Syntheses and Behaviour Activity of Conjugated Polyenic Pheromone Components

ILME LIBLIKAS

Doctoral Thesis Stockholm, Sweden 2004

Ilme Liblikas

Doctoral Thesis

Stockholm 2004

———————————————————————————————————

Akademisk avhandling som med tillstånd av Kungliga Tekniska Högskolan i Stockholm framlägges till offentlig granskning för avläggande av filosofie doktorsexamen i kemi, tisdagen den 8 juni 2004 kl. 14 i Kollegiesalen, Valhallavägen 79, KTH, Stockholm. Avhandlingen försvaras på engelska. Opponent är Professor Dr. Wittko Francke, Institute of Organic Chemistry, Hamburg University, Germany.

Cover: Main pheromone component of codling moth Cydia pomonella (L.), (8E,10E)-8,10-dodecadien-1-ol

ISBN 91-7283-787-X ISRN KTH/IOK/FR-04/91-SE ISSN 1100-7974 TRITA-IOK Forskningsrapport 2004:91 © Ilme Liblikas 2004 Liblikas, Ilme. Syntheses and behaviour activity of conjugated polyenic pheromone components. Royal Institute of Technology, Stockholm, June 2004.

ABSTRACT

This thesis deals with the synthesis of conjugated dienic and trienic pheromone components- C12-C16 alcohols, acetates and aldehydes and their applications in pheromone communication studies. Insect pheromones are widely used in modern pest management. The development of effective tools for plant protection is a result of complex broad, interdisciplinary basic and applied research. A practical use of pheromones for pest management usually requires that specific active chemicals must be isolated, identified and produced synthetically. The creation of pheromone materials besides requires the identification of the active components, determination of the roles of other possible isomers or degradation products, the design of dispensers of suitable substrates and evaporation parameters. The aim of the thesis was critically to survey methods for the syntheses of conjugated polyenic compounds and to focus to the synthesis of several conjugated polyenic pheromone components providing sufficient quantities of chemically and isomerically pure compounds for bioassaying. The principal objective of the thesis was concerned with the further aim of developing effective synthetic materials for plant protection. The work was focussed: ⇒ To identify the compounds collected from the leafminer moth Phyllonorycter emberizaepenella (Lepidoptera: Gracillaridae) and perform a detailed identification of the pheromones of the Brazilian apple leafroller Bonagota cranaodes (Meyrick) (Lepidoptera: Tortricidae) and of the codling moth Cydia pomonella (L.) (Lepidoptera: Tortricidae). ⇒ To study the effect of non-pheromonal isomers of the main pheromone component on the sex attraction of B. cranaodes and C. pomonella in wind tunnel and field tests. ⇒ To study the role of identified pheromone components in the pheromone communication of the currant shoot borer Lampronia capitella (Cl.) (Lepidoptera: Prodoxidae), and to design and optimize the dispenser for effective trapping. ⇒ To investigate the presence of sex pheromone components in the tobacco hawk moth Manduca sexta (L.) (Lepidoptera: Sphingidae). The syntheses of four geometrical isomers of 3,5-dodecadienyl acetate; (10E,12E,14Z)- and (10E,12E,14E)-(10,12,14)-hexadecatrien-1-als; (E,Z)-, (Z,E)-, and (Z,Z)-8,10-dodecadienol, codlemone acetate and aldehyde; (9Z,11Z)-9,11-tetradecadienol, corresponding acetate and aldehyde; all geometrical isomers of 8,10-and 10,12-tetradecadienols and corresponding acetates were performed to ensure all these bioassays with synthetic pheromone components. All compounds were made in high chemical and stereochemical purity, most of them more than 99% isomerically pure or by using several separation and purification techniques. The suitable synthetic schemes for the synthesis of all four geometrical isomers of four conjugated dienic pheromone components are presented in this thesis. The reactions and purification methods used here exemplify the wide range of possibilities realized for such syntheses: they present the successful application of many synthetic methods. The importance of this synthetic work is that the compounds synthesized are not commercially available or on the other hand the purity of the purchasable compounds is not satisfactory for research in the field of chemical ecology. Key words: Lepidoptera, sex pheromone, pheromone dispenser, 3,5-dodecadienyl acetate, 10,12,14-hexadecatrienal, 8,10-dodecadienol, codlemone atsetate, codlemone aldehyde, 8,10- tetradecadienol, 8,10-tetradecadienyl acetate, 10,12-tetradecadienol, 10,12-tetradecadienyl acetate, Lampronia capitella, Manduca sexta, Bonagota cranaodes, Cydia pomonella, Phyllonorycter emberizaepenella, conjugated polyenic pheromone, conjugated diene, MPLC, (9Z,11Z)-9,11-tetradecadien-1-ol, (9Z,11Z)-9,11-tetradecadien-1-al APPENDICES AND PAPERS

Appendices:

1. Synthetic scheme of the synthesis of four geometric isomers of 10,12- tetradecadienol and of their acetates 2. Synthetic schemes of the syntheses of EZ-, ZE-, and ZZ-isomers of 8,10- dodecadienol 3. Synthetic schemes of the syntheses of EEE- and EEZ-isomers of 10,12,14- hexadecatrienal 4. Synthesized compounds

This thesis is based on the following publications and manuscripts, which will be referred to by their Roman numerals I-X.

I Effect of Codlemone Isomers on Codling Moth (Lepidoptera: Tortricidae) Male Attraction. El-Sayed, A., Unelius, R.C., Liblikas, I., Löfqvist, J., Bengtsson, M. and Witzgall, P. Environ. Entomol. (1998) 27(5): 1250-1254.

II Synthesis and Characterization of the Four Geometrical Isomers of 3,5- Dodecadienyl Acetate. Unelius, C.R., Liblikas, I. and Mozuraitis, R. Acta Chem. Scand. (1998) 52: (7): 930-934.

III Sex Pheromone of the Brazilian Apple Leafroller, Bonagota cranaodes Meyrick (Lepidoptera, Tortricidae). Eiras, A.E., Kovaleski, A., Vilela, E.F., Chambon, J.P., Unelius, C.R., Borg-Karlson, A.-K., Liblikas, I., Mozuraitis, R., Bengtsson, M. and Witzgall, P. Z. Naturforsch., C: J. Biosci. (1999) 54(7/8): 595-601.

IV Flight and Molecular Modelling Study on the Response of Codling Moth, Cydia pomonella (Lepidoptera: Tortricidae) to (E,E)-8,10-Dodecadien-1-ol and Its Geometrical Isomers. El-Sayed, A., Liblikas, I. and Unelius, R. Z. Naturforsch., C: J. Biosci. (2000) 55(11/12): 1011-1017.

V Detection of Sex Pheromone Components in Manduca sexta (L.). Kalinova, B., Hoskovec, M., Liblikas, I., Unelius, C.R. and Hansson, B.S. Chem. Senses (2001) 26(9): 1175-1186.

VI Identification of Further Sex Pheromone Synergists in the Codling Moth, Cydia pomonella. Witzgall, P., Bengtsson, M., Rauscher, S., Liblikas, I., Bäckman, A.-C., Coracini, M., Anderson, P. and Löfqvist, J. Ent. Exp. Appl. (2001) 101(2): 131-141.

VII Parthenogenesis, Calling Behaviour and Insect Released Volatiles of the Leaf Miner Moth Phyllonorycter emberizaepenella. Mozūraitis R., Būda V., Liblikas I., Unelius C.R. and Borg-Karlson A-K. J. Chem. Ecol. (2002) 28(6): 1191-1208.

VIII Activity Sex Pheromone components on the Currant Shoot Borer males (Lampronia capitella (Cl.) (Lepidoptera: Prodoxidae). Liblikas, I., Mõttus, E., Kännaste, A., Kuusik, S., Ojarand, A., Mozuraitis, R., Santangelo, E., Mõttus, M., Unelius, R.C. and Borg-Karlson, A.-K. Manuscript for Chemoecology.

IX On the Variability of Genitalia and Pheromone Communication Channel of Archips podana (Scopoli) (Lepidoptera: Tortricidae). Liblikas, I., Mõttus, E., Nikolaeva, Z.V., Ojarand, A., Ovsyannikova, E.I., Grichanov, I.Ya., Ivanova, T.V. and Yemeljanov, V.A. Proc. Estonian Acad. Sci. Biol. Ecol (2004) 53(2): in press.

X Synthesis of Some Alkenoic Plant Volatiles. Liblikas, I., Mõttus, E. and Nilson, A. Proc. Estonian Acad. Sci. Chem. (1996) 45(3/4): 146-151.

LIST OF CONTENTS

ABSTRACT LIST OF APPENDICES AND PAPERS ABBREVIATIONS I. INTRODUCTION 1 II. BACKGROUND AND AIM OF THE THESIS 2 1. Pheromones in insect pest management 2 1.1. General characterization of the Lepidopteran sex pheromones 2 1.2. Methods of application of insect sex pheromones in pest management 3 1.3. The advantages of pheromone-based methods in plant protection 5 2. Aim of the thesis 6 III. SYNTHESES OF CONJUGATED POLYENIC COMPOUNDS 11 1. Selection of methods for the syntheses of conjugated polyenic compounds 11 2. General reactions, methods and transformations 13 2.1. Methods of purification 13 2.1.1. Methods of chemical purification 13 2.1.2. Methods of isomeric purification of conjugated polyenic compounds 13 2.2. Oxidation reactions 14 2.2.1. Oxidation of alcohols to aldehydes 14 2.2.2. Oxidation of bromides to aldehydes 16 2.3. Semihydrogenations 16 2.3.1. Semihydrogenations of triple bonds in alkenynes 16 2.3.2. Semihydrogenations of triple bonds in diynes 17 2.4. Monobromination and monotetrahydropyranylation 18 3. Results of syntheses of polyenic pheromone components 18 3.1. General aspects of the Wittig reaction 1 18 3.2. Wittig reactions in the synthetic schemes of conjugated dienic pheromone components 21 3.2.1. Syntheses of (Z,E)-isomers 21 3.2.2. Syntheses of (E,Z)-isomers 22 3.3. Modifications of the Wittig reaction 24 3.3.1. Schlosser`s modification of the Wittig reaction 24 3.3.2. Horner-Wadsworth-Emmon’s reaction 24 3.4. Alternative syntheses of the trienic pheromone components of Manduca sexta 26 3.5. Syntheses of (3E,5Z)-3,5-dodecadienyl and (3E,5E)-3,5-dodecadienyl acetates via Horner-Wadsworth-Emmon’s condensation 27 3.6. Alternative synthetic methods for the syntheses of (3E,5Z)- and (3Z,5E)-isomers of conjugated dienic pheromone components 27 3.6.1. Synthesis of (3E,5Z)-3,5-dodecadienyl acetate using the coupling reaction of alkenyl cuprates 27 3.6.2. Alkenyl-alkynyl coupling of vinylorganoboranes and alkynyllithiums 27 3.6.3. Pd-catalyzed cross-coupling reactions of free acetylene with (Z)-1-haloalkene or (E)-1-haloalkene 28 3.7. Syntheses of (Z,Z)-conjugated dienic pheromone compounds 30 3.7.1. Cadiot-Chodkiewicz reaction 30 3.7.2. Pd-catalyzed cross-coupling of two alkenyl moieties 31 3.8. Syntheses of (9Z,11Z)-9,11-tetradecadien-1-ol and the corresponding acetate and aldehyde 34 IV. CALCULATION OF PHEROMONE COMMUNICATION CHANNEL PARAMETERS 36 V. CONCLUDING DISCUSSIONS 37 REFERENCES 40 ACKNOWLEDGEMENTS 49 APPENDICES 51

ABBREVIATIONS

Ac2O acetic anhydride AIBN 2, 2’-azobisisobutyronitrile n-BuLi or BuLi n-butyl lithium Bz benzene Catecholborane 1,3,2-benzodioxaborole DHP 3,4-dihydro-2H-pyran DIBAL or DIBAH diisobutylaluminium hydride DMSO dimethyl sulfoxide Disiamylborane bis(3-methyl-2-butyl)borane HMPT hexamethylphosphorous triamide HOAc acetic acid KOt-Bu or t-BuOK potassium tert-butoxide LDA lithium diisopropylamide LAH lithium aluminium hydride MPLC medium pressure liquid chromatography NaHMDS or NaHDMS sodium bis(trimethylsilyl)amide NMO 4-methylmorpholine N-oxide ORN olfactory receptor neuron PCC pyridinium dichromate PDC pyridinium chlorochromate P-2 nickel or P-2-Ni borohydride-reduced nickel PPTS pyridinium p-toluenesulfonate THF tetrahydrofuran THPO– tetrahydropyranyl ether TLC thin-layer chromatography TMEDA N,N,N’,N’-tetramethylenediamine

I. INTRODUCTION

At least one third potential food harvest of the world is lost to pests. The vulnerability of crops to pests makes pest control one of the major management components of the total crop production system. Integrated pest management (IPM) is the optimization of pest control in an economically and ecologically sound manner, accomplished by the coordinated use of multiple tactics to assure stable crop production and to keep the pest damage below the economic injury level, while hazards to humans, animals, plants and the environment are minimized. In its broadest form, an IPM program encompasses all significant components of the agro ecosystem - soil, crops, water and air, insects, pathogens, weeds, nematodes and other organisms, which interact among themselves and with other components of the system. The concept of IPM is based on the recognition that no single approach to pest control offers a universal solution, and that the best crop protection can be provided by a fusion of various tactics and practices based on sound ecological principles. A considerable attention has been focused on finding alternative approaches to the use of conventional pesticides in the control of insect pests. One of the important developments in this direction has been the evaluation of pheromones in the integrated pest management. A large number of pheromones have been investigated, but of these, the ones that pertain to agricultural pests have received the greatest attention. Several new tactics in crop protection, such as the use of hormones, allelopathy and molecular genetic manipulation, will probably be of limited use during the next decades, although they have great potential for the later future. One exception will probably be the use of pheromones to control insects, a technique that has presently become widely adopted in sustainable agriculture. In discussing the use of pheromones and other behaviour modifying chemicals in pest control, it is important to point out that these chemicals will never eradicate pests completely, and that they will not be the only means applied. In the overall effort to control pests, these chemicals will probably play a significant role along with pesticides, sterilized males, natural enemies, resistant plant strains and numerous techniques, which are chemical, biological and agrotechnical in nature.The new identifications of the pheromones and the development of a practical chemical synthesis allow the necessary biological research to be carried out in order to determine whether or not the compounds can be used to interfere with the normal behaviour of the insect species and to aid in a successful pest control. Like in the advances in chemical identification, there has been tremendous progress in chemical synthesis, mass production and formulation for prolonged, controlled release. Tiny amounts of a pheromone have been found to work in a monitoring program. Progressively, more compounds are needed as we focus on mass trapping and mating disruption. Each step in this process has enhanced the integrated pest management by refining the application of control techniques or offering new, non-insecticidal control options. The research work demonstrates the importance of fundamental and interdisciplinary research dealing with the chemistry of biologically active compounds in combination with biological studies. Such combined studies provide tools for future applications of the compounds for the benefit of mankind. Research works in organic chemistry, both analytical and synthetic, play essential roles in such research efforts. This thesis deals with the syntheses of conjugated polyenic pheromone components and the application of Lepidopteran sex pheromones as a part of IPM.

II. BACKGROUND AND AIM OF THE THESIS

1. Pheromones in insect pest management

1.1. General characterization of the Lepidopteran sex pheromones

Pheromones (Gk. phereum, to carry; horman, to excite or stimulate) are chemicals emitted by one member of a species to cause a specific reaction with another member of the same species (Karlson and Butenandt, 1959). Pheromones may be further classified on the basis of the interaction mediated, such as alarm, trail, aggregation or sex pheromones (Jutsum and Gordon, 1989). The class the most widely explored are the sex pheromones excreted by females to attract conspecific males for mating. They are the sex pheromones of insects that are of particular interest to agricultural integrated pest management (IPM) practitioners. The research on insect pheromones has flourished in the last 40 years because of advances in the collection, isolation and chemical identification of very small quantities of pheromones produced by living insects (Millar and Haynes, 1998). Among the insect taxa, in which mate-finding is mediated by pheromones, the Lepidoptera is the order that has been best investigated (Tumlinson, 1988). Up to the end of 20th century, Lepidopteran sex pheromones have been identified from more than 500 species and sex attractants reported for approximately 1500 species (Arn et al., 2000). The largest chemical group of Lepidopteran pheromones (about three-quarters of them) is composed of C10–C18 saturated or unsaturated straight-chain fatty alcohols and their derivatives (acetates and aldehydes), having up to three conjugated or non-conjugated double bonds with E- or Z-configuration. The double bonds occur on various locations, from the methyl terminal to next to the functional group, i.e. at almost all possible positions. This type of pheromone components are biosynthesized from saturated fatty acids in the pheromone glands by a combination of chain-shortening, desaturation, reduction and acetylation reactions (Bjostad et al., 1987; Ono et al., 2002). The second major group of moth pheromone components consists of saturated, mono- or poly-unsaturated (one to four double bonds of Z- and E-types) linear and branched hydrocarbons with 15-29 carbon atoms in the molecule and their monoepoxides and are probably biosynthesized from linolenic acid and linoleic acid (Rule and Roelofs, 1989; Millar, 2000). The third group are C7-C9 straight chain, mono- and diunsaturated alcohols or ketones, with a functional group in position 2 and either S- or R-configuration (Arn et al., 2000). Each species has established a species-specific communication system for mating strictly with a partner of the same species utilizing the unique sex pheromone components. Among moths it has usually been the female that has released a species-specific pheromone blend with variations in chain length, degree of unsaturation, functional group and total number of compounds to attract the male for mating (Linn and Roelofs, 1989; Roelofs, 1995). The identification of the sex pheromone of the silkworm moth, Bombyx mori (in 1959 by Butenandt), can be regarded as a start of the decoding of the chemical language which plays a highly significant role between male and female insects. In addition to bombykol, the first pheromone identified from insects, many pheromone components with a conjugated dienyl structure have been identified from numerous Lepidopteran species. Conjugated dienes constitute one of the essential chemical groups of the pheromones, but their biosyntheses have been investigated in only five species (Ono et al., 2002). It was originally thought that pheromones found in the Nature were single compounds, but it was then discovered that most pheromones investigated were mixtures of several components. The first pheromone identified in the silkmoth had a single component that elicited a response in males. This led to the misconception that all pheromones were made from a single component unique to its own species. The discovery of multiple compound pheromones was

made when individual components of pheromones were tested on insects and received no response (Sorensen, 1996). In most lepidopteran species there are at least two distinct pheromone compounds that are obligatory to cause a response by the opposite sex (Kaissling, 1996). Furthermore, several moth species have pheromone mixtures of up to seven components that function in attraction (Sorensen, 1996). The numbers of chemicals within pheromones attribute to its uniquity against other insect pheromones. The components of pheromones are similar among many insect species but present in different relative concentrations. Within the 4500 Lepidopteran species native to Central Europe, only 150 chemically distinct pheromone components have been identified (Arn et al., 2000). Thus the narrowly defined difference in component ratio makes pheromones of each species distinct from each other. Pheromones are a primary method of communication in insects, by which the chemicals serve to identify members of the same species and thus possible mates. Pheromones have an evolutionary importance for insects, as they inform other members of the species of their availability to mate, and prevent attempted copulation between different species or individuals of the same sex. The receptor neurons are highly sensitive to the concentrations of the pheromone components (Kaissling, 1996), which allows nature to utilize fewer chemicals and still elicit a specific response only by members of the same species. Pheromones have both inter- and intraspecific effects. One role of the interspecific effect is to maintain reproductive isolation between sympatric species (Mustaparta, 1996; Tamaki, 1985). Intraspecific effect means that pheromone produced by individuals of a species modify the behaviour of individuals of the same species. Intraspecific pheromones are used to signal the readiness of an individual to mate, thus inducing a pre-copulatory behaviour (Ferveur, 1997). Specificity of male attraction is achieved by precisely regulated proportions of blend components and by synergistic as well as antagonistic compounds (Roelofs and Wolf, 1988; Linn et al., 1988). There is evidence that male flight behaviour changes with pheromone blend composition (Palaniswamy et al., 1983; Willis and Baker, 1988; Quartey and Coaker, 1993). The effectiveness of behavioural studies depends on the purity of compounds because small amounts of contaminants may have considerable effects.

1.2. Methods of application of insect sex pheromones in pest management

There are four main uses of pheromones in the integrated pest management of insects.

1) Detection and Monitoring. The principal use of insect sex pheromones is to attract insects to traps for detection and determination of their temporary distribution. In most instances, it is the males who respond to female-produced sex pheromones. Trap baits, therefore, are designed to reproduce the ratio of chemical components and the emission rate of calling females. Ideally, the trap bait should uniformly distribute its pheromone content over time and not permanently degrade the pheromone in the process. Attractant-baited traps are used in monitoring programs for at least three purposes: ⇒ to estimate the relative density of a pest population at a given site; ⇒ to indicate the first emergence or peak flight activity of a pest species in a given area, often to time an insecticide application and eliminate unnecessary spraying or to signal the need for additional sampling efforts; ⇒ to detect the presence of an immigrant pest. For pests that cause unacceptable levels of damage even at low population densities, such as the codling moth or apple maggot in commercial apple orchards, traps can be used as the only sampling method for determining the dates to begin and end insecticide application programs.

Consistent trapping protocols are essential for population evaluations, spray thresholds and year to year comparisons. The information from trap catches can be very useful for decision making on insecticide applications or other control measures. Monitoring pest populations with pheromone-baited traps is one of the most useful applications derived from basic research in chemical ecology. This monitoring function is the keystone of integrated pest management.

