Patent Application: US-201313745509-A

Abstract:
an unsaturated hydroxyl - ester pheromone for the neotropical root weevil diaprepes abbreviatus has been isolated , identified and synthesized . it is useful for trapping the weevil to reduce or prevent damage to plants .

Description:
for purposes of the invention , the following are definitions of certain terms to be used hereinafter . as used in the specification and claims , the singular form “ a ”, “ an ”, and “ the ” include plural references unless the context clearly dictates otherwise . for example , the term “ a cell ” includes a plurality of cells , including mixtures thereof . the term isolated , purified , or biologically pure as used herein , refer to material that is substantially or essentially free form components that normally accompany it as found in its native state . in an exemplary embodiment , purity and homogeneity are determined using analytical chemistry techniques such as polyacrylamide gel , electrophoresis , or high performance liquid chromatography . unless defined otherwise , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs . the terms “ about ” and “ approximately ” are defined as plus or minus ten percent ; for example , about 100 ° f . means 90 ° f . to 110 ° f . although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention , the preferred methods and materials are now described . we report here the isolation , identification and synthesis of a pheromone from male d . abbreviatus that attracts female d . abbreviatus . isolation and identification of the pheromone was obtained from adult d . abbreviatus weevils from a laboratory colony maintained at the united states horticultural research laboratory , a unit of the agricultural research service of the usda , located in ft . pierce , fla . these insects have been supplemented annually with field - collected adults . neonate larvae were placed on an artificial diet ( product no . f1675 , bio - serv , inc ., frenchtown , n . j .) and reared as described by lapointe et al . ( 2008 ). adults were held in separate 60 × 60 × 60 cm mesh cages and fed young citrus leaves ( citrus macrophylla wester ). males and females were caged separately . thereafter groups of about 20 to 30 adults of about 4 to 6 weeks of age were held separately without food , but allowed water via a water - saturated cotton dental wick for approximately 24 hours prior to aeration assays . after the assay , adults were returned to their respective cages and provided food and water . individual unmated adults were used in tests no more than once a week over a period of about 3 months . cohorts of known age were caged separately . in a first assay employed to find an attractive chemical of insect origin , a gas chromatograph - coupled electroantennogram detection ( gc - ead ) system was used to analyze male aeration samples . this system included an agilent 7890a gc equipped with a split / splitless injector , an hp - 1 capillary column ( approximately 30 m × 0 . 32 mm × 0 . 25 μm , agilent technologies , inc ., santa clara , calif ., usa ), a post column glass y - tube ( supelco , bellefonte , pa ., usa ) splitter for dividing column effluent in an approximately 1 : 1 ratio between a flame ionization detector ( fid ) and a heated ( 200 ° c .) ead transfer line . two lengths of deactivated column ( approximately 0 . 32 mm id ) were used to carry effluent to the fid and ead port after the split . at the start of gc - ead runs , the gc oven temperature was held at about 35 ° c . for 3 minutes and then increased to about 260 ° c . at a rate of about 15 ° c ./ min and held at about 260 ° c . for about 10 min . injector and fid temperatures were set at about 220 ° c . and about 300 ° c ., respectively . splitless injection was used with helium as the carrier gas at a flow rate of about 2 . 3 ml / min . a heated transfer line emptied into a charcoal - filtered , humidified air stream ( about 200 ml / min at about 30 cm / sec ) that carried the effluent over an antennal preparation . the antennal preparation can be made by plucking the antenna from either a male or female insect ( grasping it firmly at the base of the antenna near the head with fine forceps ) and placing the antenna between two metal electrodes of a universal eag probe , to which small amounts of salt - free electrode gel ( spectra 360 , parker laboratories , fairfield , n . j ., usa ) had been applied . the probe was connected to a type prg - 2 amplifier ( universal eag probe , syntech , hilversum , the netherlands ). the