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Purification, Stereoisomeric Analysis and Quantification of Biologically Active Compounds in Extracts from Pine Sawflies, African Butterflies and Orchid Bees
Mid Sweden University, Faculty of Science, Technology and Media, Department of Natural Sciences, Engineering and Mathematics. (Organisk kemi)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Stereochemistry plays an important role in nature because biologically important molecules such as amino acids, nucleotides and sugars, only exist in enantiomerically pure forms. Semiochemicals carry messages, between the same species (pheromones) and between different species (allelochemicals). Both pheromones and allelochemicals can be used as environmentally friendly pest management. Many semiochemicals, i.e. behaviour modifying chemicals, consist of pure or well-defined mixtures of stereoisomers, where some of the other stereoisomers can be repellent. It is therefore important to be able to separate them to produce a synthetic pheromone in a mixture that is attractive.

Pine sawflies are a family of insects that in some cases can be severe defoliators of conifer trees. Diprion pini, Diprion similis and Neodiprion sertifer are severe pests for these trees and have got the most attention in pine sawfly pheromone studies. The pheromone precursors are stored in the female body as long-chain secondary alcohols, which, when released, are esterified to acetates or propionates. The alcohols are chiral, and normally one of the stereoisomer is the main pheromone component, sometimes possible together with other stereoisomers as essential minor components.

Bicyclus is a genus of African butterflies, and especially Bicyclus anynana has become a popular model for the study of life history evolution, morphology, mating choice and genetics. The wing pattern of Bicyclus differs depending on the season, with large eyespots during the rain-season and small or absent spots during the dry season.

 Euglossa is one of the genera among the orchid bees in the Neotropics that does not produce its own pheromone. Instead, the males collect fragrances from orchids and other sources and store them in a pocket in their hind legs. Both Bicyclus and Euglossa use semiochemicals similar to pine sawflies, and thus can be analysed by the same methods.

Pheromones and other semiochemicals in insects are often present in low amounts in a complex matrix, and purification of the sample before chemical analysis is often required. A common method is gradient elution on a solid phase silica column. Separation of stereoisomers can be achieved either by using a column with a chiral stationary phase (CSP) or with pre-column derivatisation using a column with an achiral stationary phase (ASP) or a combination of both, with mass detection as the dominant detection method. The purpose of this work has been to improve the purification method, find suitable methods to separate the stereoisomers of secondary alcohols, and to apply this on extracts of insects.

By selecting the right fractions to collect during gradient elution the purification method was optimised. To reduce plasticizer contamination from ordinary columns, solid phase columns of Teflon or glass were used. For pre-column derivatisation of different chiral alcohols various acid chlorides were tested. For the pine sawfly pheromone precursors enantiopure (2S)-2-acetoxypropionyl chloride was the best choice. To separate some of the stereoisomers achiral 2-naphthoyl chloride was used. For derivatisation of 6,10,14-trimethylpentadecan-2-ol (R)-trans-chrysanthemoyl chloride was the best choice. The derivatised alcohols were separated on different columns, both chiral and non-chiral. Varian FactorFour VF-23ms was chosen as a general-purpose column, the Agilent HP-88 column was the best column with an ASP of those tested, and the Chiraldex B-PA column (CSP) was the only one that could separate all eight stereoisomers of derivatised 3,7-dimethylundecan-2-ol, 3,7-dimethyldodecan-2-ol, and 3,7-dimethyltridecan-2-ol.

To determine the stereoisomeric purity of standard solutions used in field experiments and extracts of different species of insects the optimised methods were applied. For extracts from B. anynana, Euglossa and Neodiprion lecontei this work describe the first determination of the stereochemistry of some of their semiochemicals.

For the determination of the stereochemistry of chiral semiochemicals the methods for purification and separation presented herein have shown to be of great value. The results will hopefully contribute to a better understanding of the communication among insects, and ultimately to a more environmentally friendly pest control.

