DOI: 10.1002/cbic.201600237
Communications
Position-Specific Mass Shift Analysis: A Systematic Method
for Investigating the EI-MS Fragmentation Mechanism of
epi-Isozizaene
Patrick Rabe, Tim A. Klapschinski, and Jeroen S. Dickschat*[a]
This work is dedicated to Prof. Wittko Francke on the occasion of his 75th birthday.
The EI-MS fragmentation mechanism of the bacterial sesquiterpene epi-isozizaene was investigated through enzymatic conversion of all 15 synthetic (13C1)FPP isotopomers with the epiisozizaene synthase from Streptomyces albus and GC-MS and
GC-QTOF analysis including MS-MS. A systematic method,
which we wish to call position-specific mass shift analysis, for
the identification of the full set of fragmentation reactions was
developed.
After the identification of the terpenoid odorants geosmin (1)
and 2-methylisoborneol (2) by Gerber in the 1960s
(Scheme 1),[1] it has been shown during the past two decades
Scheme 1. Terpenes from bacteria.
that terpenes are widespread in bacteria.[2] These compounds
are produced from the precursors geranyl diphosphate (GPP,
monoterpenes), farnesyl diphosphate (FPP, sesquiterpenes), or
geranylgeranyl diphosphate (GGPP, diterpenes) through the
action of terpene cyclases that catalyse substrate ionisation to
initiate multistep domino reactions via cationic intermediates.
After the discovery of the first bacterial terpene cyclases for
pentalenene (3) and epi-isozizaene (4),[3] today more than 50
enzymes of this class have been described.[4] Their complex
cyclisation mechanisms are difficult to address, but recently detailed insights have been obtained from structural biology in
combination with site-specific mutations,[5] quantum chemical
calculations,[6] feeding experiments and enzymatic conversions
of carefully designed isotopically labelled substrates.[7] We have
recently synthesised all 15 (13C1)FPP isotopomers and completely labelled (13C15)FPP in order to use these precursors in
[a] Dr. P. Rabe, T. A. Klapschinski, Prof. Dr. J. S. Dickschat
Kekul¦-Institut fìr Organische Chemie und Biochemie
Rheinische Friedrich-Wilhelms-Universit•t Bonn
Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany)
E-mail: [email protected]
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cbic.201600237.
ChemBioChem 2016, 17, 1333 – 1337
the enzymatic conversion with (1(10)E,4E,6S,7R)-germacradien6-ol (5) synthase from Streptomyces pratensis.[8]
Product 5 exists in the form of two interconverting conformers, resulting in complex NMR spectra with overlapping signals
that prevented a full assignment of NMR data to the two conformers in previous studies, but with the enzymatically obtained labelled sesquiterpene alcohols in hand the assignment
was possible. The synthetic (13C1)FPPs can also be used for systematic investigation of the EI-MS fragmentation mechanisms
of terpenes, which has never previously been performed, due
to the lack of isotopically labelled material. We have now developed a method that involves enzymatic conversion of all 15
(13C1)FPPs and product analysis by GC-MS and high-resolution
GC-QTOF including MS-MS (MS2) for position-specific mass shift
analysis (PMA). Here we present the application of this method
in a detailed EI-MS fragmentation study for the widespread
bacterial sesquiterpene epi-isozizaene (4).
