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. Ips Confusus, 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, Dendroctonus brevicomis [1]. Later on multistriatin, frontalin, and
isomeric brevicomins and hydroxy brevicomins were reported as active components in the beetle
species (Scheme 1.1) [2-4].
o
o
Multistriatin
o
o
o
o
Frontalin
Brevicomin
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]. Dendroctonus brevicomis emits both
exo brevicomin and endo brevicomin 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 [814]. 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 diastereomeric bicyclic
compounds; 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 C4H7
radical 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).
COOR1
R
R
COOR1
4
6
5
7
4, 5 ;
6, 7 ;
R = H, R1 = H
R = H, R1 = CH3
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
+ .
R
H
O
R1
O
R
.
H
+
O
R
.
O
R1
O
+
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.
O
O
N
X
()
n
X= -CH=CH-
n =2
N
()
N
S
N
n =1
X
n
S
x= -CH 2 -CH 2 -
endo
8
12
exo
9
13
endo
10
14
exo
11
15
Scheme 1.5
Partanen et al. attempted the isomeric differentiation of norbornane/ene di-endo and diexo 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.
O
O
N
N
X
X=O
X=S
16
18
X
17
19
O
O
N
N
X
X=O
X=S
20
22
21
23
X
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.
H
H3CO
H
24
endo
OCH3
25
exo
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.
endo
CD 3CN-CI
+
D
H
O
CH3
exo
CD 3CN-CI
[MD+CD 3CN] +
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 hydroxy brevicomins 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 hydroxy brevicomins. 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 hydroxy brevicomins (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
OH
OH
RO
O
O
O
O
O
O
(1R,2R,5S,7R)-2-hydroxyexo-brevicomin
1
(1R,2R,5S,7S)-2-hydroxyendo-brevicomin
2
(1S,2R,5R,7S)-2-hydroxyexo-brevicomin
R=H
R=D
3
3-d
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 collisioninduced 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
at m/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.
C7H13 O2 (m/z 129)
C7H12 DO 2 (m/z 130)
C9H15 O2 (m/z 155)
C9H15 O2 (m/z 155)
C6H10 O2 (m/z 114)
C6H9DO 2 (m/z 115)
.
-COCH 3
.
-OH
-C3H6O
OR
C7H14 O2 (m/z 130)
C7H13 DO 2(m/z 131)
-COCH 2
-C2H4
O
O
+.
-C2H4O2
C7H12 O (m/z 112)
C7H12 O (m/z 112)
.
-C2H5
C5H7O (m/z 83)
C5H7O (m/z 83)
M ion
C9H16 O3 ( R=H m/z 172)
C9H15 DO 3 (R=D m/z 173)
.
-C2H5
C7H11 O3 (m/z 143)
C7H10 DO 3 (m/z 144)
-COCH 2
C5H9O2 (m/z 101)
C5H8DO 2 (m/z 102)
C7H12 O3 (m/z 144)
C7H11 DO 2 (m/z 145)
-COCH 2
C5H10 O2 (m/z 102)
C5H9DO 2 (m/z 103)
-H2O
C5H8O (m/z 84)
C5H8O (m/z 84)
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].
+.
OH
OH
O
O
O
M
+.
o
OH
+.
O
O
+
.
+ C2H5-CHO
m/z 114
ion of 1-3
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 hydroxy bevicomins, 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 3d, whereas other fragment ions are shifted as expected (Figure 1.3).
83
100
43
80
60
40
102
112
55
74
20
144
130
0
40
60
80
100
120
140
160
173
180
m/z
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 exo
brevicomins, 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 hydroxy brevicomin (2) is expected to be more energetic than the corresponding exo
isomer (1). The repulsive interactions in endo isomer (2) can be clearly viewed in Newmann
projections (Scheme 1.12).
1
2
3
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 exo isomers 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.
With a view to checking the order of stability for compounds 1-3, theoretical calculation
studies have been performed on the molecular ions of 1-3. Optimized geometries of the isomeric
compounds is given in Figure 1.5. The energies obtained are -577.95093679, -577.94322181 and
-577.95215873 for the compounds 1, 2 and 3 respecively, in atomic units.These values show the
compound 2 is highly unstable with 5.67 kcal/mol excess energy than the stable isomer. It is
interesting to note that among the exo isomers, compound 1 is found to have 0.77 kcal/mol
excess energy than compound 3. Thus the order of the stability is 3>1>2 which is in agreement
of the experimental data.
.
Figure 1.5 Optimized geometries of the compounds 1-3
1.1.4. Chemical ionization (CI)
The CI-MS technique is also a proven method to distinguish cyclic stereoisomers,
wherein the isomers with substituent in axial/equatorial position show stereoselective
fragmentation that enable discrimination of one isomer from another [25-26]. With a view to
studying the effect of axial/equatorial hydroxyl groups in 1-3, we extended the study under CI
conditions. The CI mass spectra of 1-3 were recorded using methane, isobutate and ammonia as
reagent gases. Before recording the spectra, the CI experimental conditions were optimized by
changing source temperature and pressure of the reagent gas. Isobutane/CI and ammonia/CI
yielded exclusively protonated molecule, [M+H]+ as the abundant ion. However, the methane/CI
spectra showed two abundant ions corresponding to [M+H]+ and [MH-H2O]+ ion (Figure 1.6).
The [MH-H2O]+ is the base peak for all the compounds, but noteworthy differences are observed
in the relative abundance of the [M+H]+ion (60, 56 and 25% in 1, 2 and 3, respectively). The
[M+H]+ ion is found to be low abundant in 3 than in 1 and 2, whereas it is almost same in 1 and
2. It indirectly indicates the [M+H]+ ion of 3 is less stable than those of 1 and 2 and leads to the
formation of relatively high abundant [MH-H2O]+ ion. It can be noted that the hydroxy group is
in equatorial position in 3, where as it is axial in 1 and 2, hence the observed difference in the CI
spectra is primarily due to the position of hydroxy group (axial/equatorial), and other
stereochemical effects are negligible. The axial hydroxy groups are known to enhance stability
for cationized/protonated species of diols and sugars [27-29]. Similarly, the [M+H]+ ion from 1
and 2 could gain better stability with the solvation of proton by axial hydroxy group and ring
oxygen, whereas such stability is not possible in 3.
155
100
80
0
a
173
60
0
40
0
20
0
0
83
80
60
99
137
100
120
140
160
180
m/z
180
m/z
155
100
b
80
60
173
40
99
20
137
115
0
80
60
100
120
140
160
155
100
80
c
0
0
60
0
0
40
0
0
173
20
83
0
Figure
60
80
97
100
137
113
120
140
160
180
m/z
2H-1 benzopyran (Chromenes) and 3, 4-dihydro-2H-1 Benzopyran (Chromans) are
important classes of oxygenated heterocycles that have attracted much synthetic interest,
O
O
because of the biological activity
of naturally occurring
representatives. Benzopyran ring system
Chromene
Chroman
is found in over 4000 natural and designed products, exhibiting an extensive range of biological
activities. Many Benzo pyran derivatives contain anti cancer and vasorelaxant properties. 4Aminobenzopyrans and their derivatives have drawn considerable attention in the last decade as
modulators of potassium channels influencing the activity of the heart and blood pressure [32].
