Capturing Volatile Natural Products by Mass Spectrometry

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Capturing volatil

Technische Universitat Braunschweig, Instit

38106 Braunschweig, Germany. E-mail: j.di

Cite this:Nat. Prod. Rep., 2014, 31, 838

Received 28th August 2013

DOI: 10.1039/c3np70080a

www.rsc.org/npr

838 | Nat. Prod. Rep., 2014, 31, 838–86

e natural products by massspectrometry

Jeroen S. Dickschat*

Covering: up to 2013

This review gives a modern methodic overview of how volatile natural products from various sources such

as plants, animals, bacteria and fungi can be trapped and how compound identification can be performed

even in cases of very low yields or within highly complex compound mixtures. A detailed discussion is

presented on how a structural proposal for an unknown analyte can be derived from GC-MS data.

Furthermore, the application of trace analytical techniques in biosynthetic studies with isotopically

labelled compounds is presented, including a discussion of the pros and cons of different kinds of stable

isotope labellings in GC-MS analyses.

1 Introduction2 Sampling techniques3 Interpretation of EI mass spectra3.1 The molecular ion3.2 Fragment ions3.2.1 Fragmentations with the cleavage of one bond3.2.2 Fragmentations with the cleavage of two bonds3.2.3 Multistep fragmentation mechanisms4 Retention time and retention index5 Examples of GC-MS based structure elucidation6 Feeding experiments7 Conclusions and outlook8 Acknowledgements9 References

1 Introduction

Volatiles are produced by nearly every organism and may havevery important functions. A prominent example is the rstpheromone ever reported. To identify the volatile attractantButenandt and coworkers extracted 500 000 pheromone glands,yielding 280 g of crude extract from which they obtained 12 mgof the pure pheromone that was identied by elemental analysisand UV spectrosocopy as a hexadecadienol with conjugateddouble bonds.1 Further structure elucidation by chemicaldegradation2 and synthesis of the four possible stereoisomers3

resulted in the identication of bombykol as (10E,12Z)-hexa-decadien-1-ol.

ut fur Organische Chemie, Hagenring 30,

[email protected]

1

Since the early days, analytical techniques have very muchimproved with basically two revolutionary developments. Thephenomenon of nuclear magnetic resonance was discoveredindependently by Bloch4 and Purcell5 in 1946, for which theyreceived the Noble Prize in Physics in 1952. NMR spectroscopywas made available to chemical laboratories in the 1960s andbecame a standard technology some years later. This techniqueallowed for the rapid structure elucidation of organic mole-cules. Another milestone was the development of the rstfunctional mass spectrometer by Aston in 1920 that he used todemonstrate that most chemical elements exist in form ofisotopes.6 For his pioneering work he was awarded the NobelPrize in Chemistry only two years later. The rst coupled systemof a gas chromatograph with a time-of-ight (TOF) mass spec-trometer was developed in the 1950s by McLafferty and Gohlke.7

Since this time, the technique has been very much improved bythe development of several ionisation techniques, such aselectron impact ionisation (EI), chemical ionisation (CI), fastatom bombardment (FAB), and matrix assisted laser desorptionionisation (MALDI), coupling of mass spectrometers to liquidchromatography (especially HPLC), and various types of massanalyzers including sector eld, TOF, quadrupole, ion trap,Orbitrap, and Fourier transform ion cyclotron resonance(FTICR) mass spectrometers. Details of these developments willnot be presented here, but are given in the accompanying articleby Carter in this themed issue.8

This article aims at demonstrating how the technique of GC-MS, that is in our days available in almost every chemical labo-ratory, can be used to rapidly elucidate the structures of all kindsof volatile natural products without isolating large quantities—as is required for chemical degradations or structure determi-nation by NMR. The most important points brought across in

This journal is © The Royal Society of Chemistry 2014

http://dx.doi.org/10.1039/c3np70080a

http://pubs.rsc.org/en/journals/journal/NP

http://pubs.rsc.org/en/journals/journal/NP?issueid=NP031006

Fig. 1 Schematic view of sampling techniques for volatiles. A) Closed-loop stripping apparatus (CLSA), and B) solid phase micro-extraction(SPME).

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this review are (I) how a structure for an unknown volatile can beproposed based on GC-MS data, and (II) how GC-MS can effi-ciently be used in biosynthetic feeding experiments that aregrounded on the usage of isotopic labellings. Rather than beingcomprehensive which is nearly impossible due to the largenumber of investigations on volatile natural products such as(insect) pheromones, aroma constituents of (ouring) plants,and volatiles from bacteria and fungi, in this article an effort hasbeen made to present a selection of historically interesting andmost recent work. In particular, no attempt has been made tocover the extremely large number of more than 3000 reports onvolatile plant constituents in essential oils.9

Recent previous reviews on volatile natural productssummarise the work on bacterial volatiles,10 plant volatiles,11

halogenated volatiles from marine algae,12 biosynthetic path-ways to volatiles in plants,13 and the importance of volatiles insoil ecosystems.14

2 Sampling techniques

The principle setups of two important sampling techniques forthe collection of volatiles from various biological probes areshown in Fig. 1. The closed-loop stripping apparatus (CLSA) wasoriginally developed by Grob and Zurcher for stripping ofvolatiles from aqueous solutions,15 but can easily be modiedfor the investigation of volatiles released by biologicalsamples,16 such as bacterial or fungal agar plate cultures(Fig. 1A).17,18 Even volatiles from plants,16 insects,16 mites,19 andspiders20 have been collected by use of a CLSA. The method isbased on a closed system in which a continuous air stream iscirculated. The air stream is directed through a small chambercontaining the biological sample and then through a charcoallter for trapping of any emitted volatiles. Aer a collection timeof several hours up to one day the charcoal lter is removed andthe absorbed volatiles are eluted with an organic solvent, e.g.,dichloromethane. The obtained headspace extract can then be

Jeroen S. Dickschat studiedchemistry at TU Braunschweig. In2005 he obtained his PhD underthe guidance of Prof. StefanSchulz, working on bacterialvolatiles and terpene biosyn-thesis as a fellow of the Fonds derChemischen Industrie. Aer apostdoctoral stay at the Uni-versitat des Saarlandes with Prof.Rolf Muller he moved to the lab ofProf. Peter Leadlay at theUniversity of Cambridge (UK) as

a fellow of the Deutsche Akademie der Naturforscher Leopoldina.Since 2008 he is a group leader and Emmy Noether fellow at TUBraunschweig. His research interests include biosynthetic pathwaysto secondary metabolites, the identication and structure elucida-tion of new natural products, and their total syntheses, cumulatingin his habilitation in 2013.


analysed by GC-MS. Since the biological sample is contained ina closed system that is well separated from the environment, theexperiments can be performed without the danger of infectingsamples, such as bacterial agar plate cultures, with microbialcontaminants. This non-invasive technique yields severalmicroliters of extract that can be used for repeated GC-MSanalyses, e.g., on chiral stationary phases, or for derivatisationsby microreactions for structure elucidation. A drawback of themethod is that due to the closed-loop principle a sample can becontaminated by compounds from a previous analytical run,and thus clean blank runs are particularly important.Mechanically damaged charcoal lters should never be used toavoid accumulation of carbon particles in the pump that maycause memory effects.21

An alternative method for the analysis of volatiles frombiological samples is solid phase microextraction (SPME).Originally developed for the analysis of trace compounds inwater,22 SPME is also very useful for headspace samplings.23 Themethod makes use of a poly(dimethylsiloxane) bre as adsor-bant that is presented to the headspace above a biologicalsample in a closed vessel such as an agar plate (Fig. 1B). Aerequilibration, usually within a few minutes, the bre can bewithdrawn into a needle that is directly inserted into the GCinjector where the bre is expelled for thermal desorption of thecollected volatiles. The method is solvent-free, fast, cheap, andnon-invasive. Initially, SPME was mainly used for the analysis ofenvironmental pollutants, food and drug analysis,24 but insectpheromones,25 plant volatiles,26 or volatiles emitted by bacterialliquid and agar plate cultures27,28 can also efficiently be ana-lysed. A drawback of the method is the differential affinities ofvolatiles from varying compound classes for the adsorbentbre.29

3 Interpretation of EI mass spectra

Large electronic databases containing EI mass spectra of manyknown volatile natural products and small synthetic organic

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molecules are available, some of them summarising specialisedknowledge, e.g., the mass spectra of terpenes.30,31 These data-bases are of utmost importance for analytical work on volatilenatural products since they allow for the rapid identication ofmany known compounds. One remaining challenge for thehighly efficient identication of already known compounds inthe future is the development of strong chemoinformatic toolsthat interconnect all the accumulated knowledge from thevarious databases and make it available to all analytical chem-ists. Herein, the chemical sciences can certainly learn frombiology that has been revolutionised during the last decade bythe development of strong bioinformatic tools and large data-bases that today make bioinformatic data available to everyscientist. However, signicant progress in the eld of naturalproducts chemistry can only be made when new compoundscan be identied. The GC/EI-MS technology delivers twodifferent kinds of important information, the mass spectrumand the gas chromatographic retention time, both of whichshould always be considered for the development of a structuralproposal for an unidentied analyte. EI mass spectra are char-acterised by a molecular ion [M+] that arises by collision of ahighly energetic electron (usually 70 eV) and the neutral analyte.The electron impact leaves the ionised analyte in a high energystate that causes a series of fragmentation reactions to give acharacteristic pattern of fragment ions of which the mostintensive fragment ion is referred to as the base peak ion (orbriey “base peak”). A major difficulty, particularly in EI-MS, isthat the molecular ion may be of very low abundance due to itsvery efficient fragmentation, e.g., aliphatic alcohols easilyundergo a neutral loss of water and their mass spectra are verysimilar to the mass spectra of the corresponding olens. Insome cases, the analyte may undergo thermal reactions duringthe chromatographic process, e.g., decarboxylations or Coperearrangements. These difficulties of the GC-EI-MS techniqueshould always be kept in mind to prevent erroneous datainterpretation. In this chapter a choice of interesting exampleswill be presented to show how structural information can beextracted from a mass spectrum. In practice, the successfuldevelopment of such a structural proposal from EI-MS data willrequire an experienced analytical chemist.

Fig. 2 Mass spectra of A) gibepyrone A (1) and B) 2,5-dimethylpyrazine(2) and isotope pattern of the molecular ions.

