Journal of Chemical Ecology, Vol. 7, No. 4, 1981

S Y N T H E S I S A N D I D E N T I F I C A T I O N OF A T H I R D C O M P O N E N T OF THE S A N JOSE S C A L E SEX P H E R O M O N E

R.J. ANDERSON, K.G. ADAMS,

2 M.J. GIESELMANN,

2 C.A. HENRICK,

1

3 H.R. CHINN, 2

2 R.E. RICE, 4 and W.L. ROELOFS

3

2Chemistry Research Laboratory, Zoecon Corporation, Palo Alto, California 94304 3Department of Entomology, New York State Agricultural Experiment Station, Geneva, New York 14456 4Department of Entomology, University of California, Davis, California 95616

(Received August 5, 1980)

A b s t r a c t - - T w o components of the San Jose scale sex pheromone had

previously been identified as 7-methyl-3-methylene-7-octen-1-yl propanoate (I) and (Z)-3,7-dimethyl-2,7-octadien-l-yl propanoate (II). An isomer and various homologs have subsequently been synthesized and tested in a greenhouse bioassay. The Eisomer of I1 (XI) was found to be attractive to male scales. This compound has now been isolated and identified from airborne extracts of virgin female scales. The composition of the natural pheromone was 48.5%, 46.7%, and 4.8% of I, II, and XI, respectively. In field tests in California and New York, synthetic XI was found to be attractive to male scales alone and in combination with I and II, but there was no obvious increase in trap catch when the synthetic isomers were present in the same ratio as in the natural blend. Key W o r d s - - S a n Jose scale, Quadraspidiotusperniciosus, sex pheromone, synthesis, 7-methyl-3-methylene-7-octen-l-yl propanoate, (E)- and (Z)3,7-dimethyl-2,7-octadien-l-yl propanoate, attractant, Homoptera, Diaspididae.

1Quadraspidiotus perniciosus (Comstock) (Homoptera: Diaspididae). 695 0098-0331/ 81/ 0700-0695503.00/09 1981PlenumPublishingCorporation

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ANDERSON ET AL. INTRODUCTION

The sex pheromone of the San Jose scale [Quadraspidiotus perniciosus (Comstock)], a commercially important homopterous pest, was isolated and initially identified (Gieselmann et al., 1979a) as a two-component mixture of 7-methyl-3-methylene-7-octen-l-yl propanoate (I) and (Z)-3,7-dimethyl-2,7octadien-l-yl propanoate (II).

I

o

oJ - . / I

II

These structural assignments were confirmed by the synthesis of each of these ester components and comparison of the synthetic samples with the naturally occurring pheromone (Anderson et al., 1979). To establish which structural features of these pheromone components are crucial for attractancy in this species, several closely related homologs of I and II and an isomer of II were prepared. We report herein the preparation and the biological activity of these new compounds, and the resultant identification of a third component of the San Jose scale pheromone. M E T H O D S AND MATERIALS

Preparative thin-layer chromatography was carried out on 1-m • 20-cm glass plates coated with 1.3 mm of Merck (Darmstadt) silica gel PF-254. Silica plates impregnated with Rhodamine 6G dye were used to visualize those compounds lacking significant UV absorbance at 254 nm. Gas-liquid chromatographic analyses were performed on model 402 Hewlett-Packard instruments equipped with hydrogen flame ionization detectors. N M R spectra were determined on a Varian T-60 spectrometer. Infrared spectra were measured on a Unicam SP-200G or a Perkin-Elmer 281 spectrophotometer. Mass spectra were measured on a Hewlett-Packard model 5985 GC-MS-DS with an all glass jet separator at 70 eV ionization potential. Elemental analyses were performed by Mr. Erich Meier at the Stanford University Chemistry Department microanalytical laboratories. All temperatures are in degrees Celsius. 7-Methyl-3-methylene-7-octen-l-yl Acetate (IIl). To 6.25 ml (10 mmol) of 1.6 M n-butyllithium in hexane at room temperature and under a nitrogen atmosphere was added 1.49 ml (1.16 g, 10 mmol) of N,N,N',N'-tetramethyl-

