biorational pesticides based on pheromone

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FINAL REPORT

Covering Period: August 15. 1991 - February 28, 1996

Submitted to the Office of the Science Advisor U.S. Agency for International Development

BIORATIONAL PESTICIDES BASED ON PHEROMONE ANALOGUES

Principal investigator: Grantee Institution:

Collaborator: Institution:

Project Number: Grant Number:

A.I.D. Grant Project Officer: Project Duration

R~c~D IN RIDJR

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Jan Vrkoc Institute of Organic Chemistry and Biochemistry Department of Natural Products Academy of Sciences of the Czech Republic Flemingovo nam. 2, 16610 Praha 6. Czech Republic

Glenn D. Prestwich Depa1iment of Chemistry State University of New Yoriv Stony Brook. N. Y. 11 794-3400, U.S.A.

936-5600 DHR-5600-G-00-1051-00

Dr. Phil Warren August 14. 1991 to February 28, 1996

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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

I 5.2. Vapor pressures determinations 4

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

I 9. Future work 14

10. Literature Cited 15

I Appendix

I I

Table 1 16

Table 2 i7

I Table 3 18

Table 4 19

I Figure 1 - 12 Cydia molesta 20

I Figure 1 - 18 Ostrinia nubilalis "? -'-

Publications 1 - 9 so

I I I . I#

I I I I I I I I I I I I I I I I I I I I I

3. Executive Summary

All new compounds were chemically characterized and biologically tested. The data from

electrophysiological and behavioral test suggested that inhibitory properties of the

pheromone analogs are not entirely 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 further biochemical as well as field studies. Pest

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

materials. In past decades attention is given to volatile natural chemicals (semiochemicals)

produced by arthropods for their chemical communication among or between species. Sex

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

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

relies on chemical inhibition of insect olfaction by irreversibly activating a receptor cell or

blocking pheromone recognition. For this strategy some synthetic pheromone analogs were

suggested and for biological tests electrophysiological and behavioral test were used.

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

were chemically characterized and biologically tested. The data from electrophysiological and

behavioral test suggested that inhibitory prope1ties of pheromone analogs are not entirely

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

fmther biochemical as well as field studies.


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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 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 of prospective biorational pesticides - analogs of insect sex pheromones which could have a potential to disrupt premating communication between the sexes and thus to 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 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 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 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 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 this effect have not yet been recognized. Mechanisms for interrupting long distance communication even by natural pheromone are very complex and probably more than 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.

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 sensilla by limited quantities of a chemical compounds and in effect a selective anosmia is resulting.

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 (l ), fluorinated ESE 10-12:Ac (2), fluorinated analogs of esters components of Diparopsis castanea (3) and trifluormethyl ketones of Spodoptera littoral is ( 4 ). 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). Oxime ether analogs of sex pheromone components of A.gratis segetum are


-.+-

pheromone mnmcs (9). Electrophysiological and morphological characteristics of pheromone receptors and the behavioral activity of analogs have been studied m Diprion pini ( 10).

The insects chosen for our study was one of key orchard pests, the Oriental fruit 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 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 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 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 found.

5. Methods and Results:

5.1. Synthetic work All pheromone mimics were synthesized by procedures reporting in project-funded

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 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 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

electrophysiological studies the saturated vapor pressures of all compounds were determined using a method based on gas chromatographic retention data. The data are summarized in Table 3.

5.3. Entomological studies 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 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 wind tunnel observation.


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Electroantennograph_1 · Two glass Agi AgCl 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 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).

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 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 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. Stimulus duration was 0.8 sec. the air flow rate was 1 l.min- 1

• Between successive 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 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 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 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 expressed as a percentage of the EAG response to the standard stimulation.

Single sensillum recording 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 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 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 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 instruments. Receptor potentials (DC recordings) and spike activity were recorded simultaneously by two independent channels of signal conditioner.


Shorr-range behavior The effect of analogs on male precopulation behavior was investigated in disposable

Petri dishes ( 10 cm i.d. ). The compound investigated was loaded on a filter paper disc ( 1 O 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 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, 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 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

0.3 high plexiglass flight-tunnel. Charcoal filtered and humidified air was pushed through the tunnel by four ventilators. The air velocity was maintained at 0.5 ms- 1

• The flight-tunnel 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 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 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, 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 were observed while responding to odor source in which the main component in pheromone standard was replaced by an appropriate amount of the analog.

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 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 (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 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 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 each day's session.


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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 attempts. The total time of obser.vation was either two minutes if the male did not take off or it lasted until its landing.

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 (a= 0.05) was uere used to compare mean responses for differences (t1i1:m1 =m2).

5.3.2. Results

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 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 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 EAG responses to the lactone derivatives were observed (Fig. 5 ON).

The spontaneous activity of neurones associated withs. trichodea was generally very 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 not. In 0. nubilalis no lactone analogs showed any ESG activity (Fig. 9 ON).

Behavioral observations 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 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 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 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 flight-tunnel equipment.


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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. molest a males 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 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 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 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 (Fig.11 ). For 5-membered lactone no significant (at p=0.05) effect was observed ( 22% and 33% reduction, Fig.12).

II. lsosteric analogs: chloroformates (Ci11 8, ON 9) and sulphur analogs (CM 10, CM 11, ON 12, ON 13)

EAG Chloroformate and sulphur analogs showed the larges EAG activities of all analogs

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.

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 of neuronal activity was very similar to that of authentic pheromone. Sulphur analogs generally showed short lasting spike activities in comparison with authentic pheromone and/or chloroformates.

Behavioral observations Shorr rnnge bioassay m C. molesta (Fig. 10 C M): Values of the"confusion

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%; 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 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 that the exposure of C. molesta males to a mixture of pheromone standard and a 10-fold


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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 ON). ON 9 showed 32, 80* and·'90%* reduction of male orientation, ON 12 even higher. i.e. 90* and 100*% ).

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 concentrations tested.. In 0. nubilalis however, EAG responses were relatively high. Measurable effects were observed at concentrations three orders of magnitude higher than 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 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 mechanisms underlying this effect.

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 ESG activity.

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 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%).

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 100%* of responding males). Strong inhibitory effect was observed also for ON 6 (80%* at both l 00 and l 000 ng levels).

IV.Saturated compounds (12:Ac, 14:Ac)

The saturated compounds generally showed rather high EAG activity in both species (Fig. 4 CM, fig. 4 ON) being within the range of activity of the isosteric analogs.


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Contrary to EAG results no ESG reactions were observed. Nevertheless the presence of strong receptor potentials was observed (Fig. CM 8), an effect accounting for a rather strong EAG activity.

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 strong disruption effect for mating behavior.

Flight-tunnel observations. Likewise saturated compounds inhibited orientation of males to pheromone in both species. 90% * disruption was observed when 100 ng of

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 to odor source (Fig. 18).

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. 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 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.

As the molecular shapes of chloroformates and lactones are identical with respect to the unsaturated hydrocarbon chain with main pheromone components, the main spatial 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 the 4-membered Iactone (Scheme b) shows the largest deviations.

\

a b

I /

~~/'/ I !/

/; 0

c


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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 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 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 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 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.

Although the replacement of the acetate methyl group by a chlorine in chloroformate analogs is not supposed to have important steric consequences, the methyl 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 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 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 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 hyperphysiological concentrations. Another explanation for the significant inhibitory activity of chloroformates could be their possible binding to antenna! proteins through a carbamate 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 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 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 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, respectively). This fact could account for the strong confusion effect observed for sulphur


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analogs in flight-tunnel experiipents both in C. moles ta and 0. nubilalis. When the 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.

The vinyl-branched analogs were supposed to substantially affect the pheromonemediated 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. 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 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 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 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 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 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. 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 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 male behavioral responses.

Conclusions The present data suggest that the inhibitory properties of the pheromone analogs

are not to be entirely connected with their mimicking capability of the pheromone. Apparently, several constitutional and configurational properties of the molecule and, 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 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 studies directed towards mechanisms controlling mating disruption.


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6. Impact, Relevance and Technology Transfer

The support from Program in Science and Technology Cooperation. U.S. Agency 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 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 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 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 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.

All the knowledge. expertise and equipment we have obtained during the project duration will positively influence the future research of individuals in both chemical and entomological laboratories of our department.

Another important contribution for the progress of this project was also the possibility to purchase chemicals. solvents and bulks.

7. Project Activities/Outputs

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.

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. Florida; Dr. J. Tumlinson.

-J.. Dr. B. Kalinova, 3 months in 1993 at Arizona Research Laboratories. University of Arizona, Tucson: prof J. Hilderbrandt,

5. Dr. J.Zdarek. 2 months in 1994 at Department of Entomology, University of Massachutes, Amherst; prof. R Carde,

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 New York at Stony Brook. New York: prof. G.D. Prestwich.


- l-1--

Publications

1. B. Koutek. M. Haskovec. K. Konecny and J. Vrkoc.: Gas chromatographic 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-diaminopropane. Synthetic Communication 23. 2399-2404 (1993).

3. M.Hoskovec. B. Koutek. J. Lazar, L. Streinz. E. Brofova. B. Kalinova and J. Vrkoc.: a.a-Disubstituted allylic sulfones: an approach to the synthesis of vinyl-branched pheromone analogues. Helv. Chim. Acta 77, 1281-1287 (1994).

4. B. Koutek. M. Haskovec. P. Vrkoeova. K. Konecny and L. Feltl.: Gas chromatographic determination of vapour pressures of pheromone-like compounds II. Alcohols.

J Chromatogr. 679, 307-317 (1994). 5. L. Streinz, A. Svatos. J. Vrkoc. J. Meinwald.: Chlorofluoroacetic acid

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 D. Deutsch.: Inhibitors of arachidonoyl ethanolamine hydrolysis. J Biol. Chem. 269. 2937-2940 (1994).

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. 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 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. Sebek. D. Saman and J. Vrkoc.: New mimics of the acetate function in pheromone-based attraction. Bioorg. .'vied. Chem . .:+. -1-79-..+88 ( 1996).

8. Project Productivity

To the best of our knowledge we did not succeed to prepare azaanalogs only. The azaanalogs we prepared were tmstable and not suitable for further biological tests

9. Future work.

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 continue the study of the vinyl-branched analogs in more detail.


-l5-

10. Literature Cited

1. Klun J.A .. Schwarz M .. Wakabayashi '.'J .. Waters R.M.: Moth responses to selectively

fluorinated sex pheromone analogs. J Chem. Ecol. 20, 2705 ( 1994 ).

2. Tellier F .. Sauvetre R.: Synthesis of a new fluorinated analog (E.E)-8.10-dodecadienol

( codlemone) Tetrahedron Lett. 33. 3643 ( 1992).

3. Tellier F., Sauvetre R.: Fluorinated analogs of ester components of red bollworm sex

pheromone. 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. Bioorg. Medicinal. Chem. Letter 2. 2593 ( 1993).

5. Tellier f., Hammoud A .. Ratovelomanana V .. Linstrumelle G .. Descoins C.: Synthesis of a

biologically active chlorinated analog of (E.E)-8, 10-dodecadienol ( codlemone ). Bioorg. JV!edicinal. Chem. Letter l, 1629 (1993).

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, 1 O-dodecadien-1-ol, the main component in codling moth ( Cydia

pomonella L.). J Chem. Ecol. 20, 489 (1994).

7. Riba M., Eizaguirre M., Sans A .. Quero C.. Guerrero A.: Inhibition of pheromone action

in Sesamia nonagriodes by haloacetate analogs. Pestic. Sci. 41, 97 (1994).

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 aggregation pheromone. Russ. Chem .. Bull. 42, 7 41 (1993)

9. Martin D., Weber B.: Oxime ether analogs of sex pheromone components of turnip moth

(Agrotis segetum Schiffermuller ). J Chem. Ecol. 20. 1063 ( 1994 ).

10.Anderbrant 0 .. Hansson B.S.: Electrophysiological and morphological characteristics of

pheromone receptors in male pine sawflies. Diprion pini (Hymenoptera: Diprionidae ), and

behavioural response to some compounds. J Insect Physiol. -+ l. 395 ( 1995)


Table I.

CM l

CM2

CM3

CM4

CM 5

CM6

C:'vl 7

CM 8

CM 9

Uvl!O

CM 11

-16-

Cyclia mo!esta pheromone analogues

·· · ·· OCOCH3

CF,

~"ocoo1,

~-rocoa

0

0

/V5~/"-0COCil3

~S~OCOCil3

(Z)-8-dodecen-l-yl acetate

(Z)-1 O-tetradecen-3-olide

(Z)-11-pentadecen-4-olide

(Z)-12-hexadecen-5-olide

7-propylnon-7-en-1-yl acetate

7-propy lnon-2-en-1-y I acetate

7-propyl-9 ,9-difluoronon-8-en

l-y I acetate

(Z)-8-dodecen- l-yl chloroformate

dodec-1-yl chloroformate

9-thiadodec-1-y l acetate

8-thiadodec- l-yl acetate

I I I I I I I I i I I I I I I I I I I I I

Table II

ON l

ON 2

ON 3

ON4

ON 5

ON6

ON7

ON 8

ON9

UN 10

ON 11

ON 12

ON13

-17-

Ostrinia m1bilalis pheromone analogues

0 0

OCXXll3

~VV'ocna~ CF1

/'~ocoa

~A/'y/'\/VOCXXJIFO

~VV'V'\/vocua1m

/vs~ , ClCOCliJ

/'-S~OCXXJiJ

(Z)-11-tetradecen- l-yl acetate

(E)-11-tetradecen- l-yl acetate

(Z)-13-hexadecen-3-olide

(Z)-14-heptadecen-4-olide

(Z)-15-octadecen-5-olide

l O-ethyldodec-11-en- l-yl acetate

l 0-ethyldodec- l 0-en- l-yl acetate

l O-ethyl-12, 12-diOuorododec-

11-en- l-yl acetate

(Z)-11-tetradecen- l-yl

chloroformate

(Z)-11-tetradecen- l-yl

(R)-chlorofluoroacetate

(Z)-11-tetradecen-l-yl

(S)-chloro i1uoroacetate

11-thiatetradec-1-yl acetate

12-thiatetradec- l-yl acetate

-

I c<J -~

T;:ible 3. Sex Pheromone Analogues of C. mo/esta and 0. nubila!is - The V<Jpour Pressures

r------~

!code of tested L

cornpound I 5o·c "6CfC 7o·cJ:

18-12 ·0/\-::----·,---, .1 10s 3 802 -

I 80'C

n.12 - 2 770 Cl\D Cl\ll Cl\\~ 2 770 2 590 2 '1•10 Cl\16 3 810 3 500 3.250 Cl\!7 2 5•10 2 360 2 200 ('f\18 2.560 2 410 (_ l\l'l 1 330 1 310 c_'idl(l

71 l-lH1/\c ()N3 Ul'l"l t'N5 ()N6

llN7 ON8 ON9 UNIO ON!! ONl2

0 610

0 560

3 509 2 660 1 956 4 054 2 300 2.990 2.060 2 320 1 290 0 770

3 756 ---

0 620 3 270 0 570 2 490

---

relative retention times ---1==-- -- --- _results--·

9o·c 1oo·c 110·c 12o·c 13o·c 14o·c 15o·c 15o·c intercept_ slope r2 [%JIPcc [P:iJ

3.265 3 066 - - - - - - 1 8249 -0. 1158 99 96 0 312

2 560 2.480 2 400 2.310 - - - - 1 1570 -0 0543 99 83 0.0548

1.912 1 877 1.837 1 816 - - - - 0 7655 -0 0308 99.51 0 00828

3 808 3.644 3 432 3 329 - - - - 1 6·192 -0 0813 99·l1 0 00281

2.170 - - - - - - - 1.2669 -0 0887 99 96 0 536

2 790 - - - - - - 1 6576 -0 11'10 99.91 0 368

1.940 - - - - - - - 1 2078 -0 0987 99 99 0 572

2 230 2.130 - - - - - 1 1076 -0 0655 99 57 0 184

1 280 1 260 - - - - - - 0 3158 -0 0182 99 22 0 135

0 780 0 810 0 820 0.830 - - - - -0 3084 0 0285 96 03 0 0305 ----- -------'-----

3 481 3 231 2 996 2 812 - - - - 1 7040 -0.1163 99 95 0 0288

- 2 720 2 540 2 440 2 360 2 290 - - 1 2021 -0 0685 98 02 0 00462

- - - 1 932 1 903 1 886 1.861 1 836 0 7229 -0 0208 99 3·1 0 000892

- - - 3 775 3 602 3 '163 3_318 3 182 1 5·186 -0 0710 99 90 0 000287

0.640 0 650 0 670 - - - - - -0 5223 0 0310 98 28 0 0382

3.050 2.860 2 720 2.560 - - - - 1 4970 -0 0965 99 89 0.0364

0.580 0.590 0.600 - - - - - -0 5982 0.0228 99.90 0 0399

2.350 2.280 2.160 2.080 - - - - 1 0725 -0 0668 99 33 0 0175

2.270 2.180 2.080 2.000 1.920 - - - 0.9753 -0.0641 99 87 0 0038-4

2 210 2.120 2.030 1.960 1 890 - - - 0 9355 -0 0596 99 96 0 00624

- 3.050 2.930 2.750 2.620 2.080 - - 1 3794 -0.0830 99.47 0 00365

standards

p/p, stand:ird P.us c [Pa

1 0 n-C,.H 30 1 8043 5 7 n-C, 5H,.. 0 1910 36.5 n-C,aHJa 0 02007 107.5 n-C 1aHJa 0 02007 0.56 n-C,.H30

1 8043 0.82 n-C,.H,0

1 80'13 0.55 n-C,.H30

1 8043 1 64 n-C,sH:n 0.5760 2 24 n-C,~HJ.< 0.1910 9.9 n-C,aH,,, 0.02007

1.0 n-C,aHJ.< 0 1910 62 n-C 1eH 3, 0 02007

32.3 n-C, 0 H<z 0 00209 100 3 n-C20H" 0 00209 0.75 n-C 1aH 30

0.02007 0.79 n-C,,H 3, 0 1910 0.72 n-C1eHJ.< 0.02007 1.65 n-C 17 H30

0.06148 7.5 n-C,,H'" 0 02007 4.6 n-C,,HJ, 0 02007 7.9 n-C 1 ~H 1~ 0.02007

--

-------- ------------


-19-

Table 4.

Differences among analogs in the complementary electrophysiological and behavioral tests

I I CM ON ! I

Analog Code I type EAG I ESG Behavior EAG ESG Behavior

I

1, OCOCl I CM8

1. -i...++ +++ ++ I

I i ON9 I i I +++ ++ ! ++ i I

I I i I

: Saturated i I I

I I 12:Ac +++ I I +++ I I -I I I i I 14:Ac I +++ ++ ' ! i I - I

' I ' I

I 9-thia I CM10 ' I I

I I I

i

I ++ i +

I ++ I

111-thia I ON12 I I +++ + -r-+ I I I

' I

I 8-thia I

CMll I ++

I I I 12-thia ON13 I +++ + ! I

I

i ! I I I

\ 4-lactone i CM2 - ++ I I I ' ' ON3 -r

: 5-lactone CM3 --r

ON4

vinyl CMS ON6 + +

+++ most active


Cydia molesta

Mating age

Percentage of copulations 1oor-~~~~~~~~~~~~~~~~~~~~~___,

80 --------- --sa---~a---~0------------50

60

40 -----

20

0 1 2 3 4 5 6 7 a

Age (in days after eclosion)

Numbers above bars = N

Fig. 1: Circadian mating activity of Oriental fruit moth, Cydia mofesta. The maximum of mating activity was observed between 12-13 hr of photophase. Within this period all behavioral experiments were made.

-20-


-21-

Cydia molesta

Circadian mating activity

No. of copulations

251 , .. 20 r---------------------------------:: r~--------------- --

1 ; f. ;:: 15 --------------------------------

Fig. 2:

5 ------------------------------- ~ . :·. : . -~. :: 't --------

I .:~ ; . : :~ :: -~ ::;

Q l~r --'--' ----'--'-'--'-'--'--'--'---'--'-'--'-' _,_r -'--'--'--'-'-"--'--'-'--'--'-'''--'--'' "'-""--""'-""'-=-""-""-'""---'"'-=-u.

2 4 6 a 10 12 14 16

Time of day (hours)

18 20 22 24

The effect of age on copulation of Oriental fruit moth, Cydia molesta. 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 used in all behavJOral experiments.

~--

I

Cl r I

I

--

l:J ..... I/) .... 0

~ ~

~ .:r; w

Cydia molesta - sex pheromone

250,00 I -----------~----------]

I 200,00 J

150,00

100,00

50,00 -

•-~-' . o o~--------10----------<o--------o~------o-

i

I

/~

o 00 l ___,_ _______________ , _____ _ '

hexan -5 -4 -3 -2 -1 0

log dose (µg)

---- ------------

Fig. 3:

--zs-12:Ac

--E8-12:Ac

-+-Z8-12:0H

-0-12:0H

I

The EAG dose-response curves of four components of female sex pheromone_

Responses are expressed in percentage of EAG reaction to standard (0.05 ug of

Z8-12:Ac). No correction for volatility of compounds was made. Different pheromone

components show different dose-response curves indicating different receptor populations.

-----·-----------··----

I r<J <·1

I

-"O +J (/) -0

~ 0

200.00 I

150,00

Cydia molesta - pheromone analogs I.

-------zs-12:Ac

--CM9

-+-CMS

100,00 (.!)

-o-12:Ac

<t w ...L-.----------------

- "' 0,00 + ·------;------------·---·-----·

-5 -4 -3 -2 -1 0 2 3

log dose (µg)

- - -~ -· -- -------------------------

Fig. 4: The EAG dose-response curves to Z8-12:Ac and chloroformate pheromone analogs

Responses are expressed in percentage of EAG reaction to standard.

No correction for volati!Hy of compounds was made.

Unsaturated chloroformate analog and 12: Ac show relatively high EAG activity.

Double bond in the moli9cule is essential for retaining the electrophysiological activity

- unsaturated chloroforrrate analog showed much smaller EA G reactions.

---------------------

I

"" '' I

200,00

I 150,00 -1J ...... Vi .._ 0

~ 0

- 100,00 (!) <(

I W

50,00

0,00 _; ___ _

-5

--------·----- -~-·----------~------- ---~ - ---

C~rdia molesta - pheromone analogs II.

/------1 / I

:====-:;:----=====·======~ ~-~--------..., -0 -- - • :=--- I 0-.--a/~~

I i

-4 -3 -2 -1 0 2 3

log dose (µg)

--zs-12:Ac --CM2

-+---CM3

-D--CM4 ------

i I I i

--- ----- ------ _ ___J

Fig. 5: The dose-response curves to Z8-12:Ac and three lactone analogs. Responses are expressed in percentage of EAG reaction to standard.

No correction for volatility of compounds was made. 5-membered lactone show the

highest EAG activity followed by 4- and 6- membered factones.

I ---------------------

I <r, Cl

'

Cydia molesta - pheromone analogs Ill.

200,00 -----.

150,00 I ""O ....... (J)

...... 0

~ 0

I --CM10 I I~"'"''' - 100,00 (9

/:.------111 -+-CM11

<l'. LU I

I

--------0, 00 ~----------· --------'·----------

-5 -4 -3 -2 -1 0 2 3

log dose (µg)

Fig. 6: The dose-response curves to Z8-12:Ac and sulfur analogs.


No correction for volatility of compounds was made. Almost similar EA G

dose-response curves for sulfur analogs were observed, however CM 10 showed

slightly higher EAGs at saturation concentrations than CM 11.

---------------------

1 -0 rl

I

"O ...... V) -0

200,00

150,00

~ e_, 100,00 ~ <1'. w

Cydia molesta - pheromone analogs IV.

~·

. ------·------1

l±J8-12:Ac

1

MG M7

M5

50,00 I -~ a:==== ..

0,00 ;___ -+---------- - - I - ... ·----- -·-·-···

-5 -4 -3 -2 -1 0 2 3

log dose (µg)

Fig. 7: The dose-response curves to Z8-12:Ac and vinyl-branched analo1gs.


No correction for volatility of compounds was made. The vinyl-branched analogs

showed the smallest EAG activities from all analogs tested. The most active was CM

5, followed by CM 6 and CM 7.

--------------·-------


-27-

. alf cm2

crn3 cm4

I I~~~. I I 'I

~1' • .. ..i. 1.1 {

r I I

cm8

~12:Ac I 0.1 mV

0.5 sec

Fig. 8,: 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, 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 pronounced.


-28-

arr Z8-12:Ac

I I ,

~ 1

cm 10 l-&-~~~~~~~~ cm5

cm 7 cm5

E8-12:Ac Z8-12:0H

0.1 mV

0.5 sec 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 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. 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 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 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 separate specialists within the same sens1/lum.

I

°' rl I

0 ..... .....

..... :r: 6 0 0

,. dose (ng) 0 0 ~ 0 0

Fig. 10:

2 u

2 u

2 u

N ..... 00 N

Pheromone analog

c: ... 0;;:; ·u; ·u 2 !E c: w 0 0 u u

Mating disruption observed in Petri dishes in atmosphere permeated by respective analogs. Results are expressed as confussion coefficient indicating U1e strengh of mating dirsuption effect. The most potent mating disruptant was ZB-12:Ac inhibiting at a dose 1 ug mating almost 80% of pairs observed followed by chloroformate (60%) and sulfur analogs (57%). Analog CM 7 was the most active from vinyl-derivatives (35% disruption effect). CM 3 (45%) was the most potent disruptant from lactone analogs, followed by CM 4 (22 %). Minor pheromone components EB-12:Ac and ZB-12:0H exhibited considerable mating disruption effect at relatively low doses. ZB-12:Ac, the main pheromone component was the most potent disruptant of mating behavior in C. molesta from all compounds tested (42% at 0.001 ug level).

----··----------------

I 0 rri

I

Behavioral response

Cydia molesta - wind tunnel Inhibition of male reactions to pheromone

-

-100%

;~~\ J~----- CMS Pheromone analog .s .E ~ -0 Cl ....... .......

.§ :+: Q CD

~ ~ ~ ~ 12:Ac g c ~ ~ 0 Ql -_. ·c

0

Fig. 11:

"C <ll ....... - (/) ro o E ....... --= 0 Q) .... c: ._,. 0 <ll

:;::: (/)

:0 c ·- 0 .i:: c. c: (/) - <ll ....

Flight-tunnel observation of effect of selected analogs on male orientation to pheromone standard (three component pheromone blend, 1 O ng). Tile behavior of males were categorized into four categories: activation, taking off, oriented flight, touching the source and landing. Analogs were applied at 100 ng doses to filter paper disc loaded with the pheromone blend_ As indicated in short-range bioassay ZB-12:Ac was the most active disruptant compound, followed by 12:Ac, CM 8, CM 2, CM 10, CM 7, CM 5 and CM 3. ---------------------

I ~

r'I I

Behavioral response (!)

..:::L <1J .....

'O (!) .... c tl)

·.:: 0

Cydia molesta .. wind tunnel Substitution of the main component in the pheromone blend

Fig. 12:

14%

12%

8%

6%

4%

II) -<1> "O - L.. (1' (1'

cu E -g C') C') co nl c ...... t: ·- U) Q.) 't:J 0 t.J i::: ...... t... 0 '"C Q) c. cu a.. II) ....., cu <U ,__

...... ~ o-

Flight-tunnel experiments designed to answer the question if any of analogs tested can replace the main pheromone component in three pheromone blend. No tested compound was able to replace ZB-12:Ac in standard pheromone, though some compounds were able to activate males.

---------------------


Ostrinia nubilalis

Circadian mating activity

Percentage of copulations

sor--~~~~~~~-.. ........ ..-~~1 ~ 40 -----------------------------1'-----------f

30 -----------------------------!-----------

20 ----------------------------- ~ ---------

~

10 ------------------------ - '--------1

I 2 4 6 8 10 12 14 16 18 20 22 24

Time of day (hours)

• Lab population 19 Field population

Fig. 1: 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 compared wild and laboratory populations. The mating period for wild population was shorter with maximum between 4-7 hr of scotophase.

-32-


-33-

Ostrinia nubilalis

Mating age

Percentage of copulations

Age (in days after eclosion)

Fig. 2: 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 were used in all behavioral experiments.


Ostrinia nubilalis

Q) 200 -------------------TI

-3 -2 -1 0

dose (log µg)

Fig. 3:

-.~-

1 2

-34-

1

--- -------- --i -•- Z lab I

[Jz wild I

lab

wild I __ _J

The comparison of El~G responses to two pheromone components of femaie sex 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 sensitive to Z isomer than the laboratory ones.

I •r, C'i

'

----- ------ - -- -- -· ·---- -- ------ --- --- --1

Ostrinia nubilalis - pheromone analogs I.

- ---- - 1 - ---- l

I 200

I I

150

-a .._, Ul -0

~ 0

- 100 (9

I /~ 1-=•-z11-14:Ac

-n-E11-14:Ac 1--__ _,._ON 9

I

-{]-14:Ac -~-

<:( w

50

0 i__ -- ----- - __J _____ - -

-5 -4 -3 -2 -1 0 2 3

log dose (µg) ________ J Fig. 4: The EAG dose-response curves to Zand E isomer of 11-14:Ac, 12·Ac and to chloroformate analog ON 9. Responses are expressed in percentage of EAG reaction to standard. No correction for volatility of compounds was made Chloroformate analog showed relatively high EAG activity, though the measurable effects was observed at doses shifted to highe concentrations in comparison with the authentic pheromone. However, the saturation amplitude was similar as for Z11-14:Ac.

---------------------

I \0 r'I

I

200

150 ~

-0 ...... !/)

..... 0

~

Ostrinia nubilalis - pheromone analogs II.

--· .~

-------1

I I I i

!

--.z11-14:Ac

-11J-E11-14:Ac

--+-ON3

- 100 -a-ON 4

e,, <l'. w

-o-ON 5 -----

50 j

-o ; __ -------'1-~ ~==t===~o--==---.o-l------- ---0----___. --------0

0 -- - ---- -- ___ ,_ .!.--------------- _,_ -- ---

-5 -4 -3 -2 -1 0 2 3

log dose (pg)

-- --- - --- - -- --------------------- -- ---- -- --- - - - - -- -----------

Fig. 5: The dose-response curves to Zand E isomer of 11-'14· Ac and lactone analogs ON 2, ON 3 and ON 4. Responses are expressed in percentage of EAG reaction to standard. No correction for volatility of compounds was made EAG activity of lactone analogs was very small_

---------------------

I r~

r'I I

--- ----- - ------------- ----------------- --------------- ----- - -- ·---

Ostrinia nubilalis - pheromone analogs Ill.

i---- - -

I

200 -!

i ! I

' 150

"O ~

Cf) --Z11-14:Ac ..... 0 --11-E11-14:Ac

...._o o' - 100 ~/<1 -+--ON 12

<.:J <( w

-D I -o--ON 13

I

I

0 .

-5 -4

---------------- ___ ,_ -

-3 -2 -1 0

log dose (pg)

Fig 6: Th8 dose-response curves to Z and E isomer of ·11-14:Ac and sulfur analogs Of\! 12 and Of\! 13. Responses are expressed in percentage of EAG reaction to standard. f\Jo correction for volatility of compounds was made. ON 12 and ON 13 exhibited almost the same EAG activity. EAG amplitudes at saturation doses were relatively smaller than for those for authentic pheromone components.

--- ----- _____ J

---------------------

I 00 rr)

I

"O +-' Vl

....... 0

:::z 0

----- ----- -------- -------------

Ostrinia nubilalis - pheromone analogs IV.

--- --------

200

150 -· ~

__.---m '-<>-·Z11-14:Ac

-l!t-E11-14:Ac

--+-ON7

- 100 --0-0N 6

(.9 <( w

-o-ON 8

_ _.-+--------~ 50

!

Q$

-{)

I 0----

I

0 '.._ .. ------ ·---------------1----- - ---- ____ J ____ --------~

-5 -4 -3 -2 -1 0 2 3

- -- ----- -- ---- -

log dose (µg) ... ______ j

Fig. 7:

The dose-response curves to Zand E isomer of 11-14:Ac and three vinyl branched

analogs of Z11-14:Ac. Responses are expressed in percentage of EAG reaction to

standard. No correction for volatility of compounds was made.

---------------------

I

°' rr, I -----------------------------------

Ostrinia nubilalis - pheromone analogs V.

---HHH --- • " ------- -- -

I

200 J

I

150

TI --Z11-14:Ac' ' . .., Ul ..... 0

~ 0

~ -:::i: w

100

50

~- --0

_.....-0----- 0 / - --{) __..--------·

---"-E11-14:Ac

-+-ON10

l-o-ON11

~-()~"._1_0R

0 -

-5 -4 -3 -2 -1 0 2 3

log dose (pg)

Fig. 8: The dose-response curves to Zand E isomer of 11--14:Ac and to optically active

analogs ON 10 and ON11 and their racemic mixture ON 1 OR. Responses are

expressed in percentage of EAG reaction to standard. No correction for volatility of

compounds was made. Note that though the main pheromone component is not

opticaffy active, the receptor is able to dircriminate between two optically active

analogs and their racemic mixture.