2) Mass trapping. A second major use of pheromones is to mass trap insects, in order to remove large numbers of insects from the breeding and feeding population. Massive reductions in the population density of pest insects ultimately help to protect resources such as food or fibre for human use. Since pheromone traps are highly effective for catching certain insects, numerous traps placed throughout the environment of a pest can sometimes remove so many insects that the local population as well as the damage it causes are essentially limited. Mass trapping of insect pests is a process, also termed removal trapping or "trap out", that uses species-specific sex pheromones that attract male moths. The success of mass trapping depends upon capturing males before the mating occurs. Although mass trapping programs using chemical attractants have targeted such important pests as bark beetles, codling moth, apple maggot, Japanese beetle and Indian meal moth, field-scale successes have been limited. For mass trapping adequately to reduce pest populations, a large number of very efficient traps are usually needed. Efficient traps capture a high percentage of the target insects. For many insects, the efficiency of commonly used traps is not known; however, low efficiency seems to be a limiting problem in some instances. Removal trapping is also most likely to succeed when the density of the target pest is low and the immigration into the area trapped is minimal.

3) Mating disruption. A third major application of pheromones aims at in the disruption of mating in populations of insects. With the commercial availability of insect sex pheromones of several agricultural pests in the 1970s, scientists and entrepreneurs turned their attention to mating disruption as a "biorational" approach to insect control. In theory, mating disruption might be accomplished in two principal ways: false trail following or confusion. False trail results from dispersing many point sources of pheromone into crops. The number of males finding females at the end of the pheromone trail must be greatly reduced. The emission of pheromone is relatively low from each source, at which a downwind trail is created and not lost in a background of released pheromone. Males following these trails are thought to spend their mating energies in pursuit of artificial pheromone sources. The false odour plumes attract males away from females that are waiting to mate. This causes a reduction of mating, and thus reduces the population density of the pests. Male confusion is thought to be the result of ambient pheromone concentrations sufficient to hide the trails of calling females. The effect is the adaptation of antennal receptor sites and/or habituation of the central nervous system of the insect. When a receptor site is continually activated by high ambient concentrations of pheromones, the resulting electrical signal diminishes, the receptor site becomes unresponsive and the insect becomes navigationally blind. Mating disruption programs are most successful where a large area is treated, where the treated area is isolated from sources of pests that might immigrate, and where the pest population is low. Pheromone-based mating disruption is an alternative management strategy for many lepidopteran pests. Mating disruption is used to control several species, including the codling moth, Cydia pomonella (L.) (Charmillot, 1990); pink bollworm, Pectinophora gossipiella (Saunders) (Baker et al., 1990); oriental fruit moth, Grapholita molesta (Busck) (Rice and Kirsch, 1990); grape berry moth, Endopiza viteana (Clemens) (Dennehy et al., 1990); European grape moth, Eupoecilia ambiguella (Arn et al., 2000); grapevine moth, Lobesia botrana (Arn et al., 2000).

Although the highest efficacy in mating disruption may be achieved when the disruptant closely resembles the pheromone blend emitted by female moths (Roelofs, 1978: Minks and Gardé, 1988), release from sympatric, coseasonal moths should also be considered for disorientation of mate-seeking males (Vakenti et al., 1988). Field studies have shown that the non-pheromonal isomers as well as an equilibrium blend of the four isomers of codlemone, disrupt male codling moth orientation to females more efficiently than pure codlemone alone (McDonough et al., 1994 and 1996).

4) Attractant bait containing an insecticide. Sex pheromones are used in attractant baits containing an insecticide. Combining insect attractants with insecticides is a practice that has been used in pest management for many years. Insects that were attracted to the lure treated, were killed by an insecticide that could not be applied safely, economically or effectively in any other manner.

Most of the present commercial formulations of pheromones for both trap baits and mating disruption mimic the natural chemical blends of females as clearly as possible. Most insect sex pheromones are multicomponent with precise ratios of components which may be expensive to manufacture. Many commercial agricultural systems use IPM methods to manage pest problems, and home gardeners can use similar methods to control pests in their gardens (Ebesu, 2003). The codling moth, L. pomonella (Lepidoptera: Tortricidae), is an excellent example illustrating the progress of application pheromones in plant protection. The pheromone monitoring began in the 1970s, mass trapping efforts in the 1980s and mating disruption in the 1990s. The latter is now an integral part of integrated pest management programs in pome fruit orchards.

1.3. Advantages of pheromone-based methods in plant protection

Insect sex pheromones are inherently different from conventional pesticides. The advantages of the pheromone-based methods are self-explanatory: ⇒ Pheromones help us to protect our plants against certain pests without the need of a pesticide or a significant reduction of the number of treatments and thus working with minimum pollution of our environment. ⇒ Pheromones act by modifying the behaviour of the pest species rather than killing it, in other words, are more target-specific than conventional insecticides. Due to the specificity of pheromones no other harmless or useful insects are killed. ⇒ Pheromones are generally effective at very low rates and used at concentrations close to those in nature. They are volatile and usually dissipate rapidly in the environment. ⇒ Pheromones are environmentally friendly and non-toxic to humans and wildlife. They are compounds of natural occurrence. ⇒ Formulated in passive dispensers that present little direct exposure to humans and non-target organisms. ⇒ Application of pheromone-based methods does not result in insect strains developing a natural resistance. ⇒ Pheromones can be used at any stage of crop growth.

2. Aim of the thesis

The development of effective tools for plant protection is a result of complex broad, interdisciplinary basic and applied research. A practical use of pheromones for pest management usually requires that specific active chemicals must be isolated, identified and produced synthetically. The creation of pheromone materials besides requires the identification of the active components, determination of the roles of other possible isomers or degradation products, the design of dispensers of suitable substrates and evaporation parameters.

The aim of the thesis is critically to survey methods for the syntheses of conjugated polyenic compounds and to focus to the synthesis of several conjugated polyenic pheromone components providing sufficient quantities of chemically and isomerically pure compounds for bioassaying.

An identification of the chemical constituents present in the female pheromone gland results in several compounds. Male insects respond both physiologically and behaviourally to these compounds. The compounds eliciting the EAD-responses are selected for studying the behavioural influence, and for working out attractive baits useful for plant protection. A few components of the glands composition are usually required to complete a characteristic behavioural sequence. The importance of other constituents can be cleared up using field trapping or wind tunnel assays or other methods. Additional compounds can act as antagonists or synergists. Many insect species utilize the same compounds in their pheromone mixtures but in different ratios, and many species produce the isomers of the same component in precisely defined ratios. For these reasons it is especially important to use chemically and isomerically very pure compounds in bioassaying and pheromone materials development. In addition to the chemical composition, the amount of substance released is of great importance in pheromone-based systems for control or monitoring of insect species. Different kinds of dispensers have been tried with respect to constancy and predictability in release rate. An ideal dispenser should have a constant release rate during the whole flight period of the target insect (Byers, 1988). The design of effective dispensers for trapping or disruption also needs chemically and isomerically pure compounds.

The principal objective of the thesis was concerned with the further aim of developing effective synthetic materials for plant protection. The work was focussed on solving the problems connected with several insects:

⇒ To identify the compounds collected from the widespread leafminer moth Phyllonorycter emberizaepenella (Lepidoptera: Gracillaridae) and perform a detailed identification of the pheromones of the Brazilian apple leafroller Bonagota cranaodes (Meyrick) (Lepidoptera: Tortricidae) and of the codling moth Cydia pomonella (L.) (Lepidoptera: Tortricidae). ⇒ To study the effect of non-pheromonal isomers of the main pheromone component on the sex attraction of B. cranaodes and C. pomonella in wind tunnel and field tests. ⇒ To study the role of identified pheromone components in the pheromone communication of the currant shoot borer Lampronia capitella (Cl.) (Lepidoptera: Prodoxidae), and to design and optimize the dispenser for effective trapping. ⇒ To investigate the presence of sex pheromone components in the tobacco hawk moth Manduca sexta (L.) (Lepidoptera: Sphingidae).

Background of the bioassays:

1. (10E,12E,14Z)- and (10E,12E,14E)-(10,12,14)-hexadecatrien-1-als are sex pheromone components of the tobacco hawk moth, Manduca sexta (L) (Lepidoptera, Sphingidae). Two of the M. sexta main sex pheromone components were planned to be prepared in an aim to find out how males detect the minor pheromone components and whether there are any pheromone-sensitive olfactory receptor neurones on female antennae or not (appendix 3, paper V).

The tobacco hornworm moth, Manduca sexta, is not only a common pest on tobacco, but its larvae also eat the leaves of a wide range of other solanaceous plants including tomato, eggplant, Jerusalem cherry, and potato. The search for the sex pheromone of M. sexta involved many investigations over several years. The isolation and identification of the female-produced sex pheromone was reported (Tumlinson et al., 1989) as a blend of twelve saturated and mono-, di-, and triunsaturated aldehydes with chain lengths of 16 and 18 carbon atoms. In the laboratory wind tunnel tests two components of this blend, 10E,12Z-16OAld and 10E,12E,14Z-16OAld, were required to stimulate M. sexta to complete a characteristic behavioural sequence: upwind orientated flight in the pheromone plume, approaching and touching the pheromone source, and bending their abdomens in apparent copulatory attempts. Although other aldehydes did not demonstrate clear behavioural activity in wind-tunnel tests, all of the 16-carbon aldehydes found in the pheromone glands elicited some response in olfactory interneurons in males, whereas 10E,12Z-16OAld, 10E,12E,14Z-16OAld and 10E,12E,14E-16OAld evoked the greatest response. The 8- component mixture was found to attract more males than the live female or the two- and four- component blends in field attraction tests (Tumlinson et al., 1994). M. sexta is one of the most thoroughly studied insect models used in olfactory research. It is the experimental insect of choice of many physiological and biochemical studies due to its rapid rate of development and a good size, and it can be reared on artificial diet easily enough to make it readily available for a variety of investigations. That makes the synthesis of M. sexta pheromone components especially valuable.

2. The synthesis of four geometrical isomers of 3,5-dodecadienyl acetate is planned to be performed for detailed identification of compounds produced in female insect sex pheromone glands of the Brazilian apple leaf roller, Bonagota cranaodes (Meyrick) (Lepidoptera: Tortricidae) and for the first field trapping tests of the pheromone candidate compounds (II, III).

The leafroller moth, Bonagota cranaodes, is the economically most important insect pest on apple in South America (Lorenzato, 1984). B. cranaodes accounts for an annual crop loss of 3-5% despite intensive insecticide spraying (Kovaleski, 1992). The damage is done by larvae, which feed on the fruit surface, are difficult to control with insecticides and need multiple treatments (Eiras et al., 1994; Coracini et al., 2001). There is a considerable interest for developing a monitoring system based on a synthetic pheromone lure, as outbreaks of B. cranaodes occur throughout the growing season and this necessitates frequent insecticide sprays. The development of the reliable synthetic lure for routine monitoring of the seasonal abundance of B. cranaodes is the first step toward a reduction of insecticide use; the future goal is the development of an environmentally safe control of B. cranaodes by mating disruption. The main pheromone component is (3E,5Z)-3,5- dodecadienyl acetate (Unelius et al., 1996). Attempts to develop pheromone materials for a mating disruption technique require additional knowledge of the female sex pheromone of B. cranaodes. Regardless of the purity of the starting material, dienic pheromone compounds are known to isomerise rapidly on rubber septa used for field trapping (Chisholm et al., 1985; Brown and McDonough, 1986; Witzgall et al., 1993).

Therefore, it was necessary to synthesize all nonpheromone isomers and to test their possible antagonistic or synergistic effect in binary blends with the main compound.

3. All geometrical isomers of 8,10-and 10,12-tetradecadienols and corresponding acetates were synthesized for use as reference compounds in identification of the compounds collected from the calling female of Phyllonorycter emberizaepenella. The synthetic compounds were also needed for the field tests of potential sex pheromone components (appendix 1; paper VII).

An investigation was planned in order to verify reproduction by parthenogenesis of the thelytoky type of the widespread leafminer moth P. emberizaepenella that has been reported earlier (Būda et al., 1991) but never confirmed, as well as to elucidate the sexual behaviour of this moth and to determine whether or not it released potential sex pheromones during the calling. It was also planned to perform field screening experiments of several Phyllonorycter species. It is known that (10Z,12Z)-10,12-tetradecadien-1-yl acetate and (10E,12E)-10,12- tetradecadien-1-yl acetate are sex pheromone components of the apple blotch leafminer, Phyllonorycter crataegella (Ferrao et al., 1998).

4. The syntheses of the geometrical isomers of codlemone, (E,Z)-, (Z,E)-, and (Z,Z)- 8,10-dodecadienol, codlemone acetate and aldehyde were planned for a study of their effect on the sex attraction of male the codling moth, Cydia pomonella (L.) (Lepidoptera, Tortricidae), to the main pheromone component, (E,E)-8,10- dodecadienol, in the field and in a wind tunnel (appendix 1; papers I, IV, VI).

C. pomonella is the most important pest of pears and apples worldwide. Larvae of the codling moth make the fruits unsuitable for most commercial markets. The codling moth is responsible for most of the insecticide sprays on apples and pears. The resistance of the codling moth towards the standard insecticides has increased to the point at which growers are forced to increase the number of sprays. Researchers have estimated that 90-95% of the male codling moths in an orchard must be trapped or prevented from finding a mate, to reduce the number of fertile eggs laid by females to an economically manageable level.

The main sex pheromone compound, (8E,10E)-8,10-dodecadienol (codlemone), was identified by Roelofs and co-workers (Roelofs et al., 1971) using electroantennographic recordings. The development of analytical instruments and techniques made it possible to identify eight minor components besides the main one, codlemone, present in the pheromone gland of Cydia pomonella (Einhorn et al., 1984; Arn et al., 1985). There have been intensive studies of the pheromonal communication of codling moth since the main pheromone component, E,E-8,10-dodecadienol, was identified. In spite of that the role of sex pheromone synergists and antagonists has remained unclear. For these reasons this study has been undertaken to re-test the effect of the minor gland components on the attractivity of codlemone.

The behavioural role of minor female-produced compounds is of importance with respect to the development of pheromone-based control techniques. The addition of behavioural synergists to the main compound enhances the male behavioural response and the species- specificity of synthetic blends. This is critical for monitoring lures. At the same time there is evidence that the absence of minor compounds increases the effectiveness of mating disruption formulations (Minks and Cardé, 1988; Cardé and Minks, 1995). A behavioural synergistic effect has been demonstrated for two saturated alcohols, 12OH and 14OH (Arn et al., 1985; Einhorn et al., 1986; Bartell et al., 1988), but in later flight tunnel tests the activity of these compounds could not be substantiated (McDonough et al., 1993). None of the tests indicated the presence of sex pheromone components other than codlemone (McDonough et al., 1995).

The further work on minor components has been to improve the pheromone-based control techniques. It has already applied the 3-component blend of codlemone, 12OH and 14OH for control of the codling moth by mating disruption techniques (Barnes et al., 1992; Howell et al., 1992; McDonough et al., 1992; and Thomson et al., 1998). Field experiments showed that addition of the E,Z- and Z,E-isomers of 8,10-dodecadienol disrupt the male orientation to females more efficiently than pure codlemone alone (McDonough et al., 1994, 1996). Addition of these isomers reduced the male attraction to codlemone in pheromone-baited traps.

Codlemone is not abundant in the female sex pheromone glands of tortricid species, and it is behaviourally active only in codling moth (Arn et al., 2000). Therefore there is a low selection pressure on the chemical communication channel in codling moth, which may explain the strong attractiveness of the codlemone as the main pheromone compound alone. In closely related species, the chestnut moth Cydia splendana, the geometrical isomer of codlemone, 8E,10Z-12OH, was identified as a sex pheromone component. The other isomer, 8Z,10Z-12OH is known as a sex pheromone component of the other three tortricides. The 8Z,10E-12OH shows no pheromonal activity among tortricid species.

The behavioural role of the codlemone isomers needs to be identified. For this purpose, optimal dispensers for mass-trapping and for further development of the mating disruption technique have to be designed. The codlemone isomers are present in sex pheromone gland extracts and elicit the male antennal response. The corresponding acetate, E,E-8,10- dodecadienyl acetate, and aldehyde, E,E-8,10-dodecadienal, are also present in the gland and show the EAD response. The occurrence of the minor components in subnanogram quantities in extracts complicated their detection and identification. Therefore an attempt was planned to find additional components from the pheromone gland extracts of the female codling moth.

Thus, there was the need to synthesize and purify (E,Z)-, (Z,E)-, and (Z,Z)-8,10- dodecadienol and their acetates, E,E-8,10-dodecadienyl acetate and E,E-8,10-dodecadienal to observe the effect of each single compound on the male attraction to codlemone in the field and in a wind tunnel.

5. (9Z,11Z)-9,11-tetradecadienol, corresponding acetate and aldehyde had to be synthesized for studies of their role in the pheromone communication of the currant shoot borer Lampronia capitella (Cl.) (Lepidoptera: Prodoxidae). Pheromone components were needed to perform the field tests to evaluate the dispenser for monitoring and mass-trapping of L. capitella (VIII).

L. capitella is a serious pest insect causing damage in black and red currant plantations. In early spring, larvae of L. capitella damage the leaves and flower buds. In the summer this pest also damages berries, causing them to fall before harvest. Three pheromone components of this insect, (9Z,11Z)-9,11-tetradecadienol and the corresponding acetate and aldehyde, were identified quite recently by Löfstedt and co-workers (Löfstedt et al., 2004). For economic reasons, it is needed to create a working dispenser for monitoring, field trapping or disruption. The pheromone source efficiency is a combination of several sequential processes: the initiation of an upwind pheromone-orientated flight, approaching the source, and trapping the attracted males. The effective trap has to catch a maximum number of the insects attracted near the trap. The insects not trapped remain hovered around the sex pheromone source, and have the opportunity to mate before being trapped during some later period of activity. In this case the number of insects trapped is high, but the effect on increasing the number of offspring is not reduced significantly. This phenomenon may be one of the explanations of the failure of mass- trapping experiments and stresses the importance of optimization of dispensers.

Due to different evaporation rates, the ratio of components in blend vary in time, and to ensure its behavioural activity we must know the rate of change in component ratio during the test period. Before starting the determination of the role of pheromone components, it is evidently essential to design a dispenser with known parameters: emission rate and ratio of components in the blend and the stability of the substances in the dispenser. This is one of the aims of this study. Field experiments to elucidate the behavioural activity of (9Z,11Z)-9,11-tetradecadienol, and the corresponding acetate and aldehyde, using different formulations to attract L. capitella males, were planned. Since very little is known about the pheromone of L. capitella, there is an obviously need of ongoing research for several years. Therefore, (9Z,11Z)-9,11-tetradecadienol is the compound needed to be synthesized and purified repeatedly. That makes it rational to create a synthetic scheme in which each step is optimized for maximum yield and purity. Reproducibility, convenience and simplicity are also factors that need consideration.

III. SYNTHESES OF CONJUGATED POLYENIC COMPOUNDS

1. Selection of methods for the syntheses of conjugated polyenic compounds

The most important goal in the selection of methods is critically to survey methods for the syntheses of polyenic components of pheromones. The general strategies and methods employed in synthesizing insect pheromones constitute a collection almost as broad as the entire scope of organic synthesis. The preparation of a polyenic pheromone compound usually consists of a sequence of 6- 10 reactions. The need for regio- and stereochemically pure compounds for biological tests is well-recognized. The effectiveness of synthesized pheromones and attractants in the field is affected by many factors; among these the chemical and steric purity of the strereoisomers plays an important role. Thus, particular care must be devoted to the regio- and stereochemical control of the reactions used for the construction of such molecules. The chemical structures of many insect sex pheromones contain a long carbon chain with terminal functionalities like alcohol, acetate or aldehyde groups, and conjugated diene systems. These systems can be of the EE-, EZ-, ZE- or ZZ-types. As the skeleton, with desired length of the carbon chain and the number, positions, and stereochemistry of the double bonds needed, are set up, the changes between alcohol, acetate and aldehyde structures proceed without difficulties. Thus, the attention must be paid to the construction of such skeletons.

Numerous methods have been developed for the stereoselective syntheses of conjugated polyenes: ⇒ Syntheses from carbonyl compounds by Wittig type approaches. ⇒ Partial reductions of diynes prepared by Cadiot-Chodkiewicz coupling reactions between alkyne and bromoalkyne compounds. ⇒ Carbon chain extension of a diene system already prepared and bearing an allylic functionality that can be substituted by a Grignard reagent. ⇒ Selective reduction of the acetylenic unit in 1,3-alkenynes which can be prepared in various ways: 1) Alkenyl-alkynyl couplings of vinylorganoboranes and alkynyllithiums. 2) Pd-catalyzed cross-couplings of alkenylboranes with haloalkynes. 3) Pd-catalyzed cross-coupling reactions of free acetylene with (E)- or (Z)-1- haloalkenes. 4) Wittig reactions. ⇒ The direct coupling of two alkenyl groups is the most straightforward and widely used approach. Most frequently used are Pd-catalyzed cross-coupling reactions between: 1) Grignard reagent and alkenyl halide, 2) Organoaluminium or organozinc compound and alkenyl halide, 3) Organoboron compound and alkenyl halide, and by 4) Cross-coupling reactions of dialkenyl cuprates.

There is no outstanding and universal method for the synthesis of most of the polyenic pheromone compounds. Many of the methods used suffer from serious drawbacks; most frequent is the formation of the undesired EE-isomer, the thermodynamically most stable one, in the synthesis of EZ- or ZE-dienic compounds. In many other cases the preparation of the starting materials is tedious and necessitates many steps under drastic reaction conditions. Organometallic compounds are very often expensive and highly air and water sensitive. The task to deliver all the four geometrical isomers with conjugated diene systems can be performed according two strategies: synthesizing all the four isomers separately and with maximum isomerical purity, or synthesizing the four isomers by routes, each yielding two isomers in a mixture.