Fused tetrahydropyranobenzopyrans and tetrahydrofuranobenzopyran derivatives are also
frequently found in naturally occurring bioactive molecules, and this attracted many organic
chemists to develop synthetic methods [33]. There have been very few reports on the synthesis of
linear tricyclic benzopyrans. Yadav et al. reported a simple, one pot synthesis of N-(aryl)
tetrahydropyrano chromenylamines and N-(aryl) tetrahydrofurano chromenylamines by treating
an appropriate salicylaldehyde’s schiffs base with dihydropyran or dihydrofuran in the presence
of a catalyst [34]. This reaction involves a Diels-Alder cycloaddition reaction and is expected to
yield a mixture of stereo isomers, viz., the isomers due to ring fusion (cis/trans fusion) and the
isomers due to generation of a new chiral centre at the linkage of N-aryl substituent. However,
the experimental products are found to be exclusively the cis fused linear tricyclic products;
hence, there were only two diastereomers, differing at the stereochemistry of N-aryl substituent
(Scheme 1.13).
R
N
R
H
HN
H
H
LiBF 4
CH3CN, RT
+
OH
R
HN
O
H
+
O
O
H
O
O
H
Scheme 1.13
The diastereomeric N-(aryl) tetrahydrofuranochromenylamines can be easily separated by
column chromatography. The diastereomeric N-(aryl) tetrahydropyranochromenylamines cannot
be separated by column chromatography, but they can be separated with techniques like gas
chromatography (GC) or reverse phase high-performance liquid chromatography (HPLC).
Therefore, the hyphenated techniques such as gas/liquid chromatography coupled to mass
spectrometry (GC-MS or LC-MS), which enables simultaneous separation and identification, are
ideal for the analysis of such reaction products to characterize the products without prior
separation, provided that the stereoisomers give distinct mass spectra. Therefore, knowledge on
the mass spectral behaviour of the diastereomeric tetrahydropyrano or tetrahydrofurano
benzopyrans is crucial in the analysis of above reaction mixtures.
Mass spectrometry is a proven technique for the differentiation of stereoisomers by
applying the appropriate methods. The isomeric pyran derivatives have been well studied by
mass spectrometry [35]. Electron ionization (EI) fragmentation patterns of pyran derivatives and
their fused cyclic compounds have been documented in detail [36-38]. The fragment ions due to
Retro-Diels Alder (RDA) reactions were found to be key to the characterization of these
compounds. The RDA reaction has been successfully used to characterize different cyclic
systems containing a double bond in a six-membered ring such as substituted cyclohexenes,
tetralins [39], fused bicyclic [40-41], tricyclic[42], polycyclic compounds [43-45], oxazines [46],
isobenzofurans, naphthofurans [47], isoquinolines [48], chromans [49,50], chromones [51] etc.,
which include differentiation of isomeric compounds also [52-55].
1.2.2. Retro-Diels-alder Reaction
It is well known that in EI mode, the electron beam will strip off an electron from the
molecule to produce a molecular cation. The presence of double bonds provides π electron for
the electron removal. The distonic species then undergoes an -cleavage and forms an alkene
and diene. This reaction is called Retro-Diels-Alder reaction (RDA). A typical example and its
mechanism are explained in the scheme 1.14.
+.
+.
+.
+
Mechanism
-e-
.
.
+
+
+
+
.
.
.
+
+
+
Scheme 1.14. Typical example of an RDA reaction in cyclohexene under EI.
The mechanism and thermochemistry of RDA reaction is extensively discussed in a
review article [56]. Some times the RDA fragmentation is associated with a transfer of one or
two hydrogens also. For example, oxygen containing heterocyclic compounds exhibited this
behaviour. Shizuko Eguchi showed that 1-chromanones yield the fragment ions due to
RDA+H [57] (Scheme 1.15).
+.
H3CO
+ . RDA
O
C
O
H3CO
O
O
H3CO
+
OH
RDA+H
C
O
Scheme 1.15. Fragmentation of 7-methoxy-2,2dimethyl chromanone
Mandelbaum et al. also showed the occurance of hydrogen transfer in norbornene and
bicyclo[2.2.2] octane systems during RDA [58] (Scheme 1.16). Thus RDA fragmentation has
been characteristic for the most of the double bond containing cyclic compounds in EI. It has
been shown that the RDA fragmentation depends upon many sterochemical grounds by which it
is used for the differentiation of stereoisomers. Mostly, the RDA fragmentation was used to
discriminate cis- and trans- fused cyclic compounds, wherein cis fused isomers undergo RDA
reaction more favourably than trans fused isomers.
Scheme 1.16. Hydrogen transfers associated with RDA fragmentation in norbrnene anhydride
and bicycle [2.2.2] octane anhydride.
Bel et al. reported the differential behaviour of the cis and trans isomers of the 1,2,3,4,9apentamethyl-1,4,4a,9,9a,10-hexahydroanthracene [59]. The cis isomer showed both possible
RDA fragmentations and resulted in most abundant ions by the cleavage of the cyclohexene ring.
The trans isomer exhibited entirely different behaviour. The RDA fragmentation of the tetralin
ring B gave the most abundant ion (Scheme 1.17).
Zitrin et al. reported the EI fragmentation of some diketones (Scheme 1.18) where cis
isomer undergoes RDA and trans isomer did not show this ion. The same behaviour is observed
with analogus systems also [60].
+ .
100%
26%
+ .
+ .
B
A
B
A
H
H
+ .
10%
100%
Scheme 1.17
+ .
+ .
+ .
(CH2)n
O
(CH2)n
(CH2) n
O
H
(CH2)n
O
(CH2) n
H
(CH2) n
O
Scheme 1.18
Lesman et al. [61] studied the mass spectra of exo (Ib-IVb) and endo (Ia-IVa) isomers of
adducts of 1,1’dicycloalkenyls with maleimide and n-phenylmaleimide (Scheme 1.19) The
spectrum of endo isomers contained (RDA-H) ion as the abundant peak, and a low abundant
RDA peak. But the RDA reaction with and without hyderogen transfer gave rise only to small
peaks in the mass spectra of exo isomers. The low abundance of the peak corresponding to ion a
(m/z 133) in the mass spectrum of exo isomers was explained by the
(H2C)n
(H2C)n
O
O
C
C
NH
NH
C
C
(H2C)n
n=1
n=2
n=3
n=4
O
(H2C)n
Ia
IIa
IIIa
IVa
Ib
IIb
IIIb
IVb
Scheme 1.19
O
restrictive distances between the hydrogens at postiotions 3 and 6 and the oxygen atoms of the
carbonyls. Assuming the absence of skeletal rearrangement in the molecular ion prior to
fragmentation, the authors had proposed a mechanism (Scheme 1.20) for the formation of (RDAH) ion. The high stability of the pentadienlyic ion ‘a’ presumably furnishes the driving force for
the reaction.