--------------------~


--+O-

•r.~INI\\ .. ~U1~1~1l~~~1)1~/r~M¥M,~/.LlfWl'il;..:.111\!r#.ttAi\1itm,•wi~NJ..t1~w,'4'i' v. ,l\~1!11 ON 13

I

I I

I I

I

I

1 mV

0.5 sec

Fig. 9 Single sensillum recordings 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 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 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 perceived by receptors within the sensillum.

--t· 1

Behavioral response

- - - - -

., .c -"' tl1 °' 100 ~ ii:: c:

" 15 H ., c:

c "' <::: ., .c «:: u 0 ::i

.8

Fig. 10:

-1·50% - . -40%

-- -30%

-- -20%

-- ·10%

,-·0%

z

E

Blend I dose (ng)

I/) CJ

"@

CJ E °' Cl ci:I c: +J ·-t:: 'O Q) c: (..) 0 ,_ c.. (]) I/) 0... Q)

..... ._ 0

Behavioral response of laboratory males of 0. nubila/is to 1, 10 and 100 ng of Z

(97%Z:3%E), E (3%Z:97%E) and hybrid H (65%Z:35%E) blends. Responses are

expressed as percentage of males tested for each blend (n::::30, ::::100%). All

blends/doses activated males in wind tunnel experiments, however only Z blend

resulted in oriented flight, finding the source and copulation attempts. The most

attractive doses were 1and10 ng. 10 ng of Z blend were used in all fwther wind

tunnel experiments.

- - - - ----------- -

I

('I -t

I

100%-

Cll :s 90%

~Ill 80%

E .8 70%-.... 0 a; 60%

c: .=.. 50% .2 Cll :t: I/) 40% .a c: ·- 0 30% .c 0. c: I/)

Cll .._

Fig. 11 - 18 :

Ostrinia nubilalis - wind tunnel Inhibition of male reaction to pheromone:

Z11-14:Ac

c Q' 100

Compound (ng) 1000

0

g "' D Ql

-"" c c ~ c

Ql

c D

"' --

------------------------

- l O> c 6 c

"' <:: .c u :J 0 --

Behavioral rnsponse

Inhibition of Cl nubilalis male reactions to standard pheromone (1 O ng of Z blend, 97%Z:3%E) by 10, 100, 1000 ng of respective analogs, Z8-14:Ac and 14:Ac. Responses are expressed as percentage of male response to standard pheromone blend (= 100%). All compound significantly reduced the attractivity of the standard pheromone blend. The most active compounds were 14:Ac, sulfur analogs and chloroformate. Relatively less active were lactones and vinyl-branched Analogs ft should be emphasized however, that the most potent inhibitor was 100 ng of the main pheromone component, Z11-14:Ac and its saturated equivalent.

---------------------

I r'I "i'

I

100%·

<11 'B 90%

- !/) !'a 0

70% E ..... ..... . I

0 Qi 60%· c: .::.. 50% .2 Ill ::: !/) 40% .0 c:

30% ·- 0 ..c: a. c: !/) 20%

- QJ ....

Fig. 11 - 18 :

Ostrinia nubHalis ~wind tunnel Inhibition of male reaction to pheromone:

14:Ac

_....-, ,...;~ _--.....__= .. ~

1000 ""l OJ c c c ~

0

<IJ

"" ~

I I I 1~-- •

··.,. /-· ' al

--..,,--,,.--- !:::: /I :E ~

Ol cu -= "" - .c u <IJ c OJ ·c 0

l) ::i 0

Behavioral response

Inhibition of 0. nubilalis male reactions to standard pheromone (1 Ong of Z blend, 97%Z:3%E) by 10, 100, 1000 ng of respective analogs, Z8-14:Ac and 14:Ac. Responses are expressed as percentage of male response to standard pheromone blend (= 100%). All compound significantly reduced the attractivity of the standard pheromone blend. The most active compounds were 14:Ac, sulfur analogs and chloroformate. Relatively less active were lactones and vinyl-branched analogs. It should be emphasized however, that the most potent inhibitor was 100 ng of the main pheromone component, Z11-14:Ac and its saturated equivalent.

- - - - - - - - - - - - -· - - - - - - - -

I tj· --t·

I

-(!) B - (/) ro o E ...... ..... _ 0 ~ c: -0 (!)

:;::; (/)

;§ 5 ..c: c. c: (/)

- (!) ,_

Fig. 11 - 18 :

100%-

90%


ON 3

~ 80% l__. ... .....--

70%

-----

--. ___ JF-:-: D

-. c -,/ c 6 c

Compound (ng) Q' t1l

<.::: "" 0 -0 t3 1000 "' "' :J

D -"' c -~ c cu "' c c c "' 01!

Behavioral response

Inhibition of 0. nubilalis male reactions to standard pheromone (10 ng of Z blend,

97%Z:3%E) by 10, 100, 1000 ng of respective analogs, Z8-14:Ac and 14:Ac.

Responses are expressed as percentage of male response to standard pheromone

blend (= 100%). All compound significantly reduced the attractivity of the standard pheromone blend. The most active compounds were 14:Ac, sulfur analogs and chloroformate. Relatively less active were factones and vinyl-branched analogs ft

should be emphasized however, that the most potent inhibitor was 100 ng of the

main pheromone component, Z11-14:Ac and its saturated equivalent. I

-·----------··---------

I •ri -t

I

- 90% "O CIJ-

80% - !/) (U 0

70% E ...... ..... -= 0 CIJ ... c: -0 CIJ

:;:::; VJ :c c: 30% ·- 0 ..c: 0.

c: !/) CIJ ....

Fig. 11 - 18 :

Ostrinia nubilalis ~wind tunnel Inhibition of male reaction to pheromone:

ON 4

Compound 1000 c.

c c c ~

<lJ

""" ~

Behavioral response

Inhibition of 0. nubilalis male reactions to standard pheromone (10 ng of Z blend, 97%Z:3%E) by 10, 100, 1000 ng of respective analogs, ZB-14:/\c and 14:Ac Responses are expressed as percentage of male response to standard pheromone blend (= 100%). All compound significantly reduced the attractivity of the standard pheromone blend. The most active compounds were 14:Ac, sulfur Elnalogs and chloroformate. Relatively less active were lactones and vinyl-branched analogs. It should be emphasized however, that the most potent inhibitor was 100 ng of the main pheromone component, Z11-14:Ac and its saturated equivalent.

---------------------

I

'° ,. I

'O Q) .....

- fl) ctJ 0 E .,... ---= o e c: -0 Q)

:.;::: fl)

;§ § .c: a. c: fl) - Q) ._

Fig. 11 - 18 :


100% r --c·. 90% \---------\~~~---( -------

50%-

0%

ON 6

.. ., Ol s c c ,"!

·l :r: Ol .:;::

0 'O

"' "' "" c ."! <1J

t: 0

O> s 'O c cu

<::: Behavioral .c u ::::J 0 response -

Inhibition of 0. nubilalis male reactions to standard pheromone (1 Ong of Z blend, 97%Z:3%E) by 10, 100, 1000 ng of respective analogs, Z8-14:Ac and 14:Ac. Responses are expressed as percentage of male response to standard pheromone blend (= 100%). All compound significantly reduced the attractivity of the standard pheromone blend. The most active compounds were 14:Ac, sulfur analogs and chloroformate. Relatively less active were lactones and vinyl-branched analogs. It should be emphasized however, that the most potent inhibitor was 100 ng of the main pheromon~ component, Z11-14:Ac and its saturated equivalent.

-------------------~-

I l~

-t I

100% -Q) B (0 !/) 80%

E .8 70% ...... 0 (ii 60% c: ..=.. 50% .!2 Q) ~II) 40% .c c:

30% ·- 0 .c: a. c: !/) 20% Q) .... 10%

Fig. 11 - 18 :

Ostrinia nubifalis - wind tunnel Inhibition of male reaction to pheromone:

10

Compound

ON 8

·-- --~=~;=~ -.~ :_ -~~--~-~--->/. (ng) 100 ·~ -~

1000 ·-- - ·r

1

~ °' OJ

·E ~ c -cu

c ~ 0 2 ~ c 0

--. -

·--

"'' c 6 c cu

"' .c u :J .'?

Behavioral response

Inhibition of 0. nubilalis male reactions to standard pheromone (10 ng of Z blend, 97%Z:3%E) by 10, 100, 1000 ng of respective analogs, Z8-14:Ac and 14:Ac. Responses are expressed as percentage of male response to standard pheromone blend (= 100%). All compound significantly reduced the attractivity of the standard pheromone blend. The most active compounds were 14:Ac, sulfur analogs and chforoformate. Relatively less active were lactones and vinyl-branched analogs. It should be emphasized however, that the most potent inhibitor was 100 ng of the main pheromone component, Z11-14·Ac and its saturated equivalent.

---------------------

I 00 --t·

I

-1:l (l) ...... - rn Ill 0 E ...... .... ._: o e c: -0 Q)

:::; Ill :0 c: ·- 0 .c c. c: Ill

- Q) .....

Fig. 11 - 18 :

60%

50%

40%

30%

20%

10%

0%


ON 9

------ 1 _____ ___

100

Compound (ng) 1000 ·---.~~ c «= c !1l

r

.. •.f :c °' ~ c2 D

"' Q.l

.>< E 2 "' §

·---...

°' c -6 c ro

"" .c u ::J

E

Behavioral response

Inhibition of 0. nubilalis male reactions to standard pheromone (10 ng of Z blend, 97%Z:3%E) by 10, 100, 1000 ng of respective analogs, Z8-14:Ac and 14.Ac. Responses are expressed as percentage of male response to standard pheromone blend(= 100%). All compound significantly reduced the attractivity of the standard pheromone blend. The most active compounds were 14:Ac, sulfur analogs and chloroformate. Relatively less active were lactones and vinyl-branched analogs. It should be emphasized however, that the most potent inhibitor was 100 ng off he main pheromone component, Z11-14:Ac and its saturated equivalent.

-----·----------------·

I

°' -t I

-Q) :s

- IJ) «! 0 E ...... ...... 0 Q.j c: ~ .2 Q) ~ IJ) ..a c: ·- 0 .!: 0. c: IJ)

Q) ...

Fig. 11 - 18 :

100%-

70%

50% I

40% I

30%

20%


ON12

~---- I ~r ~ J ~ I I

~I ~I :::=! I r

. _ __,,~ =--:K.---~ ------Compound (ng)

100 -.·rc' .. _~0

1000 0

<lJ -" "'

:c .~l <::: 0 <lJ

OJ ~ c c J"

c <lJ c ()

I

I

r----

r·----

----OJ c -6 c "' "" .c l) ::i 0

Behavioral response

Inhibition of 0. nubilalis male reactions to standard phernmone ('1 Ong of Z blend, 97%Z:3%E) by 10, 100, 1000 ng of respective analogs, Z8-·14:Ac and ·14:Ac. Responses are expressed as percentage of male response to standard pheromone blend (= 100%). All compound significantly reduced the attractivity of the standard pheromone blend. The most active compounds were 14:Ac, sulfur analogs and chforoformate. Relatively less active were lactones and vinyl-branched analogs. It should be emphasized however, that the most potent inhibitor was 100 ng of the main pheromone component, Z11-14:Ac and its saturated equivalent.

---------------------

--I·-. ~ , . - -",. . - -

<~-~·:1~~~~~~:z~_~,:;_·5:/.~~~:-~- .. ;~:

--~~'1:~~:;' .··· " •» ,'.

·I

I·

:I

I I

I I

·1 I

'i \

I.· I

I I I

Journal of Chromatography, 626 (1992) 215-221 Elsevier Science Publishers B.V., Amsterdam

CHROM. 24 516

Gas chromatographic determination of vapour pressures of pheromone-like acetates

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 (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 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 Clausius-Clapeyron equation. Also, for structurally similar subseries of acetates, e.g., for w - 3 or w - 5 unsaturated derivatives, the vapour pressures were shown to have a simple dependence on the number of carbon atoms per molecule. The vapour pressures at 25'C ranged from 2.633 Pa for (Z)-5-decenyl acetate to 0.005 Pa for (E)-13-hexadecenyl acetate.

INTRODUCTION

In recent years, research on the sex pheromones of moths and butterflies has opened up new possibilities for developing ecologically safe strategies for insect pest control as alternatives to the use of conventional insecticides [1]. Most of these pheromones are volatile, multi-component mixtures of unsaturated even-carbon (C10-C18) acetates or alcohols with one or two double bonds at various positions in the molecule in either the Z or E geometric configuration, and a precise ratio of the components is required for the full insect response [2]. In order to mimic a pheromone-releasing insect in practical applications, it became necessarito develop controlled-release systems for use in monitoring, mass trapping and aerial dissemination control programmes. The successful applications of synthetic pheromone blends however, require volatility con-

Correspondence to· B. Koutek, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Flemingovo nam. 2, 16610 Prague 6, Czechoslovakia.

siderations to be put on a quantitative basis. In this context, a knowledge of evaporative characteristics of the individual blend components is of great importance.

Among the physico-chemical properties that determine the transport and fate of chemicals in the environment, vapour pressure is one of the most important. Clearly, a compound's vapour pressure will affect its partitioning between the vapour and liquid (particulate-bond) phases and, in turn, its effectivity. For many organic chemicals of environmental relevance, including pheromones, low pressures cause difficulties [3] in direct measurements by conventional Knudsen effusion [4] and gas saturation [5,6] methods. As a consequence, the literature interlaboratory data often disagree by factors of 2-3 or more. Gas chromatography (GC) is an alternative method for measuring vapour pressures [7,8], offering advantages in terms of speed, solute sample size, purity and stability requirements.

The original idea of relating GC retention times to solute vapour pressure [9] has been improved by introducing a latent heat ratio term for unknown

0021-9673/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved

•,_:__-•.

'• 1.·

I I I I-I I I I I I I I I I I I I

..

I

, __ _ -,- ·:,,,..·· . '

- . - - . - '

-·-'g"='--'-····-·~~·-· -----· ... · _____ ,_ :.. . ...: .. ~..:. ___ ...... -··-- -~----·~···- ..: .. ·~.:....:;.~~~;,;,,."""""'"""'""";;...;:=:.::.~;:..,.:;~

.; l

'

216

and reference compounds [10] and subsequently applied to estimate the vapour pressures of many polychlorinated biphenyls and dioxins [11, 12], herbicide esters [10] and organophosphorus pesticides [8]. In addition, two different GC approaches have been used [13-15] to treat the retention time (or volume) - vapour pressure relationships for pheromone-like compounds. One [13] uses the substance under study directly as the liquid stationary' phase in packed glass chromatographic columns, whereas the other [14, 15] makes use of a cholesteryl p-chlorocinnamate-coated capillary column, suggesting that on liquid crystal phases the elution order expressed as equivalent chain length (ECL) is determined by the length-to-breath ratio of the compounds. Disregarding the fact that, for restricted sets of compounds, both these approaches have produced good vapour pressure estimates, there are two problems connected with them. The first approach is very time consuming as it requires a separate column for each substance, and the efficient use of liquid crystal columns is limited to a narrow temperature range of ca. ± 10°C about the mesophase transition temperature.

The purpose of this work was to examine if the rapid and simple GC method reported previously [7,10,16] to be useful for environmentally hazardous chemicals would provide~, -. alternative to more sophisticated methods for det .mining vapour pressures of pheromone-like acf. ,tes with the same degree of accuracy.

EXPERIMENTAL

Chromatography and chemicals Samples were analysed on a Hewlett-Packard HP

5880 chromatograph equipped with a flame ionization detector and a 2-m fused-silica capillary column (cross-linked 5% methylsilicone, HP-1, film thickness 0.52 µm) with splitless injection. Chromatography was carried out isothermally at l0°C intervals from 90° to 140°C with a hydrogen flow-rate of 10 ml/min. n-C 18 and n-C20 hydrocarbons were used as reference standards. Retention times were determined using an HP 3396A integrator. As recommended [7], long retention times of compounds producing asymmetric peaks at low temperatures were not taken at the peak maximum, but were calculated at the mid-point between the beginning and the end of the peak.

B. Koutek et al./ J. Chromatogr. 626 ( 1992) 215-221

The acetates were either obtained from Sigma (St. Louis, MO, USA) and used as received or were synthesized in our laboratory. In the latter instance the purity of the compounds was at least 97% as determined by capillary GC. In the abbreviated nomenclature used for the acetates, the letters after the colon indicate the functional type (Ac = acetate); the number between the dash and colon indicates the number of carbon atoms in the chain and the letters and numbers before the dash indicate the configuration and position of the double bonds.

Data treatment The equations for calculating vapour pressures

from GC retention data were derived by Hamilton [I OJ. Briefly, pressures of the substances at the same temperature are related through

(1)

where the subscripts t and r refer to the test and reference compounds, respectively, and His the latent heat of vaporization. These vapour pressures are also related to their retention times (t):

In P1 = In P, - In (ttft,) (2)

Combining eqns. I and 2 and rearranging yields

ln (ttf t,) = (1 - Hi/H,) ln P, - c (3)

Therefore, a plot of ln (ttf t,) versus ln P, would have a slope 1 - Ht/H, and an intercept c. Eqn. 1 can then be used to determine the vapour pressure of a test compound at any temperature given the vapour pressure of the reference compound at that temperature.

RESULTS AND DISCUSSION

Table I gives the GC retention time data measured isothermally at six temperatures. Before using these data in the vapour pressure calculations, it seemed useful to analyse them with respect to the molecular structure of the analytes. As the Kovats system of retention indices is known to permit correlations of this type, the measured retention times in Table I were first converted into retention indices I defined by the equation

I= lOON + lOOn [(log ta - log tN)/(log tN+n - log tN)]

""\ ~

.I. ~ ', '

- ' ' -I.,.·

·rolSigma ve r were te stance ast 97% as ·eted noer ter the = acetate);

mlicates 1i nd the nd ate the e bonds.

I r pressures

v lmilton at e same

I (1)

1e st and His the Ia-

r _,ssures lf).

(2)

1gllds

(3)

l tould t qn. 1 Jr pressure ~ g,. n the .m t that

I data mea

~fo-using tla ns, it Jee to the he Kovats

1e.tt.cortl times on indices

.v+l log

I I I

-_ -~:~· --

--~<

:._--···. -

B. Koutek et al. / J. Chromatogr. 626 ( 1992) 215-221 217

TABLE I

GC RETENTION TIMES (min) OF THE PHEROMONE-LIKE ACETATES

Compound 90°C 100°C ll0°C 120°C 130°C

lO:Ac 1.074 0.688 0423 0.285 0.188 12:Ac 4.023 2.341 1.338 0.822 0.509 14:Ac 14.496 7.554 4.391 2.555 1.506 Z5-IO:Ac 0.986 0.604 0 395 0.266 0.175 £5-IO:Ac 0.916 0.582 0.365 0.237 0.161 Z7-l2:Ac 3.522 2.108 1.194 0 744 0.465 £7-12:Ac 3.526 2.055 1.178 0.728 0.454 Z9-l2:Ac 3.845 2 228 1.272 0.791 0.484 £9-12:Ac 3.826 2.235 1.26! 0.782 0.478 Z9-l4.Ac 13.186 6.771 4.09! 2.471 1.514 £9-14:Ac 13.051 7.045 4.049 2.463 1.484 Zll-14:Ac 13.032 6.784 4.215 2.423 1.433 £ll-l4:Ac 13.724 6.975 4.195 2.422 1.441 Z9-16.Ac 44.199 22.145 l l.127 6.233 3.395 £9-16:Ac 45.026 22.638 11.505 6.291 3.458 Zll-16:Ac 46.495 23.522 11.706 6.423 3.567 Ell-l6:Ac 49.529 24.865 12.193 6.689 3.622 Zl3-16:Ac 52.051 26.513 12.935 7.066 3.839 £l3-16:Ac 52.639 26.435 12.922 7.087 3.821 11-C 18H 38 15.521 8.395 4.441 2.571 1.483 n-C,0H42 58.501 26.909 14.277 7.424 3.994

where tA, tN and tN+n are the adjusted retentions of the analyte and of n-alkanes possessing N and N + n carbon atoms, respectively.

The correlations between the retention indices corresponding to ll0°C, 1110

, and the number of the carbon atoms Nin the acetate chain were established by means of the equation I= a + bN and are shown in Figs. 1 and 2. For n-alkenyl compounds, the position of the double bond relative to the nonpolar end of the molecule will affect the GC retention times. Thus, considering the different structural features of thew - 3 (9-12:Ac, l l-14:Ac and 13-16:Ac) and w-5 (5-lO:Ac, 7-f2:Ac, 9-14:Ac and 11-16:Ac) unsaturated acetates, both subseries were treated separately. The linear dependence of the retention indices of a homologous series with normal alkyl chain on carbon number N represents a common trend among the GC data. Therefore, also given in Figs. 1 and 2 are similar dependences of 3- and 5-alkenes based on literature data [17, 18]. The retention indices of Kuningas et al. [18] were

140°C

0.128 0.568 0.913 0.121 0.108 0.298 0.284 0.307 0.303 0.951 0.922 0.881 0.886 1.961 1.955 1.997 2.047 2.156 2.156 0.884 2.223

2000

1900

1800

1700

= 1600 ~ -~ 1500 0

~ 1400

~ 1300

1200

1100

1000

900

10 11 12 13 14 15 16

Fig. 1. Retention indices (/110) for (Z)-alkenyl acetates and (Z)

alkenes plotted against the respective carbon number. 6 = w- 3-Acetates; A = w - 5-acetates; D = w - 3-alken~s; • = w- 5-alkenes.

I

I I I-I I I I·

I I I I I I I I I I I

-~::. •.,,

-'-:.· '' ,,..,....;;.<d.>~~-""-"'·-~-"-- ~-~-----

·:

·-I j -.,

218

2000

1900

1800

1700

~ 1600

" ~ 1500

~ 1400

-e 1300

1200

1100

1000

900

10 11 12 13 14 15 16

Fig. 2. Retention indices (!1 10) for (£)-alkenyl acetates and (£)

alkenes plotted against the respective carbon number. Symbols as in Fig. I.

TABLE III

B. Koutek et al./ J. Chromatogr. 626 (1992) 215-221

TABLE II

COEFFICIENTS OF THE EQUATION I= a+ bN

In all instances r2 = 0.999 or better.

Compound Configuration a b

w - 5-Acetates z 409.23 97.61 E 366.23 100.71

5-Alkenes z 15.39 97.66 E 5.88 98.54

cv - 3-Acetates z 396.38 99.31 E 390.95 99.64

3-Alkenes z 4.69 99.21 E 0.96 99.36

used for this purpose, as both the stationary phase (OV-101) and temperature (l l0°C) that they used are comparable to our experimental conditions.

Tlie linearity of the plots provides evidence for the incremental nature of the sorbate-sorbent interaction energy irrespective of the homologous series considered. Inspection of the least-squares regres-

PARAMETERS OF EQN. 3 AND VAPOUR PRESSURES (25°C) OF THE PHEROMONE-LIKE ACETATES

Compound" HJH, c P(Pa) Err ore (%)

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 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 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 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 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 £13-16:Ac !.0992 -1.4623 0.0041 0.005

" Standard: n-octadecane. b Standard: n-eicosane. ' Ref. 13. d Refs. 14 and 15. e Error = IOO(p0 c-Pexo)fp,,

0•

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--; ~-. -

B. Kowek et al. f J Chromatogr. 626 ( !99:C) 215-221

sion coefficients of the equation I= a + bN (Table II) further reveals that (i) the values of the coefficient b vary only slightly (by ca. ± 1.5%) around a mean value of99.03 i.u., demonstrating an approximate constancy of the methylene group contribution to the sorbate-stationary phase interaction energy within all subseries of compounds compared; slightly higher slopes, however, are invariably found for the E isomers; (ii) specific interactions between the sorbent and terminal functional groups, in addition to double bonds, are reflected almost exclusively in the intercept a; and (iii) reasonable statistics of the I vs. N linear dependences (r > 0.999 in all instances) permit the I values of nonavailable acetates to be predicted with good accuracy by prudent extrapolation.

As the method used for vapour pressure determination is a comparative one, vapour pressures being calculated from that of a standard compound, it became important to assess the values given in the literature. The vapour pressures of n-octadecane over the temperature range 40-130°C have been shown [7,19] to fit the equation ln P (Torr) = A + B/T with A = 25.548 and B = - 10 165. On the other hand, the Antoine equation, log P (Torr) = A + B / (t + C), with A = 7.99897, B = - 2607.622 and C = 177 .32, has been proposed [20] for the vapour pressure-temperature (80-170°C) dependence of n-eicosane. Similar parameters have also been found by other workers [21]. Therefore, the above-mentioned constants A, Band C were used to calculate the vapour pressures of the reference standards in this work. Accordingly, the vapour pressure values for n-octadecane and n-eicosane extrapolated to 25°C are 0.02546 and 0.00172 Pa, respectively.

QC-determined vapour pressures PGc of all nineteen acetates at 25°C were calculated from relative retention times (Table I) by using eqns. 1 and 3. The results are given in Table III. It should be noted that the difficulties in separating the C18 hydrocarbon and C14 acetates resulted in a substantial scatter of the points of the In t, 0 1. vs. P, plot. Therefore, neicosane was used as reference compound for all C1.i. acetates.

To test the validity of this approach, it was useful to compare the PGc values from Table III (eqn. 1) with the limited amount of vapour pressure data on unsaturated acetates (PL) that have been published

219

previously [13-15]. When literature values were selected for this comparison, some judgement was necessary, as different reports for a single compound sometimes agreed very well but in other cases poorly. Of the three data sources [13,14,22] dealing with vapour pressures of acetates in Table III, we favoured the more recent results. The older data [22], based on a gas saturation approach, had been already questioned [13,23], and were not considered further. The literature PL values in Fig. 3 and Table III are therefore those based on refs. 13, 14 and 15. They correspond to two different experimental techniques, (i) the GC method [13] which uses the substance under study as the stationary phase, if necessary these data being extrapolated 5°C below the temperature range at which they were measured, and (ii) the GC method [14,15] which uses a liquid crystal stationary phase (as the vapour pressures obtained by the latter method were measured at 30°C they were recalculated to 25°C by making use of the Clausius-Clapeyron equation and the corresponding heats of vaporization given in ref. 23). The results of comparison of our PGc data with those taken from the literature (PL) are depicted in Fig. 3. As can be seen, the regression line obtained parallels they = x line. The equation of the regression line by a linear least-squares fit is

In P1. (Pa) = 1.0126 In PGc + 0.444 (11 = 8, r = 0.9991, S.E. = 0.094)

0

·1

0:: -2 :l Q'.

-= -3

.4

·S

-6

/

-6 -5

/ /

/ /

a/ /

-4 -3 -2

In p(GC)

/ ~

-1

/ /

(4)

0

Fig. 3. Logarithmic plot of literature vapour pressures PL versus P Ge data from the present work. The regression line (solid) and the y = x line (dashed) are shown.

I I

- -

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I I I I I I I I I

_: ,_,~,.

j ·j l

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220

Although both data sets for lO:Ac, 12:Ac. 14:Ac, £5-IO:Ac, Z5-10:Ac, Z7-12:Ac, Z9-14:Ac and Zl1-16:Ac are well correlated on the HP-1 column, the PGc underestimated PL by a factor of about 1.6. Two reasons have been identified [16] that might cause this inequality. One could occur if the difference in activity coefficients y among the test and reference compounds were related to compound volatility, while the other could be due differences in y which were not correlated with volatility. As no significant scatter of points about the regression line in Fig. 3 was observed, the deviations in the present instance are probably attributable to a systematic error connected with the GC column. This systematic error can be eliminated and the accuracy improved by using Fig. 3 as a calibration plot to correlate PGc with PL. Hence, the final vapour pressures PL of test compounds (Table III, eqn. 4) were obtained from measured P Ge data by correcting them according to eqn. 4. It is shown that this correction provides vapour pressures within a factor of about 1.17 of average literature values, thus achieving better precision of vapour pressure determinations than reported interlaboratory results. The mean relative error for the testing set of eight acetates was found to be less than ± 7%.

To extend further the scope of the method, the effect of temperature on vapour pressures was also investigated. The Clausius-Clapeyron equation

ln P = - Hv/RT + C (5)

where Hv is the enthalpy of vaporization and Rand Care the gas (8.3144 J mo1- 1 K - 1) and integration constants, respectively, was found to be adequate for describing the vapour pressure-temperature dependence over the range 25-45°C.

The method ofleast squares was used to fit eqn. 5 to the data using Hv and C as parameters. The resulting parameter estimates were further found to depend systematically on the carbon number N within the saturated and the monoenic w-3 and w-5 acetate subseries. As a consequence, an empirical relationship given by the equation

In P (Pa) = - (AN+ B)/T + (CN + D) (6)

resulted, where the constants A-D varied depending on the subseries -type. The numerical values of the constants are summarized in Table IV. Tests of this empirical equation are provided by comparison of

B. Koutek et al./ J. Chramatogr. 626 ( 1992) 215-221

TABLE IV

PARAMETERS OF EQN. 6

Acetate subsenes A B c D

Saturated 679.7 1154 3 1.218 !4.853 w-3-(Z) 499.4 3297.2 0.725 20.768 w-3-(E) 500.7 3360.5 0.726 20.966 w- 5-(Z) 536.3 2572.4 0.833 18.759 w- 5-(E) 533.8 2742.9 0.822 19.299

both the vapour pressures at 25°C and heats of vaporization as they are predicted by this equation with original PGc data based on eqn. 1 and vaporization enthalpies obtained previously (23] from independent measurements. The agreement found between PGc (eqn. 1) and P (eqn. 6) values with a relative error not exceeding 2% demonstrates the predicative validity of eqn. 6. Additional support for its reliability comes from a comparison between estimated and experimental Hv data. For compounds in common with ours, the Hv values (kJ mol - 1

) found by McDonough et al. [23] and ourselves are, respectively: IO:Ac, 67. 78 and 66. l; 12:Ac, 77.58 and 77.4; 14:Ac, 87.37 and 88.7; Z7-12:Ac, 75.91 and 74.91 and 74.9; Z9-14:Ac, 85.7 and 83.8; Zll-16:Ac, 95.46 and 92.7. Hence the experimental enthalpies are reproduced to within about± 3%'.

At a constant temperature of 25°C, eqn. 6 with parameters from Table IV can be substituted into the correction eqn. 4, yielding corrected vapour pressure relationships as shown in Table V. These equations enable vapour pressures at 25°C to be

TABLEV

PROPOSED RELATIONSHIPS FOR PREDICTING VAPOUR PRESSURES AT 25'C

Acetate subseries

Jn PL= a+ bN"

a b

Saturated 11.563 - l.075 w-3-(Z) 10.275 -0.962 w- 3-(E) 10.261 -0.965 w-5-(Z) 10.702 -0.978 w- 5-(£) 10 67 -0.981

a Vapour pressure in Pa.

' . -·

I ~ -

I I I I I I I I I I I I I I I I I I I

B. Koutek et al. I J. Chro/11({/ogr. 626 ( 1992) 215-2]1

predicted for selected homologous subseries of acetates from the carbon numbers N. Based on a restricted set of available experimental data, it is believed that these equations should predict vapour pressures with an average error of less than 10%.

A specific comment is needed regarding the intercepts and slopes in Table V. Whereas significant differences in these parameters are observed among the saturated. w-3 and w-5 monoenic subseries. the same differences in parameters between Z and E isomeric subseries (although they might be real) are all within the 95% confidence interval limits.

The conclusion reached from the results is that the present GC method can provide vapour pressures of pheromone-like acetates with average deviations of less than ±I 0% of the literature values, well within the interlaboratory precision of other techniques. Taking into account its other advantages, e.g., simplicity, speed, sample size and purity requirements, the approach presented here can be considered as a viable means for calculating vapour pressures of a large variety of compounds. In this respect, we believe the present results for acetates to be prototypical for a number of related systems. We are currently examining the potential of this method for determining vaporization properties of other classes of pheromone components, such as alcohols and dienes.

ACKNOWLEDGEMENT

This research was supported in part under grant No. DHR-5600-G-00-1051-00, Program in Science and Technology Cooperation, US. Agency for International Development.

221

REFERENCES

l E. D. Morgan and N. B. Mandava (Editors), C RC Handbook of Natural Pesticides, CRC Press, Boca Raton, Fl, 1988.

2 R. L. Ridgway, R. M. Silverstein and M. N. Inscoe (Editors), Behavior-Modifying Chemicals for Insect Afanagement-Applications of Pheromones and Other Attractants, Marcel Dekker. New York, 1990.

3 R. C. Reid, J. M. Prausnitz and B. E. Poling, The Properties of Gases and Liquids, McGraw-Hill, New York, 4th ed., 1987.

4 J J. Murray, R. F. Pottie and C. Pupp, Can. J. Chem., 52 (1974) 557.

5 W. F. Spencer and M. M. Cliath. Residue Rev., 85 (1983) 57. 6 W. J. Sonnefeld, W. H. Yoller and W. E. May, Anal. Chem.,

55 (!983) 275. 7 T. F. Bidleman. Anal. Chem., 56 (1984) 2490. 8 Y.-H. Kim, J. E. Woodrow and J. N. Seiber, J. Chromatogr.,

314 (1984) 37. 9 D. J. Jensen and E. D. Schall, J. Agric Food Chem., 14 (1966)

123. 10 D. J. Hamilton, J. Chromatogr., 195 (1980) 75. l J J. W. Westcott and T. F. Bidleman. J. Chromatogr., 210

(!981) 331. l 2 B. D. Eitzer and R. A. Hites, Environ. Sci. Technol., 22 (1988)

1362. 13 A. M. Olsson. J. A. Jonson, B. Thelin and T. Liljefors. J.