The synthetic routes to polyenic pheromone compounds are usually sequences of several steps. Therefore, is not easy and economical to synthesize each of the four geometrical isomers separately in the stereoselective mode. Instead, synthetic routes are examined in which only one of two C=C double bonds is furnished highly stereoselectively and the other one rather nonspecifically (Ando et al., 1985). Separation of each component from the mixture of four isomers with conjugated diene systems must be difficult, but the two isomers obtained according to Ando et al. can be separated successfully. Ando et al. (1985) synthesized four geometrical isomers of 5,7-, 6,8-, 7,9- and 8,10- dodecadien-1-ols systematically by two routes: one involved the Wittig reaction between (E)-2- alkenal and a phosphorane with an appropriate carbon chain, and yielded a mixture of (E,Z)- and (E,E)-isomers in the ratio 3:2. The other route involved the Wittig reaction of a 2-alkynal and the stereoselective reduction of the triple bond in the resulting enyne to a (Z)-double bond by hydroboration with dicyclohexylborane to yield a mixture of (Z,Z)- and (Z,E)-isomers. Both of the mixtures were chromatographed separately on silver nitrate impregnated silica gel, to give four geometrically pure isomers. A de novo stereospecific approach for each geometrical isomer would only be time- consuming (Coffelt et al., 1979)! They also used the 2+2 principle.

As similar conception was used in the following papers presented here (appendix 1 and papers II, III and VII). The E,Z- and E,E-isomers of 3,5-dodecadienyl acetates, 8,10- tetradecadienols and 10,12-tetradecadienols, were synthesized as mixtures of two isomers. The synthesis of long-chain aldehydes with several conjugated double bonds in the molecules is certainly a troublesome preparation, because the compounds easily undergo degradation and extensive isomerization. Therefore, the reaction procedure has to be carefully chosen according to the properties of the individual compounds and the possibility of separating products from by-products. The decisive moment in the selection of the synthetic method is often either the commercial availability or the simplicity of preparation of the starting materials. Finally, important factors in selecting the methods for the syntheses of pheromone compounds are the technical situation and the equipment available in the laboratory, the traditional usage of certain reactions and procedures in the working group, and the experimental skills of the experimentarian.

The laboratory methods available for the syntheses of pheromone components are in principle different from biosynthetic pathways. The biosynthesis of sex pheromones in moths usually begins with the production of palmitic (C16) and stearic (C18) acids. The fate of these fatty acids depends on the chain length, degree of unsaturation and functional group of the pheromone blend in each species of moths. The production of the pheromone components in insects proceeds by the action of enzymes. The key enzymes involved in pheromone biosynthetic pathways are: ⇒ Acetyl-CoA carboxylase and fatty acid synthetase, to make fatty acids with 16 or 18 carbon atoms; ⇒ Specific desaturases, to make mono- and diunsaturated fatty acids; ⇒ Chain-shortening enzymes to make the right chain length fatty acid; ⇒ Depending upon the functional group of a particular pheromone, a reductase, an acetyltransferase or an oxidase is used, sometimes in combination, to make the final pheromone product. The order in which these enzymes are used determines the final pheromone components (Jurenka, 1997).

2. General reactions, methods and transformations

2.1. Methods of purification

2.1.1 Methods of chemical purification

As stated above, the synthetic route leading to polyenic pheromone compounds usually consists of 6-10 reaction steps. To be able to run each step without complications, it is important to use pure starting materials. The usual work-up procedure of the reaction mixture consists of treatment with water to remove water soluble materials, extraction of the product into an organic solvent, drying the extract and removing the solvent. The crude material can be purified either by distillation, crystallization or chromatographic methods.

In the syntheses presented here, most of the purifications are performed chromatographically on silica gel. That method is applicable to compounds having high enough boiling points to ensure the evaporation of the solvent without a great loss of the product. Chromatography has some very important advantages over distillation: the processes caused by high temperature (decomposition, polymerization or isomerization) are excluded. Chromatography is applicable even to so small amounts of the compound that distillation is technically unrealizable; 100 mg is not a problem for chromatographic separations. A high boiling point of the compound does not set the limits of using chromatographic methods. The loss of compound during a chromatographic procedure is minimal. In the reaction sequence, the compounds were usually purified before the next step, using medium pressure liquid chromatography (MPLC) with a SEPARO MPLC equipment (Bæckström Separo AB, Lidingö, Sweden). SEPARO variable-length glass columns with an inner diameter of 15, 25, 35 or 50 mm were dry packed with silica gel Merck 60 (0.040-0.063 mm). The columns were eluted with continuous gradients running from nonpolar to polar solvent medium afforded by a SEPARO constant-volume mixing chamber, combined with an open reservoir. Gradient elution with hexane and increasing amounts of ethyl acetate was performed as described by (Bæeckström et al., 1987; Jirón, 1996). A prerequisite was the solubility of the compound in a nonpolar solvent to apply the simplest method for loading by pump. Compounds not soluble in nonpolar solvents had to be preabsorbed on the silica gel.

2.1.2. Methods of isomeric purification of conjugated polyenic compounds

Crystallization at low temperature, chromatography on silver nitrate impregnated silica gel, formation of urea inclusion complexes and Diels-Alder adducts and high performance liquid chromatography are the methods of choice.

a) Crystallization at low temperature. Conjugated dienic alcohols, acetates and aldehydes can be purified by crystallization from hydrocarbon solvents (pentane, hexane) at -30 °C or lower. The method works well with alcohols having 12 or more carbon atoms in their chains. The lower the temperature, the quicker is the crystallization and the more impurities and minor isomers crystallize together with desired crystals. Better results are obtained with (E,E)- and (Z,Z)-alcohols, but (E,Z)- and (Z,E)-alcohols are also purified by crystallization. The smallest workable amount seems to be 0.2-0.3 grams of compound; less is too difficult to handle. Crystallization works with aldehydes and acetates as well.

b) Argentated silica gel chromatography. MPLC on pure silica gel does not differentiate the geometrical isomers of dienic pheromone compounds; therefore, silica gel impregnated with 15-20% of silver nitrate is used. The separation is based on the ability of olefins to form co-ordination compounds with silver ions. The procedures published give little experimental information; they do not publish an exact methodology for the separation of pheromone type compounds, although the method is widely applicable. A preparative-scale separation of alkene geometric isomers by MPLC, described by Evershed et al., (1982), gives no description of a methodology for the separation of conjugated dienic alcohols or aldehydes. It is very difficult to get baseline separation of the geometric isomers. Better results are obtainable with low loadings, which mean comparably large amounts of expensive silver nitrate. The overlapping fractions can be rechromatographed. The number of regenerations of the columns packed with the silver nitrate impregnated silica gel is limited due to the formation of the inactive silver species. In conclusion, it is tedious work to purify the pheromone compounds using argentated silica gel chromatography; the method is preferable from some hundred milligrams to some grams of compounds.

c) Urea inclusion complexes. The (E,E)-isomers of dienes are known to form urea inclusion complexes (clathrates) (Leadbetter and Plimmer, 1979). From the hot solution of urea in methanol, the (E,E)-isomers crystallize together with urea whereas the other isomers remain in solution. Filtration of the clathrate crystals, dissolving in brine and extraction give the pure (E,E)-isomer. The method is applicable for preparation of pure (E,E)-isomers and also for the isolation of the (E,E)-isomer as impurity from the (E,Z)- or (Z,E)-compounds. (8E,10E)-8,10-tetradecadien-1-ol, (E10,E12,E14)-10,12,14-hexadecatrien-1-ol, and (10E,12E)-10,12-tetradecadien-1-ol were purified by this method (appendix 1, papers V and VII).

d) Formation of the Diels-Alder adduct from the (E,E)-isomer with the powerful dienophile tetracyanoethene. The method is selective, as only less sterically hindered (E,E)-isomers can form this adduct and the others remain unaffected. This reaction has been applied in the purification of conjugated dienic and trienic compounds to remove the unwanted (E,E)- or (E,E,E)-isomers as impurities from the other isomers. A selective Diels-Alder reaction is considered being more expeditious and efficient than repeated crystallizations. The method is not used in this work.

e) Of the silver-complexing methods used for pheromone isomer separation, Heath et al., (1977) used AgNO3-coated microparticulate silica HPLC columns. Impressive resolution of insect pheromone geometrical isomers (non-conjugated dienes) was achieved due to highly efficient columns utilizing small particle sizes and a high pressure packing technique. 50-fold increase in efficiency over slurry-packed columns was reported. Geometric isomers of several mono-unsaturated and non-conjugated di-unsaturated pheromone components were successfully separated using reversed-phase HPLC and a polar mobile phase containing silver nitrate (Phelan and Millar, 1981). The HPLC method, needing special apparatus and applicable mainly for the separation or purification of small amounts of compounds was not used in this work.

2.2. Oxidation reactions

2.2.1. Oxidation of alcohols to aldehydes

The oxidation of alcohols to aldehydes is an important procedure in the preparation of insect pheromone components, as many of them belong to this class of compounds. Hence an

oxidation procedure is often needed at the end of the synthetic scheme, but also within the scheme, for instance in the preparation of an aldehyde to be used for Wittig couplings. In selecting the method, special care must be taken in case it is necessary to oxidize a compound already having a double bond. Mild conditions and greatly selective and effective reagents have to be selected for oxidation of highly sensitive substances. Several reagents can be used: pyridinium chlorochromate (PCC; Corey and Suggs, 1975; Corey and Boger, 1978; Agarwal et al., 1990), pyridinium dichromate (PDC; Corey and Schmidt, 1979), activated DMSO (Mancuso et al., 1978; Mancuso and Swern, 1981; Tidwell, 1990), Dess-Martin Reagent (Schwarz et al., 1986), and manganese dioxide. A method selected for oxidation has to meet the main requirements: no effect on the stereochemistry of the double bonds block, high yield of the aldehyde, simplicity of the procedure, acceptable cost of the reagents.

1. PCC allows an efficient oxidation of a wide range of alcohols to carbonyl compounds in methylene chloride with only a modest excess of a reagent. The mildly acidic character of PCC precludes its use with acid sensitive substrates.

2. A nearly neutral PDC reagent was preferred for the oxidation of dienic alcohols. In conditions for PDC oxidation (dry methylene chloride as a solvent, room temperature) the isomerization of the dienic block generally did not occur and aldehydes could be obtained in modest yields (50-70%). The use of PDC had been attempted for oxidation of the (9Z,11Z)- 9,11-tetradecadien-1-ol to the corresponding aldehyde, (9Z,11Z)-9,11-tetradecadien-1-al, the sex pheromone component of L. capitella. The oxidation gave the aldehyde in a yield a bit above 60% with isomerization of the double bonds block in a range of 2-6% (VIII). Oxidation of sorbinol using PDC gave the aldehyde together with by-products, which were successfully separated by MPLC on silica gel (V).

3. Swern reagent, DMSO-oxalyl chloride, is the most widely used of the DMSO-based reagents for the oxidation of primary alcohols. Swern oxidation is known to use even milder conditions for sensitive dienic blocks and therefore to have less isomerizing factors. This method results in generally high and frequently quantitative yields with short reaction times and minimal formation of by-products, but requires more accurate experimental techniques than other methods and very dry solvent environment. Swern oxidation was used successfully for the preparation of (9Z,11Z)-9,11- tetradecadien-1-al (VIII) and the (10E,12E,14E)- and (10E,12E,14Z)-isomers of hexadecatrienal (V). In the first case the yield of the oxidation reaction was very good, 81%, and only a minor loss in isomeric purity (from 99.1% to 98.5%) occurred during the procedure. Oxidation of codlemone to the corresponding aldehyde proceeded without isomerization of double bonds using Swern oxidation procedure.

4. Dess-Martin reagent (a periodinane, 1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol- 3(1H)-one) was used with remarkable success in the oxidation of (E)- and (Z)-allylic and (E)- and (Z)-homoallylic alcohols to the corresponding aldehydes with nearly quantitative yields (Schwarz et al., 1986). Numerous methods were used for the preparation of (E)-2-alkenals. Preparation of (Z)-2-alkenals was more problematic due to their tendency to isomerise under acidic or basic conditions to the far more stable E forms. The high cost of the reagent limited its use mainly to cases where the other methods gave unsatisfactory results. The reagent was used with great success for the oxidation of 2,13- and 3,13-octadecadienols, both EZ- and ZE-isomers (I. Liblikas, personal data). Swern oxidation of these compounds did not result in the expected aldehyde. Reagent was not used in this work.

5. Manganese dioxide became a valuable reagent, enabling selective oxidation of the conjugated alcohol in the presence of a nonconjugated alcohol. In the synthetic scheme of (10E,12E,14Z)-10,12,14-hexadecatrien-1-ol, the 14-hydroxy-E2,E4-tetradecadienol was oxidized to 14-hydroxy-E2,E4-tetradecadienal (V). MnO2 was also used for preparing aldehydes

from (E)-2-alcohols in the schemes for preparation of (9Z,11Z)-9,11-tetradecadien-1-ol (VIII), (10E,12Z)-10,12-tetradecadien-1-ol (appendix 1) and (8E,10Z)-8,10-dodecadienol (appendix 2).

2.2.2. Oxidation of bromides to aldehydes

The preparation of aldehydes using amine N-oxides was a method under increased investigation during last decade. An overview appeared (Albini, 1993). The use of amine N-oxides as selective oxidants in the mild conversion of primary aliphatic bromides and iodides into aldehydes became an important method in organic synthesis. 4-methylmorpholine N-oxide (NMO) was not used for that conversion before. NMO was used in the synthetic schemes of (10E,12E,14Z)-10,12,14-hexadecatrien-1-al (V) and (8E,10Z)-8,10-tetradecadien-1-ol (VII).

2.3. Semihydrogenations

2.3.1. Semihydrogenations of the triple bonds in alkenynes

There are various methods for the preparation of (E)- or (Z)-alkenynes, which in turn can be transferred, into the dienic compounds either by anti-hydrogenation (LiAlH4, alkali metals) or by syn-hydrogenation (Lindlar’s semihydrogenation, use of dialkylboranes, P-2 nickel, zinc- copper couple or activated zinc) of the triple bond.

1. LiAlH4 (lithium aluminium hydride) is a stereoselective reducing agent that makes it possible to convert the triple bond to a (E)-double bond. The method works for reduction of β- or γ-alkynes. It has been used for the reduction of 1(2-tetrahydropyranyl)-8-yne-decan-10-ol to the 1(2-tetrahydropyranyl)-8-decen-10-ol (appendix 2). LiAlH4 has been used even in the presence of (Z)-double bonds in the molecule, leaving these unaffected (Attugalle et al., 1995). Out of two conjugated triple bonds of 3,5-tetradecadiyn-1-ol only the triple bond in γ-position has been reduced by LiAlH4 (Yadav et al., 2001).

2. The system alkali metal/liquid NH3 is recommended for the stereospecific reduction of isolated triple bonds to (E)-olefins. One of the latest investigations to develop efficient laboratory procedures is that of Brandsma et al., (1999). An illustrative example of the successful application of this method is the stereoselective conversion of an en-yne system to (E,E)-diene in the synthesis of (10E,12E)-10,12-hexadecadienal (Yadav et al., 1989). The method is not used in this thesis.

3. Lindlar’s catalyst, Pd-CaCO3-PbO has been used for many hydrogenations although many modified catalysts have been investigated to improve the selectivity for semihydrogenation (Rajaram et al., 1983). The method has not been used in this work.

4. Procedures for reduction with dialkylboranes are mainly derived from work presented by Zweifel and Polston (1970). A review of the literature showed that both disiamylborane (bis-[3-methyl-2-butyl]borane) (Negishi et al., 1973; Negishi and Yoshida, 1973; Negishi and Abramovitch, 1977; Dang and Linstrumelle, 1978; Miyaura et al., 1979; Commercon et al., 1980; Ratovelomanana and Linstrumelle, 1981a and 1981b; Rossi et al., 1982; Svirskaja et al., 1984; Cabezas and Oehlschlager, 1999; Yadav et al., 2001) and dicyclohexylborane (Descoins and Henrick, 1972; Bestmann et al., 1981a and 1981b; Ando et al., 1985; Ferrao et al., 1998; Tellier and Descoins,

1990; McElfresh and Millar, 1999; Nishida et al., 2003) were very often applied for reduction of the triple bond in enynes. There seemed to be no reason to prefer one of the two to the other. The method was stereoselective, only the Z-isomer was produced during a reduction procedure. Catecholborane (1,3,2-benzodioxaborole) was also used for the Z-stereoselective reduction of the triple bond (Bestmann et al., 1981b). Method applied to reduce the (Z)-enyne into (Z,Z)-diene in the synthetic procedure of (9Z,11Z)-9,11-teradecadien-1-ol (VIII). Enyne was added to a dicyclohexylborane solution prepared just before use, followed by protonolysis with acetic acid and oxidation of the dicyclohexylborinate with hydrogen peroxide in alkaline medium to facilitate the isolation of the (Z,Z)-diene product. A good yield (81%) of the (9Z,11Z)-9,11-teradecadien-1-ol was obtained. It turned out from experiments that, when preparing the dicyclohexylborane, one should avoid an excess of borane as it could later attack the double bond of the enyne and, consequently, decrease the yield of the product desired. It was important to consider the cyclohexanol formation during the reduction process with dicyclohexylborane. Cyclohexanol could not be isolated from the dienic alcohol desired, by using chromatographic techniques. Therefore, the alcohol should be protected or acetylated before reduction.

5. Borohydride-reduced nickel (P-2 nickel), used with ethylenediamine, is highly stereospecific for the hydrogenation of alkynes to pure olefins with stereoisomeric purity >99% (Brown and Ahuja, 1973a and 1973b). There are examples of successful reductions of triple bonds in conjugated enynes using this reagent (Yadav et al., 1989). Attempts to use this method for reduction the enyne in the incurvaria pheromone scheme, gave poor results compared to dicyclohexylborane. It was less stereoselective and some overhydrogenation appeared during the procedure. The hydrogen uptake was too fast to make it possible to follow the enyne consumption by TLC.

6. Z-selective reduction of the triple bond in the (E)-enyne acetate using a Zn-Cu couple as a reducing agent to produce the (Z,E)-dienic acetate in 87% yield and 97.4 % stereoisomeric purity is presented in the synthetic pathway to (3Z,5E)-3,5-dodecadien-1-yl acetate (II, III). Method developed by Sondengam et al., (1980).

7. The use of activated zinc powder has been considered to have some advantages over the reduction with hydrogen and “poisoned” Pd-catalysts: short reaction times and experimental simplicity. Stereospecific reductions of a variety of acetylenic derivatives have been carried out in absolute ethanol with zinc powder activated with 1,2-dibromoethane (Aerssens and Brandsma, 1984; Aerssens et al., 1990). Zn-powder in propanol in the presence of KCN has been used for the reduction of the triple bond in (10E,12E)-10,12- hexadecadien-14-yne-1-yl acetate to produce the corresponding (E,E,Z)-acetate (Tellier, 1991). The method has not been used in this work.

2.3.2. Semihydrogenations of the triple bonds in diynes

Diynes, easily prepared by the Cadiot-Chodkiewicz coupling reaction, are converted to (Z,Z)-dienes by reduction with dicyclohexylborane. Whereas addition of the second equivalent of the dialkylborane is very slow, the less hindered dicyclohexylborane is preferable compared with disiamylborane. The method is exemplified by the synthesis of (3Z,5Z)-3,5-dodecadien-1- yl acetate (II, III), (8Z,10Z)-8,10-tetradecadien-1-ol (VII), (9Z,11Z)-9,11-hexadecadienal (Svirskaja et al., 1980), (8Z,10Z)-8,10-dodecadien-1-ol (Chisholm et al., 1985).

2.4. Monobromination and monotetrahydropyranylation

It is very common to start the syntheses of conjugated dienic alcohols, acetates and aldehydes from α,ω-diols. In that case one OH-group has to be reserved at the end of the final molecule, the other one can be used to build up the structure needed. For that reason it is necessary to differentiate these equivalent OH-groups from each other in some ways. As α,ω- compounds with different functional groups are very important building blocks for the preparation of dienic pheromone compounds, some attention must be paid to find out the most convenient and economical ways of preparing this type of compounds. The most common method seems to be monobromination using a continuous extraction technique to increase the yield of monobrominated product. In that case, the other OH-group can be protected or saved unattached and the bromine can be used for further transformations. The method has been used at the beginning of very many synthetic schemes including the synthetic routes presented in this thesis: appendixes 1, 2, and 3; papers I, IV, V, VI, and VII. The other possibility is the monoprotection of α,ω-diol as tetrahydropyranyl ether. Bestmann and Gunawardena (1992) use p-toluenesulfonic acid monohydrate as a catalyst and run the reaction in diethyl ether for 16h. The yield of monoprotected alcohol is a little over 40%. The method using extraction between a hydrocarbon solvent and a water phase is further studied by Nishiguchi et al., (2000). In case it is needed to repeat this procedure several times, it is rational to find out the best conditions for this reaction, i.e. temperature, catalyst, reaction time, and co-solvent which are all variable to some extent depending on the chain length of the diol. Under optimal conditions the yield of monoprotected α,ω-diol can exceed 90%. An extraction technique has been used in the scheme for L. capitella pheromone synthesis (VIII), and for the synthesis of (10E,12Z)-10,12-tetradecadien-1-ol (appendix 1). The advantage of monotetrahydropyranylation compared to monobromination is the short reaction time (2 hours versus 70 hours), the use of less harmful and corrosive reagents (3,4- dihydro-2H-pyran versus concentrated hydrobromic acid), and no needs of special apparatus (monobromination is better performed in a special continuous extraction apparatus while for monoprotection only a flask with stirring device is needed). The author’s preference is the monotetrahydropyranylation, of course, if the bromine is not absolutely necessary for future reactions.

3. Results of syntheses of polyenic pheromone components

None of the dienic pheromone compounds synthesized here was made in order to study the chemical aspects of its synthesis in detail. They were all made to meet the needs of biological or technological aspects. In most cases it was more important to get the compound than to modernize or improve any reaction in the synthetic scheme. Consequently these were not optimized or not always the best routes for preparing the corresponding dienic pheromone components. It was difficult to estimate the value of the method, to see the advantages over the others or to get the best possible results if it was used just once in the reaction scheme.