3.1 The molecular ion

Several important pieces of information can be extracted froman EI mass spectrum. The molecular ion [M+] points towardsthe elemental formula of an analyte. Unambiguous informa-tion about the elemental formula can be concluded from highresolution (HR-MS) data that are, due to recent advances inmass spectrometer developments, of good availability. To givean example, the molecular formulae of acetone (C3H6O) andbutane (C4H10), both resembling a molecular weight of 58 Da,can easily be distinguished from their accurate masses(58.041316 Da versus 58.077601 Da; note that the electronmass, 0.000549 Da, has to be subtracted from the exactmolecular weight since the positively charged molecular ionand not the neutral molecule is detected by HR-MS). A majorlimitation in the interpretation of HR-MS data results from

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the strongly increasing number of possible molecularformulae with increasing molecular weight, but this is usuallynot a problem when analysing volatiles that are per de-nitionem low molecular weight compounds. The most recentinnovations that keep pushing the boundaries of what ispossible in terms of mass resolution include the develop-ments of Fourier transform ion cyclotron resonance massspectrometry (FT-ICR) and Orbitrap mass spectrometry.These techniques are certainly of high relevance for modernmetabolimics and proteomics studies, but only of limitedinterest for studying volatile natural products for which theGC-MS coupling is still the best method. However, the veryinteresting GC-TOF technology may soon become affordableto every chemistry lab for high-resolution MS analyses ofvolatiles.

If no high resolution data are available, every suggestedstructure of an unidentied compound must accord with theobserved molecular ion. The detected isotope pattern of themolecular ion gives very useful information about the elementalformula of an unknown analyte, as will be exemplied here forgibepyrone A (1). Its mass spectrum (Fig. 2A) shows a molecularion at m/z ¼ 164 with an isotope pattern of m/z ¼ 164 (100%),165 (11.2%), and 166 (1.0%). Assuming that the analytecontains only carbon (natural isotope distribution of 12C/13C ¼98.9% : 1.1%), hydrogen (1H z 100%), and oxygen (16O/18O ¼99.8% : 0.2%), the expected isotope patterns for differentelemental formulae matching the molecular ion at m/z ¼ 164can be calculated (Table 1). The best match is obtained forC10H12O2, which is equal to the molecular formula of 1, thusdemonstrating the robustness of the method.



Scheme 1 Polythiocycloalkanes from marine bacteria (3–9) and fromshiitake mushrooms (10–18).

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Other frequently occurring elements in natural productsinclude sulfur, chlorine and bromine, which can also easily berecognised from their isotope patterns (32S/33S/34S ¼95.0% : 0.8% : 4.2%; 35Cl/37Cl ¼ 75.8% : 24.2%; 79Br/81Br ¼50.7% : 49.3%). The characteristic isotope pattern of sulfur wasused to delineate the sulfur content of a series of poly-thiacycloalkanes from marine bacteria (3–9) and from shiitakemushrooms (Lentinus edodes, 10–18) including lenthionine 16(Scheme 1).32,33 Selenium and tellurium compounds exhibiteven more complex isotope patterns that can be very useful inthe identication of volatiles, such as dimethyl diselenide,dimethyl triselenide, or dimethyl ditelluride, which are releasedby some bacteria during growth on selenium or telluriumoxyanions.28,34,35

Considering nitrogen as a component of an unidentiedanalyte complicates the situation, but careful analysis of themolecular ion still delivers useful information about themolecular formula. The presence of an odd number of nitrogenatoms in a natural product containing only the usual elements(C, H, O, N, S, Cl, and Br) is indicated by an odd molecular ion,while no nitrogen or an even number of nitrogen atoms resultsin an even molecular ion (nitrogen rule, this rule is also strictlyvalid for molecules containing F, P, and I, but these elementsoccur very rarely in volatile natural products). Furthermore, thenatural isotope pattern of nitrogen (14N/15N ¼ 99.6% : 0.4%)can be used for delineating the nitrogen content of an unknownvolatile. This isotope pattern leads to slightly decreased [M + 1]+

and [M + 2]+ ions as compared to a hydrocarbon of the samemolecular weight, whereas oxygen results in a slightly decreasedintensity of the [M + 1]+, but increased [M + 2]+ signal (Table 2exemplarily summarises the possible molecular formulae for acompound with a molecular weight of 108 Da containing C, H,N, and O).

An example is given by the mass spectrum of 2,5-dime-thylpyrazine (2, Fig. 2B). From the isotope pattern of themolecular ion the presence of sulfur, chlorine, and bromine canbe excluded. Considering only C, H, and O does not give asatisfactory match of the calculated and measured isotopepatterns (Table 2), suggesting that an even number of nitrogenatoms is contained in the analyte. Indeed, the calculatedisotope pattern for C6H8N2 perfectly agrees with the analyticaldata.

In the case of high quality EI mass spectra, the accuracy ofsuch an in depth analysis of the isotope pattern of themolecularion is very high. The method reaches its limits in the case ofmass spectra with a molecular ion of low intensity, resulting incomparably large measuring errors for the isotope pattern due

Table 1 Calculated isotope patterns for compounds with themolecular ion [M]+ ¼ 164, composed of C, H, and O

Formula m/z ¼ 164 m/z ¼ 165 m/z ¼ 166

C12H20 100% 13.3% 0.8%C11H16O 100% 12.2% 0.8%C10H12O2 100% 11.1% 0.9%C9H8O3 100% 10.0% 1.0%C8H4O4 100% 8.9% 1.1%


to a larger signal-to-noise ratio. Furthermore, in the case ofcompounds of high molecular weight, a large number ofpossible molecular formulae composed of the elements C, H, N,and O matches this particular molecular weight, and conse-quently, the calculated isotope patterns of two or more of theseformulae may be in accordance with the measured data.However, in the case of volatiles that have a molecular weightbelow 300 Da the isotope pattern of the molecular ion gives inmost cases valuable structural information.

3.2 Fragment ions

More detailed structural information can be obtained from thefragment ions that must, however, always be interpreted in thelight of the molecular ion. A very detailed and didacticallyperfectly structured discussion of all kinds of fragmentationreactions with importance for EI mass spectra is summarised ina text book by two leading analytical chemists in the eld,McLafferty and Turecek.36 Some of the most important frag-mentation reactions and their use for interpretations of massspectra will also be presented here.

The large body of information that can be extracted from thefragmentation pattern is one of the major advantages of EI-MScompared to other mass spectrometric methods. In this chapteronly analytes that have an even number of electrons prior toionisation will be discussed, since this is the usual case foralmost all volatile organic molecules. Ionisation proceeds withthe loss of one electron, producing an odd-electron molecularion [M]+c from a non-radical analyte. This initially formedmolecular ion is obtained in a high energy state due to theionisation process that is usually performed at an electronenergy of 70 eV. In consequence, several fragmentation reac-tions lead to a characteristic pattern of fragment ions via wellknown mechanisms. Aer ionisation of a molecule A–B to theinitial (A–B)+c radical cation, fragmentation by the breaking ofone of the bonds results in the fragments Ac and B+ or A+ and Bc,

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Table 2 Calculated isotope patterns for compounds with themolecular ion [M]+ ¼ 108, composed of C, H, N, and O

Formula m/z ¼ 108 m/z ¼ 109 m/z ¼ 110

C8H12 100% 8.8% 0.3%C7H8O 100% 7.7% 0.4%C6H4O2 100% 6.6% 0.6%C6H8N2 100% 7.3% 0.2%C4H4N4 100% 5.8% 0.1%C5H4N2O 100% 6.2% 0.3%

Scheme 2 Fragmentation mechanisms with the cleavage of onebond.

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yielding an even-electron cation and an odd electron radical inboth cases. As has been recognised already in 1968 by Audier,the fragmentation proceeds preferably with the positive chargeremaining on the fragment with the lowest ionisationpotential.37

3.2.1 Fragmentations with the cleavage of one bond

Fragmentation mechanisms by the cleavage of one bond aresummarised in Scheme 2. Ionisation of saturated hydrocarbonsresults in a radical cation with subsequent bond-breaking of theionised s-bond (Scheme 2A). The fragment ions that areobserved with highest intensities are formed by bond breaksnext to chain branches, thus resulting in the most stable, i.e.,tertiary or secondary carbocations. An interesting example isgiven by the doubly alkyl-branched fatty acid methyl ester(FAME) 19. A structural proposal for this compound, that wasobserved in headspace extracts from the actinomycete Micro-monospora aurantiaca, was derived from its EI mass spectrum(Fig. 3A).38 The molecular ion of 19 was very weak, but thepattern of fragmentations of the alkyl chain pointed to amolecular ion atm/z¼ 256. Alkyl chain fragmentations resultedin intense fragment ions of m/z ¼ 87 and 115, but not of m/z ¼101, pointing to a g-methyl branch (the high intensity of the ionm/z ¼ 87 is due to fragmentation via double hydrogen rear-rangement as discussed in chapter 3.2.3, while the fragment ionat m/z ¼ 74 by McLafferty rearrangement is in agreement with amethyl ester, chapter 3.2.2). Furthermore, fragment ions wereobserved at m/z ¼ 157 and 199, but not at m/z ¼ 171 and 185,that seemed to suggest an h-ethyl branch (position 8) that wasfurther corroborated by a weak fragment ion atm/z ¼ 227 ([M �C2H5]

+). However, this is a good example that shows the dangersof a premature interpretation of mass spectra. As was describedby Ryhage and Stenhagen,39 based on extensive labellingexperiments and investigation of various alkyl branched methylesters, the main contribution to the fragment ion at m/z ¼ 199is due to a loss of the C2- to C4-portion, including any attachedalkyl substituents, plus a proton, and not due to the alpha-fragmentation as shown in Fig. 3. Compound 19 was the onlyethyl-branched compound identied among a large number ofother FAMEs emitted by M. aurantiaca and was suggested toarise from 2-ethylhexanoic acid, the oxidation product of thewidespread contaminant 2-ethylhexanol that originates fromplasticisers.

