This document is the doctoral thesis of Frans C. Griepink analyzing the sex pheromones of two moth species, Symmetrischema tangolias and Scrobipalpuloides absoluta. The thesis includes an introduction outlining lepidopteran sex pheromones and their importance in pest control. It then describes research isolating, identifying, and synthesizing the sex pheromones of the two moth species through various chemical analysis techniques. Field tests were also conducted. The thesis concludes with a general discussion of three-dimensional sex pheromone structures and their activity, potential for pest resistance, biosynthesis, and linking laboratory research to practical application in pest control.

1. AnalysisoftheSexPheromonesof
Symmetrischema tangolias andScrobipalpuloides absoluta

2. Promotor: dr.Ae.deGroot
hoogleraarindebio-organische chemie
Co-promotoren: dr.J.H.Visser
leideronderzoeksgroep signaalstoffen
InstituutvoorPlanteziektenkundig Onderzoek
dr.T.A.vanBeek
universitairhoofddocent fytochemie

3. FransC.Griepink
AnalysisoftheSexPheromonesof
Symmetrischema tangolias andScrobipalpuloides absoluta
(withasummaryinEnglish)
(meteensamenvattinginhet Nederlands)
(conunresumenenEspanol)
Proefschrift
terverkrijgingvandegraadvandoctor
opgezagvanderector magnificus
vandeLandbouwuniversiteit Wageningen,
dr.C.M.Karssen,
inhetopenbaarteverdedigen
opwoensdag6november1996
desnamiddagstevieruurindeAula

4. ISBN90-5485-573-8
l-JIt
• '
ipo-dlo
Agricultural University
Wageningen
The research described in this thesis was part of the research program of the DLO
Research Institute for Plant Protection (IPO-DLO), Wageningen. The work was a
concerted effort ofIPO-DLO,the Department of OrganicChemistry of the Wageningen
AgriculturalUniversity(OC-WAU)andtheInternationalPotatoCenter(OP),Lima,Peru.
Theprojectwasfinancially supportedbytheNetherlands'Ministerfor Development
Co-operation(DGIS).

5. Stellingen
1. DeDMDS-methodeisgeschiktervoorhetidentificeren vanseksferomonendandepartiële
reductie methode.
Attygalle,A.B.,Jham,G.N.,Svatos,A.,Frighetto,R.T.S.,Meinwald,J.,Vilela,E.F.,Ferrara,F.A.and Uchôa-
Fernandes,M.A.1995.TetrahedronLett.,36,5471-5474.
Svatos,A.,Attygale,A.B.,Jham,G.N.,Frighetto,R.T.S.,Vilela,E.F.,èaman,D.andMeinwald,J.1996.
ƒ.Chem.EcoL,22,787-800.
2. Hetisonwaarschijnlijk datderatiovangeëmitteerde seksferomooncomponenten
gedurendehet'roepen'vanhetvrouwtje,waarbijersprakeisvaneenactieftransport van
dezeverbindingenveranderd,alsgevolgvanhetonderlingeverschilinvluchtigheid van
dieseksferomooncomponenten zoalsHuntetal.suggereert.
Hunt,R.E.andHaynes,K.F.1990.J.Insect.Physiol, 36,769-774.
3. Bijhetonderlingvergelijkenvanverschillendeverbindingeninwindtunnelsenbijhet
makenvanEAG'swordteronvoldoenderekeninggehoudenmethetverschilin
vluchtigheidvandeze verbindingen.
i. Dathetinjecteren vanintacteseksferomoonklieren viaeen'solid-phase'GC-injector de
levensduurvandekolomtengoedezalkomen,zoalsAttygalleetal.beweert,mag
betwijfeld worden.
Attygalle,A.B.,Herrig,M.,Vostrowsky,O.and Bestmann,H.J.1987.ƒ.Chem.Ecol.,13,1299-1311.
5. Hetbepalenvandeeffectiviteit vaneenverwarringstechniek aandehandvanhet aantal
gevangeninsecteninvallenmethetzelfde seksferomoondatgebruiktwordtomte
verwarren,kanleidentotverkeerdeconclusies.
Tsai,R.S.and Chow,Y.S.1992.ƒ.oftheAgriculture AssociationofChina,New SeriesNo.157,76-80.
6. PovolnyconcludeerttenonrechtedatScrobipalpuloidesabsolutaeeninsectisdathooginde
bergen leeft.
Povolny,D.1975.Acta Univ.Agric. (Brno),II,379-393.
7. Jemoetookvaneenmotgeenolifant maken.
Rasmussen,L.E.L.,Lee,T.D.,Roelofs,W.L.,Zhang,A.and DavesJr,G.D.1996,Nature, 379,684.
3. Goedkunnentellen,iseenvereistebijhetontwikkelenvan seksferomoonsyntheseroutes
voor lepidoptera.

6. 9. Hetstoppenvandekinderbijslag isbetervoorhetmilieudandeverhogingvan de
brandstofaccijns.
10. Omdatdeuitslagvaneenreferendum inkangaantegenhetbeleidvandedemocratisch
gekozenregering,kanmenzichafvragen ofeenreferendum welzodemocratischis.
11. Deverkeersveiligheid zouerbijgebaatzijnomnaastdete-hardrijdersook te-langzaam
rijderste bestraffen.
12. Vegetarismeiseenluxe.
13. JimDavisgeeftinzijnstripGarfield eengoedekarakteriseringvanhetverschiltussen
hondenenkatten.
14. Dankzijhetbroeikaseffect zittenwemomenteelnietineennieuwe ijstijd.
15. ZoalshetCPU'tjethuistikt,tikthetnergens.
Stellingenbehorendebijhet proefschrift
"AnalysisoftheSexPheromonesof
Symmetrischema tangolias andScrobipalpuloides absoluta"
Wageningen,6november1996
FransC.Griepink

7. CONTENTS
Chapter page
Generalintroduction 1
1.1 Insectsandpests 1
1.2 Butterflies andmoths 2
1.3 Lepidopteransexpheromones 2
1.3.1 General 2
1.3.2 Isolationtechniques 6
1.3.3 Identification techniques 6
1.3.4 Chemicalsynthesis 8
1.4 Insectcontrol 9
1.4.1 Pesticidesversusalternativecontrolmethods 9
1.4.2 Sexpheromonesinpestcontrol 10
1.5 Symmetrischematangolias 11
1.5.1 Nomenclature. 11
1.5.2. Biology,occurrenceandimpact 12
1.6 Scrobipalpuloidesabsoluta 13
1.6.1. Nomenclature 13
1.6.2 Biology,occurrenceandimpact 14
1.7 Motivationandscopeforthisthesis 15
1.8 Referencesandnotes 16
Massspectrometryofdimethyldisulphidederivativesasatool 23
forthedeterminationofdoublebondpositionsinlepidopteransex
pheromonesandrelatedcompounds
2.1 Introduction 23
2.2 Methodsandmaterials 26
2.3 TheanalysisofDMDSderivatiseddoublebonds 27
2.3.1 Mono-unsaturated molecules 27
2.3.2 Double-unsaturated molecules 29
2.3.3 Triple-unsaturated molecules 41
2.4 Conclusionsanddiscussion 46
2.5 Referencesandnotes 47
Isolation,identificationandsynthesisofthesexpheromoneof 49
Symmetrischema tangolias
3.1 Introduction 49
3.2 Methodsandmaterials 49
3.3 Resultsanddiscussion 52
3.4 Referencesandnotes 59

8. Scrobipalpuloides absoluta
5.1
5.2
5.3
5.4
5.5
Introduction
Methodsand materials
Resultsand discussion
5.3.1 Identification and synthesis
5.3.2 Fieldtests
Discussion
Referencesand notes
Determinationofthesexpheromoneglandcontentof 61
Symmetrischema tangolias bymeansofdirectglandintroduction
intoatwo-dimensional gasChromatograph
4.1 Introduction 61
4.2 Methodsandmaterials 62
4.3 Resultsanddiscussion 65
4.4 References 70
Isolation,identificationandsynthesisofthesexpheromoneof 73
73
73
75
75
87
89
90
6 WindtunnelbioassaysoftheSymmetrischema tangolias 93
sexpheromone
6.1 Introduction 93
6.2 Methodsandmaterials 94
6.3 Resultsanddiscussion 99
6.4 Referencesandnotes 101
7 Generaldiscussion 103
7.1 Three-dimensionalsexpheromonestructuresand 105
consequencesfortheiractivity
7.2 Sexpheromonesandtheprobabilityofresistance 107
7.3 Biosynthesis 108
7.4 Thelinkbetweenlaboratoryresultsandpracticalapplication 111
7.5 Somenotesonthecommercialsynthesisofpheromone 112
7.6 Referencesandnotes 114
8 Summary 117
9 Resumen 121
10 Samenvatting 125
CurriculumVitae 131

9. Voorwoord
Hetboekje datvooruligt,ishetuiteindelijke resultaatvanruimvierjaarwerk,uitgevoerd op de
vakgroep voor Organische Chemie van deLandbouwuniversiteit, Wageningen (OC-WAU), het
Instituut voor Planteziektenkundig Onderzoek, Wageningen (IPO-DLO) en het International
PotatoCenter,Lima,Peru (CIP).
Het beschreven onderzoek aan insectenferomonen is een combinatie van chemie enerzijds, en
biologie anderzijds. Het samenbrengen van deze twee totaal verschillende expertises was een
bijna op zichzelf staand deel van dit project, dat overigens niet alleen plaatsvond op het
onderzoeksvlak maar eveneens op het organisatorische en overlegtechnische vlak. Om u een
indruktegevenvandeinitiëlebenaderingvanhetoptelossenprobleemdoordebeidebetrokken
Wageningsegroepenhetvolgende.
VakgroepOrganischeChemie:"Dangaanweduswatvandiebeestjes uitknijpen om vervolgens
uitgebreid tegaankijken naar detoetepassenchemischeanalysemethoden omdestructuur van
de seksferomonen op te helderen. Nadat we ook de chemische aspecten en de synthese ervan
grondighebbenbekeken,kunnenwe(ohja)ooknog'eventjes'kijkenofhetwerktinhetveld."
Het IPO-DLOdachteraanvankelijk hetvolgendevan:"Na uitvoerig onderzoek aan de biologie
en fysiologie van debeestjes zullen we metwat technieken de structuur van de seksferomonen
'eventjes' ophelderen.Dieseksferomonen zullenwedan 'eventjes' gaanmaken omze vervolgens
tekunnengebruikeninuitgebreidgedragsonderzoek en veldwerk."
CIP:"Waarblijvendieferomonen nou toch?"
Bovenstaande voorstelling is te simplistisch weergegeven. Desalniettemin geeft ze een globaal
beeld van deverschillende invalshoeken waarmee diverse onderzoeksdisciplines een probleem
kunnen beoordelen. De vier jaar die dit project duurde, hebben geleerd dat er geen 'eventjes'
bestaatinonderzoek endat slechtsdoornauwe samenwerking tussen degenoemdegroepen het
complexeendisciplineoverschrijdende vraagstukzoalsbeschrevenindetitelvandit proefschrift,
kanworden opgelost.
Hetgebruik vanhetwoord 'groepen' inhetvoorgaande impliceert aldat dit onderzoek niet het
resultaat is geweest van de inspanningen van een enkel individu maar dat van een groter
gezelschapvanmensenvanwieikereenaantalgraagspeciaalzouwillen bedanken.
Prof. dr. Aede de Groot, dr. Teris van Beek, dr. Hans Visser en drs. Simon Voerman, voor de
mogelijkheid diejullie me hebben gegeven om dit onderzoek te kunnen doen. Teris en Hans,
vanwegejulliedirectebetrokkenheidbijhetonderzoekendedaarbijbehorendediscussies hebben
jullieinbelangrijkematebijgedragen aanhetbehaalde resultaat.
Gert Romeijn (Planteziektenkundige dienst) voor de moeite die hij zich heeft getroost om de
identiteitvandeonderzochtemottente verifiëren.

10. Frank Ciaassen voor het synthetiseren van de eerste (ennog steeds de lastigst te synthetiseren)
feromooncomponent.
Sander Houweling voor de dappere poging die hij gedaan heeft om de concentraties van
feromonen indeluchttemeten.
FalkoDrijfhout diemetzijn onderzoek eensubstantiëlebijdrage heeft geleverd aanhet behaalde
eindresultaat.
Dr. Romano Orru voor zijn hulp bij het synthetiseren van die ene lastige feromooncomponent
waarbij hij mocht ondervinden dat die op het oog simpele seksferomonen soms toch lastig in
elkaarteknutselen zijn.
Dr.Janvan der Persvan Syntech Laboratories teHilversumvoor hetmogen gebruiken van zijn
prachtige apparatuur. De resultaten die hieruit zijn voortgekomen waren essentieel voor het
onderzoek.
Dr.Stefan Schulz and Prof.dr.WittkoFrancke of theUniversity of Hamburg who taught me to
makedimethyldisulphide derivativesofsexpheromonecompounds.
Dr. Maarten Posthumus voor je enthousiaste hulp bij het verkrijgen en interpreteren van de
massaspectra van diverse complexe, jouw apparatuur danig vervuilende dimethyldisulfide
derivaten.Ikhoopdatjedureapparaatweereenbeetjeoverdeschrikheenis.
Mariannevooralles,maarwatditonderzoekbetreft vooralvoordemaandendiejehebt geholpen
bijdeverzorging van debeestjes enhetwindtunnelwerk. Zonder jouwas ikmisschienjuist die
maandenaantijdtekort gekomen.
Diverse mensen van het lab Organische Chemie die mij de beginselen van het synthetiseren
hebbenbijgebracht, maarmetnamedr.BenJanssen,ing.HenkSwartsendr.Hans Wijnberg.
Dr.ElenaPinellivoorhetvertalenvandesamenvattingincorrectSpaans.
Ing.JesüsAlcazar,dr.FaustoCisneros,RosaGhilardi,ing.ManuelDelgado,ing.MiquelChevedo,
ing.MariaPalacios,ing.Oder Fabianand manyothernicepeopleinPerufortheirhelpduring the
fieldwork aswellastheirsuccessful effort providingmewithaniceremembrance of Peru.
Dr.GaryJudd oftheResearchStationofSummerland, B.C.Canada,forhislastcriticalnotes with
respecttothelanguageand content.
BroerThijsvoorhetomslagenmavoorhetkritischbeoordelenvandeNederlandse tekst.
Enalslaatstenatuurlijk niettevergetendekornuitenenkornuitinnenmetwieikmijaltijd goed
kononderhouden inhetonsbekendeetablissementonderhetgenotvaneenlekker glaasje.
p"/-^/">S
WijkbijDuurstede, 12september 1996

