I p /}'->{ ~(3, v-Ca q c I qo:r~o FINAL REPORT I I Covering Period: August 15. 1991 - February 28, 1996
I Submitted to the Office of the Science Advisor U.S. Agency for International Development I I BIORATIONAL PESTICIDES BASED ON PHEROMONE I ANALOGUES I
I Principal investigator: Jan Vrkoc Grantee Institution: Institute of Organic Chemistry and Biochemistry Department of Natural Products I Academy of Sciences of the Czech Republic Flemingovo nam. 2, 16610 Praha 6. Czech Republic I Collaborator: Glenn D. Prestwich Institution: Depa1iment of Chemistry I State University of New Yoriv Stony Brook. N. Y. I 11 794-3400, U.S.A. I
Project Number: 936-5600 I Grant Number: DHR-5600-G-00-1051-00
I A.I.D. Grant Project Officer: Dr. Phil Warren I Project Duration August 14. 1991 to February 28, 1996 m f:i ~i I R~c~D IN RIDJR JU~J ? f 199~ I BEST AVAfLABLE COPY ~f~~~Tf~ - ...... ~ ..···'" '-·- {~ : ' I L' _-:;rt:''•r ---- I I
I 2. Table of Contents
I 3. Executive Summary 2 ,.., I 4. Research Objectives .) 5. Methods and Results 4 I 5.1. Synthetic work 4 5.2. Vapor pressures determinations 4 I 5 .3. Entomological studies 4 I 5 .3 .1. Methods 5 5.3.2. Results 7 I 5.3.3. Discussion 10 I 6. Impact, Relevance and Technology Transfer 13 7. Project Activities I Outputs 13 I 8. Project Productivity 14 9. Future work 14 I 10. Literature Cited 15 I Appendix I Table 1 16 I Table 2 i7 I Table 3 18 Table 4 19 I Figure 1 - 12 Cydia molesta 20
Figure 1 - 18 Ostrinia nubilalis -'-"? I Publications 1 - 9 so I I I . I# I I I 3. Executive Summary I All new compounds were chemically characterized and biologically tested. The data from I electrophysiological and behavioral test suggested that inhibitory properties of the pheromone analogs are not entirely connected with their mimicking capability of the I pheromone. Some of new inhibitors ( chloroformates, sulfur analogs and 4-membered lactones) may prove useful as tools in further biochemical as well as field studies. Pest I control is a dynamic field, and changes in its technology come about frequently. New chemical compounds that are continually being made available usually displace older I materials. In past decades attention is given to volatile natural chemicals (semiochemicals) produced by arthropods for their chemical communication among or between species. Sex I pheromones, one of many semiochemicals. have been used often to effectively monitor or control a number of pests. One of the control strategy is based on the disruption of the I communication between sexes by permeating the atmosphere of the area under treatment by synthetic pheromone. The second strategy of mating disruption has been proposed which I relies on chemical inhibition of insect olfaction by irreversibly activating a receptor cell or I blocking pheromone recognition. For this strategy some synthetic pheromone analogs were suggested and for biological tests electrophysiological and behavioral test were used. I Eleven analogs were prepared for Cydia molesra and thirteen for Ostrinia nubilalis. Both species are worldwide serious pests and occur also in Czech Republic. All new compounds I were chemically characterized and biologically tested. The data from electrophysiological and behavioral test suggested that inhibitory prope1ties of pheromone analogs are not entirely I connected with their mimicking capability of the pheromone. Some of new inhibitors ( chloroformates. sulfur analogs and 4-membered lactones) may prove useful as tools in I fmther biochemical as well as field studies. I I I I I I
-.)- I I I 4. Research Objectives: Chemical communication is the most important information channel in insects. Pheromones can be used as biorational means for pest control and they already found I his place in different areas of Integrated Pest Management. Hygienically safe, selective biorational pesticides based on pheromones need further development including their methods of application. In our research program we studied one class I of prospective biorational pesticides - analogs of insect sex pheromones which could have a potential to disrupt premating communication between the sexes and thus to I impair reproduction of populations. The searching of new methods in the strategy of pest control based on natural compounds or their analogs is in agreement with public aversion to chemical insecticides. As in other countries also in Czech Republic there I exists a need for the development of alternatives in existing pest management. The main aim of our proposal was to synthesize new types of pheromone mimics. provide physiological and behavioral testing on laboratory colonies. and in case of I finding an active analog to start preliminary investigation of antennal proteins and mating disruption field trials in small scale. The strategies of sensory disruption by analogs were supported by published data that some pheromone mimics already I known before this project started have shown significant inhibitory activity on premating behavior. From the analysis of the known structures of sensory disruptants and taking into account inconsistency of some results obtained in different species it I was concluded that further research in this field was needed. Mating disruption control strategy by permeating the area under the treatment by synthetic pheromone is still complicated and the mechanisms involved in achieving I this effect have not yet been recognized. Mechanisms for interrupting long distance communication even by natural pheromone are very complex and probably more than I one are involved at the same time and some of them may act synergistically. The ignorance of the mechanisms slow down the development of optimal design of this method and its practical implementation. I In contrast chemical inhibition of insect olfaction is based on irreversibly activating a receptor cell (hyperagonism ), or blocking pheromone recognition in the receptor cell (antagonism). Both modes are based on disruption of biochemical events in the I sensilla by limited quantities of a chemical compounds and in effect a selective anosmia is resulting.
I The problem of our project was at the same time studied in several research teems in USA, France, Spain. Russia and Switzerland. The topics of research were mainly fluorinated analogs as for example mono-, di- and tri- fluoro analogs of Zl 1-14:0Ac I (l ), fluorinated ESE 10-12:Ac (2), fluorinated analogs of esters components of Diparopsis castanea (3) and trifluormethyl ketones of Spodoptera littoral is (4 ). I Chlorinated analogs of ESE 10-12 :OH ( 5 .6) have been found as biologically active and halogen acetate analogues of Sesamia nonagrioides are inhibitors (7). 2.6-Dimethyloctyl formate was found as an attractant of Tribolium confusum (8). I Oxime ether analogs of sex pheromone components of A.gratis segetum are I I I -.+-
I pheromone mnmcs (9). Electrophysiological and morphological characteristics of pheromone receptors and the behavioral activity of analogs have been studied m I Diprion pini ( 10). The insects chosen for our study was one of key orchard pests, the Oriental fruit I moth ( (vdia moles ta), and a serious pest on maize in Europe and North America European com borer ( Ostrinia nubilalis). While Oriental fruit moth was taken as a model of pest species where mating disruption with pheromone was successfully used I both in Australia and United States. mating disruption strategy for 0. nubilalis has not been so far studied in detail. Our approach to the mating disruption by pheromone mimics was based on the use of new reactive mimics of both known and new I structural types. All our studies were done on the chemical and biological level, where mainly electroantennographical (EAG) and electrosensillographical (ESG) investigation of new pheromone analogues were studied. Both the most EAG active I pheromone mimics and some of EAG inactive compounds were then used for flight-tunnel behavioral investigations. Taking into account EAG responses and inhibitory effect of the selected analogs interesting structure-activity relationship was I found. I 5. Methods and Results:
I 5.1. Synthetic work All pheromone mimics were synthesized by procedures reporting in project-funded I publications. All structures of the synthesized compounds were proved by MS, IR and NMR spectra. Eleven compounds were synthesized for Cydia molesta tests and thirteen I compounds for Ostrinia nubilalis tests. The chemical structures of the compounds are summarized in Table 1 and Table 2. From the list of compounds we wanted to prepare I we did not achieve the success only with azaanalogs. 5.2. Vapor pressures determinations The above prepared analogs show high differences in volatility. For dose response I electrophysiological studies the saturated vapor pressures of all compounds were determined using a method based on gas chromatographic retention data. The data are I summarized in Table 3. 5.3. Entomological studies I Oriental fruit moths, Cydia molesta, and European Com Borer, Ostrinia nubilalis. originated from a colony maintained under laboratory conditions. Larvae were reared on an semiartificial diet under a 16:8 light:dark regimen. Pupae were sexed and males I were kept separately from females under the same light and temperature conditions. Newly emerged adults were collected daily and provided with water and sugar solution. 2-4 days old males were used for EAG experiments, 3-4 days old males for I wind tunnel observation. I I I I - ..,_ I I Electroantennograph_1 · I Two glass AgiAgCl rnicroelectrodes filled with physiological saline were used for EAG recordings: the ground electrode was placed into the head capsule of an intact male moth and the recording electrode was connected with the distal end of the male antenna. tip I of which had been cut off. Ante1mal responses were amplified (signal conditioner CyberAmp 320, Axon Instruments), digitized (Metrabyte DAS-16 AID. sample period 250 msec) and analysed by a PC 486 computer (Stand Alone Acquisition System, Run Teclmologies). I The main pheromone components (Z8-12:Ac for C. molesta. Zl l-14:Ac for 0. nubilalis) and their analogs were dissolved in hexane forming a series of dilutions from 5 ng to 5 mg per µl. Five µl of aliquots was pipetted onto a filter paper disc (10 mm dia, Whatman I N°2) and each loaded disc was inserted into a Pasteur pipette after solvent evaporation. The odor cartridges were stored deeply frozen in closed glass vials when not used for experimentation. The cartridges conditioned in laboratory temperature for at least lhr were I used for stimulation. Stimuli were delivered onto the antennal preparation by air puffs blown through the cartridge, outlet of which was positioned at a distance 2.5 cm from the antenna. 1 Stimulus duration was 0.8 sec. the air flow rate was 1 l.min- • Between successive I stimulations a continual stream of clean and humidified air was blown over the antennal preparation. Intervals between two successive stimuli ranged from 1 to 20 minutes depending I on the type and intensity of the stimuli. Typically, 1-4 min were adequate for complete recovery of the antenna! sensitivity to the original level at lower doses. while 10 -20 minutes were necessary when doses > 10 ~Lg were used. Three EAG replicates were recorded for each I serial dilution of each odorant. Recordings were repeated on three male antennae. Main pheromone components served as a standard to normalize EAG responses from different individuals and to control over viability and constancy of the preparation. Stimulation with I the standard both preceded and followed each experimental session. The EAG responses to solvent were subtracted from the overall EAG response. EAGs to test chemicals were then I expressed as a percentage of the EAG response to the standard stimulation.
Single sensillum recording I Receptor potentials and nerve impulses were recorded extracellularly from receptor cells associated with the sensilla trichodea using modified tip-cutting teclmique. A whole animal preparation was used. The head and one protruding antenna of a male placed in a I disposable pipette tip was fixed by small droplets of molten wax. The antenna was carefully bent dorsally and fixed by wax. The tips of sensilla trichodea were cut by means of two glass I microknives (microelectrodes with broken tips. -30 µm i.d.) mounted in micromanipulators. The recording electrode ( 10 .um in diameter) slipped over cut sensilla trichodea was filled with receptor lymph saline, the reference electrode, inserted in the head, contained saline I approximating the ionic composition of the moth haernolymph. Prior the slipping, the tip of the recording electrode was dipped into heated vaseline to prevent it from drying out. Electrical activity of the receptor cells was recorded similarly as EAG recordings on the same I instruments. Receptor potentials (DC recordings) and spike activity were recorded simultaneously by two independent channels of signal conditioner. I I I I I I Shorr-range behavior The effect of analogs on male precopulation behavior was investigated in disposable I Petri dishes ( 10 cm i.d. ). The compound investigated was loaded on a filter paper disc ( 1O mm dia) placed in the center of the dish housing the calling female. After 30 min of equilibration a male was introduced into the dish and its behavior was observed for a 30 min I period. Experiments were performed simultaneously with six pairs of dishes (one test and one control) in four replicate series. Mating efficiencies of males in the test and control dishes were expressed in the form of confusion coefficients, CC [%] = {Cc I Nc - -CE / NE).100, I where CC is the confusion coefficient, Cc no. of copulations in controls, Nc no. of pairs in controls, CE no. of copulations in the experimental group, and NE no. of pairs in the I experimental group. The total amount of pairs used for each treatment was at least 24. Flight-tunnel experiments The C. molesta and 0. nubilalis males were flown in a 1.86 m long x 0.3 m wide x I 0.3 high plexiglass flight-tunnel. Charcoal filtered and humidified air was pushed through the 1 tunnel by four ventilators. The air velocity was maintained at 0.5 ms- • The flight-tunnel I conditions used were: 22-26 °C, -J.0-60% relative humidity and 700 and 10 lux light intensity for C. molesw and 0. nubilalis respectively. In preliminary series of experiments male reactions to the calling female, to main I pheromone components alone and to the respective pheromone blends were determined. Males were allowed to respond to 1, 10 and 100 ng of pheromone blend to ascertain which odor source is behaviorally comparable with calling female. 10 ng of respective pheromone I blend loaded on filter paper disc (1 cm dia) was fully comparable with the female and therefore was used as standards in all flight-tunnel experiments. Using pheromone analogs, two different types of observation were performed. Firstly, I male reactions to pheromone standard masked by 100 ng of the respective analog were observed to see if the analog has an ability to modify the male orientation to odor source. Secondly, to determine, if the analog can substitute the main pheromone component, males I were observed while responding to odor source in which the main component in pheromone standard was replaced by an appropriate amount of the analog. I Based on preliminary studies. circadian rhythms of mating activity (Fig. 1-2 CM, 1-2 ON) and initial mating age (Fig. 3-..J.CM. ON) were established in laboratory stocks of both experimental species. The maximum of mating activity was observed 13 hr after the onset of I photophase in C. molesta and 6 hr after the onset of scoptophase in 0. nubilalis under LD 16: 8 hr regimen. The optimum age for mating was 3 days after adult eclosion in both sexes. Prior each flight session (3-..J. days old) males were placed individually into glass tubes I (release cages. l 0 cm long, 4 cm dia). After 15 min acclimatization period males were released from central part of the tunnel into an odor plume which was created by pinning the filter paper disc ( 10 mm dia) loaded with odor onto a holder placed centrally near upwind I end. The filter paper disc created turbulence and so structured the plum (its parameters and orientation was checked using TiCl~ prior and after each flight session). Each male was tested once and then discarded. Five males were tested for each filter paper source. In six replicate I series. altogether 30 males were flown for each treatment. To assure a convenient state of the males. additional five males were flown in response to the standard pheromone blend after I each day's session. I I I - / - I
I Male behavior was classified into four categories: i) activation (walking and wing fanning), ii) take off. iii) oriented flight iv) touching the odor source. landing and copulation I attempts. The total time of obser.vation was either two minutes if the male did not take off or it lasted until its landing.
I Statistical anal_vsis. The data were subjected to statistical analyses utilizing the StatgraphicrM_Plus software package (Manugistic, Rockville, MD, USA). Student's t-test and Single factor Anova analysis ). I (a= 0.05) was uere used to compare mean responses for differences (t1i1:m1 =m2
I 5.3.2. Results I I. Lacton derivatives (CML 4, CML 5, CML 6, ONL 4, ONL 5, ONL 6)
In C. molesta all lactone derivatives showed EAG act1v1ty (Fig. 5 CM), though I marginally measurable effects were observed at concentrations several orders of magnitude higher then those needed to produce the same effect using authentic pheromone. The most I active compound was 5-membered lactone, followed by 4-membered one. Only small EAG activity was observed for 6-membered derivative of Z8-12:Ac. In 0. nubilalis very small I EAG responses to the lactone derivatives were observed (Fig. 5 ON).
The spontaneous activity of neurones associated withs. trichodea was generally very I small (< 0.5 Hz) in both species tested. All neurones responsive to the main pheromone component were tested for their responsiveness to the lactone analogs. In C. molesra (Fig. 3. 9 CM) only 5-membered lactone elicited spike activity, while 4- and 6-membered lactones did I not. In 0. nubilalis no lactone analogs showed any ESG activity (Fig. 9 ON).
Behavioral observations I Short range bioassay in C. molesta (Fig. 10 CM): The values of "confusion coefficients" determined at 3 concentration levels demonstrate that the 4-membered lactone I possesses a strong disruption effect for mating behavior (30, 37 and 42% at doses 10, 100 and 1000 ng, respectively). Disruption effect of 5-membered lactone was less pronounced f 0,13 and 53% at doses 10, 100 and 1000 ng resp.), while 6-membered lactones had only I weak activity (0, 0, 20% at doseslO, 100 and 1000 ng resp.). This is why the last compound was not studied in the flight tunnel. It should be noted, however, that none of the analogs was found to be as active as the moth's own main pheromone component in causing disruption of I normal pheromon-induced behavior (CC = 78.8 , 45 and 44 % at doses 10, 100 and l 000 ng resp., Fig. 11 CM). Short range bioassay in 0. nubilalis was not used due to our failure to design a suitable bioassay setup. Hence all behavioral investigations in this species utilized I flight-tunnel equipment. I I I I -8- I
I Flight-runnel observarions. 10 ng of a three component blend proved to be comparable with calling females and was used as a standard in all behavioral experiments. The behavior profiles presented in Fig. l l CM indicate that the exposure of C. molesta males I to a 1: 10 mixture of standard and 4-membered lactone resulted in a significant reduction (75% *) in touch/landing responses relative to the standard alone (note that 100 ng of the I main pheromone component caused 80%* inhibition!). Under the same conditions. addition of 5-membered lactone caused 30% * (significant at p=O.O 1) reduction of males finding the odor source. When behavior of males responding to pheromone was compared with behavior I of males responding to pheromone-analog ( 1: 10) mixture. the most affected behavioral element was the oriented flight, i.e. the activated males logged in the odor plume and performed the oriented flight less frequently. Males that eventually found the source needed I significantly longer periods to accomplish this than the males responding to the pheromone. In 0. nubilalis 4-membered lactone reduced significantly ( 48% * and 88% * reduction at doses l 00 and 1000 ng, significant at p=0.05) male orientation to pheromone standard I (Fig.11 ). For 5-membered lactone no significant (at p=0.05) effect was observed ( 22% and 33% reduction, Fig.12).
I II. lsosteric analogs: chloroformates (Ci11 8, ON 9) and sulphur analogs (CM 10, CM 11, I ON 12, ON 13) EAG Chloroformate and sulphur analogs showed the larges EAG activities of all analogs I investigated in both species (Fig. 4 and 6 CM, fig. 4 and 6 ON), although they did not reach activities of the main pheromone components.
I ESG All neurones responsive to the main pheromone components responded to all isosteric analogs tested in both species (Fig. 8.9 CM. 9 ON ). In the chloroformate analog the pattern I of neuronal activity was very similar to that of authentic pheromone. Sulphur analogs generally showed short lasting spike activities in comparison with authentic pheromone I and/or chloroformates. Behavioral observations Shorr rnnge bioassay m C. molesta (Fig. 10 C M): Values of the"confusion I coefficient" determined at four concentration levels demonstrate that the isosteric analogs possess a strong disruption effect for mating behavior ( CC for CM 8 was 40, 42 and 67%; I for CM l 0 20,25, and 70% at doses 10. 100 and 1000 ng respectively). However, none of the analogs was found to be as active as the moth's own main pheromone component in causing disruption of normal pheromone-induced behavior (cf. CC for Z8-12:Ac: 44, 46 and I 78.8% at doses 10. l 00 and 1000 ng, resp .• Fig. 10 CM).
Flight-tunnel observations. The behavior profiles presented in Fig. 11 CM indicate I that the exposure of C. molesta males to a mixture of pheromone standard and a 10-fold I I I I -9- I
I excess of the isosteric analogs resulted in a significant reduction in touch/landing responses (CM 8: 75%*, CM 10: 70%* ) relative to pheromone alone. Again the oriented flight was most affected. Basically the same activity pattern was observed in 0. nubilalis (Fig 12.13 I ON). ON 9 showed 32, 80* and·'90%* reduction of male orientation, ON 12 even higher. i.e. 90* and 100*% ). I I III. Vinyl branched analogs (CM 7, CM 5, CM 6, ON 7, ON 6, ON 8,)
The vinyl derivatives showed very weak EAG actIV1ty in C molesta at all I concentrations tested.. In 0. nubilalis however, EAG responses were relatively high. Measurable effects were observed at concentrations three orders of magnitude higher than I those needed to produce the same effect using authentic pheromone (Fig. 7 CM, fig. 7 ON ) When blended with the pheromone ( 1: 100 pheromone:analog), vinyl analogs CM 5 and CM 7 decreased the EAG response to pheromone (studied on C. molesta only, not showed). It I should be emphasized that these vinyl analogs were the only compounds tested in this study, which showed inhibitory effect at the receptor level. It suggests that they may react directly with the pheromone receptor. Further studies are needed to understand biochemical I mechanisms underlying this effect.
I Single cell reactions to some vinyl-branched analogs (CM 5 and CM 6) were observed in C molesta (Fig. 7CM). Other analogs (CM 7, ON 8, ON 7 and ON 6) did not showed any I ESG activity.
I Behavioral observations Short range bioassa.v in C. molesta (Fig. 10 CM). Values of the "confusion coefficient" determined at two concentration levels ( l 00 and l 000 ng) demonstrate that I vinyl-branched analogs possess a disruption effect on the mating behavior at relatively higher doses (CC for CM 5 was determined 8 and 14% resp., for CM 7 - 9 and 45%).
I Flight-tunnel observations. For C. molesta 100 ng of CM 5 inhibited 45%* of responding males and CM 7 40%* (Fig. 11 CM). In 0. nubilalis 100 ng of vinyl-branched analog ON 8 showed 60% * reduction of male responses to the pheromone ( 1000 ng inhibited I 100%* of responding males). Strong inhibitory effect was observed also for ON 6 (80%* at both l 00 and l 000 ng levels).
I IV.Saturated compounds (12:Ac, 14:Ac)
I The saturated compounds generally showed rather high EAG activity in both species I (Fig. 4 CM, fig. 4 ON) being within the range of activity of the isosteric analogs. I I I -10- I I Contrary to EAG results no ESG reactions were observed. Nevertheless the presence I of strong receptor potentials was observed (Fig. CM 8), an effect accounting for a rather strong EAG activity.
I Behavioral observations Short range bioassay in C. molesta (Fig. 10 CM). Values of the "confusion coefficients" determined at three concentration levels demonstrate that l 2:Ac possesses a I strong disruption effect for mating behavior.
Flight-tunnel observations. Likewise saturated compounds inhibited orientation of I males to pheromone in both species. 90% * disruption was observed when 100 ng of
I 12:Ac was added to standard pheromone in C. molesta (Fig.I I). In 0. nubilalis addition of 10, 100 and 1000 ng of 14:Ac resulted in 61*, 90*, and 100%* inhibition of male orientation I to odor source (Fig. 18). I 5.3.3. DISCUSSION The most interesting feature of our results are striking differences among analogs in the complementary electrophysiological and behavioral tests as apparent from Table 4. I Although we have little experimental evidence to propose the exact mode of action of the tested analogs. several mechanisms can be hypothesized. It is generally accepted that molecular size and shape are important for insect I pheromone chemoreception. Apart from stereochemical requirements, however, electronic charge-charge attraction, hydrogen bonding, hydropathic bonding, and van der Waals forces are potentially important in binding to proteinaceous macromolecules. I As the molecular shapes of chloroformates and lactones are identical with respect to the unsaturated hydrocarbon chain with main pheromone components, the main spatial I differences among them should originate from the polar groups. Based on energy- -minimized molecular geometries chloroformate (Scheme a) and, to a lesser extent. also the 5-membered lactone (Scheme c)) show a high degree of similarity to acetate group, while I the 4-membered Iactone (Scheme b) shows the largest deviations. I I \ ~~/'/ I I !/ /; I / 0 c I a b I I I -11- I
I Accordingly. it is more probable that the acetate receptor would reject the 4-membered rings due to its size or shape. The fact that -t-membered lactones posses relatively high disruption I effect on male orientation to pheromone is rather surprising since these compounds produced much weaker EAG responses in comparison with authentic pheromone and failed to elicite spikes, even though small receptor potentials were observed. However, if I specificity of pheromones and pheromone receptors is coupled to specificity of the ---- -phemmone clearing enzyme system, analogs that are racking sufficient pheromone mimicry would be cleared from the receptor less effectively producing an aberrant response of I receptors and thus acting as behavioral inhibitors. On the other hand, it is known that 4-membered lactones as ambient electrophiles may undergo, in the presence of nucleophiles, oxygen-alkyl or oxygen-acyl bond cleavage. Therefore, 4-membered lactones might be I able to disrupt the normal pheromone-induced behavior also via non specified interactions with proteinaceous structures involved in transduction process. A lower inhibitory activity of the 5-membered lactones may in this case be related to its lower chemical reactivity. I Although the replacement of the acetate methyl group by a chlorine in chloroformate analogs is not supposed to have important steric consequences, the methyl I group has a higher hydrophobicity than the chlorine atom and, also, the possibility to engage in short-range binding through dispersion forces with the receptor structure complementary to the acetate methyl is probably reduced for the chloro-derivative. Beside this. the IR carbonyl I frequencies n (C=O) for authentic pheromone and chloroformate analogs (Ref. 9 - 1740 cm·' vs. 1778 cm·') differ significantly indicating the different ability to form the hydrogen bonds. The hydrogen bond is widely regarded as being the most important intra- and I intermolecular cohesive force and a major contributor of non-covalent interaction energy in biological systems. All these differences may account for the slightly reduced electrophysiological activity of the chloroformate in comparison to authentic pheromone I molecules. Regardless of its reduced activity, the chloroformate analogs elicite sufficiently high electrophysiological responses in the pheromone receptor neurons and could theoretically overload the olfactory system acting as inhibitors when present in I hyperphysiological concentrations. Another explanation for the significant inhibitory activity of chloroformates could be their possible binding to antenna! proteins through a carbamate I linkage (under evolution of HCl !), thereby locking the sensory transduction mechanism and/or inactivating the pheromone catabolizing enzymes (see details in Ref. 9). A double bond environment appears essential for good inhibitory activity since saturated I cloroformate equivalents are only very weak behavioral inhibitors. - The replacement of CH=CH groups by -S- or -CH2 groups is considered to be
bioisosteric. However. the replacement of a C=C bond by S-CH2 moiety cause substantial I perturbation of the conformation. charge and electron density in analog in comparison with the autentic molecule, The resulting small differences in bond length and angles might influence the interaction of thioanalogs with the pheromone receptors resulting in lower I electrophysiological activities. However. the existence of more conformers having energy minimum similar to the authentic molecule implicates that sulphur analogs might perhalps excite also other sensory cells (E8-12:Ac in C. molesta and Ell-14:Ac in 0. nubilalis, I respectively). This fact could account for the strong confusion effect observed for sulphur I I I I
I - t 2- I
analogs in flight-tunnel experiipents both in C. moles ta and 0. nubilalis. When the I differences in volatilities are considered the confusion effect of sulphur analogs were comparable to that of pheromones. Due to the low cost synthesis and stability. sulphur analogs represent the promissing candidates for mating disruption techniques. I The vinyl-branched analogs were supposed to substantially affect the pheromone mediated behavior in both C. moles ta and 0. nubilalis. All vinyl-branched analogs investigated in this study showed only inhibitory effect on male orientation to pheromone. I This inhibitory effect was more pronounced in 0. nubilalis than in C. molesta. No synergism as observed in previous study on Trichoplusia ni was observed. Our finding that some vinyl I analogs have the ability to inhibit EAG response to pheromone at the antenna! level is interesting and deserves furhter investigation. Behavior of saturated acetates (12:Ac, 14:Ac) deserves a special comment. The I major component of the Oriental fruit moth pheromone, (Z)-8-dodecenyl acetate, was identified in 1969. Recently, the composition of the pheromone blend was reexamined and significant amounts (3.44 ± 1.16%) of 12:Ac have been identified in the effluvia of calling I females. The same is true for 0. nubilalis (viz publikaci o reidentifikaci feromonu 0. nubilalis). It is possible that the role of these compounds in the natural pheromone of C. molesta and 0. nubilalis might have been overlooked. Our electrophysiological results I demonstrating that, although eliciting no spike activity in pheromone receptor cell types, both 12:Ac and 14:Ac elicite a relatively high receptor potentials, may not necessarily be in I contradiction with this hypothesis. because ESG studies performed up to date did not detect any specialized receptor cells for 12:Ac in C. molesta nor for 14:Ac in 0. nubilalis. As minor pheromone components these compounds, when added to standard pheromone blend. I might alter the balance of sensory input to such an extent that the insects no longer respond appropriately to pheromone stimlulus. A similar inhibitory effect of imbalance in the pattern of sensory input had been previously observed on the behavior of A.wographa gamma. a I species that uses a simple two-constituent pheromone blend. It was observed that increasing the level of the minor component in the binary blend resulted in a substantial decrease of I male behavioral responses. I Conclusions The present data suggest that the inhibitory properties of the pheromone analogs I are not to be entirely connected with their mimicking capability of the pheromone. Apparently, several constitutional and configurational properties of the molecule and, I in turn, its chemical reactivity are of special significance to the inhibitory process. More specific studies are required to elucidate the role these factors may play for effective binding to proteinaceous substrates. At present. the inhibitory mechanisms remain speculative. In I spite of this, the new inhibitors described (chloroformates, sulfur and. vinyl analogs and 4-membered lactones) may prove useful as tools in further biochemical as well as field I studies directed towards mechanisms controlling mating disruption. I I I -13- I
I 6. Impact, Relevance and Technology Transfer The support from Program in Science and Technology Cooperation. U.S. Agency I for lntemational Development has been great contribution for our research in many ways. First of all main contribution was for scientific development of all coworkers on this project. During training programs six researchers spent 3 months and one I researcher 2 months at renowned universities and/or in research institute of USDA. For three young scientists this training was the first training abroad. During the training in USA and discussions with professors at New York State University at I Stony Brook. Cornell University at Ithaca. University of Arizona in Tucson, University of Massachusetts at Amherst and USDA-ARS Laboratories at Gainesville we have gained many knowledge and new ideas not only for continuation of our I research but also for future career of individuals. Secondly, the money from budget for equipment was spent for purchase of PC computers, middiepressure iiquid chromatography (MPLC), rotavapors, aguisition and I evaluation system for EAG. and GC spare parts. All this equipment was used to strengthen the capacity of chromatographic works and EAG and ESG measurements and interpretations. I All the knowledge. expertise and equipment we have obtained during the project duration will positively influence the future research of individuals in both chemical I and entomological laboratories of our department. Another important contribution for the progress of this project was also the I possibility to purchase chemicals. solvents and bulks.
I 7. Project Activities/Outputs I Trainings 1. Dr. B. Koutek. 3 months in 1993 at Department of Chemistry, State University of New York at Stony Brook. New York: prof. G.D .. Prestwich. I 2. Dr. L. Streinz. 3 months in 1993 at Department of Chemistry, Cornell University, Ithaca. New York: prof. J. Meinwald. 3. Dr. M. Haskovec. 3 months in 1993 at USDA-ARS Laboratories at Gainesville. I Florida; Dr. J. Tumlinson. -J.. Dr. B. Kalinova, 3 months in 1993 at Arizona Research Laboratories. University of I Arizona, Tucson: prof J. Hilderbrandt, 5. Dr. J.Zdarek. 2 months in 1994 at Department of Entomology, University of Massachutes, Amherst; prof. R Carde, I 6. Dr. B. Koutek. 3 months in 1,994 at Department of Chemistry, State University of New York at Stony Brook, New York: prof. G.D. Prestwich, 7. Dr. M. Rejzek. 3 months in 1994 at Department of Chemistry, State University of I New York at Stony Brook. New York: prof. G.D. Prestwich. I I I I - l-1-- I
I Publications 1. B. Koutek. M. Haskovec. K. Konecny and J. Vrkoc.: Gas chromatographic I determination of vapour pressures of pheromone-like acetates. J Chromatogr. 626, 215-221 (1992). 2. I. Kovafova and L. Streinz.: Preparation ofE-alkanes using lithium and 1.3- I diaminopropane. Synthetic Communication 23. 2399-2404 (1993). 3. M.Hoskovec. B. Koutek. J. Lazar, L. Streinz. E. Brofova. B. Kalinova and J. I Vrkoc.: a.a-Disubstituted allylic sulfones: an approach to the synthesis of vinyl-branched pheromone analogues. Helv. Chim. Acta 77, 1281-1287 (1994). I 4. B. Koutek. M. Haskovec. P. Vrkoeova. K. Konecny and L. Feltl.: Gas chromatographic determination of vapour pressures of pheromone-like compounds II. Alcohols. I J Chromatogr. 679, 307-317 (1994). 5. L. Streinz, A. Svatos. J. Vrkoc. J. Meinwald.: Chlorofluoroacetic acid I derivatization for analysis of chiral alcohols. J Chem. Soc., Perkin Trans. l, 3509-3512 (1994). 6. B. Koutek. G.D. Prestwich. A.C. Howlett, S.A. Chin, D. Salehani, N. Akhavan and I D. Deutsch.: Inhibitors of arachidonoyl ethanolamine hydrolysis. J Biol. Chem. 269. 2937-2940 (1994).
I 7. B. Kalinova. A. Minaif. L. Kotera.: Sex pheromone characterization and field trapping of the European com borer, Ostrinia nubilalis (Lepidoptera: Pyralidae) in South Moravia and Slovakia. I Eur. J Enromol. 2.1, 197-203 (1994) 8. M. Haskovec. D. Saman and B. Koutek.: Synthesis of (Z)-14-heptadecen-4-olide and (Z)-11-pentadecen-4-olide, sex pheromone analogues of Ostrinia nubzlalis and I Cydia molesta. Collect. C::ech. Chem. Commun. 59, 1211-1218 (1994). 9. M. Haskovec. 0. Hovorka. B. Kalinova. B. Koutek. L. Streinz. A. Svatos. P. I Sebek. D. Saman and J. Vrkoc.: New mimics of the acetate function in pheromone-based attraction. I Bioorg. .'vied. Chem . .:+. -1-79-..+88 ( 1996). I 8. Project Productivity To the best of our knowledge we did not succeed to prepare azaanalogs only. The I azaanalogs we prepared were tmstable and not suitable for further biological tests 9. Future work.