The gene for the epi-isozizaene synthase (EIZS) from Streptomyces albus J1074 (accession number EFE83901, phylogenetic
trees of all characterised bacterial terpene cyclases and of epiisozizaene synthases are shown in Figures S1 and S2 in the
Supporting Information) was amplified by PCR and cloned into
the Escherichia coli expression vector pYE-Express by homologous recombination in yeast.[9] The protein was expressed, purified and used to convert FPP into the single product 4, which
was identified by GC-MS and comparison with an authentic
standard obtained from FPP with the known EIZS from Streptomyces coelicolor A3(2)[3b] (Figure S3). The EIZS from S. albus exhibits the highly conserved aspartate-rich motif 98DDQHD, the
pyrophosphate sensor R193, the NSE triad 239NDLCSLPKE, and
the 343RY dimer that together make up the first coordination
sphere of the substrate and the Mg2 + cofactor.[5h] The enzyme
is composed of 365 amino acid residues, shares only 61 %
identical sites with its homologue from S. coelicolor and is phylogenetically fairly distant from this enzyme, but as we show
here also produces 4 from FPP. We have previously reported
on the production of 4 by S. albus agar plate cultures.[2c]
The EI mass spectrum of 4 is shown in Figure 1. To investigate the mechanisms by which the most prominent fragment
ions are formed, all 15 (13C1)FPPs were converted through the
action of the S. albus EIZS, and the obtained products were analysed by GC-MS [mass spectra of the corresponding (13C1)-4
isotopomers are shown in Figure S4]. The labelling in each of
the isotopomers of 4 can be located precisely; this is a prerequisite for the following analysis, because of the unequivocally
established mechanism (by means of experimental work[3b, 10]
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Communications
Figure 1. EI mass spectrum of 4.
Scheme 3. PMAs for A) m/z 189 (PMA189), B) m/z 175 (PMA175), and C) m/z
161 (PMA161) with information extracted from Figure S4 (a: a-cleavage. i:
inductive cleavage. rH: rearrangement of hydrogen.) Black and red circles
indicate carbon atoms that contribute fully or partially to a fragment ion.
Scheme 2. Enzymatic cyclisation of FPP through the action of EIZS. Carbon
numbering of 4 (not systematic) indicates the origin of each carbon from
FPP by identical number.
and quantum chemical calculations[6b, e]) of the cyclisation to 4
(Scheme 2). This includes the origin of the diastereotopic
methyl groups C12 and C15 in 4, which is the same for S. albus
EIZS as reported for S. coelicolor EIZS, on the basis of 13C NMR
analysis of the product obtained from (12-13C)FPP (Figure S5).
Each carbon atom of an analyte that contributes to the formation of a particular fragment ion can be identified through
enzymatic conversion of the corresponding 13C-labelled FPP
and EI-MS analysis of the product by a mass shift of +1 relative
to the mass for the unlabelled analyte. With all 15 (13C1)FPP isotopomers this PMA leads to a full picture of which carbon
atoms of the analyte form a fragment ion, summarised for the
ion m/z 189 by the black circles (PMA189), each representing an
individual experiment (Scheme 3 A; data in Figure S4 are the
basis for all PMAs). In some cases the mass of a fragment ion
might be partially increased by +1, as observed for m/z 189 in
the experiments with (12-13C)- and (15-13C)FPP, pointing to
multiple mechanisms for the formation of the investigated
fragment ion and indicated by red circles in PMA189. This analysis shows that the fragment ion at m/z 189 is clearly formed
by loss of C12 or of C15, whereas cleavage of either of the
ChemBioChem 2016, 17, 1333 – 1337
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other two methyl groups in 4 is not important. This is explained in terms of EI ionisation of 4 with loss of one p electron to yield radical cation 4C + and subsequent a-cleavage
either of C12 (path a) or of C15 (path b), both resulting in the
same fragment ion A + . Similarly, PMA175 reveals the formation
of the fragment ion at m/z 175 by loss of C4 and C5, which
can proceed by two sequential a-cleavages of 4C + with neutral
loss of ethene to BC + , followed by a third a-fragmentation with
loss of hydrogen to afford C + (Scheme 3 B).
The formation of the fragment ion m/z 161 is more complex.
As indicated by PMA161, all 15 carbon atoms of 4 contribute to
this fragment ion, but for C3, C4, C5, C12 and C15 only a partial
contribution is observed (Scheme 3 C). The best explanation for
this finding is a combination of two fragmentation mechanisms, either with loss of C4 and C5 plus one methyl group
(C12 or C15), or of the C3-C12-C15 portion. The first mechanism might proceed either through initial loss of ethene—that
is, via BC + , followed by loss of C12 (path a) or C15 (path b)
through another a-cleavage—or by a reversed order of steps,
through loss of one of the methyl groups first to yield A + and
subsequent cleavage of ethene, giving rise to D + in both
cases. The neutral loss of ethene from A + might be a concerted
process of two inductive (heterolytic) bond cleavages as
shown, or of two homolytic a-fragmentations; this cannot be
distinguished by our labelling experiments. To investigate
which of the discussed orders of steps contributes to the formation of D + , MS2 experiments with m/z 189 and m/z 176
were performed (Figures S7 and S8). The MS2 data showed
that both ions A + and BC + are direct precursors of D + . That is,
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Ó 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communications
both pathways are important, but with more pronounced formation of D + via BC + . Formation of the fragment ion at m/z
161 with loss of the C3-C12-C15 portion of 4 is explainable in
terms of two a-cleavages from 4C + to afford EC + , with subsequent rearrangement of a hydrogen (rH) with a third a-cleavage to give F + .