+.
H
O
OH
+
+
NH
O
NH
O
.
ion ' a'
Scheme 1.20
Similarly RDA ions dominated the EI mass spectra of few cis-annulated isomers of
diketone and diethers (Scheme 1.21). The trans annulated isomers of these compounds though
show abundant molecular ions but RDA fragments are negligible or absent [62-65].
OR
O
R
R
O
X
OR
O
OR
O
OR
Scheme 1.21. Skeletal structures of the various isomeric compounds studied
RDA reactions have also been shown in the negative mode. David et al. observed that the
quinolone antimicrobial agent, ofloxacin, 9-fluoro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo2,3-dihydro-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid underwent cyclo reversion
in negative ion chemical ionization (NCI) conditions [66]. The mass spectrum revealed a radical
anion at m/z 361 and the fragment ion at m/z 319 (relative abundance 20%) (Scheme 1.22).
-.
O
-.
O
O
F
F
OH
OH
-C3H4
N
N
H3C
O
N
N
O
CH3
N
N
O
H3C
Scheme 1.22
This observation had been an inspiration to Etinger et al. who further extended the
technique for isomeric differentiation [67]. Cis annulated diones exhibited sterospecific RDA
reactions in their positive mode of EI and CI conditions. The corresponding trans isomers did not
undergo RDA fragmentation. The NCI mass spectrum of diones exhibited practically no
fragmentation below laboratory collision energy of 60 eV. At higher collision energies, like 80
eV, RDA fragmentation occurred only for cis annulated isomers.
In another work, cis-and trans-Diels-Alder adducts of p-benzoquinones and
glucofuranodienes were studied under negative ion mode. The RDA process enabled the
stereoisomers to be differentiated. The cis-isomers undergo a highly favoured RDA reaction
[68].
Recently, Morlender et al. reported the differentiation of the stereoisomers (2, 3 cis and
2,3 trans 3-methoxytricylco [6.2.2.02,7] dodeca-9-enes) generated by a chiral centre remote from
the ring junction [69] (Scheme 1.23). The isomers exhibited different behaviour under EI. The
expected cyclohexa-1,3-diene radical cation (m/z 80) formed by RDA fragmentation was the
most abundant ion in the 70 eV mass spectrum of endo isomer. This ion was much less important
in the spectrum of exo isomer where m/z 111 corresponding to O-methylcyclohex-2-en-1-one,
which is formed by an RDA fragmentation accompanied by a hydrogen migration (RDA-H) with
the charge retained in the dienophile moiety was the major peak.
In the part 1 of this chapter, isomers of substituted conduramines have been successfully
differentiated by isomeric specific fragmentations under EI conditions. The differences observed
are consistent, but smaller in few cases. It is known that the fragmentation of protonated
molecular ions, produced under chemical ionization conditions is different to that from
molecular ions produced under EI conditions.
Hence, the study is extended to chemical
ionization (CI).
2.2.1.1. Brief introduction to CI
CI technique is especially useful when the molecular ions are absent in the EI spectra of
compounds, and it is also being used to confirm the molecular weight of a compound. The source
components of CI technique are nearly the same as that of EI, except, CI uses tight ion source,
and a CI reagent gas (Figure 2.7).
CI reagent gas
(pressure 1 torr)
Figure 2. 7. Schematic diagram of CI interface
Reagent gas (e.g. methane, iso-butane and ammonia) is first subjected to electron impact
to yield reagent gas ions. Because of the high pressure in the ion source, these initial reagent gas
ions further undergo ion-molecule reactions with neutral reagent molecules (G) to yield reagent
selective ions (reagent plasma, e.g., GH+). When sample is introduced, the sample molecules (M)
undergo ion-molecule reactions with reagent plasma to produce sample ions. In general, reagent
gas molecules are present in the ratio of about 100:1 with respect to sample molecules. Pseudomolecular ions, [M+H]+ (positive ion mode) or [M-H]- (negative ion mode) are often observed.
Unlike in EI method, the CI process is soft ionization and yields abundant quasi-molecular ions,
with less fragment ions.
Positive ion mode: GH+ + M ------> MH+ + G
Negative ion mode: [G-H]- + M ------> [M-H]- + G
In CI mass spectrometry the molecules of a vaporized sample are ionized by a set of
reagent ions (reagent plasma) in a series of ion-molecule reactions. The energy transferred by
these reactions is lower than the energy imparted by electrons in EI source, and therefore
fragmentation of the sample molecules is greatly decreased. For this reason CI mass
spectrometry has been finding increasing use as a tool for the molecular weight confirmation and
for elucidation of structure of variety of organic compounds including differentiation of isomeric
compounds. Generally hydrogen (H2), methane (CH4), isobutane (iso-C4H10) and ammonia
(NH3) are used as reagent gases in CI mass spectrometry [42-43]; with all these CI gases the
compounds form protonated molecule ion in their CI spectra. For example, when CH4 is used as
the reagent gas in CI mass spectrometry, the reacting species formed in CH4 plasma are CH5+,
C2H5+ and C3H5+. These three ionic species undergo ion-molecule reactions with the neutral
sample molecules and produce [M+H]+, [M+C2H5]+ and [M+C3H5]+ ions, respectively (where M
is the neutral analyte molecule). In general, the CI mass spectra recorded using CH4 as the
reagent gas includes fragment ions in addition to above mentioned pseudo-molecular ions.
Specially designed apparatus enables use of many other gases as CI reagents. Acetone, dimethyl
ether, ammonia, trimethyl borate, trimethyl silyl ion, formaldehyde dimethyl acetal and
methylene chloride are some examples [44-48].
Chiral recognition has been demonstrated by the use of suitable chiral CI reagents [4950]. Isomeric differentiation was also attempted under CI conditions. But the studies were
limited to a limited variety of compounds. CI mass spectra of cyclic alcohols were extensively
studied and their fragmentation patterns were elaborately discussed. One of the early studies
related to the stereochemical effects in the CI mass spectra was reported by Winkler and
McLafferty [51]. The authors reported the isobutane CI mass spectra of cis and trans isomers of
(1, 2), (1,3) and (1,4) cyclohexane diols. The spectra of cis- and trans- 1,4 diols showed distinct
mass spectra. The [MH-H2O]+ and [MH-2H2O]+ ions were found to be relatively more abundant
in trans isomer than in cis isomer. And the [M-H]+ peak was abundant in the spectra of trans
isomer, where as it was absent in the cis isomer. The relative abundance of dimeric species,
[2M+H]+ was more abundant in cis isomers. The effect of temperature and pressure on the CI
mass spectra was also studied.