I Both in 0. nubilalis and C. molesta the vinyl-branched analogs showed significant reduction of male responses to pheromones. even the responses in C. molesta were slightly weaker. In the case we will be able to get a financial support, we would like to I continue the study of the vinyl-branched analogs in more detail. I I I -l5- I I 10. Literature Cited 1. Klun J.A .. Schwarz M .. Wakabayashi '.'J .. Waters R.M.: Moth responses to selectively I fluorinated sex pheromone analogs. J Chem. Ecol. 20, 2705 ( 1994 ). 2. Tellier F .. Sauvetre R.: Synthesis of a new fluorinated analog I (E.E)-8.10-dodecadienol (codlemone) Tetrahedron Lett. 33. 3643 ( 1992). I 3. Tellier F., Sauvetre R.: Fluorinated analogs of ester components of red bollworm sex pheromone. I Syn. Commun. 21, 395 ( 1991 ). 4. Duran I., Parrilla A., Feixas j., Guerrero A.: Inhibition of antennal esterases of the Egyptian armyworm Spodoptera littoralis by trifluoromethyl ketones. I Bioorg. Medicinal. Chem. Letter 2. 2593 ( 1993). 5. Tellier f., Hammoud A .. Ratovelomanana V .. Linstrumelle G .. Descoins C.: I Synthesis of a biologically active chlorinated analog of (E.E)-8, 10-dodecadienol (codlemone ). Bioorg. JV!edicinal. Chem. Letter l, 1629 (1993). I 6. Lukas P., Renou M., Tellier F .. Hammond A., Audermard H., Descoin C.: Electrophysiological activity and field activity of halogenated analogs of (E,E)-8, 1O-dodecadien-1-ol, the main component in codling moth ( Cydia I pomonella L.). J Chem. Ecol. 20, 489 (1994). 7. Riba M., Eizaguirre M., Sans A .. Quero C.. Guerrero A.: Inhibition of pheromone I action in Sesamia nonagriodes by haloacetate analogs. Pestic. Sci. 41, 97 (1994). I 8. Gamalevich G.D .. Serebryakov E.P.: Pheromones of Coleoptera 12. Synthesis of (+/-)-2,6-dimethyloctyl formate. the biologically active analog of the smaller flour beetle I aggregation pheromone. Russ. Chem .. Bull. 42, 741 (1993) I 9. Martin D., Weber B.: Oxime ether analogs of sex pheromone components of turnip moth (Agrotis segetum Schiffermuller ). I J Chem. Ecol. 20. 1063 ( 1994 ). 10.Anderbrant 0 .. Hansson B.S.: Electrophysiological and morphological characteristics of I pheromone receptors in male pine sawflies. Diprion pini (Hymenoptera: Diprionidae ), and behavioural response to some compounds. I J Insect Physiol. -+ l. 395 ( 1995) I I I I I -16-
I Table I. Cyclia mo!esta pheromone analogues I I (Z)-8-dodecen-l-yl acetate I CM l (Z)-1 O-tetradecen-3-olide I CM2 (Z)-11-pentadecen-4-olide I CM3 0 (Z)-12-hexadecen-5-olide I CM4 0
7-propylnon-7-en-1-yl acetate CM 5 I ·· · ·· OCOCH3
7-propy lnon-2-en-1-y I acetate I CM6 CF,
I 7-propyl-9 ,9-difluoronon-8-en C:'vl 7 ~"ocoo1, I l-y I acetate CM 8 (Z)-8-dodecen- l-yl chloroformate I CM 9 dodec-1-yl chloroformate I ~-rocoa I Uvl!O /V 5 ~/"-0COCil 3 9-thiadodec-1-y l acetate
I CM 11 ~S~OCOCil3 8-thiadodec- l-yl acetate I I I I I I -17- I Table II Ostrinia m1bilalis pheromone analogues I I ON l (Z)-11-tetradecen- l-yl acetate I ON 2 (E)-11-tetradecen- l-yl acetate I ON 3 0 0 (Z)-13-hexadecen-3-olide I ON4 (Z)-14-heptadecen-4-olide i I ON 5 (Z)-15-octadecen-5-olide I ON6 l O-ethyldodec-11-en-l-yl acetate
ON7 l 0-ethyldodec- l 0-en- l-yl acetate I OCXXll3
ON 8 l O-ethyl-12, 12-diOuorododec- I ~VV'ocna~ 11-en- l-yl acetate
CF1 (Z)-11-tetradecen- l-yl ON9 /'~ocoa I chloroformate
~A/'y/'\/VOCXXJIFO (Z)-11-tetradecen- l-yl I UN 10 (R)-chlorofluoroacetate
~VV'V'\/vocua1m (Z)-11-tetradecen-l-yl I ON 11 (S)-chloro i1uoroacetate /vs~ I ON 12 , ClCOCliJ 11-thiatetradec-1-yl acetate I ON13 /'-S~OCXXJiJ 12-thiatetradec- l-yl acetate I I I I c T;:ible 3. Sex Pheromone Analogues of C. mo/esta and 0. nubila!is - The V r------~ standards code of tested L relative retention times ---1==------_results--· 2 stand:ird ! cornpound I 5o·c "6CfC7o·cJ: I 80'C 9o·c 1oo·c 110·c 12o·c 13o·c 14o·c 15o·c 15o·c intercept_ slope r [%JIPcc [P:iJ p/p, P.us c [Pa - - - 1 8249 -0. 1158 99 96 0 312 1 0 n-C,.H 30 1 8043 18-12 ·0/\-::----·,---,.110s 3 802 -3 509 3.265 3 066 - - - -0 0543 99 83 0.0548 5 7 n-C, H,.. 0 1910 - 2 770 2 660 2 560 2.480 2 400 2.310 - - - - 1 1570 5 n.12 0 02007 1 956 1.912 1 877 1.837 1 816 - - - - 0 7655 -0 0308 99.51 0 00828 36.5 n-C,aHJa Cl\D 0 02007 4 054 3 808 3.644 3 432 3 329 - - - - 1 6·192 -0 0813 99·l1 0 00281 107.5 n-C 1aHJa Cl\ll 1 - - - - 1.2669 -0 0887 99 96 0 536 0.56 n-C,.H 30 8043 Cl\\~ 2 770 2 590 2 '1•10 2 300 2.170 - - - 99.91 0 368 0.82 n-C,.H, 1 80'13 Cl\16 3 810 3 500 3.250 2.990 2 790 ------1 6576 -0 11'10 0 99 99 0 572 0.55 n-C,.H 1 8043 Cl\!7 2 5•10 2 360 2 200 2.060 1.940 ------1 2078 -0 0987 30 99 57 0 184 1 64 n-C,sH:n 0.5760 ('f\18 2.560 2 410 2 320 2 230 2.130 - - - - - 1 1076 -0 0655 - - - 0 3158 -0 0182 99 22 0 135 2 24 n-C,~HJ.<0.1910 (_l\l'l 1 330 1 310 1 290 1 280 1 260 - -- 0 0285 96 03 0 0305 9.9 n-C,aH,,, 0.02007 c_'idl(l 0 770 0 780 0 810 0 820 0.830 - - - - -0 3084 ------'------99 95 0 0288 1.0 n-C,aHJ.< 0 1910 71 l-lH1/\c 3 756 3 481 3 231 2 996 2 812 - - - - 1 7040 -0.1163 98 02 0 00462 n-C eH , 0 02007 ()N3 - - 2 720 2 540 2 440 2 360 2 290 - - 1 2021 -0 0685 62 1 3 -0 0208 99 3·1 0 000892 32.3 n-C, 0 H ------I I -19- I I Table 4. Differences among analogs in the complementary electrophysiological and behavioral tests I I I I I CM ON ! I Analog Code I type EAG I ESG Behavior EAG ESG Behavior I I -i...++ +++ ++ 1, OCOCl CM8 I I 1. I i i I ON9 I I +++ ++ ++ I i i I ! I I I I I : Saturated i 12:Ac I +++ - I +++ I I I I I I i I I 14:Ac I +++ - ++ I ! i I I ' ' I ' I I I i 9-thia I CM10 ++ i + ++ I I I 111-thia ON12 I I +++ + -r-+ I I I I ' I I I I I I 8-thia CMll ++ I I 12-thia ON13 +++ + I ! I I I I I i I I \ 4-lactone i CM2 I ! - ++ I I I ' I ON3 -r : 5-lactone CM3 --r ON4 I vinyl CMS + I ON6 + I +++ most active I I I I I I I I -20- I I Cydia molesta I Mating age I Percentage of copulations 1oor-~~~~~~~~~~~~~~~~~~~~~___, I 80 ------sa---~a---~0------50 I 60 I 40 ----- I 20 0 I 1 2 3 4 5 6 7 a Age (in days after eclosion) I Numbers above bars = N I Fig. 1: I Circadian mating activity of Oriental fruit moth, Cydia mofesta. The maximum of mating activity was observed between 12-13 hr of photophase. I Within this period all behavioral experiments were made. I I I I I I I I -21- I I I Cydia molesta I Circadian mating activity I No. of copulations I 251 ,.. 20 r------:: r~------ 1 ; f. ;:: I 15 ------I I 5 ------~ . :·. : . -~. :: 't ------ .:~ ; . : :~ :: -~ ::; I I Q l~r--'--' ----'--'-'--'-'--'--'--'---'--'-'--'-'_,_r -'--'--'--'-'-"--'--'-'--'--'-'''--'--''"'-""--""'-""'-=-""-""-'""---'"'-=-u. 2 4 6 a 10 12 14 16 18 20 22 24 I Time of day (hours) I Fig. 2: The effect of age on copulation of Oriental fruit moth, Cydia molesta. I Though some individuals were able to mate immediately after emergence, the optimal age for mating was 3 days after eclosion. Therefore 3-days old moths were I used in all behavJOral experiments. I I I I I -----·------··---- ~-- Cl r I I I -- .... ~ l:J w .:r; ~ ~ ..... 0 I/) 100,00 200,00 150,00 250,00 50,00 o I ' Z8-12:Ac). The populations. Fig. components Responses 00 hexan l J o - •-~- I I ' 3: EAG ---- dose-response No are show -5 o~------10------ . correction expressed different ------ -4 for curves in dose-response Cydia volatility percentage of ___,_ molesta four -3 log ______ of dose components compounds of - sex curves (µg) EAG -2 pheromone reaction indicating was of female made. -1 , ------~------] _____ to standard different /~ sex Different _ pheromone_ 0 (0.05 receptor pheromone ug I i of --E8-12:Ac -+-Z8-12:0H --zs-12:Ac -0-12:0H ------ r I I - w (.!) - ~ "O 0 (/) 150,00 100,00 200.00 0,00 The Fig. Responses No - Double Unsaturated unsaturated - - -5 + I correction -~ 4: EAG -· bond -- ------ dose-response are -4 - chloroformate in chloroforrrate for expressed the volati!Hy moli9cule "' -3 curves Cydia of in analog analog compounds ·------;------·---·-----· is percentage molesta essential -2 to and Z8-12:Ac showed log - 12: dose pheromone was for of -1 Ac EAG retaining much and (µg) made. show reaction chloroformate smaller analogs ...L-.------ 0 relatively the electrophysiological to EA I. standard. G high ----- reactions. pheromone EAG 2 activity. analogs activity 3 --zs-12:Ac -o-12:Ac --CM9 -+-CMS ------ '' "" I I I I W ~ (!) - 0 <( .._ 1J - ...... Vi 0 100,00 150,00 200,00 50,00 0,00 The Fig. highest Responses No -5 _; ___ _ correction 5: dose-response EAG -4 : ..., ====-:;:----=====·======~ are activity for expressed volatility -0 -3 curves followed C~rdia -- - of I in to molesta compounds -2 • percentage by Z8-12:Ac 4- log and - dose pheromone -1 :=--- 6- and ------·----- was of (µg) membered EAG three made. analogs 0 reaction lactone 5-membered ~-~------ factones. II. -~-·------~------ to analogs. 0-.--a/~~ / standard. /------1 lactone 2 ---~ show - --- 3 I I I i --- the ------ -+---CM3 --zs-12:Ac -D--CM4 --CM2 ----- ------ _ ___J i I i I ------ Cl I ' ...... ~ 0 LU (9 (J) 0 200,00 150,00 100,00 0, 00 The Fig. slightly dose-response Responses No -5 ~------· correction 6: dose-response higher -4 ------'·------ are for EAGs curves expressed ------ volatility -3 curves at Cydia for saturation sulfur of in to molesta compounds percentage -2 Z8-12:Ac analogs concentrations log - pheromone dose -1 and was were of (µg) EAG sulfur made. observed, analogs reaction than 0 analogs. /:.------ Almost CM Ill. however to 11. similar standard. -----. CM EA 11 2 1 G 10 showed 3 I I I I I I~"'"''' -+-CM11 --CM10 ------·------ rl -0 1 I w ~ ~ - <1'. e_, "O ...... 0 V) 150,00 200,00 100,00 50,00 0,00 The Fig. Responses showed No 5, -5 I ;___ followed correction 7: dose-response the -~ -4 a:==== by are smallest for CM expressed volatility -+------ 6 -3 .. curves and EAG Cydia CM of activities from in to molesta compounds 7. percentage -2 Z8-12:Ac log - pheromone dose -1 and all was of (µg) analogs EAG vinyl-branched made. analogs reaction 0 ~· tested. The IV. vinyl-branched to analo1gs. The standard. - - most I - ... ·----- 3 2 active -·-·-··· analogs was . ------·------1 l±J CM 8-12:Ac M5 M7 MG 1 I -27- I I I I . I alf cm2 I I crn3 cm4 I I 'I I I • .. ..i. 1.1 { I ~1' I I~~~. r I I cm8 I I ~12:Ac I I I 0.1 mV I 0.5 sec Fig. 8,: I Spike activity recorded from olfactory neuron of male C. molesta specialized for perception of Z8-12:Ac. Stimulation: 50 ug of pheromone analogs CM 2, CM 3, CM 4, CM 9 and 12:Ac, 5 ug of CM 8, 5 ng of Z8-12:Ac. Stimulus duration was 0.8 sec, I interstimulus intervals > than 2 minutes, spontaneour activity of the cell was < 0.5 Hz. Except of pheromone. chloroformate and 5-membered lactone elicited highly reproducible response in Z8-12:Ac cell, while responses to other analogs were less I pronounced. I I I -28- I I I I arr Z8-12:Ac I I , I 1 ~ cm 10 cm5 I l-&-~~~~~~~~ I I cm 7 cm5 I I I E8-12:Ac Z8-12:0H I 0.1 mV 0.5 sec I Fig. 9: Spike activity recorded from olfactory neuron of male C. molesta specialized for perception of Z8-12:Ac. Stimulation: 50 ug of CM 10, CM 6, CM 7, CM 5, 50 ng of I minor pheromone components E8-12:Ac, and Z8-12:0H, 5 ng of Z8-12:Ac. Stimulus duration, interstimulus intervals and the sensilla was the same as in previous fig. I The ZB-12:Ac receptor responded to vinyl-branched analogs CM 6, CM 5 and to sulfur analogs CM 10. Pheromone component EB-12:Ac did not elicited any (DC, AC) response. This indicate that EB-12:Ac receptor is not located within the same I type of sensilla trichodea as ZB-12:Ac cell. Z8-12:0H elicited relatively high depolarization (DC recording) of Z8-12:Ac cell and in some cases few spikes was observed. The amplitude-of spikes recorded after Z8-12:0H stimulation was the I same as those recorded after Z8-12:Ac. Futher experiments are necessary to figure out if Z8-12:Ac and Z8-12:0H are perceived by the same receptor types or two I separate specialists within the same sens1/lum. I ----··------ °' °' rl rl I I I I ,. ,. dose dose (ng) (ng) 0 0 ...... disruptant disruptant low low 0.001 0.001 from from EB-12:Ac EB-12:Ac mating mating at at vinyl-derivatives vinyl-derivatives (60%) (60%) analogs. analogs. Mating Mating Fig. Fig. ...... a a 6 6 ...... doses. doses. dose dose 10: 10: lactone lactone 0 0 0 0 ug ug and and dirsuption dirsuption disruption disruption 0 0 0 0 0 0 level). level). Results Results and and 1 1 of of sulfur sulfur ZB-12:Ac, ZB-12:Ac, :r: :r: 0 0 ~ ~ ug ug mating mating analogs, analogs, ZB-12:0H ZB-12:0H mating mating (35% (35% u u 2 2 analogs analogs are are effect. effect. observed observed behavior behavior expressed expressed the the followed followed almost almost disruption disruption exhibited exhibited The The main main (57%). (57%). in in most most in in 80% 80% Petri Petri pheromone pheromone by by u u 2 2 C. C. as as effect). effect). Analog Analog CM CM considerable considerable potent potent of of molesta molesta confussion confussion dishes dishes pairs pairs 4 4 (22 (22 CM CM CM CM mating mating observed observed in in component component from from %). %). 3 3 7 7 u u 2 2 atmosphere atmosphere (45%) (45%) was was coefficient coefficient Pheromone Pheromone mating mating Minor Minor all all disruptant disruptant N N N N 00 00 ...... the the compounds compounds followed followed was was pheromone pheromone was was disruption disruption most most the the indicating indicating permeated permeated analog analog the the was was active active most most by by most most tested tested ZB-12:Ac ZB-12:Ac chloroformate chloroformate effect effect components components potent potent from from U1e U1e potent potent by by (42% (42% at at strengh strengh respective respective disruptant disruptant inhibiting inhibiting relatively relatively at at of of ·u; ·u; 2 2 u u 0;;:; 0;;:; c: c: 0 0 c: c: !E !E ·u ·u ...... u u 0 0 w w ------ rri rri 0 0 I I I I Behavioral Behavioral response response ZB-12:Ac ZB-12:Ac paper paper touching touching CM CM males males pheromone pheromone Flight-tunnel Flight-tunnel Fig. Fig. ;~~\ ;~~\ .§ .§ .s .s ~ ~ -0 -0 _. _. 0 0 g g 10, 10, ·c ·c :+: :+: .E .E ~ ~ 11: 11: c c 0 0 Ql Ql Cl Cl disc disc were were ~ ~ Q Q ...... ~ ~ CM CM Inhibition Inhibition the the ~ ~ was was - ~ ~ ~ ~ CD CD ...... loaded loaded standard standard categorized categorized 7, 7, source source observation observation the the 12:Ac 12:Ac CM CM Cydia Cydia J~----- most most with with 5 5 of of and and and and (three (three the the male male molesta molesta active active into into landing. landing. CM CM - of of pheromone pheromone component component effect effect four four 3. 3. CMS CMS reactions reactions disruptant disruptant categories: categories: Analogs Analogs of of - wind wind selected selected blend_ blend_ pheromone pheromone compound, compound, to to were were tunnel tunnel pheromone pheromone activation, activation, As As analogs analogs applied applied Pheromone Pheromone indicated indicated blend, blend, followed followed on on at at taking taking male male 100 100 in in 1 1 O O analog analog short-range short-range by by ng). ng). ng ng off, off, orientation orientation 12:Ac, 12:Ac, doses doses -100% -100% Tile Tile oriented oriented behavior behavior CM CM ·- .i:: .i:: :0 :0 - :;::: :;::: --= --= - c: c: 0 0 c: c: 0 0 ro ro E E to to bioassay bioassay ._,. ._,. "C "C ...... ...... c c c. c. 0 0 (/) (/) ...... (/) (/) to to Q) Q) o o (/) (/) filter filter flight, flight, 8, 8, CM CM of of 2, 2, ------ r'I r'I ~ ~ I I I I Behavioral Behavioral response response ..:::L ..:::L ...... <1J <1J (!) (!) ·.:: ·.:: 'O 'O 0 0 ...... tl) tl) c c (!) (!) Substitution Substitution compounds compounds compound compound can can Flight-tunnel Flight-tunnel Fig. Fig. Cydia Cydia replace replace 12: 12: in in was was were were the the the the experiments experiments molesta molesta able able main main able able pheromone pheromone of of to to pheromone pheromone to to replace replace the the activate activate designed designed .. .. main main ZB-12:Ac ZB-12:Ac wind wind males. males. component component to to blend blend component component answer answer in in tunnel tunnel standard standard in in the the three three question question pheromone, pheromone, pheromone pheromone if if any any though though of of blend. blend. 4% 4% 6% 6% 8% 8% 12% 12% 14% 14% analogs analogs No No some some a.. a.. t: t: Q) Q) t... t... t.J t.J Q.) Q.) nl nl C') C') cu cu tested tested tested tested ...... o- 't:J 't:J ·- ,__ ,__ cu cu II) II) c. c. 0 0 i::: i::: - c c C') C') E E (1' (1' <1> <1> II) II) ....., ....., '"C '"C ~ ~ ...... ...... cu cu -g -g 0 0 U) U) "O "O - co co (1' (1' L.. L.. I -32- I I I I I Ostrinia nubilalis Circadian mating activity I Percentage of copulations I sor--~~~~~~~-...... -~~1 40 ------1'------~ I f 30 ------!------I 20 ------~ ------I ~ 10 ------'------1 I I 2 4 6 8 10 12 14 16 18 20 22 24 I Time of day (hours) I • Lab population 19 Field population Fig. 1: I Circadian mating activity of European corn borer, Ostrinia nubifafis. The maximum of mating activity was observed between 2-6 hr of scotophase. A considerable difference in the width of the mating period was observed when I compared wild and laboratory populations. The mating period for wild population was shorter with maximum between 4-7 hr of scotophase. I I I I I I I -33- I I I Ostrinia nubilalis I Mating age Percentage of copulations I I I I I I Age (in days after eclosion) Fig. 2: I The effect of age on copulation of 0. nubila/is. The optimal age for mating was 3 days after eclosion. Therefore 3-days old moths I were used in all behavioral experiments. I I I I I I I I I -34- I I I I Ostrinia nubilalis I Q) 200 ------ -.~- I TI ------i I -•- Z lab I [Jz wild I 1 I lab wild I I __ _J I -3 -2 -1 0 1 2 I dose (log µg) I Fig. 3: The comparison of El~G responses to two pheromone components of femaie sex I pheromone of wild and laboratory population. Responses are expressed in percentage of EAG reaction to standard (0.5 ug of Z11-14:Ac). No correction for different volatility of compounds was made. The wild population was slightly more I sensitive to Z isomer than the laboratory ones. I I I I I ------ •r, C'i ' I - 0 (9 - <:( ~ w .._, -a Ul 0 100 150 200 50 0 -5 The reaction effects Z11-14:Ac. chloroformate Fig. authentic Chloroformate i__ 4: EAG was to -4 pheromone. dose-response standard. observed analog analog -3 No at ON showed However, Ostrinia doses correction curves 9. Responses -2 relatively nubilalis shifted the to log Zand for saturation dose to volatility -1 - pheromone high are highe E (µg) isomer -- ----- expressed EAG concentrations amplitude - of __J 0 _____ compounds of activity, analogs - 11-14:Ac, - in was percentage though I. similar in was 12·Ac comparison ----- the /~ made ------ as 2 1--__ - of and measurable ---- - for EAG -- to - ---- -- with -· 1 - ·---- I 3 I I I l I -- 1-=•-z11-14:Ac the -n-E11-14:Ac -{]-14:Ac _,._ON ------ ______ -~- --- 9 --- J --1 ------ r'I \0 I I w ..... -0 e,, ~ ...... - ~ !/) 0 100 200 150 50 0 -5 ~ -- j - The lactone 2, Fig. standard. -- ---- --- ON - --- 5: dose-response 3 - -- -4 analogs and ------ No ON correction was 4. -3 Responses curves very Ostrinia for small_ to -2 volatility ~==t===~o--==---.o- -- nubilalis ---- Zand are log -- -- ___ expressed dose ,_ of E - -1 --- .!.------ l------ pheromone isomer compounds - - (pg) - - -- ------ of in ---0----___. _,_ 0 11-'14· percentage --· analogs -- --- was -o .~ Ac made II. and of ------0 lactone EAG EAG ; 2 activity __ reaction ------1 analogs ------'1- of to I I 3 ! I i ON --.z11-14:Ac -11J-E11-14:Ac --+-ON3 -a-ON -o-ON ----- 5 4 ------ r~ r'I I I --- ----- <.:J ..... w "O <( o' ...._o - ~ Cf) 0 150 200 100 - 0 ------ -5 -! ! I i I i---- I I . ' smaller Th8 almost and f\Jo Fig correction Of\! 6: dose-response the than 13. -4 same Responses ------ for for those EAG volatility -3 curves ------ Ostrinia for activity. are authentic of ___ to expressed -2 compounds ,_ nubilalis - Z EAG and log pheromone amplitudes dose E ------ - -1 isomer pheromone in was (pg) percentage made. of components. at ·11-14:Ac 0 saturation analogs ----- ON of EAG - 12 -- Ill. and ·--- and ~/<1 doses reaction sulfur ON were -D 13 analogs to exhibited standard. relatively - Of\! - I --Z11-14:Ac --11-E11-14:Ac -+--ON -o--ON 12 --- ----- 12 13 _____ J ------ rr) 00 I I :::z ...... w (.9 - 0 "O <( +-' Vl 0 150 200 100 50 ----- 0 -5 - 0---- Q$ '.._ ------ The Fig. standard. analogs -- .. ----- ------ ------ ------ -- 7: dose-response ---- - of -4 No Z11-14:Ac. correction ·------1----- -3 curves Ostrinia Responses for ------ to volatility -2 nubilalis Zand - ---- log are dose ____ of E - -1 isomer pheromone J expressed compounds ____ (µg) ------~ of 11-14:Ac 0 ~ in analogs was percentage __.---m -· made. _ _.-+------~ IV. and three of EAG -{) vinyl 2 reaction branched 3 ! I I '-<>-·Z11-14:Ac to -l!t-E11-14:Ac -o-ON --0-0N --+-ON7 ... ______ 6 8 j ------~ °' rr, I I TI . ..... 0 ~ -:::i: ~ w .., Ul 0 ------ 200 150 100 50 0 Fig. The opticaffy compounds analogs expressed analogs J -5 I ~- I - 8: dose-response ---HHH and ON active, --0 -4 in was 10 their percentage and the made. racemic receptor -3 curves ON11 Ostrinia Note of --- mixture. and to • EAG " is ------ that -2 Zand nubilalis able their -- - reaction though log to racemic E dose dircriminate isomer - -1 pheromone the to (pg) standard. mixture main of 11--14:Ac 0 pheromone between analogs ON No 1 / OR. and correction _.....-0----- __..------· V. two Responses to component optically optically - --{) for 0 2 volatility active active are is not 3 of l --Z11-14:Ac' ---"-E11-14:Ac -+-ON10 -o-ON11 ~-()~"._1_0R ' I I --+O- I I I I 1 •r.~INI\\ .. ~U ~ ~1l~~~1) ~/r~M¥M,~/.LlfWl'il;..:.111\!r#.ttAi\1itm,•wi~NJ..t1~w,'4'i' v. ,l\~1!111 1 I ON 13 I I I I I I I I I I I I 1 mV I I 0.5 sec I I Fig. 9 Single sensillum recordings I from sensilla trichodea in 0. nubilalis .. The tip cutting technique was used. The doses used to elicite the response were: 0.5 ug of Zand E11-14:Ac, 500 ug of analogs. Note that there are no differences in spike amplitudes of E and Z cells. Except I of Zand E11-14:Ac, also chloroformate (ON 9) and sulfur analogs (ON 12 and ON 13) elicited spikes in cells associated with s. trichodea. Because ofsimilar spike amplitudes of Zand E cells, it is not clear, if the active analogs stimulate Zand/or I E11-14:Ac cell. The compounds such 14:Ac, ON 7 and ON 6, though did not elicited spikes. caused significant depolanzation (DC) in receptor cells. This observation suggests that these compounds are perhaps also I perceived by receptors within the sensillum. I - --t· 1 - - - - Behavioral response - - - - tunnel attractive resulted expressed blends/doses (97%Z:3%E), Fig. Behavioral -"' ~ ., 10: .c " ii:: c «:: ., ., tl1 0 experiments. 15 .c <::: .8 °' c: "' u c: ::i in doses oriented as response 100 E activated percentage (3%Z:97%E) were flight, H of 1and10 males laboratory finding of ------ males and in ng. wind the hybrid males tested 10 source tunnel ng H E of of for (65%Z:35%E) 0. Z and experiments, each blend nubila/is copulation Blend blend were to I z (n::::30, blends. dose used however 1, attempts. 10 (ng) in and ::::100%). - -- -- -- -1·50% Responses . all ,-·0% -20% -30% -40% ·10% only 100 fwther The Z ng All most blend +J 0... °' CJ ci:I t:: Q) ,_ (..) (]) of wind "@ ·- 'O are ._ E I/) CJ c: c: Cl c.. 0 Q) I/) ..... 0 Z - ------ -t -t ('I ('I I I I I pheromone pheromone chloroformate. chloroformate. blend blend should should main main 97%Z:3%E) 97%Z:3%E) Responses Responses Fig. Fig. Inhibition Inhibition 11 11 ·- .c .c .a .a :t: :t: .2 .2 ...... ~Ill ~Ill pheromone pheromone E E c: c: 0 0 c: c: Cll Cll (= (= be be :s :s .=.. .=.. a; a; .8 .8 - .._ .._ Cll Cll 0 0 0. 0. I/) I/) c: c: Cll Cll I/) I/) 100%). 100%). 18 18 of of emphasized emphasized are are blend. blend. : : Cl Cl by by 100%- 90% 90% 80% 80% 70%- 60% 60% 50% 50% 40% 40% 30% 30% Relatively Relatively nubilalis nubilalis 10, 10, expressed expressed All All component, component, Compound Compound The The 100, 100, compound compound Inhibition Inhibition however, however, most most male male 1000 1000 Ostrinia Ostrinia less less as as active active (ng) (ng) reactions reactions percentage percentage ng ng active active Z11-14:Ac Z11-14:Ac reduced reduced significantly of of that that of of 100 100 male male compounds compounds respective respective nubilalis nubilalis were were the the Z11-14:Ac Z11-14:Ac to to and and most most reaction reaction of of lactones lactones standard standard male male its its potent potent analogs, analogs, were were 1000 1000 - saturated saturated wind wind response response and and pheromone pheromone the the to to 14:Ac, 14:Ac, inhibitor inhibitor vinyl-branched vinyl-branched attractivity attractivity pheromone: pheromone: Z8-14:Ac Z8-14:Ac tunnel tunnel -- equivalent. equivalent. "' "' c c g g c c to to sulfur sulfur was was standard standard -"" -"" ~ ~ "' "' 0 0 (1 (1 O O and and of of analogs analogs 100 100 ng ng D D c c c c Ql Ql Q' Q' D D c c Ql Ql the the - l l of of 14:Ac. 14:Ac. Analogs Analogs ng ng pheromone pheromone --- --- ----- .c .c <:: <:: -- -- ---- --- 6 6 0 0 :J :J ---- u u "' "' c c c c O> O> standard standard Z Z of of and and blend, blend, the the Behavioral Behavioral rnsponse rnsponse ft ft ------ "i' "i' r'I r'I I I I I should should main main pheromone pheromone chloroformate. chloroformate. blend blend Responses Responses 97%Z:3%E) 97%Z:3%E) Fig. Fig. Inhibition Inhibition 11 11 pheromone pheromone ·- - ..c: ..c: .0 .0 ::: ::: .2 .2 ...... - c: c: c: c: 0 0 E E (= (= !'a !'a <11 <11 be be - Qi Qi .::.. .::.. 'B 'B ...... QJ QJ ...... 0 0 !/) !/) Ill Ill a. a. c: c: !/) !/) !/) !/) 0 0 100%). 100%). . . 18 18 of of emphasized emphasized are are blend. blend. : : 0. 0. by by 100%· 100%· Relatively Relatively 90% 90% 70% 70% 60%· 60%· 50% 50% 40% 40% 30% 30% 20% 20% nubilalis nubilalis 10, 10, expressed expressed All All component, component, I I The The 100, 100, compound compound Inhibition Inhibition _....-, _....-, however, however, most most male male 1000 1000 less less Ostrinia Ostrinia as as ,...;~ ,...;~ active active reactions reactions Z11-14:Ac Z11-14:Ac percentage percentage active active ng ng significantly significantly _--.....__= _--.....__= that that of of of of compounds compounds respective respective male male were were nubHalis nubHalis the the to to and and most most of of lactones lactones standard standard 14:Ac 14:Ac reaction reaction reduced reduced male male its its potent potent analogs, analogs, were were .. .. 1000 1000 saturated saturated ~wind ~wind response response and and pheromone pheromone the the ~ ~ to to 14:Ac, 14:Ac, inhibitor inhibitor vinyl-branched vinyl-branched attractivity attractivity ""l ""l pheromone: pheromone: Z8-14:Ac Z8-14:Ac tunnel tunnel equivalent. equivalent. ~ ~ c c c c OJ OJ c c to to sulfur sulfur was was standard standard -· -· "" "" ~ ~ 0 0 (1 (1 /I /I --..,,--,,.--- Ong Ong I I and and of of analogs analogs 100 100 ·c ·c I I u u - c c -= -= :E :E OJ OJ 0 0 Ol Ol /-· /-· the the ··.,. ··.,. ------ I I of of analogs. analogs. 14:Ac. 14:Ac. ' ' ng ng pheromone pheromone 1~-- "" "" .c .c ~ ~ 0 0 l) l) standard standard ::i ::i cu cu !:::: !:::: al al Z Z of of and and • • blend, blend, the the Behavioral Behavioral response response It It -·------··------ --t· tj· I I pheromone Fig. 97%Z:3%E) blend Inhibition main chloroformate. should Responses 11 - ;§ :;::; ..... ..c: - c: c: 0 E 0 (!) ro pheromone (= - - _ B ...... ,_ (/) (!) ~ 5 (/) (/) (!) o c. be - 100%). 18 of emphasized are blend. : 0. by 100%- 90% 80% 70% Relatively nubilalis 10, expressed All l__. Compound component, ... .....-- The 100, Inhibition compound however, most Ostrinia male 1000 less as (ng) active reactions percentage ng Z11-14:Ac active significantly of that of male nubilalis compounds respective were the to reaction and most ON of factones standard reduced the male its 3 analogs, potent 1000 - were saturated wind ~ response and pheromone to 14:Ac, inhibitor pheromone: I attractivity vinyl-branched tunnel Z8-14:Ac 01! D c c c equivalent. to ,/ sulfur ___ was standard -"' cu "' -. 0 (10 JF-:-: - and of analogs 100 -0 c <.::: c "' "' Q' "' c ng the of 14:Ac. analogs ng ------. "" t3 -~ D 6 c c pheromone :J t1l standard Z of and blend, the Behavioral response ft ------ •ri •ri -t -t I I I I main main should should pheromone pheromone chloroformate. chloroformate. blend blend Responses Responses 97%Z:3%E) 97%Z:3%E) Inhibition Inhibition Fig. Fig. :c :c ·- ..c: ..c: ...... :;:::; :;:::; - 11 11 c: c: 0 0 0 0 E E c: c: (U (U CIJ- pheromone pheromone (= (= - - -= -= ...... "O "O ...... 0. 0. !/) !/) CIJ CIJ 0 0 c: c: be be VJ VJ CIJ CIJ CIJ CIJ ...... !/) !/) 0 0 - 100%). 100%). 18 18 of of emphasized emphasized are are blend. blend. : : 0. 0. by by 90% 90% 80% 80% 70% 70% 30% 30% Relatively Relatively nubilalis nubilalis 10, 10, expressed expressed All All Compound Compound component, component, The The 100, 100, compound compound Inhibition Inhibition however, however, most most Ostrinia Ostrinia male male 1000 1000 less less as as active active percentage percentage reactions reactions Z11-14:Ac Z11-14:Ac active active ng ng reduced reduced significantly of of that that of of male male compounds compounds nubilalis nubilalis respective respective were were the the to to and and most most reaction reaction ON ON of of lactones lactones standard standard male male its its 4 4 potent potent analogs, analogs, 1000 1000 ~wind ~wind were were saturated saturated response response and and to to pheromone pheromone the the inhibitor inhibitor 14:Ac, 14:Ac, pheromone: pheromone: vinyl-branched vinyl-branched attractivity attractivity tunnel tunnel ZB-14:/\c ZB-14:/\c ~ ~ c c c c c c c. c. equivalent. equivalent. to to sulfur sulfur was was """ """ ~ ~ standard standard (10 (10 and and of of Elnalogs Elnalogs 100 100 ng ng the the of of analogs. analogs. 14:Ac 14:Ac ng ng pheromone pheromone standard standard Z Z of of and and blend, blend, the the Behavioral Behavioral response response It It ------~- '° '° ,. ,. I I I I should should main main chloroformate. chloroformate. pheromone pheromone blend blend Responses Responses 97%Z:3%E) 97%Z:3%E) Inhibition Inhibition Fig. Fig. 11 11 pheromon~ pheromon~ - .c: .c: ;§ ;§ :.;::: :.;::: ---= ---= - c: c: 0 0 c: c: o o E E ctJ ctJ (= (= Q) Q) be be - - .,... .,... 'O 'O ._ ._ ...... Q) Q) § § a. a. fl) fl) fl) fl) e e Q) Q) 0 0 fl) fl) 100%). 100%). 18 18 of of emphasized emphasized are are blend. blend. : : 0. 0. by by 100% 100% Relatively Relatively 90% 90% 50%- nubilalis nubilalis 10, 10, expressed expressed 0% 0% All All component, component, r r \------\~~~---( \------\~~~---( The The 100, 100, compound compound Inhibition Inhibition however, however, most most male male 1000 1000 less less Ostrinia Ostrinia as as --c·. --c·. active active ------ Z11-14:Ac Z11-14:Ac reactions reactions percentage percentage active active ng ng significantly significantly that that of of of of compounds compounds respective respective male male were were nubilalis nubilalis the the to to and and most most of of lactones lactones standard standard reaction reaction ON ON reduced reduced male male its its potent potent analogs, analogs, 6 6 were were saturated saturated - response response wind wind and and pheromone pheromone the the to to inhibitor inhibitor 14:Ac, 14:Ac, .. .. vinyl-branched vinyl-branched attractivity attractivity ., ., pheromone: pheromone: Z8-14:Ac Z8-14:Ac tunnel tunnel ,"! ,"! equivalent. equivalent. c c s s c c Ol Ol to to sulfur sulfur was was standard standard "" "" 0 0 ."! ."! "' "' (1 (1 ·l ·l Ong Ong and and of of analogs analogs 100 100 'O 'O .:;:: .:;:: c c :r: :r: 0 0 t: t: "' "' <1J <1J Ol Ol the the of of analogs. analogs. 14:Ac. 14:Ac. ng ng pheromone pheromone - .c .c <::: <::: 'O 'O 0 0 ::::J ::::J u u cu cu c c O> O> s s standard standard Z Z of of and and blend, blend, the the Behavioral Behavioral response response It It ------ -t -t l~ l~ I I I I main main should should chforoformate. chforoformate. pheromone pheromone blend blend Responses Responses 97%Z:3%E) 97%Z:3%E) Fig. Fig. Inhibition Inhibition 11 11 ·- .c: .c: .c .c ~II) ~II) .!2 .!2 ...... (0 (0 pheromone pheromone c: c: c: c: 0 0 E E Q) Q) (= (= be be ..=.. ..=.. (ii (ii .8 .8 - ...... !/) !/) B B Q) Q) a. a. 0 0 Q) Q) - c: c: !/) !/) 100%). 100%). 18 18 of of emphasized emphasized are are blend. blend. : : 0. 0. by by 100% 100% 80% 80% 60% 60% 70% 70% 20% 20% 40% 40% 50% 50% 30% 30% 10% 10% Relatively Relatively nubilalis nubilalis 10, 10, expressed expressed All All component, component, Compound Compound The The 100, 100, compound compound Inhibition Inhibition however, however, 10 10 most most male male 1000 1000 Ostrinia Ostrinia less less as as (ng) (ng) active active reactions reactions percentage percentage Z11-14·Ac Z11-14·Ac ng ng active active ·-- reduced reduced significantly --~=~;=~ --~=~;=~ of of that that of of 100 100 compounds compounds respective respective male male were were nubifalis nubifalis the the -.~ -.~ :_ :_ to to ·~ ·~ and and most most of of lactones lactones standard standard reaction reaction ON ON -~~--~-~--->/. -~~--~-~--->/. male male -~ -~ its its potent potent analogs, analogs, 8 8 1000 1000 were were saturated saturated - response response wind wind ·-- and and pheromone pheromone the the to to inhibitor inhibitor 14:Ac, 14:Ac, - ·r ·r vinyl-branched vinyl-branched attractivity attractivity pheromone: pheromone: Z8-14:Ac Z8-14:Ac tunnel tunnel ·E ·E cu cu c c °' °' equivalent. equivalent. to to sulfur sulfur 1 1 was was standard standard ~ ~ ~ ~ - OJ OJ (10 (10 and and of of analogs analogs 100 100 ~ ~ 2 2 0 0 0 0 c c c c ~ ~ ng ng the the --. --. - of of 14:Ac. 14:Ac. analogs. analogs. ng ng "' "' .c .c .'? .'? pheromone pheromone :J :J u u ·-- cu cu c c "'' "'' 6 6 c c standard standard Z Z of of and and blend, blend, the the Behavioral Behavioral response response It It -----·------· -----·------· 00 00 --t· --t· I I I I pheromone pheromone should should main main chloroformate. chloroformate. blend(= blend(= 97%Z:3%E) 97%Z:3%E) Responses Responses Fig. Fig. Inhibition Inhibition 11 11 ·- - :0 :0 .c .c :::; :::; ...... - pheromone pheromone c: c: 0 0 c: c: o o Ill Ill E E (l) (l) be be - ._: ._: - 1:l 1:l ...... ...... Ill Ill Q) Q) ...... 0 0 c: c: c. c. - e e Q) Q) Ill Ill 0 0 rn rn 100%). 100%). 18 18 of of emphasized emphasized are are blend. blend. : : 0. 0. by by 60% 60% 50% 50% 40% 40% 30% 30% 20% 20% 10% 10% Relatively Relatively nubilalis nubilalis 0% 0% 10, 10, expressed expressed All All component, component, Compound Compound The The 100, 100, compound compound Inhibition Inhibition however, however, most most ------ male male 1000 1000 Ostrinia Ostrinia less less as as 1 1 ______ active active (ng) (ng) reactions reactions percentage percentage Z11-14:Ac Z11-14:Ac ng ng active active significantly significantly ______ of of that that of of 100 100 compounds compounds male male respective respective nubilalis nubilalis were were the the to to and and most most of of reaction reaction lactones lactones ON ON standard standard reduced reduced male male its its potent potent 9 9 analogs, analogs, 1000 1000 were were saturated saturated - response response wind wind and and ·---.~~ ·---.~~ pheromone pheromone the the to to 14:Ac, 14:Ac, inhibitor inhibitor vinyl-branched vinyl-branched pheromone: pheromone: attractivity attractivity Z8-14:Ac Z8-14:Ac tunnel tunnel !1l !1l «= «= c c c c equivalent. equivalent. to to sulfur sulfur r r was was standard standard 2 2 ~ ~ .>< .>< "' "' (10 (10 •.f •.f .. .. and and of of analogs analogs 100 100 :c :c E E D D c2 c2 § § "' "' °' °' Q.l Q.l ng ng the the of of analogs. analogs. 14.Ac. 14.Ac. ng ng ·---... ·---... .c .c "" "" pheromone pheromone -6 -6 E E u u °' °' c c ro ro ::J ::J c c standard standard Z Z off off and and blend, blend, he he Behavioral Behavioral response response It It ------ °' -t I I blend pheromone 97%Z:3%E) Fig. should Responses main Inhibition chforoformate. 11 .2 .!: ...... ·- ..a ~ - c: E «! c: 0 Q) pheromone (= :s - ...... Q.j ~ be Q) ... 0. c: 0 0 IJ) IJ) Q) IJ) - 100%). 18 of emphasized are blend. : 0. by 100%- 70% 50% 40% 30% 20% Relatively nubilalis 10, expressed All I component, I Compound The 100, ~---- compound ~I Inhibition I however, most male 1000 Ostrinia less ~r ~I as (ng) active reactions percentage ng Z11-14:Ac active were active significantly ~ of that of 100 male compounds respective nubilalis the J . to -.·rc' _ __,,~ and most ON12 reaction of standard lactones reduced .. =--:K.---~ male _~0 its analogs, potent 1000 - were saturated wind response and phernmone to the 14:Ac, inhibitor :::=! ~ pheromone: vinyl-branched attractivity ------ Z8-·14:Ac tunnel J" c c OJ ~ equivalent. to sulfur was standard -" "' 0 ('1 I I Ong and of analogs r I 100 :c .~l <::: c () 0 c the I I of ·14:Ac. analogs. ng r·---- ---- r---- "" .c -6 pheromone "' c 0 OJ ::i c l) standard Z of and blend, the Behavioral response It ~ --I·-., . - -",. . - - <~-~·:1~~~~~~:z~_~,:;_·5:/.~~~:-~- ..;~: --~~'1:~~:;' .··· " •» ,'. ·I I· Journal of Chromatography, 626 (1992) 215-221 Elsevier Science Publishers B.V., Amsterdam CHROM. 24 516 :I Gas chromatographic determination of vapour pressures of pheromone-like acetates I Bohurnir Koutek, Michal Hoskovec, Karel Konecny and Jan Vrkoc Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Flemingovo nam. 2, 16610 Prague 6 I (Czechoslovakia) (Received May 27th, 1992) ABSTRACT The vapour pressures of nineteen Zand Emonounsaturated C10-C16 even-carbon acetates were determined using a method based on I gas chromatographic (GC) retention data. Experimental measurements were carried out at six temperatures in the range 90-140'C on a 2-m HP-! capillary column by utilizing n-C 18 and n-C20 hydrocarbons ·as vapour pressure reference compounds. Corrections for the systematic errors were made by relating the experimentally determined vapour pressures P oc to the literature values PL through a linear regression relationship. Over a narrow temperature range of 25-45'C, the GC-measured vapour pressures were found to satisfy the I Clausius-Clapeyron equation. Also, for structurally similar subseries of acetates, e.g., for w - 3 or w - 5 unsaturated derivatives, the ·1 vapour pressures were shown to have a simple dependence on the number of carbon atoms per molecule. The vapour pressures at 25'C I ranged from 2.633 Pa for (Z)-5-decenyl acetate to 0.005 Pa for (E)-13-hexadecenyl acetate. 