Conformational Analysis, Thermal Rearrangement, and EI-MS Fragmentation Mechanism of (1(10)E,4E,6S,7R)-Germacradien-6-ol by 13C-Labeling Experiments

Article abstract of DOI:10.1002/anie.201507615

An uncharacterized terpene cyclase from Streptomyces pratensis was identified as (+)-(1(10)E,4E,6S,7R)-germacradien-6-ol synthase. The enzyme product exists as two interconvertible conformers, resulting in complex NMR spectra. For the complete assignment of NMR data, all fifteen (¹³C₁)FPP isotopomers (FPP=farnesyl diphosphate) and (¹³C₁₅)FPP were synthesized and enzymatically converted. The products were analyzed using various NMR techniques, including ¹³C, ¹³C COSY experiments. The (¹³C)FPP isotopomers were also used to investigate the thermal rearrangement and EI fragmentation of the enzyme product. All 15 isotopomers of (¹³C₁)- and fully labeled (¹³C₁₅)farnesyl diphosphate were synthesized and converted to the title compound by a newly characterized bacterial terpene cyclase from Streptomyces pratensis. The enzyme products were used for structure elucidation of the sesquiterpene alcohol, full assignment of NMR data and a conformational analysis, and investigation of its Cope rearrangement and of its EI-MS fragmentation mechanism.

Full text of DOI:10.1002/anie.201507615

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International Edition: DOI: 10.1002/anie.201507615
German Edition: DOI: 10.1002/ange.201507615
Bacterial Terpenes
Conformational Analysis, Thermal Rearrangement, and EI-MS
Fragmentation Mechanism of (1(10)E,4E,6S,7R)-Germacradien-6-ol
by 13C-Labeling Experiments
Patrick Rabe, Lena Barra, Jan Rinkel, Ramona Riclea, Christian A. Citron, Tim A. Klapschinski,
Aron Janusko, and Jeroen S. Dickschat*
Abstract: An uncharacterized terpene cyclase from Strepto-
myces pratensis was identified as (+)-(1(10)E,4E,6S,7R)-
germacradien-6-ol synthase. The enzyme product exists as
two interconvertible conformers, resulting in complex NMR
spectra. For the complete assignment of NMR data, all fifteen
(13C1)FPP isotopomers (FPP = farnesyl diphosphate) and
(13C15)FPP were synthesized and enzymatically converted.
The products were analyzed using various NMR techniques,
including 13C, 13C COSYexperiments. The (13C)FPP isotopom-
ers were also used to investigate the thermal rearrangement and
EI fragmentation of the enzyme product.
The genome of S. pratensis ATCC 33331 encodes five
terpene cyclases, four of which show close homology to the
synthases for geosmin,[4c] 2-methylisoborneol,[4f, g] 7-epi-a-
eudesmol,[4n] and epi-cubenol,[4l] in agreement with the
production of these terpenes by the bacterium.[2a] The gene
of the fifth uncharacterized terpene cyclase (accession
number ADW03055; exhibiting the aspartate-rich motif
86DDEYCD and the NSE triad 227NDLVSYHKE) was
cloned into the expression vector pYE-Express by homolo-
gous recombination in yeast.[4o] The purified protein con-
verted farnesyl diphosphate (FPP) into (1(10)E,4E)-germa-
cradien-6-ol (1), identified by GC–MS (Figure 1A), while
geranyl and geranylgeranyl diphosphate were not accepted.
Two of the Cope rearrangement products of 1 (2a and 2b)
were also observed as a result of the thermal impact of the GC
analysis (the mass spectra of 1, 2a, and 2b are shown in
Figure S1 in the Supporting Information). The 1H and
13C NMR spectra of 1 recorded in CDCl3 at room temper-
ature showed broad and poorly resolved signals (Figure S2).
