Abstract
In a situation of danger, search of a partner/food, insects communicate through the way of chemicals called ‘phereomones’. Pheromones are the substances that are secreted by an individual bio-organism and are received by a second individual of the same species and produce a specific reaction. In other words, pheromones are chemical substances used for intraspecific communication. There are a variety of pheromones like ‘alarm pheromones’, used to call for help by an insect when it is in danger and ‘trial pheromones’ used to get the attention of other insects when food is found. Queen of honey bees releases a pheromone to attract the worker bees. The pheromone released from the mandibular glands of the queen induces the worker bees to feed and groom her and inhibit ovary development in the workers. There are ‘sex attractants’ also with which one sex of an insect (usually females) attracts the opposite. But in the case of insects, these sex attractants are often fatal to the host plants. One such is the attack on a tree by bark beetles. Initially a few beetles attack and bore into the tree to construct a nuptial chamber, and expel frass, which ia a mixture of fecal pellets and wood fragments. This frass contains an attractant that triggers massive secondary invasion that kills the tree. IpsConfusus, a male forest pest, produces an attractant that can attract three females for mate. This behaviour had killed billions of board feet of timber in USA and Europe. Hence, it has become important to know the composition of the pheromones released by insects in order to save the plants. Alkyl substituted 6,8-dioxabicyclo [3.2.1] octane skeleton is a common structural subunit found in the pheromones of a variety of bark beetle species, which act as ‘sex attractants’ in their communication system[1-2]. Several researchers focused their study to isolate and identify chemical structures of pheromones of these pests. Brevicomin (7-ethyl-5-methyl-6, 8-dioxabicyclo[3.2.1]octane, Scheme 1.1) was the first pheromone of the 6,8 -dioxabicyclo [3.2.1] octane skeleton identified from western pine beetle, Dendroctonusbrevicomis [1]. Later on multistriatin, frontalin, and isomeric brevicomins and hydroxybrevicomins were reported as active components in the beetle species (Scheme 1.1) [2-4].
Scheme 1.1
These pheromones are optically active, and they exist either in optically pure form or in a mixture with enantiomeric excess of one compound [5-7]. Dendroctonusbrevicomis emits both exobrevicomin and endobrevicomin but only the exo isomer is found to be active. Hence, knowledge of absolute configuration is essential for successful pest management. Structural assignment of the identified pheromones have been done based on optical rotation, infrared, nuclear magnetic resonance and mass spectral data; along with their unambiguous synthesis [8-14]. Most of the times, these natural compounds are obtained in traces, hence mass spectrometry is the ideal technique to analyze these compounds because the mass spectrometry is a proven technique for not only compounds of low quantity but also for mixture by using hyphenated techniques such as gas-chromatography-mass spectrometry (GC-MS). Many researchers tried electron ionization (EI) conditions for structural elucidation of pheromones, and the EI mass spectra of multistriatin, frontalin, brevicomin and some other substituted brevicomins have been reported in the literature. But the studies were not focused on the differentiation of isomeric pheromones.
Mass spectrometry has been used as a tool for the differentiation of diastereomers (acyclic and cyclic) and enantiomers. The pheromones belong to diastereomericbicycliccompounds; hence, the literature studies focused in such type of compounds are discussed below. A very few reports could be found in the literature that covered the differentiaion of endo-exo isomers by mass spectrometry. One of the earliest studies was done by Peters et al. [15], who studied 3-Oxabicyclo[3.3.1]Nonane (1), its derivatives, viz. 7-exo alkyl derivatives (2a-2c) and 7-endo-alkyl derivatives (3a-3c) (Scheme 1.2).
Scheme 1.2
The most important difference was found in the EI mass spectral behavior of 2c and 3c. In the spectrum of 2c the loss of ‘t-Bu’ yielded the ion at m/z 125. In the spectrum of 3c, in addition to the peak at m/z 125, peaks are found at m/z 126 and 127 (loss of C4H8 and C4H7radical from molecular ion, respectively). This spectral behaviour was explained by the fact that the t-Bu group in compound 3c was able to approach the ‘O’ atom by the conversion of the boat ring into a flattened chair, allowing a transannular hydrogen transfer. Then elimination of C4H8 by a simple cleavage reaction afforded the ion at m/z 126.
Curcuruto et al. studied the EI mass spectral behaviour of mono and di substituted exo (4 and 6) and endo (5 and 7) norbornanes (Scheme 1.3) [16]. Clear differences are observed in the relative abundances of fragment ions in the EI spectra of the methyl ester derivatives 6 and 7. The greatest difference is in the relative abundances of the ion at m/z 122 due to primary methanol loss, which are 84%, for 7 and 7% for 6. Such behaviour is analogous to that observed for compounds 4 and 5, related to water loss (50% in 5 and 25 % in 4).