humidified air stream was directed to pass over the antennal preparation generating a signal . the effluent sent through to the fid line also caused a signal to be generated and signals from the amplifier and the fid were conditioned using a syntech idac - 2 interface . eag and fid signals acquired from the idac - 2 were displayed and stored on a computer running the gc - ead 2011 software program ( syntech ). as shown in fig2 , a single peak of a chemical compound , labeled d , of insect origin was detected by gc - ead from aerations of male weevils . no response was observed at the retention time observed for compound d from either male or female antennae when exposed by gc - ead to aerations of female weevils or citrus leaves alone ( data not shown ). the aerations from male weevils elicited consistent antennal responses from both male and female d . abbreviatus . although the amount of this compound in 24 - hour aerations of ten d . abbreviatus males was sufficient to elicit a response from male and female antennae by gc - ead , the small amounts approached the detection limit of the gc as shown in fig2 . nevertheless , as stated above , the effluent containing aeration samples elicited consistent antennal responses from both female and male d . abbreviatus . peaks a through c of fig2 correspond respectively to the plant volatiles linalool , geraniol , and geranial , and peak d of fig2 corresponds to an insect - generated chemical . the asterisks in fig2 indicate positive antennal responses typical of those consistently produced in multiple runs with multiple antennae . in a separate assay to further identify the insect - generated compound , multiple groups of approximately 20 to 30 male and female d . abbreviatus , held separately , were placed in separate glass aeration chambers without plant material and provided with a continuous flow ( about 500 ml / min ) of filtered , humidified air for about 24 hr at about 27 ° c . in an environmental chamber ( about 12 : 12 hr l : d ). volatiles from the headspace were collected on super q filters ( alltech deerfield , ill ., usa ) connected to the exit port of the aeration chambers . after collection , the filters were eluted with approximately 5000 of methylene chloride . separate from the volatile collections , the accumulated feces in the aeration chambers at the end of the about 24 - h collection period were collected by washing the chambers with a minimum amount of methylene chloride . the resulting extract was filtered and concentrated under nitrogen . as an initial purification , methylene chloride ( ch 2 cl 2 ) eluates ( about 200 μl ) from the super q filters and washes of the aeration chambers were passed through supelclean lc - si solid phase extraction ( spe ) columns containing approximately 200 mg of packing ( supelco , bellefonte , pa .) previously conditioned with about 15 ml of methylene chloride . the methylene chloride eluates plus a filter wash of about 2 ml of methylene chloride were saved to check for the presence of biologically active insect - derived compound . the spe column was eluted with about 2 ml each of pentane containing approximately 15 % ethyl acetate ( etoac ), approximately 30 % etoac and approximately 50 % etoac . the three spe column fractions and the saved eluates were analyzed by ei gc - ms ( electron impact ) for the presence of the biologically active insect - derived compound . only the approximately 15 % etoac fraction contained the compound of interest . this fraction was concentrated to about 100 μl under a fine stream of n 2 and subjected to fractionation by preparative gc . initial fractionation was accomplished using an agilent 6890 gc ® with cool - on - column injector and fitted with an approximately 20 cm length of deactivated fused silica attached to an approximately 30 m × 0 . 53 mm inner diameter ( approximately 0 . 5 μm film thickness ) db1 column . the analytical column was split using a “ y ” capillary connector between equal lengths of approximately 0 . 1 mm inner diameter and approximately 0 . 25 mm inner diameter lengths of deactivated fused silica column . the effluent from the approximately 0 . 1 mm column ( about 13 . 8 %) went to the gc fid while the 0 . 25 mm column ( about 86 . 