'i \ INTRODUCTION siderations to be put on a quantitative basis. In this context, a knowledge of evaporative characteristics In recent years, research on the sex pheromones of the individual blend components is of great im I.· I of moths and butterflies has opened up new possi portance. bilities for developing ecologically safe strategies Among the physico-chemical properties that de for insect pest control as alternatives to the use of termine the transport and fate of chemicals in the conventional insecticides [1]. Most of these phero environment, vapour pressure is one of the most mones are volatile, multi-component mixtures of important. Clearly, a compound's vapour pressure unsaturated even-carbon (C 10-C18) acetates or al will affect its partitioning between the vapour and cohols with one or two double bonds at various liquid (particulate-bond) phases and, in turn, its ef I positions in the molecule in either the Z or E geo fectivity. For many organic chemicals of environ metric configuration, and a precise ratio of the com mental relevance, including pheromones, low pres ponents is required for the full insect response [2]. sures cause difficulties [3] in direct measurements by I In order to mimic a pheromone-releasing insect in conventional Knudsen effusion [4] and gas satura practical applications, it became necessarito devel tion [5,6] methods. As a consequence, the literature op controlled-release systems for use in monitoring, interlaboratory data often disagree by factors of mass trapping and aerial dissemination control pro 2-3 or more. Gas chromatography (GC) is an al I grammes. The successful applications of synthetic ternative method for measuring vapour pressures pheromone blends however, require volatility con- [7,8], offering advantages in terms of speed, solute sample size, purity and stability requirements. The original idea of relating GC retention times Correspondence to· B. Koutek, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Flemin to solute vapour pressure [9] has been improved by govo nam. 2, 16610 Prague 6, Czechoslovakia. introducing a latent heat ratio term for unknown 0021-9673/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved , __ _ -,- ·:,,,..·· . ' •,_:__- •. - . - - . - ' -·-'g"='--'-····-·~~·-·-----· ...· _____ ,_ :...... : .. ~..:. ___ ...... -··-- -~----·~···- ..: ..·~.:....:;.~~~;,;,,."""""'"""'""";;...;:=:.::.~;:..,.:;~ 1.·'• I 216 B. Koutek et al./ J. Chromatogr. 626 ( 1992) 215-221 and reference compounds [10] and subsequently ap The acetates were either obtained from Sigma plied to estimate the vapour pressures of many (St. Louis, MO, USA) and used as received or were I polychlorinated biphenyls and dioxins [11, 12], her synthesized in our laboratory. In the latter instance bicide esters [10] and organophosphorus pesticides the purity of the compounds was at least 97% as [8]. In addition, two different GC approaches have determined by capillary GC. In the abbreviated no been used [13-15] to treat the retention time (or vol menclature used for the acetates, the letters after the I ume) - vapour pressure relationships for phero colon indicate the functional type (Ac = acetate); mone-like compounds. One [13] uses the substance the number between the dash and colon indicates under study directly as the liquid stationary' phase the number of carbon atoms in the chain and the I- in packed glass chromatographic columns, whereas letters and numbers before the dash indicate the the other [14, 15] makes use of a cholesteryl p-chlo configuration and position of the double bonds. rocinnamate-coated capillary column, suggesting that on liquid crystal phases the elution order ex Data treatment I pressed as equivalent chain length (ECL) is deter The equations for calculating vapour pressures mined by the length-to-breath ratio of the com from GC retention data were derived by Hamilton pounds. Disregarding the fact that, for restricted [I OJ. Briefly, pressures of the substances at the same I sets of compounds, both these approaches have temperature are related through produced good vapour pressure estimates, there are (1) two problems connected with them. The first ap proach is very time consuming as it requires a sep where the subscripts t and r refer to the test and I arate column for each substance, and the efficient reference compounds, respectively, and His the la use of liquid crystal columns is limited to a narrow tent heat of vaporization. These vapour pressures temperature range of ca. ± 10°C about the meso are also related to their retention times (t): I phase transition temperature. In P = In P, - In (ttft,) (2) The purpose of this work was to examine if the 1 rapid and simple GC method reported previously Combining eqns. I and 2 and rearranging yields [7,10,16] to be useful for environmentally hazard I ln (ttf t,) = (1 - Hi/H,) ln P, - c (3) ous chemicals would provide~, -. alternative to more sophisticated methods for det .mining vapour pres Therefore, a plot of ln (ttf t,) versus ln P, would sures of pheromone-like acf. ,tes with the same de have a slope 1 - Ht/H, and an intercept c. Eqn. 1 I gree of accuracy. can then be used to determine the vapour pressure of a test compound at any temperature given the EXPERIMENTAL vapour pressure of the reference compound at that temperature. I Chromatography and chemicals Samples were analysed on a Hewlett-Packard HP RESULTS AND DISCUSSION 5880 chromatograph equipped with a flame ioniza .; I l tion detector and a 2-m fused-silica capillary col Table I gives the GC retention time data mea ' umn (cross-linked 5% methylsilicone, HP-1, film sured isothermally at six temperatures. Before using thickness 0.52 µm) with splitless injection. Chroma these data in the vapour pressure calculations, it I tography was carried out isothermally at l0°C in seemed useful to analyse them with respect to the tervals from 90° to 140°C with a hydrogen flow-rate molecular structure of the analytes. As the Kovats of 10 ml/min. n-C 18 and n-C20 hydrocarbons were system of retention indices is known to permit cor used as reference standards. Retention times were relations of this type, the measured retention times I determined using an HP 3396A integrator. As rec in Table I were first converted into retention indices ommended [7], long retention times of compounds I defined by the equation producing asymmetric peaks at low temperatures were not taken at the peak maximum, but were cal I= lOON + lOOn [(log ta - log tN)/(log tN+n - log I culated at the mid-point between the beginning and tN)] I the end of the peak. ""\ I ~ .. I .I. -_ -~:~· -- --~< .,.·~ ', ' - ' ' - :._-- I ···. - B. Koutek et al. / J. Chromatogr. 626 ( 1992) 215-221 217 ·rolSigma TABLE I ve r were GC RETENTION TIMES (min) OF THE PHEROMONE-LIKE ACETATES te stance ast 97% as Compound 90°C 100°C ll0°C 120°C 130°C 140°C ·eted no 0423 0.285 0.188 0.128 er ter the lO:Ac 1.074 0.688 12:Ac 4.023 2.341 1.338 0.822 0.509 0.568 = acetate); 14:Ac 14.496 7.554 4.391 2.555 1.506 0.913 mlicates Z5-IO:Ac 0.986 0.604 0 395 0.266 0.175 0.121 1i nd the £5-IO:Ac 0.916 0.582 0.365 0.237 0.161 0.108 nd ate the Z7-l2:Ac 3.522 2.108 1.194 0 744 0.465 0.298 £7-12:Ac 3.526 2.055 1.178 0.728 0.454 0.284 e bonds. Z9-l2:Ac 3.845 2 228 1.272 0.791 0.484 0.307 £9-12:Ac 3.826 2.235 1.26! 0.782 0.478 0.303 I Z9-l4.Ac 13.186 6.771 4.09! 2.471 1.514 0.951 r pressures £9-14:Ac 13.051 7.045 4.049 2.463 1.484 0.922 Zll-14:Ac 13.032 6.784 4.215 2.423 1.433 0.881 v lmilton £ll-l4:Ac 13.724 6.975 4.195 2.422 1.441 0.886 at e same Z9-16.Ac 44.199 22.145 l l.127 6.233 3.395 1.961 £9-16:Ac 45.026 22.638 11.505 6.291 3.458 1.955 Zll-16:Ac 46.495 23.522 11.706 6.423 3.567 1.997 (1) Ell-l6:Ac 49.529 24.865 12.193 6.689 3.622 2.047 Zl3-16:Ac 52.051 26.513 12.935 7.066 3.839 2.156 1e I st and £l3-16:Ac 52.639 26.435 12.922 7.087 3.821 2.156 His the Ia- 11-C 18H 38 15.521 8.395 4.441 2.571 1.483 0.884 2.223 r _,ssures n-C,0H42 58.501 26.909 14.277 7.424 3.994 lf). (2) 1gllds (3) 2000 where tA, tN and tN+n are the adjusted retentions of l tould t qn. 1 the analyte and of n-alkanes possessing N and N + 1900 Jr pressure n carbon atoms, respectively. 1800 ~ g,.n the The correlations between the retention indices 110 1700 .m t that corresponding to ll0°C, 1 , and the number of the carbon atoms Nin the acetate chain were estab = 1600 ~ lished by means of the equation I= a + bN and are -~ 1500 shown in Figs. 1 and 2. For n-alkenyl compounds, 0 ~ 1400 the position of the double bond relative to the non ~ I 1300 data mea polar end of the molecule will affect the GC reten tion times. Thus, considering the different structur 1200 ~fo-using al features of thew - 3 (9-12:Ac, l l-14:Ac and 13- tla ns, it 1100 Jee to the 16:Ac) and w-5 (5-lO:Ac, 7-f2:Ac, 9-14:Ac and 1000 he Kovats 11-16:Ac) unsaturated acetates, both subseries were treated separately. The linear dependence of 900 1e.tt.cor tl times the retention indices of a homologous series with 10 11 12 13 14 15 16 on indices normal alkyl chain on carbon number N represents a common trend among the GC data. Therefore, 110 Fig. 1. Retention indices (/ ) for (Z)-alkenyl acetates and (Z) also given in Figs. 1 and 2 are similar dependences alkenes plotted against the respective carbon number. 6 = .v+l log of 3- and 5-alkenes based on literature data [17, 18]. w- 3-Acetates; A = w - 5-acetates; D = w - 3-alken~s; • = The retention indices of Kuningas et al. [18] were w- 5-alkenes. I I I I -~::. •.,, -'-:.· '' ,,..,....;;. I 218 B. Koutek et al./ J. Chromatogr. 626 (1992) 215-221 2000 TABLE II 1900 COEFFICIENTS OF THE EQUATION I= a+ bN 2 I 1800 In all instances r = 0.999 or better. 1700 Compound Configuration a b ~ 1600 w - 5-Acetates z 409.23 97.61 I- " ~ 1500 E 366.23 100.71 5-Alkenes z 15.39 97.66 ~ 1400 -e E 5.88 98.54 1300 cv - 3-Acetates z 396.38 99.31 I E 390.95 99.64 1200 3-Alkenes z 4.69 99.21 1100 E 0.96 99.36 I 1000 900 used for this purpose, as both the stationary phase (OV-101) and temperature (l l0°C) that they used 10 11 12 13 14 15 16 ·: are comparable to our experimental conditions. I Tlie linearity of the plots provides evidence for 10 Fig. 2. Retention indices (!1 ) for (£)-alkenyl acetates and (£) the incremental nature of the sorbate-sorbent inter alkenes plotted against the respective carbon number. Symbols ·-I as in Fig. I. action energy irrespective of the homologous series I· -.,j considered. Inspection of the least-squares regres- TABLE III I PARAMETERS OF EQN. 3 AND VAPOUR PRESSURES (25°C) OF THE PHEROMONE-LIKE ACETATES Compound" HJH, c P(Pa) Errore (%) I Eqn. I Eqn. 4 Exp' Exp.d IO:Ac 0.7821 3.1977 1.392 2.179 2.262 2.181 -1.9 12:Ac 0.8996 1.5954 0.1827 0.279 0.276 1.1 I 14:Acb 0.8575 1.5142 o.029e 0.0201 0.0349 -14.6 Z5-IO:Ac 0.7685 3.3373 1.678 2.633 2.659 I £5-IO:Ac 0.7846 3.354 1.609 2.523 2.524 0 Z7-12:Ac 0.8854 I. 7581 0.235 0.359 0.319 0.337 9.5 £7-12:Ac 0.8958 1.7363 0.212 0.324 I Z9-12:Ac 0.8995 1.6427 0.191 0.292 E9-12:Ac 0.9035 !.6342 0.1862 0.284 Z9-14:Acb 0.8147 1.6744 0.0309 0.0461 0.0495 7 E9-14:Acb 0.8261 1.6486 0.0279 0.0416 I Zll-14:Ac0 0.8364 I 6367 0.0259 0.0386 EI 1-14:Acb 0.8489 1.5524 0.0228 0.0339 Z9-16:Ac 1.0723 - !.2107 0.0058 0.0085 E9-16:Ac 1.0781 - 1.2511 0.0055 0.008 I Zll-16:Ac l.0812 -1.2909 0.0052 0.0076 0.0065 16.9 £11-16:Ac 1.0944 -1.2864 0.0045 0.0066 Z13-16:Ac 1.0947 -1.4442 0.0042 0.0061 I £13-16:Ac !.0992 -1.4623 0.0041 0.005 " Standard: n-octadecane. b Standard: n-eicosane. ' Ref. 13. d Refs. 14 and 15. I e Error = IOO(p c-Pexo)fp,, • 0 0 I I I --·--... : '·_:.. .,,.~;i, • I" .. -·-· .~ -- , --; i- _- . .,..._- -,,' -.,_-- '_-., _-' --; ~-. - I r~ I I B. Kowek et al. f J Chromatogr. 626 ( !99:C) 215-221 219 sion coefficients of the equation I= a + bN (Table previously [13-15]. When literature values were se I II) further reveals that (i) the values of the coeffi lected for this comparison, some judgement was cient b vary only slightly (by ca. ± 1.5%) around a necessary, as different reports for a single com mean value of99.03 i.u., demonstrating an approxi pound sometimes agreed very well but in other mate constancy of the methylene group contribu cases poorly. Of the three data sources [13,14,22] I tion to the sorbate-stationary phase interaction en dealing with vapour pressures of acetates in Table ergy within all subseries of compounds compared; III, we favoured the more recent results. The older slightly higher slopes, however, are invariably data [22], based on a gas saturation approach, had I found for the E isomers; (ii) specific interactions be been already questioned [13,23], and were not con tween the sorbent and terminal functional groups, sidered further. The literature PL values in Fig. 3 in addition to double bonds, are reflected almost and Table III are therefore those based on refs. 13, exclusively in the intercept a; and (iii) reasonable 14 and 15. They correspond to two different experi I statistics of the I vs. N linear dependences (r > mental techniques, (i) the GC method [13] which 0.999 in all instances) permit the I values of non uses the substance under study as the stationary available acetates to be predicted with good accu phase, if necessary these data being extrapolated I racy by prudent extrapolation. 5°C below the temperature range at which they were As the method used for vapour pressure determi measured, and (ii) the GC method [14,15] which us nation is a comparative one, vapour pressures being es a liquid crystal stationary phase (as the vapour calculated from that of a standard compound, it pressures obtained by the latter method were mea I became important to assess the values given in the sured at 30°C they were recalculated to 25°C by literature. The vapour pressures of n-octadecane making use of the Clausius-Clapeyron equation over the temperature range 40-130°C have been and the corresponding heats of vaporization given I shown [7,19] to fit the equation ln P (Torr) = A + in ref. 23). The results of comparison of our PGc B/T with A = 25.548 and B = - 10 165. On the data with those taken from the literature (PL) are other hand, the Antoine equation, log P (Torr) = A depicted in Fig. 3. As can be seen, the regression line I + B / (t + C), with A = 7.99897, B = - 2607.622 obtained parallels they = x line. The equation of and C = 177 .32, has been proposed [20] for the the regression line by a linear least-squares fit is vapour pressure-temperature (80-170°C) depend In P1. (Pa) = 1.0126 In PGc + 0.444 (4) ence of n-eicosane. Similar parameters have also (11 = 8, r = 0.9991, S.E. = 0.094) I been found by other workers [21]. Therefore, the above-mentioned constants A, Band C were used to calculate the vapour pressures of the reference 0 / I standards in this work. Accordingly, the vapour / pressure values for n-octadecane and n-eicosane ex ·1 / trapolated to 25°C are 0.02546 and 0.00172 Pa, re ~ spectively. 0:: -2 I QC-determined vapour pressures PGc of all nine :l / Q'. teen acetates at 25°C were calculated from relative / -= -3 retention times (Table I) by using eqns. 1 and 3. The a/ / results are given in Table III. It should be noted that I .4 / / the difficulties in separating the C 18 hydrocarbon / and C14 acetates resulted in a substantial scatter of ·S the points of the In t, 0 1. vs. P, plot. Therefore, n I eicosane was used as reference compound for all -6 .i. C1 acetates. -6 -5 -4 -3 -2 -1 0 To test the validity of this approach, it was useful In p(GC) to compare the PGc values from Table III (eqn. 1) I Fig. 3. Logarithmic plot of literature vapour pressures PL versus with the limited amount of vapour pressure data on P Ge data from the present work. The regression line (solid) and I unsaturated acetates (PL) that have been published the y = x line (dashed) are shown. I I I - - I _: ,_,~,. I 220 B. Koutek et al./ J. Chramatogr. 626 ( 1992) 215-221 I Although both data sets for lO:Ac, 12:Ac. 14:Ac, TABLE IV £5-IO:Ac, Z5-10:Ac, Z7-12:Ac, Z9-14:Ac and PARAMETERS OF EQN. 6 Zl1-16:Ac are well correlated on the HP-1 column, the PGc underestimated PL by a factor of about 1.6. Acetate subsenes A B c D I Two reasons have been identified [16] that might Saturated cause this inequality. One could occur if the differ 679.7 1154 3 1.218 !4.853 w-3-(Z) 499.4 3297.2 0.725 20.768 ence in activity coefficients y among the test and w-3-(E) 500.7 3360.5 0.726 20.966 I reference compounds were related to compound w- 5-(Z) 536.3 2572.4 0.833 18.759 volatility, while the other could be due differences in w- 5-(E) 533.8 2742.9 0.822 19.299 y which were not correlated with volatility. As no significant scatter of points about the regression line I in Fig. 3 was observed, the deviations in the present both the vapour pressures at 25°C and heats of va instance are probably attributable to a systematic porization as they are predicted by this equation error connected with the GC column. This system with original PGc data based on eqn. 1 and vapor I atic error can be eliminated and the accuracy im ization enthalpies obtained previously (23] from in proved by using Fig. 3 as a calibration plot to corre dependent measurements. The agreement found be late PGc with PL. Hence, the final vapour pressures tween PGc (eqn. 1) and P (eqn. 6) values with a I PL of test compounds (Table III, eqn. 4) were ob relative error not exceeding 2% demonstrates the tained from measured P Ge data by correcting them predicative validity of eqn. 6. Additional support according to eqn. 4. It is shown that this correction for its reliability comes from a comparison between provides vapour pressures within a factor of about estimated and experimental Hv data. For com I 1.17 of average literature values, thus achieving bet pounds in common with ours, the Hv values (kJ 1 ter precision of vapour pressure determinations mol - ) found by McDonough et al. [23] and our than reported interlaboratory results. The mean rel selves are, respectively: IO:Ac, 67. 78 and 66. l; I ative error for the testing set of eight acetates was 12:Ac, 77.58 and 77.4; 14:Ac, 87.37 and 88.7; Z7- found to be less than ± 7%. 12:Ac, 75.91 and 74.91 and 74.9; Z9-14:Ac, 85.7 To extend further the scope of the method, the and 83.8; Zll-16:Ac, 95.46 and 92.7. Hence the ex effect of temperature on vapour pressures was also perimental enthalpies are reproduced to within investigated. The Clausius-Clapeyron equation about± 3%'. At a constant temperature of 25°C, eqn. 6 with (5) ln P = - Hv/RT + C parameters from Table IV can be substituted into I where Hv is the enthalpy of vaporization and Rand the correction eqn. 4, yielding corrected vapour 1 1 Care the gas (8.3144 J mo1- K - ) and integration pressure relationships as shown in Table V. These constants, respectively, was found to be adequate equations enable vapour pressures at 25°C to be for describing the vapour pressure-temperature de I pendence over the range 25-45°C. The method ofleast squares was used to fit eqn. 5 TABLEV to the data using Hv and C as parameters. The re PROPOSED RELATIONSHIPS FOR PREDICTING VA I sulting parameter estimates were further found to POUR PRESSURES AT 25'C depend systematically on the carbon number N within the saturated and the monoenic w-3 and w-5 Acetate Jn PL= a+ bN" subseries acetate subseries. As a consequence, an empirical a b I relationship given by the equation Saturated 11.563 - l.075 In P (Pa) = - (AN+ B)/T + (CN + D) (6) w-3-(Z) 10.275 -0.962 w- 3-(E) 10.261 -0.965 I j resulted, where the constants A-D varied depending ·j w-5-(Z) 10.702 -0.978 l on the subseries -type. The numerical values of the w- 5-(£) 10 67 -0.981 constants are summarized in Table IV. Tests of this empirical equation are provided by comparison of a Vapour pressure in Pa. I - -I _{ I I ---- ' . _-• --· : -.•;:" - I -· ~ - I I I B. Koutek et al. I J. Chro/11({/ogr. 626 ( 1992) 215-2]1 221 predicted for selected homologous subseries of ace REFERENCES tates from the carbon numbers N. Based on a re I stricted set of available experimental data, it is be l E. D. Morgan and N. B. Mandava (Editors), C RC Handbook lieved that these equations should predict vapour of Natural Pesticides, CRC Press, Boca Raton, Fl, 1988. 2 R. L. Ridgway, R. M. Silverstein and M. N. Inscoe (Editors), pressures with an average error of less than 10%. Behavior-Modifying Chemicals for Insect Afanagement-Ap I A specific comment is needed regarding the in plications of Pheromones and Other Attractants, Marcel tercepts and slopes in Table V. Whereas significant Dekker. New York, 1990. differences in these parameters are observed among 3 R. C. Reid, J. M. Prausnitz and B. E. Poling, The Properties of Gases and Liquids, McGraw-Hill, New York, 4th ed., the saturated. w-3 and w-5 monoenic subseries. the 1987. I same differences in parameters between Z and E 4 J J. Murray, R. F. Pottie and C. Pupp, Can. J. Chem., 52 isomeric subseries (although they might be real) are (1974) 557. all within the 95% confidence interval limits. 5 W. F. Spencer and M. M. Cliath. Residue Rev., 85 (1983) 57. The conclusion reached from the results is that 6 W. J. Sonnefeld, W. H. Yoller and W. E. May, Anal. Chem., I 55 (!983) 275. the present GC method can provide vapour pres 7 T. F. Bidleman. Anal. Chem., 56 (1984) 2490. sures of pheromone-like acetates with average de 8 Y.-H. Kim, J. E. Woodrow and J. N. Seiber, J. Chromatogr., viations of less than ±I 0% of the literature values, 314 (1984) 37. I well within the interlaboratory precision of other 9 D. J. Jensen and E. D. Schall, J. Agric Food Chem., 14 (1966) techniques. Taking into account its other advantag 123. 10 D. J. Hamilton, J. Chromatogr., 195 (1980) 75. es, e.g., simplicity, speed, sample size and purity re l J J. W. Westcott and T. F. Bidleman. J. Chromatogr., 210 I quirements, the approach presented here can be (!981) 331. considered as a viable means for calculating vapour l 2 B. D. Eitzer and R. A. Hites, Environ. Sci. Technol., 22 (1988) pressures of a large variety of compounds. In this 1362. 13 A. M. Olsson. J. A. Jonson, B. Thelin and T. Liljefors. J. I respect, we believe the present results for acetates to Chem. Ecol., 9 (1983) 375. be prototypical for a number of related systems. We 14 R. R Heath and J. H. Tumlinson. J. Chem. Ecol., 12 (1986) are currently examining the potential of this meth 2081. od for determining vaporization properties of other 15 R.R. Heath, P. E. A. Teal, J. H. Tumlinson and L. J. Men classes of pheromone components, such as alcohols gelkoch, J. Chem. Ecol .. 12 (1986) 2133. I 16 D. A. Hinckley, T. F. Bidleman, W T. Foreman and J. R. and dienes. Tushall, J. Chem. Eng. Data, 35 (1990) 232. 17 S. Rang, K. Kunmgas, T. Strenze, A. Orav and 0. Eisen, J. ACKNOWLEDGEMENT Chromatogr., 406 (1987) 75. I 18 K. Kuningas, S. Rang and T. Kailas, J. Chromatogr., 520 (1990) 137. This research was supported in part under grant 19 A. B. MacKnick and J.M. Prausnitz, J. Chem. Eng. Data, 24 No. DHR-5600-G-00-1051-00, Program in Science (1979) 175. I and Technology Cooperation, US. Agency for In 20 K. Sasse, J. Jose and J. C. Merlin, Fluid Phase Equilib., 42 ternational Development. (1988) 287. 21 V. Piacente, T. Pompili. P. Scardala and D. Ferro, J. Chem. Thermodyn, 23 (1991) 379. I :12 Y. Hiraoka and M. Suwanai, Appl. Entomol. Zoo!., 13 (1978) 38. 23 L. M. McDonough, D. F. Brown and W. C. Aller, J. Chem. I Ecol., 15 (1989) 779. I I I I I ______, ______---- SYNTHETIC COMMUNICATIONS, Z3(li), :!397-:404 (1993) t') i PREPARATION OF E·ALKENES USING LITHIUM AND l,3·DIA."1INOPROPANE i ~ ,, 'i Irena Kovarova"and Ludvik Streinz Institute of Organic Chemistryand Biochemistry Academy of Science:;of Czech Republic Fl.emingovonamesti 2, 166 10 Praha 6. Czech Republic ! Abstract : A new selective reducing system, litld"!n i1t J,J-diami11opropane. is ·l dacribed. whiclt enablu du: preparation of E-014fi1is from acl!!ykna in high .I I purity. 'l ! The preparation of compounds with double bonds in high stereoselectivity is of great importance. for example. in the synthesis of pheromones. We therefore j report a new and convenient method for the preparation of (E)-.alkenes. I 1 The most commonly used method of (£)-double bond synthesis is the 1 reduction of acetylenes by allcali metals and liquid ammonia. Warthen et aL srudied this system and considered it to be the best method in terms of stereoselectivity and yield. However. thls method is not suitable for compounds with longer carbon chains. The yield ls probably poor in these cases becau.'ieof • to whomcorrespondence ~hould be addressed I 2397 ·ti~ V).._ •$ ~I - Copyright ii:) 1993 hy Marcel Dekker, Inc. - - - 2398 their propyl-) A:s alkylamines Reggel resulted selectivity. double and stops with calcium in because bond side also 1,3-diamincpropane sufficient necessitates 1.2-diaminoethane. and results be 10-tetradecyne first bonds 1-hexyne a As an u.red succeeded mixture at lower products. reduction calcium. requires in alternative and/or substrates We to bonds) et room in of in are the the as al. saturated to poor (V). either solub1licy found these Methylamine a a solubility of led ~ obtain used - an temperarure. alkene high cosolvent. and stage We in then milder both Unlike yields, phenol cm. for aromatic to methylamine ro reducing in reductions that found excess reported good replaced carbon the this the The amines (OAP) by stage. combination 3-hexyn-1--01 111 of perhaps conditions the - reduction controlling the (Vn. utilisation mechod. liquid The results. reaction that subsmues and of ring depending various chains, lithium-methylamine 5 The has it reduction .b. ethylamine amine system four are benzene as 1,2-diaminoethane or ammonia. due : many authors The low a we 1.2-diaminoethane - - - - - TIIP-0-5-nonyn-l-ol does ~ummansed than with and very but unsaturated of to and (III), the in used reduction aliphatic on low advantages higher is OAP even of reaction (YID high it not the with lithium powerful the found Jnd apparently 5-nonyn-l-yl various solvatation is 1.2-dialkylacetylenes reaction less triple is possible 1,2-diaminoethane the require thus and primary primary compounds proceeds high. conditions. in that 3 bifunctiona.l equivalents system, • were over compounds metal-amine a phenylacerylene bond KOV Table ~he alone cosolvent the temperatures. Reduction of capable to a that also amines, a.mines (0, AROV acetate high qualities. stop fast lithium reducing high the were l. in (containing used 4-methyl-3.S-dioxa~ enough of liquid containing The the l.2-diaminoethane excess reduction of A should volatility system (mechyl-, unequal which Li (IV), in in method.. in AND by reducing reaction TilF properties and giving lithium-OAP these reduction combination ammonia (VIIJ). both lithium of be STREINZ with l-phenyl- however OAP triple can to of solvent a always used:_ media. ethyl-. at rise which in triple those triple low low The also low are the or of or in of to ;; I I - - - - compounds presence product aromatic stage. PREPARATION 1-phenylhexane. was substrate mixture indicator _VDI) group difference gave not bond cyclohexanone. phenol substituted negligible distilled molecular spectrometer 1.3-Diaminopropane A All observed yet The =H-NMR different of characteristic with were been reactions (VI) obtained reached ring, of ·compound described for was in amounts from I. sieve11. the (Z)-isomers not olefins all successfully during obtained yield ll is products.~ complete. slow. OF spectr.i of This and reduced in easily a were sodium-benzophenone after the between E-ALKENES dark change method CDC!, of the under vm Ill subsequent substrate, (OAP) substrates (Z}-isomer. carried in reduc~d. reductions yielded the was at the purple EXPERIMENT explained. also Unfonunately were of mild low with all, the is substrate was reduction less colours was out an which reductions even 68-98% yield recorded reduction and or (Ill. terrame1hyb1lane conditions. distilled effective than of not under purple-brown the ar compounds IV, Fmally. first has (from was reduced. higher - - 1.5%, of· AL of this reduction VI). a on of nitrogen. of from ketyl underwent the tool triple compound identified yellow compound SECTION The colour the :i the not temperature. The -:orresponding calcium The Varian for I. released immediately bond aromatic colour resulting does JS included II Tetrahydrofuran reaction to product the change a and an purple by reduction in IV V not hydride Unity-:oo preparation the imemal alcohol. V. GCJMS conjugation at substrates was The stop of (E)-olefms of in - - - - After -30 is or reduction (E}-alkem:s the the Table prior and purple-brown) not terminal identified in and of Th~ reference. acetate the reduction (200 the analysis the stored (THF) a 60"C of (VI. I). reaction with extreme to general alkene acetate contain of 1,2-di 2399 triple ;...1Hzl The (IV) (the VD. over has the use. was an as of as IR ~ ff. ' j ·• J. ~~~! ;t -~ !': .I ., J : ' :;4-03 :;4-03 ). ). 95. 95. 29. 29. 77. 77. n. n. 29. 29. 29. 29. 27. 27. 114. 114. .. .. 73; 73; 96. 96. 41. 41. 27. 27. 91. 91. 29, 29, 41. 41. 100% 100% 27. 27. 55. 55. 138. 138. ( ( 133. 133. 105, 105, 97. 97. 124. 124. 55. 55. Analysis Analysis 31. 31. 41, 41, 29. 29. 85 85 67, 67, (100%), (100%), 104. 104. - 149. 149. 69. 69. 27. 27. 39. 39. MS MS m/z m/z 29. 29. 162. 162. 57, 57, 55. 43 43 129. 129. GC GC 41. 41. 91(100%). 91(100%). 183, 183, 129. 129. 82. 82. 129. 129. 96. 96. ). ). ). ). 69. 69. -1-1. -1-1. .. .. 45. 43, 43, 45. .. .. 54. 54. 39. 39. -l-3. -l-3. for for 167.. 167.. 92. 92. (Mi. (Mi. (100%). (100%). (M··). (M··). (M (M (M (M (M"). (M"). (M"), (M"), (M°·). (M°·). 55. 55. 55, 55, 67. 67. 51. 51. 55. 55. (100%), (100%), 100 100 160 160 183. 183. 162 162 117 117 105. 105. 184 184 83. 83. 81. 226 226 85(100%). 85(100%). 226 226 81. 81. 242 242 69. 69. 69. 69. 41 41 63. 63. 65. 65. OH OH OH OH Standards Standards 1120. 1120. 1120. 1120. 3370 3370 3343 3343 and and 3 3 ) ) 1 - 1138. 1138. C=C C=C lR lR 1090. 1090. 1137. 1137. 1090. 1090. 3480. 3480. 3490. 3490. C-0 C-0 (cm' =C-H =C-H =C-H =C-H v v TABLE TABLE Products Products 1185. 1185. =C-H =C-H cococ cococ =C-H =C-H =C-H =C-H 970 970 =C-H =C-H THPO- 1041 1041 1622 1622 1103. 1103. 1185. 1185. 1102. 1102. C-0 C-0 C=C C=C cococ cococ C-0 C-0 3591. 3591. C=O C=O C=C C=C =C-H =C-H =C-H =C-H 721 721 =C-H =C-H 3588. 3588. =C-H =C-H THPO- =C-H =C-H C-C=C C-C=C 969 969 1201, 1201, 1742 1742 1407 1407 1643. 1643. 1060 1060 1135. 1135. 1035 1035 1656 1656 1035 1035 1050 1050 1403. 1403. 1060 1060 1656 1656 1050 1050 1239. 1239. 1135. 1135. 1201. 1201. 574 574 3010 3010 3007 3007 3635. 3635. 969 969 3025 3025 3028. 3028. 3007 3007 3002 3002 3019 3019 3629. 3629. Reduction Reduction l-ol l-ol l-ol l-ol £-ALKENES £-ALKENES or or acetate acetate OF OF ' ' l-hexen l-hexen Data Data l-yl l-yl l-ol l-ol MS MS Compound Compound and and -10-terradecen -10-terradecen -10-teradecen -10-teradecen IR IR 1-phenylhexane 1-phenylhexane (Zl-1-phenyl- (Z)-3-llexen- (Z)-5-nonen- (E)-4-methyl-3.5-dioxa- (E)-3-hexen-l-ol (E)-3-hexen-l-ol (Z)-THP-0-5-nonen- (Z)-4-methyl-3,5-dioxa- (E}-THP-0-5-nonen- PREPARATION PREPARATION ------ ' ' .. .. .. .. l l I I I I _, _, (dt (dt lH. lH. lH. lH. 3H. 3H. (m. (m. 4H. 4H. 3H. 3H. IH. IH. 4H. 4H. 2H. 2H. 3H. 3H. J=7. J=7. 1=6.5. 1=6.5. =CH}. =CH}. Cff:C), Cff:C), Cli:O), Cli:O), (s. (s. CCff.:). CCff.:). (t. (t. OCff.:), OCff.:), OCHO). OCHO). STREINZ STREINZ (m. (m. 3.39 3.39 CH:C=). CH:C=). 1=7. 1=7. J=7. J=7. Cff:C=). Cff:C=). =CC!i:). =CC!i:). 1=2. 1=2. (dt. (dt. 2.45-2.26 2.45-2.26 2 1=5.5. 1=5.5. 1=5.5. 1=5.5. H. H. Analysis Analysis (t. (t. CH,). CH,). CH,). CH,). 2H. 2H. I I =CH). =CH). 1=6.4. 1=6.4. IH.OCHO}. IH.OCHO}. 6H. 6H. 12H, 12H, 12H.CH:C). 12H.CH:C). 0.90 0.90 2.05 2.05 2H. 2H. 2H. 2H. CH,). CH,). 2H. 2H. 1.51-1.27 1.51-1.27 (k. (k. (k. (k. 4H. 4H. AND AND 3.74 3.74 (m.IH. (m.IH. l.20(t l.20(t lH. lH. (t. (t. 3H. 3H. GC GC 3H, 3H, OCH). OCH). (m. (m. 1=12. 1=12. A A (m. (m. (m, (m, 1.20 1.20 (m. (m. 1 2.14-l.94(m. 2.14-l.94(m. 3H. 3H. (m. (m. (m. (m. (m. (m. 1=6. 1=6. 2.08-l.86 2.08-l.86 CCH=>. CCH=>. 4.67 4.67 4.66 4.66 1=3.4, 1=3.4, 1=3.0. 1=3.0. (m, (m, for for Cli:C). Cli:C). lH. lH. J=8, J=8, (t. (t. (td. (td. (t. (t. (d. (d. 4.06 4.06 OCH). OCH). J=8. J=8. lH. lH. AROV AROV (t. (t. (Hz) (Hz) Cff:C). Cff:C). C!-'~C=C). C!-'~C=C). .+H. .+H. CH.iC). CH.iC). (t. (t. CH,). CH,). (m. (m. Cli:O}. Cli:O}. J J • • 5.40-5.26 5.40-5.26 1.88-1.25 1.88-1.25 IH. IH. Clf:Cff:O). Clf:Cff:O). 1.65-1.19 1.65-1.19 3.63 3.63 6.36 6.36 2.10-1.20 2.10-1.20 4.56 4.56 CH,). CH,). Cff:O). Cff:O). CH,). CH,). 3.55-3.33 3.55-3.33 4.58 4.58 2.37-2.24 2.37-2.24 KOV KOV 0.99(t.1=7. 0.99(t.1=7. 0.98 0.98 2.32-2.20 2.32-2.20 2.26-2.08 2.26-2.08 CH=CH}. CH=CH}. 4H. 4H. CH=CH). CH=CH). :J=5. :J=5. 13H. 13H. 3H. 3H. 5.45-5.29 5.45-5.29 0.97 0.97 13H. 13H. 4H. 4H. 2H. 2H. 3.50 3.50 3H. 3H. 3H. 3H. 4H. 4H. Standards Standards 10.4. 10.4. ). ). 2H. 2H. 2H. 2H. 3 (!!!. (!!!. OCH). OCH). CH=CH}, CH=CH}, (ppm) (ppm) (m. (m. CH=). CH=). (m. (m. (m. (m. (m. (m. Cli:O). Cli:O). J=l2. J=l2. (m. (m. J=9, J=9, =CCff.:). =CCff.:). 8 8 :irom.H). :irom.H). 1=1.5. 1=1.5. OCHJ. OCHJ. 1 OCH:). OCH:). CH=CH}. CH=CH}. CH CH=CH). CH=CH). OCff.:). OCff.:). 2 2 Cff:C=C}. Cff:C=C}. CH=CH). CH=CH). CH=>. CH=>. and and =C~). =C~). =CCff.:). =CCff.:). 7.4. 7.4. IH. IH. CCff.:). CCff.:). 1.60-1.16 1.60-1.16 OCH). OCH). (t (t CH,). CH,). (t. (t. lH. lH. 2H. 2H. CH,). CH,). 2H. 2H. 5H. 5H. 4H. 4H. (td. (td. 2H. 2H. 3H. 3H. 2H. 2H. 2H. 2H. lH. lH. 2H. 2H. 1=11.0. 1=11.0. 4H. 4H. 1=15.0. 1=15.0. 2H. 2H. 2H. 2H. 2H: 2H: 2H. 2H. 6H. 6H. IH. IH. 3H. 3H. 3H. 3H. 0.88 0.88 0.88(t.1=9. 0.88(t.1=9. 0.90 0.90 Z.!7-!.91 Z.!7-!.91 10.4. 10.4. 1.70-1.29 1.70-1.29 (m. (m. 1.65-1.29 1.65-1.29 1=3.0. 1=3.0. (m. (m. (m. (m. 3.62-3.34 3.62-3.34 1.n-1.57 1.n-1.57 (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. (m. 5.67 5.67 3:70-3.32 3:70-3.32 1=6. 1=6. TABLE TABLE 1=6. 1=6. CH:C=l. CH:C=l. 1=7.3. 1=7.3. 1=1, 1=1, Products Products 1=1. 1=1. 6.5. 6.5. (t. (t. (ddd. (ddd. 10.4. 10.4. (t. (t. (t (t (t (t (t. (t. CCff.:), CCff.:), ZH. ZH. 500MHz: 500MHz: 3.87 3.87 5.45-5.35 5.45-5.35 5.45-5.29 5.45-5.29 2.16-1.92 2.16-1.92 3.93-3.69 3.93-3.69 6.5. 6.5. 0.88 0.88 CH:C=C). CH:C=C). OCHO). OCHO). 5.46-5.28 5.46-5.28 0.90 0.90 1=6.5. 1=6.5. CH,Cli:O), CH,Cli:O), CH:C=C). CH:C=C). OCHO). OCHO). 2.17-2.01 2.17-2.01 5.46-5.26 5.46-5.26 S.51-5.30 S.51-5.30 5.69-5.54 5.69-5.54 CH,Cli:O), CH,Cli:O), 1.80-0.95 1.80-0.95 3.70-3.58 3.70-3.58 2.12-1.96 2.12-1.96 5.65-5.51 5.65-5.51 3.63 3.63 CH,CO~). CH,CO~). 5.41-5.36 5.41-5.36 C!'~OCO). C!'~OCO). 2.06-1.88 2.06-1.88 4H. 4H. 3.68 3.68 0.87 0.87 PhCH=). PhCH=). 7.45-7.15 7.45-7.15 (m. (m. l~H.CH,). l~H.CH,). .: .: Reduction Reduction l-ol l-ol or or acetate acetate . . 1-hexen 1-hexen Data Data l-yl l-yl I- Compound Compound 0-teradecen 0-teradecen -10-cerradecen -10-cerradecen H-NMR H-NMR 1 - I (E)-THP-0-5-nonen- (Z)-THP-0-5-nonen-l-ol (Z)-THP-0-5-nonen-l-ol (E)-4-methyl-3.5-dioxa- (E)-3-hexen-l-ol (E)-3-hexen-l-ol (Z)-4-methyl-3.5-dioxa- (Z)-5-nonen- (Z)-3-hexen-l-ol (Z)-3-hexen-l-ol (E)-5-nonen-1-ol (E)-5-nonen-1-ol (Z)-l-pheny (Z)-l-pheny 5-nonyn-1-ol 5-nonyn-1-ol 24-02 24-02 :,_sj. :,_sj. v--~ v--~ ------_, PREPARATION OF E-ALKENES . 2401 ------2400 KOVAR.ovA AND STREINZ ------c ~ TABLE l Conl!nm:d TABLE l \ Temp. Rcacti1m Product Yield 1 R1.-cuveredI Reduction by Lithium in l,J-Diaminopropane '2 c. time Starting ' c .s Q ~ Material ·1 u ::e Temp. Reaction Product Yield• Recovered ·c min % % ci. 'i Startin2 I ,... .: time l A -30 4P cyclohexanone 0.4 97.1 =Q 1! Material• so· 97.0 u :; ! VI A 25 2.0 ·c min % % A 60 60 l.3 98.0 21.9 75.3 A -30 60 (E)-THP-0-5-nonen-1-ol 14.0 58.3 B 25 60 I A 25 105 51.3 18.l VII A -30 45 - - 100° 34 40.2 15.l A 60 vm A -30 45 96 ° B 25 21 68.4 b 3.6 - - Method: A - reaction perfonned in a mixture ofTHF/DAP. B - reaction without THF A -30 60 (E)-4-methyl-3.5-dioxa- - 100 II A 25 720 -10-tettadecene 64.0 31.2 • GC data b isolated yield • HPLC data B -30 60 - 100 B 25 47 98.4b - A 25 120 (E)-3-hexen-l-ol 93.1 b - m A 60 90 (E)-3-hexen- l-ol 76.2 - spectra were recorded on. a Brucker IFS-88 infrared spectrophotometer. Mass 1-hexanol 8.0 .. . B 25 95 (E)-3-hexen-l-ol 37.2 - . spectra .were recorded on a VG Anal~-ZAB~EQnws spectrometer by EI 1-hexanol - 53.3 (70 eV) and on a F'JSOnsMD 800 GCJMSSystem (DB 1 column. 30 m.0.25mm B 2S 45 · (E)-3-hexen-l-ol 38.0 - 1-hexanol 62.0 .. i:d;). ~aschromatographic analy~Werecarried out on-a· Hewlett-Packard -- ·- B 60 80 (E)-3-hexen-l-ol 45.6 - . 5890A gas chromatograph with HP-5 column (5% phenylmethylsilicon. 25 m. 1-hexanol 28.9 4-hexyn-1-ol 7.3 0.3 lmm i.d.). HPLC chromatography wu performed on a Hewlett-Packard I090 A -30 60 S-nonyn-1-ol 33.4 1.8 chromatograph equipped with a HP 8~Bcomputer and DAD UV detector with (E)-5-nonen-l-ol 52.Z IV Tessek silica gel SGX 5µ. column. 3x (3mm i.d.. x 150mm). Starting materials 5-nonen-1-yl acetate 3.4 A 25 260 5-nonyn-l-ol 32.4 - not commercially· available were prepared from acetylenic hydrocarbons and (E)- 5-nonen-1-ol - 31.7 alcohols using methods of pheromone synthesis, namely, alkylation of the A 60 80 S-nonyn-1-ol 67.6 - (E)-5-nonen- l-ol 10.9 terminal triple bond. protection of the hydroxyl group and acetylation. The B 25 170 5-nonyn-1-ol 34.4 - (El-5-nonen-1-ol 51.2 standards with corresponding (Z)- ~~ (0.8mmo() in THF (0.36 mil. lichiumwire (ll.8 mmol) cut into ~mallpieces was ti - - :'.;- - -- COPY COPY ...... ...... AVAILABLE AVAILABLE ~--. ~--. ...... --- BEST BEST - I I i i r-- ( ( a a co co by by for for in in ml) ml) of of were were The The The The under under same same using using aqueous aqueous Science Science (1983). (1983). analysis analysis (0.5 (0.5 the the ml). ml). in in STREINZ STREINZ l l 3378 3378 acidified acidified analysed analysed Valierova Valierova removal removal removed removed (1957). (1957). washed washed (0. (0. 2796 2796 reactions reactions llliing llliing 11, 11, TLC TLC wacer wacer alkaline alkaline AND AND then then 891 891 was was 48. 48. remperacure. remperacure. A A Irena Irena were were after after Development Development Program Program the the and and were were and and these these until until 22. 22. in in ethanol ethanol Dr. Dr. the the distilled distilled and and of of AROV AROV 1979). 1979). solvent solvent ( ( by by layers layers water water desired desired lost lost phenol. phenol. 96% 96% gel gel chromatography. chromatography. thank thank similarly similarly 616. 616. in in and and the the KOV KOV of of the the J.Org.Clrem. J.Org.Clrem. 