Another important fragmentation mechanism thatfrequently results in fragment ions with high intensities is the

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a-cleavage (Scheme 2B). In this process a C–C single bond of thecarbon bound to a heteroatom X via a C–X single or C]X doublebond is cleaved (a confusing situation of nomenclature is thatby this process the a,b-carbon bond in C–X systems is cleaved,while in C]X systems the bond from the sp2 carbon to thea-carbon is cleaved; this is not equal to cleavage of the a,b-carbon bond). The cleavage is initiated by ionisation at theheteroatom X yielding a radical cation, followed by a cleavagereaction that is driven by recombination of the radical electronwith an electron of an adjacent C–C single bond. This results inthe loss of a radical Rc under formation of a cation that canusually be observed with high intensity.

An example is given by the mass spectrum of 1-phenyldecan-1-one (20), the major volatile compound released by the myx-obacterium Stigmatella aurantiaca Sg a15.40 Fragment ions withhigh intensities are observed at m/z ¼ 77 and 105 that can beexplained by two different a-fragmentations to either the le orthe right side of the carbonyl group (Fig. 3B). The occurrence ofintensive fragment ions at m/z ¼ 120 and m/z ¼ 133 is due to aMcLafferty rearrangement (chapter 3.2.2) and a doublehydrogen rearrangement (chapter 3.2.3).

Several other volatile compounds in which strong a-frag-ment ions have been observed are shown in Fig. 4. The alcohol21 from the myxobacterium Myxococcus xanthus shows ana-fragment ion atm/z¼ 59 as is typical for 3-alcohols.17 Cleavageof the other C–C bond from the carbinol carbon results inm/z¼143. Both fragment ions together point to the molecular ion at[M]+c ¼ 59 + 143 � 30 ¼ 172 (sum of masses of both a-fragmentions�mass of carbinol function that is counted twice) that is incase of aliphatic alcohols usually hard to detect. The methylketone 22, a pheromone from the desert spider Agelenopsisaperta, shows an intensive a-fragment ion at m/z ¼ 43.20



Fig. 3 EI mass spectra of selected volatile secondary metabolites.

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However, interpretation of the expected a-fragment ions ofaliphatic ketones has to be taken with care, because the samefragment ions can arise by cleavage of a C–C s-bond fromaliphatic alkyl chains (i.e., C2H3O

+ and C3H7+ both yield m/z ¼

43). HR-MS data can be used to distinguish between bothpossibilities, while it is not possible in the case of fragment ionsto draw any conclusions from their isotope patterns, as dis-cussed above in detail for the molecular ion, because a contri-bution of other fragment ions to the respective ions cannot beexcluded. The lactone 23 was rst identied from the black-tailed deer Odocoileus hemionus columbianus41 and is alsoreleased as a volatile trace component by the marine bacteriumDinoroseobacter shibae DFL-27.42 Its mass spectrum shows anintensive base peak ion atm/z¼ 85 that is very characteristic forg-lactones and arises from a-cleavage of the g-lactone moiety. Aprominent a-cleavage is also realised in the isomeric lactone 24that is known as buibuilactone, a pheromone component of the

Fig. 4 Volatile compounds showing prominent a-fragment ions intheir EI mass spectra.


cupreous chafer beetle Anomala cuprea.43 Buibuilactone is alsoreleased by D. shibae and the closely related bacterium Lokta-nella. Its a-fragmentation results in a base peak ion of m/z ¼ 111.42

The a-cleavage next to heteroatoms is a very useful frag-mentation reaction for a detailed structure elucidation of vola-tile alkenes. The position of an olenic double bond cannotusually be determined from the mass spectrum of unknownalkenes, but the iodine-catalysed addition of dimethyl disulde(DMDS) yields a 1,2-bis(methylsulfanyl) adduct that showscharacteristic fragment ion formation via a-cleavage of the C–Cbond between the two methylsulfanyl groups (Scheme 3). Themethod was rst reported for the localisation of olenic doublebonds in simple alkenes44 and was subsequently also success-fully applied to determine double bond positions in function-alized molecules such as unsaturated acetate esters,45

alcohols,46 aldehydes,46 ketones,47,48 fatty acids,49 methylesters,46,50–53 lactones,54 and terpenes.55 Quantities as low as10 ng are sufficient for the procedure.46 It has been pointed outthat the derivatization proceeds as an anti-addition, therebyresulting in the formation of threo and erythro adducts from Zand E olens, respectively, that can be gas-chromatographicallyseparated.50 However, since reference DMDS adducts will berequired for stereochemical assignments by means of GC-MS,and these will have to be obtained from the respective Z and Eolens, determination of the double bond conguration seemsto be more straightforward by direct comparison of thegeometrical isomers of the alkenes.

Only a few exemplary cases of double bond localizations byDMDS addition will be specically discussed here, includingthe identication of the female sex pheromone from the currantstem girdler Janus integer, (Z)-octadec-9-en-4-olide (25, Scheme3A).54 The mass spectrum of the DMDS adduct 26 showedintensive fragment ions at m/z ¼ 201 and 173 due to a-frag-mentation of the C–C bond between the two methylsulfanylgroups, which is in agreement with an olenic double bond atthe 9-position of 25. The authors did not comment on thepossibility of the alkene function in position 7 for which thesame fragment ions would be expected, but synthetic 25matched the natural product in terms of mass spectrum and GCretention time, and showed bioactivity in males. A similarproblem occurred in the identication of a series of volatileunsaturated methyl ketones from marine arctic bacteria of thecytophaga-avobacterium-bacteroides group, exemplied bythe representative compound (Z)-hexadec-10-en-2-one (27,Scheme 3B).47 The low resolution EI mass spectrum revealedintensive fragment ions atm/z¼ 201 and 131, in agreement witha C]C double bond either in the 5- or 10-position. The problemwas resolved by HR-EIMS, showing accurate masses for thediagnostic a-fragment ions at m/z ¼ 201.129 and 131.090 thatallowed for the determination of the elemental compositions ofthe fragment ions and hence in the localization of the doublebond in the 10-position.

The reaction of polyunsaturated compounds with DMDSonly yields the expected polyadducts when the double bondsare separated by four or more methylene units, whereasconjugated dienes result in 1,4-monoadducts, and dienes with

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Scheme 3 Double bond localization by DMDS derivatization.

Scheme 4 Volatiles showing fragment ion formations by inductivecleavage.

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double bonds separated by one to three methylene groupsyield cyclic structures that likely arise via the bis-adducts(Scheme 3C).56–58 Another possibility is given by the partialreduction of olenic double bonds using in situ-generateddiimine followed by DMDS addition which can also be per-formed on a ng scale.59

The last fragmentation mechanism involving bond breakingof one bond, discussed here, is the inductive cleavage. Ionisa-tion of a group X, either bound via C–X single bond(s) or a C]Xdouble bond, induces heterolytic cleavage of a C–X or a C–Cbond with charge migration. In the case of methyl esters theformation of [M � 31]+ fragment ions can be explained by aninductive cleavage resulting in the loss of a methoxide radical(Scheme 4A). An example is shown for the methyl ester 19,38 inFig. 3A, in which the low-intensity fragment ion at m/z ¼ 225 isdue to the loss of a methoxide radical by inductive cleavage.This fragment ion is of high diagnostic value, because themolecular ion of 19 is of very low intensity, but can be deduced

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from the [M� 31]+ fragment ion. A recently reported example ofa sesquiterpene that was suggested to undergo an inductivecleavage in the formation of the fragment ion m/z ¼ 109 is tri-chodiene (29), the main volatile emitted by the fungus Fusariumsporotrichioides (Scheme 4B).60 A detailed discussion of thefragmentation mechanisms for blastmycinone (30) and severalstructurally related lactones from Streptomyces ambofaciens wasalso recently presented, including the loss of the acyloxy sidechain by inductive cleavage (Scheme 4C).61

3.2.2 Fragmentations with the cleavage of two bonds

Some fragmentation mechanisms proceed with the breaking oftwo bonds, as shown in Scheme 5. The most important reac-tions of this type are the retro-Diels–Alder fragmentation andthe McLafferty rearrangement. The retro-Diels–Alder fragmen-tation can proceed via two alternative mechanisms, both initi-ated by ionization of a double bond of a cyclohexene followed byan a-fragmentation to a radical/allyl cation species. Subse-quently, either a second a-cleavage takes place resulting in theneutral loss of an alkene and a radical cation representing the“diene” portion of the molecule, or an inductive cleavage yieldsunder neutral loss of a diene a radical cation that refers to the“dienophil” portion of the molecule. Both charged fragmentsmay be observed in a mass spectrum and are easily identieddue to their even masses (if no nitrogen is contained in theanalyte), but the formation of one of the two charged fragmentsis usually strongly favoured.



Scheme 6 Examples of volatile natural products showing prominentretro-Diels–Alder fragment ions in their EI mass spectra.

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A classical textbook example for two structurally very similarcompounds, one of these showing an intensive retro-Diels–Alder fragment ion, while the related molecule is not,62 is shownin Scheme 6A. Retro-Diels–Alder fragmentation of a-ionone (31)yields by neutral loss of isobutene a fragment ion at m/z ¼ 136that is not observed in the mass spectrum of b-ionone (32).Dehydrogeosmin (33) was rst identied in the ower scent ofRebutia marsoneri and a few other Cactaceae,63 and has recentlybeen shown to be present in headspace extracts of severalstreptomycetes.64 Its mass spectrum is dominated by a retro-Diels–Alder fragment ion at m/z ¼ 126. Its rst identicationaer isolation of 6 mg of impure material solely relied on theinterpretation of the mass spectrum followed by synthesis of areference compound. The homomonoterpene 34 is released intrace amounts by several 2-methylisoborneol producing acti-nomycetes.65 The structure of this compound was also sug-gested from its mass spectrum exhibiting a retro-Diels–Alderion at m/z ¼ 82, and unambiguously established by synthesis.Themass spectrum of tricho-acorenol (35) that was rst isolatedfrom Trichoderma koningii presents a retro-Diels–Alder ion atm/z ¼ 84.66 This fragment ion was relevant for locating the sideof incorporation from various deuterated mevalonolactoneisotopologues in a biosynthetic study as is discussed in detail inchapter 6.67

A second fragmentation reaction in which two bonds arebroken is the McLafferty rearrangement (Scheme 6B). This typeof reaction is initiated by ionisation of an unsaturated C]Xfunctional group to yield a radical cation, followed by hydrogentransfer via a six-membered transition state that shis theradical centre into the g-position. A subsequent a-cleavageresults in neutral loss of an olen under formation of a conju-gated allylic radical cation. The rearrangement step may also be

Scheme 5 Fragmentation mechanisms with the cleavage of twobonds.


succeeded by an inductive cleavage to yield an alternativeradical cation. Which of the two cleavage reactions is preferreddepends on the ionisation potential of the two fragments.37

McLafferty ions are oen easily recognised, because they haveeven masses (if no nitrogen is contained in the molecule).