2 %) exited the wall of the gc and into the heated block ( 200 ° c .) of a brownlee - silverstein collector ( brownlee and silverstein 1968 ). samples ( approximately 10 μl each ) were injected onto the column at an initial temperature of about 30 ° c ., after about 2 min the oven temperature was increased to a final temperature of about 225 ° c . at about 10 ° c ./ min . the fractions were collected in approximately 30 cm cooled glass capillaries ( brownlee and silverstein 1968 ). after collection , samples were recovered by washing the capillaries with 3 aliquots of approximately 25 μl of methylene chloride . fractions were analyzed by gc - ms for the presence of the compounds having daughter ions at approximately m / z 154 , 142 and 140 , all of which maximize within about 0 . 01 min of each other . these fractions from replicated collections to amass sufficient material for nmr analysis were combined , concentrated and re - fractionated using a db35 column ( approximately 30 m × 0 . 53 mm id , approximately 0 . 5 μm film thickness ) as above . the fractions from this separation were eluted from capillaries using deuterated chloroform ( cdcl 3 cambridge isotopes 99 . 96 %), analyzed by coupled gas chromatography - mass spectrometry ( gc - ms ). the fractions containing the compound having the 172 mw were combined , concentrated under n 2 and submitted form nmr analysis . gc - ms was conducted using instruments operated in the electron impact ( ei ) and chemical ionization ( ci ) mode . ei spectra were obtained using an agilent 5973 ms interfaced to a 6890 gc equipped with a cool on - column injector . the injector was fitted with an about 10 cm length of about 0 . 5 mm id deactivated fused silica tubing connected to about a 1 m ( about 0 . 25 mm id ) length of deactivated fused silica tubing as a retention gap . the retention gap was connected to an about 30 m × 0 . 25 mm id with an about 0 . 25 μm coating thickness dbsms ® analytical column . the temperature program was : initial oven and injector temperatures = about 30 ° c ., about 5 min ; oven and injector temperatures increased at about 10 ° c ./ min ; final temperature = about 225 ° c . spectra were obtained between 60 - 300 atomic mass units . chemical ionization spectra were obtained using an agilent 5975 ms interfaced to a 7890 gc . the gc was equipped with a cool on - column injector fitted with retention gaps as above . the analytical column used was an about 30 m × 0 . 25 mm id , about 0 . 25 μm coating thickness db1ms ®. the gc was operated using the same program as for ei spectra and the ci spectra were obtained by scanning from m / z 60 - 300 using isobutane as a reagent gas . tentative chemical identification of the putative pheromone peak was deduced from fragmentation patterns obtained from both ei - and ci - ms analyses ( fig3 a and 3b ). the peak corresponding to the putative pheromone was also recovered by washing the glass aeration jars to recover the excreta ( frass ) of males in methylene chloride . combining multiple collections of the headspace over males provided sufficient material to obtain ci and ei mass spectra ( fig3 a and 3b ). initial gc analyses indicated the presence of numerous compounds eluting in the area of the peak with gc - ead activity ( fig2 ). ei gc - ms analyses indicated that several of these , including the ead active , had approximately m / z = 154 in common and fragmentation patterns corresponding to monoterpene alcohols . although the ei mass spectrum of the compound of interest showed an approximately m / z 154 as a potential molecular ion ( fig3 b ), the additional presence of an approximately m / z 142 ( m / z 154 - 12 amu ) fragment indicated either a mixture , or that approximately m / z 154 might not be the molecular ion . a maximization of approximately m / z 140 , 142 and 154 within ± 0 . 01 min suggested all ions to be fragments of the same molecular ion and thus m / z 154 could not be a molecular ion . ci - ms analysis ( fig3 a ) gave clear m + 1 ions at approximately m / z 173 , thus supporting a molecular weight of approximately 172 daltons . in addition , the ci analysis also gave a base peak at approximately m / z 141 ( m + 1 - ch 3 oh ) and approximately m / z 155 ( m + 1 - 18 ), thus approximately m / z 154 in the ei spectra being the corresponding m - 18 fragment . the ci analysis did not give any direct explanation of the approximately m / z 142 fragment in the ei spectra , but the loss of about 30 amu ( m / z 172 - m / z 142 ) suggested a long range proton transfer to a carbonyl group followed by a neutral loss of ch 2 ═ o characteristic of a hydrocarbon chain with a terminal alcohol as well as a carbonyl . this was congruent with a methyl ester as suggested by the ( m + 1 - ch 3 oh ) loss in the ci spectrum . a characteristic neutral loss of about 32 amu