3.1. General aspects of the Wittig reaction

The Wittig reaction of aldehydes with phosphorus ylides is an excellent and most frequently used key step in pheromone synthesis. The Wittig reaction lacks full stereochemical control and the yields are often moderate. However, careful control of the reaction conditions makes it possible to enhance the (Z)- or (E)-selectivity and to obtain good yields.

In general, this method is applicable mainly for the preparation of mixtures of isomers and in cases of inexpensive starting materials it makes it possible to prepare larger amounts of compounds and allow the losses of material during purification. This olefination method enjoys widespread prominence and recognition because of its simplicity, convenience, and efficiency. The reaction of a phosphorus ylide with an aldehyde or ketone introduces a carbon- carbon double bond in place of the carbonyl bond: 1 1 R R X + X + 2 O R3 P 2 R3 P O R Y R Y aldehyde phosphorus alkene phosphine or ketone ylide oxide

The Wittig reaction involves phosphorus ylides as the nucleophilic carbon species. Phosphorus ylides are usually stable, but quite reactive compounds. The synthetic potential of phosphorus ylides initially derives from G. Wittig. Phosphorus ylides have been loosely classified according to their general reactivity. “Stabilized” ylides have strongly conjugating substituents (e.g., COOMe, CN or SO2Ph) on the ylidic carbon and usually favour the production of (E)-alkenes. “Semistabilized” (or moderated) ylides bear mildly conjugating substituents (e.g., Ph or allyl) and often give no great preference one way or the other, and “nonstabilized” ylides lack such functionalities and usually favour (Z)-alkenes (Maryanoff and Reitz, 1989; Ideses and Shani, 1989). Phosphorus ylides are usually prepared by deprotonation of phosphonium salts. The phosphonium salts used are most often alkyltriphenylphosphonium halides, which can be prepared by the reaction of triphenylphosphine and an alkyl halide (Tomida et al, 1983; Vinczer, 1987). However, the Wittig reaction lacks full stereochemical control. The product is generally contaminated with the other isomer and their separation can be difficult. The Z:E isomeric ratio of the Wittig product depends mainly on the structure of the reagents (aldehyde and phosphorane) and less on the reaction conditions. The effect of the base used for the ylide generation, the influence of the solvent and reaction temperature on the stereochemistry and the yield of Wittig condensation have been studied in more detail by Vinczer et al., (1988a).

Effect of the temperature of addition. Lower temperature enhances Z-selectivity. At low temperature the reaction results in a single isomer, which decomposes by the elimination of Ph3PO with nearly 100% stereospecificity to the Z-olefin.

Effect of the base. Alkyltriphenylphosphonium halides are only weakly acidic and strong bases are used for deprotonation. As hard base sodium or potassium alcoholates, as borderline KOt-Bu can be applied. n-BuLi, NaHMDS and K/HMPT can be regarded as soft ones. The character of the base applied for the ylide generation significantly influences the yield of Wittig condensation. Hard bases are found to ensure the best results; lower yields are obtained when borderlines are applied, and soft bases afford the poorest yields (Vinczer et al., 1988b). Very similar yields and stereoselectivities were obtained with NaHMDS and K dissolved in amine. Z-selectivities of 97-98% were achieved at low temperature, but the yields were moderate (30-40%). NaHMDS was a very useful base for the generation of lithiumsaltfree solutions of alkylidenetriphenylphosphoranes to carry out Z-stereoselective Wittig reactions (Bestmann et al., 1976). Acceptable or excellent yields were obtained when alkoxides were used as base, On the other hand, the different alkoxides gave slight differences in diastereomeric purity under the

same reaction conditions. The use of approximately equimolecular quantities of KOt-Bu, afforded the highest Z-selectivity (98%), yield 50-60%. The highest yields (60-85%) but somewhat lower Z-selectivity were obtained with NaOEt with a diastereomeric purity of 90% at room temperature and 95% at -78 ˚C. Only when using n-BuLi, was the Z:E ratio significantly dependent on the molar ratio of base to phosphonium salt. Reaction of the phosphonium salt with one equivalent of n-BuLi resulted in the high inhomogeneity of the product (85:15 Z:E). If less than one equivalent of n- BuLi was used for ylide generation, the deprotonation of the resulting ylide was negligible, and a high Z-selectivity was achieved (92:8). On the other hand, the Z-selectivity was significantly reduced (60:40) if an excess (3 equiv.) of n-BuLi was used.

Solvent effects. The Z/E ratio of the Wittig products is dramatically dependent on the polarity of the solvent used. From a variety of data, Schlosser (Schlosser et al, 1985) generally concluded that THF, 1,2-dimethoxyethane, ethyl ether, and t-butyl methyl ether were solvents of choice with respect to furnishing a higher yield and Z-stereoselectivity, while protic solvents such as alcohols and DMSO should be avoided. According to Vinczer et al., (1988a) THF and toluene were equally suitable.

Variation of the alkyl chain of the phosphonium salt. The yield of the Wittig reaction is only slightly affected by variation of the alkyl chain of the phosphonium salt. When the substituent of the phosphonium salt contains more than five carbon atoms, higher temperature during the ylide generation increases the yield 10-15%. On the other hand, phosphonium salts having base-sensitive groups (carboxyl and acetoxyl) result in poor yields due to the reaction between the base and the functional group. High yields of the olefin (60-80%) can be obtained, if the alkyl chain of the phosphonium salt has no base- sensitive group (Vinczer et al., 1988a).

The stereoselectivity of the Wittig reaction strongly depends on the structure of the ylide. The broadest generalisation is that unstabilized ylides predominantly give the Z-alkene, whereas stabilized ylides mainly give the E-alkene. Ideses and Shani (1989) compared the reaction of reactive (nonstabilized) saturated ylide with an α,β-unsaturated aldehyde and the opposite approach, the reaction of moderate (semistabilized) allylic ylide with a saturated aldehyde. The results clearly showed that for synthesis of a conjugated diene with a specific configuration was better to react a reactive (saturated) ylide with unsaturated aldehyde in order to minimize the number of isomers and to get one of them as pure as possible. The same observation was made by Goto et al., (1975) synthesizing the 9E,11Z-14OAc. An isomeric purity of 85-90% was obtained from triphenylphosphonium salt of 1-bromo-9- acetoxynonane and (E)-2-pentenal (eq. 1). The isomeric purity was lower from triphenylphosphonium salt of (E)-1-bromo-2-pentene and 9-acetoxynonanal (eq. 2).

+ O + AcO eq. 1 AcO P (Ph)3 Br

+ O OAc eq. 2 P (Ph) Br + AcO 3

The same principle resulted from the work presented by many other chemists (Hall et al., 1975; Bestmann et al., 1977; Bestmann et al., 1981b; Chisholm et al., 1981; Huang et al., 1987; Chen and Millar, 2000). Stereoisomeric purity up to 94% was achieved. Under regular conditions with different bases and in several solvents the Z-isomers will predominate. If the E-isomer is needed, two equivalents of Li base are needed. If a mixture of

all four isomers is required, the other system, in which a moderate (E or Z) allylic ylide reacts with saturated aldehyde, is recommended. Teich et al., (1979) found it better to run the Wittig reaction with the phosphonium salt of the bare bromohydrin without any protecting group on the hydroxyl group. For better results, two equivalents of base were recommended. The same suggestion was given by Lo and Shiao (1986).

3.2. Wittig reactions in the synthetic schemes of conjugated dienic pheromone components

3.2.1. Syntheses of (Z,E)-isomers

For the synthesis of ZE-isomers, it is reasonable to use the building blocks in which the E-double bond is already stereoselectively fixed and to obtain the Z-double bond via a Wittig reaction. An analysis of the structures of the molecules as (8Z,10E)-8,10-dodecadienol, (8Z,10E)-8,10- and (10Z,12E)-10,12-tetradecadienols shows that these compounds can be built up from an (E)-2-unsaturated aldehyde and a saturated ylide bearing a functionality at the end (eq. 3-5), (appendices 1 and 2; papers I, IV, VI and VII). The other possibility is to run the Wittig reaction between an unsaturated aldehyde with ω-functionality and an (E)-2-unsaturated phosphorus ylide. Considering the data available from revision of the use of the Wittig reaction in the preparation of dienic pheromone compounds and the conclusions given above, the choice has to be made in favour to the first approach.

The significantly important factor selecting which part of the diene that has to be derived from aldehyde and which from the phosphorus ylide was that the aldehydes needed, (E)-2- butenal and (E)-2-hexenal, were commercially available inexpensive compounds. (E)-2-alkenals with longer carbon chains are not commercially available. However, general and versatile method was reported for the preparation of (E)-2-alkenols, the precursors of aldehydes, in which the condensation reaction is the key element. The widely applicable Doebner modification of Knoevenagel condensation in polar basic solvents such as pyridine- piperidine solution using about equal amounts of aldehyde and malonic acid gave (E)-2- alkenoic acids. Lithium aluminium hydride reduction yielded the corresponding alcohols in high stereochemical purity (95-100%) and in good yields. The method had the advantage of being a simple reaction of commercially available inexpensive reagents (X).

All abovementioned conditions were taken into account while the Z,E-isomers of 8,10- dodecadienol, 8,10- and 10,12-tetradecadienol were synthesized. The choice of the Wittig coupling reaction conditions depended on the aim of the synthesis.

Whereas the (E,E)-isomer of 8,10-dodecadienol was commercially available, it was planned to synthesize the (Z,E)-isomer as (Z)-stereospecifically as the method allowed. For this purpose NaHDMS was selected as a base because it was known to afford a high (Z)-selectivity (98% by Vinczer et al., 1988b). The expected moderate yield was not a limiting factor due to relatively low costs and simple preparations of the starting materials for this Wittig reaction. Smaller loss in purification compensated the compound missing due to the lower yield of the reaction. Thus the reaction of the ylide from the acetate of 1,8-bromohydrin with crotyl aldehyde using NaHDMS-base resulted in 43% of (8Z,10E)-8,10-dodecadienyl acetate besides 4 % of (E,E)-isomer. Replacing the base by NaOEt increased the outcome up to 61%. Final purification by chromatography with silver nitrate impregnated silica gel resulted in >99% isomerically pure (8Z,10E)-8,10-dodecadienol (eq. 3), (appendix 2, papers I, IV, VI).

O + H KOH P (Ph) Br eq. 3 AcO 3 AcO HO NaN[Si(CH3)3]2 EtOH THF In the preparation of 8,10- and 10,12-tetradecadienols, both the (Z,E)- and the (E,E)- isomer were required. Considering that it was easy to make quite big amounts of these compounds and that the (E,E)-isomer was the most easily separated from the others, it was decided to go to the mixture of two isomers. n-BuLi was a base of choice for the Wittig reaction leading to the mixture of (E)- and (Z)-isomers (Vinczer et al., 1988b; Maryanoff and Reitz, 1989; Lo and Shiao, 1986) . A higher temperature of addition of the aldehyde was also known to lower the (Z)-selectivity (Vinczer et al., 1988a, Maryanoff and Reitz, 1989). The statement that phosphonium salts from nonprotected bromohydrin resulted in higher yields was proven in comparison of the syntheses of (8Z,10E)-8,10- and (10Z,12E)-10,12- tetradecadienol. The Wittig reaction of phosphonium ylide from unprotected 1,8-bromohydrin yielded a mixture of (Z,E)- and (E,E)-isomers in the ratio 61:39. The (E,Z)- and (Z,Z)-isomers were not detected by GC-MS. The separation of the isomeric mixture was carried out by chromatography on silica gel impregnated with 15% of AgNO3. Finally, 99.0% isomerically pure (8E,10E)-8.10-tetradecadienol derived from urea inclusion complex and 99.4% isomerically pure (8Z,10E)-8,10-tetradecadienol by crystallization from pentane at -30°C was obtained (eq. 4), (VII). O H + HO P (Ph)3 Br + eq. 4 HO BuLi, THF HO The second approach was the synthesis of the mixture of two isomers of 10,12- tetradecadienol where 1,10-bromohydrin was monoprotected as tetrahydropyranyl ether before coupling its phosphorus ylide with crotyl aldehyde. The yield of diene was somewhat lower, 56% of a mixture of 28% of (10E,12E)-1-(2-tetrahydropyranyloxy)-10,12-tetradecadiene and 72% of (10Z,12E)-1-(2-tetrahydropyranyloxy)-10,12-tetradecadiene. After deprotection the isomers were separated by AgNO3-SiO2 chromatography. Finally, 99.1% isomerically pure (10E,12E)-10,12-tetradecadienol derived from urea inclusion complex and 98.9% isomerically pure (10Z,12E)-10,12-tetradecadienol well obtained by crystallization from pentane at -30 °C (eq. 5), (appendix 1). O THPO PPTS HO + H eq. 5 P (Ph) Br + + THPO 3 BuLi, THF EtOH THPO HO

3.2.2. Synthesis of (E,Z)-isomers

The preparation of (E,Z)-isomers of 8,10-dodecadienol, 8,10- and 10,12-tetradecadienols using the Wittig coupling reaction of (E)-2-unsaturated aldehyde with saturated phosphonium ylide was expected to give the best results. This time the functionality had to lie on the aldehyde. Thus, (E)-2-unsaturated aldehydes had to be prepared and coupled with alkyl phosphonium ylides.

LiAlH HO 4 MnO2 OTHP OTHP OTHP eq. 6 HO CH Cl O THF 2 2 In the synthetic route to (E8,Z10)-8,10- dodecadienol the aldehyde needed, (E)-1-(2- tetrahydropyranyloxy)-8-decenal, was prepared from 1-(2-tetrahydropyranyloxy)-8-decyn-10-ol by reduction of the double bond with LiAlH4 and oxidation of the resulting alcohol with MnO2 (eq. 6). This procedure was technically easier and gave better yields than the one used in our laboratory before: preparation of (Z)-1-(2-tetrahydropyranyloxy)-8-decenol by hydrogenation of the same alkynol over a Pd/BaSO4 catalyst and oxidation of the resulting (Z)-alcohol with PCC. (E)-1-(2-tetrahydropyranyloxy)-8-decenal was coupled with ethyl phosphonium ylide under the conditions leading predominantly to the (Z)-stereoselective coupling (NaOEt as a base and temperature of addition of the aldehyde -78°C) to yield after deprotection a (8E,10Z)-8,10- dodecadienol in 78% yield and 94% of isomeric purity. AgNO3-SiO2 chromatography enriched the isomeric purity to over 99% (appendix 1).

(8E,10Z)-8,10- and (10E,12Z)-10,12-tetradecadienols were obtained following the same principle, whereas the preparation of the aldehyde unit was different (eq.7 and 8). Coupling reactions of the aldehydes with formylmethylenetriphenylphosphorane gave 2-alkenals, the C=C double bonds of which were revealed to have (E)-geometry. (Trippett and Walker, 1961; Ando et al.,1985).

+ P (Ph)3 Cl NMO O OTHP OTHP OTHP Br O O DMSO Et3N, Toluene + P (Ph)3 Br PPTS OTHP OH eq. 7 NaOEt, THF MeOH

4-Methylmorpholine N-oxide was used in the synthetic sequence of (8E,10Z)-8,10- tetradecadien-1-ol (eq.7) as oxidizing reagent to obtain 8-(2-tetrahydropyranyloxy)-octanal. The use of amine N-oxides as selective oxidants in the mild conversion of primary aliphatic bromides and iodides into aldehydes was reviewed (Albini, 1993). 4-methylmorpholine N-oxide had not been used for that conversion before. The Wittig reaction preparing (8E,10Z)-8,10-tetradecadien-1-ol yielded 75% of crude product from which 97% isomerically pure dienic alcohol was obtained by low temperature crystallization from pentane. AgNO3-SiO2 chromatography increased the purity up to 99.5% (VII).

+ P (Ph)3 Cl PDC O OTHP OTHP OTHP HO O O CH2Cl2 Et3N, Benzene

+ 15 P (Ph)3 Br Amberlyst OTHP OH eq. 8 NaOEt, THF MeOH The preparation of (10E,12Z)-10,12-tetradecadien-1-ol yielded 79% of crude product containing 2.5% of (E,E)-isomer and 8.5% of monoenic compound (eq. 8). Purification was performed by chromatography on AgNO3 impregnated silica gel. Recrystallization from hexane at low temperature gave 99.3% isomerically pure alcohol (appendix 1).

3.3. Modifications of the Wittig reaction

3.3.1. Schlosser’s modification of the Wittig reaction

The reaction of the nonstabilized ylides with aldehydes can be induced to yield (E)- alkenes with high stereoselectivity by a procedure known as Schlosser modification of the Wittig reaction. The “betaine-ylide reaction” entails the addition of a strong base to the Wittig intermediates at low temperature (before they collapse to products) and often leads to a high proportion of (E)-alkene. In Schlosser`s procedure the ylide is generated as a lithium halide complex and allowed to react with an aldehyde at low temperature, forming a mixture of diastereomeric betaine- lithium halide complexes. At low temperature, fragmentation to an alkene and triphenylphosphine oxide does not occur. This complex is treated with a strong base such as phenyllithium to form ß-oxido ylide. Stereoselective protonation of the ß-oxido ylide by addition of t-BuOH yields the more stable threo-betaine as a lithium halide complex, which gives E-alkene under warming. An over-stoichiometric concentration of the lithium halide is crucial for achieving stereocontrol (Schlosser and Christmann, 1966; Schlosser et al., 1985; Reitz et al., 1986).

In this thesis the Schlosser modification of the Wittig reaction has been used for the synthesis of the (E,E,E)-isomer of 10,12,14-hexadecatrienal (appendix 3, paper V).

This trienic aldehyde was previously separated as a by-product of the synthesis of the (E,E,Z)-isomer via Wittig’s reaction under (Z)-stereoselective conditions (NaHMDS-base, THF- HMPA solvent) by Doolittle et al., (1990). Tellier (1991) and Ando et al., (1988) produced small quantities of the target compounds heavily contaminated with isomeric impurities. Retro-synthetic analysis of (E,E,E)-10,12,14-hexadecatrienal revealed a convenient disconnection between carbon atoms 10 and 11. Chen and Millar (2000) brominated (2E,4E)- 2,4-hexadecadienol and treated the resulting bromide with triphenylphosphine to afford (2E,4E)-2,4-hexadienyltriphenylphosphonium bromide. A Wittig reaction of the latter with 10- acetoxy-decanal gave a mixture of 10,12,14-hexadecatrienyl acetates (EEE:ZEE=64:36). An additional portion of (E,E,E)-isomer was prepared by addition of a catalytic amount of iodine and exposure to light. In the synthetic scheme presented in this thesis, the (2E,4E)-2,4-hexadiene unit was used as an aldehyde partner of the Wittig coupling. Thus, the readily available (2E,4E)-2,4-hexadien- 1-al was coupled with 10-hydroxydecyl-triphenylphosphonium bromide under the Schlosser conditions (Schlosser et al., 1985) to give the mixture of (E,E,E)- and (Z,E,E)-isomers. The desired (E,E,E)-isomer of very high isomeric purity was isolated by urea complex inclusion procedure and then oxidized to (E10,E12,E14)-10,12,14-hexadecatrien-1-al by Swern oxidation. Despite the lower overall yield of (E,E,E)-isomer from this synthetic route this method can be considered as convenient as the Chen’ one. Both methods are suitable for the preparation of the trienic aldehyde on a preparative scale.

3.3.2. Horner-Wadsworth-Emmons reaction

Horner and co-workers were the first to let phosphoryl-stabilized carbanions react with aldehydes and ketones to produce olefins. Wadsworth and Emmons (1961) published the paper which popularized this method in the organic synthetic community. Horner was the first to use phosphine oxides. Wadsworth and Emmons developed the phosphonate modification of the Wittig reaction. Therefore the phosphonate-mediated olefinations are referred to as the “Horner- Wadsworth-Emmons” reaction, and the phosphine oxide variant is called the “Horner”-reaction.

Horner-Wadsworth-Emmons reaction is the reaction of phosphonate carbanions with carbonyl compounds (eq. 9). Phosphonate carbanions are generated by treating alkylphosphonate esters with bases (NaH, n-BuLi, NaOEt). The alkylphosphonate esters are made by reaction of an alkyl halide with a phosphite ester. The Horner-Wadsworth-Emmons reaction is generally restricted to phosphonates bearing an α- substituent that can stabilize a carbanion. (e.g., COO¯, COOMe, CN, aryl, vinyl, SOOR, P(O)(OR)2, SR, OR, and NR2). The absence of such groups usually results in poor yields of alkene products. When R is an electron-attracting group, the formation of an (E)-olefin is preferred. When R is an alkyl group, the intermediate does not undergo elimination.

base RCH X + P(OC H ) - 2 2 5 3 RCH2P(=O)(OC2H5)2+C2H5X RC HP(=O)(OC2H5)2 O

O P(OC2H5)2 R'2C=O R' R R' C=CHR + (C H O) P(=O) eq. 9 2 2 2 5 2 O

The use of the method is illustrated in the synthetic scheme of (10E,12E)-10,12- hexadecadien-1-ol (Vig et al., 1990). The synthesis is based on the combination of the Horner- Wadsworth-Emmons synthesis of alkyl 2,4-dienoates with copper-catalyzed Grignard reaction to obtain conjugated E,E-dienic compound (eq. 10).

O O Na LiAlH4 Py, PBr3 O OH Dimethylethyl- phosphonoacetat

O-THP MgBr Br O-THP eq. 10 Li2CuCl4

Baeckström et al., (1988) prepared 93% isomericaly pure methyl ester of (2E,4E)-2,4- decadienoic acid by treatment of methyl 2(E)-4-dimethylphosphonate-2-butenoate with hexanal. The purity was increased >99% using a urea complex inclusion procedure.