Chem. Ecol., 9 (1983) 375. 14 R. R Heath and J. H. Tumlinson. J. Chem. Ecol., 12 (1986)

2081. 15 R.R. Heath, P. E. A. Teal, J. H. Tumlinson and L. J. Men

gelkoch, J. Chem. Ecol .. 12 (1986) 2133. 16 D. A. Hinckley, T. F. Bidleman, W T. Foreman and J. R.

Tushall, J. Chem. Eng. Data, 35 (1990) 232. 17 S. Rang, K. Kunmgas, T. Strenze, A. Orav and 0. Eisen, J.

Chromatogr., 406 (1987) 75. 18 K. Kuningas, S. Rang and T. Kailas, J. Chromatogr., 520

(1990) 137. 19 A. B. MacKnick and J.M. Prausnitz, J. Chem. Eng. Data, 24

(1979) 175. 20 K. Sasse, J. Jose and J. C. Merlin, Fluid Phase Equilib., 42

(1988) 287. 21 V. Piacente, T. Pompili. P. Scardala and D. Ferro, J. Chem.

Thermodyn, 23 (1991) 379. :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.

Ecol., 15 (1989) 779.

- - - -t')

V).._ -

_______ , _________ _ SYNTHETIC COMMUNICATIONS, Z3(li), :!397-:404 (1993)

i

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PREPARATION OF E·ALKENES

USING LITHIUM AND l,3·DIA."1INOPROPANE

Irena Kovarova" and Ludvik Streinz

Institute of Organic Chemistry and Biochemistry

Academy of Science:; of Czech Republic

Fl.emingovo namesti 2, 166 10 Praha 6. Czech Republic

Abstract : A new selective reducing system, litld"!n i1t J,J-diami11opropane. is

dacribed. whiclt enablu du: preparation of E-014fi1is from acl!!ykna in high

purity.

The preparation of compounds with double bonds in high stereoselectivity is

of great importance. for example. in the synthesis of pheromones. We therefore

report a new and convenient method for the preparation of (E)-.alkenes.

The most commonly used method of (£)-double bond synthesis is the

reduction of acetylenes by allcali metals and liquid ammonia. Warthen et aL 1

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.'ie of

• to whom correspondence ~hould be addressed

2397

Copyright ii:) 1993 hy Marcel Dekker, Inc.

!

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- - - -2398 - - - - - -KOV AROV A AND STREINZ

their lower solub1licy 111 liquid ammonia. Jnd thus a cosolvent should be used:_

A:s an alternative ro this mechod. low aliphatic primary a.mines (mechyl-, ethyl-.

propyl-) are used in combination with lithium 3• ~he high volatility of low

alkylamines led to the utilisation of higher primary amines, which however

resulted in poor yields, perhaps due to low solvatation of lithium in these media.

Reggel et al. ~ then replaced ethylamine with the bifunctiona.l l.2-diaminoethane

and succeeded in reducing various unsaturated compounds (containing triple or

double bonds) and reported it as a very powerful metal-amine system with low

selectivity. Methylamine and 1,2-diaminoethane were also used in combination

with calcium. Unlike the lithium-methylamine system, the reduction always

stops in the alkene stage. The authors found that the reducing properties of

calcium in either methylamine or 1.2-diaminoethane alone were unequal to those

in a mixture of both amines 5.b.

We found that the reduction of 1.2-dialkylacetylenes by lithium in

1,3-diamincpropane (OAP) has many advantages over that in liquid ammonia or

1.2-diaminoethane. The reaction does not require a high excess of solvent

because the solubility of subsmues in OAP is high. Reduction in lithium-OAP

also requires milder conditions than the 1,2-diaminoethane method.. which

necessitates a high excess of amine and high reaction temperatures. giving rise to

side products. We found that four and even less equivalents of Li and OAP are

sufficient to obtain good results. The reduction proceeds fast enough both at low

and at room temperarure. depending on the triple bond qualities. TilF can also

be u.red as a cosolvent. The system is apparently capable of reducing triple

bonds to saturated carbon chains, but it is possible to stop the reaction in the

first reduction stage by controlling the reaction conditions.

As substrates for the reduction we used various compounds containing a triple

bond and/or an aromatic ring : TIIP-0-5-nonyn-l-ol (0, 4-methyl-3.S-dioxa~

10-tetradecyne cm. 3-hexyn-1--01 (III), 5-nonyn-l-yl acetate (IV), l-phenyl-

1-hexyne (V). phenol (Vn. benzene (YID and phenylacerylene (VIIJ). The

results of these reductions are ~ummansed in Table l. The reduction of

;;

I

I

- - - -PREPARATION OF E-ALKENES - - - - - -2399

compounds I. ll and Ill yielded 68-98% of the -:orresponding (E}-alkem:s (the

presence of (Z)-isomers was less than 1.5%, not included in Table I). The

product obtained after the reduction of· compound V was identified as

1-phenylhexane. This substrate, which has a triple bond in conjugation with an

aromatic ring, is easily reduc~d. and the reduction does not stop in the alkene

stage.

A characteristic change of colours (from yellow to purple or purple-brown)

was observed during the reductions of compounds I. II and V. After the reaction

mixture reached a dark purple or purple-brown colour the reduction of the

substrate was complete. Unfonunately this colour change is not a general

indicator for all of the substrates (Ill. IV, VI). The reaction of the acetate (IV)

gave different products.~ the substrate first underwent a reduction of the acetate

group with the slow. subsequent reduction of the released alcohol. Th~ extreme

difference in yield between the reductions of compound IV at -30 and 60"C has

not yet been successfully explained. Fmally. the aromatic substrates (VI. VD.

_VDI) were not reduced at all, even ar higher temperature. The terminal triple

bond of ·compound vm also was not reduced. The product of the reduction of

phenol (VI) obtained in low yield was identified by GCJMS analysis as

cyclohexanone.

The described method is an effective tool for the preparation of 1,2-di

substituted olefins under mild conditions. The resulting (E)-olefms contain

negligible amounts of (Z}-isomer.

EXPERIMENT AL SECTION

All reactions were carried out under nitrogen. Tetrahydrofuran (THF) was

distilled from sodium-benzophenone ketyl immediately prior to use.

1.3-Diaminopropane (OAP) was distilled from calcium hydride and stored over

molecular sieve11.

=H-NMR spectr.i were recorded on :i Varian Unity-:oo (200 ;...1Hzl

spectrometer in CDC!, with terrame1hyb1lane JS an imemal reference. IR

~

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24-02 KOV AROV A AND STREINZ

TABLE 2

1H-NMR Data or Reduction Products and Standards for GC Analysis

Compound 8 (ppm) J (Hz)

(E)-THP-0-5-nonen- l-ol 500MHz: 5.45-5.35 (m. 2H. CH=CH). 4.56 (t. 1=3.0. I H. OCHO). 3.87 (ddd. 1=3.0. 7.4. 10.4. IH. OCH). 3.74 (dt. 1=6.5. 6.5. 10.4. IH. OCH). 3.50 (m. lH. OCH). 3.39 (dt

. 1=6.5. 6.5. 10.4. IH. OCH). 2.10-1.20 (m. 12H, Cff:C), 0.88 (t 1=7.3. 3H. CH3).

(Z)-THP-0-5-nonen-l-ol 5.45-5.29 (m. 2H. CH=CH). 4.58 (d. 1=3.4, IH.OCHO}. 3.93-3.69 (m. 2H. Cli:O). 3.55-3.33 (m, 2H. Cli:O), 2.16-1.92 (m. 4H. Cff:C=C}. 1.88-1.25 (m. 12H.CH:C). 0.90 (t 1=1, 3H. CH,).

(E)-4-methyl-3.5-dioxa- 5.46-5.28 (m. 1=11.0. 2H. CH=CH}. 4.67 (k. 1=5.5. lH.

-10-cerradecen .: OCHO). 3.62-3.34 (m. 4H. Cli:O}. 2.14-l.94(m. 4H. CH:C=C). 1.70-1.29 (m. 13H. Cff:C). l.20(t 1=7. 3H. CH,Cli:O), 0.88 (t J=9, 3H. CH,).

(Z)-4-methyl-3.5-dioxa- S.51-5.30 (m. 1=15.0. 2H. CH=CH). 4.66 (k. 1=5.5. lH.

- I 0-teradecen OCHO). 3:70-3.32 (m. 4H. Cff:O). 2.08-l.86 (m. 4H. CH:C=C). 1.65-1.29 (m. 13H. CH.iC). 1.20 (t. J=7. 3H. CH,Cli:O), 0.88(t.1=9. 3H. CH,).

(E)-3-hexen-l-ol 5.69-5.54 (m. lH. CH=>. 5.45-5.29 (m, lH. =CH). 3.63 (t. 1=6. 2H. OCH:). 2.32-2.20 (m. 2H. CH:C=). 2.12-1.96 (m. 2H. =C~). 0.99(t.1=7. 3H. CH,).

(Z)-3-hexen-l-ol 5.65-5.51 (m. lH. CH=). 5.40-5.26 (m.IH. =CH}. 3.70-3.58 (m. 2H. OCHJ. 2.37-2.24 (m. 2H. Cff:C=). 2.17-2.01 (m. 2H: =CCff.:). 0.98 (t. J=8, 3H, CH,).

(Z)-5-nonen- l-yl acetate 5.46-5.26 (m. 2H. CH=CH}, 4.06 (t. 1=6.4. 2H. C!'~OCO). Z.!7-!.91 (!!!. 4H. C!-'~C=C). 2.05 (s. 3H. CH,CO~). 1.n-1.57 (m. 2H. Clf:Cff:O). 1.51-1.27 (m. 4H. CCff.:), 0.90 (t. 1=1.5. 3H. CH,).

(E)-5-nonen-1-ol 5.41-5.36 (m. 2H. CH=CH}. 3.63 (t. 1=6. 2H. OCff.:), 2.06-1.88 (m. 4H. =CCff.:). 1.65-1.19 (m. 6H. CCff.:). 0.87 (t. 1=1. 3H. CH,).

5-nonyn-1-ol 3.68 (t. 1=6. 2H. OCff.:). 2.26-2.08 (m. 4H. =CC!i:). 1.80-0.95 (m. 6H. CCff.:). 0.97 (t. J=8. 3H. CH,).

(Z)-l-pheny I- 1-hexen 7.45-7.15 (m. 5H. :irom.H). 6.36 (td. 11=12. 21=2. IH. PhCH=). 5.67 (td. 1J=l2. :J=5. lH. CCH=>. 2.45-2.26 (m. ZH. CH:C=l. 1.60-1.16 <m • .+H. Cli:C). 0.90 (t. J=7. l~H.CH,).

I ' ..

I l

..

PREPARATION OF £-ALKENES :;4-03

TABLE 3

IR and MS Data or Reduction Products and Standards for GC Analysis

Compound lR MS v (cm'1) m/z

(E}-THP-0-5-nonen- l-ol 3019 =C-H 226 (M··). 96. 85 ( 100% ). 1201. 1185. 1137. 1120. 69. 55. -1-1. 29. 1035 THPO-969 =C-H

(Z)-THP-0-5-nonen- l-ol 3007 =C-H 226 (M .. ). 129. 97.

' 1201, 1185. 1138. 1120. 85(100%). 69. 55. 41. 29. 1035 THPO- ..

574 C-C=C

(E)-4-methyl-3.5-dioxa- 3025 =C-H 183. 167.. 149. 138. 95.

-10-terradecen 1135. 1103. 1090. 83. 81. 69. 57, 55. 41. 29. 1060 cococ 969 =C-H

(Z)-4-methyl-3,5-dioxa- 3002 =C-H 242 (M .. ). 183, 105, 73;

-10-teradecen 1656 C=C 69. 55, 45. 43, 41, 29, 27. 1135. 1102. 1090. 1060 cococ

(E)-3-hexen-l-ol 3629. 3588. 3480. 3370 OH 100 (M"), 82. 67, 55. 1050 C-0 41 (100%), 39. 31. 27. 3028. 970 =C-H

(Z)-3-llexen- l-ol 3635. 3591. 3490. 3343 OH 1050 C-0 -1656 C=C 1403. 721 =C-H

(Z)-5-nonen- l-yl acetate 3007 =C-H 184 (M"). 129. 124. 96. 1742 C=O 81. 67. 54. 43 (100%), 29. 1239. 1041 C-0

(Zl-1-phenyl- l-hexen 3010 =C-H 160 (M°·). 129. 1643. 1622 C=C 117 (100%). 104. 91. 77. 1407 =C-H 63. 51. 39. 27.

1-phenylhexane 162 (Mi. 162. 133. 114.

- 105. 92. 91(100%). n. 65. 55. -l-3. 41. 29. 27.

- - - - - - - - - - _, - - - - - - - . - - -2400 KOV AR.ovA AND STREINZ

TABLE l

Reduction by Lithium in l,J-Diaminopropane

'i Temp. Reaction Product Yield• Recovered ci. time Startin2 ,... .: = 1! Material• Q

u :; ·c min % %

A -30 60 (E)-THP-0-5-nonen-1-ol 14.0 58.3

I A 25 105 51.3 18.l

A 60 34 40.2 15.l

B 25 21 68.4 b 3.6

A -30 60 (E)-4-methyl-3.5-dioxa- - 100

II A 25 720 -10-tettadecene 64.0 31.2

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 -1-hexanol 8.0

B 25 95 (E)-3-hexen-l-ol 37.2 -1-hexanol - 53.3

B 2S 45 · (E)-3-hexen-l-ol 38.0 -1-hexanol 62.0 --

B 60 80 (E)-3-hexen-l-ol ·- 45.6 -1-hexanol 28.9 4-hexyn-1-ol 7.3

A -30 60 S-nonyn-1-ol 33.4 1.8

IV (E)-5-nonen-l-ol 52.Z 5-nonen-1-yl acetate 3.4

A 25 260 5-nonyn-l-ol 32.4 -(E)- 5-nonen-1-ol - 31.7

A 60 80 S-nonyn-1-ol 67.6 -(E)-5-nonen- l-ol 10.9

B 25 170 5-nonyn-1-ol 34.4 -( El-5-nonen-1-ol 51.2

B 25 45 5-nonyn- l -ol 4.4 -(E)-5-nonen-1-ol 72.0

A -30 38 1-phenylhexane 55.1 29.3

v A 25 23 57.8 12.2

A 60 7 90.3 -B 25 IO 25.0 6.1

I l !

. '

i ~

. ~~

ti

PREPARATION OF E-ALKENES 2401

TABLE l Conl!nm:d

'2 Temp. Rcacti1m Product Yield 1 R1.-cuvered I c. c .s time Starting ' Q ~ Material ·1 u ::e ·c min % %

A -30 4P cyclohexanone 0.4 97.1 VI A 25 so· 2.0 97.0

A 60 60 l.3 98.0 B 25 60 21.9 75.3

VII A -30 45 - - 100°

vm A -30 45 - - 96 °

Method: A - reaction perfonned in a mixture ofTHF/DAP. B - reaction without THF

• GC data b isolated yield • HPLC data

spectra were recorded on. a Brucker IFS-88 infrared spectrophotometer. Mass .. .

. spectra .were recorded on a VG Anal~- ZAB~EQ nws spectrometer by EI

(70 eV) and on a F'JSOns MD 800 GCJMS System (DB 1 column. 30 m. 0.25mm

.. i:d;). ~as chromatographic analy~ Were carried out on- a· Hewlett-Packard

. 5890A gas chromatograph with HP-5 column (5% phenylmethylsilicon. 25 m.

0.3 lmm i.d.). HPLC chromatography wu performed on a Hewlett-Packard I 090

chromatograph equipped with a HP 8~B computer and DAD UV detector with

Tessek silica gel SGX 5µ. column. 3x (3mm i.d.. x 150mm). Starting materials

not commercially· available were prepared from acetylenic hydrocarbons and

alcohols using methods of pheromone synthesis, namely, alkylation of the

terminal triple bond. protection of the hydroxyl group and acetylation. The

standards with corresponding (Z)-<iouble bonds for identification of the

re~uction products were prepared from the starting materials by cacalycic

hydrogenation using deactivated palladium on calcium carbonate (10% Pd) ..

Gener.ii procedure for reduction by lithium in OAP:

To a stirred-solution of substrate (0.2 mmol) and I .J-diaminopropane

(0.8mmo() in THF (0.36 mil. lichium wire (ll.8 mmol) cut into ~mall pieces was

c ~

\

-

~ --

- - -2404 - - - - - - -KOV AROV A AND STREINZ

added and the m1x.ture cooled or heated to the desired remperacure. The

reacuon mixture was maintained at :such temperature until TLC analysis

indicated no further change and then quenched with 96% ethanol (0. l ml). The

remaining lithium was removed from the mixture, and distilled wacer (0.5 ml)

was added 7• After extraction with ether the organic layers were washed using

water and brine. The solution was dried (MgSOJ, the solvent was removed in a

vacuum and ttie,crude product was analysed by gas chromatography.

The experiments without THF were performed similarly and llliing the same

rates. though on a larger sea.le. The crude products of these reactions were

purified by column chromatography on silica gel and were analysed by

1H-NMR. IR and MS.

Acknowledgement: This work was in part supported by the Program in Science

and Technology Cooperation. U.S. Agency for International Development under

Geant DHR-5600-0--00-1051-00. We also wish to thank Dr. Irena Valierova for

carrying our all of the GC/MS analyses.

REFERENCES AND NOTES

1. Warthen J.D .• Jacobson M.: Syntl&esis 1973. 616.

2. Rossi R.. Carpita A.; Synthesis 1977. 561.

3. Benkeser R.A.. Schroll G .• Sauve D.M.: I.Am.Chem.Soc. 11, 3378

(1955).

4. Reggel L .• Friedel R.A .• Wender I.: J.Org.Clrem. 22. 891 (1957).

5. Benkeser R.A.. Kang J.; J. Org. Cl1em. 44. 3737 ( 1979).

6. Benkeser R.A.. Belmonte F.G .• Kang I.: J.Org.Clrem. 48. 2796 (1983).

7. The ~tion mix.rures from rhe reduction of phenol. after removal of

the metal lithium. were evaporated. dissolved in water and then acidified co

prevenr any acidic organic mareria1s from being lost in the alkaline aqueous

portion.

f'l!.., ... ;.,,.,i i.., ,..,,,. N .. ci.~rlands 14 April 1993)

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- - - .. epar- - - - - -HELVETICA CHIMICA ACTA - Vol. 77 (1994)

116. <X,<X-Disubstituted Ally! Sulfones: An Approach to the Synthesis of Vinyl-Branched Pheromone Analogues

by Mkh:tl lloskovcc, Bohumir Koutck •,Josef Lazar, Ludvik Strcinz, Eva Brofova, Bianka Kalinova, and .Jan Vrkoc

lnsl1lulc of Organic Chcnmtry and Biochemistry, Academy of Sciences of the Czech Republic, Flcmrngovo rn\m 2, CZ--16610 Praha 6

(14 11.94)

1281

The two-step alkylat1on of phenyl prop-2-cnyl sulfone (I) with protected w-bromoalkanols and l-10doalkanes ( ~ 3, sec Sd1e111c I) followed by a Pd-catalyLed dcsulfonylation with L1BH 4 affords a 96:4 mixture of vinylhranchcd, protected alcohols and con c'pond111g ethyhdenc-hranchcd isomers (see Sche111e 2; 4 and 5, respectively) By ulili11ng the large difference 111 react1v1ty of mono- and tnsuhstituted C=C bonds towards smglet oxygen, the clhyhdene dcnvalivcs arc easily 1cmovcd from the mixture by photo-oxygenation The vinyl-branched compounds are inert to tlm reaction and can he convcn1cntly 1sol«lcd in highly pure form (99 5"!.i) and ca 4S'V,, overall yield.

Racemic 7-propylnon-8-cn- l-yl acetate (8a; Scheme 2) was recently found to effectively disrupt the pheromone-mediated attraction of the false codling moth Cryptophlehia !e11cotret11 ( Lcpidoptera) to virgin females or to synthetic lures [ l]. This compound was isolated by prep. GC as an impurity in the commercial synthesis of the actual pheromone, which 1s a blend [2] of(£)- and (Z)-dodec-8-enyl acetates in a 1: 1 ratio. Note that both the total number ofC-atoms (12) and the position of the C=C bond at C(8) are the same in analog Sa as in the actual pheromone components. The mode of action of Sa and its inhibitory threshold have not been determined so far. Further, it might be advisable to know if the vinyl-branched analogues of other lepidopteran pheromones could similarly act as inhibitors nf the pheromone-mediated msect attraction.

Since it appeared that the access to vinyl-branched pheromone analogues might provide further insight into the structure-activity relationships within the insect chemoreception, we examined a new synthetic strategy that would potentially lead to a variety of different analogues. We sought for a general synthesis that would lead quantities on the gram scale required for extensive behavioral tests. Detailed herein is the synthesis of two vinyl-branched compounds Sa and Sb. While the former compound represents a branched analog of (£)/(Z)-dodec-8-enyl acetate (a pheromone component of Cryptophle/Jia !eucotreta [2] and Cydia mole.It a [3]), the latter compound is derived from (£)/(Z)tetradec-11-enyl acetate (a pheromone component r4J of Ostrinia 1111/Ji!a!is and many other pests).

Our strategy illustrated Ill Schemes 1 and 2 was generally based on the chemistry of ally! 'Sulfones. The key step was the generation of ally! sulfonyl carbanions (5] [6] from ally! phenyl sulfone (I) and their reactions with haloalkanes, leading to the formation of new C-C bonds, Sulfone I was conveniently prepared by the reaction of ally! bromide with sodium henzencsulfinatc according to the procedure described in [7].

-

- - - - - - - - - - ., - - - - - - - - - • 1282 HELVETICA CHIMICA ACTA - Vol 77 (1994)

Thus, the o:,o: -dialkylated ally! sulfones 3 were generally synthesized by treating

sulfone I with I.I equiv. of BuLi in THF/N,N,N',N'-tetramethylethylenediamine (TMEDA) at -60 to 0°, followed by reaction with an appropriate alkylating agent (Scheme I). Typically, I was first reacted with a protected bromoalcohol (6-bromcihexan-1-ol or 9-bromonomrn-1-o[) and the obtained monoalkylatcd derivatives 2a, b alkylated

with 1-iodopropane or 1-iodoethane to produce the d1alkylatcd sulfones 3a, bin ca. 57% yield (with respect to 1 ).

s,fw111<· I

~S02Ph 1 Bul1 /THF, TMEDA

2 Br(CH2)nOY

PhS02'((CH2)nOY

2a Y = EtOCH(Me), n = 6

b Y = EtOCH(Me), n = 9

1 Bul1 I THF, TMEDA

2 RI

S02Ph

R-t(CH2)nOY

3a R = Pr, Y = EtOCH(Me), n = 6 b R = Et, Y = EtOCH(Me), n = 9

For the reductive removal of the PhS02 group, a large variety of reagents are available [8]. Sodium amalgam either in alcohol or buffered with anhydrous Na 2HP04 is most frequently employed, among other electron-transfer reagents such as alumrnium amalgam 111 aqueous THF, Grignard reagents with Ni or Pd catalyst~. Li in aliphatic amines [9]. and Na or Mg in alcohols [IO]. The excellent ability of low-valent Pd complexes to facilitate the displacement of the PhS02 group at an ally lie po~ition by a hydride inn as nucleophilc is also well documented [11] [ 12]. This reaction may proceed l'la a rr-allyl

intermediate and, as a con seq ucnce, the hydride transfer may occur either at the o: - or the )'-position yielding a regioisomcr mixture. However. by this method, for the generation of terminal olefins, having the C=C bond either <;tabihzcd by conjugation with a Ph group or disubsututcd with Mc groups or else for fl -tosyl homoallyhc alcohols, dcsulfonylation

occurred without any migration or the C=C bond [12] [13]. In our hands, attempts to prepare isomerically pure terminal okfins from the dialkylatcd sulfones 3a, b by this way failed. Depending on the reaction conditions examined (reducing agents: Na8H4, Li8H4,

LiBHEt,, HC02NH 4 ; catalyst: [Pd(PPh,),j or [PdCl,(PPh,)J), terminal olefins were obtained in only ca. 60 96"/i, selectivity. The combination [PdCl,(PPh 1) 2]/LiBH4 at -60° proved to be the best one, affording an unscparablc 96:4 mixturc of vinyl- and ethyhdenehranched compounds 4 and 5 (Scheme 2). However, as the mode of phcromonal action is

L1BH4, THF

[PdCl2(Ph3PhJ

Ry(CH2)nOY

' (96%)

4a R = Pr, Y ~ EtOCH(Me), n = 6

b R =Et, Y = EtOCH(Me), n = 9

+

Scheme:!

R'((CH2)pY 1 Dowex(HO!) R"((CH 2)nOY

I (4%) 2 102, hv, TBAB

""" +

Sa R = Pr, Y ~ EtOCH(Me), n ~ 6

b R =El, Y = EIOCH(Me). n = 9 6a R "'""Pr, Y ""H, n = 6}

b R = El, Y = H, n = 9 J Ac20 Py

Sa A= Pr, Y =Ac, n = 6} b R = Et, Y = Ac, n = 9

OH

Rt(CH2)pY

7a R = Pr, Y = H, n = 6

b R = Et. Y = H, n = 9

generally based on highly rcgioselective or stcrcosclccllvc proccs~cs [ 14], even the best

isomeric purity of 96 'Yo was not sufficient for our purposes. Since a large difference in reactivity towards singlet oxygen should exist [15] between

compounds 4 (monosubstituted C=C bond) and S (trisubstituted C=C bond), we employed the singlet photo-oxygenation to achieve their separation. Thus, the alcohol

- ~-

HELVETICA CHIMICA ACTA - Vol. 77 (1994)

mixture, obtained by deprotection with Dowex (H+ form) from 4a/Sa 96:4, was photooxygenated by the method of simultaneous oxidation and reduction [16], i.e. in the presence of (Bu4N)BH4 (TBAH), using Bu4N-solubilized Rose Bengal as sensitizer and

CHC13 as solvent. In a clean reaction and under total conversion (GC) of the ethylidenebranched alcohol derived from Sa, diol 7a was obtained and identified by 1H- and

"C-NMR. The formation of this tertiary alcohol was anticipated, as it is known [17] that 10 2 prefers to react with t.risubstituted olefins on the more crowded side (syn-addition). In contrast, the vinyl-branched alcohol 6a derived from 4a remained intact and could be

conveniently isolated in highly pure form ( > 99.5 by cap. GC). The loss of material associated with this procedure only amounted to some 5-10%. No attempt was made to isolate 7b from the mixture 6b/7b obtained similarly from 4b/Sb. The overall yields of the desired vinyl-branched alcohols 6a and 6b were 42 and 45 %, respectively. Subsequent acetylation of 6a and 6b yielded the target compounds Sa and Sb, respectively.

Another, synthetically also interesting transformation of the disubstituted allyl sulfones 3 consists in the desulfonylation to produce pure ethylidene isomers. It is known [18] that o:-monoalkylated /J,y-unsaturated phenyl sulfones are easily isomerized to

o:/J-unsaturated derivatives with a catalytic amount of t-BuOK. This suggests a possible control of regioselective product formation via tandem desulfonylation/isomerization of the sulfones 3. Indeed, exposure of 3a, b to NaHg, and t-BuOK in dry MeOH at 0°

resulted in a smooth one-pot conversion to the isomers Sa, bin generally high yield (ca.

85 % ) and a purity exceeding 99 % (Scheme 3 ). Subsequent deprotection with Dowex (H+

form) and acetylation led 1·ia 9a, b to 10a, b, the double-bond isomers of Sa, b.

Scheme3

NaHgxi 1-BuOK Do-...ex (HEil) 3a,b Sa,b R"((CH 2)nOY

9a R = Pr, Y = H, n = 6 }

b R = Et, Y = H, n = 9 J Ac,O/Py

10a R = Pr, Y =Ac, n = 6 } b R = Et, Y = Ac, n = 9

In conclusion, the o:,o: -disubstituted ally! sulfones are intermediates in the synthesis of

racemic vinyl-branched alkanols. The two-step transformation includes a Pd-catalyzed reductive desulfonylation leading to a mixture of terminal and non-terminal olefins, and the removal of the latter from the mixture by photo-oxygenation; the method makes the difficult separation of vinyl and ethylidene isomers unnecessary. One of the attractive features of the o:,o: -disubstituted ally! sulfones is that they may alternatively provide pure ethylidene isomers when using sodium amalgam/t-BuOK in the desulfonylation step.

Although this approach is primarily connected with pheromone research, its scope is

by no means restricted only to this area.

This work was financially supported, 1n part, by grant No. DHR-5600-G-00-1051-00, Program 1n Science and

Technology Cooperation, from the US. Agency for /11tematio11al D<'1•e/opme111

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~84- - --ETIC~AA~l.77- - - -Experimental Part

General. GLC: Hewlett-Packard-HP-5880A chromatograph, FID detector; 25-m capillary column (internal

diameter 0.3 mm, HP5-5% phenyl methylsilicone, cross-linked). Prep. medium-pressure liquid chromatography

(MPLC): Merck 60 silica gel (0.040-0.063 mm); Biichi-B-680-Prep-LC system with stepwise gradient of EtP in

light petroleum ether. 1H-and 13C-NMR Spectra: CDCl3solns.; Varian Unity-500 spectrometer, operating at499.5

MHz; chemical shifts fJ in ppm rel. to TMS; data in Tables 1-3.

9-( 1-Ethoxyethoxy )-3-(phenylsulfonyl)non-l-ene (2a). To a stirred soln. of dry THF (240 ml), TMEDA (10.5

g, 90 mmol), and phenyl prop-2-enyl sulfone (I; 16.4 g, 90 mmol) under Ar, BuLi (2.5M in hexanes; 38 ml, 96 mmol)

was added over 15 mm at -60°. After l h, l-bromo-6-(1-ethoxyethoxy)hexane (23.0 g, 90 mmol) was added and

stirring continued at -60° for 3 h. The temp. was raised to 0° and the mixture poured into ice-water and extracted

with Et20 (3 x 150 ml). After washing w1th brine, drying (K2C03), and evaporating, 2a was isolated by prep.

MPLC: 23.3 g (73 %). Anal. calc. for C 19H300 4S (354.5): C 64.4, H 8.5, S 9.0; found: C 64.2, H 8.3, S 8.9.

12-( 1-Ethoxyethoxy )-3-(phenylsu/fonyl)dodec-1-ene (2b) was synthesized analogously from 1 (25.2 g, 138

mmol), BuLi (2.5M; 58 ml, 145 mmol), and l-bromo-9-(\-ethoxyethoxy)nonane (40.8 g, 138 mmol): 41.3 g (74%).

Anal. calc. for C 22H 360 4S (396.6): C 66 6, H 9.2, S 8.1; found: C 66.8, H 9.3, S 8.0.

9-( 1-Ethoxyethoxy)-3-(pheny/.1·u(fonyl)-3-propy/non-3-ene (3a). BuLi (2.5M in hexanes; 8 8 ml, 22 mmol) was

added dropwise (15 min), under Ar, to a stirred and cooled (-60°) soln. of2a (7.14 g, 20 mmol) and TMEDA (2.3 g,

20 mmol) in dry THF (70 ml). After 1 h, Prl (3.7 g, 22 mmol) was added and stirring continued at -60° for 3 h.

Then, the reaction mixture was quenched with ice-cold H20 (500 ml) and extracted with Et20 (3 x 100 ml). The

combined Et20 extract was washed with HP and brine, dried (K2C03), and evaporated and the residue purified by

prep. MPLC: 6.3 g (79%) of3a. Anal. calc for C22H360 4S (396.6): C 66.6, H 9.2, S 8.1; found: C 66.3, H 9.1, S 7.9.

12-( J-Ethoxyethoxy)-3-ethyl-3-(pheny/sulfony/}dodec-J-ene (3b). In the same manner as described above, 2b

(7.93 g, 20 mmol) was alkylated with Et! (3.4 g, 22 mmol)· 6.5 g (76%) of3b. Anal. calc. for C24H4004S (424.6):

C 67.9, H 9.5, S 7.6, found: C 68.l, H 9.4, S 7.4.