11. voorMarianne
voormijnouders,ElsVerhaarenHenkGriepink

12. Chapter 1
General introduction
1.1 Insectsandpests
Withmorethanamillionspecies,insectsarebyfarthemostabundant form,innumbers,
ofanimal life1
.From thecoldness of thepolar snow capstotheheat ofthedeserts, and
from the aridity of the salt lakes to the dampness of the jungle, insects survive
everywhere. An ability to fly, a short life cycle, and ahigh reproductive rate are the
foundations forthembecomingthemostabundantformofanimallifeknown.
Many insect species are herbivorous and when they feed on plants meant for human
purposes, they become potential threats. In those cases where a significant part of the
harvestislost,insectsareconsideredapestandmustbedestroyed,oratleast controlled.
Some common known pest species are, for example, the migratory locust{Locusta
migratoria),and the Coloradopotatobeetle {Leptinotarsadecemlineata).These insects are
pestsbecausetheyactuallyeattheplants.Other insectsareconsidered pestsnotbecause
they eattheplant,but mainlybecause they,ortheir immature stages,arethevector for
viruses,fungi orbacteria which dotheactual damage.Examples areaphids and thrips,
which transmit viruses, or certain species of bark beetles which employ the fungus
CeratostomellaulmiwhichisresponsiblefortheDutchElmdisease.
Atpresent,mankindisincapableofcontrollingmanypestinsectsinawaythatis effective
and alsosustainable and compatible with the environment. Insectsevolved long before
higher animals and man appeared on earth. Whatever ended the era of the dinosaurs
apparentlydidnotstoptheoccurrenceoftheinsects.Therefore,itislikelytoassume that
humanity can be considered more capable of destroying higher organisms and itself,
beforeexterminatingasinglepestinsectspecies.
1.2 Butterfliesandmoths
Butterflies and mothsbelong totheClassInsecta and totheOrder Lepidoptera. Various
species have been reported to be crop pests. Whereas most (coloured) butterflies fly

13. Generalintroduction
during the daytime, most (non-coloured) moths restrict their activities to the night.
Becausetheyliveinthedark,thereisnoneedforthemtocarryexcessivecolours.Instead,
mothshaveevolved aremarkable way of recognising and locatingeachother.In moths
females usually, but sometimes males, release a blend of volatile chemicals which is
detected and recognised by conspecific members of the opposite sex. The partner is
specifically attractedtothisspecialvolatilechemicalblend.Throughthismechanism, the
probability of mating is highly increased, and thereby the existence of the species is
secured.
1.3 Lepidopteransexpheromones
1.3.1 General
The word pheromone is a contraction of the Greek words 'pherein', which means to
transfer and 'hormön',which means to excite. Pheromones are defined as substances,
which are secreted to the outside by an individual and when perceived by a second
individualofthesamespecies,theytriggeraspecific response2
.Severaltypeslikealarm,
trail and aggregation pheromones are known toexist for insects.When apheromone is
released with the intention of attracting members of the opposite sex for mating, it is
calledasexpheromone.Inmoths,mostsexpheromones arereleased byfemales to attract
conspecific males.Insomeprimitivemothspecies,males,orboth themalesand females
releaseasexpheromone3
.
To date, sexpheromones have been characterised for more than 400 species and
subspeciesofLepidoptera4,5
.Inaddition,forover900otherspeciesand subspecies,male
sexattractants havebeen found. The latter are called sexattractants,because although
theymightbestronglyattractivetothemalesofaparticularspecies,thereisnoproof that
the individual compounds are actually released by the females. In this thesis, a sex
pheromoneisdefined asthemixtureofchemicalcompounds,proventobepresentinthe
females, which isthe most attractive toconspecific males in the natural habitat of the
insect.Itisassumed thatthefemale mothdoesnotreleaseotherchemicalcompounds as
partofthesexpheromonethanthosewhicharepresentinhersexpheromone gland.
Thefirstinsectsexpheromonewasisolatedandidentified in1959byButenandt6
.Heand
his co-workers extracted and purified about 12milligrams of a, to the males, highly
attractivecompound from 500,000females oftheorientalsilkmoth (Bombyxmon). They
identified thiscompound as(E,Z)-10,12-hexadecadienol (Bombykol) (figure 1.1).Inthese
early pioneering years itwas never considered that a sex pheromone might consist of

14. Chapterone
morethanonecompound.Lateritbecameobviousthatmultiplecomponent pheromones
weremorearulethananexception.In1978itwasdiscovered thatsexpheromone gland
extractsofBombyxmoricontained,inaddition,thecorrespondingaldehydeofBombykol,
namelyBombykal(figure1.1),whichwaspartofthesexpheromone7
.
16
10 12 10 12
Bombykol Bombykal
Fig.1.1 SexpheromonecomponentsofBombyxmori,(E,Z)-10,12-hexadecadienol (Bombykol)and
(E,Z)-10,12-hexadecadienal(Bombykal).
Malemothsareextremelysensitivetotheirsexpheromones.Forexample,amountsofless
than 10pg (10"n
gram) ofthesexpheromone ofBombyxmoriwhenoffered onapieceof
filter paper tothemaleselicitabehavioural response8
.Other research shows that male
moths are able to detect and to respond to sexpheromone concentrations as low as
picograms per litre of air. Experiments have been carried out with Adoxophyesorana,
markedwithradioactive 32
P,todeterminethedistanceoverwhichthesemothswereable
tolocateasourcewithvirginfemales.Itturnedoutthatthemaleswereabletolocatethe
femalesoveradistanceof75metreinjustonenight.Measuredoverseveralnights,males
wereevencapableofreachingsourcesthatwereseveralhundredsofmetresaway9
.
Theindividualcomponentsthatoccurinasexpheromonearenotnecessarily chemically
specific forasinglemothspecies,however, inpracticefemales ofonespeciesattract and
mate only with males of the same species. One of the reasons is that the correct ratio
betweenthedifferent componentsofthesexpheromoneblend isalsoanimportant factor
fortheattractiveness10
.Toshowthis,acomparisonhasbeenmadeof34specieswith the
same two-component attractant/sexpheromone system, namely (Z)-9- and (Z)-ll-
tetradecenyl acetate (figure 1.2). A3:1mixtureof (Z)-9-tetradecenyl acetateand (Z)-ll-
tetradecenyl acetate, for example, is attractive toAdoxophyes orana but not to Clepsis
spectrana.Ifthe ratio of thesetwo components isinverted, the attractiveness forClepsis
spectrana and Adoxophyesoranais reversed as well11
. Several species as indicated in
figure 1.2usethesame,or almostthesame,ratioof individual components. Becauseof
this,and considering thefact thattheratioofsexpheromone components always shows
variation from individual to individual12
"14
, it could be expected that certain species
respond to other species. In practice this does not happen because these species are
separated geographically,ortheiractivity differs inthetimeoftheseasonortimeof the
day15
-16
.

15. General introduction
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Fig. 1.2 The same two pheromone compounds in a two-component attractant/sex pheromone
system for34species. (A)attractant, (I)chemical identification only,no behavioural tests,
(O)optimisedattractant,(P)sex pheromone.
Someofthereferenceswhichwereusedforfigure1.2areratherold.Itispossiblethatthe
sexpheromone contained more than just these two components, but due to the less
sensitiveanalyticalequipment,minute,butbiologicallyimportant,componentsmayhave
beenoverlooked (seeforanexample41
).Moreover,theactualrecognition and acceptance
occurs at the moment when males and females approach each other very closely, and
probably alsoby other means thanjust sexpheromone recognition. Itcanbe seen from
figure 1.2, that the identified sexpheromone composition in the insect sex pheromone
glandisnotinevitablythemostattractiveblend (for example,entry 12and 21).A reason
for thismaybethatalong-maintained laboratory colonyhas altered thesex pheromone
composition slightly,but biologically significant when compared to the wild species42
.
Sexpheromone producing glands may probably contain antagonists as well, meant to
repelotherspecies,and precursors,whicharenotreleased aspart ofthesex pheromone
blend.Thismustallbekeptinmindwhenexamininginsectsand theirsexpheromones.
Up to now, all the identified sexpheromones and attractants for Lepidopteran are
compoundswith alinear carbon chainwith lengthsvarying from 10to23carbons. The

16. Chapterone
sexpheromones originatefrom thefatty acidbiosynthesis.Therefore,mostofthem have
an even number of carbon atoms in the chain, however, exceptions are known. For
examplethemothsPhthorimaeaoperculella43
andKeiferialycopersicellaAi
, which both are
closelyrelated toSymmetrischematangolias and Scrobipalpuloidesabsoluta,havesexphero-
mones with chain lengths of 13 carbon atoms. The majority of lepidopteran sex
pheromones have an acetate as terminal functional group, nevertheless alcohols,
aldehydes,andoccasionallyformates,propionates,(iso)butyrates,and (iso)valerates have
been found. In one insect, Bucculatrix thurberiella,anitrateester was identified as the
terminalfunctional group45
.Thechainitselfmaycontainzerotofourdoublebonds,triple
bonds,(chiral)methylgroups,ketonesor(chiral)epoxides4
.Todate,only non-branched
straight chaincompounds with alengthof 10to16carbons,zerotothreedouble bonds
and an alcohol, acetate or aldehyde as functional groups, have been identified as sex
pheromonesorsexattractantsinGelechiidae(table l.l)4
.
Table1.1 Allthesexpheromones,andrelatedstructureswhichhavebeenidentified formembersof
theGelechiidae family .(A)attractant, (I)chemical identification only, (O) optimised
attractant,(C)possibleattractant,(P)sexpheromone.(xno.ofpublications)
chainlength
functional group
saturated
(E)-3-
(Z)-3-
(E)-4-
(Z)-4-
(E)-5-
(Z)-5-
(E)-7-
(Z)-7-
(E)-8-
(Z)-8-
(E)-9-
(Z)-9-
(E)-10-
(E)-ll-
(Z)-ll-
(E,E>-3,5-
(E,Z)-3,5-
(Z,E)-3,5-
(E,Z)-4,7-
(E,Z,Z)-4,7,10-
(Z,E)-7,11-
(Z,Z)-7,11-
10
OH
lxP
Ac
lxP
lxP
lxP
lxP
3xA
lxP
lxP
11
Ac
lxA
12
Ac
2xP,2xA
lxP,lxO, lxA
5xA
2xA
2xA
lxC
lxA
lxA
13
Ac
lxP
lxP
lxA
lxA
3xA
lxP
lxP
14
Ac
2xA
lxO,5xA
lxA
lxA
lxA
lxA
lxC, lxA
2xA
lxC, lxA
lxA
lxA
16
Aid
lxO
OH
lxP
lxP
Ac
lxA
lxP
2xP, lxO,3xA
lxP,6xA

17. Generalintroduction
1.3.2 Isolationtechniques
Therearetwowaystocollectsexpheromonesfrom aninsect:1)byextracting(partof)the
insectwithasuitablesolventlikehexaneordichloromethane,or2)bycollecting airborne
volatilecompounds from (part of)the insects onto asuitable adsorbent likePorapakQ,
Tenax, activated charcoal, or directly onto the column of a gas Chromatograph. The
secondapproachgiveslesschanceofdegradation,however,themethod islimitedbythe
amount of sexpheromone that is released by individual insects. The latter is species
dependent and varies from approximately 5to160ng/hr46,47
. The extraction of sex
pheromone glandsiseasiertoscaleup,however,with thisapproach much non-relevant
materialisco-extractedand itisnotalwaysapparentwhichcompound isactuallypartof
the sexpheromone. Sexpheromone gland extracts are examined directly or can be
subjected topurification first.Purification canbedonebycolumnchromatography, high
pressureliquidchromatography (HPLC)orpreparative gaschromatography. Amore or
lesscombined method involvesthedirectintroduction ofthesexpheromone gland into
thegasChromatograph (GC).Theintactsexpheromone gland isheated intheGC which
causesthevolatilecompounds toevaporate.Theyarethenfocused atthestartoftheGC
column(seefordetailsaboutthisapproach,chapter4).
1.3.3 Identification techniques
The GC is an excellent, sometimes underestimated, tool for the analysis of complex
mixtures of volatile compounds, like insect sexpheromones in asexpheromone gland
extract.GCanalysisisverysensitive.Amountsoflessthanonenanogramcanbedetected
withthecommonlyused flame ionisationdetector (FIdetector orFID).TheGCisableto
separate complex mixtures into the individual components. The retention time for a
particular compound depends on the type of column that isused. The retention times
obtained are often converted into their retention indices (RI's)by comparing them toa
standard range of alkanes thereby improving the accuracy48
. The comparison of the
calculated RI's of the sexpheromone compounds with those calculated for reference
compounds on several columns, provides information about the length of the carbon
chain, the presence and number of double bonds, sometimes even the position and
configuration of double bonds, and the functional group present, like an alcohol,
aldehyde, acetate, etc. If more than one double bond is present in the molecule, it is
possible to determine whether some, or all,double bonds are conjugated. In case the
sampleisverycomplex,orcontaminated,itispossibletoresolvetheindividualpeaksby
using atwo-dimensional GC(2D-GC)made up by two interconnected GC's.Instead of