Abstract [sv]

Många naturligt förekommande kemiska ämnen finns som två spegelbilder av varandra, ungefär som höger och vänster hand. Dessa kan ha helt olika egenskaper och det är därför viktigt att kunna separera dem. Insekter och andra djur använder olika doftämnen för att kommunicera med varandra, om det är inom samma art kallas de för feromoner. De kan bestå av ett ämne eller en blandning av flera. Dessa doftämnen kan man även använda för att på ett miljövänligt sätt bekämpa skadeinsekter. En fälla med syntetiskt feromon för en viss insekt lockar endast till sig den arten, medan alla andra är opåverkade. Eftersom dessa ämnen ofta finns som spegelbilder där kanske bara den ena är aktiv och den andra rent av frånstötande, måste man kunna separera dem för att framställa ett syntetiskt feromon som är attraktivt.

Målet med detta arbete har varit att bestämma feromonet hos olika arter av tallsteklar som kan vara svåra skadedjur på tallskog. De metoder som tagits fram har även tillämpats på några arter av afrikanska fjärilar samt orkidébin från Centralamerika eftersom de använder snarlika doftämnen.

Att få fram feromonet från en insekt är lite som att leta efter in nål i en höstack eftersom de ofta bara innehåller några miljarddels gram per individ. Provet behöver först renas, och en del av arbetet i det här projektet har gått ut på att ta fram en lämplig reningsmetod. Huvudfokus har dock varit på att ta fram metoder som kan separera och identifiera det eller de ämnen, och spegelbilder av dessa, som doftämnena består av. När lämpliga metoder tagits fram har extrakt av olika insektsarter analyserats. I några fall är det första gången som deras feromon bestämts i detalj. Resultaten kan förhoppningsvis bidra till en ökad kunskap om insekters sätt att kommunicera, och i slutändan till miljövänligare bekämpning av skadeinsekter.

Place, publisher, year, edition, pages
Sundsvall: Mittuniversitetet , 2011. , 66 p.
Series
Mid Sweden University doctoral thesis, ISSN 1652-893X ; 116
Keyword [en]
Semiochemicals, sex pheromone, pine sawflies, Bicyclus, Euglossa, chiral separation, derivatisation, GC-­MS
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:miun:diva-14662ISBN: 978-­91-­86694-­58-­6 (print)OAI: oai:DiVA.org:miun-14662DiVA: diva2:452872
Public defence
2011-10-28, O111, Sundsvall, 10:15 (Swedish)
Opponent
Supervisors
Available from: 2011-11-01 Created: 2011-10-31 Last updated: 2012-08-02Bibliographically approved
List of papers
1. The Male Sex pheromone of the Butterfly Bicyclus anynana: Towards an Evolutionary Analysis
Open this publication in new window or tab >>The Male Sex pheromone of the Butterfly Bicyclus anynana: Towards an Evolutionary Analysis
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2008 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 3, no 7, e2751- p.Article in journal (Refereed) Published
Abstract [en]

Background: Female sex pheromones attracting mating partners over long distances are a major determinant of reproductive isolation and speciation in Lepidoptera. Males can also produce sex pheromones but their study, particularly in butterflies, has received little attention. A detailed comparison of sex pheromones in male butterflies with those of female moths would reveal patterns of conservation versus novelty in the associated behaviours, biosynthetic pathways, compounds, scent-releasing structures and receiving systems. Here we assess whether the African butterfly Bicyclus anynana, for which genetic, genomic, phylogenetic, ecological and ethological tools are available, represents a relevant model to contribute to such comparative studies.

Methodology/Principal Findings: Using a multidisciplinary approach, we determined the chemical composition of the male sex pheromone (MSP) in the African butterfly B. anynana, and demonstrated its behavioural activity. First, we identified three compounds forming the presumptive MSP, namely (Z)-9-tetradecenol (Z9-14:OH), hexadecanal (16:Ald) and 6,10,14-trimethylpentadecan-2-ol (6,10,14-trime-15-2-ol), and produced by the male secondary sexual structures, the androconia. Second, we described the male courtship sequence and found that males with artificially reduced amounts of MSP have a reduced mating success in semi-field conditions. Finally, we could restore the mating success of these males by perfuming them with the synthetic MSP.