7-Propy/non-8-en-1-o/ (6a) and 7-Propy/non-8-ene-1,7-diol (7a). A soln of [PdC12(PPh3h] (175 mg, 0.25 mmol)

in dry THF (10 ml) was added dropwise, under Ar, to a stirred and cooled (-60°) soln. of3a (2.00 g, 5.04 mmo])

and LiBH4 (540 mg, 25.0 mmol) in dry THF (25 ml). The mixture was stirred for 8 hat r.t. Then, the reaction

mixture was quenched with ice-cold HzO ( l 00 ml) and extracted with Et20 (3 x 50 ml). The combined Et20 extract

was washed with H,O and brine, dried (K,C01), and evaporated and the residue purified by prep. MPLC' 1.15 g (90%) of 4a/5a 96:4 (GLC and 1H-NMR)-. Th.is mixture was dissolved in MeOH (50 ml) and stirred with DoH·ex

50 W (H" form; l g) for 24 h. The ton exchanger was filtered off, the solvent evaporated, and the residue mixed with

a ~tock soln. of Bu4N-solubilized Ro.1e Bengal in CHCI3 (250 ml) and irradiated with a constant flow of02 bubbling

through the soln. using a Hanau 150-W high-prcssu1e Hg lamp in a commercial quartz-glass photoreactor

(Normag ), At 0, 15, and 45 mm of irradiation, 1.42 g (5.8 mmol) of (Bu4 N)BH4 was added in 3 portions. After 60

min of irradiation, the CHC13 wa' evaporated Et20 (50 ml) and Kl (l g) dissolved 111 a minnnum amount of H20

were added with stirring to precipitate Bu4Nl. After 30 min, the solids were filtered and washed with Et20. The

Et20 layer was dried (MgS04) and evaporated and the residue separntcd by prep. MPLC: 758 mg (82 °!f,) of 6a and

32 mg of7a. 6a: Anal. calc. for C 12H240 ( 184.3): C 78.2, H 13.1; found: C 78.0, H 13.3. 7a. Anal. calc for C1 2H240 2

(200.3): C 72.0, H 12.1; found: C 72.1, H 12.2.

10-Ethyldodec-I l-en-1-o/ (6h) wa' synthesized analogously from 3b (2 0 g, 4.7 mmol), LiBH4 (510 mg, 23.5

mmol), and [PdC1 2(PPh 3h] (175 mg, 0.25 mmol): 907 mg (80'\'<>). Anal. calc. for C 14 H2RO (212.4): C 79.2, H !3.3;

found: C 79.0, H 13.2.

7-Pmpybwn-7-en-l-o/(9a) Sodium amalgam (2.5'Yo, 22 g) and 1-BuOK (150 mg) were added to a cooled (0°)

soln of3a (2.2 g, 5.57 mmol) and anh. NaH 2P04 (2.64 g, 22 mmol) 111 dry MeOl-1 (70 ml). After stirring for 2 h, the

mixture was decomposed by H20 (250 ml) and extracted with Et20 (3 x 80 ml). The combined extract was washed

with H20 and brine, dried (K 2CO_i). and evaporated: 1.5 g of Sa. The yellow oil was dissolved in MeOH (50 ml)

and treated with Down 50 W (H + form; 1.5 g) for 24 h. The ion exchanger was filtered off and McOH evaporated.

Pun ilea lion oflhe residue by prep. MPLC gave 0.87 g (85%) of9ll Anal. calc forC 12 H240 (184 3): C 78.2, H 13.1,

found: C 78.3. H 13.0.

IO-Ethyldodcc-IO-c11-/-o/ (9b) wa' synthesized analogously from 3b (1.5 g, 3.5 mmol), &odium amalgam

(2 5%; 15 g), and 1-BuOK (100 mg): 650 mg (87.5%). Anal. calc for C 14 H280 (212.21): C 79.2, H 13 3; found:

C 79.4, H 13.5.

7-P10pr/11011-8-e11-/-y/ Acl'tatc (Sa), lrl-Dhy/dodec-l l-c11-l-yl Acetate (Sb), 7-Pmp,J'/n1111-7-en-l-yl Acetate (IOa). and 10-Ethl'idoda-/()-en-l-rl Al'<'tatc (I Ob). A typical procedure was as follows: a!cohol 6a (500 mg, 2.71

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- - - - - •• - - -HELVETICA CHJMICA ACTA - Vol. 77 ( 1994) 1287

Table 3. 13C-NMR Chemical Sh(fls [ppm] o,{Compounds Sa, IOa, Sb, and I Ob. For numbering, see Tables 1 and 2.

Sa JO a Sb IOb

C(l2) 64.66 (1) 64.69 (1)

C(l l) 28.60 (1) 29.49.(1)

C(IO) 25.89 (I) 28.13(1)

C(9) 64 65 (1) 64 64 (I) 29.73 (1) 29.73 (I)

C(8) 28.59 (1) 29.50 (1) 29.52(1) 29.51 (I)

C(7) 25.88 (1) 29.31 (1) 29.50 (1) 29.62 (I)

C(6) 29.35 (1) 25.83 (I); 25.88 (1) 29.24 (t) 29.26 (t)

C(5) 27.02 (I) 28.03 (I); 28.08 (t) 27116(1) 28.61 (I)

C(4) 34.90 (I) 36.83 (I) 34.63 (t) 36.67 (I)

C(3) 43.80 (d) 140.16 (s) 45.79 (d) 142.13 (s)

C(2) 143.54 (d) 118.36 (d); 118.44 (d) 143.37 (d) 116.92 (d); 117.48 (d)

C(l) 113.86 (1) 13.16 (q); 13.17 (q) 113.99 (I) 12.85 (q); 12.88 (q)

Pr-C(3) 37.29 (I); 20.23 (I); 39 15 (I); 21.28 (1); 14 15 (q) 21 28 (/), 13 91 (q);

14 12 (q)

Et-C(3) 27.67 (I); 11.62 (q) 29.82 (t); 13.00 (q), 13.16 (q)

AcO 171.24 (s); 21 02 (q) 171.24 (.1); 20.99 (q) 171.25 (s); 21.02 (q) 171.28 (.1 ); 21.03 (q)

mmoi) w.ts added to AczO ( 1.33 g, 13 mmol), pyridme (2.04 g, 23 mmol), and 4-(dimethylamino)pyridine (25 mg) at -10° and left 10 the refrigerator overnight. The mixture was then poured into ice-cold H 20, extracted with Et20, and chromatographed (prep. MPLC) almost quant. yield of pure (99.5% by GLC) Sa. Anal. calc. for C 14H260 2

(la; 226 4): C 74.3, H 11 6, found: C 74.4, H 11.6. Sb· Anal. calc. for C 16 H300 2 (254.4): C 75.5, H 11.9; found: C 75.7, H 12 0 IOa: Anal. calc. for C 14H260 2 (226 4): C 74.3, H 11.6; found: C 74.1, H 11.4. I Ob: Anal. calc. for C 16 H3ti02 (254.4). C 75.5, H 11.9: found· C 75.3, H 11.8 .

REFERENCES

[I] B. V. Burger, M. lc Roux, W. M. Mackenroth, H. S.C. Spies, J. H. Hofmeyr, Tetrahedron Le//. 1990, 31, 5771 [2] C.J. Persoons, F.J. Ritter, W.J. Nooyen,J. Chem. Ecol. 1977,3, 717. [3] W. L. Roelofs, A. Comeau, R. Selle, Nature (London) 1969, 224, 723. [4] Y. Tamak1, ll1 'CRC Handbook of Natural Pesticides', Vol. IV, 'Pheromones', Eds. E. D. Morgan and

N. B. Mandava, CRC Press, Boca Raton, 1988, pp. 35-94. [5] P. D. Magnus, Tetrahedron 1977, 33, 2019. [6] B. M. Trost, Bull. Chem. Soc. Jpn 198S, 61, 107. [7] M. Julia, J.P. Stacino, Tetrahedron 19S6, 42, 2469. [8] R. C. Larock, in 'Comprehensive Orga01c Transformations', VCH Publishers, New York, 1989, p. 33, and ref.

cit. theretn . [9] N. K. N. Yee, R. M. Coates, J. Org. Chem. 1992, 57, 4598.

[IO] G. H. Lee, E. B. Choi, E. Lee, Ch.S. Pak, Tetrahedron Leu. 1993, 34, 4541. [I I] M. Mohri, H. Kmoshita, K. Inomata, H. Kotake, Chem. Lei/. 19S5, 451. [12] K. lnomata, S. lgarashi, M. Mohri, T. Yamamoto, H. Kinoshita, H. Kotake, Chem. Leu. 19S7, 707 . [13] H. Kotake, T. Yamamoto, H. Kinoshita, Chem. Lett. 19S2, 1331. [14] T.C. Baker, Experientia 19S9, 45, 248. [15] K. Goll01ck, H.J. Kuhn, in 'Singlet Oxygen', Eds. H. H. Wasserman and R. W. Murray, Academic Press, New

York, 1979, p. 298 [16] P. Baeckstriim, S. Okecha, N DeSilva, D. W1jekoon, T. Norin, Acta Chem. Scand., Ser. B 19S2, 36, 31. [17] J.R. Hurst, S.L. Wilson, G.B. Schuster, Tetrahedron 19S5,41, 2191. [18] D. Savoia, C. Trombini, A. Umani-Ronchi, H. Chem. Soc., Perkin Trans. I 1977, 123.

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I I I I I I I I i I I 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, CZ-128 40 Prague 2, Czech Republic

Received 21 April 1994

Abstract

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 C16 alkanols) whose polarities

approximated that f the test compounds and melting point corrections for compounds that are solids at ambient

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

a parameter.

1. Introduction

The saturation properties of pure liquids play a major role in both the understanding of fluid phase behaviour and the design and operation of a multitude of industrial processes [1,2]. Such properties are essential not only when used directly in calculations, but also when

• Corresponding author. "For Part I, see Ref. [20].

used as input to variety of models and applications.

At present, there is an increasing need for vapour pressures of high-molecular-mass organic compounds at ambient temperatures [3,4]. One of the most important reasons for this is the increased public sensitivity to the effect of chemicals on health and the environment generally. As the vapour pressure of an organic chemical exerts a large influence on its dispersal in the environment, a knowledge of the vapour pressures should allow one not only to model the fate of organic pollutants [5,6] but

0021-9673/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0021-9673(94)00521-A .


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308 B. Koutek et al. I J. Chromatogr. A 679 (1994) 307-317

also to optimize the use of ecologically friendly behaviour-modifying chemicals (7]. An impetus for developing more effective design for practical applications of these compounds is the continuing value of pheromones for monitoring insect flight activity and the recent commercial success in controlling several pests by permeating the air with their sex pheromones [7]. Both the release rates and, in the case of blends, the release ratios of pheromone components from dispensers are governed, for the most part, by the vapour pressures of the compounds. It appears that environmental concerns are weighing against the use of traditional pesticides and expectations are [8] that pheromones will capture about 15-40% of the insecticide market within 10 years. Thus, an understanding of the pheromone evaporative process can aid in the optimization of selectivity conditions and the minimization of the loss of the biological activity of synthetic pheromone blends.

The vapour pressures of compounds of low volatility are commonly determined by either gas saturation (9,10] or effusion (11 J methods. Gas chromatography ( GC) is an alternative method for measuring vapour pressures (12,13}, offering advantages in terms of speed, solute sample size, purity and stability requirements. It is based on the use of a non-polar stationary phase and isothermal conditions such that a compound's GC retention time is related directly to its vapour pressure. The GC method has been used to study polychlorinated biphenyls and dioxins [14,15}, herbicide esters [12], organophosphorus pesticides (16], tetraorganostannanes [17], linear alkylbenzenes [18] and fatty acid methyl esters (19]. Using this approach, we have obtained [20] some vapour pressure data on pheromone-like acetates.

In this paper we show that the GC method yields equally good results in determining equilibrium vapour pressures of more polar compounds, viz. monounsaturated (C 10-C18 )

pheromone-like alcohols. The extensive set of 98 compounds studied also allowed the influence of subtle structural differences in chain length and the positions of double bonds on vapour pressures to be revealed.

2. Experimental

2.1. Chromatography

Samples were analysed on a Hewlett-Packard HP 5890 chromatograph equipped with a 3 m x 0.31 mm I.D. fused-silica capillary column (cross-linked 5% methylsilicone, HP-1, film thickness 0.52 µ,m) with split injection and a flame ionization detector. The length of the column employed (3 m) is a compromise between the need for acceptable resolution when working with mixtures and the need to avoid prohibitively long retention times, particularly at lower temperatures. The chromatograph was operated isothermally with a hydrogen flow-rate of 5 ml/min at l0°C intervals in the range 50-1600C as specified. Cw C 12 , C 14 and C 16 alkanols were used as reference standards. Retention times were determined on a HewlettPackard HP 5895A ChemStation. Adjusted retention times were calculated by subtracting the retention time of methane from the retention time of the chemical. As recommended (13], long retention times of compounds producing unsymmetrical peaks at low temrt· ,.itures were not taken at the peak maximri ., but were estimated at the centre of gravit:· of the peak. The reproducibility of retention cime measurements expressed as the relative standard deviation of at least three measurements for each compound was 0.03%.

2.2. Chemicals

The alcohols were either obtained from the Research Institute for Plant Protection (IPODLO) (Wageningen, Netherlands) and used as received or synthesized in our laboratory. In the latter instance, the purity of the chemicals was at least 97% as determined by capillary GC. Condensed nomenclature for alcohols is used: the letters after the colon indicate the functional type (OH= alcohol), the number between the dash and colon indicate the number of carbon atoms in the chain and the letters and numbers before the dash indicate the configuration and

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B. Koutek et al. I J. Chromatogr. A 679 (1994) 307-317 309

position of the double bonds, e.g., Z3-10:0H is (Z)-3-decenol.

2.3. Data treatment

The method has been discussed in detail by Bidleman [13) and Hinckley et al. [21) and will therefore be only briefly reviewed here. At a constant temperature, the vapour pressures of a test and of a reference compound (subscripts T and R, respectively) are related by the ratio of their latent heats of vaporization:

6.HT ln PT = !1H · In PR + C

R (1)

where 6.H is the latent heat of vaporization and C is a constant. A similar equation has been developed for the GC (adjusted) retention times t':

( t~) ( 6.HT) ln t~ = 1 - t:i..HR In PR - C (2)

Hence, a plot of ln(t~/t~) versus ln PR should have a slope 1- 6.HT/!1HR and an intercept -C. Eq. 1 can then be used to determine the vapour pressure of the test compound at any temperature if the vapour pressure of the reference compound at that temperature is known.

Since the GC method gives the subcooled liquid vapour pressure (defined as the liquid vapour pressure extrapolated below the melting point) [22], it was necessary to convert the literature-based solid vapour pressures (P5 ) into subcooled-liquid vapour pressures (Pd by using the equation developed by Mackay et al. [23):

(3)

where TM and T are the absolute melting and ambient temperatures, respectively, R is the gas constant and !1SF is the entropy of fusion. The usually employed "average" value of !1SF = 56.5 J /K ·mo! (or the corresponding value 6.SF/ R = 6.79) seems to be too low for alcohols, however. Based on the value of enthalpy of fusion (6.HF) published [24) for 1-hexadecanol (34.286 kJ I mol), !1SF for this compound amounts 106.34

. ..,-, --· _- -- _-.-:

J /K · mol and, as a consequence, 6.SF/ R = 12.789. Hence this constant was used to convert literature P8 values of 1-hexadecanol and 1-pentadecanol into PL.

2.4. Statistical analysis

The data were subjected to statistical analyses utilizing the Statgraphics Plus 7.0 software package (Manugistic, Rockville, MD, USA).

3. Results and discussion

The accuracy of the GC method as represented by Eqs. 1 and 2 depends to a large extent on two factors: (i) the accuracy of the PR values and (ii) the similarity of infinity dilution activity coefficients y in the stationary phase between the test and reference compounds to which Eq. 2 is applied. Strictly, an additional term, -ln(yT/ l'R), should appear (see discussion in Ref. [21]) on the right-hand side of this equation and only when l'T - l'R (or at least l'T /yR - constant) can the use of Eq. 1 lead to reasonable results. As values of y on a squalane liquid p'· ase have been found [25) to range from 0.48 tr J.73 for hydrocarbons and from 17 to 34 :. r alcohols, the frequently employed reference hydrocarbons seemed to be disqualified for our purposes. Therefore, we chose to use n-alkanols (i.e., compounds of the same chemical class as the test compounds) as the reference standards. When literature vapour pressure values were being selected for the reference n-alkanols, some judgment was. necessary. We favoured recent static measurements [26] that have been especially focused on the low vapour pressure field. The literature values of the four reference compounds given in Table 1 in the form of the Antoine equation are thus from a single report. In most instances vapour pressures were calculated from the Antoine constants by interpolation. In those instances where some extrapolation was necessary (14:0H, 15:0H and 16:0H), the temperature range of extrapolation was usually less than 40 K.

As the choice of the PR is critical for the

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Table 1 Vapour pressures (PR) of the reference n-alkanols

Alkana!

ll:OH 12:0H 14:0H 16:0H

Constants of the Antoine equation•

A B

7.094 2105.005 6.860 2011.634 6.916 2217.995 5.964 1781.618

c

176.145 162.769 165.381 120.726

a Ref. [26]; log P (kPa) =A - Bl(t + C).

PR (25°C) (Pa)

0.4255 0.1402 0.01844 0.00207b

b Vapour pressure is for the subcooled liquid; it was calculated from the original solid vapour pressure (5.4726 · 10-• Pa) using Eq. 3 and m.p. 56°C.

accuracy of vapour pressures determined by the comparative GC method, the literature PR data were checked for internal consistency. Examination of the logarithm of vapour pressure (corrected for melting point) as a function of the number of carbon atoms in alkanol series (Fig. 1) confirms that an excellent linear correlation, ln P = (-1.0675 ± 0.0058)nc + (10.8973 ± 0.0734) (n = 7, S.E. = 0.0374, r2 = 0.9999), does exist.

0

-1

-2

-4

-5

-6 D

-7

-8+-~--.-~~~~~~~~~~~--.-~--;

9 10 11 12 13 14 15 16

Fig. 1. Liquid vapour pressures (Pa) (25°C) of alkanols [25] as a function of the number of carbon atoms. D =Original (solid) vapour pressures of 15:0H and 16:0H.

Table 2 Adjusted GC retention times (min) of the n-alkanols

Alkana! 80°C 90°C 100°C 110°c 120°c

9:0H 0.773 0.482 0.332 0.237 0.172 lO:OH 1.553 0.926 0.605 0.409 0.283 ll:OH 3.162 1.800 1.123 0.724 0.481 12:0H 6.557 3.541 2.122 1.313 0.837 14:0H 26.696 13.305 7.385 4.242 2.256 15:0H 53.448 25.600 13.698 7.600 4.379 16:0H 110.337 50.162 25.846 13.914 7.757

3.1. Validation of the method

Six n-alkanols with known PL [26] were chromatographed along with the 14:0H reference, and Pac values at 25°C were calculated from the relative retention data (Table 2) using Eqs. 1 and 2. In Fig. 2, these Pac values are compared with PL' As can be seen, the regression line obtained closely parallels the y = x line. The equation of the regression line by a linear leastsquares fit is

ln [PL (Pa)]= (1.02536 ± 0.0049) In Pac ( 4)

(n = 6, S.E. = 0.0402, r 2 = 0.9999)

0

-1

-2

a.~ -3 ,;

-4

-5

-6

-7 -7 -6 -5 -4 -3 -2 -1 0 2

In PGC

Fig. 2. Logarithmic plot of the literature vapour pressures PL of alkanols (25°C) vs. the corresponding P Ge data (Eq. 1) from the present work. The regression line (solid) and y =x line (dashed) are shown.

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m red on line e.lhe Lr St-

I I


with a slope nearly equal to 1. Note that in Eq. 4 the intercept has not been included at the 0.05% probability level. The quality of fit produced by the proposed vapour pressure correlation is excellent, thus demonstrating the validity of the GC method even for polar compounds.

Another noteworthy feature of this correlation is that it appears to be applicable over a range of pressures that covers three orders of magnitude. Table 3 presents a comparison between the GCbased and literature vapour pressure data. Our corrected (Eq. 4) vapour pressures differ from those given by N'Guimbi et al. [26] by values ranging from 0.6% (for lO:OH) to 5.3% (for 12:0H). In addition to the original database [26] employed in deriving Eq. 4, Table 3 also includes a complete data set [3] obtained from the Chebyshev-type polynomial in x of degree 3 by extrapolation. This polynomial has been proposed to allow extrapolation for about 150 K with fair confidence. The slightly lower (about 10%) but consistently similar vapour pressure values following from the use of this equation might be regarded as a notable agreement between the two literature data sets. It is noticable that vapour pressures of lO:OH (1.233 Pa) and 12:0H (0.1328 Pa) following from the use of Eq. 4 compare favourably with the values 1.190 and 0.1397 Pa obtained [27] for these compounds from a simultaneous correlation of vapour pres-

Table 3

sure and thermal data. Hence it appears that the GC method is capable of yielding vapour pressures of saturated alcohols with an error below 10%.

This conclusion finds further support in the estimated heats of vaporization. In deriving vapour pressures from GC retention time data, AHT/AHR, the ratio of the enthalpies of vaporization of a test to that of the reference compound is obtained. Hence, by utilizing the literature [28] experimental AHR value for our reference standard, 14:0H (102.2 ± 2.4 kJ mol- 1

),

the remaining enthalpies of vaporization of alkanols may be calculated from the AHT/AHR ratios given in Table 3. The results calculated by this approach are 72.17 kJ mol- 1 for 9:0H, 79.27 kJ mol- 1 for lO:OH and 91.69 kJ mol- 1

for 12:0H. These values compare well with the corresponding calorimetric data, viz. 76.86 ± 0. 75, 81.50 ± 0. 75 and 91. 96 ± 0.59 kJ mol-1, respectively, yielding a maximum error of 6.1 % .

3.2. Vapour pressures of alkenols

Vapour pressures of all measured alkenols were determined by the same approach as described above for saturated compounds. Taking advantage of the internal consistency of the vapour pressure data for saturated derivatives demonstrated above, the test compounds were

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) Ip EXP; p EXP taken from Ref. [25].

'1~ / . . '

I '•·

·I I I I I I I I -~

I I I I I I I I I I I I


Table 4 GC data and vapour pressures (25°C) of decenols

Alcohol Relative retention time"

50°C 60°C 70°C 80°C

Z3-10:0H 0.128 0.146 0.168 0.189 E3-10:0H 0.125 0.142 0.163 0.183 Z4-10:0H 0.135 0.153 0.174 0.193 E4-10:0H 0.141 0.160 0.179 0.198 Z5-10:0H 0.144 0.162 0.183 0.202 E5-10:0H 0.147 0.165 0.185 0.205 Z6-10:0H 0.147 0.166 0.186 0.206 E6-10:0H 0.147 0.165 0.185 0.205 Z7-10:0H 0.153 0.171 0.192 0.212 E7-10:0H 0.153 0.171 0.191 0.210 Z8-10:0H 0.175 0.195 0.217 0.237 E8-10:0H 0.165 0.183 0.204 0.224

'Standard 12:0H.

chromatographed using four reference standards: ll:OH (for C 12 alkenols), 12:0H (for C 10 , C 13 and C 14 alkenols), 14:0H (for C 15 and C 16 alkenols) and 16:0H (for C 18 alkenols).

The relative retention times and calculated

Table 5 GC data and vapour pressures (25°C) of dodecenols

Alcohol

Z2-12:0H Z2-12:0H Z3-12:0H E3-12:0H Z4-12:0H E4-12:0H Z5-12:0H E5-12:0H Z6-12:0H E6-12:0H Z7-12:0H E7-12:0H Z8-12:0H E8-12:0H Z9-12:0H E9-12:0H

Z10-12:0H E10-12:0H

•Standard ll:OH.

Relative retention time'

60°C

2.034 2.083 1.768 1.719 1.793 1.896 1.864 1.944 1.867 1.913 1.901 1.951 2.002 2.002 2.078 2.096 2.402 2.246

70°C

1.961 2.002 1.726 1.682 1.743 1.832 1.803 1.870 1.800 1.839 1.829 1.871 1.922 1.914 1.986 1.998 2.273 2.128

80°C

1.894 L933 1.691 1.646 1.701 1.775 1.753 1.811 1.753 1.787 1.780 1.813 1.855 1.855 1.920 1.924 2.171 2.041

90°C

1.827 1.854 1.649 1.606 1.652 1.715 1.698 1.745 1.697 1.726 1.720 1.748 1.789 1.784 1.840 1.837 2.065 1.946

P (Pa)

90°C Eq. 1 Eq. 4

0.210 1.669 1.691 0.204 1.704 1.727 0.214 1.536 1.553 0.218 1.432 1.445 0.223 1.406 1.418 0.225 1.367 1.378 0.227 1.380 1.391 0.225 1.365 1.376 0.232 1.311 1.320 0.229 1.294 1.303 0.258 1.109 1.112 0.244 1.187 1.192

vapour pressures for CIO' cl2' C13, C14, cl5> c16 and C 18 alkenols are listed in Tables 4-10. Inspection of these tables reveals that the vapour pressures of all alkenols are similar to those of the corresponding alkanols. In spite of this, two

100°c

1.780 1.806 1.624 1.591 1.612 1.665 1.662 1.701 1.664 1.673 1.672 1.700 1.738 1.732 1.789 1.775 1.979 1.886

P (Pa)

Eq. 1

0.167 0.162 0.204 0.212 0.196 0.181 1.187 0.175 0.187 0.178 0.181 0.174 1.169 0.169 0.161 0.156 0.132 1.145

Eq. 4

0.160 0.155 1.196 0.204 0.188 0.173 0.179 0.167 0.179 0.170 0.173 0.166 0.162 0.162 0.154 0.149 0.125 0.138

I

I I I I I I

'js' c16 le 4-10. h apour > those of · t,, two

-t-

I I I I I I I


Table 6 GC data and vapour pressures (25°C) of tridecenols


70°C 80°C 90°C 100°c

Z7-13:0H 1.762 1.721 1.675 1.633 E7-13:0H 1.817 1.769 1.713 1.666 Z9-13:0H 1.890 1.839 1.776 1.719 E9-13:0H 1.939 1.856 1.790 1.728

Zll-13:0H 2.266 2.177 2.077 1.999 Ell-13:0H 2.167 2.077 1.982 1.900

"Standard 12:0H.

subtle trends are apparent in all series considering the influence of double bond position: (i) the vapour pressures of series members with a double bond located near the centre of the carbon chain are generally higher than those of the corresponding saturated compounds and (ii) the

Table 7 GC data and vapour pressures (25°C) of tetradecenols


Z2-14:0H E2-14:0H Z3-14:0H E3-14:0H Z4-14:0H E4-14:0H Z5-14:0H E5-14:0H Z6-14:0H E6-14:0H Z7-14:0H E7-14:0H Z8-14:0H E8-14:0H Z9-14:0H E9-14:0H

Z10-14:0H E10-14:0H Zll-14:0H Ell-14:0H Z12-14:0H E12-14:0H

80°C

3.820 3.883 3.389 3.286 3.358 3.526 3.408 3.551 3.343 3.462 3.340 3.485 3.412 3.616 3.512 3.622 3.682 3.731 3.881 3.834 4.346 4.102

"Standard 12:0H.

90°C

3.538 3.586 3.186 3.083 3.157 3.289 3.217 3.308 3.138 3.243 3.134 3.259 3.209 3.301 3.285 3.376 3.473 3.464 3.587 3.548 4.024 3.774

100°c

3.299 3.345 3.013 2.912 2.966 3.079 3.017 3.096 2.954 3.050 2.959 3.057 3.004 3.087 3.079 3.157 3.223 3.221 3.335 3.300 3.714 3.495

110°c

3.113 3.140 2.847 2.767 2.811 2.902 2.843 2.917 2.798 2.865 2.798 2.883 2.840 2.922 2.898 2.961 3.016 3.025 3.117 3.079 3.447 3.250

P (Pa)

110°c Eq. 1 Eq. 4

1.590 0.0673 0.0628 1.619 0.0639 0.0596 1.674 0.0607 0.0565 1 678 0.0575 0.0534 1.920 0.0472 0.0436 1.833 0.0491 0.0455

vapour pressures of isomers with a double bond positioned on the second carbon atom of the chain (irrespective of the end of the molecule from which the numbering starts) are either close to or lower than those of the saturated compounds. It appears that the double bond position

P (Pa)

120°C Eq. 1 Eq. 4

2.911 0.0211 0.0191 2.925 0.0203 0.0184 2.685 0.0256 0.0234 2.609 0.0267 0.0243 2.650 0.0256 0.0233 2.723 0.0234 0.0213 2.663 0.0246 0.0224 2.729 0.0230 0.0209 2.634 0.0257 0.0234 2.686 0.0240 0.0218 2.634 0.0258 0.0235 2.704 0.0239 0.0217 2.665 0.0246 0.0224 2.719 0.0220 0.0200 2.725 0.0236 0.0215 2.768 0.0222 0.0202 2.819 0.0217 0.0196 2.815 0.0211 0.0191 2.909 0.0200 0.0181 2.864 0.0201 0.0182 3.185 0.0169 0.0153 3.004 0.0180 0.0163

I ·I I I I I I I I I I I I I I I I I I I I

·'

- I 1 ,, l


Table 8 GC data and vapour pressures (25°C) of pentadecenols

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 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 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 El3-15:0H 1.955 1.880 1.811 1.744 1.695 0.00654 0.00576

"Standard 14:0H.

Table 9 GC data and vapour pressures (25°C) of hexadecenols

Alcohol Relative retention time" P (Pa)

100°e 110°e 120°e B0°e 140°e Eq. 1 Eq. 4

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 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 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 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 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 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

"Standard 14:0H.

I

I I I I I

I 1-I I I I I I I I I I


Table 10 GC data and vapour pressures (25°C) of octadecenols

Alcohol Relative retention time•

120°c 130°C 140°C 150°C

Z3-18:0H 2.676 2.500 2.400 2.288 E3-18:0H 2.583 2.448 2.346 2.244 Z9-18:0H 2.465 2.339 2.254 2.160 E9-18:0H 2.596 2.455 2.366 2.247

Zll-18:0H 2.534 2.405 2.311 2.216 Ell-18:0H 2.637 2.497 2.391 2.275 Z13-18:0H 2.712 2.555 2.439 2.326 El3-18:0H 2.753 2.585 2.463 2.342

a Standard 16:0H.

relative to both the polar and non-polar ends of the molecule is significant. As illustrated in Figs. 3 and 4, these trends may be observed both for the Z- and £-isomers in all the series investigated.

For homologous subseries such as Z- or £isomers of 7-lO:OH, 9-12:0H, 11-14:0H and 13-16:0H (w-3 unsaturation), 5-lO:OH, 7-12:0H, 9-14:0H and ll-16:0H (w-5 unsatura-

7

6

5

2

0+--.~-,--,~-,---.~..,---.-~,--.-~-,--

2 3 4 5 6 7 8 9 10 11 12 13 (Z)-double bond pos1t1on

Fig. 3. Vapour pressures (Eq. 4) for (Z)-alkenols plotted against the respective double bond position. • = Decenols; • = dodecenols; & = tetradecenols; <> = pentadecenols; 0 =

hexadecenols. Dashed lines show the vapour pressures of the corresponding saturated compounds.

P (Pa)

160°C Eq. 1 Eq.4

2.200 0.000377 0.000309 2.149 0.000406 0.000333 2.083 0.000453 0.000373 2.157 0.000402 0.000330 2.130 0.000431 0.000354 2.181 0.000386 0.000316 2.221 0.000363 0.000296 2.234 0.000345 0.000282

tion) and 3-lO:OH, 5-12:0H, 9-14:0H and ll-16:0H (w-7 unsaturation), the double bond is at a constant position with respect to the non-polar end of the molecule. Analysis of the double bond effect in these subseries reveals that the vapour pressures of alkenols having different positions of unsaturation decrease in the order w-7 > w-5 > w-3 > saturated. The empirical relationships given by the In P vs. nc expression

a 0 0

6

5 ................... .

x 4 ~ .s

3

2

Q-\---,~...,----,~...,---.~-,--,~-,--,~-,----1

2 3 4 5 6 7 8 9 10 11 12 13 (E)-double bond position

Fig. 4. Vapour pressures (Eq. 4) for (E)-alkenols plotted against the respective double bond position. • = Decenols; • = dodecenols; & = tetradecenols; <> = pentadecenols; 0 = hexadecenols. Dashed lines show the vapour pressures of the corresponding saturated compounds.

I -I I I I I I I I I I I I I I I I I I

i

I .,

I

~ . ---

316 B. Kowek et al. I J. Chromatogr. A 679 (1994) 307-317

Table 11 Proposed relationships for predicting vapour pressures at 25°C

Alcohol Ln[P (Pa)]= a - bn, S.E.b r' subseries'

a b

Saturated 10.772 ± 0.135 1.058 ± 0.010 0 0458 0.9998 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 w-5-(Z) 10.805 ± 0.018 1.046 ± 0.001 0.0061 1.0000 w-5-(E) 10.849 ± 0.020 1.053 ± 0.002 0.0067 1.0000 w-7-(Z) 10.995 ± 0.222 1.053 ± 0.017 0.0751 0.9995 w-7-(E) 11.178 ± 0.307 1.071 ± 0.023 0.1040 0.9991

"Number of data points. n = 6. b S.E. =standard error of estimate.

were obtained from analyses of the calculated (Tables 4-10) ln P data. The relevant equations, listed in Table 11, may be used to estimate vapour pressures for any set of w-3, w-5 and w-7 alkenols. The quality of the fit obtained with MAD (mean average deviation) 2.3% (w-3), 1.9% (w-5) and 4.8% (w-7) combined with the convenience of only one substance-specific input variable makes this an attractive approach in predicting vapour pressures of some other structurally similar derivatives.