PMA147 reveals a mechanism for the generation of the ion at
m/z 147 with a clear loss of the C7-C8-C14 moiety and a partial
loss of either C12 or C15 of 4 (Scheme 4 A). This process could
Scheme 4. PMAs for A) m/z 147 (PMA147), B) m/z 133 (PMA133), and C) m/z
119 (PMA119).
involve a sequence of two successive a-cleavages with neutral
loss of propene from 4C + , followed by two additional a-cleavages with ring opening of the strained bridgehead radical
cation GC + and abstraction either of C12 (path a) or of C15
(path b) to afford intermediate H + . As an alternative, the reaction could proceed by loss of C12 or C15 first—that is, via A +
—and a series of three consecutive or concerted inductive
cleavages to give the same cation H + . Whereas the second
mechanism is supported by MS2 analysis of the fragment ion
at m/z 189 that gives evidence for the formation of H + from
A + (Figure S7), evidence for participation of the first mechanism was more difficult to obtain. The high-resolution mass
spectrum of 4 (Figure S6) seemed to suggest that the fragment
ion at m/z 162 is only composed of [12C1113C1H17] + (calculated:
m/z 162.1358; found: m/z 162.1352) and is thus the 13C-isotopic peak of D + —[12C12H17] + —whereas no signal for [12C12H18] +
ChemBioChem 2016, 17, 1333 – 1337
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corresponding to GC + could be detected (calculated: m/z
162.1403). However, MS2 analysis of the fragment ion at m/z
162 revealed the formation of a fragment ion [12C11H15] + (calculated: m/z 147.1168; found: m/z 147.1171, Figure S10), which is
only explainable in terms of a loss of 12CH3 from GC + , and not
of loss of 13CH2 from the 13C-isotopic peak of D + . Obviously,
the accurate masses of the 13C-isotopic peaks of D + and of GC +
are not resolved, but the presence of the masked ion GC + is
evident from its daughter ion m/z 147.1171, confirming
a minor degree of participation of the first mechanism in the
formation of H + .
PMA133 indicates a clear formation of the fragment ion m/z
133 with loss of C4–C5 and of C7-C8-C14 (Scheme 4 B). This
finding is explainable in terms of neutral loss of ethene from
4C + to yield BC + and cleavage of HC to form C + as discussed
above, followed by elimination of propene to result in I + (such
a diradical cation might rearrange to a more stable species).
This mechanism is supported by MS2 analysis showing a strong
formation of I + as a daughter ion of m/z 175 (Figure S9). The
MS2 analysis of m/z 176 (Figure S8) seems to suggest that BC +
might also cleave off propene first to result in the ion m/z 134
with subsequent hydrogen abstraction to give m/z 133, but
the fragment ion m/z 134 in the low-resolution mass spectrum
of 4 is of low abundance (Figure 1), and its MS2 analysis shows
very weak formation of a m/z 133 daughter ion (Figure S13).
Furthermore, HRMS data reveal that m/z 134 is a 13C-isotopic
peak of m/z 133 (calculated for [12C913C1H13] + : m/z 134.1045;
found: m/z 134.1048). All these data indicate that the formation of m/z 133 through loss of propene from BC + is a process
of subordinate importance. The alternative formation of m/z
133 by loss of ethene from D + to yield J + cannot be deduced
from the labelling experiments, but is a low-priority mechanism as demonstrated by MS2 of m/z 161 (Figure S11).