Conclusions/Significance: This study provides one of the first integrative analyses of a MSP in butterflies. The toolkit it has developed will enable the investigation of the type of information about male quality that is conveyed by the MSP in intraspecific communication. Interestingly, the chemical structure of B. anynana MSP is similar to some sex pheromones of female moths making a direct comparison of pheromone biosynthesis between male butterflies and female moths relevant to future research. Such a comparison will in turn contribute to understanding the evolution of sex pheromone production and reception in butterflies.

National Category
Other Basic Medicine
Identifiers
urn:nbn:se:miun:diva-8149 (URN)10.1371/journal.pone.0002751 (DOI)000264302900022 ()2-s2.0-50249165893 (Scopus ID)
Available from: 2009-01-09 Created: 2009-01-09 Last updated: 2017-12-14Bibliographically approved
2. Field Response of Male Pine Sawflies, Neodiprion sertifer (Diprionidae), to Sex Pheromone Analogs in Japan and Sweden
Open this publication in new window or tab >>Field Response of Male Pine Sawflies, Neodiprion sertifer (Diprionidae), to Sex Pheromone Analogs in Japan and Sweden
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2010 (English)In: Journal of Chemical Ecology, ISSN 0098-0331, E-ISSN 1573-1561, Vol. 36, no 9, 969-977 p.Article in journal (Refereed) Published
Abstract [en]

The pine sawfly Neodiprion sertifer (Geoffroy) uses the acetate or propionate of (2S,3S,7S)-3,7-dimethyl-2-pentadecanol (diprionol) as pheromone components, with the (2S,3R,7R)-isomer being antagonistic, synergistic, or inactive according to the population tested. In this study, we tested the attraction of males to the acetates of three analogs of diprionol, each missing one methyl group, viz. (2S,7S)-7-methyl-2-pentadecanol, (2S,6S)-2,6-dimethyl-1-tetradecanol, and (2S,3S)-3-methyl-2-pentadecanol. None of the analogs alone, or in combination with diprionol acetate, was attractive in Sweden, even at 100 times the amount of diprionol acetate attractive to N. sertifer. In Japan, the acetate of (2S,3S)-3-methyl-2-pentadecanol attracted males when tested in amounts 10–20 times higher than the acetate pheromone component. The acetate esters of the (2S,3R)-analog and the (2S,3R,7R)-isomer of diprionol also were tested in combination with the pheromone compound (acetate ester). Both compounds caused an almost total trap-catch reduction in Sweden, whereas in Japan they appear to have relatively little effect on trap capture when added to diprionol acetate. Butyrate and iso-butyrate esters of diprionol were unattractive to N. sertifer in Sweden. In summary, there exists geographic variation in N. sertifer in responses to both diprionyl acetate and some of its analogs.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:miun:diva-11946 (URN)10.1007/s10886-010-9834-y (DOI)000281971400006 ()2-s2.0-77956886375 (Scopus ID)
Available from: 2010-09-01 Created: 2010-09-01 Last updated: 2017-12-12Bibliographically approved
3. (6R,10R)-6,10,14-Trimethylpentadecan-2-one, a Dominant and Behaviorally Active Component in Male Orchid Bee Fragrances
Open this publication in new window or tab >>(6R,10R)-6,10,14-Trimethylpentadecan-2-one, a Dominant and Behaviorally Active Component in Male Orchid Bee Fragrances
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2010 (English)In: Journal of Chemical Ecology, ISSN 0098-0331, E-ISSN 1573-1561, Vol. 36, no 12, 1322-1326 p.Article in journal (Refereed) Published
Abstract [en]

6,10,14-Trimethylpentadecan-2-one (Hexahydrofarnesyl acetone; HHA) previously has been found to be a major component in tibial fragrances of male orchid bees, Euglossa spp. HHA is a chiral molecule with four possible stereoisomers, (6R,10R)-, (6R,10S)-, (6S,10R)-, and (6S,10S)-6,10,14-trimethylpentadecan-2-one. In the present study, we characterized HHA extracted from Euglossa as the pure enantiomer (6R,10R)-6,10,14-trimethylpentadecan-2-one. During bioassays in Mexico and Panama, the synthetic RR-isomer attracted males of six species of orchid bees, including three that were known to contain HHA in their tibial fragrances. Possible sources of HHA for wild bees are flowers of euglossophilous orchids and aroids. With a molecular weight of 268, HHA is the largest natural molecule known to attract male orchid bees in pure form. Its attractiveness to males suggests that low-volatility compounds have a function in male signals, e.g., serve as a "base note" in complex odor bouquets. © 2010 Springer Science+Business Media, LLC.