18. — Chapterone
thenormally used FID,other detection or analytical techniques canbe applied on-line
withtheGC,suchasanelectroantennographic detector (GC-EAD),amass spectrometer
(GC-MS)oraFouriertransform infrared spectrometer(GC-FT-IR).
Electroantennography (EAG) is a technique which relies upon the specificity and
sensitivity of the olfactory system of the insect, the set of olfactory receptors on the
antenna. In moths, the antenna is covered with thousands of sensory sensilla, each of
whichcontainstwoormoresensoryneurones,sensitivetoparticularcompoundsortoa
group of chemically related compounds. A neurone recognises a particular molecule
through itsbinding with areceptor protein inthedendritic membrane. The subsequent
depolarisation,thereceptorpotential,causestheneuronetofireactionpotentials,which
aretransmitted tothebrain.Partofthereceptorpotentialleaksintothehaemolymphof
theantennaanditisthoughtthatthesumoftheseleakingreceptorpotentialsismeasured
withEAG49
.EAGisrestrictedtotheobservationwhetherornotaninsectisabletodetect
aparticularcompound and inwhatintensity.Theeffect ofaperceivedcompound on the
behaviour of the insect has to be determined by other methods. For an electro-
antennogram, the antenna from a male moth iscut off and usually connected to glass
electrodes filled with electrolyte. The electrodes are connected to an amplifier and
recording equipment50
. A continuous air-flow isblown over the antenna to which a
sample of an extract or a reference compound isadded for ashort moment. When the
EAG technique isused as the detector of a GC,the retention times (or retention time
intervals)aremeasured ofthecompounds whichareEAG-active.Thesecompounds are
physiologically perceived by the insect and thus, are sexpheromone candidates. By
duplicating theexperimentalconditions oftheGC-EADtotheGC-MS,massspectra are
acquired ofthecompounds thathaveproven tobeperceivedbytheinsect.Inthis way,
the molecular mass and elemental composition of the sexpheromone candidate are
obtained. The configuration of the double bonds can be determined in various ways.
Whenthesexpheromonecandidateisonlymono-unsaturated,thecomparisonoftheRI's
with those calculated for reference compounds isusually sufficient. However, if more
thanonedoublebond ispresent inthemolecule,itbecomesmore difficult to determine
the position and configuration just by comparison of RI's. If the double bonds are
separated by at least two methylene groups, the EAG measurements of all mono-
unsaturated reference compoundscanprovideuseful information abouttheposition and
theconfiguration ofthedoublebonds (seealsochapter3).Ifthedoublebondsinthesex
pheromone are conjugated, or homo-conjugated (separated by zero or one methylene
group), the EAG measurements give no unambiguous results (see also chapter5).
Anotherapproachforthedetermination ofthedoublebondpositionsistoderivethesex
pheromone compounds with dimethyl disulphide (DMDS) and subsequent analyse the
obtained derivatives with MS(chapter2).It isalsopossible topartially reduce the sex

19. Generalintroduction
pheromonecompound and analysetheobtainedmono-unsaturated compounds51
"53
.The
configuration ofdoublebonds inasexpheromone compound canalsobededuced from
Fouriertransform gasphaseinfrared (FT-IR)spectroscopy52,54
'55
.Thistechniquewas not
availablefortheresearchdescribed inthisthesis.Ifenoughpurematerialisisolated,this
can be examined with nuclear magnetic resonance (NMR)56
"58
. NMR is a powerful
analyticaltechniquewhichprovidesinformation aboutthestatusofthehydrogen atoms
inthemolecule.Becauseofthelow sensitivity of theNMRequipment, thistechnique is
not always useful for sexpheromone analysis (in practice,tens of micrograms of pure
compound areneeded).Themoststraightforward, and most labour-intensive, approach
todeterminethedoublebond configuration ofthesexpheromone,istosynthesiseallthe
possiblestructuralcandidates(seealsochapter5).Theultimatestageinthe identification
ofthesexpheromone, istodeterminewhether theidentified and synthesised molecules
really arecapable of attracting male moths.With thebioassays this,and the (optimal)
ratio of the identified compounds isdetermined. For this research, thebioassays were
carriedoutinthewindtunneloftheIPO-DLO(chapter6)andinfieldsandstorehousesin
Peru.
1.3.4 Chemical synthesis
Itisessentialtoconfirm theanalytical resultsbysynthesis ofthe (tentatively) identified
compounds.Forthis,so-called,analyticalsynthesis,onlysmallamounts ofproducts are
needed.Normallyinsynthesis,stereoselectivereactionsarepreferred whichproduceonly
one (E/Z) isomer per reaction step.Nevertheless,iftheE/Z configuration of the target
sexpheromone molecule isnotyet clear, itmight beadvantageous touse a non-stereo-
selectivesteptoproducebothisomersinonestep.Ofcourse,theproductmixtures should
not exceed the level of complexity where the different components can no longer be
separated. Thecostofreagents doesnothavethefirst priority when synthesising on an
analytical scale. This changes when the structure elucidation of the sexpheromone is
completed.Bythattimetherewillbeademand forgramquantitiesofthesexpheromone,
for example to start field tests or for sales.Then,the emphasis willbe on cost control.
Effective exploitationofhuman resources,thenumberofsyntheticsteps,and thepriceof
reactantsmustbeoptimised inrelation tothequality sothat theproduct salesare most
profitable. The greater part of the chemical reactions needed in the synthesis of sex
pheromonesarerelativelysimpleand easytoscaleup59
"61
.Unfortunately, most chemical
reactionsarenotasstereoselectiveaswewould wish,therefore alwaysafew percentof
undesired isomers will be present in the product. When it appears that the
contaminationsaredeterioratingtheeffect ofthesexpheromone,extensivepurificationof

20. Chapterone
the final product is necessary. This is usually done on a silver-loaded ion-exchange
chromatographiccolumn62
.After suchapurification stepthefinalproductcouldhavean
(isomeric)purityofmorethan99%.
1.4 Insectcontrol
1.4.1 Pesticidesversusalternativecontrolmethods
Today, it has been recognised that the use of pesticides isnot the all-comprehending
answer totheproblem of insectpestsasitwasoncethought tobe.Persistent pesticides
accumulate in non-target animals higher in the food-chain. Insects seem to become
resistant faster than new pesticides canbedeveloped (thisincludes thetimeneeded for
registrationprocedures).Inthird-world countries,resistancedevelopsfaster compared to
first-world countries,becauseofthethoughtlessandimproper useofagro-chemicals.For
example, farmers using herbicides or fungicides against insect pests have been
encountered in Peru. In contrast to the large scale tomato farming, the growing of
potatoesinPeruismostlyrestrictedtofarmershavingonehectareofgroundoroftenless.
Ifafarmer isusingpesticidesforexampleandhisneighboursarenot,itturnsoutthatthe
pests simply move to the neighbour's land. Surviving insects find there an untouched
sourceoffood torecoveron.Intomatocultivationsthisproblem existslessbecause this
typeoffarming isdoneatamuchlargerscale.Inthesecasesthelargescale monoculture
is the problem. Such cultures are known to promote the development of pests. The
International Potato Center (CIP) in Peru has been working on alternative ways of
controlling different insectpests,likedeveloping plant resistance,pre-and post-harvest
management for crop and seed-potatoes, biological control and the use of sex
pheromones63
.ForthepotatomothPhthorimaeaoperculella,aneffective biological control
hasbeendevelopedbymeansofthePhthorimaeaBaculoviruswhichisadded tothestored
potatoes64
. When larvae eat the potatoes they get infected with the virus and will
subsequently die. The disadvantage of this type of pest management is that infested
larvaeliveforanother12-21daysandthus,stillcauseconsiderabledamagetothestored
potatoes.The same problem occurs when, for example, parasitoids are used as control
agent.ForSymmetrischematangoliasand Scrobipalpuloidesabsoluta,onehastriedtodevelop
similarstrategies.Itseems,however,thatneitherofthesemothspeciesisverysensitiveto
themethodsdeveloped sofar.

21. Generalintroduction
1.4.2 Sexpheromonesinpestcontrol
Incontrasttopesticides,sexpheromones aresubstancesthat areproduced and used by
insects themselves. Therefore, it is unlikely that resistance against them will develop.
Whensexpheromones arechemically identified and available,theycanbe used in pest
controlinfour different ways:(1)monitoring, (2)masstrapping, (3)mating disruption65
and(4)theattractionandsubsequentkillingoftheinsectswithouttrappingthem,known
asattract-and-kill.
Monitoring is the most common use of pheromones. As a monitoring tool, sex
pheromonesareusedtoattractexclusivelythespeciesofinterestand,therefore, provides
data about the presence and abundance of the insect pest. The appropriate time for
pesticideapplicationcanbecalculated,sothatpesticideswillonlybeusedatthe moment
whentheyaremosteffective and needed.
Thesecondway inwhich sexpheromones canbeused ismasstrapping.Thismethod is
not used very often, especially not infirst-world countries. One reason for this is that
masstrapping islessthorough than theapplicationofpesticides.Another reasonisthat
theapplication ofsexpheromones formasstrapping isarathertimeconsuming wayof
controlling a pest because one needs a lot of traps which have to be installed and
maintained.Infirst-world countrieswherelabourisexpensive,theuseofsexpheromones
inmasstrappingiscommerciallyconceivableonlyinfewcases.
Thethirdapproachismatingdisruption.Here,thesexpheromoneisappliedinsuchhigh
concentrationsontothecroporinstorehousesthatthemalepestinsectsarenolongerable
tolocatethefemale insects.Inthisway,nocopulation willoccurand,asaconsequence,
nonewoffspring willdevelop.Thismethod hasadvantagesovermasstrapping because
it isrelatively easy-to-use. In practice however, there are still few cases where mating
disruptionhasshowntobeofpracticalvalueinpestcontrol66
.Notallinsectsaresensitive
tothismethod and insectsexpheromones areoften tooexpensivefor theapplication as
mating disruptant. Another important cause is the commitment to register the sex
pheromones in many countries before they may be applied for mating disruption67
,
whichisanexpensiveandtimeconsumingprocedure.
Thefourth method which involves insect sexpheromones inthe control of insect pests
wasdeveloped as"AttractandKill"68
.Thesexpheromone isformulated intoa glue-like
liquidUV-absorber (forlightprotection)withasmallamountofaverypotent insecticide.
It isapplied in droplets onto the plants that have to be protected. The male insect is
attractedtothesexpheromone,touchesthesourceandpicksupsomeofthegluetogether
witha(sub)lethaldoseoftheinsecticide.Ifsuchamalecopulateswithafemale lateron,
thereisagoodchancethatsheispoisoned aswell.Thismethod isusedwithsuccess,for
10

22. Chapterone
example, against Pectinophoragossypiellaincotton fields inEgypt69
and againstEphestia
kuehniellainflourmillsinItaly70
.
In developing countries, the newer, expensive pesticides are not always available.
Becausethe threshold for damage ismuch higher than infirst-world countries,and the
costs of labour are much lower, the application of sexpheromones in pest control
programs could be asolution. Sexpheromones are already used inthecontrol of some
insectpestspeciesinthird-world countries.Oneestablishedexampleistheuseofthesex
pheromone ofPhthorimaeaoperculella,whichwasidentified in 1976byPersoonsetal.43
.
The IPO-DLO synthesises this sexpheromone on a commercial scale71
. This sex
pheromonehasbeenapplied inPeru,VenezuelaandTunisiaforyearswithgreatsuccess
inmasstrapping ofPhthorimaeaoperculella72
.Itappears tobecheaper and more effective
thantheformerly usedpesticides.
1.5 Symmetrischema tangolias
Fig.1.3
Photographic imageof
Symmetrischema tangolias
onthesurfaceofapotato
tuber.Themillimetre paper
atthelowerleftsideof the
picturegivesan impression
oftheinsect's dimensions.
1.5.1 Nomenclature
ThemothSymmetrischematangolias(Gyen) (figure 1.3) wasdescribed for thefirst time as
Phthorimaeaplaesiosema by Turner in 191973
.Several synonyms for this moth have been
usedsince:PhthorimaeamelanoplinthaandGnorimoschematuberosella7i
.Themost commonly
usednamefor thismothhasbeenSymmetrischemaplaesiosema(Turner)75
.In1990,Hodges
noticed thatSymmetrischemaplaesiosemahad already been described asSymmetrischema
11

23. Generalintroduction
tangoliasbyGyenand,therefore,changed thespeciesnamefromplaesiosematotangolias76
.
Untilnow,thenameSymmetrischematangoliasisstillvalid.Aspecific Englishname does
not exist for this moth but inPeru, it issimply referred toas 'Symmetrischema'. Local
farmers inPerualsonamethismoth:'lapolilladelapapa' (translation:thepotatomoth),
which is confusing because this name is also used for another devastating pest on
potatoes,Phthorimaeaoperculella.Thelatterisclosely related to Symmetrischematangolias
andoccursinthesameregions77
.
1.5.2 Biology,occurrenceandimpact
Thepotatotubermoth Symmetrischematangoliasisaseverepest on potatoes inthe field
and in storehouses in Peru. In 1952,this moth was described as a potential threat to
potato78
,butitwasnotuntil1982thatitbecameamajorpest79
.Thebiologyofthisspecies
hasbeenexamined indetail75
.Thetotallife-cycle isstronglytemperaturedependent and
variesbetween40and 75days.Thepupae ofthisspeciesareeasilyseparated into males
andfemalesbytheexternalcharacteristicswhichareshowninfigure 1.4.Theadultscan
besexedbytheirreproductiveorgans.
mostdistinguishingmark
Fig.1.4 Externalcharacteristicstodistinguishbetweenmaleandfemalepupaeof Symmetrischema
tangolias.Thelasttwosegmentsofthemalepupaearegrowntogether.
Themain distribution areas for Symmetrischema tangoliasare the higher regions of the
Peruvian Andes63
and,althoughthisspecieshasbeenreported inAustralia,itseemsthat
itwasintroduced thereratherthanbeinganendemicspecies63,80
.In1993,Symmetrischema
tangoliasappeared inBoliviaforthefirsttime81
.Inthefield thelarvaeboreintothe stems
12

24. Chapterone
ofpotatoplants,whichcausestheplantstobreakanddie.Instorehouseslarvaemineinto
potato tubers making them unsuitable for human consumption. Nevertheless, infested
tubersareoften planted,whichcausesfurther spread ofthepest.InPeru,seed potatoes
are generally stored in large storehouses of co-operatives where they are sometimes
literallycovered withpesticides.Amounts of1.3gmalathionper kgpotatoeshavebeen
observed. In the Peruvian Andes, small farmers keep the potatoes indoors or in small
open storehouses. These potatoes are not treated with pesticides and are therefore an
idealfood for Symmetrischema tangolias. Crop losses can reach up to 100%81
. Today
Symmetrischematangoliasis considered to be an even greater pest than Phthorimaea
operculellainPeru82
.
1.6 Scrobipalpuloides absoluta
1.6.1 Nomenclature
Themoth Scrobipalpuloidesabsoluta(Meyrick) (figure 1.5) was described by Meyrick in
1917forthefirst timeasPhthorimaeaabsoluta83
.Povolnynamed thisspeciesScrobipalpula
absoluta7
*.Inhispaper,heremarkedthatScrobipalpulaabsolutaisfrequently confused with
'the tomato pinworm' Keiferialycopersicella (Walsingham), which isclosely related and
sometimes occursinthesame regionsasScrobipalpulaabsoluta74
.Clarke transferred this
speciestothegenusGnorimoschema84
,however,in1975Povoln^changedthegenus name
back to Scrobipalpula85
. The present name for this species was established in 1987by
Povolny86
as Scrobipalpuloidesabsoluta.Povolnyindicated that this species differed too
much from the genus Scrobipalpula and therefore, he placed this species in the genus
Scrobipalpuloides. The commonly used English name for Scrobipalpuloides absoluta is
'tomatoleafminer' and theSpanish name for thisspecies is:'Oruga minadora dehoja y
tallo'(translation:leafandstemminingcaterpillar).
1.6.2 Biology,occurrenceandimpact
The tomato leafminer, Scrobipalpuloides absoluta, is presently considered the most
devastating pest of tomatoes in Peru, Chile, Brazil, Argentina, Bolivia, Venezuela and
Colombia87
"97
.Itprefers thelower,warmer regions,although theholotypeofthisspecies
has been collected in Huancayo, 3500 metres above sea level86
. The biology and
occurrence has been studied in many countries89
"97
. The total life cycle is strongly
13