was also seen in the ei spectra ( approximately m / z 142 - m / z 110 ), thereby supporting the structure of a methyl ester . furthermore , the neutral loss of about 15 amu ( approximately m / z 142 - m / z 127 as well as approximately m / z 110 - m / z 95 ) suggested the presence of a methyl branch . thus , the fragmentation patterns indicated a formula of c 9 h 16 o 3 with 2 degrees of unsaturation , the presence of a methyl ester and at least one methyl branch in addition to a terminal c — oh . further aerations of groups of up to about 30 males and fractionation by preparative gc using both polar and nonpolar capillary columns resulted in collection of a sufficient amount of the pheromone for nmr analysis . fractions containing the insect derived chemical were combined , concentrated under n 2 and submitted for nmr analysis . one and two - dimensional nmr spectroscopy , including double - quantum filtered correlation spectroscopy ( dqcosy ), heteronuclear single - quantum coherence ( hsqc ), heteronuclear multiple - bond correlation ( hmbc ) and nuclear overhauser enhancement spectroscopy ( noesy ) were used to characterize the pheromone . nmr spectra of the natural product were acquired at about 27 ° c . using an approximately 5 - mm txi cryoprobe and a bruker avance ii 600 console ( about 600 mhz for about 1h , approximately 151 mhz for 13 c ). the combined fractions containing the insect - derived compound were dissolved in approximately 150 μl of deuterated chloroform ( cdcl 3 ) ( cambridge isotope laboratories inc .) and placed in an about 2 . 5 mm nmr tube ( norell ). residual chloroform ( chcl 3 ) was used to reference chemical shifts to δ ( chcl 3 )= approximately 7 . 26 ppm for 1h and δ ( chcl 3 )= approximately 77 . 36 ppm for 13 c ( gottlieb et al . 1997 ). bruker topspin 2 . 0 and mestrelab mnova nmr ( mestrelab research sl ) software packages were used to process nmr spectra . 1 h nmr spectra of synthetic materials including a noe difference spectrum for 1e were obtained on a bruker aviii - 600 mhz spectrometer . pheromone nmr analysis is set forth in table 1 . methyl ( e )- 3 -( 2 - hydroxyethyl )- 4 - methyl - 2 - pentenoate was synthesized according to scheme shown in fig1 . unless otherwise specified all reagents and solvents were purchased from aldrich chemical co . tetrahydropyranyl ether ( thp ) protection of 3 - butyn - 1 - ol was conducted following a procedure by rama rao et al . ( 1986 ). a solution of 3 - butyn - 1 - ol ( approximately 14 . 0 g , approximately 0 . 2 mol ) in methylene chloride ( approximately 75 ml , dried by distillation from cah 2 ) was placed in a flask under n 2 . the mixture was cooled to approximately 0 ° c ., and pyridine p - toluenesulfonate ( approximately 0 . 28 g ) was added . 3 , 4 - dihydro - 2h - pyran ( about 20 . 1 ml ) was added via a dropping funnel maintaining the temperature between approximately 0 and approximately 5 ° c . the mixture was stirred at this temperature for about 1 h , then warmed to about 25 ° c . and stirred for additional about 2 h . thin layer chromatography ( silica gel , hexanes / ethyl acetate , approximately 3 : 1 ) showed that the reaction was complete . the reaction mixture was taken into cold water ( approximately 70 ml ) and transferred to a separatory funnel . the layers were separated and the aqueous phase was extracted with methylene chloride . combined organic extracts were washed with nahco 3 ( aq ), brine , and dried with na 2 so 4 ( anhydrous ). distillation produced approximately 23 g of thpo - protected 3 - butyn - 1 - ol with a by of about 50 - 55 ° c ./ 4 mm hg with gc - ms ( m / z , relative intensity ): 153 ( 2 , m + - 1 ), 99 ( 9 ), 85 ( 100 ), 79 ( 9 ), 67 ( 20 ), 53 ( 42 ), 41 ( 33 ). the mass spectrum matched the spectrum presented in the nist ms library for this intermediate compound . a solution of the thpo - ether of 3 - butyn - 1 - ol ( approximately 2 . 88 g , approximately 18 . 7 mmol ) in thf ( about 40 ml , dried with sodium benzophenone ketyl ) was placed under n 2 into a four - neck flask and cooled to about − 75 ° c . butyl lithium ( approximately 18 . 7 mmol , approximately 9 . 4 ml of approximately 2 . 0 m in hexanes ) was added slowly via a dropping funnel at about − 75 ° c . the mixture was stirred at this temperature for about 30 min , and then methyl chloroformate ( clcoome , about 1 . 4 ml , about 18 . 