The designing of the synthetic pathway for making (10E,12E,14Z)-10,12,14- hexadecatrien-1-al started with the analysis of the retro synthetic possibilities. In order to make the saturated phosphonium ylide result in better yield and higher stereoisomeric purity, it seemed reasonable to synthesize the ω-protected (2E,4E)-aldehyde with fourteen carbon atoms in the chain and to couple it with ethyltriphenylphosphonium bromide under the (Z)- stereoselective coupling conditions. For the preparation of the type of aldehyde needed, the Horner-Wadsworth-Emmons reaction was the procedure of choice. On following the synthetic scheme (appendix 3, paper V), the (10E,12E,14Z)-10,12,14-hexadecatrien-1-ol was obtained in 90% stereoisomerical purity. Repeated crystallization from hexane at low temperature increased the purity up to 97%. Unfortunately, there was a loss of purity by 2% during Swern oxidation. The oxidation of (10E,12E,14Z)-10,12,14-hexadecatrien-1-ol to the aldehyde proved problematic because the conjugated trienes were sensitive to acids, heat and electrophilic attacks. Here the choice of oxidation method was especially important. Doolittle et al., (1990) reported that oxidizing agents such as PDC and PCC resulted in degradation and isomerization of the conjugated triene aldehyde. Swern oxidation procedure (Mancuso et al., 1978; Mancuso and Swern, 1981; Tidwell, 1990) afforded the best results in oxidation of both (E,E,E)- and (E,E,Z)-isomers of 10,12,14-hexadecatrienol (appendix 3, paper V).

3.4. Alternative synthesis of the trienic pheromone components of Manduca sexta

An alternative synthetic route of considerable value leading to (10E,12E,14Z)-10,12,14- hexadecatrien-1-al has been presented by Chen and Millar (2000) (eq. 11). Cl Cl MgBr (HO)2 B OH Cl OH

(Ph3P)4Pd,K2CO3 (Ph3P)4Pd, Bz eq. 11 Swern OH O oxidation

Syntheses of the (E,E,Z)-isomer were also presented by Ando et al., (1988). They used the principle of synthesizing two of three double bonds stereospecifically and the third one by a Wittig reaction rather non-specifically to obtain a mixture of isomers.

Synthesis of the isomers of the trienic pheromone components of Manduca sexta by Doolittle et al., (1990) (eq. 12):

1. Dicyclohexyl- borane 2. Li t-BuMe2 S O t-BuMe Si O 2 O 3.Bu3 SnCl t-BuMe2 S 4. HOAc, H O , 2 2 eq. 12 NaOH

The ratio of (E,E,Z)- to (E,E,E)-isomers was 94:6 after the reaction, but during deprotection the isomerization occurred, yielding the 82:18 ratio of the isomers.

Tellier (1991) described the stereospecific syntheses of (E,E,Z)- and (E,E,E)-10,12,14- hexadecatrien-1-al (eq. 13). The key steps in the synthesis of the (E,E,Z)- isomer were two sequential Pd-catalyzed cross-coupling reactions: the first reaction occurred between the organoaluminium reagent and (E)-1,2-dibromoethylene and the second one was the alkylzinc acetylene coupling with 1-alkenyl halide.

1. DIBAL 1. Ac O, FeCl t-BuO t-BuO Br 2 3 2. CHBr=CHBr o Et2O Pd , Et2O

ZnBr Zn, KCN AcO Br AcO Pdo ProOH, H2O

DMSO, (COCl)2 HO O eq. 13 Et N 3

3.5. Syntheses of (3E,5Z)-3,5-dodecadienyl and (3E,5E)-3,5-dodecadienyl acetates via Horner-Wadsworth-Emmon’s condensation

Horner-Wadsworth-Emmons’s condensation was the key step in the synthetic scheme of (3E,5Z)-3,5-dodecadienyl and (3E,5E)-3,5-dodecadienyl acetates (II, III). The syntheses were

based on the Horner-Wadsworth-Emmons’s condensation of methyl (E)-4-dimethylphosphono- 2-butenoate with octanal followed by stereoselective low-temperature deconjugations of the resulting (2E,4Z)- and (2E,4E)-2,4-dodecadienoates by LDA (Ikeda et al., 1987). Although the condensation reaction yielded the ratio of (Z,E)-isomer to (E,E)-isomer 80:20, the content of (E,E)-isomer was not over 10% after the deconjugation step. A DIBAL reduction of the 3,5- dodecadienoates and subsequent acetylation of the alcohols yielded the mixture of acetates desired. Over 99% isomerically pure (E,Z)- and (E,E)-isomers were obtained by liquid chromatography on silver nitrate impregnated silica gel.

3.6. Alternative synthetic methods for the syntheses of (3E,5Z)- and (3Z,5E)- isomers of conjugated dienic pheromone components

3.6.1. Synthesis of (3E,5Z)-3,5-dodecadienyl acetate using the coupling reaction of alkenyl cuprates

An alternative method for the synthesis of (3E,5Z)-3,5-dodecadienyl acetate was presented by Simonelli et al., (1998). (3E,5Z)-3,5-dodecadienyl acetate was made according to their synthetic scheme with a key step of coupling of alkenyl cuprate with 4-iodo-3-butyn-1-ol, followed by (E)-selective reduction of the triple bond by LiAlH4 (eq. 14). I

1. LiAlH4, Diglyme OH reflux

Hex 2 CuMgBr TMEDA OAc 2 OH 2. Ac2O/ Py eq. 14

3.6.2. Alkenyl-alkynyl coupling of vinylorganoboranes and alkynyllithiums

Alkenyl-alkynyl coupling of vinylorganoboranes and alkynyllithiums is a general and highly stereoselective (>99%) method for the synthesis of trans-enynes (eq. 15). The treatment of borate complexes derived from vinylorganoboranes and alkynyllithiums with iodine results in an alkyl migration from boron to carbon, followed by a spontaneous deiodoboronation of the intermediate, to provide a highly convenient synthesis of olefins. The method is applicable to both (E,Z)- and (Z,E)-diene synthesis.

2 I2, NaOAc 1 Li R Sia B 1 + 2 2 1 Sia2BH R Sia B 1 R R R 2 R eq. 15 2 R Negishi et al., (1973b) found, that by treatment with iodine and sodium hydroxide or acetate, these borate complexes gave conjugated trans-enynes in a highly stereoselective manner (>99%). These were readily convertible into the corresponding cis-trans-dienes. In case R1 was a functionalized alkyl (i.e. protected alcohol) and R2 was an alkyl, (E,Z)-dienes were obtained, whereas the opposite case gave (Z,E)-dienes from the appropriate enynes. Negishi and Abramovitch (1977) synthesised (3E,5Z)-3,5-decadienyl acetate by treating 4- acetoxy-1-butenyl-disiamylborane sequentially with 1-hexenyllithium, iodine, and sodium acetate followed by the hydroboration with disiamylborane and protonolysis with acetic acid, in over 98% isomeric purity.

Doolittle and Solomon (1986) were modified method originated from Zweifel and Backlund (1978) using tri-n-butyltin chloride as electrophile in the electrophile-induced rearrangement (eq. 16). 98% isomeric purity was obtained after recrystallization of dienic alcohol from pentane. The (Z,E)-isomer was synthesized in an analogous manner in which 1-decyne was the starting material.

+ C Li

THPO + THPO BH B 2 Bu SnCl THPO + 3 THPO B Li B SnBu3

1. CH COOH 3 PPTS THPO H O eq. 16 2. NaOH, H O MeOH 2 2

Synthesizing the (3E,5Z)-3,5-dodecadienyl acetate, Park et al., (1998) used trimethyltin chloride as suitably bulky electrophile with highly controlled stereochemistry.

3.6.3. Pd-catalyzed cross-coupling reactions of free acetylene with (Z)-1-haloalkene or (E)-1-haloalkene

Direct stereospecific synthesis of 1,3-enynes by coupling of 1-alkynes or ω- functionalized 1-alkynes with easily available (Z)- or (E)-1-alkenyl halides in the presence of catalytic amounts of Pd0 or PdII compounds, a copper(I)halide and an excess of an amine to “neutralize” the hydrogen halide, is the method of choice to prepare the precursors of conjugated (E,Z)-, (Z,E)- or (Z,Z)-dienic pheromone components. As usual, the reactivity order with respect to halogen in C=C-X is I>Br>Cl.

One of the first procedures was developed by Sonagashira et al., (1975): acetylenic hydrogen could be readily substituted by bromoalkenes, in the presence of a catalytic amount of dichlorobis(triphenylphosphine)palladium and a cocatalyst, copper(I)iodide, in a large excess of diethylamine under very mild conditions. A system of cuprous iodide and tetrakis(triphenylphosphine)palladium(0) or bis(diphenylphosphino)ethane-palladium dichloride can also catalyze the substitution in diethylamine. In either case, no catalytic substitution of acetylenes occurs at room temperature in the absence of cuprous iodide, indicating that the role of cuprous iodide facilitating the substitution reaction is important. The reactions proceeded with total retention of configuration in the case of (E)-1-iodo-1-alkenes and with high stereoselectivity (≥97%) in the case of (Z)-1- bromo-1-alkenes. Low yields (~ 30%) resulted from the reaction of 1-halo-1-alkenes with diethylamine.

The Pd-catalyzed coupling reactions presented by Ratovelomana and Linstrumelle (1981b) were realized by Sonagashira’s et al., (1975) procedure modification. Ratovelomana modified this method, using Pd(PPh3)4 as catalyst in benzene containing n-propylamine. Under the reaction conditions the acetate function was not cleaved and pure enynol acetate was obtained in good yield. Reduction of the triple bond with disiamylborane gave (9Z,11E)-9,11- tetradecadienyl acetate, the pheromone component for several insects, in 90% yield. (E)-1- iodoalkenes gave better yields in this procedure than (E)-1-chloroalkenes (86% versus 76%). Similarly, Descoins et al., (1984) used cuprous iodide and tetrakis(triphenylphosphine)-

palladium as a catalyst for reacting (Z)-1-iodo-butene with propargyl alcohol in benzene containing n-propylamine (2 equivalents).

Better yields (50-90%) than these of Sonogashira were obtained by Rossi et al., (1982), who carried out these reactions under phase transfer conditions employing benzyltriethyl ammonium chloride as phase transfer agent, an excess of 2.5N aqueous NaOH as base and a mixture of Pd(PPh3)4 and cuprous iodide as catalyst. The stereoselectivity of the reactions of (Z)- and (E)-1-iodo-1-alkenes was almost complete, but in some experiments with (Z)-bromo-1- alkenes up to 6% of the corresponding (E)-isomers were obtained. Rossi et al., (1982) used disiamylborane to reduce the triple bond in enye alcohols (eq.17). The advantage of the phase transfer method is the simple procedure. The method is better than those involving the use of either organometallic compounds (Rossi et al., 1981; Cassani et al., 1979) or amines as solvents (Sonagashira et al., 1975; Ratovelomana and Linstrumelle, 1981). The phase transfer procedure tolerates the presence of a functional group such as –OH and, therefore, is a simple and economical way to prepare several precursors of natural products. The coupling reaction under phase transfer conditions was employed to prepare some pure insect sex pheromone components in high overall yields. Chong and Heuft (1999) synthesized (3E,5Z)-3,5-dodecadienyl acetate with high (>99%) stereoselectivity using palladium-catalyzed coupling of the E-4-iodo-3-buten-1-ol with 1-octyne under phase transfer conditions. E-4-iodo-3-buten-1-ol was obtained by hydroalumination-iodinolysis.

Pd-cat., CuI, aq.NaOH OH OH I + + Benzene, BzEt3 N Cl 87% yield, 99% stereoisomeric purity

Pd-cat., CuI, aq.NaOH OH OH eq. 17 + I + Benzene, BzEt N Cl 3 85% yield, 99% stereoisomeric purity

A similar reaction was used in the synthetic route to (3Z,5E)-3,5-dodecadienyl acetate (II, III). 3-Butyn-1-ol was coupled with (E)-1-octenyl iodide in phase transfer conditions and in the presence of Pd-catalyst to yield (E)-5-dodecen-3-yn-1-ol. Unfortunately this reaction didn’t work very well in our hands: it was running very slowly and finally the yield of enyne was moderate. However, the stereoisomeric purity was excellent (no Z-isomer was detected by 1H NMR). The low yield might be due to the other Pd-catalyst, tris(dibenzylideneacetone)- dipalladium(0), or the other phase transferring agent, tetrabutylammonium bromide, used. Z- selective reduction of the triple bond in the (E)-enyne acetate, using a Zn-Cu couple as a reducing agent, produced the (3Z,5E)-3,5-dodecadienyl acetate in 87% yield and 97.4% stereoisomeric purity.

3.7. Syntheses of (Z,Z)-conjugated dienic pheromone compounds

3.7.1. Cadiot-Chodkiewicz reaction

A widely used method for the synthesis of Z,Z-conjugated dienic pheromone components is a route, involving the Cadiot-Chodkiewicz coupling reaction and subsequent dialkylborane reduction as the key steps.

The heterocoupling of a terminal alkyne with 1-bromoalkyne in the presence of an aliphatic amine and a catalytic amount of a copper(I) salt affords unsymmetrically substituted diynes. This useful reaction is often advantageously employed for the synthesis of several pheromone components. The bromoalkyne is generally introduced into a mixture of the alkyne, aliphatic amine and methanol in the presence of a catalytic amount of copper(I)chloride and a small amount of hydroxylamine hydrochloride (eq. 18).

MeOH, H2O, CuCl 1 2 1 2 2 EtNH .Br eq. 18 R +Br R + EtNH2 R R + 2 NH OH.HCl 2

The formation of the symmetrical diyne can be suppressed by maintaining the concentration of the bromoalkyne low. This side reaction is particularly serious in case less acidic alkynes, such as alkylalkynes, are used. The reducing agent, hydroxylamine hydrochloride, serves to maintain the copper in the reduced state.

The conjugated diyne was usually stereoselectively reduced to Z,Z-alkadiene by treatment with two equivalents of dicyclohexyl- or disiamylborane and protonolysis of the intermediate organoborane with acetic acid. The addition of a second equivalent of disiamylborane to the monohydroboration product derived from diyne was very slow. This difficulty could be circumvented by using the sterically less hindered dicyclohexylborane. Whereas monohydroboration of unsymmetrically substituted diynes produced a mixture of organoboranes, the dihydroboration-protonolysis procedure was suitable for conversion of unsymmetrical conjugated diynes to the corresponding Z,Z-dienes. The yields and the stereospecificity were usually good. The Cadiot-Chodkiewicz reaction is used for preparation of several Z,Z-dienic pheromone compounds: Z8,Z10-12-OH (Chisholm et al., 1985), Z3,Z5-14-OH (Doolittle and Solomon, 1986; De Jarlais and Emken, 1987), Z10,Z12-16-OH (Raina et al., 1986), Z9,Z11-16-OAld (Svirskaja et al., 1980). Unusual was the approach by Yadav et al. (2001), who prepared 3,5-tetradecadiyn-1-ol following the standard procedure for the Cadiot-Chodkiewicz cross-coupling reaction. An E,Z- dienic block was created by using two sequential stereoselective reductions: first was the E- selective reduction with LiAlH4 (97% stereoselectively) and second a reaction with disiamylborane (99% stereoselectively).

A Cadiot-Chodkiewicz reaction was used for preparation of the (3Z,5Z)-3,5-dodecadienyl and (8Z,10Z)-8,10-tetradecadienyl acetates (II, III and VII, respectively). The method worked very well in the synthesis of (8Z,10Z)-8,10-tetradecadienyl acetate, where a THP-protected alcohol was reduced with dicyclohexylborane. In the case of (3Z,5Z)-3,5-dodecadienol, isolation difficulties existed. It became apparent that the protection of the alcohol before reduction with dicyclohexylborane was necessary because of difficulties in separation of the dienic alcohol and cyclohexanol liberating during protonolysis of the organoborane. THP-protected alcohols or acetates were easily separated from cyclohexanol by MPLC. Distillation of cyclohexanol was not recommendable because of the risk of isomerization of the diene.

The Cadiot-Chodkiewicz reaction is a good method for the synthesis of Z,Z-dienic pheromones, but there is a limitation in the synthesis of compounds, where the double bond block is too close to the end of molecule. In such cases one of the components has to possess a very short carbon chain. This means that it is a highly volatile or gaseous compound, which is not commercially available or is difficult to handle. The Cadiot-Chodkiewicz reaction must run at room temperature or a little higher to reach a reasonable reaction rate.

3.7.2. Pd-catalyzed cross-coupling of two alkenyl moieties

Syntheses by controlled carbon-carbon bond formation with organometallic reagents are a useful methods leading directly to Z,Z-dienic pheromone compounds. The cross-coupling reaction between alkenyl halides and organometallic compounds represents a process of special synthetic value, as it enables the stereodefined production of a variety of systems with carbon-carbon double bonds. The cross-coupling reaction between organometallic species with organic halides and related electrophiles is one of the most straightforward methods that are known for carbon-carbon bond formation. An elegant route to E,Z- and Z,Z-dienes via cross-coupling reaction of (Z)-1- alkenylcuprates, obtained by the carbocupration of alkynes, derives from Jabri et al., (1981 and 1983) (eq. 19). The readily available (Z)-dialkenyl cuprates obtained by carbometallation of acetylene are good candidates for a straightforward synthesis of conjugated dienes.

2 3 R R

4 4 2 X R R 1 1 R2CuLi R R 2 CuLi 2 3 eq. 19 R R This reaction has to be catalyzed by Ni0 or Pd0 catalysts. The main difficulty is that a low temperature (0…+5 °C) is necessary to prevent decomposition of the cuprate, but the coupling is very slow at that temperature. In many cases of alkenyl-alkenyl coupling via Pd0, the catalysis must be run at room temperature or even reflux. The Ni0 catalysis is faster but less stereoselective. The transmetallation reaction from a lithium cuprate to another organometallic reagent, which is more reactive towards alkenyl halides in the presence of Ni0 or Pd0 catalysts was used by analogy of Negishi et al., (1978) and Negishi (1982) who used a transmetallation from an alkenyl alane to an alkenyl zinc halide for promoting the coupling with alkenyl halides in the presence of a Pd0 catalyst. Upon addition of ZnBr2 the transmetallation occurred, producing the alkenyl zinc species, which proved to be excellent reagents for the coupling of alkenyl halides in the presence of a Pd0 catalyst (eq. 20).

CuI 2 ZnX2 1 1 1 1 + 1 2 R Li R 2CuLi R ZnX R 2 CuLi R Cu, LiX 2 3 R R

4 4 X R 1 R R Pd0 2 3 eq. 20 R R

Excellent yields of the expected dienes with an extraordinary stereoisomeric purity were obtained. Essentially complete retention of configuration of the substrate and organometallic was the main feature of this approach. Iodides are generally more reactive than the corresponding bromides and trans-halides are more reactive than their cis-analogues. Conclusion: lithium dialkenyl cuprates are not able to couple with alkenyl halides. However, upon in situ transmetallation to the corresponding alkenyl zinc reagent they couple very easily in the presence of catalytic amounts of Pd0. Only one alkenyl group of the cuprate was used in this reaction. Jabri et al., (1983) found that both alkenyl groups were transferred if the lithium cuprate was first transformed into a magnesium cuprate (eq. 21):

MgCl ZnBr 2 + 2 LiCl + 1 1 1 R 1 Cu, MgX R ZnX R 2 CuLi R 2 CuMgX 2 2 I R 2 2 1 2 R R eq. 21 Pd0

Exploring the above-mentioned reactions, Gardette (1984) set up his general methodology for the syntheses of conjugated dienic insect sex pheromones. The methodology was based on three useful reactions: 1) the carbocupration of acetylene, 2) the coupling of the obtained (Z)-alkenyl cuprates with alkenyl iodides, 3) the iodination of the functionalized (Z)-dialkenyl cuprates, which affords the often required functionalized (Z)-dialkenyl cuprates.

Considering that the diene system in pheromones is more frequently (E,Z)- or (Z,E)- type, Gardette divided the molecule into (Z)- and (E)-part and presented two synthesis strategies according the position of the terminal functionality: 1) E-Z type with the functionality lying on the (E)-part 2) Z-E type with the functionality lying on the (Z)-part.

Gardette exemplified his methodology by the synthesis of E7,Z9-12OAc according to the following one-pot procedure (eq. 22):

2 ZnBr2 + Et2CuLi Et 2 CuLi THF Et Cu, LiX Et ZnX Et2O I OAc eq. 22 Pd(PPh ) OAc 3 4

The yield of the reaction was 78% and the isomeric purity of the product was 97% (depending on the purity of alkenyl iodide). Noteworthy was the fact that the acetate functionality was not affected by the organometallic reagent used.

To avoid the oxygenated functionality in the hydroalumination process, a halogen which tolerated a strong Lewis acid such as DIBAH was used to synthesize the bombykol (eq. 23):

1. ZnBr2 NaI Cl I Pro 2 CuLi 2. I Cl Acetone

Pd(PPh3)4

OMgCl ClMg OH eq. 23 CuX

In this case the organometallic reagent completely discriminated between the alkenyl halogenides in favour of the iodide. The chloro-diene was obtained >99.5% stereoisomerically pure. Before the carbon chain extension with the functionalized Grignard reagent, the chloride was transformed into the more reactive iodide. Because of this very slow transformation with some (10%) isomerization of the dienic block, another similar route was investigated, in which the transhalogenation step preceded the key coupling reaction (eq. 24):

Pro CuLi NaI 2 I I Cl I I Acetone ZnBr2, Pd(PPh3)4 OMgCl ClMg OH eq. 24 CuX

The reaction between the diiodide and the dialkenyl zinc species proceeded quantitatively, attacking exclusively at the alkenyl iodide group. The bombykol synthesized showed a 99.8% stereoisomeric purity.

The syntheses of the Z-E type of pheromones were illustrated by the synthesis of (9Z,11E)-9,11-tetradecadienyl acetate. According to Gardette’s general methodology, the functionality lay on the (Z)-part of the molecule and had to be introduced by the (Z)-dialkenyl cuprate moiety (eq. 25): + I (CH ) - CH eq. 25 Y- (CH ) 2 CuLi 2 m 3 Y- (CH ) (CH ) - CH 2 n 2 n 2 m 3

In contrast to the previous case, the more valuable of the two partners here was the functionalized (Z)-alkenyl cuprate. For that reason it was important to use both alkenyl groups of the cuprate. As this was shown above, it was possible when the lithium cuprate was first transferred into the magnesium cuprate and then transmetallated into the zinc reagent. This experiment gave the product with 97.2% stereoisomeric purity and 60% overall yield.