Keyword
(6R, 10R)-6, 10, 14-trimethylpentadecan-2-one; Euglossa; Euglossini; Hexahydrofarnesyl acetone; Hymenoptera; Pheromone; Stereoisomer; Synthesis
National Category
Chemical Sciences
Identifiers
urn:nbn:se:miun:diva-12203 (URN)10.1007/s10886-010-9873-4 (DOI)000285986500006 ()2-s2.0-78650518354 (Scopus ID)
Available from: 2010-11-03 Created: 2010-11-03 Last updated: 2017-12-12Bibliographically approved
4. Purification, Stereoisomeric Analysis and Quantification of Sex Pheromone Precursors in Female Whole Body Extracts from Pine Sawfly Species
Open this publication in new window or tab >>Purification, Stereoisomeric Analysis and Quantification of Sex Pheromone Precursors in Female Whole Body Extracts from Pine Sawfly Species
2011 (English)In: Journal of Chemical Ecology, ISSN 0098-0331, E-ISSN 1573-1561, Vol. 37, no 1, 125-133 p.Article in journal (Refereed) Published
Abstract [en]

A GC-MS method to analyze the stereoisomeric composition of chiral secondary alcohols found in whole body extracts of pine sawfly females was developed. The tested alcohols were derivatized with optically pure (S)-2-acetoxypropionyl chloride prior to GC-MS analysis. Baseline separation was obtained for all sixteen stereoisomers of 3,7,9-trimethyltridecan-2-ol and for the four 3-methylpentadecan-2-ol stereoisomers. For 3,7-dimethyltridecan-2-ol, 3,7-dimethyltetradecan-2-ol and 3,7-dimethylpentadecan-2-ol baseline separation was obtained for 6 of the possible 8 stereoisomers. When a mixture of 16 stereoisomers of 3,7,11-trimethyltridecan-2-ol was tested, baseline separation of 7 peaks out of 16 possible was obtained. The investigated alcohols are pheromone precursors for some pine sawfly species that are severe defoliators of pine. Females from several Diprion, Neodiprion, Macrodiprion, Microdiprion, and Gilpinia species emit esters of such secondary alcohols as sex pheromones that attract males for mating. To quantify the small amounts of the precursor alcohol and its stereoisomeric composition found in whole body extracts from female pine sawflies, a purification method was optimized. An extract of 20 females of D. pini contained about 8 ng of (2 S,3 R,7 R)-3,7-dimethyltridecan-2-ol per female, and three extracts of 18, 20, and 90 females of N. sertifer contained between 5 and 13 ng of (2 S,3 S,7 S)-3,7-dimethylpentadecan-2-ol per female.

Keyword
3, 7, 11-trimethyltridecan-2-ol; 3, 7, 9-trimethyltridecan-2-ol; 3, 7-dimethylpentadecan-2-ol; 3, 7-dimethyltetradecan-2-ol; 3, 7-dimethyltridecan-2-ol; 3-methylpentadecan-2-ol; Analysis; Diprion pini; GC-MS; Gilpinia frutetorum; Gilpinia pallida; Gilpinia socia; Macrodiprion nemoralis; Microdiprion pallipes; Neodiprion sertifer; Pine sawflies; Sex pheromone; Stereoisomers
National Category
Chemical Sciences
Identifiers
urn:nbn:se:miun:diva-12640 (URN)10.1007/s10886-010-9886-z (DOI)000286673900013 ()21110221 (PubMedID)2-s2.0-79251645596 (Scopus ID)
Available from: 2010-12-13 Created: 2010-12-13 Last updated: 2017-12-11Bibliographically approved
5. Sex Pheromone of the Introduced Pine Sawfly, Diprion similis (Diprionidae), Revisited: No Activity of Earlier Reported Synergists
Open this publication in new window or tab >>Sex Pheromone of the Introduced Pine Sawfly, Diprion similis (Diprionidae), Revisited: No Activity of Earlier Reported Synergists
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Extracts of Diprion similis females were found to contain about 15 ng of the sex pheromone precursor 3,7-dimethylpentadecan-2-ol per female. When analysing the extracts after derivatisation with (2S)-2-acetoxypropionyl chloride, we found that the major stereoisomer in the extract was (2S,3R,7R)-3,7-dimethylpentadecan-2-ol. Also other stereoisomers of 3,7-dimethylpentadecan-2-ol were identified in the extract namely, 1% of (2R,3S,7S) , 0.3% (2R,3R,7R) and 0.4% of (2R,3R,7S). An unknown fifth substance showed an identical spectrum to 3,7-dimethylpentadecan-2-ol, both in SIM and full scan mode. In field tests in Ontario, Canada, the earlier identified main pheromone component, viz. the propanoate of (2S,3R,7R)-3,7-dimethylpentadecan-2-ol, was tested alone and in combination with other stereoisomers, earlier reported to be synergistic. In none of the tests were any synergistic effects detected and the threo four-isomer blend was as attractive as the pure main compound. Thus, one of the few examples of a diprionid sawfly using more than one substance in its sex pheromone could not be confirmed. The results also suggest that monitoring programs can use the more easily synthesized threo-blend without losing efficiency.

