed by alternative chiral synthesis of (2S',122)-2-acetoxy-12-heptadecene (6) using hydrolytic kinetic resolu- tion (HKR) as the key step (FIG. 6), as follows: 37.5 mmol of butyl lithium (15 ml of 2.5 M solution in hexane) was added under argon at -70 ⁰ C to 3.45 ml (30.0 mmol) of 1-hexyne (9) in 50 ml THF. After stirring 0.5 hours, 5 ml of HMPA and 1.8 ml (8.20 mmol) of 11-bromo-l-undecene (10) was added. The reaction mixture was then warmed to room temperature, stirred 24 hours, quenched with aq. NH ₄ Cl, and extracted with hexane (2 x 50 ml). Combined extracts were washed with brine, dried (anh. MgSO ₄ ), and solvents evaporated. The crude product was filtered through 10 g of silica eluting with hexane, yielding 2.05 g of hydrocarbon 11 (85%

pure by GC; 7.65 mmol). Without further purification compound 11 was treated with 13.1 mmol of rø-chloroperoxybenzoic acid at 0°C in 20 ml of dichloromethane. After 3 hours, the mixture was warmed to room temperature, and after a subsequent 30 min quenched with 50 ml of 2N NaOH and treated with 100 ml of ether. The organic layer was separated, washed with water and brine, and dried (anh. MgSO ₄ ). After filtration, removal of solvents in vacuo and flash chromatography, 0.82 g (87% pure by GC; 2.85 mmol) of unsaturated epoxide 12 was obtained. Epoxide 12 in 3 ml of dry THF with 0.030 ml of water was subjected to HKR with freshly prepared R,R-1 Jacobsen catalyst (7,8) [25 mg of (R,R)-N,N ^N -bis(3 ,5-di-tert-butylsalicylidene)-l ,2-cyclohexanediaminocobalt(II) (Aldrich Chemical Company, Milwaukee, WI) stirred 1 hour with 0.20 ml of toluene and 0.050 ml of acetic acid, and solvents removed in vacuo]. After stirring the reaction mixture 7 days, the recovered (R)-epoxide 13 was separated by flash chromatography [15 g of silica with hexanerether (80:20) as eluent] from the diol 14 and the catalyst, affording 0.4 g of 85% pure (R)-epoxide 13. After removal of solvents, 13 was dissolved in 2 ml of ether and added to a lithium aluminum hydride suspension (10 mmol) in 20 ml of ether. After stirring 3 hours, the reaction mixture was quenched with 2ν NaOH, and the organic phase removed and evaporated. The resulting acetylenic alcohol 15 was hydrogenated in 30 ml of hexane with Lindlar catalyst (5% palladium on calcium carbonate poisoned with lead, 100 mg) and 1 ml of quinoline, yielding 0.350 g of (25',12Z)-2-hydroxy-12-heptadecene (4). Acetylation of 4, as described above, and further flash column purification gave 0.350 g (1.18 mmol) of (25,12Z)-2-acetoxy-12-heptadecene (6) (yield 14.5% based on 11-bromo-l-undecene 10) with 85% ee.

FIG. 6 illustrates the scheme for an alternative synthesis of

(25, 12Z)-2-hydroxy-12-heptadecene and {IS, 12Z)-2-acetoxy-12-heptadecene.

The synthesis of 6 that could readily be scaled up employed yet another procedure, as follows (FIG 7): Methanesulfonyl chloride (1.62 ml, 21.0 mmol) was added dropwise at 0 ⁰ C to the stirred mixture of (Z)-9-tetradecen-l-ol (16; 3.95 g, 18.6 mmol; Bedoukian Research Inc., Danbury, CT) and triethylamine (4.20 ml, 30.0 mmol) in dichloromethane (25 ml). The reaction mixture was stirred for 30 min, warmed to room temperature, and water (30 ml) was added. The organic phase was separated, washed with water and brine, and dried. Solvent was evaporated, and DMSO (50 ml) was added to the product. The mixture was stirred and lithium bromide (4.85 g, 55.8 mmol) was added in one portion. After stirring for 3 hours at

4O ⁰ C, hexane (100 ml) and water (100 ml) were added. Hexane extracts were washed 4 times with water and brine, dried, and evaporated, yielding (2)-l-bromo-9-tetradecene (17; 4.00 g 14.5 mmol, > 98% pure, 78% yield).