PMA119 reveals formation of the fragment ion m/z 119 by
loss of C4–C5, C7-C8-C14 and either C12 or C15 (Scheme 4 C).
The most important mechanism for its generation is related to
the mechanism for I + : that is, cleavage of ethene from 4C + to
give BC + followed by loss of C12 or C15 to give D + and elimination of propene to afford K + . Consistently with this mechanism the MS2 analyses of both m/z 176 and m/z 161 strongly
yield m/z 119 (Figures S8 and S11). For the formation of D +
a reversed order of steps by cleavage of C12 or C15 preceding
the neutral loss of ethene was discussed above, and consequently m/z 189 is also a precursor of K + as shown by MS2
(Figure S7). Furthermore, MS2 of m/z 147 shows that H + (or
a related species) is a parent ion of m/z 119 (Figure S12).
In the low m/z range some mechanisms for the formation of
important fragment ions can be identified from the MS2 analyses, including the formation of m/z 105 from m/z 147 by loss
of C3H6 and from m/z 133 by cleavage of C2H4, or the formation of m/z 91 from m/z 133 by elimination of C3H6 and from
m/z 119 by loss of C2H4 (Figures S12, S14, and S15), but because of the multiple pathways that lead to the ions of low
masses their PMAs are inconclusive and precise mechanisms
for their formations cannot be delineated.
The main pathways for the EI-MS fragmentation of 4 are
summarised in Scheme 5. Each box above an arrow shows the
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Communications
detail. Thus, our experimental study on the fragmentation
mechanism of this sesquiterpene is deeply rooted in textbook
biochemistry.
In summary, we have described the first systematic investigation into the EI-MS fragmentation mechanism of a terpene
by EI-MS and MS2 analysis of the complete set of its singly 13Clabelled isotopomers, enzymatically generated from the corresponding (13C1)FPP isotopomers. The same approach might
also be interesting for natural products from other compound
classes, but can be comparably easily applied to terpenes that
are made from linear precursors through the action of just one
enzyme. We will systematically analyse the fragmentation
mechanisms of other sesquiterpenes in due course.
Acknowledgements
This work was funded by the DFG (DI1536/7-1). We thank Jos¦
Salas (Oviedo, Spain) for the kind gift of S. albus J1074.
Keywords: fragmentation mechanisms · isotopic labeling ·
isozizaene · mass spectrometry · terpenoids
Scheme 5. Fragmentation reactions identified by PMAs and MS2 experiments
(MS2n indicates MS2 analysis for m/z n). Normal arrows indicate main reactions; dashed arrows indicate reactions of subordinate importance.
analytical basis for the identification of the reaction of a parent
ion including the PMA and MS2 analysis and the lost portion of
4. Two initial fragmentations each lead to a series of daughter
ions. Either a methyl group (C12 or C15) is lost first, followed
by cleavage of ethene (C4 + C5) or propene (C7 + C8 + C14) in
different sequential orders of steps, thus explaining the ions at
m/z 189, 161, 147, 133, and 119 in the mass spectrum of 4, or
ethene is first cleaved off to yield m/z 176, followed by loss of
a proton to give m/z 175 and then propene to afford m/z 133
(no evidence for the reversed sequence was obtained), or by
loss of a methyl group or propene to provide m/z 161 and
133, respectively.
Feeding experiments with isotopically labelled precursors in
combination with EI-MS have occasionally been used to investigate terpene biosynthetic pathways. In such experiments special care must be taken to avoid a circular argument. The
increased masses of fragment ions in the mass spectrum of a
labelled analyte from a feeding experiment might be in agreement with a fragmentation mechanism that allows the labelling to be localised, whereas knowledge about the incorporation site might give insights into mechanistic key steps of the
biosynthesis. If the biosynthetic mechanism is then used to validate the EI-MS fragmentation, the logic short-circuit is closed.
The work presented here is based on the profound knowledge
about epi-isozizaene biosynthesis that has been studied in
ChemBioChem 2016, 17, 1333 – 1337
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Manuscript received: April 20, 2016
Accepted article published: April 28, 2016
Final article published: June 2, 2016
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