1993) 1993) to to 3737 3737 I.Am.Chem.Soc. I.Am.Chem.Soc. International International NOTES NOTES gas gas being being with with to to products products I.: I.: temperature temperature organic organic J.Org.Clrem. J.Org.Clrem. 1973. 1973. 44. 44. by by silica silica 561. 561. for for supported supported wish wish from from mixture, mixture, the the I.: I.: dissolved dissolved D.M.: D.M.: April April AND AND on on (MgSOJ, (MgSOJ, performed performed reduction reduction Kang Kang :such :such crude crude part part also also 1977. 1977. the the Cl1em. Cl1em. heated heated 14 14 .• .• quenched quenched in in ----- at at ether ether Agency Agency rhe rhe analysed analysed or or were were Syntl&esis Syntl&esis dried dried We We The The Sauve Sauve Org. Org. F.G F.G mareria1s mareria1s Wender Wender from from was was then then J. J. .• .• analyses. analyses. was was U.S. U.S. with with was was M.: M.: from from .• .• G G evaporated. evaporated. THF THF J.; J.; Synthesis Synthesis sea.le. sea.le. and and work work R.A R.A cooled cooled ci.~rlands ci.~rlands organic organic REFERENCES REFERENCES .. .. were were A.; A.; maintained maintained N N GC/MS GC/MS removed removed product product chromatography chromatography Belmonte Belmonte Kang Kang Schroll Schroll This This solution solution without without larger larger mix.rures mix.rures extraction extraction change change Jacobson Jacobson the the a a was was was was acidic acidic ,..,,,. ,..,,,. MS. MS. .• .• Friedel Friedel Cooperation. Cooperation. m1x.ture m1x.ture The The of of -- R.A.. R.A.. R.A.. lithium. lithium. R.A.. R.A.. Carpita Carpita .• .• on on i.., i.., J.D J.D After After any any column column and and all all L L ttie,crude ttie,crude the the • • further further 7 R.. R.. ~tion ~tion IR IR brine. brine. lithium lithium by by our our metal metal no no and and mixture mixture ;.,,.,i ;.,,.,i experiments experiments and and DHR-5600-0--00-1051-00. DHR-5600-0--00-1051-00. though though ...... portion. portion. and and prevenr prevenr the the The The Benkeser Benkeser Benkeser Benkeser Reggel Reggel Benkeser Benkeser Rossi Rossi Technology Technology Warthen Warthen (1955). (1955). added added The The f'l!.., f'l!.., 7. 7. 6. 6. 5. 5. 4. 4. 3. 3. 1. 1. 2. 2. H-NMR. H-NMR. carrying carrying Geant Geant and and 1 Acknowledgement: Acknowledgement: purified purified rates. rates. vacuum vacuum water water was was remaining remaining indicated indicated reacuon reacuon added added 2404 2404 --- -- ~ ~ - - a in the to 1281 its of of of on the was yield. vinyl effec from might many of of and variety same chemo to Crypto both - oxygen, compounds similarly [6] a (£)/(Z) bromide respectively) overall l-10doalkanes Sa and of the 5, to pheromone, (5] synthesis advisable represents that mixture of chemistry quantities formation Cryptophlehia and Republic, 4S'V,, insect ally! smglet from found and could are compound be the 4 lead ca the of analogues 96:4 the Synthesis Brofova, the is Note 2; lead - actual [7]. a Czech moth to and C(8) 1111/Ji!a!is This action on vinyl-branched towards Eva in the might the the derived at component l]. of recently it carbanions 5"!.i) ratio. [ of to The is Sche111e compound of herein within affords would 1 reaction bonds 4 based (99 codling 1: (see Strcinz, leading pheromones potentially w-bromoalkanols was (1994) - bond 6 Ostrinia a lures pheromone the mode that 77 C=C L1BH Sciences 2) described form in former of false sulfonyl by Further, of Analogues Ludvik isomers Detailed Praha Vol. synthesis C=C The would with Vrkoc pure pheromone compound Approach r4J protected - the the generally attraction. far. ally! the (a relationships - of .Jan Lazar, Scheme synthetic that with An tests. synthesis photo-oxygenation acetates so highly of of Academy haloalkanes, was lepidopteran latter ACTA tnsuhstituted procedure and (I) to by CZ--16610 in prepared 2 While msect 11.94) (8a; 2, the or and •,Josef the vinyl-branched acetate with Pheromone (14 Sb. other and general dcsulfonylation sulfone strategy component components. to - rn\m mixture attraction to 1 1sol«lcd [3]), position a the commercial the of Kalinova, ethyhdenc-hranchcd Sulfones: mono- behavioral a CHIMICA Koutck the and acetate generation Biochemistry, in determined of the females for Sa and from reactions the Bianka l-yl conveniently access structure-activity Ally! prop-2-cnyl mole.It been (Z)-dodec-8-enyl synthetic and Schemes c'pond111g Flcmrngovo Bohumir Pd-catalyLed according convcn1cntly virgin the the a pheromone pheromone was analogues was react1v1ty extensive sought their Ill impurity not con he and epar- I (12) new to 111 by 1cmovcd phenyl HELVETICA (a Cydia a Chcnmtry for an Vinyl-Branched into We and can of pheromone-mediated step that and as have lloskovcc, actual easily compounds and (£)/(Z)-dodec-8-enyl and .. (I) the of(£)- key followed arc pheromone-mediated of Sulfone [2] difference Organic GC the acetate nf alcohols illustrated [2] insight I) of Mkh:tl alkylat1on required in The the ofC-atoms examined 7-propylnon-8-cn- large reaction by appeared henzencsulfinatc vinyl-branched analogues. Lcpidoptera) prep. sulfone as threshold ( we blend the bonds, it analog - tlm scale dcnvalivcs protected Sd1e111c a by the lnsl1lulc further two-step Sa strategy to !eucotreta 1s if 116. disrupt inhibitors sec number pests). vinyl-branched The phenyl sodium Racemic 'Sulfones. C-C as inert Since gram 3, ulili11ng Our different ~ By ( hranchcd, clhyhdene are tively !e11cotret11 isolated total which inhibitory analog know provide act reception, of the two branched - tetradec-11-enyl other phle/Jia ally! new ally! with - ' ::) '.\ ~·1 ,, :•1 '~ f.1 -~ «.\~;.;~ • ,: •• , .•. , .... COPY ', '······.· ,'• 1 :,I .. 1' .' •,: '" I'" \l : AVAflASLE •• ~vr·,;':~ ';',, BES1 ,.1 :~· .. ,' ' l•":,. '• ··,·it\~~, ·' '• ' 1.,;'' ,' ,, ;:~ . '::; ' ·: ~ ~' is is 0° 0° to to of of to to of of In In be be and and the the the the the the sul (ca. (ca. (H+ (H+ and and and and and and • • that that pure pure at at of of in in photo known known H- scope scope step. step. possible possible 1 made made [17] [17] material material allyl allyl could could yield yield Science Science is is attractive attractive a a i.e. i.e. makes makes its its 1n 1n Dowex Dowex of of was was synthesis synthesis Subsequent Subsequent olefins, olefins, It It by by yields yields was was b. b. provide provide Pd-catalyzed Pd-catalyzed MeOH MeOH and and the the sensitizer sensitizer - isomerized isomerized high high ethylidene the a a the the known known Ac,O/Py Ac,O/Py Sa, Sa, loss loss with with [16], [16], of of as as of of dry dry in in (syn-addition). (syn-addition). is is Program Program method method suggests suggests of of overall overall J J it it intact intact research, research, in in isomers. isomers. attempt attempt easily easily The The One One side side the the respectively. respectively. identified identified as as disubstituted disubstituted = = 6 } = = 9 (GC) (GC) 96:4, 96:4, 4a/Sa This This = = 6 } includes includes = = 9 The The - desulfonylation generally generally Bengal Bengal n n n n No No respectively. respectively. are are n n n n non-terminal non-terminal )nOY )nOY alternatively alternatively Sb, Sb, 2 the the reduction reduction isomers isomers GC). GC). Ac, Ac, H, H, H, H, and and the the %, %, bin bin = = =Ac, =Ac, = = = = from from of of deprotection deprotection t-BuOK t-BuOK and and Rose Rose Y Y Y Y remained remained Y Y Y Y in in and and may may 4b/Sb. 4b/Sb. 45 45 crowded crowded and and ethylidene ethylidene intermediates intermediates Sa, Sa, cap. cap. pheromone pheromone Et, Et, Pr, Pr, (1994) (1994) Et, Et, Pr, Pr, desulfonylation/isomerization desulfonylation/isomerization 5-10%. 5-10%. t-BuOK. t-BuOK. 4a 4a Sa Sa and and R"((CH sulfones sulfones 77 77 conversion conversion anticipated, anticipated, unnecessary. unnecessary. R = = R by by they they form) form) R R = and and of of from from more more pure pure b b = R obtained obtained b b = R D<'1•e/opme111 D<'1•e/opme111 with with 9a 9a Vol. Vol. 10a 10a terminal terminal 42 42 some some from from was was DHR-5600-G-00-1051-00, DHR-5600-G-00-1051-00, - transformation transformation (H+ (H+ total total that that isomers isomers the the double-bond double-bond 99.5 99.5 tandem tandem Subsequent Subsequent of of to to (HEil) (HEil) was was oxidation oxidation is is NaHg, NaHg, phenyl phenyl No. No. photo-oxygenation; photo-oxygenation; > > on on ). ). isomers isomers the the are are sulfones the the N-solubilized N-solubilized ( ( via via were were amount amount ACTA ACTA 3 3 4 transformation transformation similarly similarly to to 7a 7a compounds compounds by by produce produce under under b, b, to to alcohol alcohol Do-...ex Do-...ex grant grant derived derived 6b 6b b b Dowex Dowex /11tematio11al /11tematio11al Bu Scheme3 Scheme3 connected connected to to ally! ally! two-step two-step amalgam/t-BuOK amalgam/t-BuOK by by mixture mixture form form 6a 6a olefins olefins for for diol diol and and 3a, 3a, sulfones sulfones 10a, 10a, CHIMICA CHIMICA a a target target and and amounted amounted with with area. area. catalytic catalytic part, part, (Scheme (Scheme to to of of using using The The Sa,b Sa,b mixture mixture to to obtained obtained Sa, Sa, ethylidene ethylidene tertiary tertiary a a formation formation 6a 6a b b 1n 1n pure pure ally! ally! the the simultaneous simultaneous % % Agency Agency interesting interesting sodium sodium this this only only the the primarily primarily conversion conversion alcohol alcohol 9a, 9a, 99 99 this this and and of of reaction reaction /J,y-unsaturated /J,y-unsaturated to to with with is is US. US. HELVETICA HELVETICA from from 6b/7b 6b/7b also also of of 1·ia 1·ia leading leading 1-BuOK 1-BuOK highly highly -disubstituted -disubstituted using using the the from from product product exposure exposure (TBAH), (TBAH), alkanols. alkanols. ---- t.risubstituted t.risubstituted alcohols alcohols supported, supported, desulfonylation desulfonylation only only vinyl vinyl 4 4 clean clean in in led led deprotection deprotection 6b 6b yielded o:,o: o:,o: one-pot one-pot a a from from procedure procedure of of method method NaHgxi NaHgxi the the mixture mixture derived derived when when with with exceeding exceeding latter latter by by In In the the -disubstituted -disubstituted approach approach and and derivatives derivatives N)BH in in Indeed, Indeed, the the this this 4 formation formation the the financially financially the the o:,o: o:,o: 3a,b 3a,b 6a 6a - this this isolated isolated vinyl-branched vinyl-branched react react synthetically synthetically restricted restricted 3. 3. smooth smooth by by of of (Bu purity purity was was of of a a the the The The to to with with isomers isomers acetylation acetylation alcohol alcohol regioselective regioselective a a o:-monoalkylated o:-monoalkylated solvent. solvent. from from the the desulfonylation desulfonylation Cooperation, Cooperation, of of obtained obtained separation separation in in vinyl-branched vinyl-branched of of consists consists vinyl-branched vinyl-branched of of work work as as 7b 7b and and means means and and conclusion, conclusion, 3 3 3 3 that that - sulfones removal removal prefers prefers In In Another, Another, This This Although Although no no % ) ) % 2 2 COPY COPY 0 1 "C-NMR. "C-NMR. oxygenated oxygenated branched branched resulted resulted presence presence associated associated fones fones CHC1 85 85 isolate isolate form) form) the the racemic racemic conveniently conveniently contrast, contrast, [18] [18] mixture, mixture, acetylation acetylation desired desired o:/J-unsaturated o:/J-unsaturated by by the the reductive reductive control control features features difficult difficult ethylidene ethylidene Technology Technology - AVAILABLE AVAILABLE ., ., BEST BEST 9 9 6 6 = = = = n n n n - H, H, H, H, 9 9 , , )pY )pY = = = = 4 2 = = = = 6 is is to to to to as as of of Y Y Y Y n n n n the the ob em best best way way Et. Et. Pr, Pr, 57% 57% -60° -60° most most agent agent amal or or inn inn group group = = = = rr-allyl rr-allyl Li8H OH OH amines amines - we we alcohol alcohol is is R R treating treating at at the the between between , , this this ca. ca. action action a a o: o: were were 4 available available 4 4 b b R alkylated alkylated Ph Ph Rt(CH 4 4 7a 7a Ph Ph 2 b b ethyhdene a a by by by by the the attempts attempts EtOCH(Me), EtOCH(Me), EtOCH(Me), EtOCH(Me), l'la l'la the the [15] [15] bin bin 0 0 are are 2 b b S0 = = generation generation complexes complexes = = even even + + HP0 hydride hydride 2a, 2a, Ac Py Py Y Y Y Y bond), bond), at at 2 and and Na8H dcsulfonylation dcsulfonylation 3a, 3a, R-t(CH2)nOY R-t(CH2)nOY with with a a (6-bromcihexan- olefins olefins aliphatic aliphatic 3a, 3a, Pd Pd ]/LiBH alkylating alkylating the the Et, Et, Pr, Pr, 2 alumrnium alumrnium exist exist 14], 14], J J Na ) Thus, Thus, [ [ 1 = = hands, hands, in in 9 9 6} 6} by by -- 9 9 6} 6} R = = R for for as as = = = = C=C C=C phcromonal phcromonal either either )nOY )nOY proceed proceed reagents reagents = = = = vinyl- 2 Li Li b b R n n n n 3a 3a n n n n agents: agents: our our of of of of synthesized synthesized of of Ac, Ac, H, H, sulfones sulfones should should alcohols, alcohols, sulfones sulfones terminal terminal such such TMEDA TMEDA = = ""H, ""H, =Ac, =Ac, = = derivatives derivatives """ """ In In may may occur occur Y Y Y Y Y Y Y Y low-valent low-valent po~ition po~ition proccs~cs proccs~cs conjugation conjugation method, method, R"((CH Pr, Pr, Et, Et, El, El, appropriate appropriate THF, THF, anhydrous anhydrous - mode mode [PdCl,(PPh I I = = of of = = separation. separation. "'""Pr, "'""Pr, bromoalcohol bromoalcohol lie lie variety variety [13]. [13]. by by catalyst~. catalyst~. may may A= A= R R mixturc mixturc (1994) (1994) oxygen oxygen this this an an (reducing (reducing the the b b R b b R RI RI Bul1 Bul1 generally generally Sa Sa 6a 6a reagents reagents 77 77 ally ally with with Pd Pd reaction reaction 2 2 (trisubstituted (trisubstituted 1 1 [12] [12] by by as as TBAB TBAB d1alkylatcd d1alkylatcd their their large large dialkylatcd dialkylatcd 96:4 96:4 homoallyhc homoallyhc S S ability ability an an 9 9 6 6 or or hv, hv, 9 9 6 6 Vol Vol a a purposes. purposes. with with , , = = ~ ~ = = = = 2 - - singlet singlet the the were were transfer transfer Dowex(HO!) Dowex(HO!) the the This This at at protected protected n n n n n n n n I I Ni Ni 10 monoalkylatcd monoalkylatcd [PdCl,(PPh,)J), [PdCl,(PPh,)J), bond bond <;tabihzcd <;tabihzcd 1 1 )nOY )nOY 3 3 and and a a our our 2 2 2 examined examined combination combination -tosyl -tosyl stcrcosclccllvc stcrcosclccllvc buffered buffered ACTA ACTA or or 12]. 12]. achieve achieve group, group, fl fl THF/N,N,N',N'-tetramethylethylenediamine THF/N,N,N',N'-tetramethylethylenediamine [ [ from from for for However. However. However, However, 2 2 with with or or group group excellent excellent with with or or )pY )pY (4%) (4%) s,fw111<· s,fw111<· Scheme:! Scheme:! reaction reaction C=C C=C to to 2 hydride hydride towards towards The The either either for for produce produce 2 2 in in EIOCH(Me). EIOCH(Me). EtOCH(Me), EtOCH(Me), bond) bond) unscparablc unscparablc 2). 2). EtOCH(Me), EtOCH(Me), EtOCH(Me), EtOCH(Me), = = ~ ~ [11] [11] obtained obtained to to = = = = by by Y Y Y Y sulfones sulfones the the CHIMICA CHIMICA The The the the electron-transfer electron-transfer PhS0 an an Y Y else else Y Y I I okfins okfins Pr, Pr, bond bond b b conditions conditions PhS0 or or PhS02'((CH the the mixture. mixture. C=C C=C =El, =El, = = reacted reacted BuLi BuLi 2a 2a or or R'((CH reagents reagents alcohol alcohol the the sufficient sufficient R R [Pd(PPh,),j [Pd(PPh,),j [IO]. [IO]. ally! ally! b b R other other the the reactivity reactivity of of (Scheme (Scheme in in Sa Sa ucnce, ucnce, of of and and first first selectivity. selectivity. not not C=C C=C 5 5 HELVETICA HELVETICA + + in in rcgioselective rcgioselective of of followed followed 9 9 6 6 terminal terminal seq seq affording affording groups groups reaction reaction = = = = documented documented the the was was was was TMEDA TMEDA n n n n and and 1-iodoethane 1-iodoethane 0°, 0°, Grignard Grignard either either I I migration migration ). ). 96"/i, 96"/i, catalyst: catalyst: con con among among alcohols alcohols 4 4 equiv. equiv. 1 1 the the Mc Mc removal removal 'Yo 'Yo ; ; one, one, pure pure regioisomcr regioisomcr a a or or highly highly to to 4 photo-oxygenation photo-oxygenation well well in in (96%) (96%) /THF, /THF, 60 60 a a to to 96 96 any any on on --- -dialkylated -dialkylated as as I.I I.I having having difference difference on on THF, THF, best best NH with with EtOCH(Me), EtOCH(Me), EtOCH(Me), EtOCH(Me), of of Br(CH2)nOY Br(CH2)nOY Bul1 Bul1 2 Mg Mg ca. ca. -60 -60 also also ' ' (monosubstituted (monosubstituted = = o:,o: o:,o: ~ ~ 2 2 1 1 Y Y Y Y displacement displacement and, and, the the amalgam amalgam 4 4 is is or or singlet singlet Ry(CH2)nOY Ry(CH2)nOY Typically, Typically, at at reductive reductive large large with with respect respect Pr, Pr, employed, employed, yielding yielding the the HC0 based based be be only only compounds compounds =Et, =Et, without without = = a a the the Ph Ph olefins, olefins, purity purity I). I). I I 2 Na Na R R isomerically isomerically - the the the the to to aqueous aqueous 9-bromonomrn-1-o[) 9-bromonomrn-1-o[) in in Depending Depending b b R 4a 4a THF THF (with (with 1-iodopropane 1-iodopropane 111 111 or or Sodium Sodium and and For For Since Since Thus, Thus, disubsututcd disubsututcd L1BH4, L1BH4, LiBHEt,, LiBHEt,, 1-ol 1-ol tained tained terminal terminal facilitate facilitate 1282 1282 nucleophilc nucleophilc proved proved failed. failed. intermediate intermediate ployed ployed occurred occurred or or prepare prepare )'-position )'-position hranched hranched frequently frequently isomeric isomeric [9]. [9]. generally generally [8]. [8]. compounds compounds gam gam yield yield (TMEDA) (TMEDA) (Scheme (Scheme with with sulfone sulfone [PdCl2(Ph3PhJ [PdCl2(Ph3PhJ ~S0 ~- - - ~ ~ ~ ~ i:: i:: g g ~ ~ ~ ~ U'l U'l 0 0 ] ] ca ca "O "O :c :c £' £' .. .. Cu' Cu' 0,. 0,. 0 0 ..::::-- .. .. ...... 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(74%). 2 2 (50 (50 H H of of Acetate Acetate washed washed H H for for (424.6): (424.6): at499.5 at499.5 EtP EtP 2 reaction reaction After After ml). ml). by by (internal (internal mg, mg, mg, mg, g g bubbling bubbling 0 0 amalgam amalgam of of extracted extracted 3; 3; 96 96 9.1, 9.1, C1 above, above, S S 2 Et 2 2 of of 4 added added for for cooled cooled purified purified mixed mixed mmol) mmol) 0.25 0.25 5.04 5.04 8.9. 8.9. was was evaporated. evaporated. with with H H the the 13 13 (25.2 (25.2 Et a a 100 100 78.2, 78.2, °!f,) °!f,) 79.2, 79.2, for for -60° -60° photoreactor photoreactor ml, ml, S S 41.3 41.3 and and (500 (500 (510 (510 g, g, 22 22 MPLC' MPLC' 1 1 of0 C C MeOH MeOH TMEDA TMEDA H H x x to to was was C C 4 4 with with mg, mg, amount amount TMEDA TMEDA at at H400 38 38 - (82 (82 chromatography chromatography 6a 6a isolated isolated 66.3, 66.3, 8.3, 8.3, in in 24 (3 (3 &odium &odium ml, ml, operating operating portions. portions. 3): 3): calc calc residue residue residue residue stirring stirring Then, Then, stirred stirred extract extract C ml), ml), (2.00 (2.00 C C flow flow McOH McOH gradient gradient 3 3 described described mg mg and and H H prep. prep. 0 0 from from (175 (175 79.2, 79.2, LiBH 2 8 8 8 mmol): mmol): added added was was the the the the in in mmol) mmol) r.t. r.t. as as combined combined C C for for (184 (184 h] h] by by ice-water ice-water Et liquid liquid and and washed washed (212.4): (212.4): and and 758 758 3 Anal. Anal. (240 (240 After After a!cohol a!cohol 2a 2a hexanes; hexanes; of3a of3a 0 0 138 138 90 90 64.2, 64.2, mmol), mmol), minnnum minnnum column column capillary and and quartz-glass quartz-glass and and found: found: RO RO 24 were were mmol) mmol) The The off off hat hat in in ml) ml) dissolved dissolved 2 C C 7a. 7a. a a into into g, g, g, g, and and calc. calc. with with stepwise stepwise constant constant H ml). ml). mmol), mmol), (PPh added added - 8 8 H combined combined 20 20 2 THF THF 3.5 3.5 a a 111 111 7-Pmp,J'/n1111-7-en-l-yl 7-Pmp,J'/n1111-7-en-l-yl 12 soln. soln. hexanes; hexanes; manner manner 8.1; 8.1; 14 (50 (50 mg) mg) ml). ml). spectrometer, spectrometer, purified purified was was 25-m 25-m g, g, MPLC: MPLC: (70 (70 (212.21): (212.21): S S 4.7 4.7 13.3. 13.3. for for g, g, C in in analogously analogously was was (2.5M (2.5M (40.8 (40.8 (23.0 (23.0 with with follows: follows: dry dry 8.0. 8.0. The The continued continued stirring 50 50 Anal. Anal. filtered filtered 4 4 with with found: found: 0 0 oil oil H H g, g, [PdC1 poured poured forC filtered filtered S S (Sb), (Sb), (150 (150 same same of of x x as as for for 28 9.2, 9.2, 0 0 (7.14 (7.14 (-60°) (-60°) (1.5 (1.5 prep. prep. evaporated evaporated of of extracted extracted ml). ml). evaporating, evaporating, H BuLi BuLi evaporated, evaporated, and and MeOH MeOH (2.5M (2.5M was was commercial commercial (3 (3 H H 9.0; 9.0; residue residue (2 (2 dissolved dissolved 9.3, 9.3, stirred stirred the the MeOl-1 MeOl-1 14 78.0, 78.0, calc calc of3b. of3b. by by N)BH was was system system medium-pressure medium-pressure were were 3b 3b S S 80 80 a a in in 0 0 detector; detector; soln. soln. calc. calc. 4 Unity-500 Unity-500 C and and yellow yellow 2 g) g) H H Ar, Ar, C C and and of2a of2a In In 3b 3b and and the the soln soln x x mixture mixture ), ), dry dry in in 66.6, 66.6, BuLi BuLi was was Et 3 Acetate Acetate cooled cooled (l (l 1-BuOK 1-BuOK irradiated irradiated ), ), (Bu 8.5, 8.5, added added synthesized synthesized A A for for (3 (3 3 solvent solvent 111 111 FID FID Anal. Anal. C C ml) ml) The The from from Prep. Prep. the the solids solids and and Anal. Anal. (76%) (76%) 66.8, 66.8, soln. soln. H H (3b). (3b). of of C0 Kl Kl 0 0 from from stirred stirred under under Varian Varian 2 and and 2 and and with with and and C0 g g the the exchanger exchanger found: found: (3a). (3a). was was C C was was a a 2 lamp lamp Sa. Sa. (7a). (7a). calc calc separntcd separntcd the the dissolved dissolved Part Part Et and and procedure procedure (500 (500 (K g) g) of9ll of9ll and and mixture mixture (K ml) ml) 6.5 6.5 of of 64.4, 64.4, mmol) mmol) ion ion 1-3. 1-3. To To off, off, 0 0 0° 0° l-c11-l-yl l-c11-l-yl Hg Hg (396.6): (396.6): (-60°) (-60°) 22 22 13.1; 13.1; 2 g g (2b) (2b) mmol) mmol) (80'\'<>). (80'\'<>). was was C C mmol) mmol) solns.; solns.; 22 22 stirred stirred min, min, with with ml) ml) S S to to H Anal. Anal. mmol) mmol) dried dried 3 found: found: H H 4 The The The The (250 (250 a a residue residue 1.5 1.5 90 90 g, g, (2a). (2a). mg mg typical typical extracted extracted 0 3 3 evaporated evaporated (85%) (85%) 30 30 (5.8 (5.8 analogously analogously 22 22 h. h. (50 (50 drying drying Tables Tables to to 36 g, g, g g filtered filtered cross-linked). cross-linked). mmol)· mmol)· analogously analogously g g A A 8.1; 8.1; cooled cooled the the ml). ml). (2.5'Yo, (2.5'Yo, g, g, H 0 0 78.2, 78.2, 24 24 in in Biichi-B-680-Prep-LC Biichi-B-680-Prep-LC CDCl 907 907 chromatograph, chromatograph, and and raised raised brine, brine, 2 and and S S (354.5): (354.5): mixture mixture (2.64 (2.64 22 22 22 Ar, Ar, C C CHCI 16.4 16.4 ice-cold ice-cold 4 4 After After was was ), ), Experimental Experimental 0.87 0.87 S S 1.42 1.42 Et and and C extracted extracted Ob). Ob). (25 (25 1 and and for for 4 (87.5%). (87.5%). g, g, (3.7 (3.7 brine, brine, ml) ml) in in was was and and 9.2, 9.2, (I (I data data 0 7.4. 7.4. P0 (I; (I; Th.is Th.is high-prcssu1e high-prcssu1e g) g) l-bromo-6-(1-ethoxyethoxy)hexane l-bromo-6-(1-ethoxyethoxy)hexane l-bromo-9-(\-ethoxyethoxy)nonane l-bromo-9-(\-ethoxyethoxy)nonane 2 mm); mm); for for Nl. Nl. 00 00 30 mg mg S S and and H H lrl-Dhy/dodec-l lrl-Dhy/dodec-l with with 4 gave gave 12.2. 12.2. Prl Prl (3.4 (3.4 mmol): mmol): under under evaporated: evaporated: ( l l ( H Spectra: Spectra: 184.3): 184.3): h, h, THF THF amalgam amalgam w1th w1th 1.5 1.5 synthesized synthesized HP HP 6, 6, stirred stirred ( ( Bu synthesized synthesized (K,C0 19 h, h, H H l l and and temp. temp. ml) ml) NaH Bengal Bengal 9.4, 9.4, 650 650 a a calc calc TMS; TMS; Et! Et! 1 1 0 0 C and and 66 66 exchanger exchanger 0.25 0.25 dry dry Al'<'tatc Al'<'tatc sulfone sulfone (Sa), (Sa), 150-W 150-W methylsilicone, methylsilicone, HzO HzO 24 H H to to 7-Propy/non-8-ene-1,7-diol 7-Propy/non-8-ene-1,7-diol wa' wa' to to with with wa' wa' irradiation, irradiation, evaporated evaporated C C MPLC MPLC evaporated evaporated The The form; form; H for for in in 72.1, 72.1, (250 (250 dried dried anh. anh. ton ton H-NMR)-. H-NMR)-. Ro.1e Ro.1e mg): mg): After After --ETIC~AA~l.77- with with mg, mg, 12 1 + + Sodium Sodium Anal. Anal. of of quenched quenched Ar, Ar, C-NMR C-NMR h. h. rel. rel. C C washing washing and and After After dropwise, dropwise, 0 0 mmol), mmol), CO_i). CO_i). C and and (9b) (9b) 2 (6h) (6h) 13 68.l, 68.l, wa' wa' 3 3 (0.040-0.063 (0.040-0.063 2 (H (H ) ) and and prep. prep. 3 3 calc. calc. H The The phenyl phenyl 4 precipitate precipitate Hanau Hanau C C (100 (100 mm mm and and Acl'tatc Acl'tatc was was ice-cold ice-cold (175 (175 washed washed brine, brine, for for 145 145 (K (6a) (6a) W W ml). ml). -60°. -60°. for for mmol) mmol) (396.6): (396.6): ppm ppm h. h. gel gel by by of3a. of3a. a a by by under under to to After After )-3-(phenylsu/fonyl)dodec-1-ene )-3-(phenylsu/fonyl)dodec-1-ene 45 45 h] h] added added S S 50 50 found: found: prop-2-enyl prop-2-enyl at at in in 3 )-3-(phenylsulfonyl)non-l-ene )-3-(phenylsulfonyl)non-l-ene 4 24 24 ml, ml, was was CHC1 (70 (70 alkylated alkylated H-and H-and and and with with Anal. Anal. calc. calc. mmol) mmol) 1 fJ fJ 0 25.0 25.0 dried dried (GLC (GLC l-en-1-o/ l-en-1-o/ (MgS0 -60° -60° found: found: silica silica Hewlett-Packard-HP-5880A Hewlett-Packard-HP-5880A ml). ml). and and 58 58 36 was was for for - min), using using mm mm mixture mixture the the (79%) (79%) N-solubilized N-solubilized (PPh 12.1; 12.1; 1-BuOK 1-BuOK HP5-5% HP5-5% residue residue was was at at %). %). H 2 4 13.2. 13.2. 13.0. 13.0. H,O H,O g) g) 60 60 g g 5.57 5.57 stirring stirring 15, 15, THF THF mg, mg, phenyl phenyl Down Down 22 15 15 7.6, 7.6, shifts shifts (15 (15 Anal. Anal. l l ml) ml) H H 150 150 96:4 96:4 extract extract ether. ether. Bu dried dried H H H H g, g, (73 (73 brine, brine, C S S 0, 0, and and 6.3 6.3 x x GLC: GLC: (2.5M; (2.5M; 0 0 soln. soln. mm, mm, g g dry dry and and of of quenched quenched 2 with with decomposed decomposed 6a: 6a: oflhe oflhe (10 (10 [PdC1 with with (540 (540 with with for for over over At At mmol) mmol) (3 (3 was was 10-Ethl'idoda-/()-en-l-rl 10-Ethl'idoda-/()-en-l-rl g), g), (2.2 (2.2 72.0, 72.0, 13.5. 13.5. reaction reaction and and in in Et 4 4 Merck Merck 9.5, 9.5, 4a/5a 4a/5a 79.0, 79.0, form; form; 78.3. 78.3. 0.3 0.3 the the J-Ethoxyethoxy)-3-ethyl-3-(pheny/sulfony/}dodec-J-ene J-Ethoxyethoxy)-3-ethyl-3-(pheny/sulfony/}dodec-J-ene ), ), 1-Ethoxyethoxy 1-Ethoxyethoxy 23.3 23.3 C C 0 0 was was was was 20 20 15 15 lion lion 0 0 and and 1-Ethoxyethoxy)-3-(pheny/.1·u(fonyl)-3-propy/non-3-ene 1-Ethoxyethoxy)-3-(pheny/.1·u(fonyl)-3-propy/non-3-ene 1-Ethoxyethoxy 1-Ethoxyethoxy H H BuLi BuLi continued continued H H 2 C C C C soln. soln. of of irradiation, irradiation, 2 chemical chemical dropwise dropwise and and THF THF the the of7a. of7a. calc. calc. MPLC: MPLC: layer layer g, g, added added of3a of3a (H" (H" Et H mmol), mmol), 10-Ethyldodec-I 10-Ethyldodec-I petroleum petroleum IO-Ethyldodcc-IO-c11-/-o/ IO-Ethyldodcc-IO-c11-/-o/ 7-Pmpybwn-7-en-l-o/(9a) 7-Pmpybwn-7-en-l-o/(9a) 12-( 12-( 12-( 12-( 7-P10pr/11011-8-e11-/-y/ 7-P10pr/11011-8-e11-/-y/ 7-Propy/non-8-en-1-o/ 7-Propy/non-8-en-1-o/ 9-( 9-( General. General. LiBH 9-( 9-( of of washed washed ilea ilea added added treated treated 0 0 mmol) mmol) mg mg 5%; 5%; W W dry dry 2 79.4, 79.4, 67.9, 67.9, 90 90 ~tock ~tock found: found: mmol), mmol), Et 32 32 Pun Pun found: found: and and was was Anal. Anal. mmol), mmol), added added (90%) (90%) (IOa). (IOa). and and in in MPLC: MPLC: C C were were (200.3): (200.3): prep. prep. min min (2 (2 with with (7.93 (7.93 through through mixture mixture MHz; MHz; 20 20 a a mixture mixture 50 50 light light (Normag (Normag soln soln diameter diameter (MPLC): (MPLC): with with was was C C g, g, stirring stirring combined combined Then, Then, ~ ~ ~84- 2 2. 0, 0 for (d) ref. 2 and mg) (q) 26 (q) New (q), 1287 5771 Et H and found: - and (25 31. 14 calc. . 1 31, C 117.48 12.88 13.00 21.03 with Press, 33, 36, 707 11.9; ); Morgan for p. Anal. (.1 (t); (q) (d); (s) (q); (I) (I) (t) (I) (I) (I) (1) 1990, H Tables D. 19S2, 19S7, Ob: calc. 1989, see E. I B Le//. 13.16 12.85 extracted 75.5, 28.61 29.82 36.67 29.73 29.51 29.62 29.26 64.69 29.49.(1) 28.13(1) Academic 142.13 116.92 171.28 IOb C Leu. 0, 2 Ser. Eds. Anal. 11.4. York, 123. H H (q) Sa. (q) Chem. Murray, New numbering, (254.4): Scand., 1977, -- Tetrahedron 2 74.1, W. I 11.62 21.02 ice-cold 0 GLC) 4-(dimethylamino)pyridine For C R. 30 by H (d) (1) (d) (s); (1) (I) (1) (1) (t) (t) (I) (I); 4541. into Kotake, Chem. Ob. 451. and 16 'Pheromones', I and 1994) Trans. C ( H. Publishers, 34, 717. 723. found: Hofmeyr, Acta IV, 29.50 29.24 27116(1) 64.66 45.79 27.67 34.63 29.73 29.52(1) 28.60 25.89 and 2191. 77 19S5, 143.37 171.25 113.99 •• for Sb H. (99.5% poured mmol), 224, 1331. VCH Perkin J. Sb, 11.6; 1993, Vol. Vol. 23 Lei/. calc. H - 1977,3, then pure Norin, Wasserman IOa, g, Kinoshita, 1969, Soc., Leu. (d) 19S2, of Spies, 19S5,41, T. (q) (q) (1) (1); (q); (t) was 4598. H. 74.3, Sa, Ecol. 35-94. . Anal. ACTA Chem. 91 (2.04 H. H. C S.C. 57, Lett. yield pp. Chem. 118.44 13.17 11.8 Sb· Pesticides', 13 20.99 21.28 25.88 28.08 H. 4): 2469. Eds. H 107. H. Chem. (London) mixture REFERENCES 1992, (.1); (q) (s) (d); (1) (q); (I); (I); (/), (I); (I) (I) (1) W1jekoon, 1988, 11.6. Kotake, Chem. Tetrahedron CHJMICA 42, Transformations', quant. Tetrahedron pyridme (226 61, 15 12 28 The D. 2 H 75.3, Yamamoto, H. Natural a 0 2019. 13.16 14 o,{Compounds 39 29.31 25.83 21 -- 28.03 36.83 64 64 29.50 C Nature Pak, T. 140.16 171.24 Chem. 118.36 JO 26 19S6, Oxygen', of 198S, Raton, 248. almost H 33, 74.4, mmol), Orga01c Nooyen,J. Mackenroth, 14 C Schuster, Org. Selle, 45, C Kinoshita, [ppm] DeSilva, 13 HELVETICA Ch.S. Jpn found· overnight. M. Inomata, Boca (q) Mohri, (I); J. Umani-Ronchi, R. 1977, g, H. N 'Singlet W.J. for 02 K. W. G.B. Lee, Soc. M. MPLC) A. in 19S9, Sh(fls 11.9: - found: 1.33 Tetrahedron Handbook 21 20.23 E. Press, ( calc. H 6, Coates, Ritter, Roux, (s); (d) (1) (d) (1) (1) (1) (1) (I); (q) (I) (I) Okecha, 11 (prep. Chem. Comeau, Kuhn, refrigerator Wilson, lc M. CRC 'Comprehensive Choi, AczO 'CRC 75.5, 15 S. 65 lgarashi, H Anal. F.J. Tetrahedron A. Yamamoto, 298 Chemical Kmoshita, Stacino, R. Trombini, in C the B. M. to ll1 S. 14 H.J. Bull. 64 25.88 29.35 27.02 37.29 34.90 43.80 28.59 Experientia p. S.L. T. 143.54 171.24 113.86 Sa H. C. 10 E. IOa: . 74.3, J.P. Yee, 0 C left 1979, added Lee, Roelofs, Trost, N. Mandava, Magnus, C-NMR Larock, Baker, Burger, (254.4). 12 Hurst, Persoons, 13 2 4): Mohri, theretn Julia, Tamak1, L. lnomata, Kotake, Savoia, H and B. Goll01ck, H. K. C. Baeckstriim, M. V. D. w.ts 3. ti0 3 B. B. P. M. D. H. R. N. P. K. M. W. C.J. N. Y. K. York, G. J.R. T.C. 226 cit. chromatographed l) H -- -10° 75.7, I] 16 [5] [6] [I] [7] [2] [8] [3] [4] [9] Table [IO] [I [16] [17] [12] [18] Pr-C(3) at [13] AcO and [14] [15] Et-C(3) mmoi) (la; C C C(3) C(9) C(2) C(8) C(7) C(l) C(6) C(l2) C(5) C(4) C(IO) C(l COPY - ' I AVAILABLE ... BEST - :;j 0 ~ ~ ~ i:: lii ~ 'O :0 ~ .~ "@ 0 ;;:-- ,....:. ;s on- ;"; ,,...:.. ... ..::;...::;. C' ' II II II II II ir) ..--.. ..., M '. r - "'! ..., --: ...-. _g. !"") \0 ..., M t-'. ..., M M _§ .--.. ~ w.< :::E '-;j) {;" ..... - II II II II . II 1r) ..., 'tj'" ,-.., .. .. '. r....'. ,..._,...... ; ..., ('! - --: ..--.. -i \0 ..., t-'. M ..., ,,..-._ 'l:;f" :i ~ ..q: 'd)' :::E w.< -ri' • ' II II II II . 0 00 ~ II t t- s rf ""' r....'. "' r-f '° N V) t;: r....'. °' ..., ~ (/) V) .i:i ,.--:., '. r- r'-! - .. - ..., --: ~~ ,-.., _g. \0 ..., r....'. MM ..., M = ~ ,-. ~ 'd? :::E w.< C' • ' •••• 11 II II II . II ~~ Ei 00 00 r-f r....'. 0 00 r....'. ob ~~ ;:: t't 0\ i.ri ..., (/) ,.--:., .i:i ""'""' M ~~~::$ r;~r;r; '° .. o--o - ..., "'! --: ..--.. ~ \0 -t C''1 ~ ..., r....'. ~~~~ lf'"iv)v)v) M ..., ~ MM .--.. ~ ~~~~;g w.< 'd) 9999<.>u.E .D-. ... N ,;:::S $:'.l 0 Cd.-. i:: c.---~ ur-r-r-r- §- O.,V)V)r"")V) ~.._tr 5 ~o-o tn u i:: "' "' E ~egeg~eg ~~~~~ ;:r:: 0 .s t5 S -;O-:o\0-:0-: ,.::::Q'\O\O"IO\ ,,...... ~ ~ ]~~~~ ~ .Sl ..t:l "0 "@ ~ C/) :::E . ~ 0 guuuu ;;--- -- II II II r- l~ r- ~ r- -h~ ...: -h ,,_,, c:i..., b:; ._. b:; c:i..., _, b:; c:i..., II II II II II r- r- lr? tj:' t- ~ r- r--'. ...... ,, .. ';;:;: -~~ ~-""' _ I ~II ..._,. c:i..., ~II c:i..., ._. ~r-: ;;1; c:i..., ~ .::;.., c:i..., ;;1; ._, ~ c:i..., ;;1; -r- 0..., _:; 8 u c £ ~ - u (1994) II ~ ...... V) r--'. 77 -- C"i II II II II II:$ ~ "' ._, V) ..., c---: ~ ""-.,i ~ ...... ,, V) r--'. Vol. . . . <:i- ...; <:i !:; £~ ~ ~r-: I .D 8 l"i ,..; ~ ~ ~ 8 ~r-: ~ - ...:::, r-i g r::: - <'I ~~~~~~ ~· c u ~ u - II II~ II~ II -:. :::::~ 0 0 <'i ...... ,, - 00 c:i ['.:' :::; ..., __:; s .. , ACTA- II ~ .,f...,....; ~-: ~ °' ""' ...t ~ V...... ,, :g,...; 0 r-- ~ ._. .,.; ~N' l - r-'. ~ :£ .._.,,,...... , ,...... , ..-.'-'r-i.:::.r- : "ci ~ - - II II II~~ II II II~ II ~ \0 '-, ,...; ,-.. ,...; oO ~ l"i N ..., ~ C"i ....; "'" r-: '° ....; "" ..., oO c:i '° CHIMICA . i:;;. ...; <:i- ._. I e'° '"ci' ~o ~II :g .,f..., ~ 0 ._.0-.-"<"I'-' '"' :<:::' .q: :ge .,f..., :-ci' .,f...,.,.;..., :g g:: "ci .,.; ~ 8 ~ ..,,- ~ u ::r:: "' II~ II~ \0 '-,....; <:i- i:;;. ._. e...o .,.; r-.: 0C: - '° 0 . II:;::: II~ II II II II \0 -., <"'-! 0 -. ~ 0 ~ 00 oO ..., '-, ..., HELVETICA 0 r-:' 'ti - ...: 0 - (o ...... 1 <:i- i:;;. i..:G'i..;G'i..;(o i..: <:i-~ ~~~~~\Cf v)~v)~.....;~ ._. :'.:: e...o :'.:: .,.; ""~ ~ ~ V) ~ ~'° ""'~ ~ V) .,.; ~ .D ,,., ~ ~II~ -,,; ""'0 ~r-: '!'i --o..: :g i . . I I !:; !:; ~~- ~ ~ ,...... , i' ,...... , ..- ._.'-co-.. ~~oo ~ .._, f ~E 1 . I I !:; !:; E E t ~ t- ~ t"'"l - ,...... , :b - V) E 00 gg i .. I~ •• I~ . . . I I,__. I I,_ E: 0 ~ :§: _§, ::: "' 00 ....; ""' °' ~~ r~ ~~ ,...... , ~_$ :b ~~ -.::t' ~~~~II~ '? - ,...... , ~ i.r) _:; 00 ~ ~ - ._....:::, ;;;;;;~~---: 2 11 £ ---- ~~~~ ~e u - uuuu --- .. .. II~~ II'.:::'.::: II II~~;;;; -~ "° 0':::; ~ ""?_,-... '° '° -EE -~?i ""? ""' '° .. ~'° ~...... ,, ~ M...... ,, '-"O\ "-'O\ ~ --o\ :;!j ..;;; "' . . .. II II II II II~ II~ II II ~ e ""? M co '° '° \0 ""? - G' '° - ?--, \Cl ""? - ""' '° .. .. ~ .. _ ;g ..q: ~-o ::g ~~ "'..., ~ .,f..., ~~ 8 ~ ""'..., ~-o ~ !"")~rt).....,,_:;_:; ~...... ,, '-0\.._,0\ ::): ~rrf~r-1~ ::g ~~~rt!_:;_:; '.;;t M...... ,, ._.O\ "-'O\ ~ M~M"'"":i,...... -iMV)"'"":i ..;;; '-'o\ £ ;:;- u - ~1.:::-: s 00 ::2:. .c °' .c in ""' .c '° "' :;, £' ..... ~ 'O ~ I ,... "' "' "' i:: § i: "' h i:l > i:: '5 "'I "' § 8 c 8 E' ~ "'I~ > t!- --"' £ ~ 1286 ::c 0 )!._ ] ~ 01~ ';:; ~I u 15' ~ ~ £ (/) 5 ~ u ::c: .D @ 00 "' "@ .,,, .c "' ..... w.. ;:: ·a I .c {l d d ""' J ~ >-; ,..; ~ ~ - I JOURNAL OF CHROMATOGRAPHY A ELSEVIER Journal of Chromatography A, 679 (1994) 307-317 Gas chromatographic determination of vapour pressures of pheromone-like compounds II.* Alcohols Bohumfr Kouteka,*, Michal Haskovec\ Pavlina Vrkoeovab, Karel Konecny\ Ladislav Feltl b a Department of Natural Products. Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, CZ-166 10 Prague 6, Czech Republic bDepartment of Analytical Chemistry, Faculty of Natural Sciences, Charles University, Albertov 2030, I CZ-128 40 Prague 2, Czech Republic I Received 21 April 1994 Abstract I The vapour pressw·es of 98 (Z)- and (E)-monounsaturated C10-C18 alcohols were determined using a method based on gas chror,H ographic retention data. This method, by utilizing a non-polar HP-1 capillary column, five experimental ~emr atures. four reference compounds (C 11' Cw C14 and C 16 alkanols) whose polarities approximated that f the test compounds and melting point corrections for compounds that are solids at ambient I temperature, provided vapour pressures that agreed reasonably well with the available literature values. For alkenols belonging to structurally similar subseries, e.g., for w-3, w-5 and w-7 unsaturated derivatives, the vapour pressures may be represented over a range of pressure by simple equations in which the number of carbon atoms is I a parameter. I 1. Introduction used as input to variety of models and applica tions. The saturation properties of pure liquids At present, there is an increasing need for I play a major role in both the understanding of vapour pressures of high-molecular-mass organ fluid phase behaviour and the design and opera ic compounds at ambient temperatures [3,4]. tion of a multitude of industrial processes [1,2]. One of the most important reasons for this is I Such properties are essential not only when the increased public sensitivity to the effect of used directly in calculations, but also when chemicals on health and the environment gen erally. As the vapour pressure of an organic chemical exerts a large influence on its disper i sal in the environment, a knowledge of the only to • Corresponding author. vapour pressures should allow one not I "For Part I, see Ref. [20]. model the fate of organic pollutants [5,6] but 0021-9673/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved I SSDI 0021-9673(94)00521-A . I I I I I 308 B. Koutek et al. I J. Chromatogr. A 679 (1994) 307-317 also to optimize the use of ecologically friendly 2. Experimental I behaviour-modifying chemicals (7]. An impetus for developing more effective design for practi 2.1. Chromatography cal applications of these compounds is the con tinuing value of pheromones for monitoring Samples were analysed on a Hewlett-Packard I insect flight activity and the recent commercial HP 5890 chromatograph equipped with a 3 m x success in controlling several pests by permeat 0.31 mm I.D. fused-silica capillary column ing the air with their sex pheromones [7]. Both (cross-linked 5% methylsilicone, HP-1, film I the release rates and, in the case of blends, the thickness 0.52 µ,m) with split injection and a release ratios of pheromone components from flame ionization detector. The length of the dispensers are governed, for the most part, by column employed (3 m) is a compromise be I the vapour pressures of the compounds. It ap tween the need for acceptable resolution when pears that environmental concerns are weighing working with mixtures and the need to avoid against the use of traditional pesticides and prohibitively long retention times, particularly at expectations are [8] that pheromones will cap lower temperatures. The chromatograph was I ture about 15-40% of the insecticide market operated isothermally with a hydrogen flow-rate '\ within 10 years. Thus, an understanding of the of 5 ml/min at l0°C intervals in the range 50- pheromone evaporative process can aid in the 1600C as specified. Cw C 12 , C 14 and C 16 al I optimization of selectivity conditions and the kanols were used as reference standards. Re minimization of the loss of the biological activi tention times were determined on a Hewlett ty of synthetic pheromone blends. Packard HP 5895A ChemStation. Adjusted re I The vapour pressures of compounds of low tention times were calculated by subtracting the volatility are commonly determined by either retention time of methane from the retention gas saturation (9,10] or effusion (11 J methods. time of the chemical. As recommended (13], Gas chromatography ( GC) is an alternative long retention times of compounds producing I method for measuring vapour pressures (12,13}, unsymmetrical peaks at low temrt· ,.itures were offering advantages in terms of speed, solute not taken at the peak maximri ., but were sample size, purity and stability requirements. estimated at the centre of gravit:· of the peak. I It is based on the use of a non-polar stationary The reproducibility of retention cime measure phase and isothermal conditions such that a ments expressed as the relative standard devia compound's GC retention time is related di tion of at least three measurements for each rectly to its vapour pressure. The GC method compound was 0.03%. I has been used to study polychlorinated bi phenyls and dioxins [14,15}, herbicide esters [12], organophosphorus pesticides (16], tetraor 2.2. Chemicals I ganostannanes [17], linear alkylbenzenes [18] and fatty acid methyl esters (19]. Using this The alcohols were either obtained from the approach, we have obtained [20] some vapour Research Institute for Plant Protection (IPO I pressure data on pheromone-like acetates. DLO) (Wageningen, Netherlands) and used as '-~ In this paper we show that the GC method received or synthesized in our laboratory. In the yields equally good results in determining latter instance, the purity of the chemicals was at equilibrium vapour pressures of more polar least 97% as determined by capillary GC. Con I compounds, ) viz. monounsaturated (C10-C18 densed nomenclature for alcohols is used: the pheromone-like alcohols. The extensive set of 98 letters after the colon indicate the functional compounds studied also allowed the influence of type (OH= alcohol), the number between the I subtle structural differences in chain length and dash and colon indicate the number of carbon the positions of double bonds on vapour atoms in the chain and the letters and numbers I pressures to be revealed. before the dash indicate the configuration and I I I B. Koutek et al. I J. Chromatogr. A 679 (1994) 307-317 309 I position of the double bonds, e.g., Z3-10:0H is J /K · mol and, as a consequence, 6.SF/ R = (Z)-3-decenol. 12.789. Hence this constant was used to convert literature P8 values of 1-hexadecanol and 1- 2.3. Data treatment pentadecanol into PL. -Plkard- a 3 mx The method has been discussed in detail by 2.4. Statistical analysis cmumn Bidleman [13) and Hinckley et al. [21) and will '-11 film therefore be only briefly reviewed here. At a The data were subjected to statistical analyses n and a constant temperature, the vapour pressures of a utilizing the Statgraphics Plus 7.0 software pack I lthe test and of a reference compound (subscripts T age (Manugistic, Rockville, MD, USA). mi be- and R, respectively) are related by the ratio of ;:m hen their latent heats of vaporization: to avoid 3. Results and discussion 6.HT :ullly at ln PT = !1H · In PR + C (1) ip was R The accuracy of the GC method as repre flow-rate where 6.H is the latent heat of vaporization and sented by Eqs. 1 and 2 depends to a large extent ml50- C is a constant. A similar equation has been on two factors: (i) the accuracy of the PR values al- developed for the GC (adjusted) retention times and (ii) the similarity of infinity dilution activity rds. Re- t': coefficients y in the stationary phase between Hllett the test and reference compounds to which Eq. 2 is re- t~) 6.HT) is applied. Strictly, an additional term, -ln(yT/ ln t~ = (1 - t:i..HR In PR - C (2) :ti the ( l'R), should appear (see discussion in Ref. [21]) ·etention on the right-hand side of this equation and only Hence, a plot of ln(t~/t~) versus ln PR should e,1_3}, when l'T - l'R (or at least l'T /yR - constant) can have a slope 1- 6.HT/!1HR and an intercept -C. ro cmg the use of Eq. 1 lead to reasonable results. As Eq. 1 can then be used to determine the vapour res were values of y on a squalane liquid p'· ase have been pressure of the test compound at any tempera ulere found [25) to range from 0.48 tr J.73 for hydro ture if the vapour pressure of the reference 1e eak. carbons and from 17 to 34 :. r alcohols, the compound at that temperature is known. n ure frequently employed reference hydrocarbons Since the GC method gives the subcooled d devia seemed to be disqualified for our purposes. liquid vapour pressure (defined as the liquid fo,each Therefore, we chose to use n-alkanols (i.e., vapour pressure extrapolated below the melting compounds of the same chemical class as the test point) [22], it was necessary to convert the compounds) as the reference standards. When literature-based solid vapour pressures (P ) into 5 literature vapour pressure values were being subcooled-liquid vapour pressures (Pd by using selected for the reference n-alkanols, some judg I the equation developed by Mackay et al. [23): ment was. necessary. We favoured recent static rol the measurements [26] that have been especially n PO- (3) focused on the low vapour pressure field. The u d as literature values of the four reference com y. In the where TM and T are the absolute melting and pounds given in Table 1 in the form of the ls.as at ambient temperatures, respectively, R is the gas Antoine equation are thus from a single report. c9=on constant and !1SF is the entropy of fusion. The In most instances vapour pressures were calcu sed: the usually employed "average" value of !1SF = 56.5 lated from the Antoine constants by interpola m1·onal J /K ·mo! (or the corresponding value 6.SF/ R = tion. In those instances where some extrapola re the 6.79) seems to be too low for alcohols, however. tion was necessary (14:0H, 15:0H and 16:0H), f carbon Based on the value of enthalpy of fusion (6.HF) the temperature range of extrapolation was usu nlbers published [24) for 1-hexadecanol (34.286 kJ I ally less than 40 K. i and mol), !1SF for this compound amounts 106.34 As the choice of the PR is critical for the I I . ..,-, --· _- -- _-.-: I ·I 1· I 310 B. Koutek et al. I J. Chromatogr. A 679 (1994) 307-317 I Table 1 Table 2 Vapour pressures (PR) of the reference n-alkanols Adjusted GC retention times (min) of the n-alkanols Alkana! Constants of the Antoine PR (25°C) Alkana! 80°C 90°C 100°C 110°c 120°c I equation• (Pa) 9:0H 0.773 0.482 0.332 0.237 0.172 A B c lO:OH 1.553 0.926 0.605 0.409 0.283 ll:OH 3.162 1.800 1.123 0.724 0.481 ll:OH 7.094 2105.005 176.145 0.4255 12:0H 6.557 3.541 2.122 1.313 0.837 I 12:0H 6.860 2011.634 162.769 0.1402 14:0H 26.696 13.305 7.385 4.242 2.256 14:0H 6.916 2217.995 165.381 0.01844 15:0H 53.448 25.600 13.698 7.600 4.379 16:0H 5.964 1781.618 120.726 0.00207b 16:0H 110.337 50.162 25.846 13.914 7.757 I a Ref. [26]; log P (kPa) =A - Bl(t + C). b Vapour pressure is for the subcooled liquid; it was calcu 3.1. Validation of the method lated from the original solid vapour pressure (5.4726 · 10-• Pa) using Eq. 3 and m.p. 56°C. I Six n-alkanols with known PL [26] were chro matographed along with the 14:0H reference, and Pac values at 25°C were calculated from the I accuracy of vapour pressures determined by the relative retention data (Table 2) using Eqs. 1 comparative GC method, the literature PR data and 2. In Fig. 2, these Pac values are compared were checked for internal consistency. Examina with PL' As can be seen, the regression line tion of the logarithm of vapour pressure (cor obtained closely parallels the y = x line. The I rected for melting point) as a function of the equation of the regression line by a linear least number of carbon atoms in alkanol series squares fit is (Fig. 1) confirms that an excellent linear corre ln [PL (Pa)]= (1.02536 ± 0.0049) In Pac ( 4) I lation, ln P = (-1.0675 ± 0.0058)nc + (10.8973 ± 0.0734) (n = 7, S.E. = 0.0374, r2 = 0.9999), does (n = 6, S.E. = 0.0402, r 2 = 0.9999) I exist. I 0 0 -1 -1 I -2 -2 a.~ ,; -3 I -4 -4 -5 .. -5 -6 D I -6 -7 ;l -7 -7 -6 -5 -4 -3 -2 -1 0 2 -1 -8+-~--.-~~~~~~~~~~~--.-~--; 9 10 11 12 13 14 15 16 In PGC I Fig. 2. Logarithmic plot of the literature vapour pressures PL Fig. 1. Liquid vapour pressures (Pa) (25°C) of alkanols [25] of alkanols (25°C) vs. the corresponding P Ge data (Eq. 1) as a function of the number of carbon atoms. D =Original from the present work. The regression line (solid) and y =x I (solid) vapour pressures of 15:0H and 16:0H. line (dashed) are shown. I I I I B. Koutek et al. I J. Chromatogr. A 679 (1994) 307-317 311 with a slope nearly equal to 1. Note that in Eq. 4 sure and thermal data. Hence it appears that the the intercept has not been included at the 0.05% GC method is capable of yielding vapour probability level. The quality of fit produced by pressures of saturated alcohols with an error ~ the proposed vapour pressure correlation is below 10%. -.;- excellent, thus demonstrating the validity of the This conclusion finds further support in the 0.283 GC method even for polar compounds. estimated heats of vaporization. In deriving 81 Another noteworthy feature of this correlation vapour pressures from GC retention time data, 837 is that it appears to be applicable over a range of AHT/AHR, the ratio of the enthalpies of vapori 1256 pressures that covers three orders of magnitude. zation of a test to that of the reference com 4.379 Table 3 presents a comparison between the GC pound is obtained. Hence, by utilizing the litera based and literature vapour pressure data. Our ture [28] experimental AHR value for our refer 1 -F corrected (Eq. 4) vapour pressures differ from ence standard, 14:0H (102.2 ± 2.4 kJ mol- ), those given by N'Guimbi et al. [26] by values the remaining enthalpies of vaporization of al ranging from 0.6% (for lO:OH) to 5.3% (for kanols may be calculated from the AHT/AHR 12:0H). In addition to the original database [26] ratios given in Table 3. The results calculated by ·e lro- 1 'erence, employed in deriving Eq. 4, Table 3 also in this approach are 72.17 kJ mol- for 9:0H, 1 1 cludes a complete data set [3] obtained from the 79.27 kJ mol- for lO:OH and 91.69 kJ mol- ·01the E . 1 Chebyshev-type polynomial in x of degree 3 by for 12:0H. These values compare well with the m red extrapolation. This polynomial has been pro corresponding calorimetric data, viz. 76.86 ± on line posed to allow extrapolation for about 150 K 0. 75, 81.50 ± 0. 75 and 91. 96 ± 0.59 kJ mol-1, e.lhe with fair confidence. The slightly lower (about respectively, yielding a maximum error of 6.1 % . Lr St- 10%) but consistently similar vapour pressure values following from the use of this equation 3.2. Vapour pressures of alkenols might be regarded as a notable agreement be tween the two literature data sets. It is noticable Vapour pressures of all measured alkenols that vapour pressures of lO:OH (1.233 Pa) and were determined by the same approach as de 12:0H (0.1328 Pa) following from the use of Eq. scribed above for saturated compounds. Taking 4 compare favourably with the values 1.190 and advantage of the internal consistency of the 0.1397 Pa obtained [27] for these compounds vapour pressure data for saturated derivatives from a simultaneous correlation of vapour pres- demonstrated above, the test compounds were Table 3 Parameters of Eq. 2 and vapour pressures (25°C) of the n-alkanols Alkanol' D.HT/D.HR c P · 1000 (Pa) Errore (%) Eq. 1 Eq. 4 Exp.b Exp.d 9:0H 0.7062 4.1446 3760 3888 3738 3334 4.0 lO:OH 0.7756 3.3008 1226 1233 1241 1087 -0.6 ll:OH 0.8379 2.4614 412.9 403.7 425.0 378.1 -5.0 12.:0H 0.8972 1.6137 139.6 132.8 140.2 136.1 -5.3 15:0H 1.0493 -0.7941 6.846 6.033 5.894' 5.335 2.4 16:0H 1.1007 -1.6180 2.446 2.100 2.069' 1.842 1.5 'Standard 14:0H. b Ref. [25]. 'Corrected by using Eq. 3. d Ref. [3]. e Error = lOO(P GC - p EXP) IpEXP; p EXP taken from Ref. [25]. . I /. '1~' I I '•· ·I I I 312 B. Koutek et al. I J. Chromatogr. A 679 (1994) 307-317 I Table 4 GC data and vapour pressures (25°C) of decenols I Alcohol Relative retention time" P (Pa) 50°C 60°C 70°C 80°C 90°C Eq. 1 Eq. 4 Z3-10:0H 0.128 0.146 0.168 0.189 0.210 1.669 1.691 I E3-10:0H 0.125 0.142 0.163 0.183 0.204 1.704 1.727 Z4-10:0H 0.135 0.153 0.174 0.193 0.214 1.536 1.553 E4-10:0H 0.141 0.160 0.179 0.198 0.218 1.432 1.445 Z5-10:0H 0.144 0.162 0.183 0.202 0.223 1.406 1.418 I E5-10:0H 0.147 0.165 0.185 0.205 0.225 1.367 1.378 Z6-10:0H 0.147 0.166 0.186 0.206 0.227 1.380 1.391 E6-10:0H 0.147 0.165 0.185 0.205 0.225 1.365 1.376 Z7-10:0H 0.153 0.171 0.192 0.212 0.232 1.311 1.320 I -~ E7-10:0H 0.153 0.171 0.191 0.210 0.229 1.294 1.303 Z8-10:0H 0.175 0.195 0.217 0.237 0.258 1.109 1.112 E8-10:0H 0.165 0.183 0.204 0.224 0.244 1.187 1.192 I 'Standard 12:0H. chromatographed using four reference standards: vapour pressures for CIO' cl2' C13, C14, cl5> c16 I ll:OH (for C 12 alkenols), 12:0H (for C 10 , C 13 and C 18 alkenols are listed in Tables 4-10. and C 14 alkenols), 14:0H (for C 15 and C 16 Inspection of these tables reveals that the vapour alkenols) and 16:0H (for C 18 alkenols). pressures of all alkenols are similar to those of I The relative retention times and calculated the corresponding alkanols. In spite of this, two Table 5 I GC data and vapour pressures (25°C) of dodecenols Alcohol Relative retention time' P (Pa) I 60°C 70°C 80°C 90°C 100°c Eq. 1 Eq. 4 Z2-12:0H 2.034 1.961 1.894 1.827 1.780 0.167 0.160 Z2-12:0H 2.083 2.002 L933 1.854 1.806 0.162 0.155 Z3-12:0H 1.768 1.726 1.691 1.649 1.624 0.204 1.196 I E3-12:0H 1.719 1.682 1.646 1.606 1.591 0.212 0.204 Z4-12:0H 1.793 1.743 1.701 1.652 1.612 0.196 0.188 E4-12:0H 1.896 1.832 1.775 1.715 1.665 0.181 0.173 Z5-12:0H 1.864 1.803 1.753 1.698 1.662 1.187 0.179 I E5-12:0H 1.944 1.870 1.811 1.745 1.701 0.175 0.167 Z6-12:0H 1.867 1.800 1.753 1.697 1.664 0.187 0.179 E6-12:0H 1.913 1.839 1.787 1.726 1.673 0.178 0.170 Z7-12:0H 1.901 1.829 1.780 1.720 1.672 0.181 0.173 I E7-12:0H 1.951 1.871 1.813 1.748 1.700 0.174 0.166 Z8-12:0H 2.002 1.922 1.855 1.789 1.738 1.169 0.162 E8-12:0H 2.002 1.914 1.855 1.784 1.732 0.169 0.162 Z9-12:0H 2.078 1.986 1.920 1.840 1.789 0.161 0.154 I E9-12:0H 2.096 1.998 1.924 1.837 1.775 0.156 0.149 Z10-12:0H 2.402 2.273 2.171 2.065 1.979 0.132 0.125 E10-12:0H 2.246 2.128 2.041 1.946 1.886 1.145 0.138 I •Standard ll:OH. I I I I B. Koutek et al. I J. Chromatogr. A 679 (1994) 307-317 313 Table 6 I GC data and vapour pressures (25°C) of tridecenols Alcohol Relative retention time" P (Pa) I 70°C 80°C 90°C 100°c 110°c Eq. 1 Eq. 4 Z7-13:0H 1.762 1.721 1.675 1.633 1.590 0.0673 0.0628 E7-13:0H 1.817 1.769 1.713 1.666 1.619 0.0639 0.0596 I Z9-13:0H 1.890 1.839 1.776 1.719 1.674 0.0607 0.0565 E9-13:0H 1.939 1.856 1.790 1.728 1 678 0.0575 0.0534 Zll-13:0H 2.266 2.177 2.077 1.999 1.920 0.0472 0.0436 I Ell-13:0H 2.167 2.077 1.982 1.900 1.833 0.0491 0.0455 "Standard 12:0H. I subtle trends are apparent in all series consider vapour pressures of isomers with a double bond ing the influence of double bond position: (i) the positioned on the second carbon atom of the vapour pressures of series members with a dou chain (irrespective of the end of the molecule ble bond located near the centre of the carbon from which the numbering starts) are either close chain are generally higher than those of the to or lower than those of the saturated com corresponding saturated compounds and (ii) the pounds. It appears that the double bond position le'js' 4-10. c16 h apour Table 7 > those of GC data and vapour pressures (25°C) of tetradecenols · t,, two Alcohol Relative retention time" P (Pa) 80°C 90°C 100°c 110°c 120°C Eq. 1 Eq. 4 -t- Z2-14:0H 3.820 3.538 3.299 3.113 2.911 0.0211 0.0191 E2-14:0H 3.883 3.586 3.345 3.140 2.925 0.0203 0.0184 Z3-14:0H 3.389 3.186 3.013 2.847 2.685 0.0256 0.0234 E3-14:0H 3.286 3.083 2.912 2.767 2.609 0.0267 0.0243 Z4-14:0H 3.358 3.157 2.966 2.811 2.650 0.0256 0.0233 E4-14:0H 3.526 3.289 3.079 2.902 2.723 0.0234 0.0213 Z5-14:0H 3.408 3.217 3.017 2.843 2.663 0.0246 0.0224 E5-14:0H 3.551 3.308 3.096 2.917 2.729 0.0230 0.0209 I Z6-14:0H 3.343 3.138 2.954 2.798 2.634 0.0257 0.0234 E6-14:0H 3.462 3.243 3.050 2.865 2.686 0.0240 0.0218 Z7-14:0H 3.340 3.134 2.959 2.798 2.634 0.0258 0.0235 E7-14:0H 3.485 3.259 3.057 2.883 2.704 0.0239 0.0217 I Z8-14:0H 3.412 3.209 3.004 2.840 2.665 0.0246 0.0224 E8-14:0H 3.616 3.301 3.087 2.922 2.719 0.0220 0.0200 Z9-14:0H 3.512 3.285 3.079 2.898 2.725 0.0236 0.0215 E9-14:0H 3.622 3.376 3.157 2.961 2.768 0.0222 0.0202 I Z10-14:0H 3.682 3.473 3.223 3.016 2.819 0.0217 0.0196 E10-14:0H 3.731 3.464 3.221 3.025 2.815 0.0211 0.0191 Zll-14:0H 3.881 3.587 3.335 3.117 2.909 0.0200 0.0181 Ell-14:0H 3.834 3.548 3.300 3.079 2.864 0.0201 0.0182 I Z12-14:0H 4.346 4.024 3.714 3.447 3.185 0.0169 0.0153 E12-14:0H 4.102 3.774 3.495 3.250 3.004 0.0180 0.0163 I "Standard 12:0H. I I I ·I I I 314 B. Koutek et al. I J. Chromatogr. A 679 (1994) 307-317 Table 8 I GC data and vapour pressures (25°C) of pentadecenols I Alcohol Relative retention time" P (Pa) 90°C 100°c 110°e 120°e B0°e Eq. 1 Eq. 4 Z9-15:0H 1.633 1.598 1.564 1.533 1.505 0.00917 0.00814 I E9-15:0H 1.692 1.649 1.605 1.568 1.536 0.00851 0.00754 Z10-15:0H 1.696 1.651 1.608 1.574 1.541 0.00845 0.00749 E10-15:0H 1.729 1.681 1.638 1.593 1.557 0.00815 0.00721 Zll-lS:OH 1.768 1.721 1.676 1.629 1.590 0.00794 0.00702 I Ell-15:0H 1.769 1.721 1.673 1.623 1.581 0.00781 0.00691 Z12-15:0H 1.841 1.790 1.735 1.677 1.639 0.00739 0.00652 E12-15:0H 1.830 1.771 1.714 1.660 1.614 0.00730 0.00644 ZB-15:0H 2.071 1.988 1.915 1.849 1.792 0.00617 0.00542 I El3-15:0H 1.955 1.880 1.811 1.744 1.695 0.00654 0.00576 "Standard 14:0H. I Table 9 GC data and vapour pressures (25°C) of hexadecenols I Alcohol Relative retention time" P (Pa) 100°e 110°e 120°e B0°e 140°e Eq. 1 Eq. 4 I Z3-16:0H 2.973 2.801 2.673 2.524 2.401 0.00329 0.00285 E3-16:0H 2.900 2.741 2.605 2.465 2.343 0.00336 0.00291 Z4-16:0H 2.919 2.758 2.629 2.486 2.364 0.00337 0.00292 E4-16:0H 3.054 2.872 2.719 2.559 2.422 0.00302 0.00261 I Z5-16:0H 2.944 2.779 2.639 2.494 2.369 0.00327 0.00283 E5-16:0H 3.033 2.851 2.703 2.546 2.412 0.00307 0.00265 Z6-16:0H 2.854 2.704 2.575 2.440 2.320 0.00348 0.00301 E6-16:0H 2.962 2.794 2.657 2.506 2.378 0.00323 0.00279 I Z7-16:0H 2.809 2.664 2.545 2.414 2.299 0.00362 0.00314 E7-16:0H 2.993 2.798 2.659 2.507 2.377 0.00312 0.00270 Z8-16:0H 2.849 2.702 2.576 2.433 2.323 0.00350 0.00303 E8-16:0H 2.967 2.799 2.656 2.510 2.377 0.00321 0.00278 I Z9-16:0H 2.880 2.711 2.586 2.447 2.335 0.00344 0.00298 E9-16:0H 3.001 2.825 2.679 2.521 2.393 0.00312 0.00270 Z10-16:0H 2.962 2.795 2.658 2.506 2.377 0.00323 0.00279 E10-16:0H 3.058 2.873 2.719 2.563 2.426 0.00302 0.00261 I Z11-16:0H 3.050 2.870 2.719 2.572 2.430 0.00308 0.00266 E11-16:0H 3.125 2.930 2.757 2.604 2.457 0.00288 0.00248 Z12-16:0H 3.191 2.997 2.822 2.665 2.510 0.00282 0.00243 E12-16:0H 3.198 2.996 2.810 2.651 2.499 0.00276 0.00238 I Zl3-16:0H 3.330 3.110 2.922 2.746 2.588 0.00261 0.00224 EB-16:0H 3.294 3.066 2.884 2.705 2.544 0.00260 0.00224 ·' I "Standard 14:0H. - I I ,,1 I l I I I B. Koutek et al. I J. Chromatogr. A 679 (1994) 307-317 315 Table 10 I GC data and vapour pressures (25°C) of octadecenols Alcohol Relative retention time• P (Pa) I 120°c 130°C 140°C 150°C 160°C Eq. 1 Eq.4 Z3-18:0H 2.676 2.500 2.400 2.288 2.200 0.000377 0.000309 E3-18:0H 2.583 2.448 2.346 2.244 2.149 0.000406 0.000333 I Z9-18:0H 2.465 2.339 2.254 2.160 2.083 0.000453 0.000373 E9-18:0H 2.596 2.455 2.366 2.247 2.157 0.000402 0.000330 Zll-18:0H 2.534 2.405 2.311 2.216 2.130 0.000431 0.000354 Ell-18:0H 2.637 2.497 2.391 2.275 2.181 0.000386 0.000316 I Z13-18:0H 2.712 2.555 2.439 2.326 2.221 0.000363 0.000296 El3-18:0H 2.753 2.585 2.463 2.342 2.234 0.000345 0.000282 a Standard 16:0H. relative to both the polar and non-polar ends of tion) and 3-lO:OH, 5-12:0H, 9-14:0H and ll- the molecule is significant. As illustrated in Figs. 16:0H (w-7 unsaturation), the double bond is at I 3 and 4, these trends may be observed both for a constant position with respect to the non-polar the Z- and £-isomers in all the series investi end of the molecule. Analysis of the double gated. bond effect in these subseries reveals that the 1- For homologous subseries such as Z- or £ vapour pressures of alkenols having different isomers of 7-lO:OH, 9-12:0H, 11-14:0H and positions of unsaturation decrease in the order 13-16:0H (w-3 unsaturation), 5-lO:OH, 7- w-7 > w-5 > w-3 > saturated. The empirical rela I 12:0H, 9-14:0H and ll-16:0H (w-5 unsatura- tionships given by the In P vs. nc expression I 7 I 6 6 5 a 5 ...... 0 0 x 4 I ~ .s 3 I 2 2 I 0+--.~-,--,~-,---.~..,---.-~,--.-~-,-- Q-\---,~...,----,~...,---.~-,--,~-,--,~-,----1 2 3 4 5 6 7 8 9 10 11 12 13 2 3 4 5 6 7 8 9 10 11 12 13 (Z)-double bond pos1t1on (E)-double bond position Fig. 3. Vapour pressures (Eq. 4) for (Z)-alkenols plotted Fig. 4. Vapour pressures (Eq. 4) for (E)-alkenols plotted I against the respective double bond position. • = Decenols; against the respective double bond position. • = Decenols; • = dodecenols; & = tetradecenols; <> = pentadecenols; 0 = • = dodecenols; & = tetradecenols; <> = pentadecenols; 0 = hexadecenols. Dashed lines show the vapour pressures of the hexadecenols. Dashed lines show the vapour pressures of the I corresponding saturated compounds. corresponding saturated compounds. I I I -I ~ .--- I I 316 B. Kowek et al. I J. Chromatogr. A 679 (1994) 307-317 Table 11 may be expressed as ln P = (1.1716 ± 0.0666) I Proposed relationships for predicting vapour pressures at ln ?Heath - (1.231±0.137) (n = 4, S.E. = 0.164, 25°C 2 r = 0.9936). Alcohol Ln[P (Pa)]= a - bn, S.E.b r' By utilizing the reliable literature [26] values I subseries' at 30°C (see Table 1) for 12:0H (0.266 Pa) and a b 14:0H (0.0366 Pa), the ratio Paikenoi / Paikanoi for compounds with the same number of carbon Saturated 10.772 ± 0.135 1.058 ± 0.010 0 0458 0.9998 atoms may be adopted as an approximate mea I w-3-(Z) 10.904 ± 0.065 1.064 ± 0.005 0.0220 1.0000 w-3-(E) 10 844 ± 0.081 1.060 ± 0.006 0.0276 0.9999 sure of the "effect of non-terminal monounsatu w-5-(Z) 10.805 ± 0.018 1.046 ± 0.001 0.0061 1.0000 ration". This vapour pressure ratio following w-5-(E) 10.849 ± 0.020 1.053 ± 0.002 0.0067 1.0000 from our data is about 1.2-1.3, which seems to I w-7-(Z) 10.995 ± 0.222 1.053 ± 0.017 0.0751 0.9995 be a reasonable value considering its similarity to w-7-(E) 11.178 ± 0.307 1.071 ± 0.023 0.1040 0.9991 the corresponding values common for non-termi nal alkene [1] and unsaturated acetate series "Number of data points. n = 6. I b S.E. =standard error of estimate. [20]. On the other hand, the ratio of 4.7-4.8 which follows from the use of the data from Ref. [29) appears to be unrealistically high. The I reasons for this discrepancy are not clear. How were obtained from analyses of the calculated ever, besides the imprecisely defined tempera (Tables 4-10) ln P data. The relevant equations, ture they used, another factor might be im listed in Table 11, may be used to estimate portant, viz. the use of cholesteryl p-chlorocinna I vapour pressures for any set of w-3, w-5 and w-7 mate as a stationary phase. It may be that the alkenols. The quality of the fit obtained with polar alcohols interact in a specific manner with MAD (mean average deviation) 2.3% (w-3), this phase and then this factor would account for I 1.9% (w-5) and 4.8% (w-7) combined with the the differences in the two studies. convenience of only one substance-specific input As the errors in the reported vapour pressures variable makes this an attractive approach in depend both on experimental uncertainties and predicting vapour pressures of some other struc on the accuracy of the literature vapour pressure I turally similar derivatives. data adopted for the reference standards, it is At this point some comment should be made difficult .Jo determine any inherent error in the regarding literature vapour pressure values of present method for ·alcohols. Some discussion is I unsaturated alcohols. To our knowledge, only possible, however: as to the latter error factor, four of the alkenols investigated in this work had recent inter-laboratory data [25,27) for lO:OH literature data available for comparison. Heath and 12:0H agree to within ±5%, which may be I and Tumlinson [29] determined the vapour regarded as a very good agreement. Hence the pressures of Z7-12:0H, Z9-14:0H, Zll-14:0H differences between our data and those taken and Z11-16:0H as 1.25, 0.177, 0.160 and 0.039 from the literature for alkanols are generally not Pa, respectively. They carried out these determi greater than the experimental errors (see Table I nations on capillary liquid crystal GC columns at 3). On the other hand, when admitting a propa "room temperature", which probably corres gation of errors, the uncertainty might reach ponded to 30°C. At that temperature our GC about 10%. Moreover, the vapour pressures for I method yields vapour pressures of 0.344, 0.044, 15:0H and 16:0H were obtained by (prudent) 0.037 and 0.0057 Pa, respectively, for the same extrapolation and additionally corrected for compounds. Hence our values are significantly melting points. It is unlikely that they are in (3.6-6.8 times) lower than those in Ref. [29]. serious error (see the internal homogeneity of I Note, however, that a high degree of correlation the alkanol data illustrated in Fig. 1), but we I exists between both data sets. The linear fit have to accept their lower accuracy. Taken .,i / I / /\) I I B. Koutek et al. I J. Chromatogr. A 679 (1994) 307-317 317 together, we assume that, at worst, the errors [4] R.D. Gray, Jr., J.L. Heidman, R.D. Springer and C. may combine to give an overall uncertainty in Tsonopoulos, Fluid Phase Equlib., 53 (1989) 355. [5] D. Mackay and S. Paterson, Environ. Sci. Technol .. 25 vapour pressures of about 15%. •] '11ues (1991) 427. [6] D. Mackay, S. Paterson and W.Y. Shiu, Chemosphere, .and 24 (1992) 695. lkanol for 4. Conclusions [7] J.H. Tumlinson, in R.L. Ridgway, R.M. Silverstein and .bon M.N. Iscoe (Editors), Behavior-Modifying Chemicals for Insect Management -Applications of Pheromones tt9nea This study has demonstrated the successful Junsatu- and Other Attractants, Marcel Dekker, New York, 1990, application of the capillary GC method to the Ch. 5. all ing determination of the vapour pressures of unsatu [8] A. Shani, Role of Pheromones in Integrated Pest Man .e s to rated alcohols whose generally low thermal agement. Institutes for Applied Research, Ben-Gurion 11 y to stability causes difficulties in direct measure University of the Negev, Beer-Sheva, Israel, 1991. ·n-termi [9] W.F. Spencer and M.M. Chath, Residue Rev., 85 (1983) ments by conventional physico-chemical meth 57. e,ries ods. The method yields reasonable vapour pres [10] W.J. Sonnefeld, W.H. Yoller and W.E. May, Anal. -4.8 sure values for both the alkanols and alkenols at Chem., 55 (1983) 275. om Ref. 25°C provided that a compound of similar struc [11] J.J. Murray, R.F. Pottie and C. Pupp, Can. J. Chem., ~h.TThe ture and polarity is used as the reference stan 52 (1974) 557. r.lfow [12] D.J. Hamilton, J. Chromatogr., 195 (1980) 75. dard. It is hoped that these new values may [13] T.F. Bidleman, Anal. Chem., 56 (1984) 2490. empera- considerably extend the database for the vapour (14] J.W. Westcott and T.F. Bidleman. J. Chromatogr., 210 pressures of alcohols, and enable entomologi (1981) 331. rin~~~ cally oriented chemists to study and model the [15] B.D. Eitzer and R.A. Hites, Environ. Sci. Technol., 22 til the physical behaviour of pheromone-like com (1988) 1362. iler with - [16] Y.-H. Kim, J.E. Woodrow and J.N. Seiber, J. Chroma pounds in th-e env!tonment -more accurately. togr., 314 (1984) 37. alt for The method is currently being used to de (17] D. Hawker, Chemosphere, 25 (1992) 427. termine vapour pressures of unsaturated alde [18] P.M. Sherblom, P.M. Gschwenct ahd R.P. Eganhouse, J. ressures hydes and will be the subject of a separate Chem. Eng. Data, 37 (1992~ :' •4. [19] S. Husam, P.N. Sarma, G.i ,,K. Swamy and K. Sita tit and report. p sure Devi. J. Am. Oil Chem. So• , 70 (1993) 149. [20] B. Koutek, M. Haskovec, K. Konefoy and J. Vrkoc, J. d , it is Chromatogr., 626 (1992) 215. r in the Acknowledgement [21] D.A. Hinckley, T.F. Bidleman, W.T. Foreman and J.R. isfn is Tushall, J. Chem. Eng. Data, 35 (1990) 232. - tor, This work was financially supported, in part, [22] W.Y. Shin and D. Mackay, J. Phys. Chem. Ref. Data, 15 lO:OH (1986) 911. by Grant No. DHR-5600-G-00-1051-00, Program [23] D. Mackay, A. Bobra, D.W. Chan and W.Y. Shin, ybe in Science and Technology Cooperation, from Environ. Set. Technol., 16 (1982) 645. :nl the the US Agency for International Development. [24] D.R. Lide (Editor), CRC Handbook of Chemistry and e taken Physics, CRC Press, Boca Raton, FL, 72nd ed., 1991- 92. ·a1 not e able [25] T. Nitta, K. Morinaga and T. Katayama, Ind. Eng. References Chem. Fundam., 21 (1982) 296. l opa- [26] J. N'Guimbi. H. Kaseghari, I. Mokbel and J. Jose, 1t reach [1] R.C. Reid, J.M. Prausnitz and B.E. Poling, The Prop Thermochim. Acta, 196 (1992) 367. ul for erties of Gases and Liquids. McGraw-Hill. New York. [27] K. Ruzicka and V. Majer, Fluid Phase Equilib., 28 )r ent) 4th ed., 1987. (1986) 253. ted for [2] R.D. Shaver, R.L. Robinson, Jr., and K.A.M. Gasem, [28] M. Mansson, P. Sellers, G. Stridh and S. Sunner, J. Chem. Thermodyn., 9 (1977) 91. e in Fluid Phase Equilib., 64 (1991) 141. [3] D. Ambrose, J.H. Ellender and C.H.S. Sprake, J. [29] R.R. Heath and J.H. Tumlinson, J. Chem. Ecol., 12 l1 y of Chem. Thermodyn., 6 (1974) 909. (1986) 2081. tlul we Taken I I I I I J CHEM. SOC. PERKIN TRANS. l 1994 3509 I Preparation of Chlorofluoroacetic Acid Derivatives for the Analysis of Chiral Alcohols I Ludvik Streinz,a Ales Svatos.a Jan Vrkol:a and Jerrold Meinwaldb a Institute of Organic Chemistry and Biochemistry, 166 10 Prague 6, Czech Republic I b Department of Chemistry, Cornell University, Ithaca, NY 14853-1301, USA (R)- and (S)-Chlorofluoroacetic acid (CFA) esters of several chiral secondary alcohols have been prepared and compared with the corresponding esters of Masher's acid. CFA itself is a readily accessible and optically stable acid which gives the expected diastereoisomeric products with chiral alcohols without epimerization. The resulting diastereoisomers are more volatile than those derived from Masher's acid, and are well resolved by both GC and HPLC. Both 1 H and 19F NMR spectra of CFA esters show characteristic signals in regions rarely overlapped by other signals. Since CFA is a • strong organic acid, it reacts with alcohols spontaneously to give esters without any additional I catalysis. Determining the optical purity of biologically active com OR I pounds and of the products of any asymmetric synthesis is important in much contemporary research. One approach used ~ ~R frequently for this purpose is the preparation of derivatives of 2 chiral alcohols and amines using 3,3,3-triftuoro-2-methoxy-2- I phenylpropioni<:; acid (Mosher's acid, MTPA), which gives rise to diastereoisomers which may be separable by GC or HPLC, 1 2 or detected by NMR techniques. • This approach has been widely applied, and has proven extremely useful. However, I 3 4 there are examples where this method failed. • New agents for the preparation of derivatives are, therefore, of potential inter 5 9 4 est, and some new examples have been described recently. - Among these oc-aryl-oc-ftuoroacetates seem to be good I 4 candidates. The presence of a fluorine atom at the chiral centre of the acetic acid moiety results in the separation of characteristic peaks in both 1 H and 19F NMR spectra. I Moreover, the peaks of interest are usually found at chemical shifts which are not overlapped by other signals. The separation of diastereoisomeric signals can be enhanced by 5 6 additional functional groups attached to the chiral C-F centre. 4 I This substitution leads, however, to decreased volatility, a R=H making the compounds less suitable for GC analysis. We report b R= OCCHCIF c R =OC(CF3)(0Me)(Ph) here the results of our study of an alternative derivatizing agent, chloroftuoroacetic acid (CFA). CFA is one of the simplest optically active compounds appropriate for this application. l O We anticipated that diastereoisomeric CfA esters might permit behaviour of both CFA and MTPA esters of alcohols la--6a, as easy GC and/or HPLC separation,_as well as NMR spectral well as the 1 H and 19F NMR spectra of these compounds. The differentiation. results are summarised in Table 3, in which we record the 11 10 12 -CFA is readily accessible, easily resolved • and the chemical shift non-equivalence (MH.F) of diastereoisomers in absolute configuration of its enantiomers has already been both 1 H and 19F NMR spectra and also differences in retention established. 13 For esterification of CFA we chose the 1,3- times (~t" GC, HPLC). In general, esters derived from MTPA · dicyclohexylcarbodiimide (DCC) method, which gives high show larger M values than do CFA esters. This is not 14 15 I yields even with sterically hindered alcohols. • In order to unexpected, in view of the increased non-bonded steric and obtain a quantitative reaction with respect to the starting electronic interactions resulting from the presence of an alcohol, we used a 3 mo! excess of the reagents. 16 Since it aromatic ring attached to the chiral carbon atom of the MTPA 1 4 is known that various chiral alcohols can be esterified esters. • I 17 spontaneously by CF 3C02 H, or by other halogen-containing However, the CHFCI proton resonance of diastereoisomeric acetic acids, 18 we have also directly esterified CFA with CFA esters typically appears as a characteristic doublet (JH,F 1-phenylethanol 4a using CFA as the solvent. Neither race 50 Hz) in a region not often disturbed by other signals mization nor kinetic resolution during these esterifications (o :;-; 6.30, see Table 1). This, along with the sharpness of these I was observed. A variety of secondary alcohols la--6a were peaks, greatly facilitates the analysis of the chiral alcohols. 4 The converted into CFA esters lb-6b (Table I). The same alcohols same favourable feature is also seen in the 19F NMR spectra were converted into MTPA esters lc--6c (Table 2) using (o :;-; -68, JF.H 50 Hz). The chromatographic behaviour of commercially available (R)- and (S)-MTPA chlorides following CFA diastereoisomers appears to be superior to that of MTPA I 1 2 a standard procedure. • We have ex_amined the GC and HPLC esters. This is important because using a chromatographic I I I 3510 J. CHEM. SOC. PERKIN TRANS. I 1994 I Table 1 Spectroscopic data for CFA esters lb-6b of alcohols la-6a Table 2 Spectroscopic data for MTP A esters lc-6c of alcohols la-6a prepared using racemic CFA Alcohol Spectroscopic data for MTPA ester Alcohol Spectroscopic data for CFA ester (R,S)-la oH 0.84 (t, J 6.4, CH3), 0.87 (t, J 6.4, CH3), 1.17-1.36 (m, I ), ), (R,S)-la JH 0.88 (t, 3 H, J 6, CH3 1.61-1.75 (m, 8 H, CH2 5.21- 6 H, CH2), 1.52-1.73 (m, 2 H, CH2 CO), 3.54, 3.55 (2 x s, 5.38 (m, 3 H, C----CH 2 , OCH); 5.71-5.88 (m, 1 H. -CH=), 3 H, OCH3), 5.16--5.38 (m, 2 H, C=CH2), 7.36--7.49 (m, 6.27 (d,JH,F 50, 1 H, CHF); JF -67.79 (d, JF,H 50, CHF), 5 H, ArH); JF 6.13 (s, CF,); Vmaxfcm- 1 2950, 1758, -67.86 (d, JF,H 50, CHF); Vmax/cm- 1 3096, 2941, 2874, 1215, 1181, 1123 and 1013; m/= 189 (BP), 139, 128, 119, I 1767, 1253, 1107,964,822;m/zl67, 151, 137,110,95,81,69 111, 105, 77, 69 (BP), 41 (R)-2a Ott 0.85 [t, J 6.6, CH" (R,S)]; 0.87 [t, J 6.6, CHiR,R)] ), (R)-2a b'tt 0.87 (t, 3 H, J 6, CH3 1.15-1.63 (m, 13 H, CH2, CH), 1.11-1.37 (m, 11 H, CH2 , CH3), 1.32 (d, 3 H, J 6.4, 5.00--5.10 (m, I H, HCO), 6.24 (d, 1 H, JH.F 50, CHClF); CHCH3 ), 1.52-1. 72 (m, 2 H, OCCH2 ), 3.49 [s, 3 H, OCH3, OF -67.75 [d, JF.H 50, CHCIF (R,R)], -67.86 [d, JF.H (R,R)]; 3.56 [s, 3 H, OCH3 . (R,S)], 5.10--5.19 (m, I H, 1 I 50, CHCIF (R,S)]; vmaxfcm- 2938, 2871, 1765, 1463, , OCH), 7.34--7.54 (m, 5 H, ArH); JF 6.14 [s, CF3 (R,R)], 1 1289, 1191, 1116, 959, 826; m/= 157, 139, 112, 83, 70, 57, 41 6.61 [s, CF3 , (R,S)]; Vmaxfcm- 2940, 2868, 1758, 1455, (BP) 1383, 1244, 1181, 1122, 1016, 846; m/z 189 (BP), 158, 119, (S)-3a oH 0.89--0.98 (m, 9 H, 3 x CH3), 1.20-1.91 (m, 9 H, CH2, I05, 89, 77, 57 CH), 5.18 (m, 1 H, OCH), 6.24 [d, JH,F 50, CHCIF, (S,R)]; (S)-3a JH 0.81 [d, J 6.6, 2 x CH , (S,R)], J I 3 0.88 [d, 6.6, 6.25 [d, JH,F 50, CHCIF, (S,S)]; OF -67.65 [d, JF.H 50, 2 x CH3, (S,S)], 0.94 [d, 3 H, J7, CH3, (S,R)]; 0.96 [d, CHCIF, (S,S)], -67.68 [d, JF.H 50, CHCIF, (S,R)]; 3 H, J 6.6, CH3 , (S,S)]; 1.01-1.81 (m, 9 H, CH, CH2), 1 Vmax/cm- 2966, 2852, 1766, 1289, 1191, 1113, 822; m/= 3.54 [s, 3 H, OCH3, (S,S)], 3.56 [s, 3 H, OCH3, (S,R)]; 207, 138, 109, 95 (BP), 67, 41 5.25-5.33 (m, I H, OCH), 7.36--7.52 (m, 5 H, ArH); JF 1 I (R)-4a JH 1.64 (d, 3 H, J 6.6, CH3), 6.06 (q, 1 H, J 6.6, HCOJ, 6.18 [s, CF3 , (S,S)], 6.59 [s, CF , (R,S)]; vmaxfcm- 3069, 3 6.32 [d, JH.F 50, CHClF, (R,S)], 6.35 [d, JH,F 50, CHCIF, 2962, 2882, 1754, 1456, 1382, 1017, 972; m/z 189, 158. 139, (R,R)], 7.42 (m, 5 H, ArH); oF -68.48 [d, JF,H 50, 127, 105, 83 (BP), 77, 69, 55 CHCIF, (R,S)], -68.51 [d, JF.H 50, CHClF, (R,R)]; (R)-4a JH 1.57 [d, J6.6, CH3 , (R,R)], 1.68 [d, J 6.6, CH3, (R,S)], 1 Vmaxfcm- 3074, 2993, 1780, 1202, 1107, 1063, 953, 822; 3.45 [s, 3 H, OCH , (R,S)], I 3 3.47 [s, 3 H, OCH" (R,R)], 216, m/z 201, 173, 146, 105 (BP), 77, 51 6.08 [q, J 6.6, CHCH3, (R, S)], 6.13 [q, J 6.6, CHCH3 , (S)-Sa JH 0.87 J ), (m, ), (t, 3 H, 6, CH3 1.21-1.99 14 H, CH2 (R,R)], 7.21-7.42 (m, IO H, ArH); oF 6.68 [s, CF3 , 1 5. 