Later, many researchers worked on the stereo chemical differentiation of cis- trans
isomers and those were discussed at length in many reviews [52-53], and also discussed in a
book covering the results up to 1992 [54]. Many scientific groups worked on isomeric
differentiation in CI conditions involving different reaction mechanishms. Cis and trans isomers
of cyclohexane ethers and esters were extensively studied and isomeric differentiation was
explained.
Shivly et al. studied cis- trans isomers of 1-ethoxy-4-methoxy cyclohexanes (Scheme
2.7) under CI conditions [55]. Cis isomers yielded a high abundant [MH]+ ion where as trans
isomers yielded an abundant [MH-ROH]+ions. The high stability of quasi-molecular ion in the
case of cis isomers was attributed to a proton bridge between two oxygen atoms. It was proposed
that the loss of a neutral alcohol in the trans isomers was due to anchimeric assistance rendered
by the intact alkoxy group leading to a bicyclic structure. The study was further supported by
computational results where the proton affinity of cis diaxial compound was 12.4 kcal/ mol
higher than the trans isomer. Removal of alcohol by anchimeric assistance was 2.7 kcal/ mol
more favourable than the non assisted elimination of alcohol.
Et
Et
O
O
+
+
OMe
H
EtO
+
OMe
H
MH + of Trans
H
EtO
+
OMe
H
EtO
+
-MeOH
-EtOH
+
O
O
MH + of Trans
Me
Me
+
H OMe
OMe
+
OEt
Cis
Scheme 2.7
+ OEt
H
Edelson-averbukh et al. (Scheme 2.8) also reported similar anchimeric assistance in the
ammonia CI fragmentation pattern of cis and trans isomers of protonated benzyl diethers [56].
Elimination of benzyl alcohol was found in the trans isomer of 1,4-bis(benzyloxy)cyclohexane
resulting in a bicyclic ion through an anchimeric assistance by one of the benzyl groups. But cis
isomer was stabilized by the intramolecular hydrogen bonding. This phenomenon was clearly
reflected from the spectra, where the protonated molecule ion is 62% in cis isomers, whereas it is
only 1% in trans isomers.
CH2Ph
+O
PhCH2O
+
OCH2Ph
H
+
OCH2Ph
H
PhCH2O
MH+ of trans
PhH2C
CH2Ph
H
O
+
O
Bridged MH+ of cis
Scheme 2.8
Denekamp and Mandelbaum studied the cis -and trans-1,4-diethers;1, 2-c and 1,2 -t
contain (Scheme 2.9) two different ether functional groups, one of which was primary and the
other was tertiary [57]. The isobutane/CI mass spectra of the cis-isomers, 1-c, exhibited only two
ions. The highly abundant ion corresponding to alcohol elimination, [MH-ROH]+ involving the
tertiary alkoxyl group, and the low abundant [MH-ROH-R’OH]+ ion corresponding to
consecutive elimination of both alcohols. In contrast to this trans isomers had exhibited
competitive elimination of ROH and R’OH affording relatively abundant [MH-ROH]+ (70-80%)
and [MH-R’OH]+ (20-30%) ions. This behaviour was attributed to the random protanation at
each of the two distant non-interacting ether groups, resulting in two isomeric MH+ ions, each of
which eliminating the corresponding alcohol. Where as it was proposed that proton transfer
between the two ether functions in the transient MH+ ions resulted in the total and exclusive
primary elimination of alcohol from the tertiary position of cis isomer. The authors extended this
study in a later publication where they reaffirmed that direct proton transfer via a strained
proton-bound transition state was most probable. The formation of a proton-bound transition
state, despite the large distance between the two alkoxyls in the trans-isomers, might be
implemented by the C-O bond elongation of the protonated primary alkoxy group. Isomeric
differentiation was successfully studied in negative CI conditions also.
OR
OR
[MH-ROH]+
4
4
CI
CI
[MH-R'OH]+
1
1
OR'
OR'
1a-t. R=CD3, R'= CH3
1b-t. R=CH3, R'=CD3
1a-c. R=CD3, R'= CH3
1b-c. R=CH3, R'=CD3
OR
OR
H
H
[MH-ROH]+
4
4
CI
CI
[MH-R'OH]+
1
1
H
OR
'
H
2a-t. R=CD3, R'= CH3
2b-t. R=CH3, R'=CD3
OR'
2a-c. R=CD3, R'= CH3
2b-c. R=CH3, R'=CD3
Scheme 2.9
Suresh Dua et al. [58] discussed the mechanism of methanol loss from the deprotanated
ions of cis- and trans-4-methoxy cyclohexanol (Scheme 2.10). Mechanistic aspects were
proposed with the help of deuterium labeled compounds and computational studies. MS/MS
spectra of the [M-D]- ion from both the isomers were visually similar apart from differences in
the relative abundances of few ions. However, the widths at half height of the peaks produced by
loss of methanol were significantly and reproducibly different. For the cis isomer, it was 29.6 V
and for the trans isomer it was 23.1 V, which means a difference in either the energetics of the
processes or the structure of the product ions. Computational studies insisted on the mechanism
for the loss of methanol in the trans isomer as shown in the Scheme 2.10. It was likely that a
similar mechanism operates for the cis isomer, except that the initial cyclization step was less
favourable in this case.
O
O
-
MeO
OMe
-
O
H
+
O
MeOH
OMe
Scheme 2.10
Edlelson-Averbukh et al. (Scheme 2.11) reported the stereospecific elimination of
dihydropyran (DHP) from protonated tetrahydropyranyl (THP) difunctional derivatives upon CI
and collision-induced dissociation [59]. The study revealed that MH+ ions of the cis isomers
were of relatively low abundant and they undergo efficient elimination of dihydropyran (DHP)
affording abundant [MH-DHP]+ ions. Trans isomers gave rise to highly abundant MH+ ions, but
the peak corresponding to elimination of dihydropyran is low abundant. A similar stereospecific
behavior was observed under CID conditions also.Tetrahydropyranylium ion (m/z 85) obtained
by a simple C-O bond dissociation, is abundant in the CI and CID mass spectra of both
stereoisomers in all the examined systems. Taking thermochemical parameters into consideration
it was expected that the activation energy of the DHP elimination to be higher than that of the
formation of the m/z 85 ion (atleast by 18 Kcal/mol), unless the presence of a stabilizing
interaction is assumed in the [MH-DHP]+ ions, resulting lowering the enthalpy of the DHP
elimination. Internal hydrogen bonding between the the two basic sites in the cis isomers in the
difunctional series was proposed for such stabilization of the [MH-DHP]+ ions which
consequently lower the activation energy of the DHP elimination t the level of of the formation
of the m/z 85 ion.