7 mmol ) was added . the resulting mixture was stirred at about − 75 ° c . for about 30 min , then slowly warmed to about 25 ° c . in approximately 2 h and poured into a cold saturated nh 4 cl solution . the organic material was extracted with hexanes / ether , approximately 1 : 1 . the combined extracts were washed with brine and dried . after removal of the solvent on a rotary evaporator , the remainder was flash chromatographed on silica gel with hexanes / ethyl acetate , approximately 4 : 1 , to isolate methyl 5 -( tetrahydro - 2h - pyran - 2 - yloxy )- 2 - pentynoate ( approximately 2 . 5 g , approximately 65 %). gc - ms ( m / z , relative intensity ): 211 ( 1 , m + - 1 ), 157 ( 4 ), 153 ( 3 ), 142 ( 4 ), 125 ( 18 ), 113 ( 9 ), 109 ( 11 ), 85 ( 100 ), 79 ( 41 ), 67 ( 18 ), 55 ( 16 ), 41 ( 26 ). 1 h nmr ( cdcl 3 ): 1 . 50 - 1 . 64 ( m , 4h ), 1 . 72 ( m , 1h ), 1 . 83 ( m , 1h ), 2 . 66 ( t , j = 6 . 6 hz , 2h , h - 4 ), 3 . 53 ( m , 1h , h - 5a ), 3 . 62 ( dt , j = 9 . 8 , 7 . 5 hz , 1h , h - 5b ). 3 . 77 ( s , 3h , och 3 ), 3 . 87 ( m , 2h ), 4 . 65 ( br t , j = 4 . 0 hz , 1h , ocho ). 1 h nmr data were in agreement with data obtained for this compound in ccl 4 ( rama rao et al . 1986 ). freshly purchased copper iodide ( cui , approximately 1 . 97 g , approximately 12 . 2 mmol ) was placed under n 2 in a four - neck flask . dry tetrahydrofuran ( thf ) ( about 35 ml ) was added , followed by n , n , n ′, n ′- tetramethylethylenediamine ( about 2 . 76 ml ). the mixture was stirred at room temperature until a green - yellow solution was obtained and then cooled to about − 70 ° c ., upon which a green suspension was formed . isopropylmagnesium chloride solution ( approximately 12 . 3 mmol ; about 6 . 1 ml of approximately 2 . 0 m in tetrahydrofuran ( thf ) was added slowly , whereupon a green suspension became colorless then turned brown . the mixture was stirred at about − 70 ° c . for about 1 h , then methyl 5 -( tetrahydro - 2h - pyran - 2 - yloxy )- 2 - pentynoate ( approximately 1 . 3 g , approximately 6 . 1 mmol ) dissolved in dry thf ( about 5 - 10 ml ) was added . the resultant mixture was stirred at about − 70 ° c . for about 3 h and quickly poured into an ice - cold mixture of saturated nh 4 cl and hexanes / ether , approximately 5 : 1 . the organic layer was separated , and the aqueous layer was extracted with hexanes / ether , approximately 5 : 1 . the combined organic extracts were thoroughly washed with saturated nh 4 cl solution until no blue color was seen . the organic extract was dried and concentrated . flash chromatography with hexanes / ethyl acetate , approximately 5 : 1 , afforded methyl 4 - methyl - 3 -[ 2 -( tetrahydro - 2h - pyran - 2 - yloxy ) ethyl ]- 2 - pentenoate ( approximately 1 . 3 g , approximately 85 %) as an approximately 92 : 8 mixture of e and z isomers as judged from gc - ms analysis . gc - ms ( e isomer , m / z , relative intensity ): 172 ( 2 ), 171 ( 3 ), 155 ( 32 ), 142 ( 8 ), 141 ( 6 ), 139 ( 6 ), 95 ( 17 ), 85 ( 100 ), 67 ( 16 ), 57 ( 10 ), 55 ( 11 ), 43 ( 11 ), 41 ( 16 ). gc - ms ( z isomer , m / z , relative intensity ): 172 ( 4 ), 155 ( 3 ), 154 ( 8 ), 142 ( 8 ), 141 ( 2 ), 139 ( 4 ), 123 ( 6 ), 95 ( 23 ), 85 ( 100 ), 67 ( 17 ), 57 ( 10 ), 55 ( 12 ), 43 ( 11 ), 41 ( 17 ). 1 h nmr ( 400 mhz , c 6 d 6 , 6 ): 0 . 86 ( d , j = 8 . 0 hz , e isomer ), 0 . 86 ( d , j = 8 . 0 hz , z isomer ), 1 . 20 - 1 . 42 ( m , 4h ), 1 . 55 - 1 . 62 ( m , 2h ), 1 . 70 - 1 . 82 ( m , 1h ), 2 . 18 - 2 . 31 ( m , 1h ), 3 . 02 - 3 . 18 ( m , 2h ), 3 . 41 ( s , och 3 ), 3 . 67 - 3 . 73 ( m , 1h ), 3 . 81 - 3 . 88 ( m , 1h ), 4 . 04 - 4 . 11 ( m , 1h ), 4 . 39 ( septet , j = 8 . 0 hz , h - 4 , z isomer ), 4 . 52 ( t , j = 4 . 0 hz , ocho , z isomer ), 4 . 66 ( t , j = 4 . 0 hz , ocho , e isomer ), 5 . 83 ( br . s , h - 2 , e isomer ), 5 . 86 ( br . s , h - 2 , z isomer ). 13 c nmr ( 101 mhz , c 6 d 6 , δ , e isomer ): 20 . 0 , 21 . 7 ( two carbons ), 26 . 3 , 31 . 4 , 32 . 9 , 37 . 3 , 50 . 9 , 61 . 9 , 67 . 3 , 98 . 8 , 115 . 1 , 167 . 2 , 167 . 8 . methyl 4 - methyl - 3 -[ 2 -( tetrahydro - 2h - pyran - 2 - yloxy ) ethyl ]- 2 - pentenoate ( approximately 256 mg , approximately 1 mmol ) was stirred with p - toluenesulfonic acid hydrate ( approximately 9 mg , approximately 0 . 