76--5.87 (m, 1 H, HCO), 6.26 [ d, JH.F 50, CHCJF, (R,R)]. (R,S)], 6.89 [s, CF3 , (R,R)]; Vmaxfcm- 3072, 2992, 6.29 [d JH.F 50, CHCIF, (R,S)]; JF -67.52 [d, JF.H 50, 1760, 1453, 1232, 1182, 1123, 1014, 860; m/z 189, 158, 119, I CHCJF, (R,R)], -67.73 [d, JF,H 50, CHCIF, (R,S)]; 105, 77 (BP) 1 Vmax/cm- 3073, 2994, 2866, 1781, 1458, 1282, 1185, 1113, (S)-Sa JH 0.87 (t, 3 H, J 6.8, CHJ), 1.09-1.40 (m, 14 H, CH2), 964, 822;m/z328, 261, 233, 201.173, 117, 104(BP),41 1.57-1.99 (m, 2 H, OCCH2), 3.43 [s, 3 H, OCH" (S,S)], (R)-6a JH 6.40 (d, 1 H, JH.F 49, CHCIF), 6.46 (d, JH,F 49, 3.53 [s, 3 H, OCH3 , (S,R)], 5.85 [d, J 6.6, OCH, (S,R)], CHClF), 7.45-8.61(m,10 H. ArH), CF3CH);JF -69.13 5.92 [d, J 6.6, OCH, (S,S)], 7.27-7.43 (m, IO H, ArH); JF I 1 (d,JF,H 49, CHClF), -69.18 (d, JF,H 49, CHClF), 5.81 (d, 6.69 [s, CF" (S,R)], 6.91 [s, CF3, (S,S)]; vmaxfcm- 2934, 1 1 H, J 7.8, HCCF3 ); vmaxfcm- 3061, 1785, 1354, 1271. 2564, 1757, 1455, 1183, 1123, 1014; m/z 217, 89, 174, 158, 1192, 1137,886, 784;m/=370(BP),301,259,238, 178, 151, 139, 123, 119, 91 (BP), 77 119, 67 (R)-6a JH 3.28 [s, 3 H, OCH3 , (R,S)], 3.67 [s, 3 H, OCH3, (R,R)], 3 I 7.18-8.60 (m, 10 H, ArH, CF3CH); JF 6.32 [d, JF,H 7.7, CF 3CH, (R,S)], 6.37 [s, CF 3, (R,R)], 6.64 [s, CF 3 , (R,S)], 6.88 [d, 3 JF,H 7.7, HCCF" (R,R)]; Vm"fcm-1" 3095, 3059, method as an analytical tool permits actual separation as well 3009, 2850, 1627, 1528, 1498, 1452, 1398, 1277, 1268, 1239, 1225;m/z259(BP),239,209, 190, 189, 174, 119, 105, I as analysis, and can be more precise than NMR spectroscopy. 19 77 It is known that halogen-containing acetates are well resolved on non-polar GC phases, where the retention times are related " IR spectrum was recorded on Bruker ISS FTIR spectrometer (in CC1 4). to the atomic weight and the number of halogen atoms in the I molecule. Fluorine is exceptional, its introduction resulting in significant shortening of retention times. 20 The relatively low Experimental column temperatures required for good resolutions of CFA 1 H (200 MHz) and 19F (188 MHz) NMR spectra were recorded diastereoisomers can be attributed to (i) their lower molecular on a Varian XL-200 spectrometer. Chemical shifts (in ) I CDC1 3 weight, (ii) the presence of fluorine in the molecule, and perhaps are expressed on the o scale measured from residual CHC1 3 21 19 also (iii) little hydrogen bonding with the liquid phase. Good (7.25) and internal CF3C02H (0.0) for F. !Values are given separations were also obtained using HPLC on silica gel; CFA in Hz. Mass spectra were obtained using a Hewlett-Packard I esters 2a and 4a can even be resolved by TLC on silica gel plates, 5890 gas chromatograph (column DB-5, 30 m x 0.25 mm i.d.) where two fully separated spots of diastereoisomers appeared coupled to an HP 5970 mass selective detector. IR spectra (none of the MTPA esters studied in this paper showed such a were recorded in the gas phase using a Hewlett-Packard 5890 II separation). gas chromatograph (column: Carbowax, 30 m x 0.25 mm i.d.) I In order to obtain reliable results in chiral alcohol analysis, coupled to an HP-5965 infrared detector. For GC analyses, a the derivative preparation must not favour one alcohol Hewlett-Packard gas chromatograph was used (columns: DB-5, enantiomer over the other, and must be free of racemization. 30 m x 0.25 mm i.d., Carbowax, 30 m x 0.25 mm i.d.) with Chiral acetic acids having an enolizable a-hydrogen atom are helium as the carrier gas. HPLC analyses were performed using I certainly capable of racemizing. Nevertheless, CFA is suffi a Hewlett-Packard HP 1090 apparatus equipped with silica ciently optically stable to be used for preparing derivatives of columns [3 x (450 mm x 3 i.d.), Tessek, Czech Republic, 3 1 chiral secondary alcohols. CFA is the least reactive of the silica 5 µm, 3% diethyl ether in hexane, flow rate: 0.4 cm min- , halogenated acetic acids towards nucleophilic substitution. 22 DAD UV detector (220 nm) controlled by an HP-85B 1 2 1 Optically pure CF A does not show any change in specific computer]. [ci] Values are given in units of 10- deg • I 0 cm g- rotation in aqueous solution and can be converted into its acid For the preparation of diastereoisomeric esters, the following chloride or methyl ester and reduced to the corresponding secondary alcohols were purchased and used without further 10 12 alcohol without any racemization. · Based on all of these purification: (R,S)-oct-l-en-3-ol la (Aldrich 0-528-4), (R)-(-) .1 considerations, CF A seems to be an attractive choice for the octan-2-ol 2a (Aldrich, 14,799-0), ( + )-isomenthol 3a (Aldrich stereochemical analysis of chiral alcohols. 24,219-5), (R)-( + )-1-phenylethanol 4a, (Aldrich 23, 742-6), (S)- I I I J. CHEM. SOC. PERKIN TRANS. I 1994 3511 I Table 3 Chromatographic and spectroscopic differences between diastereoisomers of esters of alcohols la---{ia with CFA and MTPA Ester GC M(Hz) HPLC b T/°Cc 19F R• I Acid Alcohol" t,/min of diast. tit, 'H t,/min of diast. s CFA (R,S)-la 32.19 33.08 95 0.89 12 23.36 24.16 1.6 (R)-2a e 30.45 (R,S) 32.17 (R,R) 95 1.72 21 21.03 (R,S) 22.29(R,R) 2.6 (S)-3a 30.47 (S,R) 31.80 (S,S) 115 1.33 0.8 62 18.59 (S, S) 20.02 (S,R) 1.9 I (R)-4af 62.90 (R,S) 64.17 (R,R) 115 1.27 6 6 18.32 (R,R) 21.57 (R,S) 4.6 (S)-5a 33.97 (S,S) 34.58 (S,R) 190 0.61 6 38 16.97 (S,R) 17.20 (S,S) 0.6 (R)-6a• 24.88 25.02 200 0.14 12 9 30.66 MTPA (R.S)-la 27.51 28.46 155 0.95 2 20.99 21.29 0.6 I (R)-2a 32.32 (R,S) 32.86 (R,R) 155 0.54 13 88 22.86 (S)-3a 32.75 170 4 86 20.49 (R)-4a 22.77 (R,S) 23.38 (R)R) 170 0.61 4 39 17.58 (R,S) 18.51 (R,R) 2.3 (S)-5a 31.70 (S,S) 32.13 (S,R) 225 0.43 20 39 20.34 I (R)-6a 26.71 (R,R) 29.84 (R.S) 235 3.13 78 50 31.86 (R,R) 36.16 (R,S) 0.6 I ::!":=~~,::,:,~::.':~~re:~';:'~; :';;:/(w"; ~~)°::::::,: .:::.":':,C~Aw::',::~,:":· .:~:;~,.::~.::,~~ gel plates, 20% diethyl ether in light petroleum. Re 0.28 (R,R). 0.31 (R,S). / Re 0.62 (R,R), 0.68 (R,S). •The configuration was not determined. I (-)-l-phenyldecan-1-ol 5a (Aldrich 33,161-9) and (R)-( -)- centrated under reduced pressure. Distillation of the residue 2,2,2-trifiuoro-l-(9-anthryl)ethanol 6a (Aldrich 21, 135-4). afforded (S)-chlorofiuoroacetic acid (0.36 g, 91%), b.p. 86- Since obtaining both M and dt, was the main objective of 94 °Cj40 mmHg; bf! 6.30 (d, Jfl,F 50, 1 H, CClFH) and 6.88- this paper, the CFA esters were prepared as mixtures of 7.25 (br s, 1 H, C02 H); i5F -68.09 (d, h.tt 50). I diastereoisomers, using racemic chlorofluoroacetic acid. To identify particular diastereoisomers in a mixture, esters of (R,R)-Octan-2-yl Chlorojluoroacetate 2b (Prepared in an optically active alcohols with either (R)- or (S)-chloroftuoroace Excess of CFA).-(R)-Chloroftuoroacetic acid (59 mg, 0.52 tic acid were also prepared. The syntheses were accomplished mmol), (R)-octan-2-ol (13.7 mg, 0.105 mmol) and Drierite (38 14 15 3 I via DCC esterification • using a 3 mo! excess of reagents. mg) were placed in a I cm conical vial. The mixture was set The reaction was worked up as soon as the starting alcohol had aside at room temperature and the reaction mixture was disappeared from the reaction mixture (TLC, typically 0.5-24 monitored either by GC or TLC. As soon as the starting alcohol h, yield: 91-97%). For GC analyses of CFA and MTPA esters, had disappeared from the reaction mixture (about 3 days), the 3 I two different columns were used, giving the best resolution of reaction mixture was diluted with light petroleum (1 cm , particular sets of diastereoisomers (see Table 3). The NMR and containing 5% of diethyl ether), transferred onto a Pasteur mass spectra of the resultant esters are given in the Tables I and pipette silica column, and chromatographed using the same 2, while the spectral and chromatographic comparisons are solvent mixture to give the product (22.8 mg, 97%). When the I summarized in Table 3. column was eluted with pure diethyl ether (R)-chlorofluoro acetic acid (36 mg, 77%) was recovered. The spectral data were Resolution of Chlorofluoroacetic Acid. 12-To a solution of identical with those for (R,R)-2b given in Table I. racemic chloroftuoroacetic acid (16.8 g, 150 mmol) in ethyl 3 I acetate (125 cm ) at 0 °C was added a 0 °C solution of(S)-(-) a-methylbenzylamine (18.2 g, 150 mmol) in ethyl acetate (125 Acknowledgements 3 cm ). After mixing thoroughly, the resulting solution was set This work was supported in part by the Program in Science aside at 0 °C for 2 h and then at room temperature for 3 h and Technology Cooperation, U.S. Agency for International I during which time the two diastereoisomeric salts were Development, under Grant DHR-5600-G-00-1051-00 (L. S.), deposited as a white solid (33 g), m.p. 115-126 °C (decomp.). and by the Czech Grant Agency (L. S., A. S., J. V., Grant Fractional recrystallization from acetone afforded the (S,R) 203/93/0102), and in part by a grant to J.M. (GM 48088) from diastereoisomeric salt (8.1 g, 35 mmol, 46%) (about five the National Institutes of Health. I recrystallizations), m.p. 143-145 °C (decomp.); [ex][/ -11.5 (c 3.80 in MeOH). Determination of the diastereoisomeric excess of the resolved salts was carried out by GC of the References corresponding ethyl chlorofluoroacetate enantiomers on I J. A. Dale and H. S. Mosher, J. Am. Chem. Soc., 1968, 90, 3732. I 2 J.A. Dale,D. L.DullandH.S.Mosher,J. Org. Chem., 1969,34,2543. Lipodex A [a sample of the ethyl ester was prepared by mixing 3 L. Streinz, I. Valterova, Z. Wimmer, M. Budesinsky, D. Saman, (S)-( - )-a-methylbenzylammonium salt (10 mg) with Dowex J. Kohoutova, M. Romanuk and J. Vrkoc, Collect. Czech. Chem. 3 50W-X8 exchange resin (50 mg) in absolute ethanol (0.5 cm ); Commun., 1986, 51, 2207. I the mixture was set aside for 48 h prior to GC analysis]. In a 4 Y. Takeuchi, H. Ogura, Y. Ishii and T. Koizumi, J. Chem. Soc., similar manner, (R)-( + )-a-methylbenzylamine afforded the Perkin Trans. I, 1989, 1721. pure (R,S)-diastereoisomeric salt, m.p. 143-145 °C (decomp.); 5 G. Hamman and M. Barrele, J. Fluorine Chem., 1987, 37, 85. 5 6 Y. Takeuchi, M. Asahina, K. NagataandT. Koizumi,J. Chem. Soc., [cx]~ 11.2 (c 3.80 in MeOH). Perkin Trans. I, 1987,2203. I The CFA was released from its a-methylbenzylammonium 7 Y. Takeuchi and N. Nojiri, Tetrahedron Lei/., 1988, 29, 4727. salt by means of acid. Thus, the (R,S)-diastereoisomeric salt 8 Y. Takeuchi, H. Ogura and Y. Ishii, Chem. Pharm. Bull., 1990, 38, (0.85 g, 3.64 mmol) was covered by methylene dichloride (2 2404. 3 3 9 Y. Takeuchi, N. Itoh, H. Note, T. Koizumi and K. Yamaguchi, J. Am. cm ) and concentrated hydrochloric acid (0.37 cm , 3. 74 mmol) Chem. Soc., 1991, 113, 6318. I was added to it. After being stirred for I h at room temp., the 10 G. Bellucci, G. Berti, A. Barraccini and F. Macchia, Tetrahedron, solution was dried over magnesium sulfate and then con- 1969, 25, 2979. I I I 3512 J. CHEM. SOC. PERKIN TRANS. I 1994 I l l J. A. Young and P. Tarrant, J. Am. Chem. Soc., 1949, 71, 2432. 18 G. S. Toole and F. J. Sowa,J. Am. Chem. Soc., 1937, 59, 1971. 12 D. L. Pearson, Ph.D. Thesis: The synthesis ofenantiomerically pure 19 D. Parker, Chem. Rev., 1991, 91, 1441. inhalation anesthetics-halothane and enfturane, Cornell University, 20 K. Komarek, L. Hornova and J. Churacek, J. Chromatogr., 1982, 1990. 244, 142. 13 G. Bellucci, C. Bettoni and F. Macchia, J. Chem. Soc., Perkin 21 I. 0. 0. Korhonen, J. Chromatogr., 1984, 288, 329. I Trans. 2, 1973, 292. 22 E. T. McBee, D. L. Christman, R. W. Johnson and C. D. Roberts, 14 A. Hassner and V. Alexanian, Tetrahedron Lett., 1978, 46, 4475. J. Am. Chem. Soc., 1956, 78, 4595. 15 H. Wiener and C. Gilon, J. Macromo!. Cata!., 1986, 37, 45. 16 A. Svatos, I. Valterova, D. Saman and J. Vrkoc, Collect. C=ech. I Chem. Commun., 1990, 55, 485. Paper 4/02810D 17 J. G. Traynham, J. Am. Chem. Soc., 1952, 74, 4277; B. H. Johntson, Received 12th May 1994 A. C. Knipe and W. E. Wats, Tetrahedron Lett., 1979, 43, 4225. Ac_cepted 2nd August 1994 I I I I I I I I I I t I ( I I I I © Copyright 1994 by the Royal Society of Chemistry I I I I THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 269, No. 37, Issue of September 16, pp. 22937-22940, 1994 Communication © 1994 by The American Society for Biochemistry and Molecular B10logy, Inc. I Printed m US.A. I Inhibitors of Arachidonoyl (3), homo-y-linolenoyl ethanolamide, and docosatetraenoyl eth anolamide ( 4, 5) are naturally occurring brain constituents that Ethanolamide Hydrolysis* bind to CBRl (6). Anandamide behaves as a cannabimimetic compound in vitro, stimulating receptor-mediated signal trans 1 Received for publication, June 25, 1994. and in revised form, duction that leads to the inhibition offorskolin-stimulated ad I July 20. 1994J enylate cyclase (5, 7, 8). In a neuroblastoma cell line, anan Bohumir Koutek:l:*, Glenn D. Prestwich:j:iJ, damide causes partial inhibition ofN-type calcium currents via Allyn C. Howlett[[, Suzette A. ChiniJ, a pertussis toxin-sensitive G protein pathway, independently of I David Salehanii!, Nima AkhavaniJ, and cAMP metabolism (9). Dale G. DeutschiI':'* An amidase activity, which degrades anandamide to arachi From the Departments of +Chemistry and donic acid and ethanolamine, has recently been detected in W3iochemistry and Cell Biology, State University of membrane fractions of the brain, but not in the heart or other New York, Stony Brook, New York 11794 and the muscles, and phenylmethylsulfonyl fluoride (PMSF) was found I !iDepartment of Pharmacological and Physiological Science, Saint Louis Unwersity School of Medicine, to be a potent inhibitor of this activity (10). Inclusion of PMSF St. Louis, Missouri 63104 in receptor binding assays increases the apparent potency of anandamide (8). We report herein the synthesis of several types I Arachidonoyl ethanolamide (anandamide) is a natu of specific inhibitors of anandamide hydrolysis, their ability to rally occurring brain constituent that binds to a specific inhibit anandamide breakdown in vitro, and their affinity as brain cannabinoid receptor ( CBRI). An amidase activity ligands for CBRl in vitro. We show that one of these analogs is (anandamide amidase) in membrane fractions of brain a very effective inhibitor of anandamide metabolism in intact and in cultured neuroblastoma cells rapidly degrades I neuroblastoma cells and also binds to CBRl. Inhibitors that anandamide to arachidonic acid (Deutsch, D. G., and block the catabolism of anandamide may be biologically signif Chin, S. (1993) Biochem. Pharmacol. 46, 791-796). In the icant in the study of anandamide in vivo. current study, analogs of anandamide representing I three classes of putative transition-state inhibitor (tri EXPERIMENTAL PROCEDURES fluoromethyl ketones, a-keto esters, and a-keto amides) were synthesized and tested as inhibitors of anandam Synthesis-Fatty acid ethanolamides (1) were prepared from fatty acid chlorides and ethanolamine as described (4). a-Ketoamides l2l ide hydrolysis in vitro and as ligands for CBRL The tri were prepared via the ethyl esters (3) as key intermediates. Thus, I fluoromethyl ketones and a-keto esters showed nearly alkylation of the sodium salt of the anion of2-carboethoxy-1.3-dithiane 100% inhibition of anandamide hydrolysis in vitro at 7.5 in dimethylformam1de/benzene with the appropriate long-chain alkyl µJ\I inhibitor and 27.7 µM anandamide. Arachidonyl tri bromide (11), followed by hydrolysis with CH3VCaCO/CH3CN!Hp (12), fluoromethyl ketone was the only synthetic compound gave the resulting a-keto ethyl esters (3). Hydrolysis of the esters with in the series of fatty acid derivatives able to displace dilute base followed by treatment of the a-keto acids with 2,3-dioxo-5,7- 3 I [ diphenyldihydrothieno[3,4-b]dioxin-6,6-dioxide (Steglich reagent) (13) H]CP-55940 binding to CBRI with a K; of 0.65 µM. It was also the most effective inhibitor in intact neuroblastoma gave an activated cyclic oxalate that, when treated with ethanolamine, cells, leading to a 12-fold increase of cellular anandam provided high yields of the a-keto ethanolamides (21. Trifluoromethyl ketones 4a-4e were synthesized by reaction of the correspondmg car ide levels at 12 µM. From the action of these inhibitors on boxylic acid with CH 0CHC1 to give the acid chloride (14) followed by I this hydrolytic enzyme, it seems likely that anandamide 3 2 an in situ reaction (15) of the acid chloride with pyridine and trifluoro is cleaved by a mechanism that involves an active-site acetic anhydride to produce, after hydrolysis during aqueous workup, serine hydroxyl group. These inhibitors may serve as the trifluoromethyl ketones 4. useful tools to elucidate the role anandamide plays in Enzyme Assay in Vitro and in Cell Culture-The assay of the anan I vivo. damide amidase in vitro and in intact neuroblastoma cells was per formed as described previously (10). To measure the in vitro mhibition of the amidase, each compound (7.5 µM) was preincubated in a buffer consisting of 300 µg of crude rat brain homogenate protein, 500 µg/ml I ..l 4-Tetrahydrocannabinol, the psychoactive marijuana plant fatty acid-free bovine serum albumin, in phosphate-buffered saline in a derived cannabinoid, binds to a specific brain cannabinoid re final volume of 1.0 ml, for 10 min at 37 °C. Substrate (27.7 µM anan 3 ceptor, CBRl 1 (1, 2l. Arachidonoyl ethanolamide (anandamide) damide + 0.2 µCi of 221 Ci/mmol [ H]anandamide ([arachidonyl- 5,6,8,9,ll,l2, l4,15-3H]ethanolamide)) from DuPont NEN (NET-1073) was then added and the samples incubated for 10 min. I ·This work was supported by Grants R01-DA03690, R01-DA06912 To measure the inhibition of anandamide amidase in intact neuro and K05-DA00182 (to A. C. H.l and R03-DA07318 (to D. G.D.) from blastoma cells, two separate experiments were performed, one with 1 x NIDA, National Institutes of Health, by Grant R01-GM44836 (to 106 and one with 4 x 106 neuroblastoma cells (N18TG2)/6-cm dish. G. D. P.1 from the National Institutes of Health, and by a United States Experimental cells were incubated in 2 ml of media, consisting of Agency for International Development traineeship lto B. K.J under Hams's F-12/Dulbecco's modified Eagle's medium (Life Technologies, I Grant DHR-5600-G-00-1051-00 from the Program in Science and Tech Inc.) with penicillin, streptomycin, and gentamicin plus 10% bovine calf nology Cooperatrnn. The costs of publication of this article were de serum (HyClone, Logan, UT), plus inhibitor for 20 min. [3H]Anandam frayed in part by the payment of page charges. This article must there 3 fore be hereby marked "advertisement" in accordance with 18 U.S.C. ide W.2 µCi of 221 Ci/mmol of [ H]anandamide) was added and the Section 1734 solely to mdicate this fact. incubation continued for 1 h. Control cells contained no inhibitor. At the end of the incubation, the cells were washed once with cell culture I )i On leave from the Institute of Organic Chemistry and Biochemistry, Academy of Sdences of the Czech Republic, 16610 Praha 6, Czech Republic. 55940, Compound Pfizer [fo,2/3CR ),50<]-(-J-5-(1,1-dimethylheptyl)-2-[5- To whom conespondence should be addressed: Dept. of Biochem hydroxy-2-(hydroxypropylcyclohexyl]phenol; PMSF, phenylmethylsul istry and Cell Biology, State University of New York, Stony Brook, NY fonyl fluoride; HU-243, Hebrew University 11-hydroxy-hexahydro I 11794-5215. Tel.: 516-632-8595; Fax: 516-632-8575. cannabinol-3-dimethylheptyl hor,1olog of .!l.9-tetrahydrocannabinol; 1 The abbreviations used are: CBRl, cannabin01d receptor 1; CP- CBR2, cannabinoid receptor 2. I 22937 I I I 22938 Inhibitors of Anandamide Breakdown media and removed from the plates, after a brief incubation with 2 ml RESULTS AND DISCUSSION of 0.05% trypsin in 0.53 mM EDTA solution at 37 °C. The amounts of 3 3 3 The anandamide analogs prepared (Fig. 1) represent three I [ H]anandamide, [ H]phospholipids, and [ H]arach1donate in the cells and media were quantified by liquid scintillation counting of the silica classes of putative transition-state inhibitors: trifluoromethyl scraped from the appropriate areas of the plate after quenching the ketones (4), a-keto ester (3), and a-keto amide derivatives (2). reaction with chloroform:methanol (1:1), extraction of the sample from The general strategy is based upon the hypothesis that polar the organic phase, and TLC analysis on channeled silica gel-coated ized carbonyls (17), such as those in trifluoromethyl ketones I plates, with a solvent system consisting of the organic layer of an ethyl and a-keto carboxylate derivatives, may form stabilized hy acetate:hexane:acetic acid:water (100:50:20:100) mixture. drates or enzyme adducts that mimic the tetrahedral interme Receptor Binding-For the CBRl ligand binding determinations, diates formed during the reaction between the nucleophilic brain membranes were prepared from frozen rat brains (Pel-Freez) residue (e.g. the active-site serine hydroxyl of the hydrolytic according to the procedure published by Devane et al. (16), with the I enzyme) and the carbonyl group of anandamide (Fig. 2). Hence, exception that PMSF was present at 10 µM during the final incubation at 30 °C. Quantitation of the binding of the fatty acid analogs to CBRl several trifluoromethyl ketones have been identified as strong was performed by incubating the analogs at the indicated concentration inhibitors of hydrolytic enzymes having varying specificity to with 30 µg of membrane protein in a buffer contaming 500 pM of the ward insect juvenile hormone esterase (18-21) and other hy I 3 bicyclic cannabinoid analog [ H]CP-55940, 20 mM Tris-Cl, pH 7.4, 3 mM drolases (22-24). A trifluoromethyl ketone analog of arachi MgC12, 1 rm1 Tris-EDTA, and 0.135 mg/ml fatty acid-deficient bovine donic acid ( 4a) was found to be a slow, tightly binding inhibitor serum albumin in a final volume of 200 µ!in Regisil-treated glass tubes. of a novel 85-kDa cytosolic human phospholipase ~ (25, 26). Specific binding was defined as that which could be displaced by 100 mr Some a-keto acid derivatives have been shown to act as inhibi I desacetyllevonantradol. After 60 min at 30 °C, the incubation was ter tors of serine and cysteine proteinases (27-29), and effective minated by the addition of 250 µl of 50 mg;lml bovine serum albumin inhibition of cathepsin B and papain by peptidyl a-keto esters, and the immediate filtration over GF/B filters and washing with ice a-keto amides, a-diketones, and a-keto acids has also been cold buffer (20 mM Tris-Cl, pH 7.4, 2 mM MgC12 ). The filters were treated I with 0.1 o/c sodium dodecyl sulfate pnor to addition of scintillation mix demonstrated (30). ture and counting in a liquid scintillation counter. When tested for their ability to inhibit the hydrolysis of anandamide in vitro, the most effective class of compounds, as 0 I 1a. R =CH 2(CH=CHCH2)<(CH2J,CH3 (anandamtde) 120 1 b. R = (CH2),4CH3 I A~NHCH CH 0H 1 c· R =(CH2)12CH3 >- 2 2 1 d R =(CH2liaGH3 f- >;::: 100' 1 0 <( UJ T I (/) <( 2a: R = (CH:i)"CH3 0 80 ~ 2b R (CH2)14CH3 = ~ 2c R =(CH2)12CH3 <( ' LL 0 z 60' I 0 2 ;::: w :;: 3a R =(CH 2)1sCHa 40 ~ 3b. R =(CH 2),4CH3 ~ T 3c. R = (GH2h2CH3 f-z 'i T UJ i I 0 a:: 20' 3 UJ 0.. ! 0 3a 3b 3c .fa 4-b -k .1-d 4e I ANANDAMIDE ANALOGS 4 Fm. 3. Inhibition of anandamide hydrolysis by synthetic com Fie. 1. Chemical structures of synthetic compounds. Four pounds. Three hundred micrograms of rat brain homogenate protein classes of compounds were synthesized: fatty acyl ethanolamides (1), was incubated with 7.5 µM inhibitor and 27.7 µM anandamide as de I a-keto ethanolamides (2), a-keto ethyl esters (3J, and trifluoromethyl scribed under "Experimental Procedures." Approximately 100 nmol of ketones (4) as described under "Experimental Procedures." All were arachidonic acid/h/mg of protein was produced in the control containing purified to homogeneity on silica gel and fully characterized by mass no inhibitor. The blank was determined by incubation in the presence of spectrometry and NMR ('H, 19F), and experimental details are available 200 µM PMSF. In this representative experiment, the value for each I from G. D. P. on request. See Table I for nomenclature. inhibitor was determined in triplicate. Amid~e H N-./'oH co,H Ethanolamine I Anandamide Arachidonic Acid Hp I 1l Enz-SerOH CF3 I HO OSerEnz First Tetrahedral Intermediate "Transition State" Analog Adduct I Fm. 2. Postulated mechanism of amidase inhibition by polarized carbonyls. Enzymatic hydrolysis of arachidonoyl ethanolamide (anandamide, la) is postulated to proceed by a tetraliedral intermediate, which can be mimicked by the adduct formed from trifluoromethyl ketone I and an active-site serine residue (boxed). ~v ·:-~cc~:;::':/~:?"'e~~ I ' I I Inhibitors of Anandamide Breakdown 22939 1200 ~~~~~~~~~~~~~~~~- TABLE I Competitive znteractions of anandamide analogs with fHJCP-55940 I 1100 - :;- binding to CBRI in rat brain membranes 0 a:: 1000 - The CBRl ligand binding determinations were as described under zf- "Experimental Procedures." Fatty acid analogs were incubated with the 0 900 - u :'! 6000 - ligand binding reaction mixture at final concentrations of 10 µM. Data "- 800 - 3 I 0 ~ 4000 - • are shown as specific binding of [ H]CP-55940, which was 100% in the z 2000 - presence of vehicle alone. The values are the mean and standard error 700 -- ~ (f) _.J of mean for three to five experiments for each compound. _.J 600 - "' w 0 2 4 6 8 10 12 14 16 u Percent CBRl [ARACHIOONYL,CF KETONE] uM Compound (10 )lM) I ;;; 500 - 3 specific binding w 0 400 - lb, stearoyl ethanolamide 99.1±4.8 ~ <( le, palmitoyl ethanolamide 85.5 ± 8.5 0 z ld, myristoyl ethanolamide 102.1±21.4 <( z 2a, 2-oxoeicosanoyl ethanolamide 102.6 ± 5.0 I <( 2b, 2-oxostearoyl ethanolamide 86.6 ± 9.5 2c, 2-oxopalmitoyl ethanolamide 80.6 ± 5.5 1__ • ___.1 __ 3a, ethyl 2-oxoeicosanoate 82.9 ± 18.0 ~-·•0 ____1 _____l . ___ § 3b, ethyl 2-oxostearate 102.7 ± 4.0 I u 3c, ethyl 2-oxopalmitate 93.0 ± 10.7 4a, arachidonyl trifluoromethyl ketone 21.4 ± 6.1 a 4b, y-linolenyl trifluoromethyl ketone 76.2 ± 14.4 4c, stearyl trifluoromethyl ketone 96.3 ± 8.2 4d, palmityl trifluoromethyl ketone 85.6 ± 7.3 I COMPOUND 4e, myristyl trifluoromethyl ketone 94.6 ± 6.0 FIG. 4. The effect of amidase inhibitors on anandamide levels " Significantly different at 0.05 by ANOVA. in neuroblastoma cells (N18TG2). The amount of [3H]anandamide was determmed in the control and experimental cultures containing 1 6 x 10 cells. after separation by thin layer chromatography and analysis 100 I of the silica gel sample scraped from the plate, by liquid scintillation counting. One hundred percent anandamide in the control cells corre cC> -6 80 sponds to 1.3'k of the total radioactivity detected in all the fractions c analyzed as descnbed under "Experimental Procedures." The inset a; shows the effect of increasing arach1donyl trifluoromethyl ketone con J! 60 I centration upon [3H]anandamide levels. Each incubation contained 4 x 5 6 Cl. 10 cells. Cf)" 40 Ci! CD u 20 Analog K, nH I shown in this representative experiment, were the trifluoro "' • ethanolam1de 16 .9 • -CF 3 ketone 650 6 methyl ketones (4a-4e) and a-keto esters (3a-3c) (Fig. 3J. 0 Arachidonyl trifluoromethyl ketone (4a) and ethyl 2-oxostear 1 10 100 1000 10000 ate (3b) were the most active members of these groups yielding [Arachidonyl analog] (nM) I nearly 100% inhibition of the enzyme. The least potent inhibi Fm. 5, -Log--dGSe-response -curve-for- arachidonyl--trifluoro tors were the a-keto amides (2a-2cJ and the saturated analogs methyl ketone and arachidonoyl ethanolamide competition of anandamide (lb-Id). The curve of velocity versus substrate with [3H]CP-55940 binding to CBRL The indicated concentrations concentration for anandamide hydrolysis by the amidase in the of the arachidonyl trifluoromethyl ketone and arachidonoyl ethanol amide were mcubated as described under "Experimental Procedures." I brain homogenate was nonhyperbolic. This might be antici Binding competition experiments were performed a minimum of three pated for an interfacial enzyme reaction, occurring in a crude times, and the data were analyzed by ANOVA and Tukey's post hoc test. homogenate, whose substrate and product have the potential to Heterologous binding data were analyzed for IC50 and slope factor (nH) values using the Graphpad Inplot program. K, values were calculated form micelles and to denature the membrane proteins. How 3 using the equation K, = IC5ofl + ([ H]CP-55940/Kd CP-55940) where the I ever, the inhibition of anandamide amidase by arachidonyl tri 3 Ka for [ H]CP-55940 was 350 pM, as determined by Scatchard analysis fluoromethyl ketone was reversible with increasing concentra using this assay. Each point on the curve represents the mean and tions of anandamide (data not shown). standard error of mean of four or five experiments. When anandamide is incubated with neuroblastoma I (N18TG2J cells, it is rapidly hydrolyzed to arachidonate, which is then converted to other lipids containing arachidonate (10). radation in cell culture. It is not known if arachidonyl triflu However, in the presence of arachidonyl trifluoromethyl ketone oromethyl ketone is metabolized in the neuroblastoma cells (4a) there is approximately 5-fold increase of anandamide lev employed in this study, but when incubated at 10 µM with I els (from 1.3% in the control cells to 6.3% in the experimental monocytic cells in culture for 10 min, 10% is converted to the cells) at 7.8 µ11-1 arachidonyl trifluoromethyl ketone (Fig. 4). The corresponding alcohol (31). amount of anandamide in the experimental cells increases to a The series of fatty acid derivatives were each tested, at 10 I 12-fold maximum, relative to the control cells, at approxi µM, for their ability to displace [3HJCP-55940 binding to the mately 12 µM arachidonyl trifluoromethyl ketone (Fig. 4, inset). tetrahydrocannabinol receptor in rat brain membranes The mechanism apparently involves inhibition of the amidase (CBRl). Arachidonyl trifluoromethyl ketone was the only syn rather than increased uptake of anandamide, since preloading thetic compound that exhibited significant competition with 3 the cells with labeled anandamide and then treating with 4a [ H]CP-55940 as shown in Table I. The competitive displace I 3 also resulted in a dramatic increase in anandamide levels in ment of[ H]CP-55940 by arachidonyl trifluoromethyl ketone at the cells. Of the two other in vitro inhibitors that were tested, various concentrations indicated that the K, was 0.65 µM, as y-linolenic trifluoromethyl ketone (4b) was a weak inhibitor shown in Fig. 5. This represents approximately a 15-40-fold I and ethyl 2-oxostearate (3b) was ineffective in cell culture. decrease in potency from that originally reported for arachido PMSF was also an effective inhibitor in the neuroblastoma noyl ethanolamide (3) by displacement of the cannabinoid re cells. The disparity between the activity of 3b in vitro and in ceptor probe [3H]HU-243 and that determined in the present I cell culture may be due to its susceptibility to enzymatic deg- assay. I I I 22940 Inhibitors of Anandamide Breakdown Ideally, a selective amidase inhibitor would antagonize the 7. Vogel, Z., Barg, J., Levy, R., Saya, D, Heldman, E., and Mechoulam, R. (1993) I J. Neurochem. 61, 353-355 enzyme at concentrations that fail to appreciably bind to can 8. Childers, S. R., Sexton, T., and Roy, M. B. (1994) Biochem. Pharmacol. 47, nabinoid receptors. Furthermore, unlike PMSF, an inhibitor 711-715 should not be toxic to the cells. Many of the synthetic com 9. Mackie, K., Devane, W. A., and Tulle, B. (1993) Mal. Pharmacol. 44, 498-503 pounds in this study fulfill this criteria in that they do not bind 10. Deutsch, D. G., and Chin, S. A. (1993) Bwchem. Pharmacol. 46, 791-796 I 11. Elie!, E. L .. and Hartmann, A. A. (19721 J Org. Chem. 37, 505-506 significantly to CBRl at concentrations that inhibit amidase 12. Sollad1e, G. and Ziam-Cherif, C. (1993) J. Org. Chem. 58, 2181-2185 activity by greater than 90% in cell-free preparations. Arachi 13. Steglich, W., Schmidt, H • and Hollitzer, 0. (1978) Synthesis 62~24 14. Ottenheim, H. J. C , Tijhuis, M. W., Last, L.A., and Coates, R. M. (1978) Org. donyl trifluoromethyl ketone is very interesting because it ex Synth 61, 1-4 I hibits dual effects on the degradative enzyme and at the recep 15. Boivin, J., El Krum, L., and Zard, S. Z. (1992) Tetrahedron Lett. 33, 1285-1288 tor in cell culture, where it may be a useful stable analog of 16. Devane, W. A., Dysarz, F. A., Johnson, M. R., Melvin, L. S., and Howlett, A. C. l1988) Mal Pharmacol. 34, 605--B13 arachidonoyl ethanolamide. The role that these inhibitors play 17. Mehdi, S. (1993) Bioorg. Chem. 21, 249-259 in different tissues, such as spleen, where a peripheral receptor 18. Hammock, B. D., Wing, K. D., McLaughlin. J • Lovell, V. M , and Sparks. T. C. (CBR2) exists (32) or as inhibitors of the cytosolic phospho (1982) Pestic. Bwchem. Physiol. 17, 76-88 I 19 Prestwich, G. D., Eng, W S., Roe, R. M., and Hammock, B. D (1984) Arch. lipase Ai (31) in brain and N18TG2 cells remains to be eluci Bwchem. Biophys. 228, 639-645 dated. Our approach provides the framework to select candi 20. Szekacs, A., Halarnkar, P. P., Olmstead. M. M , Prag, K A , and Hammock, B. date drugs for animal studies. The development of inhibitors D (1990) Chem. Res. Toxicol. 3, 325-332 21. Shiotsuki, T., Huang. T. L , Uematus, T.• Bonning, B C , Ward, V. K.. and I that block the breakdown of anandamide may be significant Hammock, B. D. (1994) Protein Exp. Punf. 5, 296-306 therapeutically in any of the areas that ll9-tetrahydrocannabi 22 Gelb, M. H .. Svaren. J. P., and Abeles, R. H., (1985) Bwchemistry 24, 1813- 1817 nol (33) and anandamide (34, 35) has been shown to play a role, 23 Allen K. N., and Abeles, R.H. (19891 Biochemistry 28, 8466-8477 including analgesia, mood, nausea, memory, appetite, sedation, 24 Brady, K .. Liang, T. C .. and Abeles, R. H. (1989) Biochemistry 28, 9066-9070 I locomotion, glaucoma, and immune function. 25 Street, I. P., Lin, H. K., Laliberte, F., Ghomashchi, F, Wang. Z., Perrier, H., Tremblay, N. M., Huang, Z., Weech, P. K., and Gelb, M. H. (1993) Biochem thank Keith Baker and Cathy Cantrell for istry 32, 5935-5940 Acknowledgments-We 26. Trrmble, L. A, Street, I. P, Perrier, H., Tremblay, N. M., Weech, P. K., and technical assistance with the CBRl radioligand binding assays and Bernstein, M. A. (1993) Bwchemistry 32, 12560-12565 I Rebecca Rowehl for the cell culture. 27. Angelastro. M. R. Mehdi, S., Burkhart, J.P., Peet, N. P, and Bey, P. 11990) J. Med. Chem. 33, 11-13 REFERENCES 28. Peet, N. P., Burkhart, J P., Angelastro, M. R., Giroux, E. L , Mehdi, S., Bey, P., 1. Howlett, A C., Bidaut-Russell, M., Devane, W. A, Melvin. L S., Johnson, M Kolb, M.. Neises, B., and Schirlin, D. (1990) J Med. Chem. 33, 394-407 R., and Herkenham, M. tl990) Trends Neurosci. 13, 420-422 29 Ocain, T D., and Rich, D. H. (1992) J. Med. Chem. 35, 451-456 2. Matsuda, L A., Lolait, S. J., Brownstem, M. J., Young, A. C • and Bonner, T. I. 30 Hu. L. Y., and Abeles, R. H. l1990) Arch. Biochem. Biophys. 281, 271-274 I 31. Riendeau, D., Guay, J .. Weech. P. K., Laliberte, F., Yergey, C. I., Desmarais. S .. (1990) Nature 346, 561-564 3. Devane, W. A., Hanus, L., Breuer, A., Pertwee, R. G., Stevenson, L.A., Griffin. Perrier, H .. Lm, S., Nicoll-Griffith, D., and Street, I. P. (1994) J. Biol. Chem. G., Gibson, D., Mandelbaum, A, Etinger, A., and Mechoulam, R. (1992) 269, 15619-15624 Science 258, 1946-1949 32. Munro. S., Thomas, K. L., and AbuShaar. M. '19931 Nature 365, 61-65 4 Hanus, L., Gopher,A.,Almog, S., and Mechoulam, R. (1993JJ Med. Chem 36, 33. Mechoulam. R (1986) Cannabmoids as Therapeutic Agents, CRC Press, Boca I 3032-3034 Raton, FL 5. Felder, C. F., Briley, E. M.,Axelrod,J., Simpson, J. T., Mackie, K.. and Devane, 34. Fride, E .. and Mechoulam, R. (1993) Eur. J. Pharmacol. 231, 313-314 W. A. (1993) Proc Natl. Acad. Sci U.S. A. 90, 7656-7660 35. Crawley, J. N., Corwm, R. L., Robinson, J. K., Felder, C. C , Devane. W. A., and I 6 Devane, W. A. ( 1994) Trends Pharmacol. Sci. 15, 40-42 Axelrod, J. (1993) Pharmacol. Bwchem Behav. 46, 967-972 I I I BEST AVAILABLE COPY I I I I I I - a a of in of to of the in al., 1994 The sco in The on - corn Ostri et (Borek 1984). phero Pyrali female the range predomi order the blend origin borer, utilizes 1210-5759 mainly established reared al., borer, the 1975). 3 In pheromone populations 197-203, acetate. (Klun isomers et being that during in Slovakia ISSN of corn 97: al., E corn were preliminary 91: as pheromone synthetic 1 Republic a occurs et recordings. A (Hubner) 1986). a Sciences, of population and and found population, of (Lepidoptera: has Z analyzed l-14:Ac) Anglade aL, mosaic this use geographical Czech strain indicated was (EAG) E111omol. European a discriminate El 6, et the to KOTERA E population European generations J. were Germany nubilalis that the amounts to detected. the as detected. of strain 11-tetradecenyl 1975; of Moravia nuhilalis and to the of the Academy (Kochansky Eur. Praha 1111bilalis local Z of acetates (Hrdy were al., 0. glands of 10 Further were showed Erlangen-Niirnberg, Ostrinia the ability LADISLAV blend attempts et occurs maximal South Czech of of 166 of Republic females l-14:Ac and blends local in whereas vakia l-14:Ac 1 ·z, 2 The i~omers Ostrinia larvae. 1984) enough a;:cording Ozechoslovakia Zll so-called (Klun males' Slo EI the. produce trapping METHODS pheromone colony electroantennographic nam. Federal al., identity hybrid 1111bilalis to of and vary world, Universi:ty and The and borer, strain. for individual et The MINAIF the 0. intermediate and AND States field former to Biochemistry, Z 8. (E)-11-tetradecenyl 11 efficient the E (abbrev. diapausing geometric Pyralidae), Z used an in known from corn to 16: and laboratory the as Erlangen, and and Moravia of not experiments. of INTRODUCTION a ALFRED dialect" are Flemingovo , found to analyzed 1 United were LD systems. Anglade Chemistry, ----- on Europe, using confirmed a (Z)- A 8520 glands fields acetates South field were old Unit, MATERIAL blend of which been 42, and 111 in throughout mixture European responding 1979; under belong corn trapping 96 Organic a days populations has ratio 3 hybrids the 4: of performed as (Lepidoptera: - KALINOV chemically to Ecology a "pheromone from pheromone females, pheromone 1961) of experiments northeastern 2 however, Organic Chemistry The Maini, of America were characterisation originating has of 11ubilalis, Individuals sex probably Henkestrasse & were distributed and Institute isomers 1111bilalis Field virgin ,q,atry, investigated 2 BLANKA Males (Nagy. Chemical S] nubilalis ). identified collected possible system. 