O
CH2O
CH2O
CH2OR
O
CH2OR
cis-3, R =CH 3
cis-4, R =CH 2C6H5
cis-5, R =H
trans-3, R =CH 3
trans-4, R =CH 2C6H5
trans-5, R =H
O
O
O
OH
OH
cis-6
trans-6
O
Scheme 2.11
The popular belief was that under the CI conditions, protonation occurs on the most basic
site in a multifunctional compound. But studies proved that protonation may not be selective in
some cases. Vais et al. (Scheme 2.12) reported the kinetic
R'
OR
NMe2
7c: R =H,
8c: R =H,
9c: R =Me,
10c: R =Me,
R' =H
R' =n-C 4H9
R' =H
R' =n-C 4H9
Scheme 2.12
R'
OR
NMe2
7t: R =H,
8t: R =H,
9t: R =Me,
10t: R =Me,
R' =H
R' =n-C 4H9
R' =H
R' =n-C 4H9
nature of the protonation process under CI [60]. This study had thrown light on the protonation
process in the bifunctional compounds. The authors studied cis and trans-1- butyl- 4- N, Ndimethyl amino cyclohexanols and their ethers under isobutane/CI condition (compounds 7-10).
All 1, 4-cis, and trans isomers showed differentiation in the abundance of [MH-ROH]+. The
trans isomers yielded [MH-ROH]+ as the base peak and the cis isomers yielded protonated ion
as the base peak, and relatively less abundant [MH-ROH]+ peak. Surprisingly, in the case of
compounds 8c and 8t, only the cis-isomer 8c exhibit significant [MH-MeOH]+ ions under CID
conditions. The non-occurrence of methanol elimination in the CID spectra of the 8t indicates the
retention of the external proton at the dimethylamino group in the MH+ ions that survive after
leaving ion source (Scheme 2.13). The presence of abundant ions in the isobutane –CI mass
spectra of the trans isomers can be understood only if both basic
H
MeO
+
NMe2
fast
100%
C4H9
NMe2
MeO
[MH-MeOH] +
CI
C4H9
+
NMe2
H
MeO
Trans
C4H9
CID
[MH-MeOH] +
NMe2
C4H9
OMe
CI
[MH-MeOH] +
C4H9
MeO
Cis
+ NMe 2
H
CID
[MH-MeOH] +
Scheme 2.13
sites of the aminoethers are involved in the protanation process, which thus affords
two isomeric ions each case, one protonated at the dimethylamino group and the other at the less
basic methoxyl. The absence of the [MH-ROH]+ ion in the CID spectra of the MH+ ions of trans
suggests quantitiative elimination of methanol in the ion source from the MH+ ions that had the
proton attached at the methoxy group. The high stability of the protonated molecule was
supposed to be due to its bridged structure.
Thus most of the CI studies were concentrated on cyclohexane derivatives. Only a few
reports are available on the study of isomers of cyclohexene derivatives. Bruins et al. (Scheme
2.14) studied cis/trans carveol under OH- negative ion CI conditions [61]. The peak
corresponding to [M-H-(H2O)]- is more in trans isomer rather than cis isomer. The faster
dehydration of the trans (M-H)- ions was apparently initiated by the transfer of the reactive cis-5proton to the O- site; subsequent 1,4 elimination of H2O yields a fully conjugated anion. The 5-H
of cis carveol is inaccessible for such a proton shift.
H
O
-
OH
-
-H2O
Scheme 2.14
-Amino acids and their esters belong to an important class of building blocks for the
synthesis of natural products [1]. Most -amino acids themselves exhibit powerful antibacterial
properties and are key constituents of many naturally occurring peptides, terpenes, alkaloids, and
macrolides [2]. These moieties are present in peptide enzyme inhibitors like pepsatin and bestatin
[3]. Taxol, a promising anticancer compound has phenyl isoserine unit in its side chain and this
is essential to its anticancer activity [4-6]. -amino acids are precursors to many pharmaceutical
agents like -lactam antibiotics, and also constituents of biologically active unnatural peptides
[7-11]. Some cyclic -amino acids are reported to exhibit remarkable anti fungal, anti neoplastic
or cytotoxic activities [12-13]. -amino esters are the underlying monomers of -peptides, which
have received considerable attention due to their unique structural properties and interesting
biological activities [14-16]. In addition, recent studies by a number of groups have
demonstrated that the -mercapto--amino carbonyl motif also display potent biological activity.
For instance, pseudotripeptides containing an -mercapto--amino acid residue have been shown
to be potent inhibitors of aminopeptidase A, tetanus neurotoxin and botulinum neurotoxin type B
[17-18]. Azitidinones derived from -mercapto--amino acid display potent inhibition of
cholestrerol absorption [19]. Inspired from the biological activity associated with this motif we
have synthesized some -amino acid derivatives. While taking the mass spectra of the
synthesized compounds we could see some diagnostic fragmentation pattern between
diastereomers of these -amino acid derivatives. The potential of these compounds in the
synthesis of -peptides and other important compounds led us to investigate the mass spectral
behaviour of these isomeric compounds. McLafferty rearrangements involving hetero atoms are
observed prominently in all these isomers, therefore a brief introduction is given below on the
different McLafferty rearragements.
-Amino acids and their esters belong to an important class of building blocks for the
synthesis of natural products [1]. Most -amino acids themselves exhibit powerful antibacterial
properties and are key constituents of many naturally occurring peptides, terpenes, alkaloids, and
macrolides [2]. These moieties are present in peptide enzyme inhibitors like pepsatin and bestatin
[3]. Taxol, a promising anticancer compound has phenyl isoserine unit in its side chain and this
is essential to its anticancer activity [4-6]. -amino acids are precursors to many pharmaceutical
agents like -lactam antibiotics, and also constituents of biologically active unnatural peptides
[7-11]. Some cyclic -amino acids are reported to exhibit remarkable anti fungal, anti neoplastic
or cytotoxic activities [12-13]. -amino esters are the underlying monomers of -peptides, which
have received considerable attention due to their unique structural properties and interesting
biological activities [14-16]. In addition, recent studies by a number of groups have
demonstrated that the -mercapto--amino carbonyl motif also display potent biological activity.
For instance, pseudotripeptides containing an -mercapto--amino acid residue have been shown
to be potent inhibitors of aminopeptidase A, tetanus neurotoxin and botulinum neurotoxin type B
[17-18]. Azitidinones derived from -mercapto--amino acid display potent inhibition of
cholestrerol absorption [19]. Inspired from the biological activity associated with this motif we
have synthesized some -amino acid derivatives. While taking the mass spectra of the
synthesized compounds we could see some diagnostic fragmentation pattern between
diastereomers of these -amino acid derivatives. The potential of these compounds in the
synthesis of -peptides and other important compounds led us to investigate the mass spectral
behaviour of these isomeric compounds. McLafferty rearrangements involving hetero atoms are
observed prominently in all these isomers, therefore a brief introduction is given below on the
different McLafferty rearragements.
Malaria is a vector-borne infectious disease caused by protozoan parasites. It is
widespread in tropical and subtropical regions, including parts of the America, Asia, and Africa.