At this point some comment should be made regarding literature vapour pressure values of unsaturated alcohols. To our knowledge, only four of the alkenols investigated in this work had literature data available for comparison. Heath and Tumlinson [29] determined the vapour pressures of Z7-12:0H, Z9-14:0H, Zll-14:0H and Z11-16:0H as 1.25, 0.177, 0.160 and 0.039 Pa, respectively. They carried out these determinations on capillary liquid crystal GC columns at "room temperature", which probably corresponded to 30°C. At that temperature our GC method yields vapour pressures of 0.344, 0.044, 0.037 and 0.0057 Pa, respectively, for the same compounds. Hence our values are significantly (3.6-6.8 times) lower than those in Ref. [29]. Note, however, that a high degree of correlation exists between both data sets. The linear fit

may be expressed as ln P = (1.1716 ± 0.0666) ln ?Heath - (1.231±0.137) (n = 4, S.E. = 0.164, r 2

= 0.9936). By utilizing the reliable literature [26] values

at 30°C (see Table 1) for 12:0H (0.266 Pa) and 14:0H (0.0366 Pa), the ratio Paikenoi / Paikanoi for compounds with the same number of carbon atoms may be adopted as an approximate measure of the "effect of non-terminal monounsaturation". This vapour pressure ratio following from our data is about 1.2-1.3, which seems to be a reasonable value considering its similarity to the corresponding values common for non-terminal alkene [1] and unsaturated acetate series [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 reasons for this discrepancy are not clear. However, besides the imprecisely defined temperature they used, another factor might be important, viz. the use of cholesteryl p-chlorocinnamate as a stationary phase. It may be that the polar alcohols interact in a specific manner with this phase and then this factor would account for the differences in the two studies.

As the errors in the reported vapour pressures depend both on experimental uncertainties and on the accuracy of the literature vapour pressure data adopted for the reference standards, it is difficult .Jo determine any inherent error in the present method for ·alcohols. Some discussion is possible, however: as to the latter error factor, recent inter-laboratory data [25,27) for lO:OH and 12:0H agree to within ±5%, which may be regarded as a very good agreement. Hence the differences between our data and those taken from the literature for alkanols are generally not greater than the experimental errors (see Table 3). On the other hand, when admitting a propagation of errors, the uncertainty might reach about 10%. Moreover, the vapour pressures for 15:0H and 16:0H were obtained by (prudent) extrapolation and additionally corrected for melting points. It is unlikely that they are in serious error (see the internal homogeneity of the alkanol data illustrated in Fig. 1), but we have to accept their lower accuracy. Taken

/ /

/\)

I

•] '11ues .and lkanol for .bon

tt9neaJunsatu-

all ing .e s to 11 y to ·n-termie,ries

-4.8 om Ref.

~h.TThe r.lfowempera-

rin~~~ til the iler with alt for

ressures

tit and p sure d , it is r in the isfn is - tor,

lO:OH

l ybe :n the e taken

·a1 not e able l opa-1t reach ul for )r ent) ted for

le in

l1 y of tlu we Taken

I I I


together, we assume that, at worst, the errors may combine to give an overall uncertainty in vapour pressures of about 15%.

4. Conclusions

This study has demonstrated the successful application of the capillary GC method to the determination of the vapour pressures of unsaturated alcohols whose generally low thermal stability causes difficulties in direct measurements by conventional physico-chemical methods. The method yields reasonable vapour pressure values for both the alkanols and alkenols at 25°C provided that a compound of similar structure and polarity is used as the reference standard. It is hoped that these new values may considerably extend the database for the vapour pressures of alcohols, and enable entomologically oriented chemists to study and model the physical behaviour of pheromone-like compounds in th-e env!tonment -more accurately.

The method is currently being used to determine vapour pressures of unsaturated aldehydes and will be the subject of a separate report.

Acknowledgement

This work was financially supported, in part, by Grant No. DHR-5600-G-00-1051-00, Program in Science and Technology Cooperation, from the US Agency for International Development.

References

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J CHEM. SOC. PERKIN TRANS. l 1994 3509

Preparation of Chlorofluoroacetic Acid Derivatives for the Analysis of Chiral Alcohols

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 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 catalysis.

Determining the optical purity of biologically active compounds and of the products of any asymmetric synthesis is important in much contemporary research. One approach used frequently for this purpose is the preparation of derivatives of chiral alcohols and amines using 3,3,3-triftuoro-2-methoxy-2-phenylpropioni<:; acid (Mosher's acid, MTPA), which gives rise to diastereoisomers which may be separable by GC or HPLC, or detected by NMR techniques. 1

•2 This approach has been

widely applied, and has proven extremely useful. However, there are examples where this method failed. 3

•4 New agents for

the preparation of derivatives are, therefore, of potential interest, and some new examples have been described recently. 5-

9

Among these oc-aryl-oc-ftuoroacetates seem to be good candidates.4 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. 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 additional functional groups attached to the chiral C-F centre.4

This substitution leads, however, to decreased volatility, making the compounds less suitable for GC analysis. We report 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 Cf A esters might permit easy GC and/or HPLC separation,_as well as NMR spectral differentiation.

-CFA is readily accessible, 11 easily resolved 10•12 and the

absolute configuration of its enantiomers has already been established. 13 For esterification of CFA we chose the 1,3-dicyclohexylcarbodiimide (DCC) method, which gives high yields even with sterically hindered alcohols. 14

•15 In order to

obtain a quantitative reaction with respect to the starting alcohol, we used a 3 mo! excess of the reagents. 16 Since it is known that various chiral alcohols can be esterified spontaneously by CF 3C02H, 17 or by other halogen-containing acetic acids, 18 we have also directly esterified CF A with 1-phenylethanol 4a using CFA as the solvent. Neither racemization nor kinetic resolution during these esterifications was observed. A variety of secondary alcohols la--6a were converted into CFA esters lb-6b (Table I). The same alcohols were converted into MTPA esters lc--6c (Table 2) using commercially available (R)- and (S)-MTPA chlorides following a standard procedure. 1

•2 We have ex_amined the GC and HPLC

OR

~ ~R 2

4

5 6

a R=H b R= OCCHCIF c R = OC(CF3)(0Me)(Ph)

behaviour of both CF A and MTP A esters of alcohols la--6a, as well as the 1 H and 19F NMR spectra of these compounds. The results are summarised in Table 3, in which we record the chemical shift non-equivalence (MH.F) of diastereoisomers in both 1 H and 19F NMR spectra and also differences in retention times (~t" GC, HPLC). In general, esters derived from MTPA · show larger M values than do CF A esters. This is not unexpected, in view of the increased non-bonded steric and electronic interactions resulting from the presence of an aromatic ring attached to the chiral carbon atom of the MTPA esters. 1

•4

However, the CHFCI proton resonance of diastereoisomeric CFA esters typically appears as a characteristic doublet (JH,F 50 Hz) in a region not often disturbed by other signals (o :;-; 6.30, see Table 1). This, along with the sharpness of these peaks, greatly facilitates the analysis of the chiral alcohols. 4 The same favourable feature is also seen in the 19F NMR spectra (o :;-; -68, JF.H 50 Hz). The chromatographic behaviour of CF A diastereoisomers appears to be superior to that of MTPA esters. This is important because using a chromatographic

I I I I I I I I I I I I I I I I I I .1 I I

3510

Table 1 Spectroscopic data for CFA esters lb-6b of alcohols la-6a prepared using racemic CF A

Alcohol Spectroscopic data for CF A ester

(R,S)-la JH 0.88 (t, 3 H, J 6, CH3), 1.61-1.75 (m, 8 H, CH2), 5.21-5.38 (m, 3 H, C----CH2 , OCH); 5.71-5.88 (m, 1 H. -CH=), 6.27 (d,JH,F 50, 1 H, CHF); JF -67.79 (d, JF,H 50, CHF), -67.86 (d, JF,H 50, CHF); Vmax/cm- 1 3096, 2941, 2874, 1767, 1253, 1107,964,822;m/zl67, 151, 137,110,95,81,69 (BP), 41

(R)-2a b'tt 0.87 (t, 3 H, J 6, CH3), 1.15-1.63 (m, 13 H, CH2, CH), 5.00--5.10 (m, I H, HCO), 6.24 (d, 1 H, JH.F 50, CHClF); OF -67.75 [d, JF.H 50, CHCIF (R,R)], -67.86 [d, JF.H 50, CHCIF (R,S)]; vmaxfcm- 1 2938, 2871, 1765, 1463, 1289, 1191, 1116, 959, 826; m/= 157, 139, 112, 83, 70, 57, 41 (BP)

(S)-3a oH 0.89--0.98 (m, 9 H, 3 x CH3), 1.20-1.91 (m, 9 H, CH2,

CH), 5.18 (m, 1 H, OCH), 6.24 [d, JH,F 50, CHCIF, (S,R)]; 6.25 [d, JH,F 50, CHCIF, (S,S)]; OF -67.65 [d, JF.H 50, CHCIF, (S,S)], -67.68 [d, JF.H 50, CHCIF, (S,R)]; Vmax/cm-1 2966, 2852, 1766, 1289, 1191, 1113, 822; m/= 207, 138, 109, 95 (BP), 67, 41

(R)-4a JH 1.64 (d, 3 H, J 6.6, CH3), 6.06 (q, 1 H, J 6.6, HCOJ, 6.32 [d, JH.F 50, CHClF, (R,S)], 6.35 [d, JH,F 50, CHCIF, (R,R)], 7.42 (m, 5 H, ArH); oF -68.48 [d, JF,H 50, CHCIF, (R,S)], -68.51 [d, JF.H 50, CHClF, (R,R)]; Vmaxfcm-1 3074, 2993, 1780, 1202, 1107, 1063, 953, 822; m/z 216, 201, 173, 146, 105 (BP), 77, 51

(S)-Sa JH 0.87 (t, 3 H, J 6, CH3), 1.21-1.99 (m, 14 H, CH2),

5. 76--5.87 (m, 1 H, HCO), 6.26 [ d, JH.F 50, CHCJF, (R,R)]. 6.29 [d JH.F 50, CHCIF, (R,S)]; JF -67.52 [d, JF.H 50, CHCJF, (R,R)], -67.73 [d, JF,H 50, CHCIF, (R,S)]; Vmax/cm-1 3073, 2994, 2866, 1781, 1458, 1282, 1185, 1113, 964, 822;m/z328, 261, 233, 201.173, 117, 104(BP),41

(R)-6a JH 6.40 (d, 1 H, JH.F 49, CHCIF), 6.46 (d, JH,F 49, CHClF), 7.45-8.61(m,10 H. ArH), CF3CH);JF -69.13 (d,JF,H 49, CHClF), -69.18 (d, JF,H 49, CHClF), 5.81 (d, 1 H, J 7.8, HCCF3 ); vmaxfcm- 1 3061, 1785, 1354, 1271. 1192, 1137,886, 784;m/=370(BP),301,259,238, 178, 151, 119, 67

method as an analytical tool permits actual separation as well as analysis, and can be more precise than NMR spectroscopy. 19

It is known that halogen-containing acetates are well resolved on non-polar GC phases, where the retention times are related to the atomic weight and the number of halogen atoms in the molecule. Fluorine is exceptional, its introduction resulting in significant shortening of retention times. 20 The relatively low column temperatures required for good resolutions of CF A diastereoisomers can be attributed to (i) their lower molecular weight, (ii) the presence of fluorine in the molecule, and perhaps also (iii) little hydrogen bonding with the liquid phase.21 Good separations were also obtained using HPLC on silica gel; CF A esters 2a and 4a can even be resolved by TLC on silica gel plates, where two fully separated spots of diastereoisomers appeared (none of the MTPA esters studied in this paper showed such a separation).

In order to obtain reliable results in chiral alcohol analysis, the derivative preparation must not favour one alcohol enantiomer over the other, and must be free of racemization. Chiral acetic acids having an enolizable a-hydrogen atom are certainly capable of racemizing. Nevertheless, CF A is sufficiently optically stable to be used for preparing derivatives of chiral secondary alcohols. CF A is the least reactive of the halogenated acetic acids towards nucleophilic substitution. 22

Optically pure CF A does not show any change in specific rotation in aqueous solution and can be converted into its acid chloride or methyl ester and reduced to the corresponding alcohol without any racemization. 10

·12 Based on all of these

considerations, CF A seems to be an attractive choice for the stereochemical analysis of chiral alcohols.

J. CHEM. SOC. PERKIN TRANS. I 1994

Table 2 Spectroscopic data for MTP A esters lc-6c of alcohols la-6a

Alcohol Spectroscopic data for MTP A ester

(R,S)-la oH 0.84 (t, J 6.4, CH3), 0.87 (t, J 6.4, CH3), 1.17-1.36 (m, 6 H, CH2), 1.52-1.73 (m, 2 H, CH2CO), 3.54, 3.55 (2 x s, 3 H, OCH3), 5.16--5.38 (m, 2 H, C=CH2), 7.36--7.49 (m, 5 H, ArH); JF 6.13 (s, CF,); Vmaxfcm-1 2950, 1758, 1215, 1181, 1123 and 1013; m/= 189 (BP), 139, 128, 119, 111, 105, 77, 69

(R)-2a Ott 0.85 [t, J 6.6, CH" (R,S)]; 0.87 [t, J 6.6, CHiR,R)] 1.11-1.37 (m, 11 H, CH2 , CH3), 1.32 (d, 3 H, J 6.4, CHCH 3), 1.52-1. 72 (m, 2 H, OCCH2 ), 3.49 [s, 3 H, OCH3,

(R,R)]; 3.56 [s, 3 H, OCH3 . (R,S)], 5.10--5.19 (m, I H, OCH), 7.34--7.54 (m, 5 H, ArH); JF 6.14 [s, CF3 , (R,R)], 6.61 [s, CF3 , (R,S)]; Vmaxfcm-1 2940, 2868, 1758, 1455, 1383, 1244, 1181, 1122, 1016, 846; m/z 189 (BP), 158, 119, I05, 89, 77, 57

(S)-3a JH 0.81 [d, J 6.6, 2 x CH3, (S,R)], 0.88 [d, J 6.6, 2 x CH3, (S,S)], 0.94 [d, 3 H, J7, CH3, (S,R)]; 0.96 [d, 3 H, J 6.6, CH3 , (S,S)]; 1.01-1.81 (m, 9 H, CH, CH2),

3.54 [s, 3 H, OCH3, (S,S)], 3.56 [s, 3 H, OCH3, (S,R)]; 5.25-5.33 (m, I H, OCH), 7.36--7.52 (m, 5 H, ArH); JF 6.18 [s, CF3 , (S,S)], 6.59 [s, CF3, (R,S)]; vmaxfcm-1 3069, 2962, 2882, 1754, 1456, 1382, 1017, 972; m/z 189, 158. 139, 127, 105, 83 (BP), 77, 69, 55

(R)-4a JH 1.57 [d, J6.6, CH3 , (R,R)], 1.68 [d, J 6.6, CH3, (R,S)], 3.45 [s, 3 H, OCH3 , (R,S)], 3.47 [s, 3 H, OCH" (R,R)], 6.08 [q, J 6.6, CHCH3, (R, S)], 6.13 [q, J 6.6, CHCH3 ,

(R,R)], 7.21-7.42 (m, IO H, ArH); oF 6.68 [s, CF3 ,

(R,S)], 6.89 [s, CF3 , (R,R)]; Vmaxfcm- 1 3072, 2992, 1760, 1453, 1232, 1182, 1123, 1014, 860; m/z 189, 158, 119, 105, 77 (BP)

(S)-Sa JH 0.87 (t, 3 H, J 6.8, CHJ), 1.09-1.40 (m, 14 H, CH2),

1.57-1.99 (m, 2 H, OCCH2), 3.43 [s, 3 H, OCH" (S,S)], 3.53 [s, 3 H, OCH3 , (S,R)], 5.85 [d, J 6.6, OCH, (S,R)], 5.92 [d, J 6.6, OCH, (S,S)], 7.27-7.43 (m, IO H, ArH); JF 6.69 [s, CF" (S,R)], 6.91 [s, CF3, (S,S)]; vmaxfcm-1 2934, 2564, 1757, 1455, 1183, 1123, 1014; m/z 217, 89, 174, 158, 139, 123, 119, 91 (BP), 77

(R)-6a JH 3.28 [s, 3 H, OCH3 , (R,S)], 3.67 [s, 3 H, OCH3, (R,R)], 7.18-8.60 (m, 10 H, ArH, CF3CH); JF 6.32 [d, 3JF,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, 3009, 2850, 1627, 1528, 1498, 1452, 1398, 1277, 1268, 1239, 1225;m/z259(BP),239,209, 190, 189, 174, 119, 105, 77

" IR spectrum was recorded on Bruker ISS FTIR spectrometer (in CC14).

Experimental 1 H (200 MHz) and 19F (188 MHz) NMR spectra were recorded on a Varian XL-200 spectrometer. Chemical shifts (in CDC13)

are expressed on the o scale measured from residual CHC13 (7.25) and internal CF3C02H (0.0) for 19F. !Values are given in Hz. Mass spectra were obtained using a Hewlett-Packard 5890 gas chromatograph (column DB-5, 30 m x 0.25 mm i.d.) coupled to an HP 5970 mass selective detector. IR spectra were recorded in the gas phase using a Hewlett-Packard 5890 II gas chromatograph (column: Carbowax, 30 m x 0.25 mm i.d.) coupled to an HP-5965 infrared detector. For GC analyses, a Hewlett-Packard gas chromatograph was used (columns: DB-5, 30 m x 0.25 mm i.d., Carbowax, 30 m x 0.25 mm i.d.) with helium as the carrier gas. HPLC analyses were performed using a Hewlett-Packard HP 1090 apparatus equipped with silica columns [3 x (450 mm x 3 i.d.), Tessek, Czech Republic, silica 5 µm, 3% diethyl ether in hexane, flow rate: 0.4 cm3 min-1

,

DAD UV detector (220 nm) controlled by an HP-85B computer]. [ci]0 Values are given in units of 10-1 deg cm2 g-1

•

For the preparation of diastereoisomeric esters, the following secondary alcohols were purchased and used without further purification: (R,S)-oct-l-en-3-ol la (Aldrich 0-528-4), (R)-(-)octan-2-ol 2a (Aldrich, 14,799-0), ( + )-isomenthol 3a (Aldrich 24,219-5), (R)-( + )-1-phenylethanol 4a, (Aldrich 23, 742-6), (S)-

I I J. CHEM. SOC. PERKIN TRANS. I 1994

Table 3 Chromatographic and spectroscopic differences between diastereoisomers of esters of alcohols la---{ia with CFA and MTPA

3511

I Ester GC M(Hz) HPLC

Alcohol" t,/min of diast. b T/°Cc tit, 'H 19F t,/min of diast. R• s Acid

CFA (R,S)-la 32.19 33.08 95 0.89 12 23.36 24.16 1.6

I (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 (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

I (R)-6a• 24.88 25.02 200 0.14 12 9 30.66

(R.S)-la 27.51 28.46 155 0.95 2 20.99 21.29 0.6 (R)-2a 32.32 (R,S) 32.86 (R,R) 155 0.54 13 88 22.86

MTPA

(S)-3a 32.75 170 4 86 20.49

I (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 (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::',::~,:":· .:~:;~,.::~.::,~~

I I I I I I I I I I I I I I

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.

(- )-l-phenyldecan-1-ol 5a (Aldrich 33,161-9) and (R)-( -)-2,2,2-trifiuoro-l-(9-anthryl)ethanol 6a (Aldrich 21, 135-4). Since obtaining both M and dt, was the main objective of this paper, the CF A esters were prepared as mixtures of diastereoisomers, using racemic chlorofluoroacetic acid. To identify particular diastereoisomers in a mixture, esters of optically active alcohols with either (R)- or (S)-chloroftuoroacetic acid were also prepared. The syntheses were accomplished via DCC esterification 14

•15 using a 3 mo! excess of reagents.

The reaction was worked up as soon as the starting alcohol had disappeared from the reaction mixture (TLC, typically 0.5-24 h, yield: 91-97%). For GC analyses of CF A and MTPA esters, two different columns were used, giving the best resolution of particular sets of diastereoisomers (see Table 3). The NMR and mass spectra of the resultant esters are given in the Tables I and 2, while the spectral and chromatographic comparisons are summarized in Table 3.

Resolution of Chlorofluoroacetic Acid. 12-To a solution of racemic chloroftuoroacetic acid (16.8 g, 150 mmol) in ethyl acetate (125 cm3) at 0 °C was added a 0 °C solution of(S)-(-)a-methylbenzylamine (18.2 g, 150 mmol) in ethyl acetate (125 cm3

). After mixing thoroughly, the resulting solution was set aside at 0 °C for 2 h and then at room temperature for 3 h during which time the two diastereoisomeric salts were deposited as a white solid (33 g), m.p. 115-126 °C (decomp.). Fractional recrystallization from acetone afforded the (S,R)diastereoisomeric salt (8.1 g, 35 mmol, 46%) (about five 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 corresponding ethyl chlorofluoroacetate enantiomers on Lipodex A [a sample of the ethyl ester was prepared by mixing (S)-( - )-a-methylbenzylammonium salt (10 mg) with Dowex 50W-X8 exchange resin (50 mg) in absolute ethanol (0.5 cm3

);

the mixture was set aside for 48 h prior to GC analysis]. In a similar manner, (R)-( + )-a-methylbenzylamine afforded the pure (R,S)-diastereoisomeric salt, m.p. 143-145 °C (decomp.); [cx]~5 11.2 (c 3.80 in MeOH).

The CF A was released from its a-methylbenzylammonium salt by means of acid. Thus, the (R,S)-diastereoisomeric salt (0.85 g, 3.64 mmol) was covered by methylene dichloride (2 cm3) and concentrated hydrochloric acid (0.37 cm3

, 3. 74 mmol) was added to it. After being stirred for I h at room temp., the solution was dried over magnesium sulfate and then con-

centrated under reduced pressure. Distillation of the residue afforded (S)-chlorofiuoroacetic acid (0.36 g, 91%), b.p. 86-94 °Cj40 mmHg; bf! 6.30 (d, Jfl,F 50, 1 H, CClFH) and 6.88-7.25 (br s, 1 H, C02 H); i5F -68.09 (d, h.tt 50).

(R,R)-Octan-2-yl Chlorojluoroacetate 2b (Prepared in an Excess of CFA).-(R)-Chloroftuoroacetic acid (59 mg, 0.52 mmol), (R)-octan-2-ol (13.7 mg, 0.105 mmol) and Drierite (38 mg) were placed in a I cm3 conical vial. The mixture was set aside at room temperature and the reaction mixture was monitored either by GC or TLC. As soon as the starting alcohol had disappeared from the reaction mixture (about 3 days), the reaction mixture was diluted with light petroleum (1 cm3

,

containing 5% of diethyl ether), transferred onto a Pasteur pipette silica column, and chromatographed using the same solvent mixture to give the product (22.8 mg, 97%). When the column was eluted with pure diethyl ether (R)-chlorofluoroacetic acid (36 mg, 77%) was recovered. The spectral data were identical with those for (R,R)-2b given in Table I.

Acknowledgements This work was supported in part by the Program in Science and Technology Cooperation, U.S. Agency for International Development, under Grant DHR-5600-G-00-1051-00 (L. S.), and by the Czech Grant Agency (L. S., A. S., J. V., Grant 203/93/0102), and in part by a grant to J.M. (GM 48088) from the National Institutes of Health.

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J. Kohoutova, M. Romanuk and J. Vrkoc, Collect. Czech. Chem. Commun., 1986, 51, 2207.

4 Y. Takeuchi, H. Ogura, Y. Ishii and T. Koizumi, J. Chem. Soc., Perkin Trans. I, 1989, 1721.

5 G. Hamman and M. Barrele, J. Fluorine Chem., 1987, 37, 85. 6 Y. Takeuchi, M. Asahina, K. NagataandT. Koizumi,J. Chem. Soc.,

Perkin Trans. I, 1987,2203. 7 Y. Takeuchi and N. Nojiri, Tetrahedron Lei/., 1988, 29, 4727. 8 Y. Takeuchi, H. Ogura and Y. Ishii, Chem. Pharm. Bull., 1990, 38,

2404. 9 Y. Takeuchi, N. Itoh, H. Note, T. Koizumi and K. Yamaguchi, J. Am.

Chem. Soc., 1991, 113, 6318. 10 G. Bellucci, G. Berti, A. Barraccini and F. Macchia, Tetrahedron,

1969, 25, 2979.


3512

l l J. A. Young and P. Tarrant, J. Am. Chem. Soc., 1949, 71, 2432. 12 D. L. Pearson, Ph.D. Thesis: The synthesis ofenantiomerically pure

inhalation anesthetics-halothane and enfturane, Cornell University, 1990.

13 G. Bellucci, C. Bettoni and F. Macchia, J. Chem. Soc., Perkin Trans. 2, 1973, 292.

14 A. Hassner and V. Alexanian, Tetrahedron Lett., 1978, 46, 4475. 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.

Chem. Commun., 1990, 55, 485. 17 J. G. Traynham, J. Am. Chem. Soc., 1952, 74, 4277; B. H. Johntson,

A. C. Knipe and W. E. Wats, Tetrahedron Lett., 1979, 43, 4225.

J. CHEM. SOC. PERKIN TRANS. I 1994

18 G. S. Toole and F. J. Sowa,J. Am. Chem. Soc., 1937, 59, 1971. 19 D. Parker, Chem. Rev., 1991, 91, 1441. 20 K. Komarek, L. Hornova and J. Churacek, J. Chromatogr., 1982,

244, 142. 21 I. 0. 0. Korhonen, J. Chromatogr., 1984, 288, 329. 22 E. T. McBee, D. L. Christman, R. W. Johnson and C. D. Roberts,

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Paper 4/02810D Received 12th May 1994

Ac_cepted 2nd August 1994

© Copyright 1994 by the Royal Society of Chemistry

t (


Communication

Inhibitors of Arachidonoyl Ethanolamide Hydrolysis*

1 Received for publication, June 25, 1994. and in revised form, July 20. 1994J

Bohumir Koutek:l:*, Glenn D. Prestwich:j:iJ, Allyn C. Howlett[[, Suzette A. ChiniJ, David Salehanii!, Nima AkhavaniJ, and Dale G. DeutschiI':'*

From the Departments of +Chemistry and W3iochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794 and the !iDepartment of Pharmacological and Physiological Science, Saint Louis Unwersity School of Medicine, St. Louis, Missouri 63104

Arachidonoyl ethanolamide (anandamide) is a naturally occurring brain constituent that binds to a specific brain cannabinoid receptor ( CBRI). An amidase activity (anandamide amidase) in membrane fractions of brain and in cultured neuroblastoma cells rapidly degrades anandamide to arachidonic acid (Deutsch, D. G., and Chin, S. (1993) Biochem. Pharmacol. 46, 791-796). In the current study, analogs of anandamide representing three classes of putative transition-state inhibitor (trifluoromethyl ketones, a-keto esters, and a-keto amides) were synthesized and tested as inhibitors of anandamide hydrolysis in vitro and as ligands for CBRL The trifluoromethyl ketones and a-keto esters showed nearly 100% inhibition of anandamide hydrolysis in vitro at 7.5 µJ\I inhibitor and 27.7 µM anandamide. Arachidonyl trifluoromethyl ketone was the only synthetic compound in the series of fatty acid derivatives able to displace [ 3H]CP-55940 binding to CBRI with a K; of 0.65 µM. It was also the most effective inhibitor in intact neuroblastoma cells, leading to a 12-fold increase of cellular anandamide levels at 12 µM. From the action of these inhibitors on this hydrolytic enzyme, it seems likely that anandamide is cleaved by a mechanism that involves an active-site serine hydroxyl group. These inhibitors may serve as useful tools to elucidate the role anandamide plays in vivo.

..l 4-Tetrahydrocannabinol, the psychoactive marijuana plantderived cannabinoid, binds to a specific brain cannabinoid receptor, CBRl 1 (1, 2l. Arachidonoyl ethanolamide (anandamide)

·This work was supported by Grants R01-DA03690, R01-DA06912 and K05-DA00182 (to A. C. H.l and R03-DA07318 (to D. G.D.) from NIDA, National Institutes of Health, by Grant R01-GM44836 (to G. D. P.1 from the National Institutes of Health, and by a United States Agency for International Development traineeship lto B. K.J under Grant DHR-5600-G-00-1051-00 from the Program in Science and Technology Cooperatrnn. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to mdicate this fact.

)i On leave from the Institute of Organic Chemistry and Biochemistry, Academy of Sdences of the Czech Republic, 16610 Praha 6, Czech Republic.

To whom conespondence should be addressed: Dept. of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215. Tel.: 516-632-8595; Fax: 516-632-8575.

1 The abbreviations used are: CBRl, cannabin01d receptor 1; CP-

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 269, No. 37, Issue of September 16, pp. 22937-22940, 1994 © 1994 by The American Society for Biochemistry and Molecular B10logy, Inc.

Printed m US.A.

(3), homo-y-linolenoyl ethanolamide, and docosatetraenoyl ethanolamide ( 4, 5) are naturally occurring brain constituents that bind to CBRl (6). Anandamide behaves as a cannabimimetic compound in vitro, stimulating receptor-mediated signal transduction that leads to the inhibition offorskolin-stimulated adenylate cyclase (5, 7, 8). In a neuroblastoma cell line, anandamide causes partial inhibition ofN-type calcium currents via a pertussis toxin-sensitive G protein pathway, independently of cAMP metabolism (9).

An amidase activity, which degrades anandamide to arachidonic acid and ethanolamine, has recently been detected in membrane fractions of the brain, but not in the heart or other muscles, and phenylmethylsulfonyl fluoride (PMSF) was found to be a potent inhibitor of this activity (10). Inclusion of PMSF in receptor binding assays increases the apparent potency of anandamide (8). We report herein the synthesis of several types of specific inhibitors of anandamide hydrolysis, their ability to inhibit anandamide breakdown in vitro, and their affinity as ligands for CBRl in vitro. We show that one of these analogs is a very effective inhibitor of anandamide metabolism in intact neuroblastoma cells and also binds to CBRl. Inhibitors that block the catabolism of anandamide may be biologically significant in the study of anandamide in vivo.

EXPERIMENTAL PROCEDURES

Synthesis-Fatty acid ethanolamides (1) were prepared from fatty acid chlorides and ethanolamine as described (4). a-Ketoamides l2l were prepared via the ethyl esters (3) as key intermediates. Thus, alkylation of the sodium salt of the anion of2-carboethoxy-1.3-dithiane in dimethylformam1de/benzene with the appropriate long-chain alkyl bromide (11), followed by hydrolysis with CH3VCaCO/CH3CN!Hp (12), gave the resulting a-keto ethyl esters (3). Hydrolysis of the esters with dilute base followed by treatment of the a-keto acids with 2,3-dioxo-5,7-diphenyldihydrothieno[3,4-b]dioxin-6,6-dioxide (Steglich reagent) (13) gave an activated cyclic oxalate that, when treated with ethanolamine, provided high yields of the a-keto ethanolamides (21. Trifluoromethylketones 4a-4e were synthesized by reaction of the correspondmg carboxylic acid with CH30CHC12 to give the acid chloride (14) followed by an in situ reaction (15) of the acid chloride with pyridine and trifluoroacetic anhydride to produce, after hydrolysis during aqueous workup, the trifluoromethyl ketones 4.

Enzyme Assay in Vitro and in Cell Culture-The assay of the anandamide amidase in vitro and in intact neuroblastoma cells was performed 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 fatty acid-free bovine serum albumin, in phosphate-buffered saline in a final volume of 1.0 ml, for 10 min at 37 °C. Substrate (27.7 µM anandamide + 0.2 µCi of 221 Ci/mmol [3H]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.

To measure the inhibition of anandamide amidase in intact neuroblastoma cells, two separate experiments were performed, one with 1 x 106 and one with 4 x 106 neuroblastoma cells (N18TG2)/6-cm dish. Experimental cells were incubated in 2 ml of media, consisting of Hams's F-12/Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with penicillin, streptomycin, and gentamicin plus 10% bovine calf serum (HyClone, Logan, UT), plus inhibitor for 20 min. [3H]Anandamide W.2 µCi of 221 Ci/mmol of [3H]anandamide) was added and the incubation continued for 1 h. Control cells contained no inhibitor. At the end of the incubation, the cells were washed once with cell culture

55940, Compound Pfizer [fo,2/3CR ),50<]-(-J-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(hydroxypropylcyclohexyl]phenol; PMSF, phenylmethylsulfonyl fluoride; HU-243, Hebrew University 11-hydroxy-hexahydrocannabinol-3-dimethylheptyl hor,1olog of .!l.9-tetrahydrocannabinol; CBR2, cannabinoid receptor 2.

22937


22938 Inhibitors of Anandamide Breakdown

media and removed from the plates, after a brief incubation with 2 ml of 0.05% trypsin in 0.53 mM EDTA solution at 37 °C. The amounts of [

3H]anandamide, [3H]phospholipids, and [3H]arach1donate in the cells and media were quantified by liquid scintillation counting of the silica scraped from the appropriate areas of the plate after quenching the reaction with chloroform:methanol (1:1), extraction of the sample from the organic phase, and TLC analysis on channeled silica gel-coated plates, with a solvent system consisting of the organic layer of an ethyl acetate:hexane:acetic acid:water (100:50:20:100) mixture.