AU bromide was converted to Grignard reagent by refluxing it for two hours under argon in dry THF (25 ml) with activated Mg (0.68 g). The mixture was cooled to -10 ⁰ C, and CuI (270 mg, 1.45 mmol) was added with vigorous stirring. After 5 min, (S)-propylene oxide (1.17 ml, 16.7 mmol; Alfa Aesar, Ward Hill, MA) in THF (5 ml) was added dropwise. The reaction mixture was kept at -10 ⁰ C for 20 min, then allowed to warm to room temperature, and quenched with saturated aqueous NH ₄ Cl (100 ml). The organic components were extracted with ether (2 x 40 ml), and extracts were washed with water and brine, dried (anh. MgSO ₄ ), filtered and the filtrate evaporated in vacuo, yielding crude alcohol 4. To the crude alcohol 4 acetic anhydride (1.42 ml, 15.0 mmol) and pyridine (1.62 ml, 20.0 mmol) were added. After the mixture was stirred for 18 hours at room temperature, solvents were removed in vacuo, and (2S',12Z)-2-acetoxy-12-heptadecene (6) was separated from Grignard by-products by flash column chromatography (5 % ether in hexane as eluent, 30 g SiO ₂ ). The yield of 6 (98.5% pure, > 99 ee) was 3.10 g (70.5% based on 17). Overall yield was 55% .

FIG. 7 illustrates the scheme for large-scale synthesis of (25',12Z)-2-acetoxy-12-heptadecene.

Attraction of male K. pistaciella to synthetic test animals Field experiments were conducted in a pistachio orchard of the Gaziantep Pistachio Research Institute in Gaziantep, Turkey, and a pistachio orchard near Rafsanjan, Kerman Province, Iran. At 10-20 m intervals, self-made sticky 2-L milk carton traps (9) were suspended from trees ~ 1.5 m above ground in complete randomized blocks. Traps were baited with a gray sleeve stopper (10) impregnated with HPLC fractionated test chemicals in solvent, or with a solvent control. All test chemicals were greater 93 % chemically and geometrically pure. The optical purity of all synthetic chemicals was > 95 % ee, as determined by methods described above.

EXAMPLE #1 In field experiment 1 in Turkey, traps baited with racemic

(12Z)-2-acetoxy-12-heptadecene captured significant numbers of male K. pistaciella (FIG. 8)

FIG. 8 illustrates graphical data of captures of male K. pistaciella in sticky traps baited with racemic (12Z)-2-acetoxy-12-heptadecene. Pistachio orchard of the Gaziantep Pistachio Research Institute in Gaziantep, Turkey; 12 April to 4 May 2001; 10 replicates. Bars with different letter superscripts are significantly different; Kruskal-Wallis analysis of variance by ranks (11) followed by comparison of means (Scheffe test), P< 0.05.

EXAMPLE # 2

In field experiment 2 in Turkey (FIG. 9), traps baited with racemic (12Z)-2-acetoxy-12-heptadecene captured large numbers of male K. pistaciella, whereas traps baited with (12Z)-2-hydroxy-heptadecene were as unattractive as unbaited control traps. Addition of (12Z)-2-hydroxy-12-heptadecene to (12Z)-2-acetoxy-12-heptadecene significantly reduced lure attractiveness.

FIG. 9 illustrates graphical data of captures of male K. pistaciella in sticky traps baited with (12Z)-2-acetoxy-heptadecene and (12Z)-2-hydroxy-12-heptadecene singly and in combination. Pistachio orchard of the Gaziantep Pistachio Research Institute in Gaziantep, Turkey; 17 April - 23 May 2001; 8 replicates. Bars with different letter superscripts are significantly different; Kruskal-Wallis analysis of variance by ranks (11) followed by comparisons of means (Scheffe test), P < 0.05.

EXAMPLE # 3

In field experiment 3 in Turkey (FIG. 10), traps baited with enantiospecific (25',12Z)-2-acetoxy-12-heptadecene captured significant numbers of male K. pistaciella, whereas traps baited with enantiospecific

(2R,12Z)-2-acetoxy- 12-heptadecene were as unattractive as unbaited control traps.

Addition of (2R,12Z)-2-acetoxy- 12-heptadecene to significantly reduced attractiveness of the latter.

FIG. 10 illustrates graphical data of captures of male K. pistaciella in sticky traps baited with (2S, 12Z)-2-acetoxy- 12-heptadecene and (2R, 12Z)-2-acetoxy-heptadecene singly and in combination. Pistachio orchard of the Gaziantep Pistachio Research Institute in Gaziantep, Turkey; 27 April to 23 May 2001; 8 replicates. Bars with different letter superscripts are significantly different; Kruskal-Wallis analysis of variance by ranks (11) followed by comparison of means (Scheffe test) P < 0.05.

EXAMPLE # 4

In field experiment 4 in Turkey (FIG. 11), traps baited with (25',12Z)-2-acetoxy-12-heptadecene captured large numbers of male K. pistaciella, whereas traps baited with (25,12Z)-2-acetoxy-12-heptadecene combined with either (2S',12Z)-2-hydroxy-heptadecene or (2R,12Z)-2-hydroxy-12-heptadecene captured significantly fewer male K. pistaciella.

FIG. 11 illustrates graphical data of captures of male K. pistaciella in sticky traps baited with (2S',12Z)-2-acetoxy-12-heptadecene alone and in combination with either (2S, 12Z)-2-hydroxy-12-heptadecene or (2R, 12Z)-2-hydroxy-12-heptadecene. Pistachio orchard of the Gaziantep Pistachio Research Institute in Gaziantep,

Turkey; 29 April to 21 May 2002; 10 replicates. Bars with different letter superscripts are significantly different; Kruskal-Wallis analysis of variance by ranks (11) followed by comparison of means (Scheffe test), P < 0.05.