McLafferty rearrangements occur in all types of compoundclasses with C]X double bond systems including aldehydes,ketones, esters, carboxylic acids, and alkenes, but also inaromatic compounds such as alkylated benzenes, pyridines,pyrazines, and many others. A few exemplary volatilecompounds with characteristic McLafferty fragment ions intheir mass spectra are shown in Scheme 7. The myxobacteriumMyxococcus xanthus releases, besides the alcohol 21 discussedabove, the structurally related ketone 9-methyldecan-3-one(36).17 As is typical for ethyl ketones, its mass spectrum shows acharacteristic McLafferty ion at m/z ¼ 72. However, the inter-pretation of the mass spectra should always be performed verycarefully, because not only ethyl ketones, but also a-methylbranched methyl ketones and a-ethyl branched aldehydes suchas compound 37 exhibit a McLafferty ion at m/z ¼ 72. The lattercompound was identied in paracloacal gland secretions ofseveral Caiman spp.68 As can be seen in the mass spectrum ofthe methyl ester 19 (Fig. 3), mass spectra of compounds fromthis class are characterized by a McLafferty ion at m/z ¼ 74. Themass spectra of linear methyl esters and alkyl branchedcompounds have been extensively studied by Ryhage andStenhagen in 1959 and 1960.39,69,70 This work was particularlyimportant, because fatty acid methyl esters have extensivelybeen used as taxonomic markers of bacteria especially in thepre-genome era. Recently, a series of volatile methyl esters havebeen detected in headspace samplings from the actinomyceteMicromonospora aurantiaca and the gliding bacterium Chitino-phaga.38,71 The ndings of Ryhage and Stenhagen served in theidentication of branched compounds, as exemplied by thestructure of methyl 2,7-dimethyloctanoate (38) that exhibits aMcLafferty fragment ion at m/z ¼ 88, as is typical for a-methylbranched methyl esters. The mass spectrum of the secondcompound in Fig. 3, the phenyl ketone 20 from Stigmatellaaurantiaca, is dominated by a McLafferty ion at m/z ¼ 120.

Another interesting and widespread class of volatiles arealkylated pyrazines. A series of mono- to tetra-alkylpyrazines

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Scheme 7 Examples of volatile natural products showing prominentMcLafferty fragment ions in their EI mass spectra.

Scheme 8 Fragmentation of esters and ketones via double hydrogenrearrangement and cleavage of the b,g-carbon bond.

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including 2,5-diisobutylpyrazine (39) was identied in Paeni-bacillus polymyxa.72 All alkylated pyrazines with side chainslonger than two carbons for which McLafferty rearrangementsvia a six-membered transition state are possible exhibited theexpected McLafferty fragment ions in high intensity, usuallyresulting in the base peak of the mass spectrum. Variousalkylated pyrazines have also been detected in the headspaceextracts of the myxobacteria Chondromyces crocatus and Nan-nocystis exedens,73,74 and the actinobacterium Corynebacteriumglutamicum.75 Structural suggestions for these compoundswere derived by interpretation of their EI mass spectra thatlikewise were dominated by McLafferty ions in the cases ofalkyl substituents of a chain length of more than two carbons.However, since the substitution pattern of di-, tri-, and tetra-alkylpyrazines cannot be derived from the mass spectrum, allstructural suggestions were unambiguously proven bysynthesis of reference compounds. Differentiating betweenthe various constitutional isomers of alkylated pyrazines isalso a perfect example of a problem that could be solved bycomparison of the analyte's mass spectrum to databasespectra, but for unambiguous compound identication themass spectra of all constitutional isomers should ideally beavailable. If this is not the case, it cannot be excluded that anisomer that is not included in the database may have a verysimilar mass spectrum to a compound that is included. Inextreme cases this may require the synthesis of all possibleisomers, as was recently performed for the identication oftwo chlorinated anisole derivatives from the fungusGeniculosporium.76

If mass spectra of unidentied volatile compounds arecommunicated in the original work, this may in some casesassist in the later identication by synthesis of referencecompounds in a totally different context. An example is given by

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the synthesis of two pyrazines, 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine and 3-methoxy-2,5-bis(1-methyl-propyl)pyrazine, for comparison to volatiles emitted byC. crocatus.73 Due to the published mass spectra thesecompounds could also be identied as bacterial attractants forthe pineapple beetle Carpophilus humeralis.77

3.2.3 Multistep fragmentation mechanisms

Compounds such as esters, aldehydes, and ketones usuallyshow the formation of intensive fragment ions via McLaffertyrearrangementation that are of high diagnostic value as theseions point to substituents attached to the a-position. A secondintensive fragment ion in these types of compounds arises via adouble hydrogen rearrangement initiated cleavage of theb,g-carbon bond (Scheme 8).78 In the case of unsubstitutedesters, this process results in the fragment ions m/z ¼ 87 (R ¼OMe, e.g., compound 19, Fig. 3A) or m/z ¼ 101 (R ¼ OEt), whileketones yield the fragment ions m/z ¼ 71 (R ¼ Me), m/z ¼ 85(R ¼ Et), or m/z ¼ 133 (R ¼ Ph, compound 20 in Fig. 3B), etc.This fragmentation is of diagnostic value to locate substituentsin the a- or b-positions, and, in conjunction with analysis of theMcLafferty fragment ions, substituents can be assignedprecisely to one of these two positions.

In a few cases, fragmentation reactions via multiple stepshave been investigated. Two examples are the fragmentationreactions that give rise to the base peak ions in the mass spectraof geosmin (40) and 2-methylisoborneol (41), two terpenoidbacterial volatiles that are both released by many actinomy-cetes, myxobacteria and cyanobacteria,10,17,74,79–82 which will bediscussed here.

A multistep fragmentation pathway for the formation of thebase peak ion atm/z¼ 112 in themass spectrum of geosmin (40,Fig. 5A) was suggested by Boland and coworkers (Scheme 9).83

Aer ionisation of the hydroxy function the molecule undergoesa preferred a-cleavage under formation of the more stablesecondary radical (a primary radical is formed by an inferiora-cleavage of the other ring). The subsequent steps reveal thatrearrangements in mass spectrometric fragmentation reactionsmay not only proceed via six-membered transition states, as inthe case of the McLafferty rearrangement, but can also proceedvia other ring sizes. First, a rearrangement via an eight-membered transition state transfers a hydrogen radical fromthe oxygen into the alkyl side chain, which is followed by thecombination of a hydrogen radical transfer via a six-membered



Scheme 9 Fragmentation mechanism for geosmin (40).

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transition state and subsequent a-cleavage, i.e., a McLaffertyrearrangement. The rst hydrogen radical transfer via the eight-membered transition state was proven by a deuterium labellingexperiment with labelling at the hydroxy function of 40.

The formation of the base peak ion at m/z ¼ 95 in the massspectrum of 2-methylisoborneol (41, Fig. 5B) was also suggestedto arise via a multistep procedure (Scheme 10).74 Ionisation of41 at the hydroxy function is followed by a-cleavage to a tertiaryradical intermediate. A subsequent inductive cleavage underneutral loss of acetone yields a radical cation that in a seconda-fragmentation results in an allyl cation of m/z ¼ 95.

4 Retention time and retention index

A volatile compound should only be regarded as unambiguouslyidentied by GC-MS, if both its mass spectrum and the reten-tion time match those of a synthetic or commercially availablereference compound. Special problems that are frequentlyobserved for structurally complex volatiles (e.g., terpenes) arethe high similarities of mass spectra of closely related structuralisomers, such as diastereoisomers. In the case of two or morecoeluting terpenes the mixed mass spectrum may even besimilar to the mass spectrum of a third unrelated terpene,further impeding the identication of compounds (particularlyfrom this class) solely based on the mass spectrum. For acomparison of GC retention times authentic standards arehighly desirable. However, since these standards are not alwaysavailable, a more practicable approach is the comparison ofretention indices84 that can be calculated from the compound'sretention time in comparison to retention times of a

Fig. 5 Mass spectra of the widespread bacterial volatiles A) geosmin(40) and B) 2-methylisoborneol (41).


homologous series of n-alkanes obtained under the sameexperimental conditions:

I ¼ 100 nþ tR � tR;n

tR;nþ1 � tR;n:

Herein, n is the number of carbons of the n-alkane directlyeluting before the analyte for which the retention index I is to bedetermined, tR is the retention time of the analyte, and tR,n andtR,n+1 are the retention times of the n-alkane directly elutingbefore and aer the analyte. The retention index of a compoundmainly depends on the type of GC column (or more specicallyon its polarity), but almost never on the precise GC settings,such as gas ow, temperature programme, or length of the GCcolumn. Therefore, in contrast to GC retention times, retentionindices of a particular compound can be directly compared,even if the GC runs were performed under slightly differentexperimental conditions. Large databases with tabulatedretention indices85,86 exist with a high value for analyticalchemists. In addition, GC retention indices are listed in manyscientic publications that can be found via publicly availableweb-based compound libraries such as the NIST ChemistryWebbook (http://webbook.nist.gov/chemistry/) or the Pher-obase (http://www.pherobase.com/). How the retention index ofa compound in conjunction with a retention index incrementsystem can be of use for GC-MS based structure elucidationswill be discussed in chapter 5.

As pointed out earlier, the comparison of mass spectra andretention indices is crucial for the identication of terpenes thatmay be regarded as the structurally most complex class ofvolatile natural products. The GC-MS analysis of terpenes in

Scheme 10 Fragmentation mechanism for 2-methylisoborneol (41).

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Scheme 11 Volatile terpenes from bacteria and fungi and theirbioactive non-volatile oxidation products.