T H I R D COMPONENT OF SAN JOSE SCALE PHEROMONE

697

ethylenediamine (TMEDA) dropwise. After 30 min, 430 mg (5.0 mmol) of 3-methyl-3-buten-l-ol in 1 ml of hexane and another 0.75 ml of T M E D A were added, and the resulting yellow mixture was stirred 3.5 hr at room temperature. The reaction mixture was cooled to 0 ~ and 745 mg (5.0 mmol) of l-bromo-3-methyl-3-butene in about 1 ml of hexane was added at once. The now colorless solution was stirred for 1 hr, and then water was added to the reaction. The mixture was poured into ether and 5% aqueous HC1. The ether fraction was separated, washed with saturated NaHCO3 and brine, and dried (Na2SO4). Solvent was carefully removed in vacuo, and the residue was stirred for 2 hr at 60 ~ with 0.66 ml (0.71 g, 7 mmol) of acetic anhydride and 0.8 ml (10 mmol) of dry pyridine under a nitrogen atmosphere. Ice was added to the reaction mixture, and after 30 min the mixture was poured into pentane and 5% aqueous HC1. The pentane fraction was washed with 2 M Na2CO3 and brine and was dried (NazSO4). Removal of solvent in vacuo gave about 1 g of crude product which was applied to two 1-m X 20-cm preparative silica plates (impregnated with Rhodamine 6G) which were developed in 5% ether in hexane. Recovery of the purified ester followed by microdistillation [bp 60 ~ (bath) 0.9 mm] gave 392 mg (2.0 mmol, 40% yield) of acetate III in a chemical purity of 97.3% as determined by GLC analysis (2 m 3% OV-17/0.4% Carbowax). IR (neat) 3075 ( C = C H 2 ) and 1740 cm -1 (C~---O); N M R (CDCI3, 6) 4.80 (br s, 2H), 4.70 (br s, 2H), 4.17 (t, 2H, J = 7 Hz), 2.33 (br t, 2H, J = 7 Hz), 2.07 (s, 3H) and 1.73 ppm (br s, 3H). MS (CI, CH4) m/e (relative intensity) 137 (100). Analysis: Calc'd for C12H2oO2: C, 73.43; H, 10.27. Found: C, 73.48; H, 10.21. (Z)-3,7-Dimethyl-2,7-octadien-l-yl Acetate (IV). A mixture of 90 mg (0.58 mmol) of (Z)-3,7-dimethyl-2,7-octadien-l-ol (VI) (Anderson et al., 1979), 150/~1 of acetic anhydride, and 250 #1 of pyridine was stirred under a nitrogen atmosphere at room temperature overnight. Ice was added to the reaction, and after 30 min the mixture was poured into ether and 5% aqueous HC1. The ether fraction was separated, washed with 2 M Na2CO3 and brine, and dried (Na2SO4). The solvent was removed in vacuo, and the residue was microdistilled [bp 55 ~ (bath) 0.25 mm] to give 88 mg (0.45 mmol, 75% yield) of acetate IV in a chemical purity of 95.5% as determined by GLC analysis (2 m 3% OV-17/0.4% Carbowax). IR (CC14) 3070 (C~---CH2) and 1740 cm -1 ( C = O ) ; N M R (CDC13, 6) 5.40 (br t, 1H, J = 7 Hz), 4.72 (br s, 2H), 4.57 (d, 2H, J = 7 Hz), 2.06 (s, 3H) and 1.75 ppm (br s, 6H)~ MS (CI, CH4) m/e (relative intensity) 137 (100). Analysis: Calc'd for C12H2002: C, 73.43; H, 10.27. Found: C, 73.15; H, 10.04. 3-Methylene-7-octen-l-yl Propanoate (VII). To a suspension of 1.0 g (4111 mmol) of magnesium turnings in 6 ml of dry ether under a nitrogen atmosphere was added dropwise 5.0 g (33.6 mmol) of 1-bromo-4-pentene in 25 ml of ether at a rate to maintain a gentle reflux of ether. Titration of the

