Synthesis and Analysis of Zingerone Analogues as Fruit Fly Attractants By Benjamin L. Hanssen Department of Chemistry and Biomolecular Sciences Date of Submission: 9 October 2015 Declaration of Originality I, Benjamin L. Hanssen, declare that this work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. Benjamin L. Hanssen Date ii Table of Contents Declaration of Originality .......................................................................................................... ii Acknowledgements ................................................................................................................... iv Table of Abbreviations ............................................................................................................... v Abstract ..................................................................................................................................... vi Chapter 1. Introduction .............................................................................................................. 1 1.1 Fruit Flies – Life Cycle and Economic Importance ......................................................... 1 1.2 Fruit Fly Control Methods................................................................................................ 3 1.2.1 Organophosphate Insecticides ................................................................................... 3 1.2.2 Alternative Control Methods: MT and MAT ............................................................ 3 1.3 The Chemical Basis of Lure Attraction ............................................................................ 5 1.4 Methyl Eugenol and Raspberry Ketone as Chemical Lures ............................................ 5 1.5 Lure Attractiveness .......................................................................................................... 6 1.5.1 Intrinsic Attractiveness .............................................................................................. 7 1.5.2 Volatility .................................................................................................................... 7 1.6 Chemical Lure and Receptor SAR Investigations ............................................................ 9 1.6.1 Methyl Eugenol Receptor .......................................................................................... 9 1.6.2 Raspberry Ketone Receptor .................................................................................... 10 1.7 Zingerone as a Chemical Lure ....................................................................................... 11 1.7.1 Reasons for Zingerone Attraction ........................................................................... 13 1.8 Implications of Zingerone for Fruit Fly Control ............................................................ 14 1.9 Aims and Scope of Study ............................................................................................... 14 Chapter 2. Experimental Methods ............................................................................................ 15 2.1 Synthesis of Zingerone Analogues ................................................................................. 15 2.2 Vapour Pressure Measurements ..................................................................................... 23 2.3 Biological Testing .......................................................................................................... 24 2.3.1 Electroantennography (EAG) .................................................................................. 24 2.3.2 Laboratory Cage Bioassays ..................................................................................... 25 Chapter 3. Results and Discussion ........................................................................................... 26 3.1 Selection and Synthesis of Zingerone Analogues .......................................................... 26 3.1.1 Selection of Zingerone Analogues .......................................................................... 26 3.1.2 Proposed Synthetic Methods ................................................................................... 29 3.1.3 Discussion of Synthetic Methods ............................................................................ 32 3.2 Vapour Pressure Measurements ..................................................................................... 40 3.3 Biological Testing .......................................................................................................... 47 3.3.1 Electroantennography (EAG) .................................................................................. 47 3.3.2 Laboratory Cage Bioassays ..................................................................................... 50 Chapter 4. Conclusions and Future Directions ......................................................................... 53 Chapter 5. References .............................................................................................................. 56 Appendix .................................................................................................................................. 62 iii Acknowledgements I would like to thank my supervisors Ian Jamie, Joanne Jamie, Soo Jean Park, and Phil Taylor who have provided much support and guidance throughout this year. They have been an invaluable source of knowledge and given me great insight into what research can involve. Soo deserves special acknowledgement for her immeasurable help, especially at the beginning as I was settling in with the project and lab. I cannot imagine how much harder this year would have been without Soo’s support and practical experience and knowledge. I would also like to thank Chris McRae for the use of and fixing the DSC amongst other things, without which the vapour pressure work would have been that much more difficult. Thank you to my friends Gin and Nathan for their company in the lab, the discussions that made periods of setbacks and monotony bearable, and their support and encouragement to make it through the year. Last but not least, I thank my family for all their never-ending support and encouragement this year. iv Table of Abbreviations ADI Acceptable daily intake APVMA Australian Pesticide and Veterinary Medicines Authority ARfD Acute reference dose ATR-IR Attenuated total reflectance-infrared DCC N,N’-Dicyclohexylcarbodiimide DCM Dichloromethane DMAP 4-Dimethylaminopyridine DSC Differential scanning calorimetry EAG Electroantennography EI Electron ionisation GC-FID Gas chromatography-flame ionisation detector GC-MS Gas chromatography-mass spectrometry MAT Male annihilation technique MT Mass trapping NMR Nuclear magnetic resonance SAR Structure-activity relationship TLC Thin layer chromatography v Abstract Fruit fly species have a devastating impact on food production in Australia and other countries. With new restrictions on the use of certain organophosphate insecticides, other control methods, such as male annihilation technique (MAT), will be more important for crop protection. MAT depends on effective male lures to attract pest insects to toxicants. Zingerone is a natural product that is attractive to males of Bactrocera jarvisi, a species that responds only weakly to other lures. In a step towards more effective lures, a series of zingerone analogues were synthesised and characterised. Many analogues were synthesised by an Aldol-hydrogenation synthetic route. Volatility is considered an important factor in lure attraction and the measurement of the volatility of the analogues by differential scanning calorimetry showed that fluorinated compounds have a 7-10-fold greater vapour pressure than corresponding non- fluorinated compounds. Unexpectedly, the acetyl and formyl esters of zingerone were half as volatile as zingerone. Electroantennography experiments with B. tryoni and B. jarvisi indicated that fluorinated compounds generally produce a greater response and the two species have vastly different preferences for the methylenedioxy moiety. Limited laboratory cage bioassays conducted with B. tryoni indicated that some of the fluorinated compounds tended to elicit a positive response. vi Chapter 1. Introduction Fruit flies are a significant pest of horticultural crops, and of particular concern are those species that have an impact upon food production. The Queensland fruit fly (Bactrocera tryoni) is the most serious pest fruit fly species in Australia with a very wide variety of host crops, ranging from citrus to nuts, tomatoes, and stone fruits.1-3 This species of fruit fly is also known to infest at least 60 wild, non-commercial plants.1 B. tryoni is present across northern Australia from Broome to north-west Queensland and eastern Australia from Cape York to south of Sydney. It is also present intermittently in Adelaide and Victoria.1, 4 In Australia, Jarvis’ fruit fly (B. jarvisi) is a moderate pest fruit fly present from northern Australia to northern New South Wales.4-6 It is known to infest at least 83 wild and commercial hosts, of which mangoes are the most susceptible commercial crop.5-6 Recent restrictions on the use of insecticides to control fruit flies,2, 7-8 such as B. tryoni and B. jarvisi, means that other control methods will