0. - old North the area strain 0.5. analysis strain. -'~nct a was diet Institute (E)-11-tetradecenyl vf (Klun Ostrinia 1 of glands (Hilbner), E Z In of pheromone widely 1991 distinct monitoring, Insect been this The Experiments 3-day is the pheromone and sex The in in areas Ostrinia for of Switz. has I- two 1.5-99.5: 35:65 individuals pheromone moths. Sex nubilalis the The Insects: strain Kalinovri, Pheromone, Abstract. nantly "Z" nia 98.5: 1973). the with dae), (Z)-1 proportion Italy, In Z ratio vestigation borers mone pheromone characterize composition from tophasc semi-synthetic & -- - -- - - -- ~. ... ~ ...... - t '\P """'='>!. f"'~ -- b:J ~ .... h. ~ h tlJ !""-- § r"l't (') ""< "tJ a - to I "' ~I ~-....:\\ observed ' contained •D O> I'- Ll.J "' N ~~1 responded ... N were c "' "' "' N Ll.J "' ... "' also ,, 9.u ~ JO . samples N -- a O> I'- Ll.J "' N ~ ' u.., ~ ;u ...... ~ ~~"l ' .. " .. ~ .. ()~-- glands. N "' u + ll-14:Ac O> z ... O> 0 b " 0 - - Ll.J '"' «; N (•in) " '"' Gland composition 2 antennae of O> O> Ll.J - N ~~~ - 0.5. lure : TIME 0 u + ... 0 - 0 I ;fl. <( N pheromone isomers in E 0 0 ;fl. N - - 1.5-99.5 experiments 0 : 50 A ~ 100 200 Zand detected rel 0.. Cii OJ u Cii Gl rel Gl c 98.5 0..150 ::i E 5 c E (/) g :E several · both - In - - were to in- range to ob GC tem fused virgin female lB). nubila and the A: 3 tricosan, Columns 14:Ac of mm Stnifoice, in 0. l-14:Ac Oven - pheromone - min significantly materials of (Fig. responses with be l-14:Ac 2 0.22 responses gland 1992. (Z)-1 not septa. C23 (Z)-ll-14:0H to female. for chromatogram (E)-1 males m x various heptacosan. are 4'C/min. baited (Z)-11-14:Ac. SP-2340. per - of 25 4-18, of - rubber at 60°C GC-EAD a acetate, and acetate, alcohol, letter traps C27 at µg pheromone BAD-active red on experiments July B: column number determined Reproducible 0.05. 95'C 1-14:Ac acetate, same JOO wing I held = l-14:Ac other active GC-FID-EAD Zl P was by the Mean to was and at 11ubilalis I. of No loaded on 2. (E)-1 Moravia, pentacosan, by from GC-EAD capillary responses - ratio ng caught Fig. all Fig. GC-EAD: 14:Ac, FID (E)-11-tetradecenyl (Z)-11-tetradecenyl tetradecyl 14:Ac. (Z)-11-tetradecenyl C25 tained Ostrinia females silica perature creased different South 3-5 in lis signed blends, Z:E -- - of or was on per the sup The - fe dis inter with the four com hex were con com 1975). glands one be were (delta injector injector tightly to samples period in spectro coupled was dose the technique flow compared The mrstream and to Captured I), responses relative al., inside were Stnifoice) were acetate were Traps information The of The an et were 1 : 1 were mass polar columns design were females (FID) 1987). sample Compounds The linear chromatography hung directly rubber 90 spectrometr. topically 0.05) analysis. hexadecanoic known 52 C22-27 into (Arn spectra mm). pheromone 1 i 1 ratio 'injection trap detached. is Traps gas precise of Antenna! locality. pheromone simultaneously B\lovice, of was SE efficiency. solid in 4°C/min. cag,e 20 (PT glands Mer1n, m. mass a MAT oven pheromone or were.dissolved x Mass dispensers during of (split 1 glandls. and 2ml/min & were dose, detection applied trap each types analyzed test used. the setup wire obtain (2 detector a components the of standar1s. the at -- (Velke esters rate (Z)-9-dodecenyl on to 2340 10 at adopt Capillary parts of 14:Ac To a were GC registered of production above. (Raina unsaturated were solid-sample female range EAG Three to In SP small at compounds synthetic pheromone made height out a two combined a a and and effect a 1. ionisation a the capillary of use. : in of methyl Moravia FINNINGAN changed. composition and those coupled led pheromone at composition, supplied into following were a inhibitors with calling were - 195'C, 2 active with extraction. evaporation, described multiple hormone a antennae 100 glass N lure modified to flame until (GC-EAD): pheromone (biological) to lure l-14:Ac the Compounds with apart : as position split spectra of South placed means activity, which amplified The of either m El direc,Iy was in regularly (EAG) 14 soda baited type, for with of inactive by was 60'C solvent solvent l-14:0H, linked old) 40 -20°C same which saturated use. Mass attraction treatments Isolated was in at and Duncan's splitless. 1984), - present. in possible ZI Zand detection was the space additional of other Traps segments olfactory set days After until respectively. under and equipped compared the an 4 spaced daily without analysts the dispenser old), were analyzed dispensers. performed obtained were 99%) was pheromonotropic column as with RESULTS to 4:Ac thm-walled effectiveness present injected l replicated traces (GC-MS): hrs a GC injector (Roelofs, and (2 were -20°C 52 field, 250'C, stored was blends. 1- l-14:Ac, preparation. were - 1987) The in not no at were chromatography chromatograph for examined. design, abdominal (24 checked analysis were SE regime Four served El minimize regime and pure(> The and (ANOVA) El instrument corn hydrocarbons, : al., IX gas to electroantennography gas that trap pheromone was of used daily. females were et spectra foil compounds detector, stored 1992. water, gland as of electrodes the and antenna! and/or and 220°C brain-released females spectra or antenna pheromone 5890A spectrometry 3400 samples pheromone type a edge electroantennographic sencs variance FID -14:Ac and experiments the virgm Of with gland ensure mass VIII encapsulated l-14:Ac, of glass temperature chemicals. column 2340 tested were Chemically the temperature l l The 3 of (MS) Capillary (Struble test recorded The of the I-14:Ac mass with Chrompack). to Solid Z ZI enabling detector. 1991 influence SP The over : was and various to Field Packard The El and with a acid. were m each VARIAN along dispenser i.d., decapitated the times absence FID the provided a acetate immediately Ag!AgCI 1987) capillary -- 1987). authentic gas. Analysis of 4:Ac analysis: identified row and the l the 14:Ac, mm on of by baited analyzed, the injection al., Hewlett within system were al., (Nitra) temperatures preparations: to spectrometry of Complete used removed of a membrane connected All of et l-14:Ac, et preparation: creating chromatograph. carrier retention 0.22 glands smgle this GC aluminium-polyethylene determine those experiments: due on a those chromatography: chromatography-mass polyethylene chromatography Ill analysis: ZI mass was in to were to gas In in either the obtained rotated the lo gale as glands: recorded of m x captures. detector Females outlet octadecanoic and wmg-type) Slovakia Pheromone Gland Gas response Gas Oas Field the Dispenser Data GC-MS: 1 i(Attygale ;(GC), were segmental with pressed formed (25 (Atty used and were metr. ,oltd-sample pared about to which The the continuously blowing were ically and placed moths and trap. pared ane (Z)-9-tctradecenyl pensers types scaled ducted moth 14:Ac, male identified. and abundance -- - + trap + + + 000 delta 7 type -- 6 dispenser trap - a 1 wing I 0 50 - 150 100 250 200 oL.._~--"-'"~~~~~-""'~'----' 10 0.. so~~~~~~~~~~~--, 0.. ~ ID Cl 40 :i c: Q) ::J ~ 30 20 Q) c: E jg m E E .:::: "iii 0 ..CJ :::J Cl) ro a.. Ol (lJ E u E ro c c (lJ Qi :::J E Qi ~50 iii 0 ..Q 1: - I 0. 0. I 00 I No. traps poly of of ++ rubber 1992. A + 000 females. transpar with red Cat. red - - - males males 5 polyethylene dispensers: 5 3 on of 1991. virgin of Co., wing-type baited 3 type 4 - -Strafoice, blend and 1-14, various 'i' B rubber; number - 'i' number on transparent "Z" Thomas 3 'i' July - delta- polyethylene; 1991; of wing-traps 6 dispenser Mean Mean I; by µg by "brown" c loaded 2 (A.H. rubber; - white - Bfiovice, 100 2 - Bfiovice, a blend (right). 4 (below). caught caught I~ I~ rubber 4 5 "grey" with Velke 0 "Z" 80 - 20 Velke polyethylene of 7 0.. Fig. Cl red () m c: :i E Fig. :i ID c m E jg .:::: Ci ..CJ - 780-J07); I A- II; ent septa. baited nubilalis µg ethylene; nubilalis - - Z The The con - were some other "Z" f the Bi.ichi hybrid· bed week j virgin differ of in f nubila on of be 000 Germany 3 explained Alps. Stnifoice, 2nd µg the and 0. pheromone (1988) be E of Z the [µg] with (1978), and 500 releasers. - 500 al. Note ede loaded present of individuals week al. !st the the et presence and 992. northern ·~1~ dose males I could et baited of of 2nd the of 100 septa. in 100 the degradation. south 0 blend, of Pena 4-18, traps of 50, Maini . moths "hybrid" only pheromone inherently 50 rubber week and number IO, July by pheromone catches wing as present 1st and red are prevalence by individuals pheromone reports by 0 Mean 10 hybrid a tests pheromone I moth of -- and localities (1985) "E" 3. reported used using Moravia, in o·- and strains 60 20 al. 40 "Z" 100~~~~~~~~~~~~ caught 1601 field Fig. E was et c: ID fij E 0 "@ previous indicating South lis females blend, were ences the several both of and southern revealed the - a simultaneously 1 2 occurrence to areas IE µg the in al., with ex- not and had The par- µg); than to 99: (100 dose from indi (Fig. 5). are septa et and 10 effec DISCUSSION confirm virgin (1975) the Barbattini did 99Z: females µg captures + 3 1780-107) presence the effective tests Analyses due However, (100 The gradually compared of al. "E" baited 14:Ac (Fig. - sporadic males most northern whether to attracted rubber strains The period, density. et µg) 2). 500 12 3Z:97E of all Strafoice No. (Struble Male more migration the area. (1984), responded virgin the 2). traps red In This generally initial in females borers. lower doses, and isolated fewer 99Z:IE be and most (100 Klun (Fig. in the 1992 al. in moths the probably to was hY,brid 4). decreased septa all to - et (Fig. In by by corn The question, addition this µg) in µg); 100, study Catalog of 1985). virgin above, compositions: caught population and were Nitra if (Fig. strain blend the caught experimental E 99Z:lE 50, The in this rubber population (100 al., and/or degradation. (100 or E Co., strain) lure with females, survey Z, were isomer. attracting Z appeared substantially "Z" the by et of instance Anglade substantially red a - moths µg); Z m1bi!alis European the raises in males the local "Z" blend. males the on for trap mentioned blend of were unchanged 0. virgin of males the 100% delta-type baited experiments: of pure (100 effectiveness during various 35Z:65E experimentation, that Male results Thomas The of an of of (1982), trapped population µg to borers effect Europe, followed the yheromone "Z" 0 loaded 97Z:3E The traps I al. In The that Field "hybrid" -- presence materials strain at (A.H. finned (Langenbruch corn periments field otherwbe, numbers slow wing-type than detected. 14:Ac; 1987). strain to in decline tive, cating of 3). during 2 "hybrid" little the blend the volved µg) (laboratory tially females; blend. 97Z:3E ~~ .. -- - .. the sex AL. !st. corn New 1993 moni of North Euro lapriky ET Prest phero Borer, phero obalece English strains. phero Alberta 33-53 artificial Boll. popolax- in for In 345-357. 30, European Entomol. Rughrge the zkusenos pheromone pheromone for R.E. various an 159-175. in - Sex 55: Corn field of 13: Insect im the of abstr.). Czech, European Can. 661-663. pp. with efficacity HlLL Prvnf with and campo. Ges. 1988: (in the Ecol. Pheromonstiimmen nubilalis releaser P., 1973: the di simplified 181: catches codlemone sessualle monitorovanf of feromonovymi M. a system: Springer-Verlag 1986: procedures Lepidoptera. on English European 51-55. of FELS P.E. accepted April zwei J. Orlando, on pro trap in 33-52 Carolina nubilalis, of Chem. Maisziinsler prove Entomol. components Pyralidae). Torn laboratory 225-231. J. 110: Ostrinia Science 125-131. von capture & Czech, den D.M., 1992; 1: Copulation Press, feromone of SoNNET North 22(1): (Hbn.) on Research. ZMREK 53: S. (in convenient 25, Ostrinia & relative in & de! formulation iiber various -- Schweiz. Ecol. age e 1987: production kodlemonu effectiveness pheromone V. pheromone M. Entomol. communication in Ceskoslovensku Appl. pheromone Rost!. attraction. MAIN! May v trap (Lepidoptera: the Mitt. DANIELSON Borer, to Academic Sex Vorkommen nubilalis, 129-140 R., variability Sex Can. Chem. Lymantriidae). Exp. trapping G.G.S. VALLO on Pheromone screening substrate Ochr. J. BEROZA ticinnost H.C., Corn in of Pyralidae) - - chemical D., 1975: (Hbn.) pheromone critical and na KtNG 1987: Received 22(2): 642-646. influencing borer nubilalis, Ostrinia & 150-156. P.W., York. of BRUNETTI Untersuchungen pheromone Entomol. Polymorphism 73: Effect Stidschweiz. W.L. insect Pyralidae). G.L. sull'identificazione CHIANG corn rapid UVTIZ F., of Rost/. 37: sympatrische Biochemistry. pheromonal isomer K.N. formulation PovoLNY factors New (Lepidoptera: European an nubilahs der Borer, 1985: Techniques lures. Ostrinia - m 891-894. of formulace L., 1989: AYRE O.L., in Das of the Shor. B. (Lepidoptera: 73-79. Ochr. 4: BIGLER regulation J. Entomol. & nub1lalis: assays. ROELOFS Some of Corn Ricerche 5: WoJTKOWSKI Vliv SLEssoR European Influence (eds): & S., Hbn, effect Hb. basis Ostrinia 1982: J. Pheromone Braun.1chw. L., T. (Lep1doptera: geometrical PosPECH 423-426. Econ. COPY intraspecific HosANG 1978: VRKOC leucostigma R. CHAPMAN K.C., 1978: D.G.R. pheromone Pyraliade), Hung. UVT!Z- 1986: J., 8: 1976: 1980: J. (The - Ostrinia Entomol. & & kukui'icneho, F., borer, Endocrine J. G. pomonella). (eds): 15-25. European sex nubilalis Genetic !008-1010. Miller Sci. K. nubilalis A.N. monitoring M. RAUSCHER Sbor DONALD T.E. 131-161. Orgyia LIEBHERR opposite nubilalis & the 5: 34: MATTES MAREK Borer, corn G.J. blends. BACA D.G.R. the McLEOD BRUNETTI 1987: of Cydia VRKOc PLATIA Entomol. pp. Environ. of 1979: MAC Acad. H.R., zavfJece & R.T., H.E. & pomonella. - & O:strinia pheromones: Electroantennogram O.L., 1359-1366. Corn J.J. Ostrinia P.L., Pj7Schutzdienst. with S. J.R., female M.:A., KONECNY WELLING (Lepidoptera: E. D., nigricana, STARRATT AVAILABLE Ostrinia K. G. ANDERSON moth, Bologna York, of I., de Entomol. 14: McLEOD Dt. amount & sex BusER European Europe. Rearing & Blomquist Environ. CARDE Cydia 1984: & component MENN G.A., MAIN! BYERS FRECH Cydia of H., & Hummel New borer, J., Phytopath. Univ. & and HRDY Ecol. CHAPMAN ANGLADE scales & nub1lalis BEST Czechoslovakia.) European PREISNER minor - G.G. In PALLOTTI codling Insect AN. KoNEcNY 1961: HuBAJSHAN experience D.G.R. W.L. in AKN Em,iron Borer. of D.L., A., G.G., Inc., .A. corn R., G.D. NachrBl. moth, Maisziinslers, monitorovanfm bolognesi Acta I., 291-299. I., J S., A.K. J.A., J.A., B. body A, s Chem. des in pea traps ti (First toring abstr.). pheromone jablecneho, 1975: Corn mones: America Ostrinia biet. 1om E111omol. pean mone diet. wich mones. J. York borer. 119: population BUcH1 GRANT HoRAK HRDY HRDY KENNEDY KLUN KLUN KLUN KoCl·IANSKY L11NGENBRUCH MAIN! McLEOD NAGY PENA RAINA ROELOFS S111RRATT STRUBLE - - in in in & of di be of at the Al for the the the vol was trap EAG How to Proc. of main in to glands phero type Struble evapor low pentaco sensitive the observed glands low for by sessuali Agency reported (ed.): pheromone by purity unsatisfac the pheromone Besides lead Attractants. influencing intact However, and Kennedy and I. their copulation sex their U.S. population. appeared 183-187. variability 295-299. high factor may those Reinvestigation on of fermoni caused and Z 1976) component. 4: Hrdy 722-725. Related 1978; pp. earlier, of Needed release injecting selective be In systems, dio tetracosane, attractancy pheromone 30: substantially plant a from and spite Advisor, for improve our local - 1-21. critical glands experiments. nubilalis in pheromones. recommended Hague, to trap host 1986). 7: could dispenser for the McLeod, Agronomie affect 0. as Starratt, DHR-5600-6-00-1051-00, as female It differs seen. and sex-pheromone most Science pheromone field of & al., low Attrazione Pyralidae). the Pub!., pheromone detector tricosane, & in pheromone chromatography: No. be found pheromone Technique Natwforsclz. et use, the ratio Lepidoptera in the the 1299-1311. Entomol. to Z. clear. of gas of regime 1985: pheromone in Acad. function be grant and females. Pyralidae). slightly dialect 1987: 13: were pheromones, field Hrdy P. (Starratt not responsible to generaly alkanes, constant isomer additional SPB synthetic to Intraspecif1c is Office (McLeod identified H.J. under F1ustu/a (Lepidoptera, Ecol. could remains for capillary an and the glands inactive scales and calling species efficient pheromone pheromones. found part prior 1984: used possibility. by as of Pheromones in when perhaps releasing they by Friuh. photoperiodic al., 1989; m Chem. (Lepidoptera: the pheromone stability, dose alkanes pp. in electroantennographic this Sex Prague nubilalis was moth J. most BESTMANN et just reported reason ZilNDIGIACOMO insect substance(s) function that were REFERENCES age, female of even & were of the & 123 of Hb. Hbn. The the Lymantriidae) the 0. L. The the pheromone behavior some List supported Lepidoptera of produced COOPERATORS Ostrinia Cooperation, (Horak considered release in in Paris, was brassicae. 1975: prove prepared ------of Academia, Wheather for repellent hydrocarbons analysis was pheromone pheromone type. nubilalis nubilalis dispenser S. 1986: not support repeatedly nubi/alis, possibility behavioral was Effect processes or not those Borer, PR11v1s11N1 ratio E. found W.G.O. were autoxidation 0. are: did Tabor. the I activity VoSTROWSKY E General, present to 1987). S., copulation did .. Mamestra been 1991: research Ostrinia (Lepidoptera: & Ostrinia Technology rubber design, Corn Z: 14:Ac M These The similar - pheromone as ratio C23-C25 of RAUSCHER pheromones di J. B. al., Similarly, 3 Ecol., dispenser a and A PREISNER has red & This of trap inhibitory at study the for to exclude sex Borer, trapping & 97: E. chromatographic serve Secretariat HERRIG dispensers an MARCHETTI have to of in Chem. than isomer M. that European 1980). pheromone complex to experiments Development. STOCKEL gas comparable confronti used of requirements Corn heptacosane KALINOV Science behavior. R., - the (Grant A.B., the leucostigma the P., & the present in the nei presence fact and STADLER seem T6TH Insect studies, acetates, degradation. other rate field efficiency and of sensg;ve in (1987). V. and analysis results male H., H., nubilalis The The The al. European tool for OILB-SROP, pheromone smtesi Conj titre though other main atility ever, Orgyia temps sane 0. activity ping Anderson, dispenser taining very dispenser ation when mone the tory formation et used ACKNOWLEDGEMENT. Program International ANGLADE ARN ARN BARBA'ITINI ATTYGALE BOREK - '-.:..,,.. ~ - - to 1993 1993 1211 our the due etha yne cross total - func- 25, 26, an (Ostri of similar acetate 4-oxo that (3-oxo in the Grignard known" of molecules, ''classical" • focused molesta (Z)-14-hep O')iron(Ill) 1 some efficient borer August a and October are and exhibit Similarly, of we expect - was ved (Z)-11-pentadecen-4- corn Cydia (Z)-alkcnylmagncs1um preparation We tested synthesis borohydride step~) could and Rece1 a Accepted position pheromone Republic we (4 the AND is 1110/esta, 4-oxo-(Z)-11-pcntadccenoate pheromones chlorides compounds, synthesis - (Via) sodium hydrolases. moiety. European (Z)-8-dodecen-1-yl sex Czech the acyl 3-oxopentanedioate Cydia vinylic parent proteinase. corresponding target methyl (Vlb), the to using The semiochemicals, and serine 6 the nubilalis and · the 6, of Therefore, 5 the and with compounds lactone - on butyrolactones of tris(2.4-pentadionato-O. at complicated of serine 1110/esta) of diethyl KoUTEK between reaction (Va) la/is) the Vlh). of 40%. I) Prague .<' or these bi 5 applied 1111/Jilalis based - Ostrinia chloride JO simple group of nu halogen (Via, (C)dia analogues - 30 a membered OF relatively one-pot inhibitors) Bohumfr (Z)-14-heptadecen-4-olide more is by applying Ostrinia reaction (Scheme than inhibitors moth and five acetate of the pesticides alkylation by a Biochemistry, Ostrinia synthesis way bearing Republic,166 ( successfully - (suicide the fruit racemic by and higher even suicide converted of the this SAMAN (Z)-11-pentadecen-4-olide butyrolactone synthesis Czech been (Z)-alkcn-4-olidcs case as not analogues of two-step ANALOGUES For acetate lactones the were the and biorational performed has Oriental - our replaced of cross-coupling inhibitors David was 4-oxo-(Z)-14-heptadecenoate Chemistry (Z)-14-HEPTADECEN-4-0LIDE of the Vb. way In enol required the 3-methoxycarbonylpropanoyl properties preparation new was was (Via) featured OF of on this the and pheromone synthesis the '"'n-1-yl methyl and Vh for method Sciences Organic in a to are Unfortunately, in ide · (~f Va sex synthesis of moment catalyzed _, effects. The to and HOSKOVEC, based This rcact10n step {Z)-11-PENTADECEN-4-0LIDE, alkylating the PHEROMONE Communication 1110/es;a), search key Va (Vlb), solution 1-teL. prepared of key nubilalis) Institute lactones Academy the mechanism-based Five-membered their -- The Short SYNTHESIS AND SEX Michal The olidc coupling bromides. (Vh) nolic In attention nia be nol to inhibition (Z)-1 (Cydia tadecen-4-ol method, 4-oxoesters glutarate)H. yidd (Fe(acac)_,) esters reagents. - COPY - _/l,l/lffJ'l.Ol.F '">C~T -- - - - - \_ ,,;?- ~_;; --- l in to m of of tn of 60 via the and and per fur- was This were with with 1213 from SOW 25 Prep NMR ) Illb mol a 1 and from Via oil. !Va tetrabro mol) continued were overnight ammonia The Merck Hydroge 0.2 -- cm- gradient ml) and removed and solution 7-undecene solvents spectrometer, Dowex 14-bromo-4- 0.2 on g, synthesis was converted in extracted relatively low g. 250 prepared yellow TMS. the JI/a liquid lactone were carbon (v, a decomposed x with 13.9 standing analyses min, to wm, prepared cross-linked). of made detector in were stepwise (4 of 5 for stirring (21.8 gave on - acetylene. were wao g with I) ethanolic from % Ila. Vb GLC four-step FID : for of UNITY-500 spectra and were treated with 45 mo!) solvents a an 11 relative Ilb 1-bromo-(Z)- mixture (I 1-bromoalkenes Jllb silicone, the IR (/Vb), and and the reagents, residue (Z)-alkenols gave (0.3 Evaporation with alkynol The and scale Varian and evaporated and were • - Va 7 The stirring using than 3 System (prepared ml) the and of dropwise methyl 2 on 1-bromoethane /Ja ml). wm, alkylation C0 IV. gave Illa separations petroleum 2 LC o!T 000 (ppm) h, and K solvents After (/Va) 12 I o added LiNH equipped Grignard (63%) 000 I - (I acetylide of phenyl Prep g m synthesis - tetrachloromethane. of (I solution for corresponding over of reduction was 3 filtered in a Ammonia two-step the Alkynols (pMPLC) 32.6 (Z)-enols catalyst Tables ether-light the a overnight advantageous . h. lithium B-680 water dried mol) of carefully. by was methanol of 10 CDCI in 4 'lirring HP5-5% 4-oxoalkenoates by gave expressed - with in in - Evaporation for cold 'uspcnsion 7 coupling V!b more were lb chromatograph the • P2-Ni 0.254 Biichi l-bromo-(Z)-7-undecene are mm, 3 added After given yields is standing a ice g, prepared 5pectrometer C0 and and furnished The 2 0.3 pMPLC are on over 'tirred and wa> K (75 extracted Treatment 5880A way 13 Ia dis,olvcd extracts a with by chromatography using contmued Via ion-exchanger determined - 11 overall c:hemistry l/b to ml) FT-IR HP (la) s~rnthesis was freshly ammonia. used. over wa.o, absorptions (/Va) was l-bromo-(Z)-10-tridecene 88 The diameter mm) of were and The liquid the (GLC). rc~iduc (400 • that and h. compounds one-step evaporated dried of liquid combined 14 were IFS alkyne of Ila lactones mixture the oil dropwise 20 - petroleum. of decomposed 0.063 stirring the (Ila) Vlb. this was MHz spectra on or >98% were - (internal The ml red step for DMSO pressure was hght and a Bruker ml). 'uspension target added yields. gl Hewlett-Packard in a 000 ml). triphenylphosphine 499.5 NMR dry reagents or ffi-bromoalkanols alkynols synthesized uJ,, I a purity final based (0.040 20 lactone 250 .. g at was extracts C column on 3-oxopentanedioate. borohydride preparation however '-iwisc of high Ammonia 13 500 in all x .u ml) is on · medium ether and 'tirred g) 40 Communication Purir1cation the gel . for (1 h. in (4 of a racemic form, the and The 4 In EXPERIMENTAL (45 000 10-Tridecyn-l-ol H Short bromo-(Z)-10-tridecene Grignard yields, diethyl (!Vb) protected 12% mide isomeric nation the !Vb sodium 1 operating recorded formed data capillary arative siltca Into diethyl (I methyl-3,5-dioxatetradecane9·10 lithium) brine for combined oil added and ether vacuo. (1-J+ 1fr,hcd -- - the - than proce Koutek: reactive with lower 0 COOMe is Saman, known 2 and/or "y=- : 6 chloride - O 9 THF yield other \_} = VI )iCOOMe, = NoOH }J. 2 -(' EtOH by Hoskovec, n -- , - n oq. 4 This ),. THF ococ 2 I Fe( CICO(CH Mg, HCI 103 CH NaBH short-chain (CH2)nCO(CH2) 1. 2. 1. 3. 2. 40%. Vb ethyl, !Vb propyl, < Vlb prepared = only = 1 Va, formulae R R about !Va, > ( < Via, easilly R> H H R H H In is b, a, where not , -·- 6 • 5 are 3-methoxycarbonylpropanoyl bromides MeDH of papers (1) - which 3 NH t , 5DW(H+), )OE ),.0H THF CH coupling esters 2 original NHJ(I) "' P2~ Dowex RBr, Li, in the LiC;; (CH (CH2)nBr oxo 1. 4. 3. 2. ~ re'• of lb < < Ilb lllb !Vb (CH2)n-OH -OCH(Me j and j 85%) )n Ia, 2 yield > - (Z)-alkenylmagnesium -- Ila, !Va, R> CH H H R H H Illa, (70 ketones I Br-( R-c=c- isolated 1 ·- Sc11hME The 1212 tionalized dures. corresponding described ~ .. - a - to m Yield 1215 19.0) 19.0) solvent 111 l m m (Z)-alke 111 m ddd m ddd 6.6, 7.8) llb. 1.78 Vlb the 7.4, x 12.7, compounds (7.3) 1.14 2.53 1.78 0.90 1.85 2 5.32 4.48 2.01 5.38 2.01 - 2.32 (5.6, dropwise of 4 h (GLC) dichloromethane (6.6, 1.14- (8.0, alkynol for Hz) dry of added pure m J, in of t m 111 - t m s m m m was 111 mo!) 1.40 stirring Vb mo!) I (7.5) - 2.44 2.72 2.58 (7.3) 1.24 ml) 1.58 1.35 0.90 3.67 5.31 2.01 5.39 2.01 (94%) g (0.258 After 1.24 0.151 g (200 h. parentheses, g, 1 standard) 30.4 (in 43.5 t m t m m m (50.l over /Vb I (7.1) 3.41 from 1.29 1.86 (7.3) internal 'C 1.40 0.90 5.30 2.02111 1.37 5.50 afforded 2.02111 -~---·-----· as 20 constants --" to dichloromethane -~--. product dry t m analogously t 111 m m m 111 --- in coupling warmed Illb I crude tetrabromomethane (6.7) 3.64 1.57 1.24 (7.3) 1.37 0.90 5.32 2.01 1.37 5.39 2.01 and mo!) the was and tetramethylsilane prepared of ppm) mo!) 0.160 was (/Va) m MHz, mixture t (o, g, t tt ll 111 m 7.1) 6.6) 1.46 0.151 /lb (IIIb) (6.6) The 3.64 - 1.33 (7.1) 1.58111 g, 1.46 2.12 0.97 (499.5 2.16 (41.8 1.50 shifts (2.5, (2.4, ----- 3 33 'C. I 3 chromatography (30.0 - CDCJ 0 Illa in 1 chemical at liquid II of - (87%). :> 4 5 6 7 8 Communication Vlb 9 10 II 12 md g 13 15 14 - Illa. Position NMR 1-Brdmo-(Z)-IO-tridecene (Z)-7,.Undecen-l-ol TABLE COOCl-1 H Short pressure nol 38.7 (300 Triphenylphosphine 1 solution /lb - - 'C. - Yield 19.0J 25 P2-Ni 19.0) Koutek: 111 medium 111 m t ddt ddd at ddd 111 m 6.6, g). m of 111 7.8) Via 7.4, x 12.7, compounds 2.53 1.24 1.58 (7.6) 1.73 1.85 2 1.44 4.48 0.96 2.32 5-f9 2.03 (5.6, 2.03 5.38 35.9 of Saman, - stirring (6.6, (8.0, mo!, Hz) Preparative suspension with a J, t m m to m [ m s 111 m 111 (0.33 Haskovec, GLC. Va - (7.1) 2.44 2.72 by 2.58 J.58 1.24 (7.6) 1.35 0.95 3.67 2.02 5.t8 5.38 2.02 added hydrogenated 1-bromo-3,5-dioxa-4-methyl-unde parentheses, I were solution - standard) and (in t m the t m m m mo!) 111 1-bromopropane from ml) !Va of I (6.8) 3.41 1.85 1.25 (7.6) 1.46 0.96 internal 5.29 2.03 5.39 2.03 and (500 0.164 2 as - constants g, aliquots LiNH ethanol analogously (32.0 t m in t m m m m coupling //a Illa - I (6.5) 3.63 1.56 1.25 (7.6) analyzing 1.37 0.95 2.03 5.29 5.39 2.03 and LiC=CH, by acetate) tetramethylsilane alkynol synthesized ppm) mo!), and - MHz, t (o, was t tt tt m m nickel(II) µI) 7.1) 6.6) (Illa) monitored Ila 0.363 (6.6) 3.63 of l.56m 1.26 (7.3) 1.11 1.40 2.13 2.16 1.47 shifts g (499.5 g, (Ilb) (600 (2.5, was (2.4, 3 - 1.25 (92.l CDCI %). chemical 3 in from I (lb) (72 2 3 4 5 6 7 8 9 Via 10 - II g 12 13 14 15 16 17 hydrogenation Position NMR - 7-Undecyn-1-ol (Z)-10-Tridecen-l-ol TABLE COOCH H 1214 cane9·10 43.5 1,2-Diaminoethane (prepared 1 The Ila ? - 3 2 0 as 0 was 53.2 32 10% then 30 1217 0.517 H mmol) t H l t t t t mmol) t t a t t d ct g, ct (268.4) q lactone 18 reaction ppm 17 °C, tempera 3 C combined C mg, 0 Vlb 0 solvent 53.2 the 4.59 28 35.36 25.18 29.28 29.21 29.58 29.05 27.11 28.84 81.02 13.78 27.99 22.86 For Then, at 177.27' crude -- The 129.83 129.80 H For g, (12.41 g, 77.00 room 16 h The (193 • = C Va. at I the Then. 4 ml). (8.0 1.36 !Vb 3 0 Via. h g 2 ( For H for 100 l s l t t t t t t s t t d d q q MgS0 from 12 0.93 x (Va) for 1 (CDCl . temperature. oxoester Vb product chloride 3 (4 4 (38%). 36.99 23.76 29.53 29.27 42.77 29.08 29.00 27.12 of over 27.70 22.86 13.78 stirring 51.75 173.31 give 209.08 129.85 129.77 temperature. g room HP0 to CDC1 ether 2 (GLC) 5.5 dried HCI, at H. continued (40%) in -·- Na same analogously h g with and t l t t t t pure t t d d q was Vlh the Yield and 6.3 11.0% of - - - - al for 5 /Vb H. evaporated 33.96 32.80 h 29.50 29.30 28.37 28.06 water, C, 27.05 22.87 13.79 give 129.91 129.72 /lb , - I extracted 3 stirring concentrated mmol) mmol). and synthe,ized to I% (52%) stirred ), 10.7% for g and 4 1.6 and 73. 5.0 with 4-oxo-(Z)-14-heptadecenoate was C, g, was NaHC0 2 l t t ml) t t t t t d 3-methoxycarbonylpropanoyl d q 0.63 mg, pMPLC - - added (MgS0 compounds (Vb) 77 - - I (0.6 - - found: I/lb by (500 71.8% 63.02 32.76 continued 25.62 29.27 29.68 29.04 22.86 27.12 13.78 of gave H; dropwise (189 129.88 129 was mixture pH aqueous mmol), dried 4 HCI to found ml) the ppm) with added purified stirring 10.9% ml), 53.2 (Via) H; pMPLC NaBH (20 (i5, and t t t t t t l s t s g, q C, 25 the was was by of aqueous x - - acidified llh washed 1-pentadecenoate 10.2% ml) 18.64 62.94 32.64 25.24 28.55 (1.32 29.04 (3 13.44 ;hifts 80.17 20.74 22.51 80.17 NaOH 10% 72.9% -- was C. :-tirring, residue (20 of were addition, solution with ether the and purification 71.6% 1 chemical 4-oxo-(Z)-l mixture with ethanol and IV magnesium mixed extracts solution calculated: 1 standard) 2 3 5 6 7 - 4 8 9 in a 10 11 complete 12 13 15 Communication 14 cooling The quenched Further tris(2,4-pentadionato-0,0')iron(III) NMR Position Methyl (Z)-14-Heptadecen-4-olide To COOCH TABLE C Short After was removed ethereal (296.5) mmol), and calculated: mmol) with aqueous Via. 13 ture. extracted internal ,_,_ g, as Br; The The and mo!) 53.2 min) l Koutek: s t l l l ct - l l t g, l t 10 t l d (60.3 ppm d q ( 34.3% calculated: 0.227 Via mmol) re,idue. pMPLC. ice-cold light temperature. H, 35.57 29.73 25.20 81.05 29.46 29.46 g. 29.43 29.31 29.22 28.85 27.99 27.06 177.29 20.48 14.37 13.82 Saman, 77.00 129.28 131.52 ( by the 53.2 = with (261.3) dropwise - 9.1% room to 3 (38.7 g. !Va at Br C, 25 t l s l /lib t l purified l l (8.0 t l t l l d d added H q q Haskovec, added wm,hed from (CDCl 13 THF Va 3 was C 56.7% triphenylphosphine 36.96 wa:- 42.77 - 23.76 29.71 29.43 29.38 was was 29.33 29.20 29.15 27.68 27.04 173.29' 20.45 14.34 209.10 above, 51.71 dry 129.25 131.47 For chloride CDCl and of ml)) ml) PO) ). prepared 3 residue Br. in ml (30 (Ph calculated: moll 84% (400 t l t t t l t l t - l ct Via d described q crude g, 250 30.7% freshly - 75 Tl-IF I as !Va in 34.02 32.83 28.16 H, 0.227 29.72 2940 in 29.40 The 28 29.21 residue 27.07 20.50 14.38 (233.2) Ila 129.26 131.55 (39.3 g, Br petroleum 21 manner 9.7% oil solid mmol) bromide H vacuo. mmol) 11 C, (75.3 ------ in light t t 1.6 t C l The t l l t t t d d q same 53.2 compounds g, ~---- (Va) and For Illa g. colorless the 59.6% 3-methoxycarbonylpropanoyl 63.05 32.77 25.71 29.73 of ---- 29.55 29.44 29.39 29.23 27.06 -- 20.48 14.36 a 129.29 131.51 (0.6 In filtered. --- of !Vb. Br. a:- (1.32 I-magnesium of ppm) % concentrated and found: (!Vb). evaporator °C (i5, t l t Br; l t 34.1 t l l was l l s solution q 0 - (85%) turning:- obtained tetrabromomethane a H. Ila g 62.94 rotary to 32.72 25.67 29.42 shift, % 18.66 29.32 29.07 29.03 28.77 12.36 79.50' 81.55 14.32 30.6% filtrate was a stirred with 45.0 9.1 a H, (Z)-7-tridecenyl- the on C, /Va cooled to give of magnesium and chemical 9.7% to 4-0xo-(Z)-14-heptadecenoate 3 I was III and standard) C, 2 3 4 56.8% 5 6 7 8 9 argon, 10 11 12 13 removed 14 15 16 17 brominated mo!) NMR Po,ition l-Bromo-(Z)-7-undecene solution Methy -- TABLE COOCH 1216 was mixture petroleum bromoalkene 59.8% was found: 0.23 A mmol) under tris(2,4-pentad1onato-O.O')iron(III) I.le internal .::..J ?_ - - - - - - - - - - J '\ 11: i\1 j: - IR of' Yield H. Koutek: Science !(C-H), 1980). ( - 1987). in Ei>evicr, m ( Attractants. 10.9% 19 1984). mmol). Academy I.+ ( 005 Saman. C, 1990). (C=C). 2053 ( 3 CQr>y (1983). 44, Program 3.73 28, Related - 4805 g, 653w C1111w/rnes. 75.4% 2270 R!F 1431 I 25, and Lett. Haskovec, Chem. 55, ~ 1971. and 1.00 spectrum: 47, (1981). ( Biochctntstry, Lett. found: 553. IR Biol. \//llL.4 - V/J H; and A H. 5459 Chem. Allrne.1 lactoneJ, /973, Commun. Lepidoptera Tetrahedron from Agric. Amsterdam 103. 11.0% of Biol. 11.4% L.: BEST Development. S.: Tetrahedron 1161. - C, Chem. !(C=O), Soc. C, Chcmi,try DHR-5600-G-00-1051-00, (C=CJ. L.: 1971) Commun. vs ( Agric. w Acety/e11e.1, Elsevier. 1985. No. 75.6% Ronz1n1 Haya,hi of' analogously S.: Czech. Chem. 76.5% 781 403 1989). 653 ( I Pherm11011es G., I S., Ronzini Organic - Chem. !11tematio11al grant 36. .. .. Am. 1992. of 575 Sex V /(Jr J. found: Synthc'i' Clle1111strl'. Collect. Hayashi Soc /Y, (~f bond], prepared calculated: Srnthesi.1· under .. H; A.: Chem. 1-1.: B.: lactonej, H Marche'e Prague J. List - D.: B. was Agency part Martina ln,titutc Chem. H.). 1c, V., Wakabayashi Org. .. in (238.4) H. E.: 11.4% J. .. G Commun. 1. Ace1y/e11ic 2 U.S. Kondo · M. E Koutek cis-double !(C=O), 0 Moon (Vlb) ( C, K I.: 26 Fhesis. Rcpubl .. 1-1.. vs D., H S E. V. 1986. - Synth. 15 Pricsner supported C Fiandanesc W. 782 author 76.6% .. ((C-H), M.: Marchc,se I CLcch Ph.D. Vcrkruij,se M Nakagawa O.rnwa Saman Pans Preparatit•e m Ahu.Ja C., Katzenellcnbogcn was For the Snyder Cooperation, .. Kim 1981. V., the .. L., M.: Y., Y., L.: .. by M A K., 005 G., Petrini Toth of' - K bondJ, .. 3 C. A. R. R S. calculated: H., research (57.4%). g Technology Arn OILB-SROP, Grant Naoshima Narn,hima Fiandane~c Brandsma Cardel11cchio Brandsma Hoskovcc Amsterdam 171is Hoskovcc Kang Brown Science' REFERENCES Weiss Translated Balhni I. 2. 1218 3. 4. (266.4) 5. 6. 7. cis-double 8. - (Z)-11-pentadecen-4-olide 0.51 spectrum: 9. and 10. 11. 12 13. 14. ~) ~ - I Pergamon Bioorganic & Medicinal C/1e111i.~t1)', Vol. 4, No. 3, pp 47ll-4KK. l'J% e> Copyright (<:') 19% Elsevier Science Ltd l'rintcd in U1cat lliitain. /\II 1ights1csc1vcd I~ 0968-0896/96 $15.00 + 0.00 S0968-0896(96)00029-6 I New Mimics of the Acetate Function in Pheromone-Based Attraction I Michal Haskovec, Oldrich Hovorka, Bianka Kalinova, Bohumfr Koutek,* Ludvik Streinz, Ales Svatos, Pavel Sebek, David Saman and Jan Vrkoc I Jnstit11te of' 01ganic Chemist1y and Bioche111ist1y, Academy of Sciences of the Czech Repu/J/ic, Flemingovo 1uim. 2, CZ-166 IO Prague 6, Czech Republic I Abstract-Several analogues or (Z)-8-dodeeenyl acetate (la), the major pheromone component of the Oriental fruit moth, Cydia 1110/esta, with chloroformate and lactone functional groups in place of the acetate moiety, were synthesized and investigated for their biological activity at four evaluation levels, i.e. by electroantennography (EAG), electrosensillography (ESG), short-range I sexual stimulation and activation in the flight-tunnel. We found very strict requirements on the shape as well as on the electron distribution of the acetate group for a productive interaction with the receptor. The behavioral results showed that, among the analogues investigated, the chloroformate lb, alken-4-olide 2a and also dodecyl acetate (le) possess significant (60-85%) inhibi to1y activities. Based on electrophysiological evidence demonstrating that (i) only lb is competing with the major pheromone I component la for the same receptor sites on the male antenna! sc11silla, (ii) le elicits moderate EAG but no ESG responses and (iii) 2a docs not produce any electrophysiologica\ response at all, three possible inhibitory mechanisms by which these analogues I arc acting could be distinguished. Copyright© 1996 Elsevier Science Ltd Introduction reduce the ability of males to mate. A similar effect has been observed5 on the exposure of olfactory organs of I ·The mating disruption technique belongs to one of the the male moth Mamestra brassicae to N-ethylmaleimide most perspective strategies for application of phero vapors. Also, some triftuoromethyl ketones have been mones and their analogues in insect pest control.'