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Grignard reagent (Watson and Eastham, 1967) gave a molarity of 0.72 M (~67% yield). To a solution of 0.68 g (3.3 mmol) of cuprous bromide-dimethyl sulfide complex (House et at., 1975) in 5 ml of dry ether and 10 ml of dimethyl sulfide cooled to - 4 0 ~ under a nitrogen atmosphere was added 4.2 ml (3.0 mmol) of 0.72 M 4-pentenyl magnesium bromide in ether while the temperature was maintained at - 3 5 ~ . After 1 hr, the mixture was recooled to - 4 5 ~ and 0.41 g (3.2 mmol) of 3,butyn-l-yl propanoate in 10 ml of ether was added while the temperature was raised to - 2 5 ~ After another 4 hr, 10 ml of saturated aqueous NH4C1 was added, and the reaction was allowed to slowly warm to room temperature overnight. The mixture was filtered through a pad of Celite, which was washed several times with additional ether. The filtrate was separated into components; and the ether fraction was washed with brine and dried (Na2SO4). Careful removal of solvent gave 700 mg of crude product which was purified by application to two 1-m • 20-cm preparative silica plates (Rhodamine impregnated) and development in 10% ethyl acetate in hexane. Removal of a band with an Rf value of about 0.4 followed by microdistillation [bp 65 ~ (bath) 1.0 mm] gave 276 mg (1.41 mmol, 47% yield) of propanoate VII in a chemical purity of 96.5% as determined by GLC analysis (2 m 3% OV- 17 / 0.4% Carbowax). IR (neat) 3075 ( C = C H 2) and 1730 cm -1 ( C = O ) ; N M R (CDC13, 6) 6.07-5.50 (m, 1H), 5.07 (m, 1H), 4.80 (br s, 3H), 4.17 (t, 2H, J = 7 Hz), and 1.13 ppm (t, 3H, J = 7.5 Hz). MS (CI, CH4) m/e (relative intensity) 197 (M + + H, 4), 123 (100). Analysis: Calc'd for C12H2002: C, 73.43; H, 10.27. Found: C, 73.66; H, !0.27. Methyl (E)-3,7-Dimethyl-2,7-Octadienoate (IX). Lithium-l% sodium wire (224 mg, 32 mmol) was washed free of grease with hexane and was cut i n t o small pieces upon addition to 5 ml of dry ether under an argon atmosphere. The suspension was cooled to 0 ~ and 2.45 g (15 mmol) of l-bromo-4-methyl-4-pentene (VIII; Anderson et al., 1979) in 10 ml of ether was added dropwise while the temperature was maintained at 0 ~ . After 2.5 hr, titration of an aliquot (Watson and Eastham, 1967) gave a molarity of 0.75 M ("~75% yield). To a suspension of 2.28 g (12 mmol) of cuprous iodide in 10 ml of dry ether at - 4 0 ~ under an argon atmosphere was slowly added 13 ml (9.75 mmol) of 0.75 M 4-methyl-4-pentenyllithium followed by 1.81 g (15.6 mmol) of T M E D A . After 30 min, the reaction was cooled to - 6 0 ~ and 980 mg (10 mmol) of methyl 2-butynoate in several ml of ether was added. The reaction was maintained at - 6 0 ~ for 1.5 hr, and then was quenched by the dropwise addition of 4 ml of methanol. The cooling bath was removed, and after the mixture had warmed to room temperature, 10 ml of saturated aqueous (NH4)2SO4 was added. After 30 min, the mixture was filtered through a Celite pad which was washed thoroughly with additional ether. The filtrate was