Each year, it causes disease in approximately 650 million people and kills between one and three
million, most of them young children in Sub-Saharan Africa. The most serious forms of the
disease are caused by Plasmodium Falciparam and Plamodium Vivax [1]. This group of humanpathogenic Plasmodium species is usually referred to as malaria parasites. No vaccine is
currently available for malaria; preventative drugs must be taken continuously to reduce the risk
of infection. The first antimalarial drug used was quinine (Scheme 4.1) isolated from the bark of
Cinchona species (Rubiaceae) in 1820 [2]. Quinine belong to the class of alkaloids, which are
the physiologically-active nitrogenous bases derived from many biogenetic precursors. In 1940,
another antimalarial drug, chloroquine (Scheme 4.1) was synthesized [3]. Until recently, this is
the only drug, used for the treatment of malaria. Several classes of the secondary plant
substances like quassinoids, sesquiterpene lactones and alkaloids are tested against malaria [4-5].
The malaria parasite has now become resistant to the best anti-malarial drugs. Therefore,
medicinal agents based on novel mode of action are required to overcome the emergence of
resistance and to control an ever-increasing number of epidemics caused by the malaria parasite.
HO
N
HN
N
O
N
Quinine
Cl
N
Chloroquine
Scheme 4.1. Chemical structures of quinine and chloroquine.
Febrifugine is another alkaloid with potent antimalarial activity isolated from the Chinese
herb chang shan (Dichroa febrifuga Lour) and Hydrangea umbellate. Febrifugine has been
employed by the local people as medicine against fevers caused by malaria parasites for a long
time.
Naturally, febrifugine is present along with its isomeric compound, isofebrifugine
(Scheme 4.2) [6-7]. Based on the experiments on ducks it was demonstrated that the activity of
febrifugine was 100 times to that of quinine against P. lophurae [8-9]. In vivo antimalarial
activity studies revealed that febrifugine was 200 times more active than its isomer,
isofebrifugine [10].
N
HO
O
N
N
HO
N
N
H
O
O
NH
O
Febrifugine
Isofebrifugine
Scheme 4.2. Chemical structures of febrifugine and isofebrifugine.
Hence, isolation and identification of the correct isomer is crucial for practical
applications. Successful isolation of both isomers from natural resources has been reported. In
addition, synthetic approaches [11] towards stereospecific synthesis of isofebrifugine and
febrifugine have been reported using appropriate precursor i.e., (2S,3S)-tert-Butyl 3-(benzyloxy)2-(3-bromo-2-oxopropyl) piperidine-1-carboxylate (1) and (2R,3S)-tert-Butyl 3-(benzyloxy)-2(3-bromo-2-oxopropyl)piperidine-1-carboxylate (2), respectively (Scheme 4.3).
O
O
O
O
X
X
N
O
X = Br
X=H
N
O
1
3
Isofebrifugine
O
X = Br
X=H
O
2
4
Febrifugine
Scheme 4.3. Chemical structures of the studied compounds 1-4
The spectra include two major product ions at m/z 218 and 190 corresponding to the loss of 108
(C6H5-CH2-OH) and 136 Da (CH2-CO-CH2Br+H) from the ion m/z 326, respectively (Scheme
4.4). The ion m/z 190 can also be formed from 218 through a Retro-Diels Alder (RDA) reaction;
if this is the case, the resulted ion should contain the bromine group. To verify this possibility,
the CID spectra of the ion at m/z 328 (81Br isotope) is recorded (Figure 4.4), wherein all the
bromine containing product ions should shift 2 Da and others appear at the same m/z value as
observed in the spectrum of the ion at m/z 326 (79Br isotope). The ion at m/z 190 did not shift in
the CID spectrum of m/z 328 confirming the absence of Br in the product ion at m/z 190.
Absence of bromine in the ion m/z 190 can also be confirmed from the source spectrum recorded
at higher declustering potential, where the ion m/z 190 does not show the expected bromine
pattern (Figure 4.4). These two experiments clearly rule out the possibility of formation of the
ion at m/z 190 from 218 through a RDA reaction.
H
O
+
H
O
O
- C4H8
O
Br
Br
N
N
O
O
OH
O
m/z 370
- CO 2
[M+H]
+
ion of 1 and 2 (m/z 426)
- (BOC-H)
O
Br
H
O
H
- C6H5CH 2OH
O
Br
+
N
+
N
H
m/z 218
- C3H5OBr
H
[M+H-(BOC-H)]
+
ion (m/z 326)
O
H
m/z 190
Scheme 4.4. Fragmentation pattern of [M+H]+ ion of 1 and 2.
+
N
H
+
Figure 4.4. a) ESI spectrum recorded for compound 1 at DP 340V.
b) CID spectrum of ion m/z 328 (CE=25) from compound 1
When the CID spectra of m/z 326 from the two isomers compared, it is very clear that the
relative abundance of the ion at m/z 190 is more in 1 and where as the ion m/z 218 is more in 2.
The spectrum of 2 also includes low abundant characteristic product ions at m/z 308, 282, 234
and 174, but we focused only on the two abundant product ions at m/z 190 and 218. The ion m/z
190 is formed through a McLafferty rearrangement involving hydrogen migration to carbonyl
group. It is reasonable to assume the structure of the precursor ion (m/z 326) as a protonated
species having proton on the nitrogen of piperidine ring. Theoretically the proton on the chiral
centre and proton on nitrogen can migrate to the carbonyl as a part of the McLafferty
rearrangement. Since the proton on the nitrogen is common for the both isomers, the migration
of the proton on the chiral centre must be crucial to the stereoselective formation of the ion at
m/z 190 in 1.
The rearrangement product formed by the involvement of the chiral hydrogen
results in a double bond between C1-C2, which not only relives the steric repulsions of the two
bulky groups in 1, but also leads to resonance stabilization of the product ion involving lone
pairs of oxygen. Moreover, the transition state of the rearrangement in the case of 1 involves a
stable trans decaline type structure, where as it is a less stable cis decaline type structure for 2
(Scheme 4.5). Thus, by invoking the migration of the hydrogen on the chiral center it is possible
to explain the favourable formation of the ion m/z 190 in 1.
O
O
O
Br
H
H
N+
H
H
O
H
N+
H
Br
m/z 326 from 1
m/z 326 from 2
Scheme 4.5 Transition state for McLafferty rearrangement
The ion at m/z 218 is formed by the elimination of benzyl alcohol from the ion at m/z 326
involving hydrogen migration to the benzyloxy group. In this case, there are three different
sources of hydrogen that can migrate to benzyloxy group, viz. one of the hydrogens on
neighbouring achiral carbon (CH2), and hydrogen of the chiral centre and hydrogen on positively
charged nitrogen. However, the steric environment of hydrogens on achiral carbon as well as on
nitrogen with respect to the benzyloxy substituent is same for both isomers. The only difference
between the two isomers is the stereochemistry of hydrogen on the chiral centre, where the
hydrogen is located in trans (1) or cis (2) with respect to the benzyloxy group. 1,2-eliminations
are well known in the decomposition of closed-shell protonated species, and in fact the ion at
m/z 218 is dominant in 2, which is resulted by 1,2-cis elimination process involving the proton
on the chiral carbon.