047 mmol ) in a thf — h 2 o solution ( about 8 + 2 ml ) at about 55 - 60 ° c . for approximately 1 h , or until tlc analysis ( sio 2 plates ; hexanes / ethyl acetate / meoh , approximately 16 : 6 : 1 ; visualization with kmno 4 solution ) showed very little starting ester present . the mixture was cooled to room temperature , treated with approximately 50 μl 1n naoh and concentrated to remove most of the thf . the mixture was extracted with ether / hexanes , approximately 1 : 1 , and the organic extract was dried with na 2 so 4 ( anh .). after evaporation of the solvent , the remainder was flash chromatographed with hexanes / ethyl acetate / meoh , approximately 16 : 6 : 1 . two fractions were obtained : a ) a first fraction of the starting thpo - ester , approximately 16 mg ; and b ) a second fraction which was a mixture of ester 1 and lactone 2 ( fig1 ). the second fraction was chromatographed again with hexanes / ethyl acetate / meoh , 16 : 6 : 1 to furnish 1 ( e / z 86 : 14 , approximately 90 mg , approximately 58 %) in the less polar fraction . 1 h nmr ( 600 mhz , c 6 d 6 , 6 ): 0 . 79 ( d , j = 6 . 6 hz , ( ch 3 ) 2 , a 0 . 91 ( d , j = 6 . 6 hz , ( ch 3 ) 2 , z ), 2 . 01 - 2 . 08 ( m , h - 4 e , ch 2 c ═ c , z ), 2 . 46 ( t , j = 5 . 4 hz , oh , e ), 2 . 76 ( t , j = 6 . 6 hz , ch 2 c ═ c , e ), 3 . 34 ( s , och 3 , e ), 3 . 36 - 3 . 38 ( m , ch 2 oh , z ), 3 . 41 ( s , och 3 , z ), 3 . 70 ( q , j = 5 . 4 hz , ch 2 oh , e ), 4 . 32 ( septet , h - 4 , z ), 5 . 71 ( br . s , h - 2 , z ), 5 . 80 ( br . s , h - 2 , e ). 13 c nmr ( 151 mhz , c 6 d 6 , e isomer ): 21 . 7 ( two carbons ), 35 . 6 , 36 . 7 , 51 . 1 , 62 . 5 , 115 . 6 , 167 . 7 , 168 . 7 ; z isomer : 20 . 9 ( two carbons ), 29 . 8 , 35 . 1 , 50 . 8 , 61 . 6 , 116 . 0 , 165 . 7 , 166 . 8 . lactone 2 ( approximately 10 mg ) was recovered from the more polar ( second ) fraction . gc - ms ( m / z , relative intensity ): 140 ( m + , 16 ), 125 ( 7 ), 110 ( 15 ), 97 ( 19 ), 96 ( 59 ), 95 ( 96 ), 82 ( 24 ), 81 ( 100 ), 67 ( 73 ), 55 ( 17 ), 41 ( 40 ). 1 h nmr ( 400 mhz , c 6 d 6 , 6 ): 0 . 57 ( d , j = 6 . 6 hz , ( ch 3 ) 2 ), 1 . 37 ( br . t , j = 6 . 5 hz , ch 2 c ═), 1 . 70 ( septet , j = 6 . 6 hz , ch ( ch 3 ) 2 ), 3 . 61 ( t , j = 6 . 5 hz , ch 2 o ), 5 . 67 ( d , j = 1 . 0 hz , chc ═). nmr data are in agreement with ones obtained for this compound in cdcl 3 ( d &# 39 ; annibale et al . 2007 ). nmr signals of the synthetic compound ie in cdcl 3 were : 1 h ( 400 mhz ): 1 . 12 ( d , j = 6 . 8 hz , ( ch 3 ) 2 , e ), 2 . 44 ( septet , j = 6 . 5 hz , c h ( ch 3 ) 2 ), 2 . 87 ( t , j = 6 . 4 hz , ch 2 c ═), 3 . 73 ( s , och 3 ), 3 . 81 ( br . q , 5 . 2 hz , c h 2 oh ), 5 . 85 ( br . s , ch ═). 13 c ( 101 mhz ): 21 . 5 ( two carbons ), 34 . 7 , 36 . 2 , 51 . 3 , 62 . 0 , 115 . 3 , 166 . 8 , 168 . 7 . at the core of the chosen synthetic route lies a stereoselective carbocupration of α , β - acetylenic esters that was exclusively cis - stereospecific when the reaction was conducted in thf at low temperatures ( corey and katzenellenbogen 1969 , bourque et al . 1999 , drew et al . 1999 ). however , a conjugate addition of a heterocuprate , formed in situ from isopropylmagnesium bromide and copper ( i ) iodide in the presence of n , n , n , n ′- tetramethylethylenediamine ( crimmins et al . 1984 ), to the acetylenic ester ( fig1 ) proceeded with some loss of stereoselectivity resulting in a mixture of e and z olefinic esters in an approximately 92 : 8 ratio . during acid - catalyzed removal of the tetrahydropyranyl ( thp ) protecting group , the ratio of 1e and 1z dropped to approximately 86 : 14 and a considerable amount of lactone 2 was formed , thus indicating instability of the putative pheromone in acidic medium . a noticeable cyclization of 1e to 2 occurred also in cdcl 3 that was used for recording nmr spectra and known for its residual acidity . using t - butyldimethylsilyl ( tbdms ) protecting group ( instead of thp ) for the acetylenic alcohol provided a similar stereochemistry at the carbocupration ( e / z 93 : 7 ) step but again resulted in significant lactonization when the tbdms group was removed with highly basic tetrabytulammonium fluoride . lactonization also occurred when a purified sample of the synthetic ( e )- 3 -( 2 - hydroxyethyl )- 4 - methyl - 2 - pentenoate was injected at 260 ° c . into the split - splitless injection port of the gc - coupled ms that resulted in a total ion chromatogram comprising 1e , 1z and 2 in a ratio of approximately 39 : 8 : 53 ; the same sample analyzed by cool - on - column gc - ms injection yielded an approximately 88 : 9 : 3 mixture of 1e : 1z : 2 . because of thermal instability , initial attempts to isolate the putative pheromone by preparative gc equipped with a conventional injection port failed . a purified sample of the synthetic compound stores indefinitely at approximately ) degrees to 25 ° c . in aprotic solvents , such as , for example , benzene , hexane , ethyl acetate , etc . the synthetic methyl ( e )- 3 -( 2 - hydroxyethyl )- 4 - methyl - 2 - pentenoate was diluted with hexane to approximately 100 ng / μl and about 1 μl of sample was injected on the gc - ead system described above . an about 1 . 