25. Generalintroduction
Fig.1.5
Photographicimageof
Scrobipalpuloidesabsoluta
ontheleafofatomato
plant.Themillimetrepaper
atthelowerrightsideofthe
picturegivesanimpression
oftheinsect'sdimensions.
temperature dependent and varies from 20 to 35 days. Adults and pupae of
Scrobipalpuloides absoluta have the same external characteristics as Symmetrischema
tangolias,and based on this,they canbeseparated into males and females. Although it
seemsthatScrobipalpuloidesabsolutaprefersthetomatoplantasitshost,itcanalsodevelop
onseveralothermembersintheSolanaceaefamily,likepotatoandtobacco95
.The moth's
larvae mine leaves and fruits of tomato plants causing considerable damage. Larvae
livinginsideleavesorfruits aredifficult toreachwithpesticides.Nevertheless,itseems
thatthisisstilltheonlyway tocontrolthispest98
. Scrobipalpuloidesabsolutaisresistant to
organophosphate pesticidesinBoliviaand itwasestablished thatapplying the synthetic
pyrethroid, fenvalerate every two weeks, is the most effective way of controlling
Scrobipalpuloidesabsoluta87
'88
. Inspiteofthis,farmers inParaguay often apply pesticides
twice every three days81
. Tomato farming is a large-scale industry in South America.
Farmsof150hectaresarecommoninPeruandinChile,theco-operativescanreachup to
10,000hectares99
. The majority of the tomato crop is processed and exported99
. This
exportisessentialfortheseSouthAmericancountriestoobtainforeign currency.
1.7 Motivationandscopeforthisthesis
The outline for the research described in this thesis was formulated when the Centro
InternacionaldelaPapa,Lima,Peru(CIP),togetherwiththeInstituteforPlant Protection
(IPO-DLO),Wageningen,TheNetherlandsdecidedtowriteajointprojectproposalonthe
isolation, identification and the application of the sexpheromones of Symmetrischema
14

26. Chapterone
tangoliasand Scrobipalpuloides absoluta. The Department of Organic Chemistry of the
Wageningen Agricultural University (OC-WAU),TheNetherlands waswilling toactas
thethird partner inthis research project. Theproject was financially supported by the
Netherlands'MinisterforDevelopmentCo-operation(DGIS).
Moths like Symmetrischema tangoliasandScrobipalpuloidesabsolutahavesexpheromones,
whichmightbeuseful asanalternativewaytocontroltheseinsectpests.Theaimof the
present study was to isolate, identify and synthesise these sexpheromones and to
determinewhether thesyntheticsexpheromonescanbeimplemented intoan integrated
pest management (IPM)program with regard tothesetwo pest species.A further aim
was to study analytical pathways for the identification of sexpheromones and related
compounds.
1.8 Referencesandnotes
1. Evans,H.E.1984.InsectBiology.Addison-Wesley,Massachusetts,USA.436pp.
2. Karlson, P. and Lüscher, M. 1959. "Pheromones", a new term for a class of
biologicallyactivesubstances.Nature,183,55-56.
3. Schulz, S. 1987. Die Chemie der Duftorgane mänlicher Lepidopteren. Thesis:
UniversityHamburg.281pp.
4. Arn,H., Tóth,M.and Priesner, E.1992.List ofSexPheromonesofLepidoptera and
Related Attractants, 2nd ed. International Organization for Biological Control,
Montfavet. 179pp.
5. Mayer, M.S. and McLaughlin, J.R. 1991.HandbookofInsect Pheromonesand Sex
Attractants.CRCPress,BocaRaton,Florida.1083pp.
6. Butenandt, A., Beckmann, R., Stamm, D.and Hecker, E. 1959.Über den Sexual-
lockstoff des Seidenspinners Bombyx mori. Reindarstellung und Konstitution.
Z.Naturforsch.,14b,283-284.
7. Kasang, G., Kaißling, K.E.,Vostrowsky, O. and Bestmann, H.J. 1978. Bombykal,
eine zweite Pheromonkomponente des Seidenspinners Bombyx mori L. Angew.
Chem.,90,74-75.
8. Kaißling,K.E.1979.Recognition ofpheromonesbymoths,especiallyinSaturniids
and Bombyx Mori. p. 43-51. In: F.J.Ritter (ed). Chemical Ecology: Odour
CommunicationinAnimals.Elsevier, Amsterdam.
9. Noordink,J.P.W.and Minks,A.K. 1970.Autoradiography: asensitivemethod in
dispersal studies with Adoxophyes orana(Lepidoptera: Tortricidae). Entomol.Exp.
Appi, 13,448-454.
15

27. Generalintroduction
10. Roelofs,W.1979.Production and perception oflepidopterous pheromoneblends,
p. 159-168. In: F.J. Ritter (ed.). Chemical Ecology: Odour Communication in
Animals.Elsevier,Amterdam.
11. Minks,A.K.1976.Sexferomonen vanLepidoptera:Onderzoeknaarhun mogelijke
toepassing in de gewasbescherming. II. Het onderzoek in Nederland.
Gewasbescherming,7,131-139.
12. Löfstedt, C , Lanne, S.L., Löfquist, J., Appelgren, M. and Bergström, G. 1985.
Individualvariationinthepheromoneoftheturnipmoth,Agrotissegetum.}. Chem.
Ecol,11,1181-1195.
13. Morse,D.,Szittner, R.,Grant, G.G. and Meighen, E.A. 1982.Rate of pheromone
releasebyindividualsprucebudwormmoths.J.Insect.Physiol,28,863-866.
14. Ono, T. 1993.Effect of rearing temperature on pheromone component ratio in
potato tuberworm moth, Phthorimaea operculella, (Lepidoptera: Gelechiidae).
J.Chem.Ecol,19,71-81.
15. Witzgall,P.,Bengtsson,M.,Buser,H.R.,Chambon,P.J.,Priesner,E.,Wildbolz,T.
and Arn, H. 1991.Sex pheromones of Spilonota ocellanaand Spilonota laricana.
Entomol.Exp.AppL,60,219-224.
16. Monti, L., Lalanne-Cassou, B., Lucas, P., Malosse, C. and Silvain, J.F. 1995.
Differences insexpheromone communication systems of closely related species:
Spodoptera latifascia (Walker) and S. descoinsi Lalanne-Cassou & Silvain
(Lepidoptera:Noctuidae).}.Chem.Ecol,21,641-660.
17. Steck, W.F., Underhill, E.W., Bailey, B.K. and Chisholm, M.D. 1982. Trace co-
attractantsinsyntheticsexluresfor22noctuidmoths.Experientia,38,94-95.
18. Foster, S.P. and Dugdale, J.S. 1988. A comparison of morphological and sex
pheromone differences in some New Zealand Tortricinae moths. Biochem. Syst.
Ecol,16,227-232.
19. Priesner,E.Personalcommunication,accordingto4
.
20. Priesner, E. 1978.A sex attractant for the pine beauty moth, Panolis flammea.
Z.Naturforsch.,33c,1000-1002.
21. Roelofs, W., Cardé, A., Hill, A. and Cardé, R. 1976. Sex pheromones of the
threelinedleafroller,Pandemislimitata.Environ.Entomol, 5,649-652.
22. Steck, W., Underhill, E.W. and Chisholm, M.D. 1982. Structure-activity
relationshipsinsexattractantsforNorthAmericannoctuidmoths,J.Chem.Ecol,8,
731-754.
23. Frérot, B.,Boniface, B.,Chambon, J.and Meritan, Y. 1982.Emploi du piégeage
sexuelavecdesattractifs desynthèsepour l'étude delarépartition dansla région
parisiennedetroisespècesdetordeusesdesvergers.Agronomie, 2,885-893.
16

28. Chapterone
24. Minks, A.K. and Voerman, S. 1973.Sexpheromones of the summerfruit tortrix
moth,Adoxophyesorana:trappingperformance inthefield. Entomol.Exp.Appl.,16,
541-549.
25. Ghizdavu, I.,Hodosan,F.P.,Oprean, I.,Gocan,A.,Ciupe,H. and Matic,S.1985.
Attractifs sexuelspour cinq espècesdeNoctuidaedéterminés par piégeage. Rev.
Roum.Biol.Ani, 30,25-29.
26. Boneß, M. 1978. Erfahrungen mit Sexualpheromonen von Lepidopteren. Anz.
Schädlingskde.,Pflanzenschutz,Umweltschutz.,51,161-166.
27. Booij,C.J.H,andVoerman,S.1984.Newsexattractantsfor35tortricid and4other
lepidopterous species, found by systematic field screening in The Netherlands.
}. Chem.Ecol.,10,135-144.
28. Struble,D.L.,Ayre,G.L..and Byers,J.R.1987.Sexattractantblendsfor strawberry
cutworm,Amphipoeainteroceanica (Smith),and acloselyrelated species,Amphipoea
americana(Speyer)(Lepidoptera:Noctuidae).Can.Entomol.,119,301-304.
29. Ghizdavu,I.,Hodosan,F.P.andOprean,1.1987.Attractifssexuelsspécifiques pour
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isomers.Science,175,1469-1470.
31. Renou, M., Lalanne-Cassou, B., Frérot, B.,Gallois, M. and Descoins, C. 1981.
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Hadeninae).C.R.Acad.Se.Paris.,292,1117-1119.
32. Ando,T.,Kuroko,H.,Nakagaki,S.,Saito,O.,Oku,T.andTakahashi,N.1978.Two-
component sex attractant for male moths of the subfamily Tortricinae
(Lepidoptera).Agric.Biol.Chem.,42,1081-1083.
33. Ando, T., Kuroko, H., Nakagaki, S., Saito,O., Oku, T. and Takahashi, N.1981.
Multi-component sex attractants in systematic field tests of male Lepidoptera.
Agric.Biol.Chem.,45,487-495.
34. Priesner, E.1980.Sexattractant system inPoliapisiL.(Lepidoptera: Noctuidae).
Z.Naturforsch.,35c,990-994.
35. Minks, A.K., Roelofs, W.L., Ritter, F.J. and Persoons, C.J. 1973. Reproductive
isolationoftwotortricid moth speciesby different ratiosofatwo-component sex
attractant.Science,180,1073-1074.
36. Horak, M., Whittle, C.P., Bellas,T.E. and Rumbo, E.R. 1988.Pheromone gland
components of some Australian tortricids in relation totheir taxonomy,}.Chem.
Ecol.,14,1163-1175.
17

29. Generalintroduction
37. Steck,W.,Underhill,E.W.,Chrisholm,M.D.,Bailey,B.K.,Loeffler, J.and Devlin,
CG. 1977.Sexattractants for malesof 12moth species found inwestern Canada.
Can.Entomol.,109,157-160.
38. Priesner,E.1984.ThepheromonereceptorsystemofmaleEuliaministranaL.,with
notesonotherCnephasiinimoths.Z.Naturforsch.,39c,849-852.
39. Roelofs, W.L., Lagier, R.F. and Hoyt, S.C. 1977. Sex pheromones of the moth,
Pandemispyrusana.Environ.Entomol, 6,353-354.
40. Roelofs,W.L.andBrown,R.L.1982.Pheromonesandevolutionaryrelationshipsof
Tortricidae.Ann. Rev.Ecol.Syst.,13,395-422.
41. Guerin, P.M.,Arn, H., Buser, H.R. and Charmillot, P.J. 1986.Sexpheromone of
Adoxophyesorana:additional components and variability inratioof (Z)-9-and (Z)-
11-tetradecenylacetate,].Chem.Ecol,12,763-772.
42. Hunt, R.E.,Zhao,B.and Haynes,K.F.1990.Genetic aspects of interpopulational
differences inpheromone blend of cabbage looper moth, Trichoplusiani. J.Chem.
Ecol,16,2935-2946.
43. Persoons,C.J., Voerman, S.,Verwiel,P.E.J.,Ritter,F.J.,Nooijen, W.J.and Minks,
A.K.1976.Sexpheromone ofthepotato tuberworm moth,Phthorimaeaoperculella:
Isolation,identification andfield evaluation.Entomol Exp.Appl, 20,289-300.
44. Roelofs,W.and Bjostad,L.1984.Biosynthesisoflepidopteranpheromones.Bioorg.
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45. Hall,D.R.,Beevor,P.S.,Campion, D.G.,Chamberlain, D.J.,Cork,A.,White,R.D.,
Almestar, A. and Henneberry, T.J. 1992. Nitrate esters: novel sex pheromone
components of the cotton leafperforator, Bucculatrix thurberiella Busck.
(Lepidoptera:Lyonetiidae).TetrahedronLett.,33,4811-4814.
46. Ma,M.and Schnee,M.E.1983.Analysisofindividual gypsymothsex pheromone
productionbysampleconcentrating gaschromatography. Can.Entomol, 115,251-
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47. Tóth,H.andBuser,H.1992.Simplemethodforcollectingvolatilecompounds from
single insects and other point sources for gas chromatographic analysis.
ƒ.Chromatogr.,598,303-308.
48. Kovats,E.1964.TheKovatsretentionindexsystem.Anal.Chem.,36,31A-35A.
49. Schneider, D. 1969.Insect olfaction: Deciphering system for chemical messages.
Science,163,1031-1037.
50. Visser,J.H.and Piron,P.G.M.1995.Olfactory antennalresponsestoplant volatiles
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37-46.
18