Receptor Binding-For the CBRl ligand binding determinations, brain membranes were prepared from frozen rat brains (Pel-Freez) according to the procedure published by Devane et al. (16), with the 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 was performed by incubating the analogs at the indicated concentration with 30 µg of membrane protein in a buffer contaming 500 pM of the bicyclic cannabinoid analog [3H]CP-55940, 20 mM Tris-Cl, pH 7.4, 3 mM MgC12, 1 rm1 Tris-EDTA, and 0.135 mg/ml fatty acid-deficient bovine serum albumin in a final volume of 200 µ!in Regisil-treated glass tubes. Specific binding was defined as that which could be displaced by 100 mr desacetyllevonantradol. After 60 min at 30 °C, the incubation was terminated by the addition of 250 µl of 50 mg;lml bovine serum albumin and the immediate filtration over GF/B filters and washing with icecold buffer (20 mM Tris-Cl, pH 7.4, 2 mM MgC12 ). The filters were treated with 0.1 o/c sodium dodecyl sulfate pnor to addition of scintillation mixture and counting in a liquid scintillation counter.

0

A~NHCH2CH20H 1

2

3

4

1a. R = CH2(CH=CHCH2)<(CH2J,CH3 (anandamtde) 1 b. R = (CH2),4CH3 1 c· R = (CH2)12CH3 1 d R = (CH2liaGH3

2a: R = (CH:i)"CH3 2b R = (CH2)14CH3 2c R = (CH2)12CH3

3a R = (CH2)1sCHa 3b. R = (CH2),4CH3 3c. R = (GH2h2CH3

Fie. 1. Chemical structures of synthetic compounds. Four classes of compounds were synthesized: fatty acyl ethanolamides (1), a-keto ethanolamides (2), a-keto ethyl esters (3J, and trifluoromethyl ketones (4) as described under "Experimental Procedures." All were purified to homogeneity on silica gel and fully characterized by mass spectrometry and NMR ('H, 19F), and experimental details are available from G. D. P. on request. See Table I for nomenclature.

Amid~e H

N-./'oH

Anandamide

1l Hp Enz-SerOH

First Tetrahedral Intermediate

RESULTS AND DISCUSSION

The anandamide analogs prepared (Fig. 1) represent three classes of putative transition-state inhibitors: trifluoromethyl ketones (4), a-keto ester (3), and a-keto amide derivatives (2). The general strategy is based upon the hypothesis that polarized carbonyls (17), such as those in trifluoromethyl ketones and a-keto carboxylate derivatives, may form stabilized hydrates or enzyme adducts that mimic the tetrahedral intermediates formed during the reaction between the nucleophilic residue (e.g. the active-site serine hydroxyl of the hydrolytic enzyme) and the carbonyl group of anandamide (Fig. 2). Hence, several trifluoromethyl ketones have been identified as strong inhibitors of hydrolytic enzymes having varying specificity toward insect juvenile hormone esterase (18-21) and other hydrolases (22-24). A trifluoromethyl ketone analog of arachidonic acid ( 4a) was found to be a slow, tightly binding inhibitor of a novel 85-kDa cytosolic human phospholipase ~ (25, 26). Some a-keto acid derivatives have been shown to act as inhibitors of serine and cysteine proteinases (27-29), and effective inhibition of cathepsin B and papain by peptidyl a-keto esters, a-keto amides, a-diketones, and a-keto acids has also been demonstrated (30).

When tested for their ability to inhibit the hydrolysis of anandamide in vitro, the most effective class of compounds, as

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40 ~ w :;:

T ~ f- ' T z i UJ i 0 20' a:: UJ ! 0..

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ANANDAMIDE ANALOGS

Fm. 3. Inhibition of anandamide hydrolysis by synthetic compounds. Three hundred micrograms of rat brain homogenate protein was incubated with 7.5 µM inhibitor and 27.7 µM anandamide as described under "Experimental Procedures." Approximately 100 nmol of arachidonic acid/h/mg of protein was produced in the control containing no inhibitor. The blank was determined by incubation in the presence of 200 µM PMSF. In this representative experiment, the value for each inhibitor was determined in triplicate.

co,H

Ethanolamine Arachidonic Acid

CF3

HO OSerEnz

"Transition State" Analog Adduct

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 and an active-site serine residue (boxed).

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Inhibitors of Anandamide Breakdown 22939

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COMPOUND

FIG. 4. The effect of amidase inhibitors on anandamide levels in neuroblastoma cells (N18TG2). The amount of [3H]anandamide was determmed in the control and experimental cultures containing 1 x 106 cells. after separation by thin layer chromatography and analysis of the silica gel sample scraped from the plate, by liquid scintillation counting. One hundred percent anandamide in the control cells corresponds to 1.3'k of the total radioactivity detected in all the fractions analyzed as descnbed under "Experimental Procedures." The inset shows the effect of increasing arach1donyl trifluoromethyl ketone concentration upon [3H]anandamide levels. Each incubation contained 4 x 106 cells.

shown in this representative experiment, were the trifluoromethyl ketones (4a-4e) and a-keto esters (3a-3c) (Fig. 3J. Arachidonyl trifluoromethyl ketone (4a) and ethyl 2-oxostearate (3b) were the most active members of these groups yielding nearly 100% inhibition of the enzyme. The least potent inhibitors were the a-keto amides (2a-2cJ and the saturated analogs of anandamide (lb-Id). The curve of velocity versus substrate concentration for anandamide hydrolysis by the amidase in the brain homogenate was nonhyperbolic. This might be anticipated for an interfacial enzyme reaction, occurring in a crude homogenate, whose substrate and product have the potential to form micelles and to denature the membrane proteins. However, the inhibition of anandamide amidase by arachidonyl trifluoromethyl ketone was reversible with increasing concentrations of anandamide (data not shown).

When anandamide is incubated with neuroblastoma (N18TG2J cells, it is rapidly hydrolyzed to arachidonate, which is then converted to other lipids containing arachidonate (10). However, in the presence of arachidonyl trifluoromethyl ketone ( 4a) there is approximately 5-fold increase of anandamide levels (from 1.3% in the control cells to 6.3% in the experimental cells) at 7.8 µ11-1 arachidonyl trifluoromethyl ketone (Fig. 4). The amount of anandamide in the experimental cells increases to a 12-fold maximum, relative to the control cells, at approximately 12 µM arachidonyl trifluoromethyl ketone (Fig. 4, inset). The mechanism apparently involves inhibition of the amidase rather than increased uptake of anandamide, since preloading the cells with labeled anandamide and then treating with 4a also resulted in a dramatic increase in anandamide levels in the cells. Of the two other in vitro inhibitors that were tested, y-linolenic trifluoromethyl ketone (4b) was a weak inhibitor and ethyl 2-oxostearate (3b) was ineffective in cell culture. PMSF was also an effective inhibitor in the neuroblastoma cells. The disparity between the activity of 3b in vitro and in cell culture may be due to its susceptibility to enzymatic deg-

TABLE I Competitive znteractions of anandamide analogs with fHJCP-55940

binding to CBRI in rat brain membranes The CBRl ligand binding determinations were as described under

"Experimental Procedures." Fatty acid analogs were incubated with the ligand binding reaction mixture at final concentrations of 10 µM. Data are shown as specific binding of [3H]CP-55940, which was 100% in the presence of vehicle alone. The values are the mean and standard error of mean for three to five experiments for each compound.

Compound (10 )lM)

lb, stearoyl ethanolamide le, palmitoyl ethanolamide ld, myristoyl ethanolamide 2a, 2-oxoeicosanoyl ethanolamide 2b, 2-oxostearoyl ethanolamide 2c, 2-oxopalmitoyl ethanolamide 3a, ethyl 2-oxoeicosanoate 3b, ethyl 2-oxostearate 3c, ethyl 2-oxopalmitate 4a, arachidonyl trifluoromethyl ketone 4b, y-linolenyl trifluoromethyl ketone 4c, stearyl trifluoromethyl ketone 4d, palmityl trifluoromethyl ketone 4e, myristyl trifluoromethyl ketone

Percent CBRl specific binding

99.1±4.8 85.5 ± 8.5

102.1±21.4 102.6 ± 5.0 86.6 ± 9.5 80.6 ± 5.5 82.9 ± 18.0

102.7 ± 4.0 93.0 ± 10.7 21.4 ± 6.1 a

76.2 ± 14.4 96.3 ± 8.2 85.6 ± 7.3 94.6 ± 6.0

" Significantly different at 0.05 by ANOVA.

100

C> c 80 -6 c a;

J! 60 5

" Cl. 40 Cf)

Ci! CD u 20 Analog K, "'

nH • ethanolam1de 16 .9 • -CF 3 ketone 650 6

0 1 10 100 1000 10000

[Arachidonyl analog] (nM)

Fm. 5, -Log--dGSe-response -curve-for- arachidonyl--trifluoromethyl ketone and arachidonoyl ethanolamide competition with [3H]CP-55940 binding to CBRL The indicated concentrations of the arachidonyl trifluoromethyl ketone and arachidonoyl ethanolamide were mcubated as described under "Experimental Procedures." Binding competition experiments were performed a minimum of three times, and the data were analyzed by ANOVA and Tukey's post hoc test. Heterologous binding data were analyzed for IC50 and slope factor (nH)

values using the Graphpad Inplot program. K, values were calculated using the equation K, = IC5ofl + ([3H]CP-55940/Kd CP-55940) where the Ka for [3H]CP-55940 was 350 pM, as determined by Scatchard analysis using this assay. Each point on the curve represents the mean and standard error of mean of four or five experiments.

radation in cell culture. It is not known if arachidonyl trifluoromethyl ketone is metabolized in the neuroblastoma cells employed in this study, but when incubated at 10 µM with monocytic cells in culture for 10 min, 10% is converted to the corresponding alcohol (31).

The series of fatty acid derivatives were each tested, at 10 µM, for their ability to displace [3HJCP-55940 binding to the tetrahydrocannabinol receptor in rat brain membranes (CBRl). Arachidonyl trifluoromethyl ketone was the only synthetic compound that exhibited significant competition with [

3H]CP-55940 as shown in Table I. The competitive displacement of[3H]CP-55940 by arachidonyl trifluoromethyl ketone at various concentrations indicated that the K, was 0.65 µM, as shown in Fig. 5. This represents approximately a 15-40-fold decrease in potency from that originally reported for arachidonoyl ethanolamide (3) by displacement of the cannabinoid receptor probe [3H]HU-243 and that determined in the present assay.


22940 Inhibitors of Anandamide Breakdown

Ideally, a selective amidase inhibitor would antagonize the enzyme at concentrations that fail to appreciably bind to cannabinoid receptors. Furthermore, unlike PMSF, an inhibitor should not be toxic to the cells. Many of the synthetic compounds in this study fulfill this criteria in that they do not bind significantly to CBRl at concentrations that inhibit amidase activity by greater than 90% in cell-free preparations. Arachidonyl trifluoromethyl ketone is very interesting because it exhibits dual effects on the degradative enzyme and at the receptor in cell culture, where it may be a useful stable analog of arachidonoyl ethanolamide. The role that these inhibitors play in different tissues, such as spleen, where a peripheral receptor (CBR2) exists (32) or as inhibitors of the cytosolic phospholipase Ai (31) in brain and N18TG2 cells remains to be elucidated. Our approach provides the framework to select candidate drugs for animal studies. The development of inhibitors that block the breakdown of anandamide may be significant therapeutically in any of the areas that ll9-tetrahydrocannabinol (33) and anandamide (34, 35) has been shown to play a role, including analgesia, mood, nausea, memory, appetite, sedation, locomotion, glaucoma, and immune function.

Acknowledgments-We thank Keith Baker and Cathy Cantrell for technical assistance with the CBRl radioligand binding assays and Rebecca Rowehl for the cell culture.

REFERENCES

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2. Matsuda, L A., Lolait, S. J., Brownstem, M. J., Young, A. C • and Bonner, T. I. (1990) Nature 346, 561-564

3. Devane, W. A., Hanus, L., Breuer, A., Pertwee, R. G., Stevenson, L.A., Griffin. G., Gibson, D., Mandelbaum, A, Etinger, A., and Mechoulam, R. (1992) Science 258, 1946-1949

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5. Felder, C. F., Briley, E. M.,Axelrod,J., Simpson, J. T., Mackie, K.. and Devane, W. A. (1993) Proc Natl. Acad. Sci U.S. A. 90, 7656-7660

6 Devane, W. A. ( 1994) Trends Pharmacol. Sci. 15, 40-42

7. Vogel, Z., Barg, J., Levy, R., Saya, D, Heldman, E., and Mechoulam, R. (1993) J. Neurochem. 61, 353-355

8. Childers, S. R., Sexton, T., and Roy, M. B. (1994) Biochem. Pharmacol. 47, 711-715

9. Mackie, K., Devane, W. A., and Tulle, B. (1993) Mal. Pharmacol. 44, 498-503 10. Deutsch, D. G., and Chin, S. A. (1993) Bwchem. Pharmacol. 46, 791-796 11. Elie!, E. L .. and Hartmann, A. A. (19721 J Org. Chem. 37, 505-506 12. Sollad1e, G. and Ziam-Cherif, C. (1993) J. Org. Chem. 58, 2181-2185 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.

Synth 61, 1-4 15. Boivin, J., El Krum, L., and Zard, S. Z. (1992) Tetrahedron Lett. 33, 1285-1288 16. Devane, W. A., Dysarz, F. A., Johnson, M. R., Melvin, L. S., and Howlett, A. C.

l1988) Mal Pharmacol. 34, 605--B13 17. Mehdi, S. (1993) Bioorg. Chem. 21, 249-259 18. Hammock, B. D., Wing, K. D., McLaughlin. J • Lovell, V. M , and Sparks. T. C.

(1982) Pestic. Bwchem. Physiol. 17, 76-88 19 Prestwich, G. D., Eng, W S., Roe, R. M., and Hammock, B. D (1984) Arch.

Bwchem. Biophys. 228, 639-645 20. Szekacs, A., Halarnkar, P. P., Olmstead. M. M , Prag, K A , and Hammock, B.

D (1990) Chem. Res. Toxicol. 3, 325-332 21. Shiotsuki, T., Huang. T. L , Uematus, T .• Bonning, B C , Ward, V. K.. and

Hammock, B. D. (1994) Protein Exp. Punf. 5, 296-306 22 Gelb, M. H .. Svaren. J. P., and Abeles, R. H., (1985) Bwchemistry 24, 1813-

1817 23 Allen K. N., and Abeles, R.H. (19891 Biochemistry 28, 8466-8477 24 Brady, K .. Liang, T. C .. and Abeles, R. H. (1989) Biochemistry 28, 9066-9070 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) Biochemistry 32, 5935-5940

26. Trrmble, L. A, Street, I. P, Perrier, H., Tremblay, N. M., Weech, P. K., and Bernstein, M. A. (1993) Bwchemistry 32, 12560-12565

27. Angelastro. M. R. Mehdi, S., Burkhart, J.P., Peet, N. P, and Bey, P. 11990) J. Med. Chem. 33, 11-13

28. Peet, N. P., Burkhart, J P., Angelastro, M. R., Giroux, E. L , Mehdi, S., Bey, P., Kolb, M .. Neises, B., and Schirlin, D. (1990) J Med. Chem. 33, 394-407

29 Ocain, T D., and Rich, D. H. (1992) J. Med. Chem. 35, 451-456 30 Hu. L. Y., and Abeles, R. H. l1990) Arch. Biochem. Biophys. 281, 271-274 31. Riendeau, D., Guay, J .. Weech. P. K., Laliberte, F., Yergey, C. I., Desmarais. S ..

Perrier, H .. Lm, S., Nicoll-Griffith, D., and Street, I. P. (1994) J. Biol. Chem. 269, 15619-15624

32. Munro. S., Thomas, K. L., and AbuShaar. M. '19931 Nature 365, 61-65 33. Mechoulam. R (1986) Cannabmoids as Therapeutic Agents, CRC Press, Boca

Raton, FL 34. Fride, E .. and Mechoulam, R. (1993) Eur. J. Pharmacol. 231, 313-314 35. Crawley, J. N., Corwm, R. L., Robinson, J. K., Felder, C. C , Devane. W. A., and

Axelrod, J. (1993) Pharmacol. Bwchem Behav. 46, 967-972

BEST AVAILABLE COPY

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- - - - - - - - - - - - - - - - -Eur. J. E111omol. 91: 197-203, 1994

ISSN 1210-5759

Sex pheromone characterisation and field trapping of the European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae), in South Moravia and Slovakia

BLANKA KALINOV A 1, ALFRED MINAIF 2 and LADISLAV KOTERA 1

1 Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Insect Chemical Ecology Unit, Flemingovo nam. ·z, 166 10 Praha 6, Czech Republic

2 Institute of Organic Chemistry, Universi:ty of Erlangen-Niirnberg, Henkestrasse 42, 8520 Erlangen, Federal Republic of Germany

Pheromone, Ostrinia 11ubilalis, trapping

Abstract. The analysis of pheromone glands from individual females of the European corn borer, Ostrinia nubilalis (Hilbner), originating 111 South Moravia and Slo

1

vakia showed that this population utilizes the "Z" pheromone system. The ratio of (Z)- and (E)-11-tetradecenyl acetates was found in the range of 98.5: 1.5-99.5: 0.5. Field experiments confirmed the identity of the local population as being predominantly of the Z strain. Individuals responding to E and hybrid blends were detected.

INTRODUCTION

The sex pheromone of the European corn borer, Ostrinia 1111bilalis (Lepidoptera: Pyralidae), has been identified as a mixture of geometric i~omers of 11-tetradecenyl acetate. The proportion of the isomers has been found to vary a;:cording to the geographical origin of the moths. In North America and Europe, 0. 1111bilalis occurs as a mosaic of populations with two distinct sex pheromone systems. The so-called Z strain has a 97: 3 blend of (Z)-1 I- and (E)-11-tetradecenyl acetates (abbrev. Zll l-14:Ac and El l-14:Ac) (Klun et al., 1973). The E strain has a 4: 96 blend of Z 11 and EI l-14:Ac (Kochansky et al., 1975). The Z strain is widely distributed throughout the world, whereas the E strain occurs mainly in Italy, Switz. -'~nct and northeastern United States (Klun et al., 1975; Anglade et al., 1984). In the areas vf S] ,q,atry, hybrids using an intermediate blend of the Z and E isomers in a ratio 35:65 (Klun & Maini, 1979; Anglade et al., 1984) were detected. A preliminary investigation of 0. 1111bilalis populations in former Ozechoslovakia indicated that the corn borers in this area probably belong to the Z strain. The attempts to use a synthetic pheromone for monitoring, however, were not efficient enough (Hrdy et aL, 1986). In order to characterize a possible "pheromone dialect" of the. local 0. nuhilalis population, female pheromone glands were chemically analyzed and males' ability to discriminate pheromone composition was investigated in field experiments.

MATERIAL AND METHODS

Insects: Experiments were performed on a laboratory colony of Ostrinia nubilalis (Hubner) established from individuals collected from corn fields as diapausing larvae. Further generations were reared on a semi-synthetic diet (Nagy. 1961) under a LD 16: 8. The pheromone glands were analyzed during the scotophasc in 3-day old virgin females, which are known to produce maximal amounts of pheromone (Borek & Kalinovri, 1991 ). Males 2 to 3 days old were used for electroantennographic (EAG) recordings.

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1

Pheromone analysis: The pheromone was analyzed by means of a solid-sample 'injection technique i(Attygale et al., 1987) enabling gland analysis without solvent extraction. Capillary gas chromatography ;(GC), mass spectrometry (MS) and electroantennography (EAG) were combined during analysis.

Gland preparation: The VIII and IX abdominal segments of a calling female were detached. The intersegmental membrane was encapsulated in a thm-walled soda glass capillary (2 x 20 mm). The samples were either used immediately or stored at -20°C until use. The active pheromone glands were compared with the glands of decapitated females (24 hrs old), in which pheromone production is known to be suppressed due to the absence of a brain-released pheromonotropic hormone (Raina & Mer1n, 1987).

Gas chromatography: Capillary gas chromatography with flame ionisation detection (FID) was performed on a Hewlett Packard 5890A instrument equipped either with a SP 2340 or SE 52 polar columns (25 m x 0.22 mm i.d., Chrompack). The injector space was modified to adopt a solid sample injector (Atty gale et al., 1987). Solid samples were injected splitless. N2 supplied at a 2ml/min linear flow was used as a carrier gas. The temperature regime was set at 60'C to 195'C, at a rate of 4°C/min. The injector and detector temperatures were 220°C and 250'C, respectively.

Gas chromatography-mass spectrometry (GC-MS): Mass spectra of compounds in pheromone glands were obtained on a VARIAN 3400 gas chromatograph linked to a FINNINGAN MAT 90 mass spectrometr. In this system a SP 2340 and/or SE 52 column was direc,Iy coupled to the mass spectrometr. The ,oltd-sample injection and temperature regime were the same as described above. Mass spectra were compared to those of authentic chemicals.

Oas chromatography with electroantennographic detection (GC-EAD): To obtain precise information about the retention times of gland compounds with olfactory activity, the EAG setup was directly coupled to the gas chromatograph. The antenna served as an additional (biological) GC detector

1(Arn et al., 1975).

The outlet of the capillary column used for GC analysts was split into two parts (split ratio 1 : I), one of which was connected to the FID detector, and the other of which led out of the oven into an mrstream continuously blowing over the antenna! preparation. Isolated antennae were used. Antenna! responses were recorded by Ag!AgCI glass electrodes (Roelofs, 1984), amplified and registered simultaneously with the response of the FID detector.

Field experiments: Field experiments were performed in South Moravia (Velke B\lovice, Stnifoice) and Slovakia (Nitra) m 1991 and 1992. Four replicated treatments were made at each locality. Traps were placed in smgle row along the edge of corn field, spaced 40 m apart at a height of 1 m. Traps were periodically rotated within each test sencs to minimize the possible position effect on trap efficiency. Captured moths were removed and recorded daily. The effectiveness of lure composition, dose, trap design (deltaand wmg-type) and dispenser type was examined. Traps baited with a synthetic pheromone were compared lo those baited with 3 virgm females (2 to 4 days old) placed in a small wire cag,e hung inside the trap. Females were provided with water, checked daily and regularly changed.

Dispenser preparations: Chemically pure(> 99%) Zand El l-14:Ac and 14:Ac were.dissolved in hexane and GC analyzed, to ensure that no traces of attraction inhibitors (Z)-9-dodecenyl acetate or (Z)-9-tctradecenyl acetate (Struble et al., 1987) were present. Compounds were applied topically to dispensers Ill creating the various pheromone blends. After solvent evaporation, the dispensers were tightly scaled in aluminium-polyethylene foil and stored under -20°C until use. Three types of rubber and four types of polyethylene were tested as pheromone dispensers.

Data analysis: Analysis of variance (ANOVA) and Duncan's multiple range test (PT 0.05) were conducted to determine the influence of trap design, dispenser type, lure composition and pheromone dose on moth captures.

RESULTS

GC-MS: Complete mass spectra were obtained for the following components of the female glands: 14:Ac, ZI l-14:Ac, El l-14:Ac, ZI l-14:0H, methyl esters of hexadecanoic and octadecanoic acid. Of the hydrocarbons, saturated and unsaturated

1

C22-27 were identified. All identified mass spectra were compared with those of standar1s. Compounds 14:Ac, ZI l-14:Ac, El I-14:Ac were not present in inactive pheromone glandls. The relative abundance of l 4:Ac : Z l l -14:Ac : El 1- l 4:Ac was 14 : 100 : 1. In 10 analyzed females the

- - - - - - - - - -Fig. I. GC-FID-EAD chromatogram ob

tained from active pheromone gland of female Ostrinia 11ubilalis on a 25 m x 0.22 mm fused silica capillary column SP-2340. Oven temperature was held at 60°C for 2 min and in-creased to I 95'C at 4'C/min. 14:Ac · tetradecyl acetate, (E)-1 l-14:Ac (E)-11-tetradecenyl acetate, (Z)-1 l-14:Ac -(Z)-11-tetradecenyl acetate, (Z)-ll-14:0H -(Z)-11-tetradecenyl alcohol, C23 - tricosan, C25 - pentacosan, C27 - heptacosan. A: GCFID responses B: GC-EAD responses to 14:Ac, (E)-1 l-14:Ac and (Z)-11-14:Ac.

A

N

" z " 0 .. '"' .. "' .. " ~ '"' '

u.., .. N ;u ~

' ~ .... "' ,, ... 9.u . ~ ...

N

~ ()~--

' 0 I 0 2 0 JO •D

TIME (•in)

Z:E ratio was determined to be in the range 98.5 : 1.5-99.5 : 0.5. Gland samples contained 3-5 ng of Zl 1-14:Ac per female.

GC-EAD: Reproducible responses to both Zand E isomers of ll-14:Ac were observed in all GC-EAD experiments (Fig. lB). In several experiments antennae also responded to 14:Ac. No other BAD-active materials were detected in pheromone glands.

Fig. 2. Mean number of males of 0. nubilalis caught by wing traps baited with 3 virgin females and JOO µg of various pheromone blends, loaded on red rubber septa. Stnifoice, South Moravia, July 4-18, 1992. Columns signed by the same letter are not significantly different at P = 0.05.

200 I

0.. g Cii 0..150 :E OJ ::i rel u (/) Gl

<ii 100 E 0 Cii .a

5 50 c c rel Gl E

N u ;fl.