Pheromones of the Z-Z type may be synthesized according to this methodology in the following two ways (eq. 26):

I (CH2)n-Y Et 2 CuLi (CH2)n-Y eq. 26 Et I Et Y- (CH ) CuLi 2 n 2

The method was exemplified by the description of the synthesis of (11Z,13Z)-11,13- hexadecadien-1-ol. The (Z)-alkenyl iodide functionalized along the first route was coupled with (Z)-dibutenyl cuprate under the usual conditions. The (Z,Z)-stereoisomeric purity was estimated at >99%. According to the other possible route, the functionalized cuprate was treated with (Z)- 1-iodobutene under the coupling conditions, where both alkenyl groups of the cuprate were used. The product was >99% stereoisomerically pure.

Another example was the synthesis of (9Z,11Z)-9,11-tridecadien-1-yl acetate. This compound was synthesized by coupling of two alkenyl moieties, one of which was a (Z)- propenyl group and the other one a functionalized (Z)-alkenyl iodide (eq.27): I ZnBr2 AcO OAc eq. 27 Li ZnBr Pd(PPh ) THF 3 4

In this case the organometallic partner, the (Z)-propenyl moiety, could not be prepared by carbocupration of acetylene, since lithium dimethyl cuprate did not add satisfactorily to acetylene. For that reason the (Z)-1-bromopropene required was prepared from crotonic acid, and converted into the lithium reagent (by Li metal). This was transmetallated into the zinc reagent for the coupling reaction with the functionalized (Z)-alkenyl iodide. The isomeric purity

of the diene was 92%. The loss of purity was assumed to be caused by isomerization during the preparation of the (Z)-propenyl-lithium. Nevertheless, Pd-catalyzed cross-coupling reaction of alkenyl-zinc bromide with (Z)-1-alkenyl iodide was the method of choice for the synthesis of (Z,Z)-dienic pheromone components, in which the double bonds block occurred close to the end of the molecule.

The syntheses of (8Z,10Z)-8,10-dodecadien-1-ol (appendix 2, papers I, IV, VI) and (10Z,12Z)-10,12-tetradecadien-1-ol (appendix 1) were realized according to the same reaction scheme.

3.8. Syntheses of (9Z,11Z)-9,11-tetradecadien-1-ol and the corresponding acetate and aldehyde

(9Z,11Z)-9,11-tetradecadien-1-ol, its acetate and the corresponding aldehyde have been identified from the calling female of Lampronia capitella (Cl.) (Lepidoptera: Prodoxidae), (Löfstedt et al., 2004). The pheromone of this insect was identified quite recently. Since very little was known about the pheromone of L. capitella, it was necessary to continue this work for several years and the compounds had to be synthesized many times. This was a sufficient reason for revising the synthetic pathway. Finally, the best synthetic scheme in my understanding was set up. Variations in the synthetic scheme were made concerning the convenience of the experimental performance, improvements of the yield and stereoselectivity, avoidance of the hazardous factors of the reagents (toxicity, allergenic properties), reproducibility and cost of the reagents.

In the first synthesis, the preparation of 1-(2-tetrahydropyranyloxy)-9-decyne was solved by bromination-dehydrobromination and protection of dec-10-enyl alcohol (eq. 28) (Bishop and Morrow, 1983; Miyaura et al., 1983). Br Br 2 DHP H O H O PPTS Br Br eq. 28 NaNH2 THPO THPO Br

In the second version (VIII), the acetylide was obtained by reaction of the tosylate of 1- (2-tetrahydropyranyloxy)-octan-8-ol with a lithium acetylenide/ethylenediamine complex. The reactions of alkyl bromides with lithium acetylenide/ethylenediamine complexes were the usual and widely applied procedures. It was known that tosylate was an even better leaving group than bromine and that tosylation of alcohols was a very easy to run, clean and complete reaction. Treatment of the tosylate with a lithium acetylenide/ethylenediamine complex led to 1- (2-tetrahydropyranyloxy)-9-decyne in good yield. There was no remarkable loss in yields using the tosylate and the decyne as such in the next step without purification.

The other important step in the synthesis of (9Z,11Z)-9,11-tetradecadien-1-ol was the hydroxymethylation of the organometallic intermediate with paraformaldehyde. The alkynyllithium could be created by abstracting the ethynyl proton in the terminal alkyne by means a strong base, such as BuLi or a Grignard reagent, usually EtMgBr. In case the reaction was carried out using BuLi, attention must be paid to the ratios of the reagents. It was important always to use about 5% less of BuLi than alkyne, to avoid the formation of the sticky insoluble material that meant the loss of compound and often a tedious work to prepare it. This never

happened when the Grignard reagent was used. Finally, the cost of these reagents was strongly in favour to EtMgBr. The conditions for the Wittig coupling in the synthetic scheme of (9Z,11Z)-9,11- tetradecadien-1-ol were chosen considering the suggestions given by Vinczer et al., (1988a, 1991). This time the Wittig coupling was intended to work under conditions leading to as much (Z)-coupling as possible. t-BuOK was, therefore, selected as a base, toluene was used as a solvent and the aldehyde was added at -78 °C. A good yield was obtained, provided that the alkyl chain of the phosphonium salt had no base-sensitive group. As t-BuOK was known to be quite an unstable reagent, it was always prepared shortly before use. The Wittig coupling gave (Z)-1-(2-tetrahydropyranyloxy)-9-yn-11-tetradecene in 83% yield. A highly specific conversion of only the triple bond in the conjugated dienyne system into a (Z)-double bond was accomplished by hydroboration with dicyclohexylborane and successive protonolysis. Attempts to use other reducing reagents such as P2-Ni, failed. (9Z,11Z)-9,11-tetradecadien-1-ol with an isomeric purity of 86% was obtained after deprotection and chromatography. Repeated crystallization of the compound from pentane at −30 °C increased the isomeric purity to 99.1 %.

IV. CALCULATION OF PHEROMONE COMMUNICATION CHANNEL PARAMETERS

Pheromone communication systems have a reliable signal with a restricted window of amounts and ratios released and perceived. The importance of ratios of pheromone components for optimum response and species specificity has long been appreciated for Lepidopteran pheromone systems (Roelofs, 1978), and more recently for coleopteran systems (Teale et al., 1991; Leal et al., 1994). The concept of the communication channel width has been introduced by Howse et al. (1998). The channel of chemical communication has always had a defined width, i.e. only certain chemical compounds in certain ratios to which the receiver is selectively sensitive have been able to carry the message. This has shown the importance of variation in ratios for optimum response and the width of the response, determined by genetic and environmental factors (Löfstedt, 1990; Svensson, 1996). However, the optimum of response has so far been determined by simply choosing the best experimental value or by visual interpolation between discrete values of the independent axis. The width of the response window has sometimes been set by visual inspection, but usually not quantified at all. A model based on a Gaussian response profile that allowed a quantification of the response peak (location and optimum) and a measure of the peak width (response window) was independently presented by a Swedish (Schlyter et al., 2001) and an Estonian (Mõttus et al., 2001) research group. Proposed model is a more analytical approach to quantify the optimal value of ratios of pheromone components, i.e. to find the correlation between the pheromone component ratio and the blend attractivity. The determination of the optimal ratio of components and the variability in pheromone communication within the species are examined in paper IX. The calculation method was used to determine the optimal pheromone component ratio and the communication channel width for Archips podana (Scopoli) (Lepidoptera: Tortricidae). Numerous field screening experiments aimed of determining the optimal ratio of (E)- and (Z)-11-tetradecen-1-yl acetates in the attractant of A. podana showed discordant results. Different authors estimated the optimal attractant as 60:40 to 40:60 mixtures of these two isomers. The difference in pheromone communication of different phenotypes of A. podana was hypothesised as the possible explanation of contradictory pheromone blend optimization results in different regions. In case the phenoptypes are signs of population variations, some differences in pheromone communication may be expected. To clear up the need for special attractant blends for different phenotypes, the trap catch data from different regions were used. All the A. podana populations investigated had identical values of their pheromone communication channel. Our experiments demonstrated the full identity of pheromone communication channel of A. podana of different phenotypes. The optimum ratio of 11-tetradecenyl acetate isomers was 61.3% of Z-isomer. Because of the lack of the differences in pheromone communication of A. podana in all tested areas, the optimized pheromone dispenser might be used for plant protection and environmental survey (IX). As regards conjugated dienic compounds, changes in attractivity of two-component blends obeying any distribution law have not been described. Field trapping results of L. capitella have demonstrated the positive effect of RAld on trap catch when added to ROH. Maximum effect has been achieved by using 30% or higher content of RAld in dispensers (equals 75% of RAld in effluvia). RAld alone has not been attractive. Results have confirmed the lack of maximum attractivity ratio for binary mixtures of ROH and RAld. A “plateau effect” has taken place instead. It means that after the content of RAld has achieved the optimal value in effluvia, further adding of RAld does not influence the trap catches (VIII).

V. CONCLUDING DISCUSSIONS

To fulfil the biological research presented in papers included in this thesis it was necessary to synthesize a range of highly chemically and isomerically pure pheromone components. All compounds needed were made in high stereochemical purity, most of them more than 99% isomerically pure, either directly from the synthesis or by using several separation and purification techniques.

Results of present theses:

1. The two pheromone components of M. sexta, (10E,12E,14E)-10,12,14- and (10E,12E,14Z)-10,12,14-hexadecadien-1-als were prepared in 98 and 97% isomeric purity, respectively (appendix 3; paper V).

Two new, previously unknown, pheromone-sensitive olfactory receptor neuron (ORN) types on male Manduca sexta (L.) (Lepidoptera: Sphingidae) antennae besides previously known EZ-, EEZ- and EEE- types were found. Two new ORNs were the EE-specific and Z11- specific ORNs. This finding suggests that also EE- and Z11- play an active role in the sexual communication of M. sexta. Our data bring the first physiological evidence that M. sexta females do respond to at least one pheromone component.

2. The four isomers of 3,5-dodecadienyl acetate, all with an isomeric purity of more than 99% were used in studies to complete the identification of compounds produced in female insect sex pheromone glands of the Brazilian apple leafroller, Bonagota cranaodes (Meyrick) (Lepidoptera: Tortricidae) and for the first field trapping tests of the pheromone candidate compounds (II and III).

A detailed identification of the compounds produced in female sex glands of the B. cranaodes confirmed the presence of (3E,5Z)-3,5-dodecadienyl acetate besides several saturated acetates and one monoenic acetate, Z9-hexadecenyl acetate. The latter was found to synergize the attraction of (3E,5Z)-3,5-dodecadienyl acetate in field trapping tests in the ratio of 1:1 in the lure. We tested the effect of the other geometric isomers (EE, ZE, ZZ) on male B. cranaodes attraction to E3,Z5–12Ac. None of these isomers had a pronounced effect on the male trap catch, and isomeric purity of E3,Z5–12OAc is thus not critical for the male attraction to traps. This finding is important with respect to the practical use of synthetic E3,Z5–12OAc for monitoring of B. cranaodes: economic synthesis is greatly facilitated, and isomerization will not have a decisive effect on the life of pheromone dispenser formulations. Poor attractivity of (3E,5Z)-3,5-dodecadienyl acetate alone in the field is accordingly rather due to missing pheromone synergists in the lure. The geometric isomers of dienic pheromone compounds are known to have a very pronounced behavioural effect in other tortricid moths. For example, several species, belonging to the tortricid subfamily Grapholitinae, use different isomers of 8,10-dodecadienyl acetate (8,10–12OAc) as main pheromone compounds. The attraction of the pea moth Cydia nigricana F. (Lepidoptera: Tortricidae) to the female sex pheromone (E,E)-8,10-dodecadienyl acetate is very strongly inhibited by geometric isomers (Witzgall et al., 1993). Male attraction is usually strongly synergized or inhibited by the presence of the other isomers, which are pheromone components in the same or in closely related species, whereas monoenic compounds produced by the females often play only a minor role in pheromonal communication (Witzgall et al., 1996). In comparison, E3,Z5–12OAc has not been reported from other tortricids, but only as a sex attractant for one gelechiid species (Reed and Chisholm 1985; Arn et al., 2000). B.

cranaodes belongs to the subfamily Tortricidae, within which most of the species communicate with monoenic compounds. Lack of a strong behavioural effect of the isomers of E3,Z5–12OAc makes it conceivable that monoenic gland compounds would be important for species specificity of pheromone signals in B. cranaodes and related species.

3. Bio assaying results using synthesized geometrical isomers of 8,10- and 10,12- tetradecadienols and corresponding acetates are presented in paper VII, synthetic scheme in appendix 1.

Volatiles collected from calling females of leafminer moth Phyllonorycter emberizaepenella (Lepidoptera: Gracillariidae) were identified as (8E,10E)-8,10-tetradecadien-1-ol and its acetate. It was possible that (8E,10E)-8,10-tetradecadien-1-yl acetate alone or in a binary mixture with a corresponding alcohol was a sex pheromone component of the Ph. emberizaepenella species. 8E,10E-14OAc and 8E,10E-14OH had not yet been identified from the species of the order Lepidoptera. Thus, our identification extended the number of taxons, in which these compounds are released by calling females. We proved that the leafminer moth Ph. emberizaepenella reproduces by parthenogenesis of the thelytoky type. As far as we know, Ph. emberizaepenella was the first case of thelytokous parthenogenesis found in the Gracillariidae family (appendix 1, paper VII).

4. Results of wind tunnel tests and field experiments with the geometrical isomers of codlemone, (E,Z)-, (Z,E)-, and (Z,Z)-8,10-dodecadienol and their acetates, codlemone acetate and aldehyde and the re-identification of the compounds extracted from the pheromone gland of the female codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), are presented in papers I, IV, and VI. A synthetic scheme of codlemone isomers is depicted in appendix 2. 5. Chemical analyses of the compounds extracted from the pheromone gland of female codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae, resulted in identification of fourteen compounds (VI). Besides the main constituent, codlemone, all of its possible geometric isomers were present. The codlemone acetate was present in quantities 0.01% of the main compound codlemone, but elicited a far stronger antennal response than other minor gland compounds. 3.9% of codlemone aldehyde was found. Two monounsaturated alcohols, (E)-8- and (E)-9- dodecenol, were found. Five saturated alcohols (C10, C12, C14, C16, C18), and a saturated aldehyde, octadecanal, were also found. Wind tunnel and field tests showed a strong antagonistic effect of large amounts of E,Z- and Z,Z-isomers when added to the codlemone. In amounts less than 5% (corresponds to gland composition) E8,Z10-12OH enhanced the male response. The Z,E-isomer had a synergistic effect (I, IV). Response tests with individual isomers of codlemone showed males flying upwind and contacting the source only of the main pheromone compound and the Z,E-isomer (IV). In wind tunnel experiments, addition of dodecanol, the E,Z-isomer of codlemone and codlemone acetate augmented the male attraction to codlemone at the proportions found in female glands. Larger amounts of the E,Z-isomer of codlemone and codlemone acetate had a strong antagonistic effect on male attraction. The identification of pheromone synergists and antagonists in codling moth was of importance for the development of pheromone-based control methods (VI).

6. Studies of the pheromone communication of Lampronia capitella (Cl.) (Lepidoptera: Prodoxidae) showed that two of the compounds identified, (9Z,11Z)-9,11-tetradecadien- 1-ol and (9Z,11Z)-9,11-tetradecadien-1-al, were obligatory for achieving high trap catches; the third compound identified in female pheromone, (9Z,11Z)-9,11- tetradecadien-1-yl acetate, had no influence on trap catch in field experiments.

This investigation provided a sex lure and dispenser that could be used to detect the presence of L. capitella males. Future work tuned to improve the attractant dispenser for mass- trapping. Silicon elastomer was recommended as a substrate for dispensers, as both alcohol and aldehyde were stable and made many-years storage of dispensers possible (VIII).

Although the compounds for biological research were made in respect of high chemical and isomeric purity, it was obvious that this purity was going to be destroyed when the compounds were loaded into the substrates with unknown properties of isomerization. Results from studies of antagonistic effects on geometrical isomers in wind tunnel suggested that it was necessary to develop formulations and release techniques that maintained extremely high isomeric purity of the synthetic pheromone for male trapping in the field. The attraction of some insect species had been shown to be extremely dependent on the presence of nonpheromonal isomers and decreased significantly (I, IV, and VI), whereas the others were quite insensitive and geometrical isomers were behaviourally benign or have slightly inhibitory effects (III). Thus, it is necessary to continue with developing the suitable non-isomerizing substrates for polyenic pheromone compounds (VIII). Very progressive is the use of spraying devices in the wind-tunnel tests, to ensure the quality and quantity of the blend released into the test area (I, IV).

The suitable synthetic schemes for the synthesis of all four geometrical isomers of four conjugated dienic pheromone components are presented in this thesis. The reactions and purification methods used here exemplify the wide range of possibilities realized for such syntheses. Although the syntheses presented in the thesis do not show outstanding results in development of synthetic organic chemistry, they present the successful application of many synthetic methods. The importance of this synthetic work is that the compounds synthesized are not commercially available or on the other hand the purity of the purchasable compounds is not satisfactory for research in the field of chemical ecology.

Useful materials for several research projects were prepared and delivered.

REFERENCES

Aerssens, M.H.P.J. and Brandsma, L. 1984. Regio- and stereo-specific reduction of conjugated and non-conjugated triple bonds by activated zinc powder. J. Chem. Soc., Chem. Commun. (12): 735-736. Aerssens, M.H.P.J., van der Heiden, R., Heus, M. and Brandsma, L. 1990. A Quick Procedure for the Partial Reduction of Triple Bonds. Synth. Comm. 20(22): 3421-3425. Agarwal, S., Tiwari, H.P. and Sharma, J.P. 1990. Pyridinium Chlorochromate: An Improved Method for Its Synthesis and Use of Anhydrous Acetic Acid As Catalyst for Oxidation Reactions. Tetrah. 46(12): 4417-4420. Albini, A. 1993. Synthetic Utility of Amine N-Oxides. Synthesis: 263-277. Ando, T., Kurotsu, Y.,Kaiya, M. and Uchiyama, M. 1985. Systematic Syntheses and Characterization of Dodecadien-1-ols with Conjugated Double Bond, Lepidopterous Sex Pheromones. Agric. Biol. Chem. 49(1): 141-148. Ando, T., Ogura, Y., Koyama, M., Kurane, M., Uchiyama, M. and Seol, K.Y. 1988. Syntheses and NMR Analyses of Eight Geometrical Isomers of 10,12,14-Hexadecatrienyl Acetate, Sex Pheromone Candidates of the Mulberry Pyralid. Agric. Biol. Chem. 52(10): 2459- 2468. Arn, H., Guerin, P.M., Buser, H. R., Rauscher, S. and Mani, E. 1985. Sex pheromone blend of the codling moth, Cydia pomonella: Evidence for a behavioural role of dodecan-1-ol. Experientia 41: 1482-1484. Arn, H., Tóth, M., and Priesner, E. 2000. List of sex pheromones of Lepidoptera and related attractants. Online version http://www.pherolist.slu.se/. Attygalle, A.B., Jham, G.N., Svatoš, A., Frighetto, R.T.S., Meinwald, J., Vilela, E.F., Ferrara, F.A. and Uchôa-Fernandes, M.A. 1995. Microscale, Random Reduction: Application to the Characterization of (3E,8Z,11Z)-3,8,11-Tetradecatrienyl Acetate, a New Lepidopteran Sex Pheromone. Tetrah. Lett., 36(31): 5471-5474. Bæckström, P., Stridh, K., Li, L., and Norin, T. 1987. Claisen rearrangements with mesityl oxide dimethyl ketal. Synthesis of ipsdienone, E- and Z-ocimenone, 2,6-dimethyl-2,7- octadien-4-one and 2,6-dimethyl-2,7-octadien-4-ol. Acta Chem. Scand. B. 41: 442-447. Bæckström, P., Jacobsson, U., Norin, T. and Unelius, C.R. 1988. Synthesis and Characterization of All Four Isomers of Methyl 2,4-Decadienoate for an Investigation of the Pheromone Components of Pityogenes chalcographus.Tetrah. 44(9): 2541-2548. Baker, T. C., Staten, R. T. and Flint, H. M. 1990. Use of pink bollworm pheromone in the southwestern United States, pp. 417-436, in R. L. Ridgway, R. M. Silverstein, and M. N. Inscoe (eds.), Behavior-Modifying Chemicals for Insect Management: Application of Pheromones and Other Attractants. Marcel Dekker, New York. Barnes, M.M., Millar, J.G., Kirch, P.A. and Hawks, D.C. 1992. Codling moth (Lepidoptera, Tortricidae) control by dissemination of synthetic female sex pheromone. J. Econ. Ent. 85: 1274-1277. Bartell, R.J., Bellas, T.E. and Whittle, C.P. 1988. Evidence for biological activity of two further alcohols in the sex pheromone of female Cydia pomonella L. (Lepidoptera, Tortricidae). J. Austr. Entomol. Soc. 27: 11-12. Bestmann, H.J., Stransky, W. and Vostrowsky, O. 1976. Darstellung Lithiumsalzfreier Ylidlösungen mit Natrium-bis(trimethylsilyl)amid als Base. Chem. Ber. 109: 1694- 1700. Bestmann, H.J., Vostrowsky, O., Paulus, H., Billmann, W. and Stransky, W.1977. Pheromone XI. Eine Aufbaumethode für Konjugierte (E),(Z)-Diene. Synthese des Bombykols, Seiner Derivative und Homologen. Tetrah. Lett. 121-124. Bestmann, H.J., Koschatzky, K.H., Schätzke, W., Süß, J. and Vostrovsky, O. 1981a. Pheromone, XXXII. Bausteine zur Darstellung zweifach ungesättigter Schmetterlings- pheromone. Liebigs Ann. Chem.: 1705-1720.