Scheme 1.3
Formation of [M-R1OH]+.ion in high abundance in endo isomer was explained by a favourable ‘H’ transfer on the ester oxygen (Scheme 1.4) and this was the easiest process in endo compound. They have also attempted to record the mass analyzed ion-kinetic energy (MIKE) spectrum of the molecular ions, but surprisingly the spectra of these isomers were
Scheme 1.4
found to be almost similar. The differences well evidenced under EI conditions were absent in this case. This behaviour of MIKES of the molecular ions was explained by invoking isomerization of the respective molecular ions in the second field free region to produce an identical structure from both isomers. It was considered that the time required to reach the second field-free region was longer than the the time required for isomerization.
Mass spectral behavior of several kinds of stereoisomerically fused diexo/diendo compounds was studied, but clear-cut differentiation between the isomers could be found only in few cases. Pihlaja et al reported some norbornane fused thiazolo[3,2-a] pyrimidin-5-ones (8-11) and norbornene fused thiazolo[3,2-a] pyrimidin-5-ones (12-15) [17] (Scheme 1.5).The unsaturated compounds showed peaks corresponding to retro diels-alder reaction (RDA) and RDA+H. The isomeric compounds showed difference in the relative abundance of RDA+H ions. Norbornane fused compounds showed difference in the relative abundances of the molecular ions. The relative abundances of molecular ions of diendo-fused isomers were approximately 3 times higher than those of diexo-fused isomers.
Scheme 1.5
Partanen et al. attempted the isomeric differentiation of norbornane/enedi-endo and di-exo fused 1,3 Oxazin-2(1H)-ones (16 and 17; 20 and 21) and corresponding 1,3-Oxazine-2(1H) thiones (18 and 19; 22 and 23) [18] (Scheme 1.6).Under EI conditions all the saturated compounds gave rise complicated fragmentation pattern that include several rearrangements. Retro-Diels-Alder (RDA) process was the most favoured fragmentation pathway, in this case rearrangement with hydrogen yielded RDA+H fragment ion. Peak corresponding to water loss from (RDA+H) product ion was also observed. However, the isomers gave closely similar mass spectra and hence clear cut isomeric differentiation was not possible.
Scheme 1.6
Under ammonia chemical ionization (CI) conditions, unsaturated compounds (16-19) yielded abundant peaks corresponding to RDA reaction at m/z 100 and elimination of aminoformic acid (NH2COOH) accompanied by a hydrogen transfer at m/z 105. Both of the reactions showed some stereospecificity being more favoured for the diexo than the diendo isomer. The same behaviour noted with compounds 18 and 19 with more obvious differences. For saturated compounds, the situation was complicated and isomeric differentiation was more difficult. The only abundant fragment ion in the CID mass spectra of the [M+H]+ ions generated from 20 and 21 was at m/z 107 corresponding to the elimination of amino-formic acid. In this case, the elimination was more favoured in diendo isomer. For 22 and 23 the protanated molecular ion decomposed mainly through the loss of NH2CSOH, and here again this elimination was slightly more favoured for the diexo than for the diendo isomer.
There were a few examples, where the exo (25) and endo (24) isomers were differentiated by CI methods. Morlendeer-vais and Mandelbaum studied exo and endo isomers of 2,3-cis and 2,3- trans 3-methoxytricyclo[6.2.2.02,7] dodeca-9-ene/ dodeca-9-ane [19] under isobutane CI conditions (Scheme 1.7). Elimination of methanol from protonated molecule was the predominant feature of exo isomer (100%), whereas this process was very less in the corresponding endo isomer (44%). This behavior reflected the greater stability of the [M+H]+ ion of endo isomer, which could be stabilized by the internal hydrogen bond with the π-electrons of the double bond.
Scheme 1.7
The stabilization (due to the increase in the proton affinity) of the proton bridged [M+H]+ ion of endo isomer was also reflected in the d3-acetonitrile (D3C-CN) /CI mass spectra of these compounds 24 and 26 (Scheme 1.8). The endo isomer (24) exclusively exhibited the [M+D]+ ion (m/z 194) while exo isomer (25) yielded an abundant [MD+CD3CN]+ ion (m/z 238). Processes of methanol elimination from [M+D]+ ion was also different for both the isomers. The exo isomer resulted in the expected [MD-CH3OD]+ ion at m/z 161, while the endo compound afforded the [MD- CH3OH]+ ion at m/z 162, in addition to the ion at m/z 161. The unusual elimination of CH3OH from [M+D]+ ion of endo compound was explained by the exchange of the deuteron in the [M+D]+ ion on the oxygen atom with the hydrogen on the interior of the organic moiety prior to the C-O bond dissociation.