In contrast, the NMR spectra at À508C and at 08C showed
two sets of sharp signals for the known conformers 1a and 1b
(as shown in Figure 1B by the “up/down” nomenclature for
the methyl group pointing upwards (U) or downwards (D)).[8]
The NMR data corresponded to reported, but incomplete,
data for (À)-1 from Santolina rosmarinifolia.[8a] The optical
rotary power of [a]D24 =+ 21.4 for the compound pointed
T
erpenoids are structurally and functionally fascinating
natural products. The first identified compounds from bac-
teria, the earthy and musty odorants geosmin and 2-methyl-
isoborneol,[1] were isolated from streptomycetes in the 1960s,
while recent research has shown that terpenes are particularly
widespread in this taxon.[2] The biosynthesis of terpenes starts
from a linear oligoprenyl diphosphate that is converted by
a terpene cyclase in a reaction cascade via cationic inter-
mediates into a (poly)cyclic hydrocarbon or alcohol, which
usually has several contiguous stereocenters. Crystal struc-
tures of terpene cyclases[3] revealed that specific residues in
the active site bind a trinuclear (Mg2+)3 cluster that binds in
turn to the substrate’s diphosphate for ionization to a highly
reactive cation. Hydrophobic residues shape a contour to
force the substrate into a conformation for directed product
formation and exclude water from the cavity to prevent
quenching of immature intermediates. The products of
several bacterial terpene cyclases have been character-
ized.[3g,4] Additionally, our structure-based mechanistic under-
standing of bacterial terpene cyclases has been substantially
refined by quantum chemical calculations,[4s, 5] site-specific
mutations,[3g,h,4c,6] and isotope-labeling studies.[4b,g,q,7] Herein,
we present a conformational analysis, the thermal rearrange-
ment, and EI-MS fragmentation (EI-MS = electron impact
mass spectrometry) of a sesquiterpene alcohol from Strepto-
myces pratensis by use of 13C-labeling techniques.
[*] P. Rabe, L. Barra, J. Rinkel, Dr. R. Riclea, Dr. C. A. Citron,
T. A. Klapschinski, A. Janusko, Prof. Dr. J. S. Dickschat
KekulØ-Institut für Organische Chemie und Biochemie
Rheinische Friedrich-Wilhelms-Universität Bonn
Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany)
E-mail: dickschat@uni-bonn.de
Figure 1. A) Total ion chromatogram of the sesquiterpene products
from the S. pratensis (1(10)E,4E,6S,7R)-germacradien-6-ol (1) synthase.
B) Structures of conformers 1a and 1b. U indicates that the methyl
group is pointing up, D that the methyl group points downwards.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201507615.
13448
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to the opposite enantiomer as in the plant ([a]D20 = À14.8),
establishing the terpene from Streptomyces pratensis as
(+)-(1(10)E,4E,6S,7R)-germacradien-6-ol. The absolute con-
figuration of the plant metabolite 1 was recently confirmed by
total synthesis.[9] Unlike the other terpene synthase products,
1 is not found in S. pratensis laboratory cultures (not shown).
The complex NMR spectra prevented a full assignment of
1
the H and 13C NMR signals to 1a and 1b, as in a previous
report.[8a] To overcome this problem, all fifteen (13C1)FPP
isotopomers were synthesized (Figures S3–6)[10] and con-
verted with germacradienol synthase. Each product was
extracted with (2H8)toluene and analyzed by 13C NMR
spectroscopy, resulting in two strong signals for the labeled
carbons of conformers 1a and 1b (Figure S7). These data
unambiguously established which 13C NMR signals of
1 belonged to which carbon center, but it was not possible
to assign which of the two 13C NMR signals observed in each
single experiment belonged to which conformer. Therefore,
completely labeled (13C15)FPP was synthesized and incubated
with germacradienol synthase and the product was analyzed
by 13C, 13C COSY NMR experiments.[11] This experiment
revealed two distinct sets of cross-peaks (Figure 2; for an
enlarged version see Figure S8) that allowed for an unambig-
uous assignment of all 30 carbon signals to each of the 15
carbon atoms of 1a and 1b (Table 1). The assignment of most
Figure 2. 13C,13C COSY spectrum of (13C15)-1 obtained by enzymatic
conversion of (13C15)FPP. The two sets of cross-peaks for the con-
formers are shown in yellow (for 1a) and red (for 1b).