Bestmann, H.J., Süß, J. and Vostrovsky, O. 1981b. Pheromone XXXIV. Synthese konjugiert- ungesättigter Lepidopterenpheromone und Analoga. Liebigs Ann. Chem.: 2117-2138. Bestmann, H.J. and Gunawardena, N.E. 1992. Pheromones; 87. An Efficient Synthesis of (6E,11Z)-6,11-Hexadecadienyl Acetate and (6E,11Z)-6,11-Hexadecadienal: Female Sex Pheromone Components of Antheraea pernyi and A. polyphemus (Lepidoptera: Saturniidae). Synthesis: 1239-1241. Bishop, C.E. and Morrow, G.W. 1983. Synthesis od (Z,Z)-11,13-Hexadecadienal, a Principal Component of Navel Orangeworm (Pamyelois transitella) Pheromone. J. Org. Chem. 48: 657-660. Bjostad, L.B., Wolf, W.A. and Roelofs, W.L.1987. Biology and ultrastructure of sex pheromone-producing glands, pp.77-120, in G.D. Prestwich and G.J. Blomquist (eds.), Pheromone Biochemistry. Acad. Press, New-York. Brandsma, L., Nieuwenhuizen, W.F., Zwikker, J.W. and Mäeorg, U. 1999. Reduction of Acetylenic Compounds to (E)-Olefins by Alkali Metals - An Investigation of the Scope. Eur. J. Org. Chem.: 775-779. Brown, C.A. and Ahuja, V.K. 1973a. “P-2 Nickel” Catalyst with Ethylenediamine, a Novel System for Highly Stereospecific Reduction of Alkynes to cis-Olefins. J.C.S. Chem. Comm. 553-554. Brown, C.A. and Ahuja, V.K. 1973b. Catalytic Hydrogenation. VI. The Reaction of Sodium Borohydride with Nickel Salts in Ethanol Solution. P-2-Nickel, A Highly Convenient, New, Selective Hydrogenation Catalyst with Great Sensitivity to Substrate Structure. J. Org. Chem. 38(12): 2226-2230. Brown, D. F. and McDonough, L. M. 1986. Insect Sex Pheromones: Formulations to Increase the Stability of Conjugated Dienes. J. Econ. Entomol. 79: 922–924. Būda, V.G., Mozūraitis, R.L., and Merčaitis, V.P. 1991. Parthenogenesis and signalling behaviour in the moth Lithocolletis emberizaepenella Bouche (Lepidoptera: Gracillariidae). Dokl. Akad. Nauk SSSR 119: 1017-1020. (In Russian). Byers, J.A. 1988. Novel diffusion-dilution method for release of semiochemicals: testing pheromone component ratios on Western pine beetle. J. Chem. Ecol. 14: 199-212. Cabezas, J.A. and Oehlschlager, A.C. 1999. Stereospecific Synthesis of (E,Z)- and (Z,Z)- Hexadeca-10,12-dienal. Sex Pheromone Components of Diaphania hyalinata. Synthesis: 107-111. Cardé, R. T., and Minks, A. K. 1995. Control of moth pests by mating disruption: successes and constraints. Ann. Rev. Entomol. 40: 559–585. Cassani. G., Massardo, P. and Piccardi, P. 1979. New Simple Synthesis of Internal Conjugated (Z)-enynes. Tetrah. Lett.: 633-634. Charmillot, P.-J. 1990. Mating disruption technique to control codling moth in western Switzerland, pp. 165-182, in R. L. Ridgway, R. M. Silverstein and M. N. Inscoe (eds.), Behavior-Modifying Chemicals for Insect Management: Application of Pheromones and Other Attractants. Marcel Dekker, New York. Chen, X. and Millar, J.G. 2000. Preparative Scale Syntheses of Isomerically Pure (10E,12E,14Z)- and (10E,12E,14E)-Hexadeca-10,12,14-trienals, Sex Pheromone Components of Manduca sexta. Synthesis: 113-118. Chisholm, M.D., Steck, W.F., Bailey, B.K. and Underhill, E.W. 1981. Synthesis of sex pheromone components of the forest tent caterpillar (Malacosoma disstri: Hübner) and of the western tent caterpillar (Malacosoma californicum: Pckd). J. Chem. Ecol. 7(1): 159-164. Chisholm, M.D., Reed, D.W., Underhill, E.W., Palaniswamy, P. and Wong, J.W. 1985. Attraction of Tortricid Moths of Subfamily Olethreutinae to Field Traps Baited with Dodecadienes. J. Chem. Ecol. 11(2): 217-230. Chong, J.M. and Heuft, M.A. 1999. Hydroalumination of 3-Butyn-1-ol: Application to a Stereoselective Synthesis of (3E, 5Z)-3,5-Dodecadienyl Acetate, the Sex Pheromone of the Leaf Roller Moth. Tetrah. 55: 14243-50.

Coffelt, J.A., Vick, K.W., Sonnet, P.E. and Doolittle, R.E. 1979. Isolation, Identification, and Synthesis of a Female Sex Pheromone of the Navel Orangeworm, Amyelois transitella (Lepidoptera: Pyralidae). J. Chem. Ecol. 5: 955-966. Commercon, A., Normant, J.F. and Villieras, J. 1980. Substitution des halogéno-1 alcynes-1 par les dérivés organométalliques du cuivre. Accès à une nouvelle classe de synthons: Application à la synthèse du bombykol. Tetrah. 36(9): 1215-1221. Coracini, M.D.A., Bengtsson, M., Reckziegel, A., Löfqvist, J., Francke, W., Vilela, E.F., Eiras, A.E., Kovaleski, A. and Witzgall, P. 2001. Identification of a Four-Component Sex Pheromone Blend in Bonagota cranaodes (Lepidoptera: Tortricidae). J. Econ. Entomol. 94(4): 911–914. Corey, E.J., and Suggs, J.E. 1975. Pyridinium chlorochromate. An efficient reagent for oxidation of primary and secondary alcohols to carbonyl compounds. Tetrah. Lett. 2647-2650. Corey, E. J. and Boger, Dale L. 1978. Oxidative cationic cyclization reactions effected by pyridinium chlorochromate. Tetrah. Lett. 2461-2464. Corey, E.J. and Schmidt, G. 1979. Useful Procedures for the Oxidation of Alcohols Involving Pyridinium Dichromate in Aprotic Media. Tetrah. Lett. (5): 399-402. Dang, H.P. and Linstrumelle, G. 1978. An Efficient Stereospecific Synthesis of Olefins by the Palladium-Catalyzed Reaction of Grignard Reagents with Alkenyl Iodides. Tetrah. Lett.: 191-194. De Jarlais, W.J. and Emken, E.A. 1987. Stereospecific Synthesis of (Z,Z)-3,5-Tetradecadienoic Acid, A Component of Attagenus elongatulus (Casey) Pheromone. J. Chem. Ecol. 13: 179-184. Dennehy, T. J., Roelofs, W. L., Taschenberg, E. F. and Taft, T. N. 1990. Mating disruption for control of the grape berry moth in New York vineyards, pp. 223-240, in R. L. Ridgway, R. M. Silverstein and M. N. Inscoe (eds.), Behavior-Modifying Chemicals for Insect Management: Application of Pheromones and Other Attractants. Marcel Dekker, New York. Descoins, C. and Henrick, C.A. 1972. Stereoselective Synthesis of a Sex Attractant of the Codling Moth. Tetrah. Lett.: 2999-3002. Descoins, C., Lettere, M., Linstrumelle, G., Michelot, D. and Ratovelomanana. 1984. Stereoselective Syntheses of (E)-7, (Z)-9 Dodecadien-1-yl Acetate. Synth. Comm. 14: 761-773. Doolittle, R.E., Roelofs, W.L., Solomon, J.D., Cardé, R.T. and Beroza, M. 1976. (Z,E)-3,5- Tetradecadien-1-ol Acetate, Sex Attractant for the Carpenterworm Moth, Prionoxystus robiniae (Peck) (Lepidoptera: Cossidae). J. Chem. Ecol. 2(4): 399-410. Doolittle, R.E. and Solomon, J.D. 1986. Stereoselective Synthesis of (Z,E)-3,5-Tetradecadienyl Acetate, Sex Attractant for the Carpenterworm Moth, Prionoxystus robiniae (Peck) (Lepidoptera: Cossidae) and Effect of Isomers and Monounsaturated Acetates on Its Attractiveness. J. Chem. Ecol. 12(3): 619-633. Doolittle, R.E., Brabham, A. and Tumlinson, J.H. 1990. Sex Pheromone of Manduca Sexta (L). Stereoselective Synthesis of (10E,12E,14Z)-10,12,14-Hexadecatrienal and Isomers. J. Chem. Ecol. 16(4): 1131-1153. Ebesu, R. 2003. Integrated Pest Management for Home Gardens: Insect Identification and Control. Insect Pests 13: 1-11. Einhorn, J., Beauvais, F., Gallois, M., Descoins, C. and Causse, R. 1984. Constituants secondaires de la pheromone sexuelle du Carpocapse des Pommes, Cydia pomonella L. (Lepidoptera, Tortricidae). Comptes Rendus de l’Académie des Sciences, Paris 299 (III): 773-778. Einhorn, J., Witzgall, P., Audemard, H., Boniface, B. and Causse, R. 1986. Constituants secondaires de la pheromone sexuelle du Carpocapse des Pommes, Cydia pomonella L. (Lepidoptera, Tortricidae). II. Premiére approche des effects comportementaux. Comptes Rendus de l’Académie des Sciences, Paris 302 (III): 263-266.

Eiras, A.E., Delmore, I.R.K., Parra, J.R.P., Pique, M.P.R., Vilela, E.F. and Kovaleski, A. 1994. Biologia comparada da lagarta encroladeira Phtheochroa cranaodes Meyrick (Lepidoptera: Tortricidae) em duas dietas artificiais. An. Soc. Entomol. Bras. 23: 251- 257. Evershed, R.P., Morgan, E.D. and Thompson, L.D. 1982. Preparative-scale separation of alkene geometric isomers by liquid chromatography. J. Chromatogr. 237: 350-354. Ferrao, P., Gries, G., Wimalaratne, P.D.C., Maier, C.T., Gries, R., Slessor, K.N. and Li, J. 1998. Sex Pheromone of Apple Blotch Leafminer, Phyllonorycter crataegella, and Its Effect on P. mesipilella Pheromone Communication. J. Chem. Ecol. 24(12): 2059-2078. Ferveur, J. 1997. The pheromonal role of cuticular hydrocarbons in Drosophila melanogaster. BioEssays 19(4): 353-358. Gardette, M., 1984. General Methodology for the Synthesis of Conjugated Dienic Insect Sex Pheromones. Tetrah. 40(14): 2741-2750. Goto, G., Shima T., Masuya, H., Masuoka, Y. and Hiraga, K. 1975. A Stereoselective Synthesis of (Z,E)-9,11-tetradecadienyl acetate: A major component of the sex pheromone of Spodoptera litura. Chem. Lett.: 103-106. Hall, D.R., Beevor, P.S., Lester, R., Poppi, R.G. and Nesbitt, B.F. 1975. Synthesis of the major sex pheromone of the Egyptian cotton leaf worm, Spodoptera littoralis. Chem. Ind. (London): 216-217. Heath, R.R., Tumlinson, J.H. and Doolittle, R.E. 1977. Analytical and Preparative Separation of Geometrical Isomers by High Efficiency Silver Nitrate Liquid Chromatography. J. Chromatographic Sci. 15: 10-13. Howell, J.F., Knight, A.L., Unruh, T.R., Brown, D.F., Krysan, J.L., Sell, C.R. and Kirsch, P.A. 1992. Control of codling moth in apple and pear with sex pheromone-mediated mating disruption. J. Econ. Ent. 85(3): 918-925. Howse, P.E., Stevens, I.D.R. and Jones, O.T. 1998. Insect pheromones and their use in pest management. Chapman and Hall, London, 369 pp. Huang, Y.-Z., Shi, L. and Yang, J. 1987. Highly Stereoselective Synthesis of Z,E Conjugated Diene Type Sex Pheromones. J. Org. Chem. 52: 3558-3560. Ideses, R. and Shani, A. 1989. The Wittig reaction: comments on the mechanism and application as a tool in the synthesis of conjugated dienes. Tetrah. 45: 3523-3534. Ikeda, Y., Ukai, J., Ikeda, N. and Yamamoto, H. 1987. Selective proton transfer of unsaturated esters. Syntheses of a trail-following pheromone for Subterranean termites and megatomic acid. Tetrah. 43: 743-753. Jabri, N., Alexakis, A. and Normant, J.F. 1981. Vinyl-Copper Derivatives XIII: Synthesis of Conjugated Dienes of Very High Stereoisomeric Purity. Tetrah. Lett. 22: 959-962. Jabri, N., Alexakis, A. and Normant, J.F. 1983. Alkenyl-copper derivatives 19. Synthesis of conjugated dienes and styrenes by coupling of alkenyl cuprates with alkenyl and aryl halides. Bull. de la Soc. Chim. de France, 321-331. Jirón, Z. 1996. Approaching Optimal Conditions for Running Liquid Adsorption Column Chromatography Using Simple Computational Models. Licentiate Thesis, Department of Chemistry, Organic Chemistry, Royal Institute of Technology, Stockholm, Sweden. Jurenka, R.A. 1997. Biosynthetic pathway for producing the sex pheromone component (Z,E)- 9,12-tetradecadienyl acetate in moths involves a ∆ 12 desaturase. Cell. Mol. Life Sci. 53: 501-505. Jutsum, A.R. and Gordon, R.F.S. 1989. Pheromones: importance to insects and role in pest management. pp. 1-13. In A.R.Jutsum and R.F.S. Gordon (Eds), Insect pheromones in plant protection. Wilely, Chishester, UK. Kaissling, K. 1996. Peripheral mechanisms of pheromone reception in moths. Chemical Senses. 21(2): 257-267. Karlson, P. and Butenandt, A. 1959. Pheromones (ectohormones) in insects. Ann. Rev. Entomol. 4: 39-58. Kovaleski, A. 1992. MIP fruteiras de clima temperado, pp. 271–287. In O. A. Fernandes, A.C.B. Correia, and S. A. Bortoli [eds.], Manejo integrado de pragas e nemato´ ides.

UNESP, Jaboticabal, Brazil. Leadbetter, G. and Plimmer, J.R. 1979. An improved preparation of some insect sex attractants: Synthesis and separation of geometrical isomers by formation of urea complexes. J. Chem. Ecol. 5:101-108. Leal, W.S., Hasegawa, M., Sawada, M., Ono, M. and Ueda, Y. 1994. Identification and field evaluation of Anomala octiescostata (Coleoptera: Scarabaeidae) sex pheromone. J. Chem. Ecol. 20: 1643-1655. Linn, C.E., Campbell, M.G., and Roelofs, W.L. 1988. Temperature modulation of behavioural thresholds controlling male moth sex pheromone response specificity. Physiol. Entomol. 13: 59-68. Linn, C.E. and Roelofs, W.L. 1989. Response specificity of male moths to multicomponent pheromones. Chem. Senses 14: 421-437. Lo, V.M. and Shiao, M.-J. 1986. A Convenient Synthesis of (11E,13E)-11,13-Hexadecadienal and (11Z,13E)-Hexadecadienal. Synth. Comm. 16: 1647-1655. Löfstedt, C., Zhu, J., Kozlov, M.V., Buda, V., Jirle, E.V., Hellqvist, S., Löfqvist, J., Plass, E., Franke, S. and Francke, W. 2004. Identification of the Sex Pheromone of the Currant Shoot Borer Lampronia capitella. J. Chem. Ecol. 30 (3): 643-658. Löfstedt, C. 1990. Population variation in moth pheromone communication systems and its genetic control. Entomol. Exp. Appl. 54: 199-218. Lorenzato, D. 1984. Ensaio laboratorial de controle da “trac¸adamac¸a” Phtheochroa cranaodes Meyrick, 1937 com Bacillus thuringiensis Berliner e inseticidas quý´micos. Agron. Sulriograndense 20: 157–163. Mancuso, A.J., Huang, S.-L. and Swern, D. 1978. Oxidation of Long-Chain and Related Alcohols to Carbonyls by Dimethyl Sulfoxide “Activated” by Oxalyl Chloride. J. Org. Chem. 43: 2480-2482. Mancuso, A.J. and Swern, D. 1981. Activated Dimethyl Sulfoxide: Useful Reagent for Synthesis. Synthesis: 165-185. Maryanoff, B.E. and Reitz, A.B. 1989. The Wittig Olefination Reaction and Modifications Involving Phosphoryl-Stabilized Carbanions. Stereochemistry, Mechanism, and Selected Synthetic Aspects. Chem. Rev. 89: 863-927. McDonough, L.M., Aller, W.C. and Knight, A.L. 1992. Performance characteristics of a commercial controlled-release dispenser of sex pheromone for control of codling moth Cydia pomonella by mating disruption. J. Chem. Ecol. 18(12): 2177-89. McDonough, L.M., Davis, H.G., Chapman, P.S. and Smithhisler, C.L. 1993. Responses of male codling moths (Cydia pomonella) to components of conspecific female sex pheromone glands in flight tunnel tests. J. Chem. Ecol. 19: 1737-1748. McDonough, L.M., Davis, H.G., Chapman, P.S. and Smithhisler, C.L. 1994. Codling moth, Cydia pomonella: Disruptants of sex pheromonal communication. J. Chem. Ecol. 20: 171-181. McDonough, L.M., Davis, H.G., Chapman, P.S. and Smithhisler, C.L. 1995. Codling Moth, Cydia pomonella, (Lepidoptera: Tortricidae): Is Its Pheromone Multicomponent? J. Chem. Ecol. 21(8): 1065-1071. McDonough, L.M., Chapman, P.S., Weissling, T.J. and Smithhisler, C.L. 1996. Efficacy of Nonpheromone Communication Disruptants of Codling Moth (Cydia pomonella): Effect of Pheromone Isomers and of Distance Between Calling Females and Dispensers. J. Chem. Ecol., 22(3): 415-423. McElfresh, J.S. and Millar, J.G. 1999. Sex Attractant Pheromone of Saturniid Moth Coloradia velda. J. Chem. Ecol. 25: 1067-1078. Millar, J.G. and Haynes, K. F. 1998. Methods in Chemical Ecology: Chemical Methods. Kluwer Academic Publishers, Norwell, MA. Millar, J.G. 2000. Polyene hydrocarbons and epoxides: A second major class of lepidopteran sex attractant pheromones. Ann. Rev. Entomol. 45: 575-604. Minks, A.K. and Cardé, R.T. 1988. Disruption of pheromone communication in moths: Is the natural blend really most efficacious? Entomol. Exp. Appl. 49: 25-36.

Miyaura, N., Yamade, K. and Suzuki, A. 1979. A New Stereospecific Cross-Coupling by the Palladium-Catalyzed Reaction of 1-Alkenyl or 1-Alkynyl Halides. Tetrah. Lett.: 3437- 3440. Miyaura, N., Suginome, H. and Suzuki, A. 1983. New Stereospecific Syntheses of Pheromone Bombykol and Its Three Geometrical Isomers. Tetrah. Lett. 24: 1527-1530. Mustaparta, H. 1996. Central mechanisms of pheromone information. Chemical Senses. 21(2): 269-276. Mõttus, E., Liblikas, I., Ojarand, A., Kuusik, S., Nikolaeva, Z.V., Ovsjannikova, E.I., and Borg- Karlson, A.-K. 2001. Calculation and using of pheromone communication channel parameters for optimization of pheromone dispensers. In Ed. Metspalu, L. Practice orientated results on the usage of plant extracts and pheromones in pest control. Proceedings of the International Workshop, Tartu, Jan. 24-25, 2001. Tartu.: 90-100. Negishi, E. and Yoshida, T. 1973. A Highly Stereoselective and General Synthesis of Conjugated trans,trans-Dienes and trans-Alkyl Ketones via Hydroboration. J.C.S. Chem. Comm.: 606-607. Negishi, E., Lew, G. and Yoshida, T. 1973. Stereoselective Synthesis of Conjugated trans- Enynes Readily Convertible into Conjugated cis,trans-Dienes and its Application to the Synthesis of the Pheromone Bombykol. J.C.S. Chem. Comm.: 874-875. Negishi, E. and Abramovitch, A. 1977. A Highly Efficient Chemo-, Regio-, and Stereoselective Synthesis of (7E, 9Z)-Dodecadien-1-yl Acetate, a Sex Pheromone of Lobesia botrana, via a Functionalized Organoborate. Tetrah. Lett.: 411-414. Negishi, E., Okukado, N., King, A.O., Van Horn, D.E., Spiegel, B.I. 1978. Selective carbon- carbon bond formation via transition metal catalysts. 9. Double metal catalysis in the cross-coupling reaction and its application to the stereo- and regioselective synthesis of trisubstituted olefins. J. Am. Chem. Soc. 100(7): 2254-6. Negishi, E. 1982. Palladium- or Nickel-Catalyzed Cross Coupling. A New Selective Method for Carbon-Carbon Bond Formation. Acc. Chem. Res. 15: 340-348. Nishida, T., Vang, L.V., Yamazawa, H., Yoshida, R., Naka, H., Tsuchida, K. and Ando, T. 2003. Synthesis and characterization of hexadecadienyl compounds with a conjugated diene system, sex pheromone of the persimmon fruit moth and related compounds. Biosci. Biotechnol. Biochem. 67(4): 822-829. Nishiguchi, T., Hayakawa, S., Hirasaka, Y. and Saitoh, M. 2000. Selective Monotetrahydropyranylation of 1,n-diols catalyzed by aqueous acids. Tetrah. Lett. 41: 9843-9846. Ono, A., Imai, T., Inomata, S., Watanabe, A. and Ando, T. 2002. Biosynthetic pathway for production of a conjugated dienyl sex pheromone of a Plusiinae moth, Thysanoplusia intermixta. Insect Biochem. and Molecular Biol. 32: 701–708. Palaniswamy, P., Underhill, E.W., Steck, W.F. and Chisholm, M.D. 1983. Responses of male redbacked cutworm, Euxoa ochrogaster (Lepidoptera: Noctuidae), to sex pheromone components in a flight tunnel. Environ. Entomol. 12: 748-752. Park, C.P., Gil, J.M., Sung, J.W. and Oh, D.Y. 1998. A Concise Synthesis of the Sex Pheromone of Phtheochroa cranaodes (Lepidoptera: Tortricidae). Tetrah. Lett. 39: 2583-2584. Phelan, P.L. and Miller, J.R. 1981. Separation of Isomeric Insect Pheromonal Compounds Using Reversed-Phase HPLC with AgNO3 in the MobilePhase. J. Chrom. Sci. 19:13-17. Preiss, R. and Priesner, E. 1988. Responses of male codling moths, Laspeyresia pomonella to codlemone and other alcohols in a wind tunnel. J. Chem. Ecol. 14: 797-813. Quartey, G.K. and Coaker, T.H. 1993. Role of sex pheromone components in the orientation behaviour of Ephestia cautella. Entomol. Exp. Appl. 66(3): 237-245. Raina, A.K., Klun, J.A., Schwarz, M., Day, A., Leonhardt, B.A. and Douglas, L.W. 1986. Female sex pheromone of the melonworm Diaphania hyalinata (Lepidoptera: Pyralidae) and analysis of male responses to pheromone in a flight tunnel. J. Chem. Ecol. 12(1): 229-238.