<( ... 0 -0 - +

N ;fl. 0 0

a

~ ~I

b ~~"l ~-....:\\

~~~ c ~~1 Ll.J u Ll.J Ll.J Ll.J - «;

"' "' I'-... "' O>

N - N N O> + I'- N

O> Ll.J O> "' "' "' "' -N O> O>

lure composition

-

b:J

~ .... h. § ~ h tlJ !""--r"l't (') a "tJ ""<

- - - - - - - - - -100~~~~~~~~~~~~

0 ID

1601 a I

"@ 60 ::J c:

be

bed

ede

Field experiments: The initial tests involved various lure compositions: 3 virgin females; 100% Z (100 µg); 99Z:IE (100 µg); 97Z:3E (100 µg); 99Z:lE (100 µg) + 10 µg 14:Ac; 35Z:65E (100 µg) and 3Z:97E (100 µg) loaded on red rubber septa (Struble et al., 1987). Male moths were most attracted to the "Z" blend and/or to virgin females (laboratory "Z" strain) (Fig. 2). The 99Z: IE blend trapped substantially fewer males than the 97Z:3E blend. The addition of 14:Ac had little effect in attracting moths to the 99: 1 blend. The local population responded par-tially to pure Z isomer. In 1992 12 "E" and 2 fij 40

<ll

·~1~ "hybrid" males were caught in Strafoice and E

2 "hybrid" males in Nitra (Fig. 2). The dose 20

of I 0 µg of the "Z" blend was the most effective, followed by 50, 100, and 500 µg (Fig. 3). The effectiveness of all doses, compared to that of virgin females, decreased gradually during experimentation, probably due to a

o·-10 50 100 500

pheromone dose [µg]

0 1st week 0 2nd week

000 f j f

Fig. 3. Mean number of males of 0. nubilalis caught by wing traps baited with 3 virgin females and by IO, 50, 100 and 500 µg of "Z" blend, using red rubber septa. Note the differences in moth catches of the !st and 2nd week

slow yheromone degradation. Male captures in traps baited with virgin females did not decline during the experimental period, indicating an unchanged population density. The wing-type trap appeared to be more effective than the delta-type (Fig. 4). In all of the ex- indicating pheromone degradation. Stnifoice, periments mentioned above, red rubber septa South Moravia, July 4-18, I 992. (A.H. Thomas Co., Catalog No. 1780-107) were used as pheromone releasers. The numbers of males caught in traps baited with the "Z" pheromone blend, loaded on other materials were substantially lower (Fig. 5).

DISCUSSION

The results of this study generally confirm previous reports of the presence of the Z strain of 0. m1bi!alis in the area. However, several "E" and "hybrid" individuals were detected.

In Europe, a survey by Klun et al. (1975) revealed the prevalence of the Z pheromone strain of the European corn borers. Analyses and field tests by Maini et al. (1978), Bi.ichi at al. (1982), Anglade et al. (1984), Barbattini et al. (1985) and Pena et al. (1988) confinned that the Z, E and hY,brid strains are simultaneously present south of the Alps. The presence of the E strain in the northern areas was reported only in northern Germany (Langenbruch et al., 1985). This sporadic occurrence of individuals of the E and hybrid· corn borers raises the question, whether the both strains are inherently present in some field population or if this isolated presence of E and hybrid moths could be explained otherwbe, for instance by the migration from southern localities .

.. ~~ --

- - - - -Fig. 4 (right). Mean number of males of 0.

nubilalis caught by delta- and wing-type traps baited with 100 µg of "Z" blend on red rubber septa. Velke Bfiovice, July 1-14, 1991.

Fig. 5 (below). Mean number of males of 0. nubilalis caught by wing-traps baited with I 00 µg of "Z" blend loaded on various dispensers: I - red rubber (A.H. Thomas Co., Cat. No. I 780-J07); 2 - "brown" rubber; 3 - red polyethylene; 4 - white polyethylene; 5 - transparent polyethylene I; 6 - transparent polyethylene II; 7 - "grey" rubber; 'i' 'i' 'i' - 3 virgin females. A- Velke Bfiovice, 1991; B -Strafoice, 1992.

80

a A

0..

I~ jg

.::::

I~ I Cl :i m ()

Ci ID c

..CJ E :i c: c 20 m <ll E

0 2 3 4 5 000

+ ++ dispenser type

- - - - -so~~~~~~~~~~~--,

~50 Qi a..

1: 40 Ol :::J ro u Cl) (lJ

iii 30 E 0 Qi

..Q

E 20 :::J c c ro (lJ

E 10

oL.._~--"-'"~~~~~-""'~'----'

wing trap delta trap

250

I a 0..

jg 200

ID 0..

.:::: Cl :i ~ 150 Cl) <ll "iii E 0 ~ 100 Q)

..CJ E ::J c: c: m

50 Q)

E

0 1 6 7 000

+ + + dispenser type

-

-

~ '-.:..,,..

- - - - - - - - -The present study did not prove the pheromone dialect of the local Z population. Although the isomer ratio found in pheromone glands slightly differs from those reported in other studies, 97: 3 Z: E ratio was the most efficient in the field experiments. Besides the main acetates, the 14:Ac was considered as an additional pheromone component. However, field experiments did not support this possibility.

The presence of C23-C25 hydrocarbons in inactive pheromone glands and their low volatility seem to exclude the possibility that they could function as pheromones. However, in Orgyia leucostigma (Lepidoptera: Lymantriidae) alkanes, tricosane, tetracosane, pentacosane and heptacosane present in the female scales were found to improve copulation attemps and to serve as copulation behavior releasing pheromones, in spite of their low EAG activity (Grant at al., 1987). Wheather alkanes identified in female pheromone glands in 0. nubilalis have a similar behavioral function remains to be seen.

The fact that trap design, pheromone dose and isomer ratio affect substantially the trapping efficiency has been repeatedly reported (McLeod & Starratt, 1978; Kennedy & Anderson, 1980). Similarly, for some moth species and pheromone systems, the type of dispenser used for pheromone release was found to be the most critical factor for maintaining the pheromone activity (Horak et al., 1989; Hrdy et al., 1986). Needed pheromone dispenser requirements are: pheromone stability, constant pheromone release and evaporation rate comparable to those produced by calling females. 0. nubilalis appeared to be very sensg;ve to dispenser type. The reason for generaly low trap attractancy observed when other than red rubber dispenser were used is not clear. It could be caused by pheromone degradation. The autoxidation of the pheromone in the dispenser may lead to the formation of an inhibitory or repellent substance(s) (Starratt & McLeod, 1976) influencing the male behavior. These processes were perhaps responsible for our earlier, unsatisfactory results in trapping 0. nubi/alis, even when synthetic pheromone of high purity was used and the dispensers were prepared just prior to field use, as recommended by Struble et al. (1987).

ACKNOWLEDGEMENT. This research was supported m part under grant No. DHR-5600-6-00-1051-00, of the Program in Science and Technology Cooperation, of the Office of the Science Advisor, U.S. Agency for International Development.

REFERENCES

ANGLADE P., STOCKEL J. & I W.G.O. COOPERATORS 1984: Intraspecif1c sex-pheromone variability in the European Corn Borer, Ostrinia nubilalis Hbn. (Lepidoptera: Pyralidae). Agronomie 4: 183-187.

ARN H., STADLER E. & RAUSCHER S. 1975: The electroantennographic detector - a selective and sensitive tool in the gas chromatographic analysis of insect pheromones. Z. Natwforsclz. 30: 722-725.

ARN H., T6TH M. & PREISNER E. 1986: List of Sex Pheromones of Lepidoptera and Related Attractants. OILB-SROP, Secretariat General, Paris, 123 pp.

ATTYGALE A.B., HERRIG M .. VoSTROWSKY 0. & BESTMANN H.J. 1987: Technique for injecting intact glands for analysis of sex pheromones of Lepidoptera by capillary gas chromatography: Reinvestigation of pheromone complex of Mamestra brassicae. J. Chem. Ecol. 13: 1299-1311.

BARBA'ITINI R., MARCHETTI S., PR11v1s11N1 L. & ZilNDIGIACOMO P. 1985: Attrazione dio fermoni sessuali di smtesi nei confronti di Ostrinia nubilalis Hb. in Friuh. F1ustu/a Entomol. 7: 1-21.

BOREK V. & KALINOV A B. 1991: Effect of the age, photoperiodic regime and host plant on sex pheromone titre of the European Corn Borer, Ostrinia nubilalis (Lepidoptera, Pyralidae). In Hrdy I. (ed.): Proc. Conj Insect Chem. Ecol., Tabor. Academia, Prague and SPB Acad. Pub!., Hague, pp. 295-299.

- - - - - - - - - -BUcH1 R., PREISNER E. & BRUNETTI R. 1982: Das sympatrische Vorkommen von zwei Pheromonstiimmen

des Maisziinslers, O:strinia nubilalis Hbn, in der Stidschweiz. Mitt. Schweiz. Entomol. Ges. 55: 33-53 ..

-GRANT G.G., FRECH D., MAC DONALD L., SLEssoR K.N. & KtNG G.G.S. 1987: Copulation releaser pheromone

in body scales of female Orgyia leucostigma (Lepidoptera: Lymantriidae). J. Chem. Ecol. 13: 345-357. HoRAK A., HRDY I., KONECNY K. & VRKOC J. 1989: Effect of substrate formulation on the efficacity of the

pea moth, Cydia nigricana, sex pheromone lures. Entomol. Exp. Appl. 53: 125-131. HRDY I., HuBAJSHAN M.:A., MAREK J., PosPECH L., PovoLNY D., VALLO V. & ZMREK J. 1986: Prvnf zkusenos

ti s monitorovanfm zavfJece kukui'icneho, Ostrinia nubilalis, v Ceskoslovensku feromonovymi lapriky (First experience with the monitoring of the European Corn Borer, Ostrinia nubilalis, with pheromone traps in Czechoslovakia.) Sbor UVT!Z- Ochr. Rost/. 22(2): 129-140 (in Czech, English abstr.).

HRDY I., KoNEcNY K. & VRKOc J. 1986: Vliv formulace na ticinnost kodlemonu pro monitorovanf obalece jablecneho, Cydia pomonella. (The effect of formulation on the effectiveness of codlemone for monitoring codling moth, Cydia pomonella). Shor. UVTIZ - Ochr. Rost!. 22(1): 33-52 (in Czech, English abstr.).

KENNEDY G.G. & ANDERSON T.E. 1980: European corn borer trapping in North Carolina with various sex pheromone component blends. J. Econ. Entomol. 73: 642-646.

KLUN J .A. & MAIN! S. 1979: Genetic basis of an insect chemical communication system: the European Corn Borer. Environ. Entomol. 8: 423-426.

KLUN J.A., CHAPMAN O.L., MATTES K.C., WoJTKOWSKI P.W., BEROZA M. & SoNNET P.E. 1973: Insect pheromones: minor amount of opposite geometrical isomer critical to attraction. Science 181: 661-663.

KLUN J.A., ANGLADE P.L., BACA F., CHAPMAN O.L., CHIANG H.C., DANIELSON D.M., FELS P., HlLL R.E. ET AL. 1975: Insect sex pheromones: intraspecific pheromonal variability of Ostrinia nubilalis in North America and Europe. Environ. Entomol. 4: 891-894.

KoCl·IANSKY J., CARDE R.T., LIEBHERR J. & ROELOFS W.L. 1975: Sex pheromone of European Corn Borer, Ostrinia nub1lalis (Lepidoptera: Pyraliade), m New York. J. Chem. Ecol. 1: 225-231.

L11NGENBRUCH G.A., WELLING M. & HosANG B. 1985: Untersuchungen iiber den Maisziinsler im Rughrgebiet. NachrBl. Dt. Pj7Schutzdienst. Braun.1chw. 37: 150-156.

MAIN! S., PALLOTTI G. & PLATIA G. 1978: Ricerche sull'identificazione de! feromone sessualle in popolax-1om bolognesi de Ostrinia nubilalis Hb. (Lepidoptera: Pyralidae) e relative prove di campo. Boll. !st. E111omol. Univ. Bologna 34: 15-25.

McLEOD D.G.R. & STARRATT A.N. 1978: Some factors influencing pheromone trap catches of the European corn borer, Ostrinia nubilalis (Lep1doptera: Pyralidae). Can. Entomol. 110: 51-55.

NAGY B. 1961: Rearing of the European Corn Borer, Ostrinia nubilalis, (Hbn.) on a simplified artificial diet. Acta Phytopath. Acad. Sci. Hung. 5: 73-79.

PENA A, AKN H., BusER H.R., RAUSCHER S., BIGLER F., BRUNETTI R., MAIN! S. & Torn M. 1988: Sex pheromone of European Corn Borer, Ostrinia nub1lalis: Polymorphism in various laboratory and field strains. J. Chem. Ecol. 14: 1359-1366.

RAINA A.K. & MENN J.J. 1987: Endocrine regulation of pheromone production in Lepidoptera. In Prestwich G.D. & Blomquist G.J. (eds): Pheromone Biochemistry. Academic Press, Orlando, pp. 159-175.

ROELOFS W.L. 1984: Electroantennogram assays. rapid and screening convenient procedures for pheromones. In Hummel H.E. & Miller T. (eds): Techniques in Pheromone Research. Springer-Verlag New York Inc., New York, pp. 131-161.

S111RRATT AN. & McLEOD D.G.R. 1976: Influence of pheromone trap age on capture of the European corn borer. Em,iron Entomol. 5: !008-1010.

STRUBLE D.L., BYERS J.R., McLEOD D.G.R. & AYRE G.L. 1987: Sex pheromone components of an Alberta population of European corn borer, Ostrinia nubilahs (Hbn.) (Lepidoptera: Pyralidae). Can. Entomol. 119: 291-299.

Received May 25, 1992; accepted April 30, 1993

BEST AVAILABLE COPY

- - - - - - - - - -

'">C~T _/l,l/lffJ'l.Ol.F COPY

\_

~_;; ,,;?- -

- - - - - -Short Communication

SYNTHESIS OF (Z)-14-HEPTADECEN-4-0LIDE AND {Z)-11-PENTADECEN-4-0LIDE,

- - -1211

SEX PHEROMONE ANALOGUES OF Ostrinia nubilalis AND Cydia molesta

Michal HOSKOVEC, David SAMAN and Bohumfr KoUTEK

Institute of Organic Chemistry and Biochemistry, Academy (~f Sciences of the Czech Republic,166 JO Prague 6, The Czech Republic

Rece1 ved August 25, 1993 Accepted October 26, 1993

The key step in the preparation of racemic (Z)-14-heptadecen-4-olide (Via) and (Z)-11-pentadecen-4-olidc (Vlb), sex pheromone analogues of Ostrinia 1111/Jilalis and Cydia 1110/esta, was efficient crosscoupling rcact10n of 3-methoxycarbonylpropanoyl chloride with corresponding (Z)-alkcnylmagncs1um bromides. The methyl 4-oxo-(Z)-14-heptadecenoate (Va) and methyl 4-oxo-(Z)-11-pcntadccenoate (Vh) prepared in this way were converted by one-pot reaction using sodium borohydride in an ethanolic solution to the required (Z)-alkcn-4-olidcs (Via, Vlh).

In the search for new biorational pesticides based on semiochemicals, we focused our attention to a synthesis of butyrolactone analogues of the European corn borer (Ostrinia nubilalis) and the Oriental fruit moth (C)dia 1110/esta) sex pheromones 1

•

Five-membered enol lactones bearing a halogen at the vinylic position are known" to be mechanism-based inhibitors (suicide inhibitors) of serine hydrolases. Similarly, ynenol lactones are featured as suicide inhibitors of serine proteinase. We expect that due to their alkylating properties even the more simple butyrolactones could exhibit similar inhibition effects. In our case the acetate group of the parent pheromone molecules, (Z)-1 1-teL. _, · '"'n-1-yl acetate ( Ostrinia nu bi la/is) and (Z)-8-dodecen-1-yl acetate (Cydia 1110/es;a), was replaced by a five membered lactone moiety.

The key moment of the synthesis (Scheme I) of the target compounds, (Z)-14-heptadecen-4-ol ide (Via) and (Z)-11-pentadecen-4-olide (Vlb), is a preparation of the 4-oxoesters Va and Vb. For the synthesis of these compounds we tested a ''classical" method, based on the two-step alkylation or diethyl 3-oxopentanedioate (3-oxoglutarate)H. Unfortunately, this way is relatively complicated (4 step~) and the total yidd of the synthesis was not higher than 30 - 40%. Therefore, the synthesis of 4-oxoesters Va and Vh was performed by applying the tris(2.4-pentadionato-O. O')iron(Ill) (Fe(acac)_,) catalyzed cross-coupling reaction5.<' between acyl chlorides and Grignard reagents. This method has been successfully applied5·6 to the synthesis of some func-

-

- ·- - - - -·- - - -1212 Hoskovec, Saman, Koutek:

tionalized ketones and oxo esters which are not easilly prepared by other known procedures.

The 1

isolated yield of the coupling of 3-methoxycarbonylpropanoyl chloride with the corresponding (Z)-alkenylmagnesium bromides is about 40%. This yield is lower than described (70 - 85%) in original papers5•6, where only short-chain and/or reactive

Br-( CH2)n -OCH(Me )OE t

Ia, lb

1. LiC;; CH , NH3(1)

2. Li, NHJ(I)

3. RBr, THF

4. Dowex 5DW(H+), MeDH

R-c=c- (CH2)n-OH

Ila, Ilb

j ~ P2~ R> < (CH2),.0H H H

Illa, lllb

j re'• "'

R > < (CH2)nBr H H

!Va, !Vb

Sc11hME I

.. ~

!Va, !Vb

1. Mg, THF

2. CICO(CH2)iCOOMe,

Fe( ococ }J. THF

R> < (CH2)nCO(CH2)2COOMe H H

Va, Vb

11. NaBH4, EtOH

2. 103 oq. NoOH

3. HCI

R O 0 > < ( CH2),. -(' - "y=-H H \_}

Via, Vlb

In formulae I - VI :

a, R = ethyl, n = 9

b, R = propyl, n = 6

- - - - - - - - - - -Short Communication 1213

Grignard reagents were used. The coupling of Grignard reagents, prepared from lbromo-(Z)-10-tridecene (/Va) and l-bromo-(Z)-7-undecene (/Vb), gave relatively low yields, however this one-step way is more advantageous than the four-step synthesis via diethyl 3-oxopentanedioate.

The preparation of l-bromo-(Z)-10-tridecene (/Va) and 1-bromo-(Z)-7-undecene (!Vb) is based on alkyne c:hemistry7 - 10. Alkynols /Ja and Ilb were prepared from protected ffi-bromoalkanols 11 Ia and lb by a two-step alkylation of acetylene. Hydrogenation of alkynols Ila and l/b over P2-Ni catalyst 12 gave (Z)-alkenols JI/a and Illb in isomeric purity >98% (GLC). Treatment of (Z)-enols Illa and Jllb with carbon tetrabromide and triphenylphosphine 13 furnished the corresponding 1-bromoalkenes !Va and !Vb in high yields.

In the final step of the s~rnthesis the 4-oxoalkenoates Va and Vb were converted to the racemic target lactones Via and V!b by a reduction using an ethanolic solution of sodium borohydride 14• The overall yields of the synthesis were 11 % for lactone Via and 12% for lactone Vlb.

EXPERIMENTAL

1H and 13C NMR spectra were determined in CDCI3 solution on Varian UNITY-500 spectrometer, operating at 499.5 MHz and absorptions are expressed m o (ppm) scale relative to TMS. The NMR data of all synthesized compounds are given in Tables I - IV. The IR spectra (v, in cm-1) were recorded on a Bruker IFS 88 FT-IR 5pectrometer in tetrachloromethane. GLC analyses were performed on a Hewlett-Packard HP 5880A chromatograph equipped with a FID detector and a 25 m capillary column (internal diameter 0.3 mm, HP5-5% phenyl methyl silicone, cross-linked). Preparative medium pressure liquid chromatography (pMPLC) separations were made on Merck 60 siltca gel (0.040 - 0.063 mm) using a Biichi B-680 Prep LC System with stepwise gradient of diethyl ether in hght petroleum.

10-Tridecyn-l-ol (Ila)

Into a 'tirred 'uspension of freshly prepared lithium acetylide7 (0.3 mo!) in liquid ammonia (I 000 ml) dry DMSO (400 ml) wa> added carefully. After stirring for 5 min, 14-bromo-4-methyl-3,5-dioxatetradecane9·10 (la) (75 g, 0.254 mol) was added dropwise and stirring was continued for 4 h. Ammonia was evaporated on standing overnight and the residue wao decomposed with brine (1 500 ml). The mixture was extracted with ether-light petroleum (I : I) (4 x 250 ml) and the combined extracts were dried over K2C03• Evaporation of solvents gave 45 g of a yellow oil. This oil (45 g) was added dropwise to a 'tirred 'uspcnsion of LiNH 2 (prepared from 13.9 g, 0.2 mol of lithium) in I 000 ml of liquid ammonia. After 'lirring for I h, 1-bromoethane (21.8 g. 0.2 mol) was added . · '-iwisc and stirring was contmued for 4 h. Ammonia wm, evaporated on standing overnight and the .u .. uJ,, was decomposed with ice cold water (I 000 ml). The mixture wm, extracted with ether (4 x 250 ml). the combined extracts were dried over K2C03• Evaporation of the solvents fur-1fr,hcd 40 g or a red oil that wa.o, dis,olvcd in methanol (I 000 ml) and treated with Dowex SOW (1-J+ form, 20 gl for 20 h. The ion-exchanger was filtered o!T and the solvents were removed tn

vacuo. Purir1cation or the rc~iduc by pMPLC gave 32.6 g (63%) of alkynol Ila.

- - - - - - - - - -1214 Haskovec, Saman, Koutek:

7-Undecyn-1-ol (Ilb) was synthesized analogously from I 1-bromo-3,5-dioxa-4-methyl-undecane9·10 (lb) (92.l g, 0.363 mo!), LiC=CH, LiNH2 and 1-bromopropane (0.33 mo!, 35.9 g). Yield 43.5 g (72 %).

(Z)-10-Tridecen-l-ol (Illa)

1,2-Diaminoethane (600 µI) and alkynol //a (32.0 g, 0.164 mo!) were added to a suspension of P2-Ni (prepared from 1.25 g of nickel(II) acetate) in ethanol (500 ml) and hydrogenated with stirring at 25 'C. The hydrogenation was monitored by analyzing aliquots of the solution by GLC. Preparative medium

TABLE I 1H NMR chemical shifts (o, ppm) and coupling constants (in parentheses, J, Hz) of compounds

Ila - Via in CDCI3 (499.5 MHz, tetramethylsilane as internal standard)

Position

2

3

4

5

6

7

8

9

10

II

12

13

14

15

16

17

COOCH3

?

Ila

3.63 t (6.6)

l.56m

1.26

1.40 m

2.13 tt (2.5, 7.1)

2.16 tt (2.4, 6.6)

1.47 m

1.11 t (7.3)

Illa

3.63 t (6.5)

1.56 m

1.25

1.37 m

2.03 m

5.29

I 5.39 m

2.03 m

0.95 t

(7.6)

!Va

3.41 t (6.8)

1.85 m 1.25

1.46 m

2.03 m

5.29

I 5.39 m

2.03 111

0.96 t (7.6)

Va

2.72 m

2.58 m

2.44 t (7.1)

J.58 m 1.24

1.35 m

2.02 111

5.t8 5.38 111

2.02 m

0.95 [

(7.6)

3.67 s

Via

2.53 111

1.85 ddd (8.0, 12.7, 19.0J

2.32 ddd (6.6, 7.4, 19.0)

4.48 ddt (5.6, 6.6, 2 x 7.8)

1.58 111 1.73 m

1.24

1.44 111

2.03 m

5-f9 5.38 111

2.03 m 0.96 t (7.6)

- - - - - - - - - - -Short Communication 1215

pressure liquid chromatography of the crude product afforded 30.4 g (94%) of pure (GLC) (Z)-alkenol Illa.

(Z)-7,.Undecen-l-ol (IIIb) was prepared analogously from 43.5 g (0.258 mo!) of alkynol llb. Yield 38.7 g (87%).

1-Brdmo-(Z)-IO-tridecene (/Va)

Triphenylphosphine (41.8 g, 0.160 mo!) in dry dichloromethane (200 ml) was added dropwise to a solution of Illa (30.0 g, 0.151 mo!) and tetrabromomethane (50.l g, 0.151 mo!) in dry dichloromethane (300 md at 0 - 3 'C. The mixture was warmed to 20 'C over 1 h. After stirring for 4 h the solvent

TABLE II 1H NMR chemical shifts (o, ppm) and coupling constants (in parentheses, J, Hz) of compounds /lb - Vlb in CDCJ3 (499.5 MHz, tetramethylsilane as internal standard)

Position

:>

4

5

6

7

8

9

10

II

12

13

14

15

COOCl-1 1

/lb

3.64 t (6.6)

1.58111

1.33

1.46 111

2.12 tt (2.5, 7.1)

2.16 ll (2.4, 6.6)

I 33 - 1.46 m

1.50 m

0.97 t (7.1)

Illb

3.64 t (6.7)

1.57 m

1.24

1.37 111

2.01 m

5.32

I 5.39 m

2.01 m

1.37 111

0.90 t (7.3)

/Vb

3.41 t (7.1)

1.86 m 1.29

1.40 m

2.02111

5.30

I 5.50 m

2.02111

1.37 m 0.90 t (7.3)

Vb

2.72 111

2.58 m

2.44 t

(7.5)

1.58 m

1.24

1.35 m

2.01 m 5.31

I 5.39 m

2.01 111

1.24 - 1.40 m

0.90 t (7.3)

3.67 s --- -~--. --" -~---·-----·

Vlb

2.53 111

1.85 ddd (8.0, 12.7, 19.0)

2.32 ddd (6.6, 7.4, 19.0)

4.48 111 (5.6, 6.6, 2 x 7.8)

1.14

1.78 m

2.01 m 5.32

5.38 m 2.01 m

1.14- 1.78 m

0.90 l (7.3)

-

?_ .::..J

- - - - - - - - -1216

Haskovec, Saman, Koutek:

was removed on a rotary evaporator and light petroleum (400 ml) was added to the re,idue. The mixture was cooled to a 0 °C and filtered. The solid residue (Ph3PO) was wm,hed with ice-cold light petroleum and the filtrate was concentrated in vacuo. The crude residue was purified by pMPLC. The bromoalkene /Va was obtained a:- a colorless oil (39.3 g, 84% ). For C

13H

25Br (261.3) calculated:

59.8% C, 9.7% H, 30.6% Br; found: 59.6% C, 9.7% H, 30.7% Br. l-Bromo-(Z)-7-undecene (!Vb). In the same manner as described above, /lib (38.7 g. 0.227 mo!)

was brominated with tetrabromomethane (75.3 g, 0.227 moll and triphenylphosphine (60.3 g, 0.23 mo!) to give 45.0 g (85%) of !Vb. For C 11 H21 Br (233.2) calculated: 56.7% C, 9.1% H, 34.3% Br; found: 56.8% C, 9.1 % H. 34.1 % Br.

Methy I 4-0xo-(Z)-14-heptadecenoate (Va)

A solution of (Z)-7-tridecenyl- I-magnesium bromide I freshly prepared from !Va ( 13.82 g, 53.2 mmol) and magnesium turning:- (1.32 g. 53.2 mmol) in Tl-IF (30 ml)) wa:- added dropwise ( 10 min) under argon, to a stirred solution of 3-methoxycarbonylpropanoyl chloride (8.0 g. 53.2 mmol) and tris(2,4-pentad1onato-O.O')iron(III) (0.6 g, 1.6 mmol) in 250 ml of dry THF at room temperature.

TABLE III

I.le NMR chemical shift, (i5, ppm) of compounds Ila - Via in CDCl3

(CDCl3

= 77.00 ppm as internal standard)

--- ---- ~---- -------

Po,ition Ila Illa !Va Va Via

62.94 t 63.05 t 34.02 t 173.29' 177.29 s 2 32.72 l 32.77 t 32.83 l 36.96 t 35.57 l 3 25.67 t 25.71 t 28.16 t 42.77 l 29.73 t 4 29.42 l 29.73 l 29.72 t 209.10 s 81.05 ct 5 29.32 t 29.55 t 2940 t 23.76 l 25.20 l 6 29.07 t 29.44 l 29.40 l 29.71 t 29.46 l 7 29.03 l 29.39 l 29.21 t 29.43 l 29.46 l 8 28.77 l 29.23 t 28 75 l 29.38 l 29.43 l 9 18.66 l 27.06 t 27.07 t 29.33 l 29.31 l

10 79.50' 129.29 d 129.26 ct 29.20 t 29.22 t 11 81.55 s 131.51 d 131.55 d 29.15 l 28.85 l 12 12.36 l 20.48 t 20.50 l 27.68 t 27.99 t 13 14.32 q 14.36 q 14.38 q 27.04 l 27.06 t 14

129.25 d 129.28 d 15

131.47 d 131.52 d 16

20.45 l 20.48 l

14.34 q 14.37 q

51.71 q

17

COOCH3

,_,_ - - - - - -·- - -Short Communication 1217

After complete addition, the stirring continued for I h al the same temperature. Then. the reaction was quenched with 10% aqueous HCI (500 ml) and extracted with ether (4 x 100 ml). The combined ethereal extracts were washed with aqueous NaHC03, water, and dried over MgS04• The solvent was removed and the residue was purified by pMPLC to give 6.3 g (40%) of oxoester Va. For C 18H320 3 (296.5) calculated: 72.9% C, 10.9% H; found: 73. I% C, 11.0% H.

Methyl 4-oxo-(Z)-l 1-pentadecenoate (Vb) was synthe,ized analogously from !Vb (12.41 g, 53.2 mmol), magnesium (1.32 g, 53.2 mmol), 3-methoxycarbonylpropanoyl chloride (8.0 g, 53.2 mmol) and tris(2,4-pentadionato-0,0')iron(III) (0.6 g, 1.6 mmol). Yield 5.5 g (38%). For C 16H280 3 (268.4) calculated: 71.6% C. 10.2% H; found 71.8% C, 10.7% H.

(Z)-14-Heptadecen-4-olide (Via)

To a mixed solution of NaBH4 (189 mg, 5.0 mmol) and Na2HP04 . 12 H20 (193 mg, 0.517 mmol) in ethanol (20 ml) was added dropwise 4-oxo-(Z)-14-heptadecenoate (Va) ( 1.36 g, 4.59 mmol) with cooling and :-tirring, and the mixture was stirred for 5 h at room temperature. Then, a 10% aqueous solution of NaOH (20 ml) was added and stirring was continued for 1 h at room temperature. The mixture was acidified to pH I - 2 with concentrated HCI, stirring for I h at 0 °C, then extracted with ether (3 x 25 ml), dried (MgS04), and evaporated to give 0.93 g the crude lactone Via. Further purification by pMPLC gave 0.63 g (52%) of pure (GLC) product Via. For C 17H300 2

TABLE IV 13C NMR chemical ;hifts (i5, ppm) of compounds /lb - Vlh in CDC13 (CDCl 3 = 77.00 ppm as internal standard)

Position llh I/lb /Vb Vb Vlb

1 62.94 t 63.02 l 33.96 t 173.31 s 177.27' 2 32.64 t 32.76 t 32.80 l 36.99 l 35.36 t 3 25.24 t 25.62 t 29.50 t 42.77 l 29.58 t 4 29.04 t 29.68 t 29.30 t 209.08 s 81.02 d

5 28.55 t 29.27 t 28.37 t 23.76 t 25.18 t

6 18.64 t 29.04 t 28.06 t 29.53 t 29.28 l

7 80.17 s 129.88 d 129.91 d 29.27 t 29.21 t 8 80.17 s 129 77 d 129.72 d 29.08 t 29.05 t 9 20.74 t 27.12 t 27.05 t 29.00 t 28.84 t

10 22.51 l 22.86 t 22.87 t 27.12 t 27.11 t 11 13.44 q 13.78 q 13.79 q 129.85 d 129.83 ct 12 - - - 129.77 d 129.80 ct 13 - - - 27.70 t 27.99 t

14 - 22.86 t 22.86 t 15 - - 13.78 q 13.78 q

COOCH1 51.75 q

- - - - - - - - - -1218 Haskovec, Saman. Koutek:

(266.4) calculated: 76.6% C, 11.4% H; found: 76.5% C, 11.4% H. IR spectrum: 3 005 m !(C-H), cis-double bondJ, I 782 vs !(C=O), lactonej, I 653 w (C=CJ. (Z)-11-pentadecen-4-olide (Vlb) was prepared analogously from V/J ( 1.00 g, 3.73 mmol). Yield 0.51 g (57.4%). For C 15 H260 2 (238.4) calculated: 75.6% C, 11.0% H; found: 75.4% C, 10.9% H. IR spectrum: 3 005 m ((C-H), cis-double bond], I 781 vs !(C=O), lactoneJ, ~ I 653w (C=C).

-171is research was supported in part under grant No. DHR-5600-G-00-1051-00, Program in Science J and Technology Cooperation, U.S. Agency /(Jr !11tematio11al Development.

REFERENCES

I. Arn H., Toth M .. Pricsner E.: List (~f Sex Pherm11011es of Lepidoptera and Related Attractants. OILB-SROP, Pans 1986. 2. Grant A. K., Katzenellcnbogcn J. A.: J. Am. Chem. Soc. 103. 5459 (1981). 3. Narn,hima Y., Nakagawa E .. Wakabayashi S., Haya,hi S.: Agric. Biol. Chem. 44, I.+ 19 ( 1980). 4. Naoshima Y., O.rnwa 1-1.. Kondo H .. Hayashi S.: Agric. Biol. Chem. 47, 1431 (1983). 5. Fiandane~c V., Marchc,se G .. Martina V .. Ronzini L.: Tetrahedron Lett. 25, 4805 ( 1984). 6. Cardel11cchio C., Fiandanesc V., Marche'e G., Ronz1n1 L.: Tetrahedron Lett. 28, 2053 ( 1987). 7. Brandsma L.: Preparatit•e Ace1y/e11ic Clle1111strl'. Elsevier. Amsterdam 1971. 8. Brandsma L., Vcrkruij,se H. D.: Srnthesi.1· of' Acety/e11e.1, Allrne.1 and C1111w/rnes. Ei>evicr, Amsterdam 1981.

9. Hoskovcc M .. Saman D., Koutek B.: Collect. Czech. Chem. Commun. 55, 2270 ( 1990). 10. Hoskovcc M.: Ph.D. Fhesis. ln,titutc of Organic Chcmi,try and Biochctntstry, Academy of' Science' of' the CLcch Rcpubl 1c, Prague 1992. 11. Kang S. K .. Kim W. S .. Moon B. 1-1.: Synthc'i' 1985. 1161. 12 Brown C. A .. Ahu.Ja V. K · J. Chem. Soc .. Chem. Commun. /973, 553. 13. Weiss R. G., Snyder E. I.: 1. Org. Chem. 36. 403 ( 1971) 14. Balhni R .. Petrini M.: Synth. Commun. /Y, 575 ( 1989).

Translated by the author ( M. H.).

BEST A \//llL.4 R!F CQr>y

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11:

i\1 j:

- - - - - - - - -

I e> Pergamon

I~ Bioorganic & Medicinal C/1e111i.~t1)', Vol. 4, No. 3, pp 47ll-4KK. l'J%

Copyright (<:') 19% Elsevier Science Ltd l'rintcd in U1cat lliitain. /\II 1ights1csc1vcd

0968-0896/96 $15.00 + 0.00 S0968-0896(96)00029-6

I I I I

New Mimics of the Acetate Function in Pheromone-Based Attraction

Michal Haskovec, Oldrich Hovorka, Bianka Kalinova, Bohumfr Koutek,* Ludvik Streinz, Ales Svatos, Pavel Sebek, David Saman and Jan Vrkoc

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

Abstract-Several analogues or (Z)-8-dodeeenyl acetate (la), the major pheromone component of the Oriental fruit moth, Cydia

I 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 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

I analogues investigated, the chloroformate lb, alken-4-olide 2a and also dodecyl acetate (le) possess significant (60-85%) inhibito1y activities. Based on electrophysiological evidence demonstrating that (i) only lb is competing with the major pheromone 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

I I I I I I I I I I I I

Introduction

·The mating disruption technique belongs to one of the most perspective strategies for application of pheromones and their analogues in insect pest control.'-2 To achieve the mating disruption effect, several basic approaches are possible,3 including: (i) the release of synthetic pheromone constituents or mixtures thereof into the environment to disrupt the male's ability to locate virgin females by the omnipresence of pheromone, (ii) the use of components emitted by closely akin species to reduce intraspecific attraction and (iii) the use of reactive pheromone mimics or inhibitors that either irreversibly and stoichiometrically block antenna! receptor proteins or specifically inactivate pheromone catabolizing enzymes. To date, however, no evidence suggests that the last two approaches are as efficacious in disruption as synthetic copies of the natural pheromone and no operational system based on any of these principles has been invented. Nevertheless, investigations concerned with modifying effects of various chemicals on insect olfaction indicate that some suhstances are able to react with specific chemical group at hinding proteins, receptor proteins and/or catabolic enzymes that comprise the olfactory chemoscnsory systems of the moths. These compounds may be structurally related to a component of the pheromone of the insect against which they arc applied, or they may he of an entirely different chemical class. For example, Berger and E~tcs4 found that some N-alkylmalcimidcs arc able to irreversibly block the elcctroantcnnographic response of the cabbage looper Trichoplusia ni to the pheromone and to significantly

479

reduce the ability of males to mate. A similar effect has been observed5 on the exposure of olfactory organs of the male moth Mamestra brassicae to N-ethylmaleimide vapors. Also, some triftuoromethyl ketones have been proven to inhibit the action of pheromone esterases in the processionary moth Thawnetopoea pityocampa6 and the Egyptian armyworm Spodoptera litoralis.1

In this study, the males of the Oriental fruit moth (C. molesta), CM, were selected as model species. The female sex pheromone includes (Z)-8-dodecen-1-yl acetate (la) as the main constituent8 of the threecomponent mixture. We describe herein the synthesis and biological activity in electrophysiological and behavioural assays of analogues lb-2 (Scheme 1 ). These analogues formally proceed from structural modifications of the acetate group, one of the three putative active sites of the parent molecule la, which could be involved in the interaction process with the antenna) proteins. Due to the suggested9 bioisostericity of CH,=>CI replacements and some type of similarity existing between the acetate and lactone functions, these analogues seemed to embody in their structures both the appropriate recognition and reactivity elements. It was anticipated that processing some of the analogues hy a target protein could result in unmasking the inherent latent reactive moiety in 1 and 2, and lead to inactivation of target acetate-recognizing antenna) proteins. Based on some earlier findings related to the inhibition of esterases by propan-3-olidc derivatives, Ill.II we hypothesized that some of the compounds represented by structures 2 might function as inhibitors of pheromone carboxylcsterases. In view


M. l losKov1:c cl al.

Scheme I.

R -CH 2-0-CO-Y

1 a -d

In formulae

1a: R = (Z)-7-undecenyl; Y = CH3

1b: R = (Z)-7-undecenyl; Y =Cl 1c: R = n-undecyl; Y = CH3

1d: R = n-undecyl; Y =Cl •

of the reported 12 inhibitory activity of dodecyl acetate (le) on the CM males under field conditions, we also included the saturated pheromone analogues le and d into our studies.

Results and Discussion Chemistry

Chloroformates lb and lei were prepared from the corresponding alcohols and triphosgene, a safe and crystalline substitute of phosgene, according to a known'-' procedure. Compounds 2a and c were synthesized as shown in Schemes 2 and 3, while the synthesis of 2b has been described previously}4 IR, NMR and elemental analyses of all synthesized compounds were fully consistent with the proposed structures.

Relative volatiles of analogues

Dose-response curves in insect electrophysiology and results of laboratory bioassays are usually based on amounts of stimulus applied to the odor source rather than the amount of stimulus to which the insect antenna is actually exposed. While such approach may be correct when the compounds to be compared arc structurally similar and hence of (approximately) the same volatility, it is certainly not valid for compounds differing significantly in their chemical characteristics and molecular mass. This implies that corrections for differences in volatility need to be taken into account when interpreting elcctrophysiological and behavioral results for analogues I and 2. Since the potential volatility of a chemical is related to its inherent vapor pressure, 15 we determined the saturated vapor pressures of I and 2 using a GC method, whose details have been described previously.'''·17 Table I lists the