identical to 2a and 2b in terms of their mass spectra and GC
retention times. Their structures were determined by one- and
two-dimensional NMR spectroscopy (Table 1), resulting in
their identification as shyobunol (2a) and 5,10-di-epi-shyo-
bunol (2b).[12] The relative configurations were determined
from key NOESY correlations (Figure 4A). The assignment
of NMR data was confirmed by Cope rearrangement of
(13C15)-1, obtained by enzymatic conversion of (13C15)FPP, and
subsequent analysis of the product by 13C, 13C COSY NMR
experiments (Figure S10). From these experiments, two
distinct sets of cross-peaks were detected for 2a and 2b that
gave direct insights into the carbon–carbon connectivities.
The absolute configurations of 2a and 2b can be deduced
from the stereocenters at C-6 and C-7 of 1 that are not
affected by the Cope rearrangement, as is known for various
1
1H NMR resonance signals was possible from H,1H COSY,
HSQC, and HMBC correlations (Figure 3) of the unlabeled
compound. For a few cases, the HSQC spectra of the relevant
(13C1)-1 isotopomers were very useful (Figure S9).
For structure elucidation of the two Cope rearrangement
products observed during GC–MS analysis, 1 was subjected to
a microwave reaction in toluene at 2258C. The products were
separable by column chromatography and proved to be
Table 1: NMR data of the conformers 1a and 1b of (1(10)E,4E,6S,7R)-germacradien-6-ol in (2H8)toluene recorded at À508C, and of 2a/2b in
(2H6)benzene at 258C.[a]
1+2
1a (DD)
1b (UD)
2a
2b
C[a]
1H
13C
1H
13C
1H
13C
1H
13C
1
2
CH 4.80 (d, J=11.6, 1H) 129.0 4.87 (t, J=7.5, 1H) 121.5 5.78 (dd, J=17.5, 10.8, 1H) 150.3 5.85 (dd, J=17.6, 10.8, 1H) 150.2
CH2 2.17 (m, 1H)
1.95 (m, 1H)
CH2 2.04 (m, 1H)
2.00 (m, 1H)
24.8 2.24 (m, 1H)
1.89 (m, 1H)
39.2 2.09 (m, 1H)
1.94 (m, 1H)
24.6 4.98 (dd, J=17.5, 1.2, 1H, E) 110.1 4.90 (dd, J=17.6, 0.8, 1H, E) 110.4
4.94 (dd, J=10.8, 1.2, 1H, Z) 4.86 (dd, J=10.8, 0.8, 1H, Z)
37.5 5.02 (br s, 1H, Z) 113.3 4.90 (br s, 1H)
3
113.9
4.91 (br s, 1H, E)
–
4.70 (br s, 1H)
–
4
5
6
7
8
Cq
–
131.8
–
132.0
146.8
145.5
57.4
71.9
44.6
22.3
CH 5.06 (d, J=7.0, 1H) 135.1 5.04 (d, J=8.5, 1H) 133.0 1.69 (d, J=1.7, 1H)
CH 4.54 (d, J=6.0, 1H) 68.5 4.55 (d, J=6.0, 1H) 68.4 3.81 (br s, 1H)
CH 0.75 (d, J=9.0, 1H) 49.7 0.66 (d, J=9.5, 1H) 52.5 0.74 (m, 1H)
56.6 2.32 (d, J=6.8, 1H)
70.2 3.94 (m, 1H)
49.8 1.54 (m, 1H)
21.1 1.56 (m, 2H)
CH2 1.95 (m, 2H)
1.39 (d, J=13.8, 1H)
30.7 1.80 (m, 1H)
1.30 (m, 1H)
25.6 1.63 (m, 1H)
1.49 (m, 1H)
9
CH2 2.44 (d, J=13.1, 1H) 36.1 2.14 (m, 1H)
41.8 1.48 (m, 1H)
1.29 (m, 1H)
41.1 1.53 (m, 1H)