30. Chapterone
51. Yamaoka, R., Fukami, H. and Ishii, S. 1976.Isolation and identification of the
female sex pheromone of the potato tuberworm moth, Phthorimaea operculella
(Zeller).Agric.Biol.Chem.,40,1971-1977.
52. Yamaoka, R., Tokoro, M. and Hayashiya, K. 1987. Determination of geometric
configuration inminuteamountsofhighlyunsaturated termitetrailpheromoneby
capillarygaschromatography incombinationwithmassspectrometryand Fourier-
transforminfrared spectroscopy,}. Chromatogr.,399,259-267.
53. Attygalle,A.B.,Jham,G.N.,Svatos",A.,Frighetto,R.T.S.,Meinwald,J.,Vilela,E.F.,
Ferrara, F.A. and Uchôa-Fernandes, M.A. 1995.Microscale, random reduction:
Applicationtothecharacterizationof(3E,8Z,llZ)-3,8,ll-tetradecatrienyl acetate,a
newlepidopteransexpheromone.TetrahedronLett.,36,5471-5474.
54. Attygalle,A.B.,Svatos,A.,Wilcox,C.and Voerman, S.1994.Gas-phase infrared
spectroscopyfordeterminationofdoublebondconfiguration of monounsaturated
compounds.Anal.Chem.,66,1696-1703.
55. Attygalle, A.B. 1994. Gas phase infrared spectroscopy in characterization of
unsaturatednaturalproducts.Pure&Appl.Chem.,66,2323-2326.
56. Rossi, R., Carpita, A., Quirici, M.G. and Veracini, C.A. 1982.Insect pheromone
components.Useof13
CNMRspectroscopyforassigningtheconfiguration ofC=C
doublebondsofmonoenicordienicpheromonecomponentsand for quantitative
determinationofZ/E mixtures.TetrahedronLett.,38,639-644.
57. Ando,T.,Kusa,K.,Uchiyama,M.,Yoshida,S.and Takahashi,N. 1983.13
C NMR
analyses onconjugated dienicpheromones of Lepidoptera.Agric. Biol.Chem.,47,
2849-2853.
58. Baker,J.D.and Heath,R.R.1993.NMRspectral assignment of lactone pheromone
components emittedbyCaribbeanandMexicanfruit flies,J.Chem.Ecol.,19,1511-
1519.
59. Mori, K. 1992. The synthesis of insect pheromones, 1979-1989. p. 1-523. In:
J.ApSimon (ed).Thetotalsynthesisofnaturalproducts.Volume9.JohnWiley&
Sons,NewYork.
60. Yadav, J.S.and Reddy, E.R. 1988.Synthesis of insect sex pheromones. Current
Science,57,1321-1330.
61. Brandsma,L.1971.Preparativeacetylenicchemistry.Elsevier,Amsterdam.207pp.
62. Houx,N.W.H.andVoerman,S.1976.High-performance liquidchromatographyof
potentialinsectsexattractantsandothergeometricalisomersonasilver-loaded ion
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63. CIP1993.CIPin1992:ProgramReport.TheInternationalPotatoCenter(CIP),Lima,
Peru.173pp.
19

31. Generalintroduction
64. Raman, K.V. and Alcazar, J. 1992. Biologicalcontrol of potato tuber moth using
Phthorimaeabaculovirus.CIPtraining bulletin2.International Potato Center (CIP),
Lima,Peru.27pp.
65. Minks,A.K.1975.Sexferomonen vanLepidoptera:Onderzoeknaarhun mogelijke
toepassing in de gewasbescherming. I. De algemene stand van zaken.
Gewasbescherming,6,65-70.
66. Cardé, R.T.and Minks,A.K. 1995.Control of moth pestsby mating disruption:
Successesandconstraints.Ann. Rev.Entomol.,40,559-585.
67. Minks,A.K.1990.Registrationrequirements and statusforpheromones in Europe
andothercountries,p.557-568.In:R.L.Ridgway,R.M.SilversteinandM.N.Inscoe.
(eds.).Behavior-modifying chemicalsfor insectmanagement. MarcelDekker,Inc.,
NewYork.
68. Hofer, D. and Brassel,J. 1992. "Attract and kill" to control Cydiapomonellaand
Pectinophoragossypiella.10BC/WPRS Bulletin,15,36-39.
69. Haynes, K.F.,Li,W. and Baker, T.C. 1986.Control of the pink bollworm moth
(Lepidoptera: Gelechiidae) with insecticides and pheromones (attracticide): lethal
andsublethaleffects,].Econ.Entomol, 79,1466-1471.
70. Trematerra, P. 1995.The use of attracticide method to control Ephestiakuehniella
Zellerinflourmills.Anz. Schädlingskde.,Pflanzenschutz,Umweltschutz.,68,69-73.
71. Voerman,S.and Rothschild,G.H.L.1978.Synthesisofthetwocomponents of the
sex pheromone system of the potato tuberworm moth, Phthorimaea operculella
(Zeiler)(Lepidoptera:Gelechiidae)andfield experiencewiththem.].Chem.Ecol.,4,
531-542.
72. Raman,K.V.and Booth,R.H. 1983.Evaluationoftechnologyfor integratedcontrolof
potatotubermothinfieldandstorage.International PotatoCenter (CIP),Lima, Peru.
18pp.
73. Turner.1919.Proc.R.Soc.Queensld.,31,p.126.accordingto74
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74. Povolny, D.1967.Genitalia of some nearctic and neotropic members of the tribe
Gnorimoschemini (Lepidoptera, Gelechiidae).Acta EntomologicaMusei Nationalis
Pragae,37,51-127.
75. Sanchez,G.A.,Aquino,V.and Aldama,R.1986.Contribución alconocimiento de
Symmetrischemaplaesiosema(Lep.:Gelechiidae).Rev.Per.Entomol.,29,89-93.
76. Hodges, R.W. and Os, V. 1990. Nomenclature of some neotropical Gelechiidae
(Lepidoptera).Proc.Entomol.Soc.Wash.,92,76-85.
77. Ewell,P.T.,Fano, H., Raman, K.V.,Alcazar,J., Palacios,M. and Carhuamaca,J.
1990.FarmermanagementofpotatoinsectpestsinPeru.International Potato Center
(CIP),Lima,Peru.87pp.
20

32. _ Chapterone
78. Wille, J. 1952. EntomologiaagricoladelPeru. Segundaedition, direction generalde
agricultura.MinisteriodeAgriculture,Lima,Peru..
79. Alcazar, J., Palacios, M. and Raman, K.V. 1982. XXV Convention National de
Entomologia.3-7October1982.Huaraz,Peru..
80. Osmelak,J.A.1987.ThetomatostemborerSymmetrischemaplaesiosema(Turner),and
the potato moth Phthorimaea operculella(Zeiler), as stemborers of pepino: first
Australianrecord.PlantProt.Quart., 2,44.
81. Personalcommunicationofing.J.AlcazarofCIP,Lima,Peru,1993.
82. PersonalcommunicationofDr.F.CisnerosofCIP,Lima,Peru,1995.
83. Meyrick,E.1917.SouthAmericanmicro-Lepidoptera.Trans.Entomol.Soc.Lond.,17,
1-52.
84. Clarke, J.F. 1969. Catalogueof the type specimensofmicrolepidopterain the British
Museum (NaturalHistory)describedbyEdwardMeyrick,volVII.TrusteesoftheBritish
Museum(NationalHistory),London.533pp.
85. Povolny,D.1975.Onthreeneotropical speciesofGnorimoschemini (Lepidoptera,
Gelechiidae)miningSolanaceae.ActaUniv.Agric.(Brno),II,379-393.
86. Povolny, D. 1987. Gnorimoschemini of southern South America. Ill: the
scrobipalpuloid genera(Insecta,Lepidoptera,Gelechiidae).Steenstrupia,13,1-91.
87. Matta, A. and Ripa, R. 1981. Avances en el control de la polilla del tomate,
Scrobipalpulaabsoluta(Meyr.) (Lepidoptera:Gelechiidae).I.Estudios de población.
AgriculturaTécnica,(Chile),41,73-77.
88. Moore,J.E. 1983.Control of tomato leafminer (Scrobipalpulaabsoluta)in Bolivia.
TropicalPestManagement,29,231-238.
89. Haji, F.N.P., De Vasconcelos Oliviera, CA., Da Silva Amorim Neto, M. and De
SordiBatista,J.G.1988.Flutuaçâopopulacionaldatraçadotomateiro,nosubmédio
SâoFransisco.Pesq.Agropec.Bras.,Brasilia,23,7-14.
90. Haji, F.N.P.,Parra, J.R.P.,Silva,J.P.and De Sordi Batista,J.G. 1988.Biologia da
traça do tomatiero sobcondiçôes de laboratório. Pesq.Agropec.Bras.,Brasilia,23,
107-110.
91. Hickel,E.R.,Vilela,E.F.,GomesdeLima,J.O.and Castro Delia Lucia,T.M.1991.
Comportamente de acasalamento de Scrobipalpula absoluta (Lepidoptera:
Gelechiidae).Pesq.Agropec.Bras.,Brasilia,26,827-835.
92. Hickel,E.R.and Vilela,E.F.1991.Comportamento dechamamento easpectos do
comportamento de acasalamento de Scrobipalpula absoluta (Lepidoptera:
Gelechiidae),sobcondiçôesdecampo.An. Soc.Entomol.Brasil,20,173-182.
93. Quiroz,C.E. 1976.Nuevos antécédentessobrelabiologia de lapolilla del tomate,
Scrobipalpulaabsoluta(Meyrick).AgriculturaTécnica(Chile),36,82-86.
21

33. Generalintroduction
94. Quiroz,CE. 1978.Utilización de trampas conhembras virgenes deScrobipalpula
absoluta (Meyrick) (Lep., Gelechiidae) en estudios de dinâmica de población.
AgriculturaTécnica(Chile),38,94-97.
95. Fernandez,S.A.1980.Estudiodelabiologîa delminadordeltomate,Scrobipalpula
absoluta(Meyrick) (Lepidoptera, Gelechiidae) en Venezuela. Thesis: Universidad
CentraldeVenezuela.57pp.
96. Fernandez, S.A., Salas,].,Alvarez,C.and Parra,A.1987.Fluctuación poblacional
de losprincipales insectos-plaga del tomate en la Depresión de Quîbor, estado
Lara.Venezuela.Agronomiatropical,37,31-42.
97. Râzuri,V.andVargas,E.1975.BiologîaycomportamientodeScrobipalpulaabsoluta
Meyrick(Lep.,Gelechiidae)entomatera.Rev.Per.Entomol.,18,84-89.
98. Leite, D., Bresciani, A.F., Groppo, A.G., Pazini, W.C. and Gravena, S. 1995.
Comparisonofintegratedpestmanagementstrategiesontomato.An. Soc.Entomol.
Brasil,24,27-32.
99. Personal communication of ing. M. Delgado, free-lance advisor for pest
managementproblems,Lima,Peru,1995.
22

34. Chapter 2
Massspectrometryofdimethyldisulphidederivativesasatool
forthedeterminationofdoublebondpositionsinlepidopteran
sexpheromonesandrelatedcompounds*
2.1 Introduction
Massspectrometry is a widely applied technique for the analysis of (volatile) organic
molecules.Thequalityofinformation obtained,incombinationwithitssensitivity makes
thistechniqueparticularly useful for the analysisofvolatile straight chain lepidopteran
sexpheromone compounds.Bylinkingthemassspectrometer toagasChromatograph a
complex sexpheromone extractcanbeexamined without theneed for prior isolationof
theindividualcomponents.Notonlythemolecularmassofasexpheromone component,
but information about itsfunctional group and number of doublebonds isobtained as
well. In cases of methyl-branched, or epoxidized sex pheromones, the position of the
methylgrouporepoxidecanbedetermined throughmassspectrometry(MS)alone1,2
.
Although attempts have been made4,5
, the determination of double bond positions in
linear (poly-)unsaturated sexpheromone components and related compounds, without
prior derivatisation of the double bonds, is difficult by MS examination alone3
. The
difficulty arisesbecauseafter eliminatingfunctional groupsinthemassspectrometer, the
radical sites in the olefins that are formed, migrate freely through the molecule
(accompanied byhydrogen rearrangement)3,6
.Only incaseswheremolecules possesses
twoconjugated doublebonds,canthepositionsbededuced from theMS fragmentation
ofthenon-derivatised molecule7
"9
.Inthesesituationstheco-endofthemolecule provides
twocharacteristicfragments asillustratedinfigure2.1.Thisapproachcanbe extrapolated
to determine the double bond positions in molecules with three10
and possibly more
conjugated doublebonds.
Partsofthischapterhavebeenpublished:Griepink,F.C.,vanBeek,T.A.,Visser,J.H.,Posthumus,M.A.,
Voerman,S.anddeGroot,Ae.1996.TetrahedronLett.,37,411-414.
23

35. Massspectrometricanalysisofsexpheromones
Rj=alkylpart
R2=partwiththefunctional group 0
fragment2
+ * ^ R 2
Fig. 2.1 Characteristic massspectrometric fragments that occur for conjugated straight chain
molecules.
Anumberofprocedureshavebeendescribed fortheindirectdetermination ofdouble
bondpositionsinstraightchainunsaturatedmolecules.Probablytheoldestoneistotreat
theunsaturatedmoleculewithozoneandanalysetheobtainedaldehydefragments by
GCandMS11
(figure2.2).
O, O—o Zn,HOAc
R, * " R , ^ +
O ^ " ^ R2
Fig.2.2 Ozonolysisofdoublebondspriortoanalysis.
Otherproceduresinvolvetheaddition ofcertainmolecules tothedoublebond(s),to
produceaderivativethatexhibitsaspecificmassfragmentation patternfromwhichthe
original position(s) of the doublebond(s) canbededuced. Asmentioned before, the
position ofanepoxidecanbedetermined directlybyMS.Adoublebond which has
reacted with,for example,m-chloroperbenzoicacid (m-CPBA),and isconverted toits
corresponding epoxide willfragment next totheepoxide and in thisway reveal the
fragment1
m-CPBA
• R/
o
R2
fragment2
Fig2.3 Theconversionofanunsaturatedstraightchaincompoundtoitsepoxide,plustheexpected
fragmentsthatwillforminthemassspectrometer.
24

36. Chaptertwo
positionoftheoriginaldoublebond(figure2.3)2
.Theepoxidescanalsobehydratedand
convertedintotheirtrimethylsilylethersbeforeanalysis(figure2.4)12
.
1)H2 0,amberlyst15
2)BSTFA
Fig 2.4 The hydration of an epoxide and reaction with bis(trimethylsilyl)trifluoroacetamide
(BSTFA),plustheexpectedfragments thatwillforminthemass spectrometer.
Methoxylation of the double bonds followed by MSanalysis isanother established
method(figure2.5)3
.
Hg(OAc):
AcOHg OMe
R
1 ^2
HgOAc ©
OAc
fragment2 ---•!
NaBH4,AcOH
r--- fragment1 fragment3 ---•;
OMe MeO
R2
••--- fragment4
Fig 2.5 The reaction of an unsaturated linear compound with mercuric acetate, methanol and
sodium borohydride toyield two methoxylated productswhich will fragment inthe mass
spectrometernexttothemethoxygroups.
Themajor drawback of the above mentioned derivatisation procedures is that they
requiremicrogramsofstartingmaterial.Therefore,theyarelesssuitablefortheanalysis
ofinsectsexpheromoneswhereoften onlynanogram quantitiesareavailable.Amore
sensitiveapproachistoderivetheunsaturatedmoleculewithgaseousnitricoxide(NO)
insidethemassspectrometeritself,however,thismethod isrestricted tostraightchain
moleculeswithatripleorquadruple (cis)homo-conjugated system (figure2.6)13
.The
positionofadoublebondclosetothealiphaticendofthemoleculecanbedetermined
25