Scheme 1.8
1.1.2. Scope of the Work
Isomeric hydroxybrevicomins are reported to be active pheromones in the beetle species. Stereospecific synthesis of three isomeric hydroxybrevicomins has been reported [23-25]. There have been few reports on the EI mass spectra of exo and endo isomers of brevicomin and hydroxybrevicomins. Though it was mentioned that the spectra of exo and endo isomers show differences, but no attempts were made to explain the differences. The authors used the Mass Spectrometry technique for structural elucidation purpose, but not for isomeric differentiation. The biological importance of these compounds is an inspiring factor to study the detailed mass spectral behaviour of these three isomeric hydroxybrevicomins. It is worthwhile to study these pheromone compounds under EI conditions considering their volatility. Moreover, as these compounds are associated with other components, especially isomeric compounds, a chromatographic separation system prior to MS is ideal for unambiguous analysis. Further, tandem mass spectrometry techniques (precursor/daughter ion scans) help in understanding the fragmentation pattern. With a view to differentiating the three isomeric hydroxybrevicomins, they are analyzed by GC-MS and MS/MS conditions under both EI and CI conditions.
1.1.3. Results and Discussion
Chemical structures of the studied isomeric hydroxybrevicomins (compounds 1-3) are shown in Scheme 1.9. The compounds 1-3 are analyzed by GC-MS. The retention times for compounds 1, 2 and 3 are 10.3, 10.5 and 11.18 min, respectively. The three isomers are well separated under the used GC conditions, which rules out the ambiguity of contribution of one isomer in the spectrum of another. The EI mass spectra of 1-3 recorded
Scheme 1.9.
at 70 eV from GC/MS analysis are given in Figure 1.1. The spectra of isomers show distinct differences in the relative abundances of fragment ions that enables differentiate one isomer from another isomer.
The molecular ions (M+.,m/z 172) are observed in the spectra but they are found to be low abundant (<1%). The [M-C2H5]+ ion (m/z 143) is the fragment ion appeared at high mass region. Even, the spectra recorded at low eV (20 eV) appear similar to that obtained at 70 eV, hence the spectra of 70 eV considered for discussion hereafter. The spectra of 1-3 do show same set of fragment ions (same m/z values), but noticeable differences can be seen in the relative abundances of diagnostic fragment ions at m/z 114, 112, and 83. Among the three isomeric compounds, the spectrum of 2 appears distinct from 1 and 3; wherein the ion at m/z 114 appears as the base peak, and this ion is less abundant in 1 and 3 (17 and 6 %, respectively). The ion at m/z 112, which is moderately abundant in 1 and 3 (37 and 39%, respectively), is less abundant in 2 (8 %). Similarly the ion at m/z 83 is the base peak in 1 and 3, while it is only 43 % in 2. Between 1 and 3, a small but consistent difference is observed in the relative abundance of the ion m/z 114, in which it is relatively higher in 1 (17%) than in 3 (6%). Apart from these, the fragment ions 143 and 101 are relatively more abundant in 1 and 3, but they are found to be lower in 2. The spectral differences are consistent and reproducible in repeated analysis at different times
Though the molecular ions are low abundant, attempts are made to record their collision-induced dissociation (CID) spectra. The CID spectra of M+.(m/z 172) from 1-3 are shown in Figure 1.2. The spectral differences observed in the EI spectra among 1-3 are more apparent in the CID spectra of their M+. ions, and the spectra are very clear with reduced secondary fragmentation. The spectrum of 2 is exclusively dominated by the ion
atm/z 114. The spectra of 1 and 3 showed the ion at m/z 112 as the base peak along with other fragment ions at m/z 143, 130, 129, 114 and 83. The characteristic
Figure 1.1. EI-mass spectra of compounds a) 1, b) 2 and c) 3
Figure 1.2. CID spectra of ion m/z 172 generated from a) 1, b) 2 and c) 3
fragment ion at m/z 114 is relatively higher in 1 than in 3 as observed in their EI spectra. Although there is no difference in the relative abundance of 143 and 101 in the EI spectra of 1 and 3, these ions are consistently more abundant in the M+.ion CID spectrum of 3 than that of 1.
In order to rationalize the observed differences among 1-3, it is important to arrive at the fragmentation pattern for those diagnostic ions. The fragmentation pattern is arrived for compounds 1-3 by using precursor/product ion spectra and HRMS data, and is summarized in Scheme 1.10. Moreover, the spectrum of 3d confirms the fragmentation pattern for some ions by showing the expected shift/retention in the mass values. The key fragment ion for differentiating the isomeric compounds, m/z 114 corresponds to the loss of 58 u from M+. ion. Precursor ion spectrum of the ion m/z 114 showed exclusively M+. ion as the precursor.