T H I R D COMPONENT OF SAN JOSE SCALE PHEROMONE

699

separated into components, and the ether fraction was washed with water and brine and then dried (Na2SO4). Careful removal of solvent in vacuo followed by microdistillation [bp 70 ~ (bath) 3.0 mm] gave 1.50 g (8.3 mmol, 85% yield) of methyl ester IX. GLC analysis (2 m 10% UCON) indicated that IX had been produced with high stereospecificity (E: Z, 99.0: 1.0). IR (neat) 3090 (C=CH2) and 1725 c m -1 (C~-~-O); NMR (CDC13, 6) 5.63 (br s, IH), 4.67 (br s, 2H), 3.67 (s, 3Hi, 2.13 (d, 3H, J = 2 Hz), and 1.70 ppm (br s, 3H). MS (70 eV) m/e (relative intensity) 182 (M +, 3), 95 (100). Analysis: Calc'd for Cll H1802: C, 72.49; H, 9.95. Found: C, 72.37; H, 9.87. (E)-3,7-Dimethyl-2,7-oetadien-l-ol (X). To a solution of 151 mg (0.83 mmol) of methyl ester IX in 7.5 ml of benzene under a nitrogen atmosphere was added 2.5 ml (4.5 mmol) of a 27.8% solution of diisobutylaluminum hydride (DIBAH) in heptane. After 6 hr, excess hydride was quenched with several ml of methanol. The reaction mixture was poured into a mixture of ether and 5% aqueous HC1. The organic layer was separated, washed with saturated NaHCO3 and brine, and dried (NazSO4). Removal of solvent in vacuo gave 116 mg (0.75 mmol, 90% yield) of alcohol X. IR (CC14) 3620 (OH) and 3075 cm -~ ( C ~ C H 2 ) ; NMR (CDCI3, 6) 5.42 (t, 1H, J = 7 Hz), 4.68 (br s, 2H), 4.12 (d,2H, J = 7 Hz), and 1.68 ppm (6H). Analysis: Calc'd for C10H180: C, 77.87; H, 11.76. Found: C, 77.72; H, 11.70. (E)-3,7-Dimethyl-2,7-octadien-l-yl Propanoate (XI). A mixture of 121 mg (0.78 retool) of alcohol X, 207 mg (1.60 mmol) of propanoic anhydride, and 0.15 ml of pyridine was heated at 85 ~ under a nitrogen atmosphere for 2 hr. Ice was then added to the reaction mixture and, after 30 min, was poured into a mixture of ether and 5% aqueous HC1. The ether fraction was separated, washed with 2 M Na2CO3 and brine, and dried (MgSO4). Removal of solvent in vacuo and purification by TLC (1-m • 20-cm preparative silica plate impregnated with Rhodamine 6G and developed twice in 5% ether in hexane) followed by microdistillation [bp 50 ~ (bath) 0.1 mm] gave 130 mg (0.62 mmol, 79% yield) of propanoate XI. GLC analysis (2 m 3% OV-17/0.4% Carbowax) of this ester gave a chemical purity of 97.7%. IR (CC14) 3070 ( C = C H 2) and 1740 cm -1 (C~---O); NMR (CDC13, 6) 5.37 (t, 1H, J = 7 Hz), 4.68 (br s, 2H), 4.60 (d, 2H, J = 7 Hz), 1.73 (br s, 3H) and 1.13 ppm (t, 3H, J = 7.5 Hz). MS (70 eV) m/e (relative intensity) 57 (100). Analysis: Calc'd for C 13H 220 2: C, 74.25; H, 10.54. Found: C, 74.14; H, 10.67.

Greenhouse Bioassay of Pheromone Components and Related Compounds. The greenhouse bioassay has been described in detail elsewhere (Gieselmann et al., 1979a). Rubber septa, impregnated with 33 #g of a synthetic chemical, were tested against each other and a solvent blank on five consecutive evenings.