37. Massspectrometricanalysisofsexpheromones
withthisapproach,however,onlythepositionofthatdoublebondisthen determined14
.
NO+
•
Fig 2.6 The reaction of a homo-conjugated triene with nitric oxide (NO) inside the mass
spectrometer.
The fragment which arises from the chemical ionization (CI) with nitric oxide is also
detected (at low relative intensities) in thenormal electron impact (EI)mass spectrum.
Thisindicateshoweasythedoublebondsintheinitiallyformed radicalmigratealongthe
chain (toform a conjugated system which subsequently fragments in a similar way as
shown in figure 2.1)3
'6
.The relative intensity of thisparticular fragment in the EImass
spectrumincreaseswhentheionizationenergyisreduced.
Asensitiveand morebroadly applicableprocedure isthederivatisation ofdouble bonds
with dimethyl disulphide (DMDS). This procedure has been described mainly for
moleculeswithjustoneor twodoublebonds15
"17
.Inonecaseithasbeen described asa
toolforthedeterminationofthedoublebondpositionsinanalkatriene18
.However,more
thanonemicrogram ofcompound wasused for thederivatisation reactionand analysis.
Moreover,nofunctional groupwaspresentintheoriginalmoleculeandthedoublebonds
were separated by more than three methylene groups which facilitates the analysis
considerably19
.
2.2 Methodsand materials
Reactionconditions
Approximately 1mg(4umol)ofacetatein140|0,1offreshly distilled DMDSand acrystal
of iodine (±5mg,or±0.5mg when mentioning low iodineconcentrations) ina4ml vial
wassealedand heated for 16hrat60°C.Thereactionwasquenched with afew dropsof
saturated aqueousNa2S203 (until the red colour oftheiodine faded). Theorganic layer
was collected and filtered and dried simultaneously by passing it through a Pasteur
pipettefilledwithdryNa2S04.
26

38. — Chaptertwo
Massspectrometry
Mostofthemassspectrometry wasperformed onaFinniganMAT95mass spectrometer
(70eV),coupled to a Varian GC equipped with a split/splitless injection system. The
injection volumes varied between 1-2ul (splitless). The column was aJ&W 25m DB-5
fused silicacolumn,0.25mm idand 0.25|xmfilm thickness.Conditionswere:Carrier gas
helium;column temperature 250or260°C.Themassspectraoffigures 2.33through 2.35
were recorded on a HewlettPackard 5970 quadropole mass selective detector (MSD).
Chromatographic conditions were the same as described for the Varian GC only a
HewlettPackardGCwasused instead.
2.3 TheanalysisofDMDSderivatiseddoublebonds
Adouble bond which has reacted with DMDSpreferably breaks at the former double
bondpositioninthemassspectrometer.Fromtheobtained fragments, thepositionofthe
originaldoublebondcanbededuced.TheDMDSderivativesarepreparedbyheating the
unsaturated compound with DMDSand iodine.Thestructure of theDMDS derivatives
depends on the number of double bonds present in the molecule, the concentration of
DMDSandiodine,andprobablyalsoontheheatingtimeand temperature.
2.3.1 Mono-unsaturated molecules
Themechanism for the addition of DMDSto a double bond isillustrated in figure2.7.
Iodineinitially reacts with DMDStoform methylthio-iodide which subsequently reacts
which the double bond. The obtained sulphonium-iodide intermediate reacts with a
second molecule of DMDS.Amolecule ofmethylthio-iodide isregenerated inthisstep,
therefore, the iodine acts in this case as a catalyst16
. The addition of DMDS to the
sulphonium-iodideintermediateisassumedtobeanti(figure2.7).Therefore,theaddition
of DMDSto (Z)-double bonds leads to the threo product whereas the addition to (E)-
doublebondleadstotheerythroproduct.
The initial attack of the methylthio-iodide to the doublebond can take place from the
upper orlower side of themolecule thus two enantiomers are formed. Thestructureof
thesulphonium-iodide intermediatehighlyfavours theattackofaDMDSmolecule from
the opposite side of the sulphonium group, therefore no diastereomer formation is
observed.Thepresence of asingleproduct peak inthegaschromatogram confirms this
mechanism.
27

39. Massspectrometric analysisofsex pheromones
Me-5—I
H ( H
(Z)-
Me-S—I
H ( R 2
) (
R, H
(E)-
Me
H'^TVH
. © ^ S - S M e
Me
Me
C S ©
H ' ^ T VR2
R A H
Q /
~ * ' S-SMe
Me
-MeSI
-MeSI
threo product
SMe
F V H ^ H / " S M e
H
SMeSMe
erythro product
Fig.2.7 Proposed reaction mechanism fortheformation ofDMDSderivatives of mono-unsaturated
straightchain molecules.
Asanexample,thereactionof (E)-3-tetradecenyl acetate(1)withDMDSisillustrated in
figure2.8.
A,
DMDS,I2,AT
X
MeS
SMe
Fig.2.8 Thereactionof(E)-3-tetradecenyl acetate(1)withDMDSgives 3,4-bis(methylthio)tetradecyl
acetate(2).Thedashedlinerepresents thepositionwherethemoleculeisexpectedtobreak
inthemassspectrometer yieldingfragments HandB.
The DMDS derivatives are rather stable, which is also expressed by a usually clearly
visiblemolecular ion(M+
).Inthemassspectrometer, theDMDSderivativewillloseone
andsometimesmoremethylthiogroups (SMe)leadingtofragments M+
- 47(orM+
-48
for HSMe) and fragment M+
- 95(=47+48).In casethat two methylthio groups leave
initiallythetotalleavingmassisalways95amu(atomicmassunits).
Themassspectrum of3,4-bis(methylthio)tetradecyl acetate(2)isshowninfigure2.9.The
relevantfragments arementioned intable2.1.
28

40. Chaptertwo
100n
(%)
ntensity
Ul
O
Relative
o
4lf
61
i
l, I L
8
il
7
97
i
1
Çl_47
201
-H
i22
-^ 2
1 1 301
i
' i i r i i
348(M+-
)
•• k
50 100 150 200 250 300 350 400 450 m/z
Fig.2.9 Massspectrumof3,4-bis(methylthio)tetradecylacetate(2).
Table2.1 Relevantmassspectrometricfragmentsof3,4-bis(methylthio)tetradecyl acetate(2).
m/z
348
301
241
composition
C18H36O2S2
C17H33O2S
C15H29S
source
M+
-- SMe
M+
'- SMe- acetate
m/z
147
87
201
composition
C6 Hii02 S
C4H7S
C12H25S
source
fl+
H+
- acetate
B+
The peak with thehighest intensity at m/z 201 represents fragment BoftheDMDS
derivative (figure2.8).FragmentsflandH- SMehavelowintensities,butfragment H-
acetate isclearly visible.Theintensity offragment M+
'- 95isvery small inthis case
(<0.1%)andconsequentlyfiltered outofthemassspectrumoffigure2.9.
2.3.2 Double-unsaturated molecules
Double-unsaturated molecules mayreact indifferent ways with DMDS.Thedistance
betweenthetwodoublebondsdeterminesthefinalproduct.Whenthetwodoublebonds
are separated bymore than three methylene groups,themolecule simply reacts twice
with DMDS toform exclusively anopen di-adduct. When thetwodouble bondsare
separatedbylessthan threemethylenegroups,acyclicthio-etherisformed exclusively.
Intheparticular casewhen thedoublebonds areseparated byexactly three methylene
groups, both types ofDMDS reaction product (open orclosed) canbepresent. Asan
example (E,Z)-3,8-tetradecadienyl acetate(3)istaken. This molecule hasexactly three
methylene groups between thetwodouble bonds andtheDMDS reaction product
29

41. Massspectrometryanalysisofsexpheromones
consists partly of the open DMDS di-adduct 3,4/8,9-tetrakis-(methylthio)tetradecyl
acetate(4)and partly ofthecyclicthio-ether 2-(methylthio-hexane-l-yl)-6-(3-methylthio-
ethylpropanoate-3-yl)-tetrahydrothiopyran (5)(figure2.10).
A-(E,Z)-3,8-tetradecadienylacetate(3)
DMDS,I2,AT
MeS
SMe MeS
B
SMe O
A
MeS
OB
5
SMe
Fig2.10 Exactlythreemethylene groupsbetween thetwodoublebonds in (E,Z)-3/8-tetradecadienyl
acetate(3)resultsinthetwotypesofDMDS derivatives:4(open)and5(closed).
Themassspectrum of theopen DMDSdi-adduct 4isshown infigure2.11.The relevant
fragments arementioned intable2.2.Theprincipleofring-closureisdiscussed further in
thischapter.
100i
c
CD
I 50
>
ra
O)
cc
87
61
43
41
i
67
345
201
153
131
105
jJLpl
197
17318
n9
~ i
213 245
237
261 285
i 309
297
50 100 150 200 250 300 350
440(M+-
)
392
400 450 m/z
Fig.2.11 Massspectrumoftheopen DMDS di-adduct3,4,8,9-tetrakis(methylthio)tetradecyl acetate
(4)from(E,Z)-3,8-tetradecadienylacetate(3).
Themassspectrometricfragmentation patternof4canbeinterpreted ina straightforward
manner. It appears that the loss of a methylthio group from an already-formed mass
30

42. Chaptertwo
spectrometric fragment resultsinthelossof48amu.Thelossoftwomethylthio groups,
therefore, resultsinthe lossof96amu and not of95amu, as isobserved when the two
methylthiogroupsarelostinitiallybythemolecularion.
Table2.2 Massspectrometricfragments oftheopenDMDSdi-adduct 3,4,8,9-tetrakis(methylthio)-
tetradecylacetate(4).
m/z
440
392
345
285
309
261
249
composition
C20H40O2S4
C19H36O2S3
C18H33O2S2
C16H29S2
C13H25O2S3
C12H21O2S2
C11H21S3
source
M+
-
M +
- H S M e
M +
- 2 x S M e
M+
-- 2xSMe- acetate
flB+
HB+
-HSMe
HB+-acetate
m/z
201
213
153
293
245
197
87
composition
C10H17S2
C11H17O2S
C9H13S
C14H29S3
C13H25S2
C12H21S
C4H7S
source
HB+- HSMe- acetate
RB+
-2xHSMe
HB+-2xHSMe-acetate
BC+
BC+
-HSMe
BC+-2xHSMe
fl+
- acetate
When the double bonds in the original molecule are separated by less than four
methylenegroups,theformationoftheDMDSderivativedevelopsinadifferent way.The
proposedreactionmechanismisillustratedinfigure2.12.Oneofthemethylthiogroupsof
the DMDS derivative of the first double bond attacks one of the carbons of the
sulphonium ionthat isformed asanintermediate from thesecond doublebond. In this
way,acyclicthio-etherisformed.Theiodineinthisreactionisnotonlythecatalystbutis
alsoconsumedasmethyliodideduringthereactionandthusconsideredasareactant16
.
" ' v V ^ v A x R
2
MeS
Me
SMe © s
MeSI(n=0)
Me ^
i I©
SMe MeS SMe
S-Me
1©
,© Me
S'
M
%© s | ö
Cs M I
R i W>y^R
2 = R1
CÖ^R2
—*
MeS ,&Z V...Me'© SMe
n=1,2or3
- M e I
X ^S
-N^-
Ri nr nr R2
MeS SMe
n=0
Fig2.12 Proposed mechanism for the formation of DMDS derivatives from molecules with two
doublebondswhichareseparatedbythreeorlessmethylenegroups.Fordetailsseetext.
31

43. Massspectrometiicanalysisofsexpheromones
Incasen=0,thus when the double bonds in theoriginal molecule are conjugated, the
mostremotemethylthio group thatisattached tothefirst doublebond attacksthe most
remotecarbon atomofthesulphonium ionthatisformed from thesecond double bond.
Inthiswayatetrahydrothiopheneisobtainedwiththetwomethylthiogroupsattached to
the ring (figure 2.12). The mass spectrum of the resulting bis(methylthio)tetrahydro-
thiophene (n=0) derivative is recognisable by two intense peaks: M+
-- 95 and M+
-
(95+functional group) due to the easy loss of the two methylthio groups under
formation ofastablethiopheneandthesubsequentlossofthefunctional group16
.During
thereactionofDMDSwithpoly-unsaturatedcompounds,diastereomers areformed, this
incontrasttothereactionofDMDSwithmono-unsaturated compounds(§2.3.1).
The derivatisation of (E,Z)-3,7-tetradecadienyl acetate(6) with DMDS is taken as an
exampleoftheformationofacyclicthio-etherwiththetwomethylthiogroupsoutsidethe
tetrahydrothiophenering(figure2.13).
O
A
MeS
* A0 ->
n=2
6
Ö
SMe
Fig.2.13 Expectedproduct2-(l-methylthio-heptan-l-yl)-5-(3-methylthio-ethylpropanoate-3-yl)-tetra-
hydrothiophene(7)fromtheDMDSderivatisationof(E,Z)-3,7-tetradecadienyl acetate(6).
Reagents:a)DMDS,I2,AT.
Themassspectrumofcompound 7isshowninfigure2.14.
183
100
CD
1 50
CD
Œ
125 173
43 85 97
61 i ~
Jul <[ikl|li I||.II.,LL
145
Lu
185
231
223
378 ( M + -
)
233
330
271 283
50 100 150 200 250 300 350 400 450 m/z
Fig.2.14 MassspectrumoftheDMDSderivativeproduct7.
32