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headspace extracts together with a comparison of retentionindices or usage of authentic reference compounds has recentlybeen performed on many bacteria and fungi. The number ofdifferent terpenes identied in these studies is too large for adetailed discussion in this review, but the main compounds areshown in Scheme 11. Geosmin (40) and 2-methylisoborneol (41)are widespread in bacteria and have been identied in manymyxobacteria,17,40,74 actinomycetes,64,65,82,87,88 and cyanobac-teria.89 The sesquiterpene alcohol (1(10)E,5E)-germacradien-11-ol (42) is a biosynthetic intermediate towards 40 and wasdetected as the main compound in the headspace extracts fromStigmatella aurantiaca.40 Aciphyllene (43) is less widespread andwas found among the volatiles emitted by two Nocardiastrains,90 while epi-isozizaene (44) and its antibiotic oxidationproduct albaavenone (45) are produced by many streptomy-cetes.64,82,91,92 In contrast, the antibiotic pentalenolactone (47)and its parent hydrocarbon pentalenene (46) are found in only afew streptomycetes.64,93 Trichodiene (29) is the parent hydro-carbon of the T2 toxin (48)94 and is released by Fusarium verti-cillioides,60 while aristolochene (49) is the precursor to the PRtoxin (50) in Penicillium roqueforti.95,96 The diterpene ent-kaur-ene (51) is the main volatile emitted by Fusarium fujikuroi97 andis in this species converted into the phytohormone gibberellicacid GA3 (52).98 Bioactivity in terpenoid compounds is in manycases only reached once the terpene backbone is heavilymodied by oxidative transformations. Oxidised terpenoids like47, 48, 50 and 52 exhibit, in contrast to their stem hydrocar-bons, a much lower volatility, but compounds such as 45 can beobserved in CLSA headspace extracts from manystreptomycetes.64

The terpene hydrocarbons are made by terpene cyclases,many of which are encoded in bacterial genomes, especially inactinomycetes. The products of terpene cyclases can be ana-lysed by GC-MS, if their mass spectra and retention indices areknown or a reference compound is available. The rst bacterialterpene cyclase that was identied by purication of the proteinand subsequent incubation experiments with the nativesubstrate was the pentalenene synthase from Streptomycesexfoliatus,99 followed by the characterisation of various otherbacterial terpene cyclases whose volatile products are shown inScheme 12.100–113 Several of these products have been identiedfrom the enzymatic reactions by a laborious and time-consuming procedure of isolation and NMR spectroscopicstructure elucidation, while others were readily identied byGC-MS. Since modern genome sequencing has turned into avery fast and fairly cheap technique, more andmore sequencingdata are made available, showing that many more uncharac-terised terpene cyclases are encoded in bacteria. A recentlydeveloped interesting approach to gain rapid insight into thefunction of these terpene cyclases is based on heterologousexpression in Escherichia coli followed by direct CLSA samplingof volatiles from the expression strain and compound identi-cation by GC-MS.114 This procedure resulted in the character-isation of six terpene cyclases as germacrene A synthase (54,from Chitinophaga pinensis, this enzyme is unrelated to thegermacrene A synthase from Nostoc), g-cadinene synthase (65,Scheme 13, from C. pinensis), a-amorphene synthase (66, from

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S. viridochromogenes), 7-epi-a-eudesmol synthase (67, fromS. viridochromogenes), selina-4(15),7(11)-diene synthase (68,from S. pristinaespiralis), and T-muurolol synthase (60, fromRoseiexus castenholzii, this enzyme is also unrelated to theT-muurolol synthase from S. clavuligerus). Comparison not onlyof mass spectra, but also of retention indices to tabulated datawas of utmost importance for compound identication inthis study.

5 Examples of GC-MS basedstructure elucidation

In the case of volatiles for which neither a reference massspectrum nor a retention index is available instantaneouscompound identication is prevented. However, these analytesare in fact the most interesting compounds, because they offer



Scheme 12 Volatile terpenes from characterised bacterial terpenecyclases.

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the chance of nding new natural products. In some cases achemical structure can be rationally suggested from the massspectrum by interpretation of the molecular ion including itsisotope pattern and relevant fragment ions that are formed viaspecic reactions, as explained in chapter 3. Positive compoundidentication will then require synthesis of a referencecompound. This is, however, only a suitable approach, if astructural proposal for a particular compound can be derivedfrom its mass spectrum with some certainty, i.e., without the

Scheme 13 Volatile terpenes identified by GC-MS in headspaceextracts under heterologous expression of bacterial terpene cyclasesin E. coli.


risk of investing a lot of rather undirected synthetic work.Knowledge of the mass spectra of structurally closely relatedcompounds is helpful, but also not always available.

Recent examples for the identication of volatiles bysynthesis of reference compounds include a series of degradedoxygenated sesquiterpenes (69–71) from the myxobacteriumChondromyces crocatus115 and a structurally related dimethy-loctalin derivative (72),116 an intermediate in the biosynthesis ofgeosmin117 that occurs in many myxobacteria and streptomy-cetes64 (mentioned in some publications that appeared beforethe synthesis of the compound by its molecular ion, m/z ¼ 164,and base peak, m/z ¼ 149, Scheme 14).40,86 The structures of thetrans-fused iridoids (4R,4aR,7R,7aS)-dihydronepetalactone (73),(4S,4aR,7S,7aR)-iridomyrmecin (74), and (4S,4aS,7R,7aS)-irido-myrmecin (75) from the parasitoid wasp Alloxysta victrix wererigorously established by synthesis of the compounds and alltheir trans-fused stereoisomers,118,119 thereby overcoming theproblem that the stereochemistry of a volatile compound is interms of its absolute conguration in principle not, and interms of its relative conguration practically never deduciblefrom the mass spectrum. The absolute conguration of tricho-acorenol (76) from the fungus Trichoderma harzianum was alsoidentied by an enantioselective synthesis.120 A series ofhomomonoterpenes (77 and 78) and homomonoterpene alco-hols (79–81) was recently reported from 2-methylisoborneolproducing bacteria.65 Their structures were deduced bycomparison of their mass spectra to the mass spectra of corre-sponding regular monoterpenes and monoterpene alcohols,showing diagnostic shis of 14 mass units. The structures ofthese new natural products were conrmed by synthesis ofreference compounds.

The mass spectra of compounds that exist in the form ofdifferent constitutional isomers can also be very similar, e.g., forsubstituted aromatic and heterocyclic compounds. A structuralproposal for the female sex pheromone from the wasp Zaspilo-thynnus nigripes was derived from its mass spectrum,121 but thesix constitutional isomers of the suggested structure of ahydroxymethyl-(3-methylbutyl)-methylpyrazine (Scheme 15)could not be distinguished from the mass spectrum. Since notabulated retention index data for these compounds wereavailable and the isolated material was insufficient for NMRspectroscopic analysis, unambiguous structure elucidationrequired the synthesis of all six isomers. Synthetic 82 wasidentical to the pheromone and was also shown to be emitted bythe orchid Drakaea livida to attract male wasps for pollination.Similarly, for unambiguous identication of a dimethyl fur-andicarboxylate in headspace extracts of Streptomyces griseo-avus and S. parvulus all four constitutional isomers wereobtained by synthesis or from commercial sources, resulting inthe identication of 83 as the natural product.122 The compoundwas previously, without comparison to a synthetic standard,tentatively identied as a volatile from another streptomycete.88

The marine bacterium Ruegeria pomeroyi from the Roseobacterclade was shown to emit the lactone 84 as a major compound,besides a few structurally related minor volatiles.123 The struc-ture of 84, including its absolute conguration, was fully sup-ported by an enantioselective synthesis and GC analysis on a

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Scheme 14 Volatile terpenes that were identified by the developmentof structural proposals from their EI mass spectra followed by synthesisof reference compounds.

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chiral stationary phase in comparison to the natural material.Similarly, the structure of a volatile released by the triatominebug Triatoma brasiliensis was suggested from its mass spectrumand identied as (4S,5S)-2,2,4-triethyl-5-methyl-1,3-dioxolane(85) by synthesis of all stereoisomers.124 An important hint forthe development of the structural proposal was the presence oflarge amounts of pentan-3-one in the headspace extracts that isthe precursor and/or degradation product of 85, demonstrating

Scheme 15 Volatiles that were identified by the development ofstructural proposals from their EI mass spectra or by isolation andstructure elucidation via NMR spectroscopy.

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that careful analysis and integrated evaluation of all volatileconstituents from one organism may be of tremendous help instructure elucidation.

However, this approach of delineating a structural proposalfrom the mass spectrum has its limitations. This is particularlytrue for terpenes as an important class of volatile naturalproducts, because their polycyclic structures are usually toocomplex. In such cases, GC-MS analysis of headspace extractsmay point to interesting new natural products for furtherinvestigation by compound isolation and structure elucidationvia NMR spectroscopic methods. One of the most interestingrecent examples is the structurally unique volatile sodorifen (86)from Serratia odorifera.125 The compound was trapped byabsorption on SuperQ in quantities that were sufficient forstructure elucidation by NMR analyses. This approach is ofcourse only suitable if the organism under investigation onlyemits one compound (or at least mainly one compound), andnot a complex mixture of volatiles. Headspace extracts of thefungus Fusarium fujikuroi contained three groups of terpenoidcompounds, and each of these groups was composed of onemain compound and various trace compounds that wereregarded as side products of the respective terpene cyclase.126

One of the three main compounds was the diterpene ent-kaur-ene, the biosynthetic precursor of the plant hormone gibberellicacid GA3, whereas the structure of one sesquiterpene alcoholwas unknown and only the relative conguration of the othersesquiterpene alcohol, a-acorenol, was known. The construc-tion of double knockout mutants resulted in simplied head-space extracts for compound isolation and identication of thevolatiles as (�)-a-acorenol (87) and koraiol (88).