4.3.1.2. [M+Na]+ ion
The CID spectra of [M+Na]+ (m/z 448) of 1 and 2 yield same set of product ions (Figure
4.5), however, consistent differences are observed in the relative abundances of few
characteristic ions. Fragmentation pattern arrived at based on HRMS and MS/MS spectra is
given in Scheme 4.6. As expected, the ions formed by the loss of isobutylene (56 Da) and 100
Da (C5H8O2), which are characteristic of BOC protecting group, are found in the spectra at m/z
392 and 348, respectively. The ion at m/z 392 is found to be abundant (75-100%), whereas the
ion at m/z 348 is low abundant (<5 %). The product
a
b
Figure 4.5. CID spectra (CE=30 V) of [M+Na]+ (m/z 448) of a) 1 and b) 2
ions corresponding to the further loss of 80 (HBr), 102 (NaBr) and 136 Da (C3H5BrO) from m/z
348 ions are also found at m/z 268, 246 and 212, respectively. Absence of halogen in m/z 268,
246 and 212 is confirmed by recording CID spectra of m/z 450 (81Br
isotope of [M+Na]+ ion), in which the ions at m/z 268, 246 and 212 remain at the same m/z
value; while the ions at m/z 392 and 348 are shifted two mass units as expected (spectrum not
shown). The same is evident when the source spectrum recorded at higher declustering potential
values (source fragmentation); the ions at m/z 392 and 348 appear as doublets (bromine pattern),
whereas the ions 268, 246 and 212 appear as single peaks. Elimination of HBr and NaBr are also
observed from the ion m/z 392 (product ions at m/z 312 and 290, respectively) in the CID spectra
of [M+Na-C4H8]+ ions. Apart from these, the spectra also include other low abundant ions at
m/z 154, 140, 138, 110 and 91, out of which some of them looks very diagnostic for the isomeric
differentiation. The ions m/z 154, 140, 138 and 110 are essentially formed by the further
fragmentation of the ion at m/z 246, because the CID spectra of the ion m/z 246 from 1 and 2
show similar trend in the formation of these product ions as that observed in the CID spectra of
[M+Na]+ ions. The ion at m/z 140 corresponds to the loss of C6H5-CHO and the ion at m/z 138
is formed by the loss of C6H5-CH2OH from the ion at m/z 246.
The CID spectra of [M+Na]+ from 1 and 2 do show selectivity in the extent of formation
of a few characteristic product ions. The [M+Na-(BOC-H)]+ ion (m/z 348) is
marginally higher in 1 than in 2. The [M+Na-C4H8]+ (m/z 392) is the base peak in 2, while it is
75% in 1. It is interesting to note that the product ions due to the further fragmentation of the ion
at m/z 348, i.e., the ion at m/z 246, 268 and 154 (loss of NaBr, HBr and C 7H8, respectively) are
relatively more abundant in 1 when compared to 2. This
Na
+
O
Na
+
O
- C3H5OBr
Na
O
- C4H8
O
O
Br
Br
N
N
O
O
O
m/z 312
[M+ Na]
+
+
N
O
O
OH
m/z 392
ion of 3 and 4 (m/z 448)
- COOH
- (BOC-H)
- (BOC-H)
Na
+
O
Na
+
O
- C3H5OBr
Na
O
- HBr
O
+
Br
N
N
H
H
m/z 212
O
m/z 268
m/z 348
H+
O
N
- NaBr
H+
- C7H8
H+
N
O
O
H+
m/z 154
O
m/z 246
(Or)
N
N
- C6H5CHO
N
- C6H5CH 2OH
N
O
O
m/z 138
O
m/z 140
Scheme 4.6. Fragmentation pattern of [M+Na]+ of 1 and 2.
spectral difference clearly reveals that the [M+Na-C4H8]+ ion (m/z 392) from 1 is not stable and
further decomposes to eject CO2 followed by HBr/NaBr, whereas the ion at m/z 392 from 2, gets
relatively better stability and do not undergo much of further decomposition. In a way, this
behavior is similar to that observed in [M+H]+ ion decomposition, where the [M+H]+ ion of 1
show a favorable loss of (BOC-H) as compared to that of 2.
The product ion at m/z 212 is relatively higher in 2 than in 1. The ion m/z 212 is formed
by the loss of C3H5OBr from [M+Na-(BOC-H)]+ via a Mclafferty rearrangement. It is worth to
make a note that the similar C3H5OBr loss is also found during the decomposition of [M+H]+ ion
but it is dominant in 1 than in 2. This differential stereoselectivity observed in the decomposition
of [M+H]+ and [M+Na]+ ions of 1 and 2 clearly suggests that the attached proton/metal ion also
playing a role in the observed stereoselectivity in the decomposition of two isomers. The
elimination of C3H5OBr from [M+H-(BOC-H)]+ and [M+Na-(BOC-H)]+ ions of 1 and 2 must
involve two different mechanistic pathways of complex nature and with the available data it is
difficult to propose a mechanism without computational data support.
In addition to the above discussed ions, the ions at 138 and 110 are relatively higher in 2
when compared to those in 1; the low abundant ions at m/z 140 and 138 are also diagnostic in the
CID spectra of [M+Na]+ ions. The ions 138 and 140 result from the stereoselective
fragmentation of the ion m/z 246, as confirmed by the CID experiments on m/z 246 ions from 1
and 2 (Figure 4.6). The ion at m/z 140 is characteristic of 1 and the ions m/z 138 and 110 are
diagnostic to the 2. The ion at m/z 246 corresponds to the loss of NaBr from [M+Na-(BOCH)]+ ion, and it should attain a bicyclic structure keeping the stereochemistry of original chiral
centers intact (Scheme 4.6). This is similar to Hofmann-Loffler-Fretyag (HLF) reaction that is
popularly observed in solution phase, where amine abstracts δ-hydrogen to form a cyclic
structure. Such intramolecular cyclizations in gas phase are widely studied [18-25]. Cyclization
in nitrogen containing compounds analogous to HLF reactions were also reported [26-28]. But
these were open chain compounds forming a cyclic structure. In the present case, the analyte is
forming a bicyclic structure with the removal of sodium halide.
a
b
Figure 4.6. MS4 spectrum (m/z 448 392 246) of a) compound 1 and b) compound 2.