1.25 (m, 1H)
33.7
1.62 (t, J=13.5, 1H)
–
1.79 (m, 1H)
–
10 Cq
11 CH 1.78 (m, 1H)
12 CH3 1.00 (d, J=6.0, 3H) 21.4 1.09 (d, J=6.3, 3H) 21.2 0.92 (d, J=6.8, 3H)
13 CH3 1.05 (d, J=6.5, 3H) 21.6 1.00 (d, J=6.0, 3H) 21.3 0.94 (d, J=6.9, 3H)
14 CH3 1.55 (s, 3H)
15 CH3 1.32 (s, 3H)
135.3
138.5
–
40.2
–
30.3
27.4
21.9
23.1
23.1
26.8
32.0 1.73 (m, 1H)
32.6 1.68 (m, 1H)
29.4 1.88 (oct, J=6.8, 1H)
20.8 0.94 (d, J=6.8, 3H)
21.3 1.20 (d, J=6.6, 3H)
20.3 0.93 (s, 3H)
22.0 1.48 (s, 3H)
16.3 1.30 (s, 3H)
16.9 1.46 (s, 3H)
16.2 1.72 (s, 3H)
27.8 1.68 (s, 3H)
[a] Carbon numbering as shown in Figure 1. Chemical shifts d in ppm, multiplicity m (s=singlet, d=doublet, t=triplet, oct=octet, m=multiplet,
br=broad), coupling constants J are given in Hertz. Carbon assignments for 1 were deduced from incubation experiments with 13C-labeled FPP
isotopomers (see the main text).
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Figure 3. Key HMBC correlations that enabled the assignment of
1H NMR signals of unlabeled 1.
Figure 5. EI-MS fragmentation of 1. Mechanisms for the formation of
fragment ion [A]+ (m/z=161) and the base peak ion [B]+ (m/z=84).
[M]+ (Figure 5A) that is observed at m/z = 222 for unlabeled
1 and at m/z = 223 for all (13C1)-1 isotopomers (Table S12).
Rearrangement of one hydrogen atom (rH) and inductive
cleavage (i) with the neutral loss of water yields the fragment
ion [MÀH2O]+ that is detected at m/z = 204 for natural 1 and
at m/z = 205 for all (13C1)-1 isotopomers. A subsequent
a cleavage (a) with loss of the isopropyl group is the only
relevant mechanism that yields fragment ion [A]+. This is
evident from the observation of [A]+ at m/z = 161 in the mass
spectrum for unlabeled 1 as well as for all isotopomers that
contain 13C labeling within the isopropyl group, that is, for the
products obtained from (11-13C)FPP, (12-13C)FPP, and (13-
13C)FPP. All other (13C1)FPP isotopomers had a shifted
signal for [A]+ at m/z = 162 (Figure S11). This mechanism
was further supported by HRMS data for [A]+, which
Figure 4. Cope rearrangement of 1. A) Key NOESY correlations for
determination of the relative configurations of 2a and 2b. B) Confor-
mations of 1 explaining the formation of 2a and 2b.
germacranes.[13] The bacterial shyobunol stereoisomers iso-
lated here are the enantiomers of plant compounds from
Acorus calamus,[12] in agreement with the fact that also
bacterial 1 is the optical antipode of a plant terpene. Whereas
2b arises from the reported conformer 1a,[8a] the formation of
2a is possible from the up/up conformer 1c (Figure 4B, UU).