For the disulde 89, released by amarine bacterium from theRoseobacter clade, a structural proposal was derived from itsmass spectrum (Fig. 6).127 The molecular ion together with itsisotope pattern (166: 100%, 167: 8.0%, 168: 8.4%) pointed to thepresence of two sulfur atoms and no (or an even number of)nitrogen atoms. The best agreements were calculated forC5H10O2S2 (166: 100%, 167: 7.2%, 168: 9.5%) or C6H14OS2 (166:100%, 167: 8.3%, 168: 9.3%). The fragment ions at m/z ¼ 135(loss of OMe by inductive cleavage) and m/z ¼ 59 (a-cleavage)suggested the structure of a methyl ester C5H10O2S2. The frag-ment ion at m/z ¼ 118 (neutral loss of MeSH) pointed to amethylsulfanyl group that was further corroborated by m/z ¼ 45(HCS+), a secondary fragment ion that arises from MeS+ by lossof H2, as is frequently observed in methylsulfan derivatives.Further fragment ions were in agreement with a linear structureof a disulde (m/z ¼ 79: MeSS+,m/z ¼ 93: MeSSCH2

+,m/z¼ 107:MeSSCH2CH2

+). The missing McLafferty ion at m/z ¼ 74 usuallyobserved for methyl esters is easily explained, because theMcLafferty rearrangement requires a g-hydrogen atom, but theg-position in 89 is occupied by a sulfur atom. However, a frag-ment ion for cleavage of the Cb–S bond at m/z ¼ 87 is observed.A synthesis of 89 and comparison of GC-MS data in terms ofmass spectrum and retention index proved the structure of thisnew natural product.

From an analytical chemist's point of view it is of utmostimportance that in cases where the synthesis of a referencecompound or a compound isolation is required for the



Fig. 6 Mass spectrum of the headspace constituent methyl3-(methyldisulfanyl)propionate (89) from a marine Roseobacter cladebacterium.

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identication of a volatile, the GC retention indices togetherwith the type of GC column should always be included in ascientic publication, so that future analytical work in whichthese compounds may be detected again will not require arepeated synthesis.

Retention indices are not only useful, because they can, asstandardized data in contrast to retention times, be used fordirect comparison, even if they were obtained under differentexperimental parameters, but they are also the basis for incre-ment systems. For the detailed analysis of the structures ofmethyl branched long chain lipids like methyl esters, alcohols,and ketones such a retention index based increment system wasdeveloped.128 The basic idea was the theoretical separation of alipid molecule into its longest alkyl chain, a functional group,and attached methyl branches that all contribute more or lessindependently to the analyte's retention index I:

I ¼ N þ FGþX

Mei

Herein, N is an increment for the longest alkyl chain with N¼ 100$n where n represents its number of carbons, FG is anincrement for the functional group, and Mei are increments forthe methyl branches that depend on their positioning i alongthe longest alkyl chain. Several functional group increments forHP-5 or similar GC capillaries have been determined fromunbranched representatives of the respective compound classesincluding methyl esters (FG ¼ 232),128 alkyl cyanides (FG ¼458),128 2-alcohols (FG ¼ 210),47 3-alcohols (FG ¼ 205),17

2-ketones (FG ¼ 200),47 and 3-ketones (FG ¼ 195).17 The incre-ments (Mei) for the methyl branches as determined in theoriginal work are summarized in Table 3. A recent renement ofthe system for retention index increments of fatty acid methylesters showed a slight dependency of the functional group andmethyl branch increments on the chain length, giving bettermatches between calculated and measured retention indices.38

The fundamental idea of the retention index incrementsystem, together with careful interpretation of mass spectra andbiosynthetic considerations, was also successfully applied to the


development of structural proposals for a series of structurallycomplex lactones from Streptomyces ambofaciens. One of theselactones was known as a secondary metabolite of Streptomycesspp.129 and its mass spectrum was included in mass spectraldatabases, thus allowing for instantaneous compound identi-cation as bastmycinone (30). Its mass spectrum (Fig. 7A)revealed a fragment ion at m/z ¼ 241 due to the loss of a methylgroup, and is likely to have arisen from a-cleavage from thelactone core, while the molecular ion could not be detected.Two even fragment ions atm/z¼ 214 and 200 could be assignedto McLafferty rearrangements, either initiated by ionisation ofthe lactone carbonyl oxygen or the carbonyl group of the esterside chain. Further intensive fragment ions were observed atm/z ¼ 57 and 85, accounting for two alternative a-cleavageswithin the ester side chain. These two a-fragment ions togetherwith the two McLafferty fragments were of high diagnosticvalue, because they directly pointed to the nature of the 2-alkylportion and the acyl portion of the ester in other blastmycinonederivatives. In the mass spectra of compounds 90 and 91 thesame set of a-fragment ions at m/z ¼ 57 and 85 were observed,suggesting that the same or an isomeric acyl moiety, as in 30,should be present in these molecules. This was furthercorroborated by the fact that in all three molecules the sameMcLafferty fragment ion at m/z ¼ 200 was found. The secondMcLafferty ion in 90 and 91 was detected at m/z ¼ 228 and 186,respectively. This loss of C3H6 in both cases, as in 30, ruled out a2-methylbutyryl or 2,2-dimethylpropionyl group, but was inaccordance with a 3-methylbutyryl or pentanoyl function. Byfollowing the further logic of the arguments, the McLaffertyions at m/z ¼ 200 supported a C5H11 2-alkyl chain for 90 and a2-ethyl side chain for 91. For compound 92 the a-fragment ionswere observed atm/z¼ 43 and 71 (butyrate or isobutyrate ester),but only one McLafferty fragment was found at m/z ¼ 186, thusruling out the possibility of a butyrate ester that should result ina second McLafferty ion at m/z ¼ 214 (neutral loss of ethene).The m/z ¼ 186 ion furthermore pointed to a C4H9 2-alkyl group.

Further structural insights into the precise nature of theattached substituents were concluded from the retentionindices of the compounds, using the logic of the retention indexincrement system as presented above. The moderate differencein the retention indices of 30 and 90 (55 units) is only inagreement with two methyl branched side chains, and thereforea 2-isopentyl group was assumed to be present in 90. Retentionindex calculations in the case of 91 were less clear, because theshorter 2-ethyl chain in 91 in comparison to a 2-butyl groupshould theoretically result in a retention index difference of200 units, but only a difference of 170 units was determined.However, the retention indices of short chain methyl esters arealso dominated by a stronger inuence of the functional group(in case of blastmycinones the lactone core can be regarded asthe “functional group”),38 and for biosynthetic reasons a leucinederived acyl group in 91 seemed more likely. Finally, thedifference between the retention indices of 30 and 92 of ca.100 units strongly supported an unbranched 2-alkyl moiety. Allstructural proposals as discussed here and of several furtherblastmycinone derivatives from S. ambofaciens and a few otherstreptomycetes were veried by total synthesis. This example

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Table 3 Increments (Mei) for methyl branches.128

Position i Mei Position i Mei

2 or u–1 60 7 or u–6 363 or u–2 73 8 or u–7 334 or u–3 56 9 or u–8 315 or u–4 46 10 or u–9 286 or u–5 40

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demonstrates the power of an integrative discussion of massspectra, retention indices, and biosynthetic considerations for asuccessful delineation of structural proposals for previouslyunidentied volatiles.

The above described in-depth analysis of GC-MS data, i.e., adetailed analysis of gas chromatographic retention indices andof the molecular ion and fragmentation pattern in the massspectra of unknown compounds, is a difficult task and requiresa lot of experience to develop a successful structural proposal.Most importantly, the proposal has to be veried by comparisonto a synthetic reference. Most analytical studies refer to theknown chemical space and compounds in such studies arefrequently identied by comparison to retention index datapublished in previous reports. The danger of this approach isthat an erroneous compound identication, e.g., in case of(stereo)isomers with highly similar mass spectra, is easilymultiplied.

6 Feeding experiments

A feeding experiment is an experiment in which a biosyntheticprecursor, a small molecule of the primary metabolism such asan amino acid, a sugar, a citric cycle intermediate, etc., or anadvanced biosynthetic intermediate, is fed to an organism,followed by investigation whether or not the fed compound isincorporated into the target natural product. A feeding experi-ment may be performed for elucidating the biosyntheticpathway to a volatile or for isotopic enrichment for structureelucidation. Today, in most cases, the isotopic labelling isrealised by use of a stable isotope, usually of deuterium or13C. Many classical biosynthetic studies have used radioactivelabels (e.g., 3H or 14C), but these techniques seem to be out offashion, mainly due to the rapid technological developments inmass spectrometry that allow for the very sensitive detection ofstable isotope incorporations into natural products. In earlierdays, the incorporation of radioactive isotopes into naturalproducts could be best detected, with very high sensitivity,using autoradiographe, but a major drawback is that thehandling of radioactive compounds requires special safetyequipment and trained personnel.

Both stable isotopes 2H and 13C have frequently been usedfor the biosynthetic investigations of volatile natural products,but their behaviour in GC-MS analyses is very different.Deuterated compounds are characterised by particularly strongisotope effects, much stronger than in 13C-labelled compounds.The shorter C–D bond length, as compared to a C–H bond,130

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leads to a stronger electric eld strength and, consequently, to alower polarisability of deuterated compounds in comparison totheir protonated analogues.131 Since weak dispersion forces playa major role in the gas chromatographic retention of non-polaranalytes,132 deuterated compounds usually have signicantlyshorter retention times.17,34,133,134 This has important conse-quences for feeding experiments on the biosynthetic pathwaysto volatile natural products, if using deuterated precursors. Theincorporation rates of the fed deuterated compound into thevolatile natural product are usually not 100%, therefore result-ing in the production of a mixture of the labelled and the non-labelled compounds by the respective organism. In the case oflow-resolution MS analysis, only the efficient gas chromato-graphic separation of the deuterated from the protonatedvolatile allows for a precise determination of the incorporationrates by simple peak integration (baseline separation of thedeuterated compound from the natural isotopomer requires theincorporation of ca. 3–5 deuterium atoms). Furthermore, adetailed interpretation of the “clean” mass spectrum of thedeuterated compound is possible, if mass spectral fragmenta-tion mechanisms are known, that may even allow for a local-isation of the site of deuterium incorporation in the naturalproduct. This is exemplied by the incorporation of deuteriumlabelling from several synthetic deuterated mevalonolactoneisotopomers129 into tricho-acorenol (35) that undergoes a retro-Diels–Alder fragmentation in MS (Scheme 6).67 The corre-sponding fragment ion at m/z ¼ 84 for the unlabelledcompound (Fig. 8A) was observed at m/z ¼ 88 in the feedingexperiment with [4,4,6,6,6-2H5]mevalonolactone (Fig. 8B), cor-responding to an uptake of four of the twelve incorporateddeuterium atoms into the retro-Diels–Alder fragment formingportion of 35. Similar localisations of deuterium atoms byinterpretation of the major fragment ions at m/z ¼ 138 and 151,together with interpretations of mass spectra with several othermevalonolactone isotopomers, allowed to precisely follow asequence of a 1,4- and a 1,2-hydride shi during terpene cycli-sation. The interpretation of mass spectra in such detail wasonly possible due to the gas chromatographic separation of thedeuterated from the non-labelled material.