-2 To proven to inhibit the action of pheromone esterases in I achieve the mating disruption effect, several basic the processionary moth Thawnetopoea pityocampa6 and approaches are possible,3 including: (i) the release of the Egyptian armyworm Spodoptera litoralis.1 synthetic pheromone constituents or mixtures thereof into the environment to disrupt the male's ability to In this study, the males of the Oriental fruit moth (C. I locate virgin females by the omnipresence of phero molesta), CM, were selected as model species. The mone, (ii) the use of components emitted by closely female sex pheromone includes (Z)-8-dodecen-1-yl akin species to reduce intraspecific attraction and (iii) acetate (la) as the main constituent8 of the three the use of reactive pheromone mimics or inhibitors component mixture. We describe herein the synthesis I that either irreversibly and stoichiometrically block and biological activity in electrophysiological and antenna! receptor proteins or specifically inactivate behavioural assays of analogues lb-2 (Scheme 1 ). pheromone catabolizing enzymes. To date, however, no These analogues formally proceed from structural I evidence suggests that the last two approaches are as modifications of the acetate group, one of the three efficacious in disruption as synthetic copies of the putative active sites of the parent molecule la, which natural pheromone and no operational system based could be involved in the interaction process with the on any of these principles has been invented. Neverthe antenna) proteins. Due to the suggested9 bioisostericity I less, investigations concerned with modifying effects of of CH,=>CI replacements and some type of similarity various chemicals on insect olfaction indicate that some existing between the acetate and lactone functions, suhstances are able to react with specific chemical these analogues seemed to embody in their structures group at hinding proteins, receptor proteins and/or both the appropriate recognition and reactivity I catabolic enzymes that comprise the olfactory chemo elements. It was anticipated that processing some of scnsory systems of the moths. These compounds may the analogues hy a target protein could result in be structurally related to a component of the phero unmasking the inherent latent reactive moiety in 1 and mone of the insect against which they arc applied, or 2, and lead to inactivation of target acetate-recognizing I they may he of an entirely different chemical class. For antenna) proteins. Based on some earlier findings example, Berger and E~tcs 4 found that some N-alkyl related to the inhibition of esterases by propan-3-olidc malcimidcs arc able to irreversibly block the elcctro derivatives, Ill.II we hypothesized that some of the I antcnnographic response of the cabbage looper compounds represented by structures 2 might function Trichoplusia ni to the pheromone and to significantly as inhibitors of pheromone carboxylcsterases. In view I 479 I I I M. l losKov1:c cl al. I R -CH 2-0-CO-Y I 1 a -d 2a -c I In formulae 1a: R =(Z)-7-undecenyl; Y =CH 3 2a: R = (Z)-7-undecenyl; n = 1 1b: R = (Z)-7-undecenyl; Y =Cl 2b: R = (Z)-7-undecenyl; n =2 I 1c: R = n-undecyl; Y = CH 2c: R = (Z)-7-undecenyl; n 3 3 = 1d: R =n-undecyl; Y =Cl I Scheme I. • of the reported 12 inhibitory activity of dodecyl acetate relative GC retention times of the compounds (le) on the CM males under field conditions, we also measured isothermally at five temperatures along with I included the saturated pheromone analogues le and d the calculated vapor pressure data. The results impres into our studies. sively demonstrate the importance of vapor pressure corrections in comparing electrophysiological and behavioral data for 1 and 2: at 25 °C, the relative I Results and Discussion liquid-vapor concentration ratio for the pheromone Chemistry component la and analogues lb, c, cl, 2a, b and c follows the order 1 : 1, 1 : 0.61, 1: 0.85, 1 : 0.45, I : 0.175, I Chloroformates lb and lei were prepared from the 1 : 0.027 and 1 : 0.0093, respectively. It means that the corresponding alcohols and triphosgene, a safe and alken-5-olide (2c) is about two orders of magnitude crystalline substitute of phosgene, according to a less volatile than the pheromone component la. Note known'-' procedure. Compounds 2a and c were synthe that corrected concentrations of all analogues arc used I sized as shown in Schemes 2 and 3, while the synthesis throughout this work. of 2b has been described previously}4 IR, NMR and elemental analyses of all synthesized compounds were Electrophysiological properties fully consistent with the proposed structures. I Two types of electrophysiological recordings were conducted to assess the effects of the pheromone Relative volatiles of analogues related chemicals on antennae. I Dose-response curves in insect electrophysiology and Electroantennography. Relative EAG act1v1t1cs of results of laboratory bioassays are usually based on compounds la-d and 2b corresponding to 50% of the amounts of stimulus applied to the odor source rather relative activity scale (Fig. 1) demonstrate that in no than the amount of stimulus to which the insect case did a pheromone analogue reach the response I antenna is actually exposed. While such approach may value of the natural pheromone component, be correct when the compounds to be compared arc (Z)-8-dodecen-1-ol acetate (la). The corrected EAG structurally similar and hence of (approximately) the activity of analogues decreases in the relative ratio I same volatility, it is certainly not valid for compounds 1400: 15.1:3.1:1.8: I for the la, b, 2b, le and d, respec differing significantly in their chemical characteristics tively. As expected, chloroformatc lb, which is the and molecular mass. This implies that corrections for closest structural relative of the natural pheromone differences in volatility need to be taken into account component and, rather surprisingly, also the aken- I when interpreting elcctrophysiological and behavioral 4-olide 2b, were the most active analogues. Their results for analogues I and 2. Since the potential activity range was between 0.05 and 500 pg of the volatility of a chemical is related to its inherent vapor stimulus. Responses elicited by different doses of these pressure, 15 we determined the saturated vapor two compounds arc not significantly different I pressures of I and 2 using a GC method, whose details (Student's t-test, o. =0.05). However, at the upper dose have been described previously.'''·17 Table I lists the I used (500 ~tg), an olfactory saturation could be a b ~ CH 3!CH~2 (CH 2l~HO 2a I 4 Scheme 2. (a) Pyridinium chlorochromatc/CH1Cl 2; (b) ketcne, BF,- Et,OffHF, -400C. I I l{oI I I New mimics of the ac1!tate function 481 observed for lb, but not for 2b. The last two analysis in this report. All neurons sensitive to la were I compounds, 2a and c, elicited nonsignificant responses also responsive, but to a lesser extent, to lb and 2b. when compared with the response of pure hexane Thus, the present data suggest that these compounds (Student's t-test, rx = 0.05), even at the highest doses interact with the same receptor sites as the major tested (500 ~tg). pheromone component la. With exception of the I saturated acetate le, the order of the activity of the Electrosensillography. The activities of olfactory compound tested paralleled their EAG activity. neurons associated with sensilla trichodea were analyzed (Fig. 2). In several of the preparations, very I small number of impluses was observed that could not Short-range behavior be reliably discriminated from.. the spontaneous responses in air (Student's t-test, rx = 0.05). For this The effect of the analogues on short-range communica reason, these responses were not considered in further tion among the CM conspecifics is shown in Figure 3. I The values of 'confusion coefficients' determined at four concentration levels (sec Experimental) demon strate that, of the compounds tested, the chloroformatc EtooclfcooEt (lb) and alken-3-olide (2a) possess a significant inhibi I 0 tory effect on the mating behavior, while the saturated chloroformate (Id) and alken-5-olide (2c) elicit only a very weak inhibitory effect. Therefore, the last two I compounds were ignored in further flight-tunnel experiments. None of the analogues were found to he as active as the moth's own main pheromone compo nent la in causing disruption of normal pheromone I induced behavior. I Flight-tunnel observations Of the analogues tested, the chloroformate lb was to some extent able to substitute for the main pheromone 0 component la in the pheromone blend, although it did I ~ ~COOMe not elicit the whole behavioral sequence (15% of males CH 3!CH 2J2 {CH 2J6 were activated and took flight, none of them orientated to the source or exhibited a precopulatory behavior). 6 Similar, but even less pronounced effect was observed I for le and 2b. The behavior profiles presented in Figure 4 indicate I that the exposure of the CM males to a mixture of the 2c pheromone (10 ng of the three-component blend, which was proved to be comparable with calling Scheme 3. (a) (i) Mg(OEt),/EtOH, (ii) (Z)-CH-'(CH,) -CH= 2 females) and a 10-fold excess of the analogues lb, c Cf [ -· (Cl l,) la 4.105 3.802 3.509 3.265 3.066 0.312" 1.0 I lb 2.560 2.410 2.320 2.230 2.130 0.184' 0.59 Id 1.330 1.31 1.290 1.280 1.260 0.135" 0.43 2a 2.770 2.660 2.560 2.480 2.400 2.310 0.0548" 0.176 2h 1.956 1.912 1.877 1.837 1.816 o.0082sc 0.027 I 2c 4.054 3.808 3.644 3.432 3.329 o.002s1c 0.0090 "E'>perimental values for le in ref. 17. •· St alone. Under the same conditions, the addition of 2b I to the pheromone did not significantly affect the 100000 percentage of males flying upwind and contacting the odor source. At a lower concentration -ratio (I : I) even 10000 !!!El corrected the analogues lb, c and 2a were only slightly effective. I ~ :~ When the behavioral spectrum of males responding to 0 .. 1000 pheromone is compared with the behavior of males Cl ci: w responding to a pheromone/analogue (I: 10) mixture, ., 100- the I > most affected maneuver was oriented flight: ~ activated males were less successful in their orientation ~ 10 in the odor plume. Males that found the source eventually needed a longer period than the males I responding to the pheromone. 1a 1b 1c 1d 2b compound The neurophysiological data presented here indicate that there is a class of receptor neurons on the antenna Figure 1. Experimental EAG activities I for compounds la-d and 2b; of CM male that significantly responds a comparison of uncorrected and vapor pressure-corrected data. The to stimulation activities are expressed as the reciprocal of the relative number of with the major pheromone component la and to stimu pmol required to elicit the same receptor response. Each bar repre lation with two other analogues, lb and 2b. Nonuni I ~ents the mean of at least six experiments SD did not exceed 11 %. form spike activities produced by the analogues le and I I a I I _J I ---, ,, 11 '.J ,, I• n I !I I!' ~I~ c I ------~~ ____I ------~------~ I I l_ e I I 0.1 mV 0.5 sec I Figure 2. Typical neural activity recorded from a single.~. 11ic/10dewn on antennae of the Oriental fruit moth in response to: (a) air. (b) 5 pg of la, (c) 50 pg of lb, (cl) 50 pg of le, (e) 50 11g of 2a and (f) 50 11g of 2b. The bar below the recording (a) indicates the duration of the olfactory stimulation (0.8 s). I I I I New mimks of the acetate function 4Rl 2a did not significantly differ from the spontaneous I ones_ Analogues ld and 2c did not elicit any responses, even when high concentrations were used. A rather good qualitative correlation was observed between our 1QI) laboratory short-range assay data and the flight-tunnel results (Figs 3 and 4). The most interesting feature of I 9() the results arc the striking differences among tlQ e...~ analogues in the complementary elcctrophysiological '10 ... i::: and behavioral tests. As illustrated in Table 2 summa .~ I 60 rizing the qualitative output of these tests for the mos! so ~q, () important analogues of the series, none of the 40 u analogues lb, c, 2a and b showed identical behavior at i::: 3Q ·~., all four (EAG, ESG, short-range behavioral assay and I .::! flight tunnel) levels of evaluation. This implies that no 20 to ul5 straightforward structure-activity relationship can be discerned and the inhibitory active analogues lb, c and 2a must operate by (at least) three different inhibiting I mechanisms. Although, at present, we have no evidence of the exact mode of action of the analogues tested, several mechanisms can be hypothesized. I Figure J. Inhibition of male C. mole.1ta behavior in a laboratory 'hnrt-1a11gc bioassay p10111oted hy different doses ol I :tnd 2. Each The mechanisms of inhibitory action derive from har i'> the mean of f8 experiment~ (SD s 15%). inhibiting chemicals - in relation to the pheromone composition of the species. It is generally acceptcd 18 I that molecular size and shape arc important for insect pheromone chemoreccption. Apart from stcreo chemical requirements, however, electronic charge charge attraction, hydrogen bonding, hydropathic I bonding and van der Waals forces are potentially 10() important in binding to proteinaceous macromolecules. 1oO 9() To account for the surprising differences in biological 9'.J 6o activity of the analogues, we first considered the possi I sn 7o e::.~ bility that some of the analogues might assume 70 6Q la (+) (+) (+) (+) I th ( +) (+) ( +) (+) le ( +) (-) ( +) (+) 2a (-) (-) (+) (+) I 2b (-) ( +) ( - ) (-) '( + )· positive response, ( - ): no or statistically non-significant (Student's I-test, ~ = 0.05) response. I I I I 484 M. HosKovEc et al. I I I I a b c Fi~nre 5. Superpositions of the energy-minimized ethyl ac.i:tate structure (bold structures) with those of (a) ethyl chlorofonnatc. (h) hut:111-J-olidc and (c) pentan-4-olide. Although the replacement of the acetate methyl group sufficient pheromone mimicry (e.g. 2a) would he I hy a chlorine in lb does not seem to have important cleared from the receptor less effectively and act as steric consequences, the methyl group has a higher behavioral inhibitors. The earlier studies of behavioral hydrophobicity than the chlorine atom and, also, the responses of male codling moths24 ·and European corn I possibility to engage in short-range binding through borers25 in a flight tunnel to their corresponding phero dispersion forces with the receptor structure that is mones and analogues led to the conclusion that the complementary to the acetate methyl is probably steric requirements for an analogue to be a pheromone 19 2 reduced for the chloro derivative. · n Beside this, the mimicking substance are much more stringent than for I IR carbonyl frequencies v(C=O) for la and b (1740 the analogue to be an inhibitor of behavioral output. 1 2 versus 1778 cm - ) differ significantly, indicating the On the other hand, it is known " that four-membered different ability to form the hydrogen bond. The lactones as amhident electrophiles may undergo, in the hydrogen bond is widely regarded21 as being the most presence of nucleophiles, oxygen-alkyl or oxygcn-acyl I important intra- and intermolecular cohesive force and bond cleavage. Therefore, 2a might be able to inhibit a major contributor of noncovalent interaction energy pheromone-catabolizing enzymes via acylation or in biological systems. All these differences may account alkylation of their nucleophilic groups. This would, in for the reduced electrophysiological activity of the turn, lead to disruption of normal pheromone-induced I chloroformate lb in comparison to la. Similarly to our behavior via prolonging high pheromone levels within results, only moderate electrophysiologic responses the peripheral sensory system. A lower inhibitory were found 22 to he evoked by formate mimics of the activity of the alken-4-olide (2b) (Figs 3 and 4) may in I aldehyde function on antenna! receptors of several part be related to its lower (in comparison to 2a) noctuid species. Regardless of its reduced activity, the chemical reactivity. Note that propan-3-olide has been analogue !-b c!idts a sufficiently high dectmphysio found 27 about 50-1{}0 tirr1cs more· reactive in rcaction logical response from the pheromone-sensitive receptor with adenosine, cytidine or guanosinc than butan- I neurons and could theoretically overload the receptor 3-olide while butan-4-olide completely failed to read system acting as an inhibitor when present in hyper with any of these nitrogen nuclcophilcs. A double-bond physiological concentrations. Another explanation for environment appears essential for good inhibitory the significant inhibitory activity of lb could be its activity since Id, the saturated equivalent of lb, is only I possible binding to antenna! proteins through a carba a very weak (Fig. 3) inhibitor. mate linkage (under evolution of HCI!), thereby locking the sensory transduction mechanism and/or The behavior of the saturated acetate le deserves a inactivating the pheromone catabolizing enzymes. special comment. The major component or the I Precedent for this analogy is found in the use of Oriental fruit moth pheromone, (Z)-8-dodecenyl (Z)-11-hexadecenoyl- and (Z)-9-tetradecenoyl fluorides acetate (la), was idcntitied28 in 1%9. Since 1979 it has as reactive mimics of the Heliotis virescens phen)mones been assumed8 that the Oriental fruit moth pheromone 2 I aldehydes. ' consists of four components, viz. (Z)-8- and (£)-8-dodecenyl acetates, (Z)-8-dodeccn- l-ol and 2 The fact that we found relatively high (and nearly dodecanol. The insect attraction was found '' to be equal) inhibitory activity to be coupled both with lb particularly sensitive to both the pheromone compo I and 2a was rather surprising since the alkcn-3-olidc nent ratios and concentrations. Mating disruption was (2a) failed to produce any significant EAG or ESG also attempted with an analogue of I a, dodecyl acetate response. If the specificity of pheromones and phero (le). For example, Rothschild found 12 that males of mone rccc.ptors was coupled to specificity of the phero this insect were not trapped at live virgin female or I mone clearing enzyme system, it would be expected synthetic pheromone sources when dodecyl acetate that analogues tha~ successfully mimic the pheromone (le) was present in large amounts (exceeding the should serve as better substrates for the pheromone amount of the unsaturated acetates) in the same trap I specific catabolic systems and be degraded more effect or within 15 cm from the bait. When the compound, ively than those compounds that arc structurally less however, was present as a background odor over a I similar to the pheromone. Thus, compounds lacking large area, an opposite (synergistic) effect was I I I function 485 New mimics or the ucetatc observed. Reasons for these results remained obscure. separations were made on Merck 60 silica gel I RcccrHly, the composition of the pheromone blend was (0.040-0.063 mm) using a Bi.ichi B-680 Prep LC system re-examined and significant amounts (3.44 ± 1.16%) of with a stepwise gradient of ethyl acetate in light le have been identified30 in the effluvia of calling petroleum. females. It has been suggested that the role of this I compound in the natural pheromone of CM rnight Syntheses have been overlooked. Our electrophysiological results demonstrating that le, although not detected by the (Z)-8-0odccen-l-yl chloroformate (1 h). Triphosgcne same receptor cell type as la, elicits a relatively high (0.367 mmol) solution in dry Tl-IF (I ml) was cooled I EAG activity, seem to support this suggestion. on an ice-bath and pyridine (45 pL, 1.2 equiv.) was Unfortunately, neither our own ESG experiment nor added. To the preformed white precipitate the the previous ESG studies31 were able to detect any (Z)-8-dodecen-1-ol (85 mg, 0.466 mmol) in Tl Ir (2 other receptor cells that could respond to le, probably mL) was added dropwisc during 20 min. After 2 h of' I due to the small size of the moths. If the proposition is being stirred at the ice-bath temperature, the reaction accepted that this compound represents a minor phero mixture was poured into ice and aq HCI (3.7%, 0.4 mone component and by itself could mediate a mL). Subsequent standard work up and PMPLC I particular clement of behavior, then a background of a afforded the chloroformate lb (69. mg, 60% yield). high concentration of this component might alter the Calcd for C 1,Hv02CI: C, 63.27; H, 9.39; Cl, 14.37. balance of sensory input to such an extent that the Found: C, 63.44; 9.50; Cl, 14.21. 1I-I NMR: S 0.90 (t, insects no longer respond appropriately. A similar 3H, 1=7.0 Hz, CH_1CH2CJ-1 2-), 1.29 (m, 1211, inhibitory effect of imbalance in the pattern of sensory -}, 21-1, .1=4x6.8 llz. I 6x-CH2 1.72 (m, 2 behavior input had been previously observed~ on the -CH2CH2C02CI), 2.01 (m, 41-1, -Cl:I2--CH=CI I - of Autogmpha gamma species that use a simple Cl::lz-), 4.31 (t, 21-I, J = 7.8 llz, --Cl-J 2C02CI), 5.38 1 two-constituent pheromone blend. Increasing the level (m, 2H, -CH=CH-). IR (cm- ): 3006 m [v I of minor component in the binary blend resulted in a (C-H), cis-double bond], 1778 vs [v(C=O), ester), substantially decrease of male responses in moth 690 w (v(C-CI)]. behavioral stages. I Dodec-1-yl chloroformate (ld). Calcd for C1.1H 2,02CI: In conclusion, the present data suggest that, of the C, 62.76; H, 10.13; Cl, 14.25. Found: C, 62.90; H, 10.22; analogues investigated, only the chloroformate (lb) Cl, 14.14. 1H NMR: o 0.88 (t, 3H, J = 7.0 Hz, and the alkcn-4-olide (2b) are. able to reasonably CH3CH2-), 1.26 (m, 181-I, -CH2 -), 1.73 (m, 2H, mimic the acetate function and thus produce responses 2H, J = 7.8 I J = 4 x 6.8 Hz, -CH2CH2C02CI), 4.31 (t, from the same class of pheromone receptor neurons as 1 Hz, -CH2C02Cl). IR (cm- ): 1779 vs [v(C=O), the major pheromone component la. The inhibitory ester], 690 w [v(C-Cl)]. properties of the analogues, however, seem not to be entirely connected with their mimicking capability. I (Z)-8-Dodecan-l-al (4). Pyridinium chlorochromate Apparently, several constitutional and configurational (PCC; 3.5 g, 16.3 mmol) was suspended in dry CH2Cl 2 properties of the molecule and, in turn, its chemical (25 mL) and (Z)-8-dodecan-l-ol (3, 2.0 g, 10.9 mmol) reactivity are of special significance to the inhibitory in 6 mL of the same solvent was added in one portion I process. More specific studies are required to elucidate to the stirred solution.-'-' After 2 h, dry ether (50 mL) the role these factors may play for effective binding to was added and the supernatant decanted from the proteinaceous substrates. At present, the inhibitory black gum. The insoluble residue was washed with mechanisms remain speculative. In spite of this, the ether (3 x 20 mL), the combined ethereal solution was I new inhibitors described (especially lb and 2a) may passed through a short pad of neutral alumina with prove useful as tools in further biochemical as well as charcoal and the solvent was evaporated. PMPLC of field studies directed towards mechanisms controlling the crude product afforded 1.64 g (83%) of the I mating disruption. aldehyde 4. Calcd for C 12H 220: 79.05; H, 12.17. Found: C, 79.11; H, 11.98. 1H NMR; o 0.95 (t, 31-l, J = 7.0 Hz, --· ), CH3CH2CH2-), 1.21-1.43 (m, 8H, 4 x -Cll2 (m, 2H, -CH CH CHO), 2.05-2.11 (111, 41I, I Experimental 1.44-1.71 2 2 -), 2.42 (dt, 2H, J == 7.0, 1.6 Chemistry -CH1-CH=CH-CH2 Hz, -CH2CHO), 5.31-5.38 (111, 2H, -CH=Cll · ). 11I NMR spectra were determined in CDCI-' solution 9.77 (t, IH, J = 1.6 Hz, -Cl-10). I on a Varian UNITY-500 spectrometer operating at 11 499.5 MHz and absorptions are expressed in o (ppm) (Z)-10-Tctradccen-3-olidc (2a). Ketenc was bubbled scale relative to TMS. The IR spectra were recorded into the stirred mixture of aldehyde 4 ( 1.6 g, 8.8 mmol) on a Bruker IFS 88 FT-IR spectrometer in CCl4 • GLC and borotriftuoride etheratc (26 ~tL, 0.2 mmol) in 1 I analyses were performed on a Hewlett Packard HP anhydrous THF (10 mL) at -40 °C: ' After I h, 5880A chromatograph. equipped with a FID detector another fresh catalyst was added (20 ~1L, 0.16 mmol). and a 25 m capillary column (0.3 mm i.d., HP5-5% When the starting aldehyde 4 disappeared (2 h), ·1he I phenyl methylsilicone, cross-linked). Preparative solution was flushed with dry nitrogen. Anhydrous medium pressure liquid chromatography (PMPLC) triethylamine (2 mL) and CI-ICI-' (20 mL) were added :I I I I I \Xfi M. Ho~KnvH· ct al. I at --40 °C followed by water (20 mL). The mixture was 2H, J = 4 x 7.3 Hz, -CH2CH2C02CH1), 2.00 (m, 41 I, then extracted with CHCI, (3 x 20 mL) and the -Ctl2-CH=CH-Cl:b-), 2.34 (t, 211, l = 7.3 Ilz, • combined organic extracts were dried over MgS04 -Cl:lzCO-), 2.38 (t, 2H, l = 7.4 Hz, --COCI-1 2 -- ), Removal of the solvent in vacuo and purification of the 2.47 (t, 2H, .I= 7.2 Hz, -CB C0 CH,), 3.67 (s, 3H, I 2 2 residue by PM PLC afforded 0.99 g (54%) of the pure -C02Cl:-h), 5.31-5.39 (m, 2H, -CH=CH-). ( GLC) lactone 2a. Ca led for CH,H 2x02 : C, 76.14; H, 1 11.18. Found: C. 76.31; H, 11.21. !-1 NMR: /5 0.90 (t, (Z)-12-Hexadecen-5-olidc (2c). To a mixed solution of 311..1=7.0 Hz, CH.,Cl-l CH --), 1.21-1.47 (m, IOH, I 2 2 NaBH4 (111 mg, 2.94 mmol) and Na 2 HP0.,·12ll~O 5x -CH2-), 1.62-1.85 (m, 21-1, --CH2-·Cl-I<), (140 mg, 0.392 mmol) in ethanol (10 mL) was added 2.02-2.12 (m, 4H, -Cl::h--CH=CH-CH2-), 3.06 the 5-oxoester (6) dropwise (830 mg, 2.94 mmol) with (dd, lH, .!= 16.2/4.3 Hz, -Cl::l2-CO-), 3.51 (dd, cooling (0 "C) and stirring, and the mixture was stirred (m, I I H, .I= 16.2/5.8 Hz, -CH2 -CO-), 5.32-5.39 at room temperature for 8 h. Then, a 10% aqueous 1 2H, ---CH=CH-). IR (cm · ): 3004 m [v(C-H), solution of NaOH (13 mL) was added and stirring was cis-double bond], 1836 vs [v(C=O), lactone], 1653 w continued for I h. The mixture was acidified to pH l -2 I [v(C=C)J. with coned HCI, stirred for 1 h at O°C, then extracted with ether (3 x 25 mL), dried (MgS043) and evaporated Diethyl 4-[ (Z)-6-decenyl]-3-oxopentanedioate (5). To to give an yellow oil. This oil was added to a stirred a stirred ethanol solution (150 mL) of the magnesium solution of triftuoroacetic acid (50 ~tL) in anhydrous CH Cl (20 mL). After 20 h being refluxed, the I chelate prepared from diethyl 3-oxopentanedionate 2 2 ( 12.7 g, 63 mmol), magnesium turnings (2.29 g, 95 reaction mixture was concentrated in vacuo. The mmol) and a trace of iodine was added 1-bromo further purification by PM PLC gave 0.51 g (94%) of (Z)-6-decene (18.0 g, 75.5 mmol) at room temperature pure (GLC) product 2c. Ca\cd for C 10H 2K0 2: C, 76.13; I and the mixture was refluxed for 18 h under argon.36 H, 11.19. Found: C, 75.91; H, 10.98. 'H NMR: 8 0.90 The reaction mixture was evaporated in vacuo to give a (t, 3H,1=7.3 Hz, CH,CH2CH2-), 1.28-1.94 (m, 161-I, -CH=CI-1-- dark oil which was acidified with 10% HCI and taken 8 x-CH2 -)' 2.01 (m ' ' 4H -CH--2 up into ether. The ethereal solution was washed with CH2-), 2.44 (ddd, IH, 1=6.8/8.8/17.6 Hz, I -), (dt, tH, J = 6.9/6.9/17.6 Hz, water and dried over MgS04 • After evaporation, >CH-CH2 2.58 chromatography (PMPLC) of the crude product gave.. >CH-CH2-), 4.28 (m, lH, J = 2.9/5.117.8/10.6 Hz, 17 .2 g (80%) of the oxoester 5. Calcd for C 1 ~H.12 0 5 : C, >CH-CH2-), 5.32-5.39 (m, 2H, -CH==CH --). I 67.03; H, 9.47. Found: C, 68.88; H, 9.39. 'H NMR: 8 IR (cm-'): 3001 m [v(C-H), cis-double bond], 1736 vs 0.90 (t, 3H, l = 7.3 Hz, CH1CH2CH2-), 1.28 (t, 6H, [v(C=O), lactone], 1654 w [v(C=C)]. .I= 7.1 Hz, 2 x OCH2CH,), 1.29-1.40 (m, 8H, 4 x -CH2-), 1.86 (m, 2H, CH,CH2CH2-), 2.00 (m, Bioassays I 4H, --Cl::l2-CH=CH---QJ.2-), 3.54 (d, 1H,l= 15.8 Hz, -Cl::l2-CO-), 3.59 (t, 1H, l = 7.3 Hz, Insects. Males of C. 1110/esta originated from the -CO--CH<), 3.61 (d, IH, 1=15.8 Hz, laboratory colony maintained on an semiartificial diet ·-Cl:-J2-CO-), 4.19 (q, 4H, 1=7.l Hz, under a 16:8 light:dark regime. Moths were sexed as I 2 x OCI:bCH,), 5.30-5.39 (m, 2H, -Cl::l=CH-). pupae and males were stored separately from females. Newly emerging adults were collected daily, provided l\Jcthyl 5-oxo-(Z)-12-hexadeccnoate (6). A solution of with water and sugar solution absorbed onto cotton I 5 ( 14.0 g, 41. l mmol) in dry 1,2-dimethoxyethanc wool under the same light and temperature conditions. (DME, 15 mL) was added dropwise to a stirred Males 2-4 days old were used for EAG experiments, solution of NaH (2.5 g of a 50% mineral oil disr>crsion, 3-4 days old males were used for flight-tunnel 41. l mmol) in dry DME (50 mL) at room temperature observation. I and stirring was continued for 1 h under argon:'7 Ethyl 3-brornopropionatc (8.9 g, 49.3 mmol) and finely Electroantennography. Two glass Ag/AgCl microclcc powdered Na! ( 1.65 g) were then added to the solution trodcs tilled with physiological saline were used for I and the mixture was relluxed for 18 h with stirring. The EAG recordings: the ground electrode was placed into reaction mixture was cooled to room temperature and the head capsule of an intact male moth anti the the solvent was evaporated. The residue was heated recording electrode was connected with the distal end with 15% aq solution of NaOH ( IOO mL) under reflux of the male antenna, the tip of which had been cut off. I for 24 h and the reaction mixture was acidified with Antenna! responses were amplified (signal conditioner coned HCI, saturated with NaCl and extracted with CyberAmp 320, Axon Instruments), digitized (Mctra ether. The dried solution (MgS04) was added into an byte DAS-16 AID, sample period 250 ms) and analyzed ethereal solution (300 mL) of freshly prepared diazo by a PC 486 computer (Stand Alone Acquisition I mcthane. After 20 h of being stirred the reaction System, Run Technologies). mixture was concentrated in vacuo and the residue was purified by PMPLC. Chromatography afforded 7.1 g The main pheromone component of the Oriental fruit ( 61 % ) of the ester 6. Ca led for C 11H,110.,: C, 72.30; H, moth, C. molesta, and its analogues lb-d and 2a-c I 10.71. Found: C, 72.19; H, 10.51. 'H NMR: 8 0.90 (t, were dissolved in hexane forming a series of dilutions 3H, 1=7.4 Hz, CH,CH2CH2-), 1.24-1.40 (m, SH, from 5 ng to 5 ~tg per ~LL Aliquots of 5 pL were 4 x --CH -), 1.56 (m, 2H, CH.1CH CH -), 1.89 (m, pipetted onto a filter paper disc (10 mm i.d., Whatman I 2 2 2 I \ I ~\ I New mimics of the acetate function 487 no. 2) and each loaded disc was inserted into a Pasteur ously with six pairs of dishes (one test and one control) I pipcttcr after solvent evaporation. The odor cartridges in four replicate series. Mating efficiencies of males in were stored deeply frozen in closed glass vials when the test and control dishes were expressed in the form not used for experimentation. The cartridges condi of confusion coefficients, CC (%) = (CdNc-Cil tioned in laboratory temperatures for at least 1 h were NE)IOO, where CC is the confusion coefficient, C(' no. I used for stimulation. Stimuli were delivered onto the of copulations in controls, Ne no. of pairs in controls, antenna! preparation by air puffs blown through the Cr; no. of copulations in the experimental group and N 1• cartridge outlet of which was positioned at a distance no. of pairs in the experimental group. I 2.5 cm from the antenna. The stimulus duration was 1 0.8 s, the air flow rate was I L min • Between succes sive stimulations the antenna! preparation was blown Flight-tunnel experiments. The CM males were flown by a continual stream of clean and humidified air. in a 1.86 m long x 0.3 m wide x 0.3 high plexiglass I Intervals used between two successive stimuli ranged flight-tunnel. Charcoal filtered and humidified air was from I to 20 min, depending on the type and intensity pushed through the tunnel by four ventilators. The air 1 of the stimuli. Typicaiiy, i-4 min were adequate for velocity was maintained at 0.5 ms • The flight-tunnel complete recovery of the EAG at lower doses, while conditions used were: 22-26 °C, 40-60% relative I I0-20 min were necessary when doses greater than 10 humidity and 700 lux light intensity. ~tg were used. Three EAG replicates were recorded for each serial dilution of each odorant. Recordings were In a preliminary series of experiments male reactions repeated on three IT)ale antennae. The main phero to the calling female to la alone and to the thrce I mone component Ia (50 ng) served as a standard to component pheromone blend (la 90%, £8-I 2:Ac 6%. normalize EAG responses from different individuals Z8-12:0H 4%) were determined. Males were allowed and to control over viability and constancy of the to respond to 1, 10 and JOO ng of pheromone blend to I preparation. Stimulation with the standard both determine which odor source is comparablc with the preceded and followed each experimental session. The calling female. The pheromone blend (Hl ng) loaded EAG responses to solvent were subtracted from the on a filter paper disc (10 mm dia) was fully comparable overall EAG response. EAGs to test chemicals were with the female and therefore was used as a standard I then expressed as a percentage of the EAG response in all flight-tunnel experiment. to the standard stimulation. Using pheromone analogues, two different types of observation were performed. Firstly, male reactions to I Single sensillum recording. Receptor potentials and the pheromone standard (10 ng) masked by 100 ng of nerve impulses were recorded extracellularly from the respective analogue were observed to see if the receptor cells associated with the s. trichodea using a analogue has an ability to modify the male orientation 3 modified tip-cutting technique. ~ A whole animal to odor source. Secondly, to determine if the analogue I preparation was used. A male in a disposable pipette can substitute the main pheromone component la, tip was fixed in place by small droplets of molten wax, males were observed while responding to the odor while the head and one antenna were protruded. The source in which la was replaced by an appropriate antenna was carefully bent dorsally and fixed by wax. amount of the analogue. I The tips of s. trichodea were cut by means of two glass microknives (microelectrodes with broken tips, ~ 30 The experiments were performed from 13 to 15 h after µm i.d.) mounted in micromanipulators. The recording the beginning of scotophase. Virgin males (3-4 days electrode (10 µm in diameter) slipped over cut s. old) were placed individually into clean glass tubes I triclwdea was filled with receptor lymph saline, the (release cages, 10 cm long, 4 cm i.d.) 15 min prior to reference electrode, inserted in the head, contained each session. After 15 min acclimatization period malcs saline approximating the ionic composition of the moth from the central part of the tunnel into 8 were released I haemolymph.' Prior to the slipping, the tip of the an odor plume which was created by pinning the filter recording electrode was dipped into heated vaseline to paper disc (10 mm dia) loaded with odor onto the prevent it from drying out. The electrical activity of the holder placed centrally near the upwind end. The filter receptor cells was recorded similarly as EAG record paper disc created turbulence and so structured the I ings on the same instruments. Receptor potentials (DC plum (its parameters and orientation was checked recordings) and spike activity (AC recording) were using TiCl4 prior to and after each flight session). Each recorded simultaneously by two independent channels male was tested once and then discarded. Due to the of signal conditioner. relatively rapid release rates of volatile chemicals from I filter paper sources, only five males were tested for Short-range behavior. The effect of analogues on each filter paper source. In six replicate series, male prccopulation behavior was investigated in altogether 30 males were flown for each treatment. To I disposable Petri dishes (10 cm i.d.). The compound assure a convenient state of the males, an additional investigated was loaded on a filter paper disc ( IO·mm five individuals were tested on the three-component dia) placed in the center of the dish housing the calling pheromone blend after each day's session. fcmalc. After 30 min of equilibration a male was intro I duced into the dish and its behavior was observed for a Male behavior was classified into four categories: ( i) 30 min period. Experiments were performed simultane- activation (walking and wing fanning), (ii) take off, (iii) I .JI I I 488 M. HosKovEc- et al. I oriented flight and (iv) touching the odor source, M.; Leber, J. Biochem. Biophys. Res. Comn11111. 1990, 169. landing and copulation attempts. The total time of 610. observation was either 2 min if the male did not take 12. Rothschild, G. H. L. Entomo/. Exp. Appl. 1974, 17, 204. off or it lasted until its landing. I 13. Eckert, H.; Foster, B. Angew. Clrem. Int. Ed. Engl. 1987. 26, 894. Statistical analysis. The data were subjected to statis tical analyses utilizing the StatgraphicTM_Plus software 14. Hoskovec, M.; Saman, D.; Koutek, B. Collect Czech. Chem. Commun. 1994, 59, 1211. package (Manugistic, Rockville, Maryland, U.S.A.). I Student's t-test (a= 0.05) was used to compare mean 15. Gcrstl, Z. Chemistry, Agrirnlture and tire F:111·irm1111c111: Richardson, M. L., Ed.; The Royal Society of Chemistry responses for differences (H 11 :m 1 = m 2). Thomas Graham House, Cambridge, 1991; Ch. 18. I !\'linimum energy calculations (MEC). MEC were 16. Hoskovee, M.; Kalinova, 13.; Konecny, K.; Koutek, B.; performed for ethyl acetate, ethyl chloroformatc, Vrkoc, J. J. Chem. Ecol. 1993, 19, 735. butan-3-olide and pentan-4-olide. These compounds 17. Koutek, B.; Hoskovec, M.; Konecny, K.; Vrkoc, J. J. represented those parts of the molecule that were Chromatogr. 1992, 626, 215. I altered. The aliphatic unsaturated chain of the 18. Liljefors, T.; Thelin, B.; Van der Pers, J. N. C. J. Chem. molecules remained constant for all analogues, -~nd was Ecol. 1984, JO, 1661. not expected to affect the total conformation of each 19. JOnsson, S.; Liljefors, T.; Hansson, B. J. Chem. Ecol. molecule differently. The PC software used was Hyper 1991, 17, 1381. I ChemTM for Windows (Autodesk, Sausalito, California, U.S.A.). Energy minimization operations were run 20. Lucas, P.; Renou, M.; Tellier, F.; Hammoud, A.; Audcr mard, J. H.; Descoins, C. J. Chem. Ecol. 1994, 20, 489. until the energy gradient for each molecule was less 1 than 0.1 kJ mo1- • 21. Jeffrey, G. A.; Saenger, W. Hydrogen Bondmg in I Biological Structures; Springer Berlin, 1991; pp 1-569. 22. Priesner, E.; Jacobson, M.; Bestmann, H. J. Z. Natur Acknowledgement forsch. 1975, 30, 283. I 23. Prestwich, G. D.; Carvalho, J. F.; Ding, Y. S.; llcndricks, We acknowledge the financial support of this work by D. E. Experientia 1986, 42, 964. grant no. DHR-5600-G-00-1051-00, Program in Science 24. Preiss, R.; Priesner, E.J. Chem. Ecol. 1988, 14, 797. and Technology Cooperation, from the U.S. Agency I for I ntcrnational Development. 25. Schwarz, M.; Klun, J. A.; Vebel, E. C. J. Chem. Emf. 1990, 16, 1591. 26. Pommier, A.; Pons, J. M. 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Chem. Senses 1994, 19, I. 34. Fieser, H. J.; Fieser, M. Reagents for Organic .S)·n1/ie.1is: 7. Duran, I.: Parrilla, A.: Feixas, J.; Guerrero, A. Bioorg. John Wiley: New York, 1967: p 528. i Med. Chem. Lett. 1993, 12, 2593. 35. Noels, A. F.; Herman, J. J.; Teyssie, P. J. Org Clrem. 8. Canle, A. M.; Baker, T. C.; Carde, R. T. J. Chem. Ecol. 1976, 41, 2527. 1979, 5, 423. 36. Naoshima, Y.; Ozawa, H.; Kondo, H.; Hayashi, S. /lgric. 9. Silverman, R. B. 1he Organic Chemist1y of Dmg Design Biol. Chem. 1983, 47, 1431. I and Drug Action; Academic: San Diego, 1992; Ch. 2. 37. Naoshima, Y.; Nakagawa, H.; Wakabayashi, S.; 1layashi, 10. Uotani, K.; Naganawa, H.; Kondo, S.; Aoyagi, T.; S.Agric. Biol. Chem. 1980, 44, 1419. Umezawa, H.J. Antibiotics 1982, 35, 1495. 38. Van der Pers, J. N. C.; Den Otter, C. J. J. Insect Physiol. I 11. M;1yer, R. J.; Louis-Flamberg, P.; Elliott, J. D.; Fisher, 1978, 42, 337. I (Received 28 August ·1995; accepted 14 September 1995) I BESTAVAILABLf COPY I ~I