0 μl injection of approximately 50 ng of linalool and approximately 100 ng / μl of the synthetic methyl ( e )- 3 -( 2 - hydroxyethyl )- 4 - methyl - 2 - pentenoate in hexane was used to determine retention times and test antennal responses . linalool was previously determined to elicit a consistent antennal response and was co - injected with methyl ( e )- 3 -( 2 - hydroxyethyl )- 4 - methyl - 2 - pentenoate to confirm that the antenna was viable . antennae from male and female d . abbreviatus were used as detectors to confirm antennal response to the synthetic compound . these experiments enabled us to assign a structure to the active peak as methyl ( e )- 3 -( 2 - hydroxyethyl )- 4 - methyl - 2 - pentenoate ( 1e ). the nmr analysis as shown in table 2 also revealed an inactive isomer ( methyl ( z )- 3 -( 2 - hydroxyethyl )- 4 - methyl - 2 - pentenoate ) and a related lactone 2 . the structure of the synthetic compound was confirmed as identical with the natural compound by mass spectra ( fig3 c ) of both isomers and nmr analysis . as with the natural material , the synthetic material contained a pair of z / e isomers of the compound methyl 3 -( 2 - hydroxyethyl )- 4 - methyl - 2 - pentenoate along with the lactone breakdown product . the nmr of the synthetic sample obtained in c 6 d 6 showed two doublets at approximately 0 . 79 and 0 . 91 ppm ( corresponding to the doublets at approximately 1 . 09 and 1 . 12 ppm in the isolated material ) due to a pair of methyl groups from both stereoisomers split by a single proton on the adjacent carbon . similarly , there was a pair of singlets at approximately 3 . 34 and approximately 3 . 41 ppm due to methyl groups from isomeric esters corresponding to the singlets at approximately 3 . 73 and approximately 3 . 71 ppm in the nmr of natural sample recorded in cdc 13 . a noe difference spectrum obtained by irradiating the resonance of the larger doublet at approximately 0 . 79 ppm resulted in enhancement of the larger singlet from the olefinic proton at approximately 5 . 80 ppm and vice versa . therefore , the olefinic proton and the isopropyl group in the major stereoisomer are in the cis ( z ) position , and this stereoisomer and the natural product have the trans ( e ) configuration . integration of these absorptions and others in the synthetic product confirmed the relative concentrations found during gc - ms analysis of this product . behavioral response to the pheromone was tested in an olfactometer ( model 4c , ars , inc ., micanopy , fla . usa ) wherein individual weevils ( starved for about 24 h ) were placed in a glass inlet and allowed to walk upwards into the center of an arena with a balanced , filtered and humidified airflow from two arms oriented at 180 ° to each other and outfitted with glass reservoirs containing an odor source or blank . the assays were conducted in the dark between about 9 am and 2 pm ; each run was terminated when the weevil moved into one of the glass receptacles or when about 15 min had elapsed . assays were run in the dark because of a strong phototropic response in this insect ( lapointe and hall 2009 ). weevil position was scored as no - response ( remaining in the inlet ), no - choice ( moving to but remaining in the central arena ) or choosing one of the two arms . responses of unmated approximately 4 to 6 - week - old males and females were recorded to various odor sources : fresh young citrus leaves ( flush ), flush fed upon for about 24 hr by male d . abbreviatus , approximately 30 μg of methyl ( e )- 3 -( 2 - hydroxyethyl )- 4 - methyl - 2 - pentenoate in approximately 10 : 1 hexanes : ethyl acetate pipetted onto a glass slide , and a clean glass slide . all glass components of the olfactometer were thoroughly washed between runs with warm soap and water , rinsed with methanol and air - dried . between replications of a given treatment , the arms of the olfactometer used for that treatment were switched to control for bias in the apparatus . the number of weevils choosing a treatment arm was compared with the control arm ( clean air ) by the g - test ( sokal and rohlf 1994 ). both male and female antennae responded to linalool ( 8 . 9 min in fig4 ) and to methyl ( e )- 3 -( 2 - hydroxyethyl )- 4 - methyl - 2 - pentenoate ( 10 . 6 min in fig4 ). neither responded to the z isomer ( not shown ) or the lactone ( 10 . 