The CID spectrum of m/z 246 from 1 (Figure 4.6) includes stereospecific but less
abundant ion at m/z 140 generated from the precursor ion at m/z 246 corresponding to the loss of
106 Da (Ph CHO). Whilst, the CID spectrum of m/z 246 from 2 show abundant fragment ions,
in which the ion at m/z 138 is stereospecifically dominant and corresponds to the loss of 108 Da
(Ph-CH2-OH). The decomposition behaviour of 246 from 1 and 2 suggests that hydrogen
migration to benzyloxy group is favourable in the case of 2 than in 1. Though there are two or
more different sources of hydrogens available for this migration, only the hydrogen on C1 chiral
carbon should make the difference for stereospecific fragmentation, as other sources of
hydrogens are common to both isomers. We propose that the loss of 108 Da is preferred from
the ion at m/z 246 from 2, because the C1-chiral hydrogen is cis to the bezyloxy group that might
makes easy hydrogen migration. The ion at m/z 110 is another diagnostic ion found in 2 that
might be formed from the m/z 138 by the loss of CO.
4.3.1.3 [M+Li]+ ion
It is very clear from the CID spectra of [M+Na]+ ion from 1 and 2 that the loss of NaBr
and HBr is observed only from [M+Na-(BOC-H)]+ ion but not directly from [M+Na]+ ion, more
over, the loss of NaBr is more favourable than the loss of HBr. It is also interesting to note that
the HBr loss is not found in the decomposition of [M+H]+ ions of 1 and 2. With a view to
checking the specificity of metal ion in the process of elimination of HBr/NaBr from [M+Na(BOC-H)]+ ions, experiments are performed on lithiated ions of 1 and 2.
Lithium Chloride is
added to the sample solution to produce [M+Li]+ ions. The CID spectra of [M+Li]+ ions (m/z
432) show the expected product ions at m/z 376 and 332 corresponding to the loss of isobutylene
and (BOC-H), respectively (Figure 4.7). The expected product ion at m/z 246 corresponding to
the loss of LiBr from [M+Li-(BOC-H)]+ ion is found in the spectra. The loss of HBr from
[M+Li-(BOC-H)]+ is also observed (m/z 252), and loss of LiBr is preferable when compared to
the loss of HBr as observed in the case of [M+Na]+ ions. The stereochemical effects on the
decomposition of [M+Li]+ ions of 1 and 2 (Figure 4.7) are almost similar with those obtained
with [M+Na]+ ions. The [M+Li-(BOC-H)]+ ion (m/z 332) and further expulsion of LiBr (m/z
246) are relatively abundant in 1, whereas the ion at m/z 196 corresponding to the loss of
C3H5OBr from [M+Li-(BOC-H)]+ ion, formed by McLafferty rearragement is dominant in 2.
Figure 4.7. CID spectra (CE=28 V) of [M+Li]+ ion (m/z 432) a) 1 and b) 2
By close comparison of the decompositions of [M+Na]+ and [M+Li]+ ions of 1 and 2, it
can be noted that the loss of CatBr, (where Cat = Na or Li) is always preferred over HBr loss
from [M+Cat-(BOC-H)]+ ions. And the abundances of characteristic ions (e.g m/z 246 and 212
or 196) are relatively higher in the decomposition of [M+Na]+ ions when compared to that of
[M+Li]+ ions, suggesting increased stereochemical effects with Na+ than with Li+ ions that could
be due to higher atomic size of Na+ than Li+.
4.3.2. Compounds 3 and 4
In order to check the contribution of bromine in stereochemical effects found in 1 and 2,
we moved to another set of isomeric compounds (3 and 4, Scheme 4.3), in which the bromine is
replaced with hydrogen. The ESI mass spectra of 3 and 4 show both [M+H]+ and [M+Na]+ ions,
and further CID experiments are performed on these ions. The CID spectra of [M+H]+ ions (m/z
348) resulted in the expected ions due to loss of C4H8 (m/z 292) and loss of (BOC-H) (m/z 248).
The CID spectra of m/z 348 from 3 and 4 do not show any significant differences, whereas the
CID spectra of the ion at m/z 248
do show distinct differences, and the trend resembles with that observed between the CID spectra
of [MH-(BOC-H)]+ ion of 1 and 2 (Figure 4.8). The spectra yield two major product ions that are
characteristic, viz. the ion m/z 190 due to the loss of 58 Da (C3H6O) and the ion m/z 140 due to
the loss of 108 Da (benzyl alcohol), apart from the other ions at m/z 91, 98, and 100. The ion at
m/z 190 is dominant in compound 3 while the ion m/z 140 is more abundant in compound 4.
Observing a similar trend between two isomeric sets (1 and 2 & 3 and 4) suggests that the
stereoselectivity observed in the decomposition of [MH-(BOC-H)]+ ion is not hampered in the
absence of bromine, which in turn shows absence of bromine contribution in the elimination
processes observed.
Figure 4.
8.
CID
spectra
(CE=20
V)
of
[MH(BOCH)]+
(m/z
248)
from a)
3 and b)
4.
The CID spectra of [M+Na]+ ion of 3 and 4 (m/z 370)(Figure 4.9) also yield the product
ions due to the loss of isobutylene (m/z 314) and loss of (BOC-H) (m/z 270). As expected, the
ion at m/z 314 is dominant in 4 and the ion at m/z 270 is dominant in 3. These are the typical
spectra in which both [M+Na-C4H8]+ and [M+Na-(BOC-H]+ ions are abundant and showing
stereochemical differences. Similar stereoselectivity is found even in further decomposition of
m/z 314 (Figure 4.10). The spectra of m/z 314 include two major product ions at m/z 270 and
212, in which the former ion is the base peak in
Scheme 4.9. CID spectra (CE =24V) of [M+Na]+ of compounds a) 3 and b) 4
Scheme 4.10. CID Spectra of {M+Na-C4H8]+ (m/z 314) of compounds a) 3 and b) 4
both 3 and 4. The ion at m/z 212, which formed due to the loss of acetone from the ion m/z 270,
is more dominant in 4 (75%) than in 3 (22%). Though, the decomposition of [M+Li]+ ion do not
show significant isomeric differentiation, the CID spectra of [M+Li-(C4H8)]+ (m/z 298) show
noteworthy differences in the relative abundances of product ions at m/z 254 and 196
corresponding to [M+Li-(BOC-H)]+ and [M+Li-(BOC-H)-C3H6O]+, respectively.
Figur
e
a
4.11.
CID
spect
ra
(CE=
20
b
V) of
[M+
Li(BO
CH)]+ ion (m/z 254) from a) 3 and b) 4.
The ion m/z 254 is the base peak and the ion m/z 196 is 35% in 3, while the ion m/z 196
is the base peak and the ion at m/z 254 is 50% in 4. The trend in the formation of m/z 196 is also
maintained in further decomposition of the ion m/z 254 (25% and 100% in 3 and 4, respectively
(Figure 4.11). Thus the stereochemical effects are maintained in the decompositions of [M+Na] +
and [M+Li]+ ions, irrespective of the presence of bromine, in turn supports the proposed
explanation for the observed stereochemical effects in 1-4.