Keyword
Hymenoptera, Symphyta, Semiochemical, Attractant, Chemical analysis, Gas-chromatography, Mass-spectroscopy, Pheromone trap
National Category
Other Chemistry Topics
Identifiers
urn:nbn:se:miun:diva-14659 (URN)
Available from: 2011-10-31 Created: 2011-10-31 Last updated: 2016-09-29Bibliographically approved
6. Stereoisomeric separation of derivatized 2-alkanols using gas chromatography – mass spectrometry: Sex pheromone precursors found in pine sawfly species
Open this publication in new window or tab >>Stereoisomeric separation of derivatized 2-alkanols using gas chromatography – mass spectrometry: Sex pheromone precursors found in pine sawfly species
2012 (English)In: Analytical Letters, ISSN 0003-2719, E-ISSN 1532-236X, Vol. 45, no 9, 1016-1027 p.Article in journal (Refereed) Published
Abstract [en]

Stereoisomers of long-chain secondary alcohols are used as sex pheromone precursors among pine sawflies, which can be severe pest to conifers. To use the pheromone in environmentally friendly pest managements, methods are needed that can determine the exact stereochemistry of the precursor alcohols. Combinations of 11 acid chloride derivatives and 10 GC columns were evaluated for separation of the stereoisomers of 3,7-dimethylundecan-2-ol, 3,7-dimethyldodecan-2-ol, 3,7-dimethyltridecan-2-ol, 3,7-dimethyltetradecan-2-ol, and 3,7-dimethylpentadecan-2-ol. Derivatisation with (2S)-2-acetoxypropionyl chloride in combination with the Chiraldex B-PA column separated all eight stereoisomers of 3,7-dimethylundecan-2-ol, 3,7-dimethyldodecan-2-ol and 3,7-dimethyltridecan-2-ol. A combination of two different derivatisation methods was needed to separate all stereoisomers of the longer chained alcohols, 3,7-dimethyltetradecan-2-ol and 3,7-dimethylpentadecan-2-ol. An extract of the pine sawfly Neodiprion lecontei was also analysed, and the stereochemistry of the sex pheromone alcohol prcursor was determined to be (2S,3S,7S)-3,7-dimethylpentadecan-2-ol, at an amount of about 7 ng/female. This paper presents the first GC-MS-separation of all eight stereoisomers of 3,7-dimethylundecan-2-ol, 3,7-dimethyldodecan-2-ol and 3,7-dimethyltridecan-2-ol in a single analytical run, and also the first determination of the stereochemistry of the sex pheromone precursor of N. lecontei.

Keyword
Stereoisomers, Derivatisation, Secondary alcohols, Sex pheromone, Pine sawflies, Neodiprion lecontei, GC-MS
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:miun:diva-14661 (URN)10.1080/00032719.2012.670789 (DOI)000305209100007 ()2-s2.0-84862506238 (Scopus ID)
Available from: 2011-10-31 Created: 2011-10-31 Last updated: 2017-12-08Bibliographically approved

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