Early feeding experiments with deuterated compounds incombination with GC-MS analysis have been performed toinvestigate the biosynthetic pathways to fatty acid derived insectpheromones. The reactions on fatty acid derivatives discussedin the next section likely proceed with coenzyme A (CoA)derivatives or with enzyme bound substrates, but since the truenature of the intermediates has not been investigated thebiosynthetic pathways will be discussed on the level of free acidsubstrates as in most of the original articles. In a pioneeringexperiment the biosynthesis of bombykol (96, Scheme 16), therst identied sex pheromone of the silkworm moth Bombyxmori,135 was investigated by feeding of [11,12-2H2]-(11Z)-hex-adecenoic acid (94, deuterated positions are marked by aster-isks) whose incorporation into 96 was readily detected byGC-MS.136 The deduced pathway starts from hexadecanoic acid(93) that is converted into (11Z)-hexadecenoic acid (94) byactivity of a D11-desaturase, followed by introduction of asecond double bond under abstraction of one hydrogen from



Fig. 7 Mass spectra of A) blastmycinone (30) and B–D) blastmycinonederivatives (90–92) detected in headspace extracts of Streptomycesambofaciens.


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C-10 and one hydrogen from C-13 to yield (10E,12Z)-hexa-decadienoic acid (95) and reduction to bombykol.

This successful labelling study promoted a series of similarGC-MS based feeding experiments on biosynthetic pathways toinsect pheromones. It was shown by feeding of [16,16,16-2H3]-palmitic acid (93, deuterated positions marked by asterisks)that the biosynthesis of the sex pheromones from Agrotissegetum, (7Z)-dodecenyl acetate (104), (9Z)-tetradecenyl acetate(103), and (11Z)-hexadecenyl acetate (102), proceeds by intro-duction of the (Z)-double bond to yield 94 (Scheme 17).137 Itsreduction to the alcohol and acetylation results in 102, whilethe shorter derivatives 103 and 104 require a chain degrada-tion by b-oxidations of 94 prior to reduction and acetylation.Similar results were obtained for the biosynthesis of 102 byfeeding [13,13,14,14,15,15,16,16,16-2H9]-93 to Mamestrabrassicae.138

The biosyntheses of all four compounds 96 and 102–104starts with a D11-(Z)-desaturation that is a key step in theformation of a large number of lepidopteran pheromones.Boland and coworkers have shown in a series of elegantexperiments that introduction of the (Z)-double bond by theD11-desaturases from three investigated lepidopterae, M. bras-sicae, B. mori, andManduca sexta, proceeds via a syn eliminationof C(11)-HR and C(12)-HR (Scheme 18).139 Feeding of (11S,12R)-[2H14]-93 to M. brassicae resulted in the incorporation of twelvedeuterium atoms into the insect pheromone 102, as determinedby GC-MS, while a complementary feeding experiment with(11R,12S)-[2H14]-93 resulted in the retention of all fourteendeuterium atoms in labelled 102.139 The same stereochemicalcourse was found for the reaction of 93 to 94 in B. mori and

Fig. 8 Mass spectra of A) unlabelled tricho-acorenol (35) and B) of[2H12]-35 obtained after feeding of [4,4,6,6,6-2H5]mevalonolactone.Asterisks indicate completely deuterated carbons.

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Scheme 16 Biosynthetic pathway to bombykol (96).

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M. sexta. The incorporation of deuterium labelling into 94 wasinvestigated by GC-MS aer conversion into the respectivemethyl ester with diazomethane and the dimethyl disuldeadduct by treatment with dimethyl disulde/iodine.140 Thestereochemical course of the lepidopteran D11-desaturases isthe same as previously determined for the D9-desaturases fromalgae,141 bacteria,142 and animals,143 suggesting a commonenzyme mechanism.

The biosynthesis of the sex pheromones (5Z)-dodecenol and(5Z,7E)-dodecadienol (110) of the pine caterpillar moth, Den-drolimus punctatus, was established by feeding experimentswith deuterated fatty acids including [16,16,16-2H3]-93,[18,18,18-2H3]octadecanoic acid (105) and [15,15,16,16-2H4]-(11Z)-octadecenoic acid (106), followed by analysis of thepheromone gland content by GC-MS (Scheme 19).144 Thepathway starts from 93, which is elongated to 105, followed byintroduction of a (Z)-double bond by a D11-desaturase to 106

Scheme 17 Biosynthesis of lepidopteran pheromones by activity of aD11-(Z)-desaturase. The deuterated positions as relevant for thebiosynthetic investigations on 102–104 in A. segetum are indicated byasterisks, whereas additionally marked carbons (asterisks in brackets)were only relevant for the feeding study on the biosynthesis of 102 inM. brassicae.

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and chain shortening by b-oxidation to (9Z)-hexadecenoic acid(107). A second (E)-double bond is introduced by a D11-desa-turase, possibly the same enzyme as that acting in the reactionof 106, to yield (9Z,11E)-hexadecadienoic acid (108). Twosubsequent b-oxidations to (5Z,7E)-dodecadienoic acid (109)and a reduction establish the pheromone 110. The secondcompound (5Z)-dodecenol is produced via the same pathway,only with the omission of the second D11-desaturation. Thebiosynthesis of the pheromones of the lappet moth Gastropachaquercifolia, (5Z)-dodecenol and (5Z)-dodecenal, was investigatedby the feeding of [7,7,8,8,10,10,11,11-2H8]-(5Z)-dodecenoicacid.145 Analysis of the pheromone bouquet by GC-MSconrmed that it is the direct precursor to the pheromonecomponents, while the saturated analogue[7,7,8,8,10,10,11,11-2H8]dodecanoic acid was not incorporated,suggesting that the unsaturation is introduced at an earlierstage of the pathway.

Such feeding experiments with insects can be difficult due tothe complex behaviour of these macroscopic eukaryotic organ-isms. In contrast, bacteria are much easier to handle and afeeding experiment can simply be carried out by adding anisotopically labelled precursor to the culture medium. One ofthe rst studies on the biosynthesis of volatile secondarymetabolites was performed with the myxobacteriumMyxococcusxanthus.17 As shown by the feeding of deuterated precursors, themain volatiles released by this bacterium, 9-methyldecan-3-one(36) and (S)-9-methyldecan-3-ol (21), are derived from L-leucine(113). The feeding of completely deuterated [2H10]leucineresulted in the incorporation of nine deuterium atoms into bothmetabolites; one deuterium is lost during transamination andoxidative decarboxylation to the branched starter unit iso-valeryl-CoA (112, Scheme 20). Aer chain elongation with twomalonyl-CoA units to the acyl carrier protein (ACP) boundintermediate 114 a nal methylmalonyl-CoA building block isincorporated, as was demonstrated by the feeding of [2H5]

Scheme 18 Stereochemical course of D11-(Z)-desaturases involved inthe biosynthesis of lepidopteran pheromones.



Scheme 19 Biosynthetic pathway to (5Z,7E)-dodecadienol in Den-drolimus punctatus.

Scheme 20 Biosynthetic pathway to 9-methyldecan-3-one and(S)-9-methyldecan-3-ol in Myxococcus xanthus.

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propionic acid. Release of the b-keto-thioester intermediate 115and decarboxylation yields 36, which is converted to 21 byreduction.

The major advantage of using deuterated precursors in thisstudy was the chromatographic separability of the deuteratedcompound from the material with a natural isotope pattern.This allowed for a location of the deuterium incorporation bycareful interpretation of fragment ions (Fig. 9). Importantfragment ions of 36 were observed at m/z ¼ 43 (cleavage of theterminal isopropyl group), m/z ¼ 57 (a-cleavage next to thecarbonyl group), and m/z ¼ 72 (arising by McLafferty rear-rangement). Feeding of [2H10]leucine resulted in a specicincrease of the fragment ion at m/z ¼ 43 to 50, while the frag-ment ions at m/z ¼ 57 and 72 remained unchanged, givingevidence for deuterium uptake into the isopropyl moiety(Fig. 9B). Feeding of [2H5]propionic acid resulted in the incor-poration of three deuterium atoms into 36 and 21; one deute-rium atom in the a-position was lost due to carboxylation tomethylmalonyl-CoA and a second one due to the increased C,H-acidity of methylmalonyl-CoA or of the intermediate 115. Theincorporation of labelling into the ethyl groups of 36 and 21could be located by the specic shis of the fragment ions atm/z ¼ 57 and 72 to m/z ¼ 60 and 75 (Fig. 9C). Since a completewashout of deuterium from the a-carbon was observed in thefeeding experiment with [2H5]propionic acid another strategyusing [13C2]acetic acid was required to prove the biosyntheticorigin of the acetate-derived carbons in 36 and 21. As discussedabove, the isotope effect of 13C is much weaker than fordeuterium, and consequently a 13C-labelled compound is notchromatographically separated from the 12C analog, resulting


in the superimposition of the mass spectra from the labelledcompound and the compound with a natural isotope pattern(Fig. 9D). Nevertheless, the incorporation of up to two units of[13C2]acetic acid was observable from the molecular ion. Thelocation of the incorporation was not discussed in the originalwork, but a slight increase in the fragment ions at m/z ¼ 58 andm/z ¼ 74 can be observed, which is in agreement with anincorporation of labelling into C-2 and C-3 (compare Fig. 9A and9D). Feeding of deuterated dimethylacrylic acid (111), that isalso linked to terpene metabolism in myxobacteria,117,146 gaveconsistent results.