Isolation and Identification of (E) 3,7-Dimethyl-2,7-octadien-l-yl Propanoate (XI). The procedures for rearing the insects, obtaining the crude

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ANDERSON ET AL.

pheromone extract from P o r a p a k Q airborne collections, liquid chromatographic fractionation on Florisil, saponification and esterification reactions, description of the gas chromatographic columns employed (OV-101 and XF-1150), and the use of Kovats' retention indices have also been described in detail in earlier work (Gieselmann et al., 1979a, and references therein). Briefly, the San Jose scale were reared on banana squash and a crude extract was obtained from a rinse of a short column of Porapak Q used in the airborne collection. This material was partially purified by fractionation on Florisil, eluting with a solvent gradient of diethyl ether in Skelly B. Aliquots were tested in a greenhouse bioassay, and those fractions attractive to male San Jose scale were combined. This partially purified extract was concentrated and injected on an OV-101 column. A single fraction was collected from the gas chromatograph which contained I, II, and XI, whose retention times were determined from available synthetic standards. The capillary tube used to collect the material was rinsed into a l-dram vial, and saponification was performed using dilute N a O H in 95% ethanol. The three saponified pheromone components were collected separately from the OV-101 column, and each was esterified with propanoyl chloride in carbon disulfide. Electron impact mass spectra were obtained on a Hewlett-Packard 5882 G C - M S data system modified to accept a 30-m wall-coated open tubular capillary column of OV-101. The ionization voltage was 70 eV and source temperature was 200 ~ C. Field Testing in 1979. For all tests the lure consisted of a total of 100 #g of the chemical(s) soaked into a rubber septum (5 • 9-mm rubber stoppers, sleeve type, Arthur H. Thomas Co.). Five treatments were tested: I, II, XI, a 50:50 mixture of I and II, and a mixture of I, II, and XI in a ratio of 48.5:46.7:4.8, respectively. Blanks were also included in the tests. In California, tent traps (7.6 cm • 25.4 cm) (Rice and Hoyt, 1980) were used, and testing was performed in a peach orchard. One set of caps (six replicates of each of six treatments) was placed in the orchard on April 4 in a randomized complete block design. A second, freshly prepared, set was put out in the same manner as the first on June 6. Trapping began on June 11 with the traps counted and rerandomized daily until June 18. Testing for San Jose scale in New York was performed in apple orchards. The tent trap was similar to that used in California except that the sticky surface measured 15.2 • 15.2 cm. Four replicates of each of the five treatments were tested between July 23 and August 14 with trap counts obtained two times a week. Raw trap counts (X) were transformed (log X + 1) and subjected to Duncan's muttiple range test at P = 0.05. To test for the effects of cap aging, the California data were also subjected to a two-way analysis of variance.

701

THIRD COMPONENT OF SAN JOSE SCALE PHEROMONE

RESULTS AND DISCUSSION

The scale pheromones which have been identified to date are either acetates or propanoates of terpenoid alcohols; thus, the two components of the California red scale sex pheromone (Roelofs et al., 1977) and the yellow scale sex pheromone (Gieselmann et al., 1979b) are acetates, whereas the sex pheromone of the white peach scale (Heath et al., 1979) and of the San Jose scale, I and II, are propanoates. To determine the importance of the acid component of esters I and II for biological activity, the homologous acetates III and IV were prepared from their corresponding alcohols as detailed in Figure 1. Although a synthesis of alcohol V has been previously described (Anderson et al., 1979), the one-step preparation of V shown in Figure 1 is more efficient and convenient. Thus, formation of the dianion of 3-methyl-3buten-1-ol (isopentenol) with two equivalents of the n-butyllithium-N,N,N',N'tetramethylethylenediamine complex (Cardillo et al., 1974, 1979) in hexane followed by alkylation of the dianion with isopentenyl bromide gave alcohol V, which was then acetylated with acetic anhydride and pyridine to produce the acetate III in a 40% distilled yield from isopentenol. The isomeric acetate IV was obtained by similar acefylation of alcohol VI, prepared as previously reported (Anderson et al., 1979). To determine the effect of structural modification in the alcohol moiety of esters I and II on attractancy, a homolog of I and the geometric isomer of II were prepared. The synthesis o f a 7-desmethyl homolog of I is summarized in

O ~"~O~

i) 2 n-BuLi-TMEDA hexane

0

P