a

2a -c

2a: R = (Z)-7-undecenyl; n = 1 2b: R = (Z)-7-undecenyl; n = 2 2c: R = (Z)-7-undecenyl; n = 3

relative GC retention times of the compounds measured isothermally at five temperatures along with the calculated vapor pressure data. The results impressively demonstrate the importance of vapor pressure corrections in comparing electrophysiological and behavioral data for 1 and 2: at 25 °C, the relative liquid-vapor concentration ratio for the pheromone 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, 1 : 0.027 and 1 : 0.0093, respectively. It means that the alken-5-olide (2c) is about two orders of magnitude less volatile than the pheromone component la. Note that corrected concentrations of all analogues arc used throughout this work.

Electrophysiological properties

Two types of electrophysiological recordings were conducted to assess the effects of the pheromonerelated chemicals on antennae.

Electroantennography. Relative EAG act1v1t1cs of compounds la-d and 2b corresponding to 50% of the relative activity scale (Fig. 1) demonstrate that in no case did a pheromone analogue reach the response value of the natural pheromone component, (Z)-8-dodecen-1-ol acetate (la). The corrected EAG activity of analogues decreases in the relative ratio 1400: 15.1:3.1:1.8: I for the la, b, 2b, le and d, respectively. As expected, chloroformatc lb, which is the closest structural relative of the natural pheromone component and, rather surprisingly, also the aken-4-olide 2b, were the most active analogues. Their activity range was between 0.05 and 500 pg of the stimulus. Responses elicited by different doses of these two compounds arc not significantly different (Student's t-test, o. = 0.05). However, at the upper dose used (500 ~tg), an olfactory saturation could be

~ CH3!CH~2 (CH 2l~HO

b 2a

4

Scheme 2. (a) Pyridinium chlorochromatc/CH1Cl2; (b) ketcne, BF,- Et,OffHF, -400C.

I l{o

I New mimics of the ac1!tate function 481

I observed for lb, but not for 2b. The last two compounds, 2a and c, elicited nonsignificant responses when compared with the response of pure hexane

I (Student's t-test, rx = 0.05), even at the highest doses tested (500 ~tg).

Electrosensillography. The activities of olfactory

I neurons associated with sensilla trichodea were analyzed (Fig. 2). In several of the preparations, very small number of impluses was observed that could not be reliably discriminated from.. the spontaneous

I responses in air (Student's t-test, rx = 0.05). For this reason, these responses were not considered in further

I I I I I I I

EtooclfcooEt 0

0

~ ~COOMe CH 3!CH 2J2 {CH 2J6

6

2c

I Scheme 3. (a) (i) Mg(OEt),/EtOH, (ii) (Z)-CH-'(CH,)2-CH= Cf [ -· (Cl l,)<Br; (b) (i) NaH/MeO(CH,),OMe, (ii) Br(CH2),COOEt/ Nal. (iii) Jccarboxylation/hydrolysis, (iv) Cl-1,NJEt,O; (c) (i) NaBH,/ EtOll, (ii) H' /CH,CL, reflux 24 h.

I Table I. GC vapor pressures of I and 2

analysis in this report. All neurons sensitive to la were also responsive, but to a lesser extent, to lb and 2b. Thus, the present data suggest that these compounds interact with the same receptor sites as the major pheromone component la. With exception of the saturated acetate le, the order of the activity of the compound tested paralleled their EAG activity.

Short-range behavior

The effect of the analogues on short-range communication among the CM conspecifics is shown in Figure 3. The values of 'confusion coefficients' determined at four concentration levels (sec Experimental) demonstrate that, of the compounds tested, the chloroformatc (lb) and alken-3-olide (2a) possess a significant inhibitory 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 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 component la in causing disruption of normal pheromoneinduced behavior.

Flight-tunnel observations

Of the analogues tested, the chloroformate lb was to some extent able to substitute for the main pheromone component la in the pheromone blend, although it did not elicit the whole behavioral sequence (15% of males were activated and took flight, none of them orientated to the source or exhibited a precopulatory behavior). Similar, but even less pronounced effect was observed for le and 2b.

The behavior profiles presented in Figure 4 indicate that the exposure of the CM males to a mixture of the pheromone (10 ng of the three-component blend, which was proved to be comparable with calling females) and a 10-fold excess of the analogues lb, c and 2a resulted in a significant reduction (60-85%) in touch/landing responses relative to the pheromone

I Compound" Relative retention times Vapor pressures

60°C 70 °C

I la 4.105 3.802 lb 2.560 2.410 Id 1.330 1.31

I 2a 2.770 2h 2c

"E'>perimental values for le in ref. 17.

I •· St<tndard 11-tetradecane. 'Standard 11-pentadecane. '1 Standard 11-hexadecane. 'Standard 11-octadecane.

I I I

80°C 90 °C

3.509 3.265 2.320 2.230 1.290 1.280 2.660 2.560 1.956 1.912 4.054 3.808

I00°C

3.066 2.130 1.260 2.480 1.877 3.644

110 °C

2.400 1.837 3.432

120°c

2.310 1.816 3.329

Poe (Pa)

0.312" 0.184' 0.135" 0.0548" o.0082sc o.002s1c

1.0 0.59 0.43 0.176 0.027 0.0090


482 M. I losKOVEc ct al.

100000

10000 ~ :~ 0 1000 .. Cl ci: w ., 100-> ~ ~

10

1a 1b 1c compound

!!!El corrected

1d 2b

Figure 1. Experimental EAG activities for compounds la-d and 2b; a comparison of uncorrected and vapor pressure-corrected data. The activities are expressed as the reciprocal of the relative number of pmol required to elicit the same receptor response. Each bar repre~ents the mean of at least six experiments SD did not exceed 11 %.

a

11 ,,

'.J ,, I• !I I!' n

c - - ---- --

--------- - ----~------ -- ---~

l _ e

alone. Under the same conditions, the addition of 2b to the pheromone did not significantly affect the percentage of males flying upwind and contacting the odor source. At a lower concentration -ratio (I : I) even the analogues lb, c and 2a were only slightly effective. When the behavioral spectrum of males responding to pheromone is compared with the behavior of males responding to a pheromone/analogue (I: 10) mixture, the most affected maneuver was oriented flight: activated males were less successful in their orientation in the odor plume. Males that found the source eventually needed a longer period than the males responding to the pheromone.

The neurophysiological data presented here indicate that there is a class of receptor neurons on the antenna of CM male that significantly responds to stimulation with the major pheromone component la and to stimulation with two other analogues, lb and 2b. Nonuniform spike activities produced by the analogues le and

I _J

---, ~I~

---~~ _ ___I

0.1 mV

0.5 sec 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).


New mimks of the acetate function 4Rl

1QI)

9()

tlQ ~ '10

e... ... i:::

60 .~

so ~ q, ()

40 u i:::

3Q ·~ ., 20 .::!

to l5 u

Figure J. Inhibition of male C. mole.1ta behavior in a laboratory 'hnrt-1a11gc bioassay p10111oted hy different doses ol I :tnd 2. Each har i'> the mean of f8 experiment~ (SD s 15%).

10() 1oO 9()

9'.J 6o sn 7o ~ e::. 70 6Q <II

rJJ So 8 '.fJ ~

40 !!! 411 3o ~ :iO ';::

~o -!! 21l !!!

10 f0

0

Figure 4. Inhibition of behavioral responses of male C. molesta by mi, tu res containing the pheromone (JO ng) and a JO-fold excess of the respective analogue, relative to male responses to the pheromone alone. Each bar is the mean of six experiments (SD s 18%).

2a did not significantly differ from the spontaneous 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 laboratory short-range assay data and the flight-tunnel results (Figs 3 and 4). The most interesting feature of the results arc the striking differences among analogues in the complementary elcctrophysiological and behavioral tests. As illustrated in Table 2 summarizing the qualitative output of these tests for the mos! important analogues of the series, none of the analogues lb, c, 2a and b showed identical behavior at all four (EAG, ESG, short-range behavioral assay and flight tunnel) levels of evaluation. This implies that no straightforward structure-activity relationship can be discerned and the inhibitory active analogues lb, c and 2a must operate by (at least) three different inhibiting mechanisms. Although, at present, we have no evidence of the exact mode of action of the analogues tested, several mechanisms can be hypothesized.

The mechanisms of inhibitory action derive from inhibiting chemicals - in relation to the pheromone composition of the species. It is generally acceptcd 18

that molecular size and shape arc important for insect pheromone chemoreccption. Apart from stcreochemical requirements, however, electronic chargecharge attraction, hydrogen bonding, hydropathic bonding and van der Waals forces are potentially important in binding to proteinaceous macromolecules. To account for the surprising differences in biological activity of the analogues, we first considered the possibility that some of the analogues might assume minimum energy conformations that were very different from the conformation of la. As the molecular shapes of compounds la,b and 2a,b are identical with respect to the unsaturated hydrocarbon chain, the main spacial differences among them should originate from the polar groups. Thus, the energy-minimized molecular geometries of ethyl acetate has been superposed on those of ethyl chloroformate, butan-3-olidc and pentan-4-olide to qualitatively compare the shapes. The results are shown in Figure 5. The chloroformate [Fig. 5(a)] and, to a lesser extent, also the alken-4-olide [Fig. 5(c)] show clearly a high degree of similarity to the acetate group. On the contrary, the alken-3-olide [Fig. 5(b)] shows the largest deviations. Accordingly, it is more probable that the receptor would reject the four-membered ring due to its size or shape.

'fohle 2. Qualitative summary of responses" for male C. 1110/esta on exposure to compounds la-c. and 2a and b

Compound

la th le 2a 2b

Elcctrophysiological activity

EAG

(+) ( +) ( +) (-) (-)

ESG

(+) (+) (-) (-) ( +)

Inhibitory effect

Short-range bioassay

(+) ( +) ( +) (+) ( - )

'( + )· positive response, ( - ): no or statistically non-significant (Student's I-test, ~ = 0.05) response.

Flight-tunnel

(+) (+) (+) (+) (-)


484 M. HosKovEc et al.

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 hy a chlorine in lb does not seem to have important steric consequences, the methyl 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 that is complementary to the acetate methyl is probably reduced for the chloro derivative. 19

·2n Beside this, the

IR carbonyl frequencies v(C=O) for la and b (1740 versus 1778 cm - 1

) differ significantly, indicating the different ability to form the hydrogen bond. The hydrogen bond is widely regarded 21 as being the most important intra- and intermolecular cohesive force and a major contributor of noncovalent interaction energy in biological systems. All these differences may account for the reduced electrophysiological activity of the chloroformate lb in comparison to la. Similarly to our results, only moderate electrophysiologic responses were found 22 to he evoked by formate mimics of the aldehyde function on antenna! receptors of several noctuid species. Regardless of its reduced activity, the analogue !-b c!idts a sufficiently high dectmphysiological response from the pheromone-sensitive receptor neurons and could theoretically overload the receptor system acting as an inhibitor when present in hyperphysiological concentrations. Another explanation for the significant inhibitory activity of lb could be its possible binding to antenna! proteins through a carbamate linkage (under evolution of HCI!), thereby locking the sensory transduction mechanism and/or inactivating the pheromone catabolizing enzymes. Precedent for this analogy is found in the use of (Z)-11-hexadecenoyl- and (Z)-9-tetradecenoyl fluorides as reactive mimics of the Heliotis virescens phen)mones aldehydes.2

'

The fact that we found relatively high (and nearly equal) inhibitory activity to be coupled both with lb and 2a was rather surprising since the alkcn-3-olidc (2a) failed to produce any significant EAG or ESG response. If the specificity of pheromones and pheromone rccc.ptors was coupled to specificity of the pheromone clearing enzyme system, it would be expected that analogues tha~ successfully mimic the pheromone should serve as better substrates for the pheromonespecific catabolic systems and be degraded more effectively than those compounds that arc structurally less similar to the pheromone. Thus, compounds lacking

sufficient pheromone mimicry (e.g. 2a) would he cleared from the receptor less effectively and act as behavioral inhibitors. The earlier studies of behavioral responses of male codling moths24 ·and European corn borers25 in a flight tunnel to their corresponding pheromones and analogues led to the conclusion that the steric requirements for an analogue to be a pheromone mimicking substance are much more stringent than for the analogue to be an inhibitor of behavioral output. On the other hand, it is known 2

" that four-membered lactones as amhident electrophiles may undergo, in the presence of nucleophiles, oxygen-alkyl or oxygcn-acyl bond cleavage. Therefore, 2a might be able to inhibit pheromone-catabolizing enzymes via acylation or alkylation of their nucleophilic groups. This would, in turn, lead to disruption of normal pheromone-induced behavior via prolonging high pheromone levels within the peripheral sensory system. A lower inhibitory activity of the alken-4-olide (2b) (Figs 3 and 4) may in part be related to its lower (in comparison to 2a) chemical reactivity. Note that propan-3-olide has been found 27 about 50-1{}0 tirr1cs more· reactive in rcactionwith adenosine, cytidine or guanosinc than butan-3-olide while butan-4-olide completely failed to read with any of these nitrogen nuclcophilcs. A double-bond environment appears essential for good inhibitory activity since Id, the saturated equivalent of lb, is only a very weak (Fig. 3) inhibitor.

The behavior of the saturated acetate le deserves a special comment. The major component or the Oriental fruit moth pheromone, (Z)-8-dodecenyl acetate (la), was idcntitied28 in 1%9. Since 1979 it has been assumed8 that the Oriental fruit moth pheromone consists of four components, viz. (Z)-8- and (£)-8-dodecenyl acetates, (Z)-8-dodeccn- l-ol and dodecanol. The insect attraction was found 2

'' to be particularly sensitive to both the pheromone component ratios and concentrations. Mating disruption was also attempted with an analogue of I a, dodecyl acetate (le). For example, Rothschild found 12 that males of this insect were not trapped at live virgin female or synthetic pheromone sources when dodecyl acetate (le) was present in large amounts (exceeding the amount of the unsaturated acetates) in the same trap or within 15 cm from the bait. When the compound, however, was present as a background odor over a large area, an opposite (synergistic) effect was

I I I I I I I I I I I I I I I I I I :I I

I I

New mimics or the ucetatc function 485

observed. Reasons for these results remained obscure. RcccrHly, the composition of the pheromone blend was re-examined and significant amounts (3.44 ± 1.16%) of le have been identified30 in the effluvia of calling females. It has been suggested that the role of this compound in the natural pheromone of CM rnight have been overlooked. Our electrophysiological results demonstrating that le, although not detected by the same receptor cell type as la, elicits a relatively high EAG activity, seem to support this suggestion. Unfortunately, neither our own ESG experiment nor the previous ESG studies31 were able to detect any other receptor cells that could respond to le, probably due to the small size of the moths. If the proposition is accepted that this compound represents a minor pheromone component and by itself could mediate a particular clement of behavior, then a background of a high concentration of this component might alter the balance of sensory input to such an extent that the insects no longer respond appropriately. A similar inhibitory effect of imbalance in the pattern of sensory input had been previously observed~2 on the behavior of Autogmpha gamma species that use a simple two-constituent pheromone blend. Increasing the level of minor component in the binary blend resulted in a substantially decrease of male responses in moth behavioral stages.

In conclusion, the present data suggest that, of the analogues investigated, only the chloroformate (lb) and the alkcn-4-olide (2b) are. able to reasonably mimic the acetate function and thus produce responses from the same class of pheromone receptor neurons as the major pheromone component la. The inhibitory properties of the analogues, however, seem not to be entirely connected with their mimicking capability. Apparently, several constitutional and configurational properties of the molecule and, 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 spite of this, the new inhibitors described (especially lb and 2a) may prove useful as tools in further biochemical as well as field studies directed towards mechanisms controlling mating disruption.

Experimental Chemistry

11 I NMR spectra were determined in CDCI-' solution on a Varian UNITY-500 spectrometer operating at 499.5 MHz and absorptions are expressed in o (ppm) scale relative to TMS. The IR spectra were recorded on a Bruker IFS 88 FT-IR spectrometer in CCl4 • GLC analyses were performed on a Hewlett Packard HP 5880A chromatograph. equipped with a FID detector and a 25 m capillary column (0.3 mm i.d., HP5-5% phenyl methylsilicone, cross-linked). Preparative medium pressure liquid chromatography (PMPLC)

separations were made on Merck 60 silica gel (0.040-0.063 mm) using a Bi.ichi B-680 Prep LC system with a stepwise gradient of ethyl acetate in light petroleum.

Syntheses

(Z)-8-0odccen-l-yl chloroformate (1 h). Triphosgcne (0.367 mmol) solution in dry Tl-IF (I ml) was cooled on an ice-bath and pyridine (45 pL, 1.2 equiv.) was added. To the preformed white precipitate the (Z)-8-dodecen-1-ol (85 mg, 0.466 mmol) in Tl Ir (2 mL) was added dropwisc during 20 min. After 2 h of' being stirred at the ice-bath temperature, the reaction mixture was poured into ice and aq HCI (3.7%, 0.4 mL). Subsequent standard work up and PMPLC afforded the chloroformate lb (69. mg, 60% yield). Calcd for C 1,Hv02CI: C, 63.27; H, 9.39; Cl, 14.37. Found: C, 63.44; 9.50; Cl, 14.21. 1I-I NMR: S 0.90 (t, 3H, 1=7.0 Hz, CH_1CH 2CJ-1 2-), 1.29 (m, 1211, 6x-CH2-}, 1.72 (m, 21-1, .1=4x6.8 llz. -CH2CH 2C02CI), 2.01 (m, 41-1, -Cl:I 2--CH=CI I -Cl::lz-), 4.31 (t, 21-I, J = 7.8 llz, --Cl-J 2C02CI), 5.38 (m, 2H, -CH=CH-). IR (cm- 1

): 3006 m [v (C-H), cis-double bond], 1778 vs [v(C=O), ester), 690 w (v(C-CI)].

Dodec-1-yl chloroformate (ld). Calcd for C1.1H 2,02CI: C, 62.76; H, 10.13; Cl, 14.25. Found: C, 62.90; H, 10.22; Cl, 14.14. 1H NMR: o 0.88 (t, 3H, J = 7.0 Hz, CH 3CH2-), 1.26 (m, 181-I, -CH2-), 1.73 (m, 2H, J = 4 x 6.8 Hz, -CH2CH2C02CI), 4.31 (t, 2H, J = 7.8 Hz, -CH2C02Cl). IR (cm- 1

): 1779 vs [v(C=O), ester], 690 w [v(C-Cl)].

(Z)-8-Dodecan-l-al (4). Pyridinium chlorochromate (PCC; 3.5 g, 16.3 mmol) was suspended in dry CH 2Cl 2

(25 mL) and (Z)-8-dodecan-l-ol (3, 2.0 g, 10.9 mmol) in 6 mL of the same solvent was added in one portion to the stirred solution.-'-' After 2 h, dry ether (50 mL) was added and the supernatant decanted from the black gum. The insoluble residue was washed with ether (3 x 20 mL), the combined ethereal solution was passed through a short pad of neutral alumina with charcoal and the solvent was evaporated. PMPLC of the crude product afforded 1.64 g (83%) of the 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, CH 3CH2CH 2-), 1.21-1.43 (m, 8H, 4 x -Cll 2 --· ),

1.44-1.71 (m, 2H, -CH2CH2CHO), 2.05-2.11 (111, 41I, -CH1-CH=CH-CH2-), 2.42 (dt, 2H, J == 7.0, 1.6 Hz, -CH2CHO), 5.31-5.38 (111, 2H, -CH=Cll · ). 9.77 (t, IH, J = 1.6 Hz, -Cl-10).

(Z)-10-Tctradccen-3-olidc (2a). Ketenc 11 was bubbled into the stirred mixture of aldehyde 4 ( 1.6 g, 8.8 mmol) and borotriftuoride etheratc (26 ~tL, 0.2 mmol) in anhydrous THF (10 mL) at -40 °C:1' After I h, another fresh catalyst was added (20 ~1L, 0.16 mmol). When the starting aldehyde 4 disappeared (2 h), ·1he solution was flushed with dry nitrogen. Anhydrous triethylamine (2 mL) and CI-ICI-' (20 mL) were added


\Xfi M. Ho~KnvH· ct al.

at --40 °C followed by water (20 mL). The mixture was then extracted with CHCI, (3 x 20 mL) and the combined organic extracts were dried over MgS04 •

Removal of the solvent in vacuo and purification of the residue by PM PLC afforded 0.99 g (54%) of the pure ( G LC) lactone 2a. Ca led for CH,H 2x02 : C, 76.14; H, 11.18. Found: C. 76.31; H, 11.21. 1!-1 NMR: /5 0.90 (t, 311..1=7.0 Hz, CH.,Cl-l 2CH 2--), 1.21-1.47 (m, IOH, 5x -CH 2-), 1.62-1.85 (m, 21-1, --CH2-·Cl-I<), 2.02-2.12 (m, 4H, -Cl::h--CH=CH-CH2-), 3.06 (dd, lH, .!= 16.2/4.3 Hz, -Cl::l2-CO-), 3.51 (dd, I H, .I= 16.2/5.8 Hz, -CH2-CO-), 5.32-5.39 (m, 2H, ---CH=CH-). IR (cm · 1

): 3004 m [v(C-H), cis-double bond], 1836 vs [v(C=O), lactone], 1653 w [v(C=C)J.

Diethyl 4-[ (Z)-6-decenyl]-3-oxopentanedioate (5). To a stirred ethanol solution (150 mL) of the magnesium chelate prepared from diethyl 3-oxopentanedionate ( 12.7 g, 63 mmol), magnesium turnings (2.29 g, 95 mmol) and a trace of iodine was added 1-bromo(Z)-6-decene (18.0 g, 75.5 mmol) at room temperature and the mixture was refluxed for 18 h under argon.36

The reaction mixture was evaporated in vacuo to give a dark oil which was acidified with 10% HCI and taken up into ether. The ethereal solution was washed with water and dried over MgS04 • After evaporation, chromatography (PMPLC) of the crude product gave.. 17 .2 g (80%) of the oxoester 5. Calcd for C1 ~H.1205 : C, 67.03; H, 9.47. Found: C, 68.88; H, 9.39. 'H NMR: 8 0.90 (t, 3H, l = 7.3 Hz, CH1CH2CH 2-), 1.28 (t, 6H, .I= 7.1 Hz, 2 x OCH 2CH,), 1.29-1.40 (m, 8H, 4 x -CH2-), 1.86 (m, 2H, CH,CH2CH2-), 2.00 (m, 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, -CO--CH<), 3.61 (d, IH, 1=15.8 Hz, ·-Cl:-J2-CO-), 4.19 (q, 4H, 1=7.l Hz,

2 x OCI:bCH,), 5.30-5.39 (m, 2H, -Cl::l=CH-).

l\Jcthyl 5-oxo-(Z)-12-hexadeccnoate (6). A solution of 5 ( 14.0 g, 41. l mmol) in dry 1,2-dimethoxyethanc (DME, 15 mL) was added dropwise to a stirred solution of NaH (2.5 g of a 50% mineral oil disr>crsion, 41. l mmol) in dry DME (50 mL) at room temperature and stirring was continued for 1 h under argon:'7 Ethyl 3-brornopropionatc (8.9 g, 49.3 mmol) and finely powdered Na! ( 1.65 g) were then added to the solution and the mixture was relluxed for 18 h with stirring. The reaction mixture was cooled to room temperature and the solvent was evaporated. The residue was heated with 15% aq solution of NaOH ( IOO mL) under reflux for 24 h and the reaction mixture was acidified with coned HCI, saturated with NaCl and extracted with ether. The dried solution (MgS04) was added into an ethereal solution (300 mL) of freshly prepared diazomcthane. After 20 h of being stirred the reaction mixture was concentrated in vacuo and the residue was purified by PMPLC. Chromatography afforded 7.1 g ( 61 % ) of the ester 6. Ca led for C 11H,110.,: C, 72.30; H, 10.71. Found: C, 72.19; H, 10.51. 'H NMR: 8 0.90 (t, 3H, 1=7.4 Hz, CH,CH2CH2-), 1.24-1.40 (m, SH, 4 x --CH 2-), 1.56 (m, 2H, CH.1CH2CH 2-), 1.89 (m,

2H, J = 4 x 7.3 Hz, -CH2CH2C02CH1), 2.00 (m, 41 I, -Ctl2-CH=CH-Cl:b-), 2.34 (t, 211, l = 7.3 Ilz, -Cl:lzCO-), 2.38 (t, 2H, l = 7.4 Hz, --COCI-1 2 -- ),

2.47 (t, 2H, .I= 7.2 Hz, -CB 2C02CH,), 3.67 (s, 3H, -C02Cl:-h), 5.31-5.39 (m, 2H, -CH=CH-).

(Z)-12-Hexadecen-5-olidc (2c). To a mixed solution of NaBH4 (111 mg, 2.94 mmol) and Na2HP0.,·12ll~O (140 mg, 0.392 mmol) in ethanol (10 mL) was added the 5-oxoester (6) dropwise (830 mg, 2.94 mmol) with cooling (0 "C) and stirring, and the mixture was stirred at room temperature for 8 h. Then, a 10% aqueous solution of NaOH (13 mL) was added and stirring was continued for I h. The mixture was acidified to pH l -2 with coned HCI, stirred for 1 h at O °C, then extracted with ether (3 x 25 mL), dried (MgS043) and evaporated to give an yellow oil. This oil was added to a stirred solution of triftuoroacetic acid (50 ~tL) in anhydrous CH2Cl2 (20 mL). After 20 h being refluxed, the reaction mixture was concentrated in vacuo. The further purification by PM PLC gave 0.51 g (94%) of pure (GLC) product 2c. Ca\cd for C 10H 2K0 2: C, 76.13; H, 11.19. Found: C, 75.91; H, 10.98. 'H NMR: 8 0.90 (t, 3H,1=7.3 Hz, CH,CH 2CH2-), 1.28-1.94 (m, 161-I, 8 x-CH -) 2.01 (m 4H -CH -CH=CI-1--

2 ' ' ' --2 CH2-), 2.44 (ddd, IH, 1=6.8/8.8/17.6 Hz, >CH-CH2-), 2.58 (dt, tH, J = 6.9/6.9/17.6 Hz, >CH-CH2-), 4.28 (m, lH, J = 2.9/5.117.8/10.6 Hz, >CH-CH2-), 5.32-5.39 (m, 2H, -CH==CH --). IR (cm-'): 3001 m [v(C-H), cis-double bond], 1736 vs [v(C=O), lactone], 1654 w [v(C=C)].

Bioassays

Insects. Males of C. 1110/esta originated from the laboratory colony maintained on an semiartificial diet under a 16:8 light:dark regime. Moths were sexed as pupae and males were stored separately from females. Newly emerging adults were collected daily, provided with water and sugar solution absorbed onto cotton wool under the same light and temperature conditions. Males 2-4 days old were used for EAG experiments, 3-4 days old males were used for flight-tunnel observation.

Electroantennography. Two glass Ag/AgCl microclcctrodcs tilled with physiological saline were used for EAG recordings: the ground electrode was placed into the head capsule of an intact male moth anti the recording electrode was connected with the distal end of the male antenna, the tip of which had been cut off. Antenna! responses were amplified (signal conditioner CyberAmp 320, Axon Instruments), digitized (Mctrabyte DAS-16 AID, sample period 250 ms) and analyzed by a PC 486 computer (Stand Alone Acquisition System, Run Technologies).

The main pheromone component of the Oriental fruit moth, C. molesta, and its analogues lb-d and 2a-c were dissolved in hexane forming a series of dilutions from 5 ng to 5 ~tg per ~LL Aliquots of 5 pL were pipetted onto a filter paper disc (10 mm i.d., Whatman

\ ~\

I I I I I I I I I I I I I I I I I I I

.JI

I

New mimics of the acetate function 487

no. 2) and each loaded disc was inserted into a Pasteur pipcttcr after solvent evaporation. The odor cartridges were stored deeply frozen in closed glass vials when not used for experimentation. The cartridges conditioned in laboratory temperatures for at least 1 h were used for stimulation. Stimuli were delivered onto the antenna! preparation by air puffs blown through the cartridge outlet of which was positioned at a distance 2.5 cm from the antenna. The stimulus duration was 0.8 s, the air flow rate was I L min 1

• Between successive stimulations the antenna! preparation was blown by a continual stream of clean and humidified air. Intervals used between two successive stimuli ranged from I to 20 min, depending on the type and intensity of the stimuli. Typicaiiy, i-4 min were adequate for complete recovery of the EAG at lower doses, while I0-20 min were necessary when doses greater than 10 ~tg were used. Three EAG replicates were recorded for each serial dilution of each odorant. Recordings were repeated on three IT)ale antennae. The main pheromone component Ia (50 ng) served as a standard to normalize EAG responses from different individuals and to control over viability and constancy of the preparation. Stimulation with 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 expressed as a percentage of the EAG response to the standard stimulation.

Single sensillum recording. Receptor potentials and nerve impulses were recorded extracellularly from receptor cells associated with the s. trichodea using a modified tip-cutting technique.3~ A whole animal preparation was used. A male in a disposable pipette tip was fixed in place by small droplets of molten wax, while the head and one antenna were protruded. The antenna was carefully bent dorsally and fixed by wax. The tips of s. trichodea were cut by means of two glass microknives (microelectrodes with broken tips, ~ 30 µm i.d.) mounted in micromanipulators. The recording electrode (10 µm in diameter) slipped over cut s. triclwdea was filled with receptor lymph saline, the reference electrode, inserted in the head, contained saline approximating the ionic composition of the moth haemolymph.'8 Prior to the slipping, the tip of the recording electrode was dipped into heated vaseline to prevent it from drying out. The electrical activity of the receptor cells was recorded similarly as EAG recordings on the same instruments. Receptor potentials (DC recordings) and spike activity (AC recording) were recorded simultaneously by two independent channels of signal conditioner.

Short-range behavior. The effect of analogues on male prccopulation behavior was investigated in disposable Petri dishes (10 cm i.d.). The compound investigated was loaded on a filter paper disc ( IO·mm dia) placed in the center of the dish housing the calling fcmalc. After 30 min of equilibration a male was introduced into the dish and its behavior was observed for a 30 min period. Experiments were performed simultane-

ously 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 (%) = (CdNc-Cil NE)IOO, where CC is the confusion coefficient, C(' no. of copulations in controls, Ne no. of pairs in controls, Cr; no. of copulations in the experimental group and N1•

no. of pairs in the experimental group.

Flight-tunnel experiments. The CM males were flown in a 1.86 m long x 0.3 m wide x 0.3 high plexiglass flight-tunnel. Charcoal filtered and humidified air was pushed through the tunnel by four ventilators. The air velocity was maintained at 0.5 ms 1

• The flight-tunnel conditions used were: 22-26 °C, 40-60% relative humidity and 700 lux light intensity.

In a preliminary series of experiments male reactions to the calling female to la alone and to the thrcecomponent pheromone blend (la 90%, £8- I 2:Ac 6%. Z8-12:0H 4%) were determined. Males were allowed to respond to 1, 10 and JOO ng of pheromone blend to determine which odor source is comparablc with the calling female. The pheromone blend (Hl ng) loaded on a filter paper disc (10 mm dia) was fully comparable with the female and therefore was used as a standard in all flight-tunnel experiment.

Using pheromone analogues, two different types of observation were performed. Firstly, male reactions to the pheromone standard (10 ng) masked by 100 ng of the respective analogue were observed to see if the analogue has an ability to modify the male orientation to odor source. Secondly, to determine if the analogue can substitute the main pheromone component la, males were observed while responding to the odor source in which la was replaced by an appropriate amount of the analogue.

The experiments were performed from 13 to 15 h after the beginning of scotophase. Virgin males (3-4 days old) were placed individually into clean glass tubes (release cages, 10 cm long, 4 cm i.d.) 15 min prior to each session. After 15 min acclimatization period malcs were released from the 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 the holder placed centrally near the upwind end. The filter paper disc created turbulence and so structured the plum (its parameters and orientation was checked using TiCl4 prior to and after each flight session). Each male was tested once and then discarded. Due to the relatively rapid release rates of volatile chemicals from filter paper sources, only five males were tested for each filter paper source. In six replicate series, altogether 30 males were flown for each treatment. To assure a convenient state of the males, an additional five individuals were tested on the three-component pheromone blend after each day's session.

Male behavior was classified into four categories: ( i) activation (walking and wing fanning), (ii) take off, (iii)

I I I I I I I I I I I I i I i I I I I I ~I

488 M. HosKovEc- et al.

oriented flight and (iv) touching the odor source, landing and copulation attempts. The total time of observation was either 2 min if the male did not take off or it lasted until its landing.

Statistical analysis. The data were subjected to statistical analyses utilizing the StatgraphicTM_Plus software package (Manugistic, Rockville, Maryland, U.S.A.). Student's t-test (a= 0.05) was used to compare mean responses for differences (H11 :m 1 = m 2).

!\'linimum energy calculations (MEC). MEC were performed for ethyl acetate, ethyl chloroformatc, butan-3-olide and pentan-4-olide. These compounds represented those parts of the molecule that were altered. The aliphatic unsaturated chain of the molecules remained constant for all analogues, -~nd was not expected to affect the total conformation of each molecule differently. The PC software used was HyperChem TM for Windows (Autodesk, Sausalito, California, U.S.A.). Energy minimization operations were run until the energy gradient for each molecule was less than 0.1 kJ mo1- 1

•

Acknowledgement

We acknowledge the financial support of this work by grant no. DHR-5600-G-00-1051-00, Program in Science and Technology Cooperation, from the U.S. Agency for I ntcrnational Development.

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