Scheme 1.10
The HRMS data for the ion m/z 114 (measured mass m/z 114.0679) confirms that the 58 u loss corresponds to a neutral C3H6O moiety (theoretical mass m/z 114.0681). The proposed mechanism for the formation of m/z 114 is given in Scheme 1.11. Similar fragmentation process was reported earlier in the EI fragmentation of brevicomins and related bicyclic compounds [9, 20].
Scheme 1.11
The other important characteristic ion at m/z 112 could be formed by the loss of 60 u from M+. ion. This interpretation is supported by the fact that the M+.ion is observed as the major precursor in its precursor ion spectrum. The loss of 60 u may correspond to the loss of C3H8O or C2H4O2. The HR spectra of 1-3 include a single peak at m/z 112.0883. It corroborates that the ion m/z 112 is formed by the loss of C2H4O2 moiety from M+.ion. The loss of CH3CH2COOH was reported earlier in multistriatin, but expected CH3COOH was not found in brevicomins [20-22]. Appearance of the [M-60]+ion in hydroxybevicomins, but not brevicomins suggests that the -OH group in 1-3 may be initiating this fragmentation process. The involvement of hydrogen of the -OH group is confirmed by labelling the hydroxyl hydrogen with deuterium (3-d). The ion at m/z 112 remains at the same m/z value in the EI mass spectrum of 3-d, whereas other fragment ions are shifted as expected (Figure 1.3).
Figure1.3. EI-Mass Spectrum of compound 3-d
Though the deuterium labelling experiment provided the evidence for involvement of hydroxyl hydrogen in the formation of the ion m/z 112, the actual mechanism for the formation of this ion appears to be a complex process, and it is difficult to propose a structure for this ion with the available data.The base peak ion at m/z 83 in 1 and 3 might be formed by the loss of ethyl radical from the ion at m/z 112, because its precursor ion spectrum include the ion at m/z 112 as the major precursor along with the other minor precursors at m/z 143 and 101 (Figure 1.4). The ions m/z 143 and 101 correspond to [M-C2H5]+ and [M-C2H5-COCH2]+ ions, respectively. Formation of the ion m/z 112 is less favored in 2, hence the ions m/z 101 and 143 might be leading to formation of the ion m/z 83 in 2; in fact the precursor ion spectrum of the ion m/z 83 did show the ion at m/z 101 as the major precursor in addition to the minor precursor ion m/z 143.
The compounds 1-3 have four chiral centers at positions 1, 2, 5 and 7. The compounds 1 and 2 are a pair of exo and endo isomers that differ in the stereochemistry of ethyl substituent at position 7. The compound 3 has enantiomeric relation with 1 with respect to the stereochemistry at position 1, 5 and 7, but configuration of the hydroxy group
Figure 1.4 Precursor ion spectrum of ion m/z 83 generated from compounds a) 1 and b) 2.
at position 2 remains same in both 1 and 3 (R configuration) [23]. Since, enantiomeric relation do not cause any spectral difference, the major difference between 1 and 3 in a three dimensional view is only the orientation of hydroxyl group. In general, endo isomers are known to be less stable than exo isomers as a result of steric repulsions between the substituent and the ring hydrogens [24]. Infact, Mundy et al. [9] computed total steric energies of endo and exobrevicomins, and showed that endo isomer has relatively more energy when compared with the exo isomer. The higher energy for endo isomer could be due to repulsive interactions between ethyl group at position 7 and substituents at position 2 (Scheme 1.12), because the difference between the two isomers is only at the stereochemistry at position 7. In a similar way, the endo isomer of hydroxybrevicomin (2) is expected to be more energetic than the corresponding exoisomer (1). The repulsive interactions in endo isomer (2) can be clearly viewed in Newmann projections (Scheme 1.12).
Scheme 1.12. Newman projection formulae for compounds 1-3.
The proposed fragmentation process for [M-58]+.ion (m/z 114) in 1-3 releases strain in the bicyclic system; hence, formation of the ion m/z 114 is anticipated to be favoured in 2 than in 1. In fact, the ion m/z 114 is dominant in spectrum of endo isomer (2) when compared with exo isomers (1 and 3). Between the isomeric pair of 1 and 3, both are exoisomers but the hydroxy group is in axial position in 1, where as it is equatorial in 3. Therefore, the compound 1 is expected to be less stable than 3, because of repulsive 1, 3- interactions [24] of the axial hydroxy group with axial hydrogen of cyclohexane ring. Based on this, it is reasonable to explain the formation of the ion m/z 114 is relatively higher in 1 than in 3. Apart from the ion m/z 114, the exo isomers (1 and 3) show relatively more abundant fragment ions due to simple cleavages leading to ions such as m/z 130, 143, 101 etc. when compared to the endo isomer (2). From the above experimental results, it can be concluded that the compound 3 is more stable and 1 and 2 follow next in the order.