An interesting study was recently performed by Rui andBoland, in which the stereochemical course of the biosyntheticpathway from arachidonic acid to dictyoene in Ectocarpus sili-culosus was followed by stereospecic deuterium labellings atC-16 (Scheme 21).147 For this purpose the synthetic deuteratedisotopologues of arachidonic acid 117 and 118 were fed toE. siliculosus. Along the known biosynthetic pathway148 theseisotopologues of arachidonic acid are oxidatively converted tohydroperoxyeicosatetraenoic acids 119 and 120 by lipoxygenaseactivity. The deprotonation induced fragmentation results in thedivinylcyclopropane 121 and (5Z,7E)-9-oxononadienoic acid(122). A nal Cope rearrangement of 122 yields dictyoene (123)that was captured by SPME and analysed by GC-MS for itsdeuterium content. Deuterium labelling from the pro-S hydrogenin position 16 of arachidonic acid was retained, whereaslabelling of the pro-R hydrogen was lost, showing that

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Fig. 9 Mass spectra and important fragment ions of A) 9-methyl-decan-3-one (36), B) [2H9]-36 after feeding of [2H10]leucine, C) [

2H3]-36 after feeding of [2H5]propionic acid, and D) superimposed massspectra of 36, [13C2]-36, and [13C4]-36 after feeding of [13C2]aceticacid. Asterisks indicate labelled positions.

Scheme 21 Stereochemical course of the biosynthesis of dictyoene(123) in Ectocarpus siliculosus.

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the deprotonation induced fragmentation of hydro-peroxyeicosatetraenoic acid proceeds with stereospecic loss ofthe pro-R hydrogen at C-16. This nding was in agreement withprevious investigations on the stereochemical course of thereaction in Gomphonema parvulum.149

A recent example for a biosynthetic study on an alkaloid byfeeding of isotopically labelled precursors followed by GC-MSanalysis of incorporation is given by stenusine (132) from the

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beetle Stenus bimaculatus.150 Incorporation of deuterated iso-topomers of L-lysine (124), L-leucine, and sodium acetate sug-gested the biosynthetic pathway shown in Scheme 22.Decarboxylation and transamination of 124 results in the aminoaldehyde 125, which is cyclised to the imine 126. The corre-sponding enamine 127 undergoes nucleophilic attack to theisoleucine-derived aldehyde 128, forming the condensationproduct 129, which is subsequently reduced to 130. Acetylation,most likely with acetyl-CoA, yields the acetamide 131 that isreduced to stenusine. The study also contained a detailedanalysis of mass spectral fragmentations to locate the sites ofincorporation.

As discussed for several examples in this article so far, thestrongest advantage of using deuterated precursors is thepossibility of gas chromatographic separation of deuteratedvolatiles from their natural analogs, allowing the localisation ofdeuterium incorporation, if fragmentation reactions for theanalyte are known or are at least very plausible following thereactions as discussed in chapter 3. This method has its limits,where the fragmentation reactions are unclear, or two or morealternative fragmentations resulting in the same fragment ionare possible. An example is given by the fragmentation mech-anism of the base peak ion at m/z ¼ 95 for the musty odorant2-methylisoborneol (41), for which two alternative terminala-cleavages with loss of stereochemical distinct methyl groupsare possible (Scheme 10). One of these methyl groups origi-nates from the methyl group in the terpene precursor meva-lonolactone (133) (in case of the myxobacterium Nannocystisexedens that uses the mevalonate pathway to terpenes) orfrom C-1 in deoxyxylulose phosphate (134) (in case of moststreptomycetes using the deoxyxylulose phosphate pathway,



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Scheme 23). However, analysis of the stereochemical course ofthe terpene cyclisation of (E)-b-methylgeranyl diphosphate(138) by feeding of an appropriately deuterated terpeneprecursor cannot be conclusive due to the uncertainties in thefragment ion formation. Feeding of a 13C-labelled precursorfollowed by compound isolation and 13C-NMR spectroscopywould give an unambiguous answer, but the isolation ofcompounds such as 41 from culture extracts is signicantlyhampered by the high volatility. Recently, a highly sensitivemethod for microscale biosynthetic investigations using thetechniques of headspace sampling was described.65 Feeding of[1-13C]deoxyxylulose to an agar plate culture, collection ofvolatiles on charcoal lters by CLSA, followed by GC-MS and13C-NMR analysis of the sample established the stereochemicalcourse of the cyclisation to 41. In this analytical procedure theadvantages of several techniques were combined: Headspacesampling (CLSA) only yielded the volatile material, and unlikeculture extractions, all extractable secondary metablites, andthe loss of volatiles was prevented. GC-MS analysis of theheadspace extract showed the content of terpenes that wereidentied from their mass spectra and retention indices (onlyterpenes were important since a terpene precursor was fed; allother volatiles did not incorporate the isotopic labelling andwere in consequence not visible in the 13C-NMR spectrum).Finally, 13C-NMR spectroscopy showed into which carbonatoms of 41 the labelling was incorporated. Due to the highincorporation rates, even micrograms of material were suffi-cient to detect the 13C-enriched carbons.

The same method was recently used to investigate the vola-tiles emitted by the PR toxin (50) producing fungus Penicilliumroqueforti.95 The parent hydrocarbon of 50 is aristolochene,which is made by aristolochene synthase.151,152 Several sideproducts of this enzyme have been identied in a series of

Scheme 22 Biosynthetic pathway to stenusine (132) in Stenusbimaculatus.


studies including GC-MS analyses of SPME headspace extractsfrom PR toxin producing strains of P. roqueforti,153 and ofvolatile products obtained from enzyme incubations with thewildtype enzyme or site-specic mutants.154–157 This includes thecompounds germacrene A (139), a-selinene (140), b-selinene(141), selina-4,11-diene (142), valencene (143), and (E)-b-farne-sene (144, Scheme 24). Interestingly, a single critical amino acidswitch (Y92A) turned the aristolochene synthase into an enzymewith (E)-b-farnesene synthase activity.157

In a recent study on the volatiles released by P. roqueforti anadditional previously unrecognised sesquiterpene alcohol wasdetected.95 Its mass spectrum (Fig. 10) revealed high similari-ties to the mass spectrum of eudesma-11-en-4a-ol (148), butsome of its stereoisomers (Scheme 25) were reported to havesimilar retention indices and could not be excluded.158 Sincealso the full 13C-NMR data of all stereoisomers were mentionedin the same report, the structure of eudesma-11-en-4a-ol couldbe proven by feeding of [methyl-13C]mevalonolactone,capturing of the volatiles and direct 13C-NMR analysis of theheadspace extract, resulting in the detection of the threeexpected 13C signals for 148. Finally, a third study making useof the technique was performed to investigate the mechanisticdetails of biosynthetic pathways to terpenes, including thevolatiles a-acorenol (87) and koraiol (88) in Fusariumfujikuroi.159

In contrast to the widespread usage of hydrogen and carbonisotope labellings, oxygen labellings are only applied in fewbiosynthetic studies, largely because 17O and 18O labelledcompounds are comparably expensive and only few suchcompounds are commercially available. An interesting recentexample is given by a series of studies on the biosynthesis ofspiroacetals in fruit ies of the genus Bactrocera.160,161 Thespecies-specic incorporation of labelling from [18O2]dioxygeninto the spiroacetals, such as 154, was followed by thecapturing of these volatiles by SPME and subsequent GC-MSanalysis (Scheme 26). In B. tryoni both oxygens in 154 were

Scheme 23 Biosynthetic pathway to 2-methylisoborneol andstereochemical course of terpene cyclisation. Asterisks indicate13C-labelled carbons.

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Scheme 24 Side products of aristolochene synthase from P. roque-forti identified by GC-MS.

Fig. 10 Mass spectrum of an eudesman type sesquiterpene alcoholfrom P. roqueforti.

Scheme 25 Structures of eudesma-11-en-4a-ol (148) and itsstereoisomers. Published retention indices on a DB-1 column157 aregiven in brackets.

Scheme 26 Biosynthesis of spiroacetals in Bactrocera. Asterisksindicate uptake of labelling into both oxygens of 154 after adminis-tration of [18O2]dioxygen to B. tryoni, whereas for B. curcumis only oneoxygen of 154 was labelled.

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found to be labelled aer administration of [18O2]dioxygen,160

whereas in B. curcumis the labelling was only introduced intoone oxygen.161 These results were explained by the introductionof both hydroxy functions in the linear precursor 153 byinvolvement of a cytochrome P450 in B. tryoni, while only one ofthe two hydroxy functions in 153 may be introduced by acytochrome P450 in B. curcumis and the other hydroxy functionis derived from water during b-oxidation of a fatty acidprecursor.

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7 Conclusions and outlook

As demonstrated in this article, GC-MS is an exquisite tool forthe identication of new volatile natural products. Carefulinterpretation of mass spectra with respect to the molecular ionand its isotope pattern and, most importantly, in light ofknown fragmentation pathways may give the experiencedanalytical chemist the possibility of delineating a structuralproposal. Retention index increment systems can providefurther valuable information. The (future) general availabilityof high-resolution mass spectrometry as a standard laboratorytechnique will certainly have a strong impact on the identi-cation of new volatile natural products as the elementalcompositions of molecular and fragment ions become easilyaccessible with this technique. This will provide an evenstronger fundament for structural suggestions, as it can beobtained from low-resolution data. However, it will not bepossible, neither based on low-resolution nor on high-resolu-tion data, to delineate the structure of a new compound fromGC-MS data alone. For verication of structural proposals thesynthesis or isolation of a reference compound is alwayscrucial. In the case of compounds with a known mass spec-trum, the use of (web-based) mass spectral libraries is a verypowerful tool for compound identication. In this sense,chemistry should clearly learn from the recent achievements inbiological sciences that have been revolutionised by thedevelopment of strong bioinformatic tools and the assembly oflarge databases.

The GC-MS technique is also very useful for biosyntheticinvestigations by application of isotopically labelledcompounds in feeding experiments. As discussed here indetail, deuterated compounds are of particularly high value,because a deuterated volatile can be separated from its non-deuterated analog by gas chromatography, thus allowing forthe interpretation of the “clean” mass spectrum of thedeuterated compound, which is especially useful whenencountering low incorporation rates. The most recent devel-opments in the analysis of volatiles includes the combinationof the highly sensitive GC-MS technique with feeding experi-ments of 13C-labelled precursors that allows for an efficient13C-NMR analysis of headspace samples containing onlymicrogram amounts of a particular analyte. The describedtechniques in this article together with future developmentsmay allow for the efficient identication of new volatiles as animportant class of natural products and detailed insights intotheir biosynthetic pathways.



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8 Acknowledgements

Funding by the Deutsche Forschungsgemeinscha (DFG) withan Emmy Noether fellowship (DI1536/1-3) and a Heisenbergfellowship (DI1536/4-1) is gratefully acknowledged.

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Capturing Volatile Natural Products by Mass Spectrometry

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