8 min in fig4 ). in two - choice olfactometer tests , female weevils consistently moved upwind more often ( α = 0 . 05 , g - test ) to the olfactometer arm containing either approximately 30 μg of methyl ( e )- 3 -( 2 - hydroxyethyl )- 4 - methyl - 2 - pentenoate or citrus leaves previously fed upon by males ( mfuf ) than to clean air ( table 3 ). males did not show a clear preference for the pheromone or mfuf compared with clean air . neither males or females showed a preference when offered a choice between clear air and citrus leaves ( flush ). methyl ( e )- 3 -( 2 - hydroxyethyl )- 4 - methyl - 2 - pentenoate , can also be used as a sole attractant or in combination with plant volatiles , such as those disclosed in u . s . pat . no . 8 , 066 , 979 to dickens et al ., herein incorporated by reference , including one or more of : ( a ) alcohol and aldehyde monoterpenes ( e . g ., linalool , citronellal , nerol , and trans - geraniol ), ( b ) green leaf volatiles ( e . g ., cis - 3 - hexen - 1 - ol and trans - 2 - hexen - 1 - ol ), and ( c ) an aromatic monoterpenoid ( e . g ., carvacrol ). nonlimiting examples of suitable carriers that could support the pheromone and or plant volatiles include , for example , corncob grits , pregel defatted corn grits ( pdcg ), diatomaceous earth , alumina , silica , clays , other suitable inorganic oxides , polymers , extruded corn , powdered carbohydrates such as corn starch , dextrans and cellulose ; and the like . preferred carriers include diatomaceous earth , alumina , silica , clays . other active ingredients , including pesticides , can be included in traps containing the pheromone . active pesticides can be any substance which kills or inhibits the reproductive capabilities of d . abbreviatus . unlimited examples of active ingredients suitable for use with the attractant composition of the present invention include for example , organophosphates , carbamates , arsenicals , pyrethroids , insect growth regulators , boric acid , silica gel , and borate as disclosed in u . s . pat . no . 5 , 104 , 658 , which is herein incorporated by reference . see also , for example , u . s . pat . no . 5 , 177 , 107 ; herein incorporated by reference . see also u . s . pat . no . 7 , 244 , 607 , which is herein incorporated by reference , for a pesticide called grandevo ™, a broad spectrum bacterial insecticide for citrus . preferred the pesticides are those currently used to control adults diaprepes . these pesticides include sevin , 1 - naphthalenol methyl carbamate ; guthion , phosphorodithioic acid o , o - dimethyl s -[( 4oxo - 1 , 2 , 3 - benzotriazin - 3 ( 4h )- yl ) methyl ] ester ; and orthene , acetylphosphoramidothioic asic o , s - dimethyl ester . these pesticides are currently used in foliar sprays . soil control pesticides could also be used in a trap along with the pheromone including bifenthrin , 2 - methylbiphenyl - 3 - ylmethyl 9z )-( 1rs )- cis - 3 -( 2 - chloro - 3 , 3 , 3 , trifluoroprop - 1 - enyl )- 2 , 2 - dimethylcyclopropoanecarboxylate , a pyrethroid . other embodiments of the invention will be apparent to those skilled in the art from a consideration of this specification or practice of the invention disclosed herein . it is intended that the specification and examples be considered as exemplary only , with the true scope and spirit of the invention being indicated by the following claims . the disclosure of patents and other references cited in the specification are herein incorporated by reference in their entirety . ambrogi , b . g ., vidal , d . m ., and zarbin , p . h . g . 2009 . feromônios de agregação em curculionidae ( insecta : coleoptera ) e sua implicação taxonômica . quim . nova 32 : 2151 - 2158 . beavers , j . b ., mcgovern , t . p ., and adler , v . e . 1982 . diaprepes abbreviatus : laboratory and field behavioral and attractancy studies . environ . entomol . 11 : 436 - 439 . blight , m . m ., pickett , j . a ., smith , m . c ., and wadhams , l . j . 1984 . an aggregation pheromone of sitona lineatus . naturwissenschaften 71 : 480 . blight , m . m . and wadhams , l . j . 1987 . male - produced aggregation pheromone in pea and bean weevil , sitona lineatus ( l .). j . chem . ecol . 13 : 733 - 739 . brichacek , m . p . and carlson , r . m . 2007 . dihydropyran as a template for lactone synthesis . synth . commun . 37 : 3541 - 3549 . bourque , e ., grenon , m ., laliberte , s ., and deslongchamps , p . 1999 . macrocyclic studies of ez and zz 12 - membered aza macrocycles ; 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