44. Chaptertwo
Therelevantfragments arementioned intable2.3.
Table2.3 Majormassspectrometricfragments oftheDMDSderivativeof (E,Z)-3,7-tetradecadienyl
acetate(6),compound7.
m/z
378
330
283
271
223
233
composition
C18H34O2S3
C17H31O2S2
C16H27O2S
C15H27S2
C14H23S
C10H17O2S2
source
M+-
M+-HSMe
M +
- 2 x S M e
M+
'- SMe- acetate
M+
-- 2xSMe- acetate
RB+
m/z
185
173
125
231
183
145
composition
C9H13O2S
C8H13S2
C7H9S
C12H23S2
C11H19S
C8H17S
source
HB+
-HSMe
RB+-acetate
RB+-HSMe-acetate
BC+
BC+
-HSMe
C+
Again,themolecular ion (M+
)m/z 378isclearlyvisible.Alsothelossofthe methylthio
andacetategroupsfrom theM+
'isobserved.Fragment BCappearstoloseits methylthio
groupveryeasilyand,inthisway,formsthefragment withthehighestintensitym/z183.
FragmentsH,H- acetateand H- SMearenotveryintense.Thesamewasobservedforthe
relatedfragmentsofDMDSderivative2(figure2.9).
Thereactionproductsdepend onthereactionconditions.Iftheconcentration ofiodineis
low, mainly the derivatives are formed as described in figures 2.10, 2.12 and 2.13.
Symmetrical cyclic thio-ethers are then formed exclusively20
. These cyclic thio-ethers
always bridge the two nearest possible carbon atoms. If the iodine concentration is
increaseditappearsthatothercyclicthio-ethersareformed aswell.Thereactionof(Z,Z)-
9,12-tetradecadienylacetate(8)withDMDSinthepresenceofahighiodine concentration
istakenasanexample.Theproposed reactionmechanismthatleadstotwo symmetrical
and thetwonon-symmetrical cyclicthio-ethers isshown infigure 2.15.RoutesIand III
lead to the formation of two symmetrical cyclic thio-ether tetrahydrothiopyran 9 and
thietane11,respectively.TheroutesIIandIVgiverisetothetwonon-symmetrical cyclic
thio-ethers,thetetrahydrothiophenes10and12.
33

45. Massspectrometricanalysisofsexpheromones
X
I
-SMe
n
-SMe
m
*-
k'
0
o
-SMe
-SMe
A0.
Fig.2.15 ProposedmechanismfortheformationofthefourpossibleDMDSderivatives(9-12)from
(Z,Z)-9,12-tetradecadienylacetate(8).Reagents:a)DMDS,I2,AT.
Thegaschromatogramofthereactionproductmixtureisshowninfigure2.16.
.£•
.*CO
d>
Q.
*=O
o
B)
O
1 2
~ir
m
— - — ) ^
I , • r i • . 1 1 1—
5 N
3
^ 4
1
L-
UV
- i , 1 - | " -, , , , 1 , , .- r 1 1 , , 1—
10 15
Time(minutes)
20 25
Fig.2.16 Gaschromatogram of the reaction mixtureof (Z,Z)-9,12-tetradecadienyl acetate (8)with
DMDSathighiodineconcentrations.Detector:massspectrometer.
34

46. Chaptertwo
Peak numbers 1and 2represent derivatives that have reacted in a different way with
DMDS,whicharediscussedlaterinthischapter.Peaknumbers3through6represent the
'normal' cyclic thio-ethers. Themassspectra of these compounds are shown in figures
2.17through2.20.
99
100
CD
c 50
CD
EC
0
81
43 67
i
41
kiJiLuyJliLniLi 1
231 330
111 J 4 7
i 123 i
171
223
4 -
283
+
297
i
378(M+-
)
50 100 150 200 250 300 350 400 450 m/z
Fig2.17 Massspectrumofpeaknumber3fromthegaschromatogramoffigure2.16.
Themassspectrum ofpeaknumber 3shows an intensepeak m/z 231which indicates a
fragmentflascanbeexpectedfrom structures11or12.Thepresenceofthefragments fl -
acetate (m/z 171), H- acetate- SMe (m/z 123), BC (m/z 147) and BC- SMe (m/z 99)
supports this hypothesis. For structure 11,fragments HB (m/z 255) and HB-acetate
(m/z195)areexpected (seealsofigure2.21).Sincethemassspectrum offigure2.17lacks
thesefragments,itisconcludedthatpeaknumber3hasstructure12.
100
c
CD
•g50
a
CD
££
41
97
43 75
,]i
111125
139
195
255
223
i
283
277!297
. . ill . I. .
378(M+
-)
331
50 100 150 200 250 300 350 400
Fig2.18 Massspectrumofpeaknumber4fromthegaschromatogramoffigure2.16.
450 m/z
The massspectrum of peak number 4is dominated by theM+
'(m/z 378) and peaks at
35

47. Massspectrometricanalysisofsexpheromones
m/z 255and m/z 195.The latter two fragments result from the lossofHSMe and HSMe
plusacetatefrom fragment HB(m/z303)possiblyfrom structures10or11.Ifpeak number
4had structure 11,fragments m/z231,m/z Y7 or m/z 123representing fragments fl, fl -
acetate or fl - acetate- HSMe of structure 11 respectively, should be present (see
figure2.21).Because these fragments are not significantly present, it isconcluded that
peak4hasstructure10.
100
V)
c
•£ 50
o
43
41
Hk,
97
iMii
111125
139
i
4>wA'
330
195
255 2 8 3
223
207
297
378(M+
')
50 100 150 200 250 300 350 400
Fig2.19 Massspectrumofpeaknumber5fromthegaschromatogramoffigure2.16.
450 m/z
The massspectrum of peak number 5shows similarities with that of peak number 4
(figure 2.18). It is assumed therefore, that peak number 5 is a diastereomer of peak
number4and,thus,hasstructure10.
100
(fl
c
c 50
tr
99 231
43
-.556
<7
41
97
81
àiiiilliiiii
195
111
125 147 1
71
m
189
'/'''I; 'Ji , i
223
207~]
i
330
283
255
Jt-vi- ui
297
315
378(M+
')
330
50 100 150 200 250 300 350 400
Fig2.20 Massspectrumofpeaknumber6fromthegaschromatogramoffigure2.16.
450 m/z
Themassspectrumofpeaknumber 6possessesallthecharacteristicsofstructure 11.The
fragmentation patternisshowninfigure2.21.
36

48. r~A
m/z231
-HSMe
M+,
378 » •
r *
+ BC
m/z147
| - acetate j - HSMe
m/z171 m/z99
f -HSMe
m/z123
m/z330
-SMe , -acetat«
» m'~°R3
1RB +
(m/z303)
| -HSMe
m/z255
| -acetate
m/z195
Chaptertwo
•*-m/z 223
1C
m/z75
Ftg2.21 Fragmentation pattern for DMDS derivative 11.Fragments between brackets were not
detected.
Forthesymmetrical tetrahydrothiopyran (9)amassspectrum would beexpected domi-
natedbypeaksM+
-- 95(m/z283)andM+
- 95- acetate(m/z223)17
.Becauseno spectrum
withthesecharacteristicscouldbefound,itisassumedthatthisDMDSderivativewasnot
present. The formation of the six membered sulphur containing ring is probably less
favourableunderthereactionconditionsused.
In the DMDS derivatisation reaction mixture of poly-unsaturated compounds,
incompletelyderivatised structurescanbedetected aswell.Thelatterareagain,strongly
dependent onthereactionconditions.Forexample,incaseofthederivatisation of(Z,Z)-
3,8-tetradecadienyl acetate (13) with DMDS, derivatives 14 and 15 (figure 2.22) were
identified inthereactionmixture.
SMe
14
A0-
MeS
15
SMe
Fig.2.22 IncompletelywithDMDSderivatised (Z,Z)-3,8-tetradecadienyl acetate(13)products3,4-
bis-(methylthio)-(Z)-8-tetradecenyl acetate(14)and 8,9-bis(methylthio)-(Z)-3-tetradecenyl
acetate(15).
The massspectra of compounds 14and 15 exhibit M+
peaks at m/z346.Because the
molecularmassoftheoriginalmolecule is252amu,theadditional 94amuoftheDMDS
derivative must havebeen the result of theaddition of two methylthio groups without
ring-closure. Fragment Hof14 (mass spectrum not shown) is similar to that of 2
37

49. Massspectrometryanalysisofsexpheromones
(figure2.9).FragmentBof14carriestwohydrogenslessthanthecorresponding fragment
in2,therefore,themassoffragment Bof14is2amuloweraswell.Themassspectrumof
15isshowninfigure2.23.Therelevantfragments arementioned intable2.4.
100-1
0)
I 50
107 . „ ! 155i 131 i
87
61 67
4 3 ^ r-
41
44y
95
iikiLV
183
346(M+-
)
199
215 o c , 298
j 239r2
-51
283 330
,l,li, il, I ,,ii, I , ,
50 100 150 200 250 300 350
Fig.2.23 MassspectrumoftheincompletelyreactedDMDSderivative15.
400 450 m/z
Table2.4 MassspectrometricfragmentsoftheincompletelyreactedDMDSderivative15.
m/z
346
298
131
composition
C18H34O2S2
C17H28O2S
C7H15S
source
M+
--HSMe
B+
m/z
215
155
107
composition
C11H19O2S
C9H15S
CsHii
source
fl+
fl+
-acetate
H+
-acetate-HSMe
Itwasobservedthatincaseofahomo-conjugated system(doublebondsseparatedbyone
methylene group) another reaction product is formed as well. Because molecular ions
(M+
) of incompletely DMDS-reacted derivatives originating from homo-conjugated
compounds are always well visible21
, the molecular ion peak at m/z 316 of peak 2of
figure 2.16 cannot be explained by assuming that the corresponding molecule is
incompletely derivatised. It seems that this peak represents an excessively reacted
derivative instead. In the product mixture of the derivatisation of (E,Z,Z)-3,8,11-
tetradecatrienylacetate (16) with DMDS, a peak is encountered with similar mass
spectrometric characteristics as peak 2 of figure 2.16.The molecular ion (M+
) of this
compound is detected at a mass of 2amu lower (m/z 314) than that of peak 2 of
figure 2.16. The massspectra of both these DMDS derivatives show several related
fragments (figures 2.24 and 2.25 respectively). It is, therefore, assumed that the
fragmentation patternsofthesetwoDMDSderivativesfollowcomparableroutes.
38

50. Chaptertwo
100-,
S
c
0)
>
cc
50-
43
4
97
85
55
i67
.11,ihllii4
111
a
145
125
i."t.m,», 4
160
316(M+-
)
195
223
255 283
267
i
50 100
i — i — I ' " I n
i
150 200 250 300 350
' i '
400 450 m/z
Fig.2.24 MassspectrumofanwithDMDSunuallyreactedderivativeoriginatingfrom (Z,Z)-9,12-
tetradecadienylacetate(8).(peak2fromthegaschromatogramoffigure2.16).
loo-.
.1 50
01
- 43
41
J j
85 i
67
J+.JUIUJ4
97
113
159
137
l i l , . !'•«
174
ki
221
314(M+-
)
1
r-79
185
207
50 100 150
Mi' f ,'
200
254
251
- i
239
281
250 300 350 400 450 m/z
Fig.2.25 MassspectrumofawithDMDSunusuallyreactedderivativefrom(E,Z,Z)-3,8,ll-tetradeca-
trienylacetate(16).
Ithasbeenconsideredthatthesetwopeakswereincompletelyderivatisedmoleculeswith
undetected molecular ionpeaksat m/z of344and 346respectively.The fragments 316
and 314must then originate from themolecular ionthathaslost afragment of30amu.
Thelossoftwoconsecutivemethylgroups isforbidden bymassspectrometric rules and
aninitiallossofthetwomethylgroupstogetherasoneethanemoleculeunder the direct
formation ofthedi-thio-ether isrejected asunrealistic.Itistherefore proposed that these
two products represent di-thio-ethers which have been formed through a second ring-
closingreactionasillustratedinfigure2.26.
39

51. Massspectrometricanalysisofsexpheromones
or
-Me-S-Me
R-j S R2
2-R2-4-Rr3,6-dithiobicyclo[3.1.1]-heptane
- Me-S-Me 3 ,—'1
4
^S-5 V
R2
3-R1-5-R2-2/5-dithiobicyclo[2.2.1]-heptane
Fig.2.26 Proposedmechanismsfortheformationofdi-thio-ethers.
Theproposedfragmentationpatternsareshowninfigures2.27and2.28respectively.
-Me"
- • m/z145
Fig. 2.27 Proposed massspectrometric fragmentation pattern for the di-thio-ether derivative
originatingfrom(Z,Z)-9,12-tetradecadienylacetate(8).
-Me'
m/z159
m/z223
{ _ C
3H
6
m/z239
- acetate
m/z179
Fig.2.28 ProposedmassspectrometricfragmentationpatternfortheexcessivelywithDMDSreacted
derivativeoriginatingfrom(E,Z,Z)-3,8,ll-tetradecatrienylacetate(16).
40

52. Chaptertwo
Itisnotknownwhether thedi-thio-etherswould havestructurebicyclo [3.1.1]orbicyclo
[2.2.1].Becauseseveralisomersareobserved,probablybothisomersarepresent.Theions
at m/z 160and m/z 145of figure2.24and at m/z 174and m/z 159of figure 2.25could be
related.Becausethelattertwofragments are14amuhigherinmass,theseions originate
then from the co-end of the derivative. The proposed fragmentation routes are not in
contradictionwiththis.
2.3.3 Triple-unsaturated molecules
Straight chain molecules with threedoublebonds reactwith DMDSinasimilar way as
the less unsaturated ones do. Also the formation of cyclic thio-ethers follow the same
rules as mentioned before. The major difference is that the products are more
complicated.Triple-unsaturated compoundshavemorepossibilitiesfor theformation of
diastereomers, consequently the gaschromatogram of the reaction mixture is more
complex.Still,themainproduct formed inthepresenceofalowiodineconcentration,is
thesymmetricalDMDSderivativewithonemethylthiogroupatbothpositionsnexttothe
cyclicthio-ethers(preferablythietanesorthiophenesratherthanthiopyranes).
o
A
O MeS
* A->
(EÄZJ-SÄll-tetradecatrienylacetate(16) 19
A
O MeS
A~>
(E,Z,E)-3,8,12-tetradecatrienylacetate(17)
A-
20
O MeS
A 21
O
Op
ob
< >
SMe
SMe
SMe
;
(E,Z)-3,8,13-tetradecatrienylacetate(18) DMDS,I2,AT
A B C D
Fig.2.29 MainDMDSderivativesformedfromthetriple-unsaturatedcompounds16,17and18.
41