Both rearrangement products are formed via chair-like
transition states, similar to the Cope rearrangements of
several other germacranes.[8b, 14]
The relative configurations of 2a and 2b gave additional
support for the syn orientation of the hydroxy and isopropyl
groups in 1. In fact, the 13C NMR chemical shifts of 1 and its
anti stereoisomer kunzeaol are very similar (Table S11), and
the syn or anti orientation of the substituents in the con-
formationally flexible compounds 1 and kunzeaol[15] are
difficult to determine. However, the NOESY spectra of the
more rigid compounds 2a and 2b unambiguously proved the
syn arrangement of the hydroxy and the isopropyl groups,
thereby giving indirect evidence for the correct assignment of
the relative configuration of 1.
Since isotopically labeled compounds are very useful in
studying MS fragmentation mechanisms,[16] the products
obtained from all fifteen isotopomers of (13C)FPP with
germacradienol synthase were also subjected to GC/EI-MS
and GC/EI-MS-QTOF analysis. The observed fragment ion
patterns for the isotopomers of (13C)-1 (Figure S11) gave
direct insight into the fragmentation mechanism. Electron
impact ionization of 1 proceeds preferably with the loss of one
electron from an oxygen lone pair to yield the molecular ion
+
established its molecular formula as C12H17 for all signals
12
+
at m/z = 161 or as 13C1 C11H17 for all at m/z = 162
(Table S12), and by MS2 analysis showing the direct formation
of [A]+ from [MÀH2O]+.
For the formation of the base peak [B]+ at m/z = 84, the
mechanism was shown to proceed by two a-cleavage reac-
tions with the loss of the neutral molecule 3-methylbut-1-ene
to a form a fragment ion at m/z = 152 and a third subsequent
a cleavage with the neutral loss of isoprene (Figure 5B). The
last step was confirmed by a shift of the signal for [B]+ to
m/z = 85 for all (13C1)-1 isotopomers in which the isotopic
labeling appears in the [B]+ forming portion, that is for
1 derived from (1-13C)FPP, (2-13C)FPP, (3-13C)FPP, (4-
13C)FPP, and (15-13C)FPP (Figure S11, Table S12). Further-
more, HRMS data established the molecular formulae for
molecular fragments with m/z = 84 (C5H8O+) and m/z = 85
(13C112C4H8O+). The formation of [B]+ as a daughter ion from
the fragment ion at m/z = 152 was investigated by MS2
analysis, but because of the low abundance of this ion this
experiment was inconclusive. The formation of [B]+ is thus
better described as a concerted process of three simultaneous
a fragmentations.
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33, 5846; b) X. Lin, R. Hopson, D. E. Cane, J. Am. Chem. Soc.
In summary we have characterized a bacterial terpene
cyclase from S. pratensis as (+)-(1(10)E,4E,6S,7R)-germacra-
dien-6-ol synthase. Only one closely related homologue with
99.4% identical sites is found in Streptomyces sp.
PAMC26508. As is typical for germacranes, 1 exists in
different well-defined conformers that are observable by
NMR at low temperatures. Extensive labeling experiments
using synthetic 13C-labeled FPP isotopomers enabled a full
assignment of 1H and 13C NMR data of 1, which had until this
point not been possible for 1 and related germacranes as
a result of their complex NMR spectra.[8] A thermal
rearrangement of 1 via chair-like transition states yielded
two products whose absolute configurations were deduced
from the absolute configuration of 1, while the relative
configurations of the rearrangement products reconfirmed
that 1 is different from its epimer kunzeaol. Using 13C-labeled
FPP isotopomers, we have also laid the groundwork for
analyses of EI-MS fragmentation patterns of sesquiterpenes,
and we present a first showcase study here. Future experi-
ments in our laboratories will include the usage of the FPP
isotopomers to address various other intricate problems of
sesquiterpene chemistry.
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Received: August 14, 2015
Published online: September 11, 2015
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