EXAMPLE #5

In field experiment 5 in Iran (FIG. 12), traps baited with

(2S',12Z)-2-acetoxy-12-heptadecene captured large numbers of insects, whereas traps baited with (2R,12Z)-2-acetoxy-12-heptadecene, or with

(2R, 12Z)-2-acetoxy-12-heptadecene plus (25, 12Z)-2-acetoxy-12-heptadecene, captured as few male K. pistaciella as unbaited control traps.

FIG. 12 illustrates graphical data of captures of male K. pistaciella in sticky traps baited with enantiospecific (2S, 12Z)-2-acetoxy-heptadecene and (2R,12Z)-2-acetoxy-12-heptadecene singly and in combination. Pistachio plantation near Rafsanjan, Kerman Province, Iran; 8-18 April 2002; 10 replicates. Bars with different letter superscripts are significantly different; Kruskal-Wallis analysis of variance by ranks (11) followed by comparison of means (Scheffe test), P < 0.05.

All compounds may become part of a commercial lure (formulation) for pheromone-based monitoring, manipulation and/or control of K. pistaciella populations. As will be apparent to those skilled in the art in light of the forgoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. Accordingly, the scope of the new invention is to be construed in accordance with the substance defined by the following claims.

W 2

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REFERENCES AND NOTES

1. Mart, C, Celik, M. Y., and Yigit, A. (1995). Biological Observations and chemical control of pistachio twig borer, K. pistaciella Ams. (Lep. Oinophilidae), injurious in pistachio orchards in Turkey. Acta Horticulture 419: 373-378.

2. Arn, H., Stadler, E., and Rauscher, S. (1975). The electroantennographic detector- a selective and sensitive tool in the gas chromatographic analysis of insect pheromones. Z. Naturforsch. 30c: 722-725.

3. Van den Dool, H., and Kratz, P. D. (1963). A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J. Chromatography 2: 463-471.

4. Xiao, L., and Kitazume, T. (1997). Optically active propargylic and allylic alcohols with a difluoromethyl group at the terminal carbon. Tetrahedron: Asymmetry 8 (21): 3597-3601.

5. Nuclear magnetic resonance (NMR) spectroscopy of synthetic compounds was conducted on a Varian AS500 (at 499.77 MHz for ¹ H and 125.68 MHz for

13C) spectrometer, with chemical shifts reported in ppm relative to TMS (IH, δ 0.00) and CDCl ₃ (13C, δ 77.00). For (2S, 12Z)-2-acetoxy-12-heptadecene: ¹ H NMR (CDCl ₃ ) δ: 0.87 (t, 3H, J=7.0Hz), 1.18 (d, 3H, /=6.3Hz) 1.21-1.35 (m, 22H) 1.50 (m, 2H), 2.00 (s, 3H), 4.86 (m, IH), 5.33 (m, 2H). ¹³ C NMR (CDCl ₃ ) δ: 170.68, 129.79, 129.77, 70.99, 35.88, 31.92, 29.72, 29.50, 29.485, 29.47, 29.42, 29.24, 27.14, 26.87, 25.37, 22.30, 21.31, 19.90, 13.94.

6. NMR data of (25',12Z)-2-hydroxy-12-heptadecene, were as follows: ¹ H NMR (CDCl ₃ ) δ: 0.89 (t, 3H, J=7.1 Hz), 1.18 (d, 3H, J=6.3 Hz), 1.21-1.49 (m, 24H), 2.05 (m, IH), 3.78 (m, IH), 5.34 (m, 2H). ¹³ C NMR (CDCl ₃ ) δ: 129.84, 129.82, 68.15, 39.34, 31.94, 29.74, 29.63, 29.59, 29.55, 29.51, 29.27, 27.16, 26.88, 25.76, 23.45, 22.32, 13.98.

7. Schaus, S.E., Branalt, J., and Jacobsen, E.N (1998). Total synthesis of Muconin by efficient assembly of chiral building blocks.. J. Org. Chem. 63:

4876-4877.

8. Tokunaga, M., Farrow, J.F., Kakuichi, F., and Jacobsen, E.N. (1997). Asymmetric catalysis with water: Efficient kinetic resolution of terminal epoxides by means of catalytic hydrolysis. Science 277: 936-938.

9. Gray, T.G., Slessor, K.N., Shepherd, R.F., Grant, G.G., and Manville, J.F. (1984). European pine shoot moth, Rhyacionia buoliana (Lepidoptera: Tortricidae): Identification of additional components resulting in an improved lure. Can. Entomol. 116: 1525-1532.

10. The West Company, Lionville, Pennsylvania USA.

11. Zar, J. H. (1984). Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, New Jersey, 718pp.