53. Massspectrometricanalysisofsexpheromones
Three structurally related triple-unsaturated acetates: (E,Z,Z)-3,8,ll-tetradecatrienyl
acetate(16), (E,Z,E)-3,8,12-tetradecatrienyl acetate(17)and (E,Z)-3,8,13-tetradecatrienyl
acetate(18)were synthesised (see chapter5),derivatised with DMDS and subjected to
massspectrometricanalysisinordertoseeiftheobtained derivativesare distinguishable
by their MS,and to see if it ispossible to locate all double bond positions. The main
productformed foreachofthesecompoundsisshowninfigure2.29.
Themassspectraof19,20and21areshowninfigures2.30through2.32respectively.
261
100n
0)
c 50
213
139
43
87
75 89
113
41 6 1 'p °?
Llui1414 li^JjyL
50 100
145
161
150
211
1871
?9
253
247
225
361
273
r
301313
i.M.
200 250 300 350
408 (M+
')
J^J400 450 m/z
Fig.2.30 MassspectrumoftheDMDSderivative19originatingfrom (E,Z,Z)-3,8,ll-tetradecatrienyl
acetate.
100
50
a
(D
rr
213
113
4
P 75
87
41
4M
89
139
ii.ill,,. LI|I.I.^,
147r
161
199
187:
4-V
261
225247
360
285
273!301
i
LL
313
i
333
50 100 150 200 250 300 350
408(M+
")
400 450 m/z
Fig.2.31 MassspectrumoftheDMDSderivative20originatingfrom(E,Z,E)-3,8,12-tetradecatrienyl
acetate.
42

54. Chaptertwo
450 m/z
Fig.2.32 Massspectrum oftheDMDSderivative21originatingfrom (Z,Z)-3,8,13-tetradecatrienyl
acetate.
All three DMDS derivatives have the same configuration with respect to the first two
doublebonds.Theexpected,andforthispartofthemoleculecharacteristic,fragments are
mentioned in table2.5.The expected fragments for the distinguishing differences are
shownintable2.6.
Table2.5 SpecificmassspectrometricfragmentssharedbyallthreeDMDSderivatives19,20and21.
m/z
408
360
313
301
253
247
199
187
composition
C18H32O2S4
C17H28O2S3
C16H25O2S2
C15H25S3
C14H21S2
C11H19O2S2
CioH1 5 02 S
C9H15S2
source
M+
-
M+
-- HSMe
M+
-- 2xSMe
M+
'- acetate- SMe
M+-- acetate- 2xSMe
RB+
RB+
-HSMe
BB+-acetate
m/z
139
147
99
87
261
213
161
113
composition
CsHiiS
C6H11O2S
C5H7O2
C4H7S
C12H21S3
C11H17S2
C7H13S2
C6H9S
source
HB+-HSMe-acetate
fl+
H+
- HSMe
fl+
- acetate
BCD+
BCB+
-HSMe
CD+
CD+
-HSMe
Thedistinguishing fragments for 20and 21are well visible in their massspectra (table
2.6).However, for 19 the key fragments are not very obvious, or are detected at low
intensities.TheratioofthepeakintensitiesofCD- HSMe:CDis10-15forcompound 19
butonlybetween2.6and4.5for theothertwocompounds 20and 21.Thishigh intensity
ratio(>6.5)offragments originating from theco-sideofthemoleculehasalsobeen found
for DMDSderivatives originating from (Z,Z)-9,12-tetradecadienyl acetate(8),(Z,Z)-3,6-
hexadecadienylacetate(22)and(E,Z,Z)-4,7,10-tridecatrienylacetate(23),butnotinDMDS
43

55. Massspectrometricanalysisofsexpheromones
derivatives of, for example,(E,Z)-3,7-and (Z,Z)-3,8-tetradecadienyl acetate (6and13
respectively).Themolecules8,22and23havethesamehomo-conjugated doublebond
systemwhich isabsent in6and13.Itappearsthatthisintensity ratiocanbeused to
discriminate between the DMDSderivatives originating from compounds with and
withouthomo-conjugationintheirdoublebondsystem.
Table2.6 Distinguishing massspectrometric fragments which areexpected for each of the three
DMDSderivatives19,20and21.
m/z composition source
Distinguishing fragments for 19
319
271
C14H23O2S3
C13H19O2S2
RBC+
HBC+
-HSMe
Distinguishing fragments for 20
333
285
C15H25O2S3
C14H21O2S2
HBC+
HBC+
-HSMe
Distinguishing fragments for 21
347
299
C16H27O2S3
C15H23O2S2
HBC+
HBC+
-HSMe
m/z
259
211
89
273
225
75
287
239
61
composition
C12H19S3
C11H15S2
C4H9S
C13H21S3
C12H17S2
C3H7S
C14H23S3
C13H19S2
C2H5S
source
HBC+
-acetate
HBC+-acetate-HSMe
D+
HBC+-acetate
HBC+-acetate -HSMe
D+
HBC+-acetate
HBC+-acetate-HSMe
D+
Itisstrikingthattherelativeintensitiesofthekeyfragmentscanchangeconsiderablyif
themassspectraof19,20and21arerecordedonaquadropolemassspectrometer.This
typeofmassspectrometerpromotestheoccurrenceinthemassspectrogramoffragments
withlowermasses(m/z<100).Themassspectraof19,20and21recordedonaquadropole
301 313 34«361
•' /' •' i ' •
408 (M+
')
300 350 400 450 m/z
Fig.2.33 MassspectrumoftheDMDSderivative19takenonaquadropolemassspectrometer.
44

56. Chaptertwo
massspectrometerareshowninfigures2.33through2.35.
75,
213
225 247i
261 285
^M^
313 360 408(M+
")
r • i ' 'i i i i |
50 100 150 200 250 300 350 400 450 m/z
Fig.2.34 MassspectrumoftheDMDSderivative20takenonaquadropolemassspectrometer.
61
100
113
CD
50-
213
139
161
i 187125
MiJwii J..,i,.1,
199
r
261
247 I 301 348 361 408 (M+-
)
50 100 150 200 250 300 350 400 450 m/z
Fig.2.35 MassspectrumoftheDMDSderivative21takenonaquadropolemassspectrometer.
The key fragments Dof compounds 20 and 21(m/z 75and m/z 61respectively), have
become the 100%intensity peaks. The relative intensity of fragment D (m/z 89)of19,
although not the 100% peak, is significantly higher in comparison to the relative
intensitiesofthisfragment inthemassspectra of 20and 21.Itistakenintoaccount that
the relative intensity of the isotope peak of fragment m/z 87, due to the presence of
sulphur (relative intensity 34
S=4.21%)22
,ispredominantly responsible for the relative
intensitiesofthefragments m/z89incaseofcompounds20and21.
45

57. Massspectrometricanalysisofsexpheromones
2.3 Conclusionsand discussion
TheDMDSderivatisation reacted isvery useful for theanalysisof sexpheromones and
related compounds. Thisanalytical approach hasproven tobe of major importance for
the identification of the sex pheromone compounds of Symmetrischema tangolias and
Scrobipalpuloides absoluta, the latter one in particular (chapter5). The derivatisation
reaction can be scaled down to sub-microgram levels and still provide sufficient
information for thedetermination ofthedoublebond positions.An extrabenefit of this
approachisthattherawbiologicalstartingmaterialdoesnothavetobeextracted before
the derivatisation reaction with DMDS.For the identification of the sex pheromone of
Scrobipalpuloidesabsoluta,sexpheromone glandswere directly collected inDMDS which
wasalsousedforthederivatisation reaction(chapter5).Itappearspossibleto determine
the position of three double bonds in sex pheromone like structures through DMDS
derivatisation ofthecompounds followed bymassspectrometric analysis.Tothebestof
knowledge, thisfact and aneverbefore reported typeof di-thio-ether which is formed
from homo-conjugated sexpheromone compounds. Thedetection of such structures is,
therefore,astrongindicationofthepresenceofahomo-conjugated systemintheoriginal
molecule.
46

58. Chaptertwo
2.4 References andnotes
1. Francke, W., Franke, S., Tóth, M., Szöcs, G., Guerin, P. and Arn, H. 1987.
Identification of5,9-dimethylheptadecaneasasexpheromoneofthemothLeucoptera
scitella.Naturwissenschaften,74,143-144.
2. Bierl-Leonhardt,B.A.,DeVilbiss,E.D.and Plimmer,J.R. 1980.Location of double-
bond position in long-chain aldehydes and acetates by mass spectral analysis of
epoxidederivatives,ƒ.Chromatogr.Sei.,18,364-367.
3. Baker, R., Bradshaw, J.W.S. and Speed, W. 1982. Methoxymercuration-
demercurationandmassspectrometry intheidentification ofthesexpheromoneof
Panolisflammea,thepinebeautymoth.Experientia,38,233-234.
4. Horiike, M. and Hirano, C. 1982. Identification of double bond positions in
dodecenyl acetates by electron impact mass spectrometry. Agric. Biol.Chem., 46,
2667-2672.
5. Kuwahara,Y.,Yonekawa,Y.,Kamikihara,T.and Suzuki,T.1986.Identification of
the double bond position in insect sex pheromones by mass spectroscopy; Trial
comparison on methyl undecenoates with the natural pheromone of the varied
carpetbeetle.Agric.Biol.Chem.,50,2017-2024.
6. Borchers, F.,Levsen, K., Schwarz, H., Wesdemiotis, C. and Winkler, H.U. 1977.
Isomerization oflinear octenecationsinthegasphase,].Am. Chem.Soc, 99,6359-
6365.
7. Ando, T., Katagiri, Y. and Uchiyama, M. 1985. Mass spectra of dodecadienic
compounds with aconjugated doublebond,lepidopterous sexpheromones.Agric.
Biol.Chem.,49,413-421.
8. Ando, T.,Takigawa, M. and Uchiyama, M. 1985.Mass spectra of deuterated sex
pheromoneswithaconjugated dienesystem.Agric.Biol.Chem.,49,3065-3067.
9. Ando, T., Ogura, Y.and Uchiyama, M. 1988.Mass spectra of lepidopterous sex
pheromoneswithaconjugated dienesystem.Agric.Biol.Chem.,52,1415-1423.
10. Seol, K.Y., Honda, H., Usui, K., Ando, T. and Matsumoto, Y. 1987. 10,12,14-
Hexadecatrienyl acetate:Sexpheromoneofthemulberrypyralid,Glyphodespyloalis
Walker(Lepidoptera:Pyralidae).Agric.Biol.Chem.,51,2285-2287.
11. March,J.1992.Advanced OrganicChemistry:Reactions,Mechanisms,and Structure.
JohnWhiley&Sons,NewYork.1495pp.
12. Yruela,I.,Barbe,A.andGrimait,J.O.1990.Determination ofdoublebond position
and geometryinlinearand highlybranched hydrocarbons and fatty acidsfrom gas
chromatography-mass spectrometry of epoxides and diols generated by
stereospecific resinhydration,].Chromatogr.Sei.,28,421-427.
47

59. Massspectrometryanalysisofsexpheromones
13. Brauner,A.,Budzikiewicz,H.and Boland,W.1982.Studiesinchemical ionization
mass spectroscopy. V—Location of homoconjugated triene and tetraene units in
aliphaticcompounds.Org.MassSpectrom.,17,161-164.
14. Budzikiewicz,H.,Blech,S.and Schneider,B.1991.Investigationofaliphatic dienes
bychemicalionizationwithnitricoxide.Org.MassSpectrom.,26,1057-1060.
15. Buser,H.,Arn,H.,Guerin,P.and Rauscher,S.1983.Determinationofdouble bond
position inmono-unsaturated acetatesbymassspectrometry ofdimethyl disulfide
adducts.Anal.Chem.,55,818-822.
16. Vincenti,M.,Guglielmetti, G., Cassani, G. and Tonini,C. 1987.Determination of
double bond position in diunsaturated compounds by mass spectrometry of
dimethyldisulfidederivatives.Anal.Chem.,59,694-699.
17. Carballeira, N.,Shalabi,F.and Cruz,C. 1994.Thietane, tetrahydrothiophene and
tetrahydrothiopyranformation inreactionofmethylene-interrupted dienoateswith
dimethyldisulfide.TetrahedronLett.,35,5575-5578.
18. Carlson, D.A., Roan, C , Yost, R.A. and Hector, J. 1989. Dimethyl disulfide
derivatives of long chain alkenes, alkadienes, and alkatrienes for gas chromato-
graphy/massspectrometry.Anal.Chem.,61,1564-1571.
19. Thischapter and16
.
20. Ownexperience and16
.
21. Yamamoto,K.,Shibahara,A.,Nakayama, T.and Kajimoto,G.1991.Determination
ofdouble-bond positionsinmethylene-interrupted dienoicfatty acidsbyGC-MSas
theirdimethyldisulfideadducts.Chem.Phys.Lipids,60,39-50.
22. Hesse,M.,Meier,H.and Zeeh,B.1991.SpektroskopischeMethodeninderorganischen
Chemie.GeorgThiemeVerlag,Stuttgart.336pp.
48

60. Chapter 3
Isolation,identification and synthesisofthesexpheromone of
Symmetrischema tangolias*
3.1 Introduction
Today,thepotatotubermoth,Symmetrischematangolias(Gyen)(figure1.3)isconsidered
themostimportantpestofpotatoesinPeru1
andisrecognisedasapestinneighbouring
countries.Thelarvaeofthismothminethestemsofpotatoplantscausingthemtobreak
and die.Inpotatostoragefacilities,larvaeoften boreintopotato tubersmaking them
unsuitableforhumanconsumption.Incontrasttoseed-potatoes,whichareprotectedby
largeamountsofchemicals,consumer-potatoesareunprotectedandthus,veryvulnerable
tothismoth.Lossesupto100%arecausedbythispest2
.
Theuseofasexpheromoneinthecontrolofapestpopulationprovedtobeveryeffective
withPhthorimaeaoperculella(Zeiler)3
whichiscloselyrelatedtoSymmetrischema tangolias.
Therefore,itisexpectedthatthesexpheromoneofSymmetrischema tangoliasmightbe
usefulinthecontrolofthispestaswell.Forthisreasonaprojectwasinitiatedtoidentify
thesexpheromoneofSymmetrischematangolias.
3.2 Methodsandmaterials
Insects
ThelaboratorycultureofSymmetrischematangoliaswasstarted from pupaewhichwere
collectedinastorehouseforpotatoesinCajamarca,Peru,inNovember1991.Themoths
wererearedonpotatotubers(cv.Bintje)underthefollowingconditions,22±1°Catday
and 17±1°C at night, 65±5%relative humidity, and a 12L:12Dphotoperiod. The
potatoeswereprovidedwithsmallpunchedholesinwhichthefemalescouldlaytheir
eggs.
Thischapterisbasedonthefollowingpaper:Griepink,F.C.,vanBeek,T.A.,Visser,J.H.,Voerman,S.and
deGroot,Ae.1995.J.Chem.Ecol.,21,2003-2013.
49