Ratovelomana, V. and Linstrumelle, G. 1981a. A Convenient Preparation of trans (or cis)-1- Chloroalkenes from trans (or cis)-1,2-Dichloroethylene: A New Synthesis of the Sex Pheromone of Lobesia botrana. Tetrah. Lett. 22: 315-318. Ratovelomana, V. and Linstrumelle, G. 1981b. New Synthesis of the Sex Pheromone of the Egyptian Cotton Leafworm, Spodoptera littoralis. Synth. Comm. 11: 917-923. Reed, D.W. and Chisholm, M.D. 1985. Attraction of Moth Species of Tortricidae, Gelechiidae, Geometridae, Drepanidae, Pyralidae, and Gracillaridae Families to Field traps Baited with Conjugated Dienes. J. Chem. Ecol. 11: 1645-1657. Reitz, A.B., Nortey, S.O., Jordan, A.D. Jr., Mutter, M.S. and Maryanoff, B.E. 1986. Dramatic Concentration Dependence of Stereochemistry in the Wittig Reaction. Examination of the Lithium Salt Effect. J. Org. Chem. 51: 3302-3308. Rice, R. E. and Kirsch, P. 1990. Mating disruption of oriental fruit moth in the United States, pp. 193-212, in R. L. Ridgway, R. M. Silverstein and M. N. Inscoe (eds.), Behavior- Modifying Chemicals for Insect Management: Application of Pheromones and Other Attractants. Marcel Dekker, New York. Roelofs, W., Comeau, A., Hill, A. and Milicevic, G. 1971. Sex Attractant of the Codling Moth: Characterization with Electroantennogram Technique. Science 174: 297-299. Roelofs, W.L. 1978. Threshold hypothesis of pheromone perception. J. Chem. Ecol. 4: 685-699. Roelofs, W.L., and Wolf, W. 1988. Pheromone biosynthesis in the Lepidoptera. J. Chem. Ecol. 14: 2019-2031. Roelofs, W.L. 1995. Chemistry of sex attraction. Proc. Natl. Acad. Sci. USA 92: 44-49. Rossi, R., Carpita, A. and Quirici, M.G. 1981. Dienic Sex Pheromones. Stereoselective Synthesis of (7E, 9Z)-7,9-Dodecadien-1-yl Acetate, (E)-9,11-Dodecadien-1-yl Acetate and of (9Z, 11E)-9,11-Tetradecadien-1-yl Acetate by Palladium-Catalyzed Reactions. Tetrah. 37(15): 2617-2623. Rossi, R., Carpita, A., Quirici, M.G. and Gaudenzi, M.L. 1982. Insect Sex Pheromones: Palladium-Catalyzed Synthesis of Aliphatic 1,3-Enynes by Reaction of 1-Alkynes with Alkenyl Halides Under Phase Transfer Conditions. Tetrah. 38(5): 631-637. Rule, G.S. and Roelofs, W.L. 1989. Biosynthesis of sex pheromone components from linolenic acid in the arctiid moths. Arch. Insect Biochem. Physiol. 12: 89-94. Schlosser, M. and Christmann ,K.F. 1966. Trans-Selective Olefin Syntheses. Angew. Chem. Internat. Edit. 5: 126-127. Schlosser, M., Tuong, H.B. and Schaub, B. 1985. The Betaine-Ylid Route to Trans-Alkenols. Tetrah. Lett. 26(3): 311-314. Schlyter, F., Svensson, M., Zhang, Q.-H., Knízek, M., Krokene, P., Ivarsson, P. and Birgersson, G. 2001. A model for peak and width of signalling windows: Ips dublicatus and Chilo partellus pheromone component proportions - does response have a wider window than production? J. Chem. Ecol. 27: 1481-1511. Schwarz, M., Waters, R.M. and Graminski, G.F. 1986. Retention of stereochemistry during the oxidation of (E)- and (Z)-2- and 3-alkenols to the corresponding aldehydes by the Dess- Martin reagent. Chem. & Industry: 789. Simonelli, F., Oliveira, A.R.M., Marques, F.A. and Silva, D.C. 1998. A Short and Stereoselective Synthesis of the Sex Pheromone of Bonagota cranaodes (Lepidoptera: Tortricidae). J. Braz. Chem. Soc. 9: 371-373. Sonagashira, K., Tohda, Y. and Hagihara, N. 1975. A Convenient Synthesis of Acetylenes: Catalytic Substitutions of Acetylenic Hydrogen with Bromoalkenes, Iodoarenes, and Bromopyridines. Tetrah. Lett.: 4467-4470. Sondengam, B.L., Charles, G. and Akam, T.M. 1980. A Novel Stereospecific Reduction of Alkynes to Alkenes. Tetrah. Lett. 21: 1069-1070. Sorensen, P.W. 1996. Biological responsiveness to pheromones provides fundamental and unique insight into olfactory function. Chemical Senses. 21(2): 245-256. Svensson, M. 1996. Sexual selection in moths: The role of chemical communication. Biol. Rev. 71: 113-135.

Svirskaya, P.I., Leznoff, C.C. and Roelofs, W.L. 1980. A Stereoselective Synthesis of a cis, cis conjugated Dienol, A Candidate Pheromone. Synth, Comm. 10(5): 391-397. Svirskaya, P.I., Maiti, S.N., Jones, A.J., Khouw, B., Leznoff, C.C. 1984. Syntheses of Pure (9Z,11Z), (9E, 11E), (9E,11Z), and (9Z,11E)-9,11-Hexadecadienals - Possible Candidate Pheromones. J. Chem. Ecol. 10(5), 795-807. Tamaki, Y. 1985. Sex pheromones, pp. 145-191, in G.A. Kerkut and L.I. Gilbert (eds.). Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 9, Behaviour. Pergamon Press Ltd., Oxford. Teale, S.A., Hager, J.H. and Webster, F.X. 1991. Pheromone-based assortative mating in a bark beetle. Anim. Behav. 48: 569-578. Teich, I., Neumark, S., Jacobson, M., Klug, J., Shani, A. and Waters, R.M. 1979. Mass Trapping of Males of Egyptian Cotton Leafworm (Spodoptera littoralis) and Large- Scale Synthesis of Prodlure. Elsevier/North-Holland Biomedical Press. Chemical Ecology: Odour Communication in Animals. F.J. Ritter, editor. 343-352. Tellier, F. and Descoins C., 1990. Stereospecific Synthesis of (Z,E)-9,11,13-Tetradecatrien-1-yl Acetate and Aldehyde, Sex Pheromone Components of Cecropia and Ectomyelois ceratoniale. Tetrah. Lett. 31: 2295-2298. Tellier, F. 1991. New Stereospecific Syntheses of (E,E,Z)- and (E,E,E)-10,12,14- Hexadecatrienal, Sex Pheromonal Components of Manducta Sexta. Bioorg. & Med. Chem. Lett. 1: 635-638. Thomson, D.R., Gut, L.J. and Jenkins, J.W. 1998. Pheromones for insect control: strategies and successes. In F.R. Hall & J.J. Menn (eds.), Biopesticides: Use and Delivery. Humana Press, Totowa NJ, pp. 385-412. Tidwell, T.T. 1990. Oxidation of Alcohols by Activated Dimethyl Sulfoxide and Related Reactions: An Update. Synthesis: 857-870. Tomida, I., Kato, Y. and Kayahara, H. 1983. Studies on Pheromones of Female Eri-Silk Moth, I. Preparation of C6-C11 2-Alkenyl Triphenylphosphonium Bromides. J. Fac. Agric. Shinshu Univ. 18: 127-130. Trippett, S. and Walker, D.M. 1961. The phosphobetaines: Preparation and Properties. J. Chem. Soc.:1266-1272. Tumlinson, J.H. 1988. Contemporary frontiers in insect semiochemical research. J. Chem. Ecol. 14: 2109-2130. Tumlinson, J.H., Brennan, M.M., Doolittle, R.E., Mitchell, E.R., Brabram, A., Mazomenos, B.E., Baumhover, A.H. and Jackson, D.M. 1989. Identification of a Pheromone Blend Attractive to Manduca sexta (L.) Males in a Wind Tunnel. Arch. of Insect Biochemistry and Physiology 10: 255-271. Tumlinson, J.H., Mitchell, E.R., Doolittle, R.E. and Jackson, D.M. 1994. Field Tests of Synthetic Manduca sexta Sex Pheromone. J. Chem. Ecol. 20: 579-591. Unelius, C.R., Eiras, A., Witzgall, P., Bengtsson, M., Kovaleski, A., Vilela, E. and Borg- Karlson, A.-K. 1996. Identification and Synthesis of the Sex Pheromone of Phtheochroa cranaodes (Lepidoptera: Tortricidae). Tetrah. Lett. 37: 1505-1508. Vakenti, J.M., Gaunce, A.P., Slessor, K.N., King, G.G.S., Allan, S.A., Madsen, H.F., and Borden, J.H. 1988. Sex pheromone components of the oblique banded leafroller, Choristoneura rosaceana, in the Okanagan Valley of British Columbia. J. Chem. Ecol. 14: 605-621. Vig, O.P., Sharma, M.L. and Gupta, R. 1990. Syntheses of Sex Pheromone Components of Sphingid Moths. J. Indian Chem. Soc. 67: 37-38. Vinczer, P., Juvancz, Z., Novák, L. and Szántay, C. 1987. Synthesis of pheromones, III. Improved and Stereocontrolled Syntheses of Isomeric Mixtures of 8-Dodecen-1-yl Acetates. Acta Chim. Hung. 124: 737-48. Vinczer, P., Juvancz, Z., Novák, L. and Szántay, C. 1988a. Synthesis of pheromones, IV. Chemistry of the Wittig Reaction, I. Effects of Reaction Conditions on the Stereoselectivity and Yield of the Wittig Reaction. Acta Chim. Hung. 125: 797-820.

Vinczer, P., Juvancz, Z., Novák, L. and Szántay, C. 1988b. Chemistry of the Wittig Reaction. The Role of Hard, Soft and Borderline Bases in Wittig Condensation Applied in Pheromone Chemistry. Eesti NSV Tead. Akad. Toim., Keemia, 37: 215-219. Vinczer, P., Z., Novák, L. and Szántay, C. 1991. Application of Potassium t-Butoxide in Toluene as a Base in the Wittig Reaction in Large-Scale Pheromone Syntheses. Org. Prep. Proced. Int. 23: 443-447. Wadsworth, W.S. and Emmons, D. 1961. The Utility of Phosphonate Carbanions in Olefin Synthesis. J. Am. Chem. Soc. 83: 1733-1738. Willis, M.A. and Baker, T.C. 1988. Effects of varying pheromone component ratios on the zigzagging flight movements of Grapholita molesta. J. Insect Behav. 1: 357-371. Witzgall, P., Bengtsson, M., Unelius, C.R. and Löfqvist, J. 1993. Attraction of Pea Moth Cydia nigricana F. (Lepidoptera: Tortricidae) to Female Sex Pheromone (E,E)-8,10- Dodecadien-1-yl Acetate, is Inhibited by Geometric Isomers (E,Z), (Z,E), and (Z,Z). J. Chem. Ecol. 19: 1917-1928. Witzgall,P., Chambon, J.-P., Bengtsson, M., Unelius, C,R., Appelgren, M., Makrancy, G., Muraleedharan, N., Reed, D.W., Hellrigl, K., Buser, H.-R., Hallberg, E., Bergström, G., Toth, M., Löfstedt, C. and Löfqvist, J. 1996. Sex pheromones and attractants in the Eucosmini and Grapholitini (Lepidoptera: Tortricidae). Chemoecology 7: 13-23. Yadav, J.S., Deshpande, P.K. and Reddy, E.R. 1989. Preparation of 1,3-Dienes, Its Application to the Synthesis of (Z,E)-9,11-tetradecadienyl Acetate and (E,E)-10,12-Hexadecadienal: Sex Pheromones of Cotton Pests. Synth. Comm. 19: 125-134. Yadav, J.S., Reddy, E. J. and Ramalingam, T. 2001. Short-route synthesis of (3E,5Z)- tetradecadienoic acid (megatomic acid), the sex attractant of the black carpet beetle. New J. Chem. 25: 223-5. Zweifel, G. and Polston, N.L. 1970. Selective Hydroboration of Conjugated Diynes with Dialkylboranes. A Convenient Route to Conjugated cis-Enynes, α,β-Acetylenic Ketones, and cis,cis-Dienes. J. Am. Chem. Soc. 92: 4068-4071. Zweifel, G. and Backlund. S.J. 1978. Synthesis of 1,4-disubstituted (E,Z)-1.3-dienes from lithium dicyclohexyl (trans-1-alkenyl) (1-alkynyl) borates. J. Organomet. Chem. 156: 159-170.

ACKNOWLEDGEMENTS

Main part of the syntheses, purifications and analyses of the polyenic pheromone components presented in this thesis were performed at the ecological chemistry group of the organic chemistry division of the Department of Organic Chemistry, Royal Institute of Technology, Stockholm, Sweden. I thank all my colleagues for their support to my work.

I would like to express my sincere gratitude to a number of people who have contributed in various ways to make my Ph D studies not only possible but also joyful. I would like to express my sincere gratitude to all my supervisors: Prof. Anna-Karin Borg- Karlson from KTH; Prof. Anne Luik from Plant Protection Institute of EAU; Enno Mõttus from the lab. of ecochemistry, EAU; and Assoc. Prof. Rikard Unelius from University of Kalmar. Thank you for valuable discussions and support, for providing excellent working facilities, and also for giving me the opportunity to find my own way. For comments and help during the writing process I want to express my greatest gratitude to Professor Anna-Karin Borg-Karlson, Dr. Gunhild Aulin Erdtmann, Professor emeritus Torbjörn Norin. Thanks to the head of the department Professor Christina Moberg and all colleagues at the organic chemistry department. Thanks to Lena, Ingvor and Henry to help and support with important matters. MISTRA, VISBY, KVA from Sweden and are greatly acknowledged for financial support. The study was also supported by the Estonian target financed projects and by the grants of the Estonian Science Foundation. Many thanks to the members of the Ecological Chemistry group: Anna-Karin Borg-Karlson, Torbjörn Norin, Ulla Jacobsson, Peter Bæckström, Sture Strömberg, Monika Persson, Johan Andersson, Katinka Pålson, Raimondas Mozuraitis, Ellen Santangelo and all others that have come and gone. Especially, I would like to thank Peter Bæckström for your inspiring discussions and support and finding time for both scientific and not so scientific discussions. And certainly for invention such a valuable MPLC equipment. I would like to express my deep gratitude to Tullio Ilomets from Tartu University, my first guide in the field of synthetic organic chemistry. I want to thank all my co-authors in Sweden, Brazil, Lithuania, Czech Republik and Estonia, for a fruitful collaboration.

APPENDICES

OH a Br b Br c P+(Ph)3Br- d HO HO THPO THPO 1 2 3 4

THPO e f + + HO HO THPO 7 8 g g 5 + 6

AcO AcO 9 10

a) HBr, Cyclohexane b) O , CH2CL2 c) (Ph)3P, Toluene O , BuLi, THF e) KOH, EtOH f) AgNO -SiO -chrom. g) Ac O, Py d) H 3 2 2

O k OH h OTHP i OTHP j O OTHP HO HO H H 11 12 13 14

l m OTHP OH OAc 15 16 17 O h) P(Ph)3+Cl- , Benzene O , Et2O i) PDC, CH2Cl2 j) Et3N, H

P+(Ph) Br- k) NaOEt, THF, 3 l) KOH, EtOH m) Ac2O, Py

n o p HO OH HO Br THPO Br 18 19 20 r s I THPO THPO THPO 21 22 23 I t u Br Li ZnBr 24 25 26

v x y 23 + 26 THPO HO AcO 27 28 29

n) HBr, Cyclohexane o) O , CH2CL2 p) LiC CH .EDA , DMSO

r) I2, Morpholine, Toluene s) Dicyclohexylborane, Et2O t) Li, Et2O x) KOH, EtOH y) Ac O, Py u) ZnBr2, THF v) Pd(PPh3)4, THF 2

Appendix 1. Synthetic scheme of the synthesis of four geometric isomers of 10,12- tetradecadienol and acetates.

OH a Br b Br c P+(Ph)3Br- d HO HO AcO AcO

e AcO HO

a) HBr, cyclohexane b) Ac2O, pyridine c) Ph3P, acetonitrile O NaN[Si(CH ) ] , THF e) KOH, EtOH d) H 3 3 2

a b c HO OH Br OH Br OTHP d e f OTHP HO OTHP OTHP HO h OTHP g OTHP OH O i)

a) HBr, cyclohexane b) O , CH2CL2 OAc c) LiC CH .EDA , DMSO

d) BuLi, paraformaldehyde, THF e) LiAlH4, THF f) MnO2, CH2Cl2

+ - g) EtP Ph3Br , NaOEt, THF h) PPTS, EtOH i) Ac2O, pyridine

a) b) c) HO OH HO Br THPO Br

d) e) I THPO THPO THPO 1 I f) g) Br Li ZnBr 2

h) i) j) 1 + 2 THPO HO AcO

a) HBr, cyclohexane b) O , CH2CL2 c) LiC CH .EDA , DMSO f) Li, Et O d) I2, Morpholine, toluene e) Dicyclohexylborane, Et2O 2

g) ZnBr2, THF h) Pd(PPh3)4, THF i) KOH, EtOH j) Ac2O, Py

Appendix 2. Synthetic schemes of the syntheses of EZ-, ZE-, and ZZ-isomers of 8,10- dodecadienol.

Br HO OH PDC (PH3)P CH2Cl2 + P (C6H5)3 Br HO O 2 eq. MeLi, LiBr

a) MeLi, LiBr b) HCl c) t-BuOK

(COCl2)2 DMSO

NMO Br O THPO DMSO THPO LDA Br N O O THF P(OMe)3 O O O (OMe) 2 Br P CCl4, AIBN O O O O 1. pTsOH O + 2. DIBAL Et P (PH)3 Br THPO HO O O 3. MnO2 NaHMDS THF

(COCl2)2 HO O DMSO

Appendix 3. Synthetic schemes of the syntheses of EEE-, and EEZ-isomers of hexadecatrienal.

Appendix 4. Synthesized compounds

(8E,10Z)-8,10-dodecadien-1-ol (8Z,10E)-8,10-dodecadien-1-ol (8Z,10Z)-8,10-dodecadien-1-ol

(8E,10E)-8,10-dodecadien-1-yl acetate (8E,10Z)-8,10-dodecadien-1-yl acetate (8Z,10E)-8,10-dodecadien-1-yl acetate (8Z,10Z)-8,10-dodecadien-1-yl acetate

(8E,10E)-8,10-dodecadien-1-al

(3E,5E)-3,5-dodecadien-1-yl acetate (3E,5Z)-3,5-dodecadien-1-yl acetate (3Z,5E)-3,5-dodecadien-1-yl acetate (3Z,5Z)-3,5-dodecadien-1-yl acetate

(10E,12E,14E)-10,12,14-hexadecatrien-1-ol (10E,12E,14Z)-10,12,14-hexadecatrien-1-ol

(10E,12E,14E)-10,12,14-hexadecatrien-1-al (10E,12E,14Z)-10,12,14-hexadecatrian-1-al

(8E,10E)-8,10-tetradecadien-1-ol (8E,10Z)-8,10-tetradecadien-1-ol (8Z,10E)-8,10-tetradecadien-1-ol (8Z,10Z)-8,10-tetradecadien-1-ol

(8E,10E)-8,10-tetradecadien-1-yl acetate (8E,10Z)-8,10-tetradecadien-1-yl acetate (8Z,10E)-8,10-tetradecadien-1-yl acetate (8Z,10Z)-8,10-tetradecadien-1-yl acetate

(10E,12E)-10,12-tetradecadien-1-ol (10E,12Z)-10,12-tetradecadien-1-ol (10Z,12E)-10,12-tetradecadien-1-ol (10Z,12Z)-10,12-tetradecadien-1-ol

(10E,12E)-10,12-tetradecadien1-yl acetate (10E,12Z)-10,12-tetradecadien-1-yl acetate (10Z,12E)-10,12-tetradecadien-1-yl acetate (10Z,12Z)-10,12-tetradecadien-1-yl acetate

(9Z,11Z)-9,11-tetradecadien-1-ol (9Z,11Z)-9,11-tetradecadien-1-yl acetate (9Z,11Z)-9,11-tetradecadien-1-al