Stereoisomeric Composition of Natural Myrtucommulone A

Article abstract of DOI:10.1021/acs.jnatprod.5b00358

Myrtucommulone A (MC A) (1), isolated from Myrtus communis (myrtle), shows the same pharmacological activity for inhibition of inflammation and induction of apoptosis as synthetic MC A, which consists of three stereoisomers, i.e., two enantiomers and one

Full text of DOI:10.1021/acs.jnatprod.5b00358

Article
pubs.acs.org/jnp
Stereoisomeric Composition of Natural Myrtucommulone A
†
†
‡
,†
#
Marcus Hans, Mael Charpentier, Volker Huch, Johann Jauch,* Torsten Bruhn,
̈
,#
,§
Gerhard Bringmann,* and Dietmar Quandt*
†
Institute of Organic Chemistry, Saarland University, Campus C 4.2, 66123 Saarbru
̈
cken, Germany
Institute of Inorganic Chemistry, Saarland University, Campus C 4.1, 66123 Saarbrucken, Germany
Institute of Organic Chemistry, University of Wurzburg, Am Hubland, 97074 Wurzburg, Germany
Nees-Institute for Biodiversity of Plants, University of Bonn, Meckenheimer Allee 170, 53115 Bonn, Germany
S Supporting Information
‡
#
§
̈
̈
̈
*
ABSTRACT: Myrtucommulone A (MC A) (1), isolated from
Myrtus communis (myrtle), shows the same pharmacological
activity for inhibition of inflammation and induction of
apoptosis as synthetic MC A, which consists of three
stereoisomers, i.e., two enantiomers and one meso form. This
led to the question of whether the natural MC A is a pure
stereoisomer or a mixture of stereoisomers. The specific
rotation and electronic circular dichroism (ECD) data of
natural MC A (1) as well as of a pentacyclic derivative 4
revealed that naturally occurring MC A (1) consists of the
racemate and the meso form in a 1:1 ratio. A probable
precursor of MC A (1), nor-semimyrtucommulone (5), was
also isolated from myrtle as a racemate. The absolute
configurations of the enantiomers of 1 and 5 were determined using a combination of experimental and quantum-chemical
calculated ECD spectra.
1
,6
Myrtus communis L. (myrtle) is an evergreen shrub growing in
the Mediterranean area. Blue and white berries of myrtle have
been used to prepare the mirto rosso and mirto bianco liquors.
Extracts from the leaves are used in traditional medicine due to
their antibiotic and anti-inflammatory activity. In 1974,
Additionally to the known antibiotic activity,
other
pharmacological properties were also discovered during the
past decade. These include anti-inflammatory activity,
cytotoxicity and induction of apoptosis in cancer cells,
antioxidative effects, bi5nding to TRH receptors, and
1
1
0
1
1
2
7
1
Kashman et al. reported the isolation, structure elucidation,
inhibition of α-glucosidase.
and antibacterial activity of myrtucommulone A (MC A) (1)
and myrtucommulone B (MC B) (2) from M. communis
We became interested in myrtucommulones due to their
anti-inflammatory and cytotoxic activities and developed a short
and efficient synthesis for MC A (1) and other members of this
2
(Figure 1).
13
Later, MC A was also found in other members of the
compound family. The synthesis afforded MC A (1) as a
mixture of all three stereoisomers, i.e., two enantiomers and
one meso form. Pharmacological tests of anti-inflammatory as
well as apoptosis-inducing properties interestingly show
identical activities for both synthetic and natural MC A (1).
This led to the question of whether the natural MC A is a pure1
stereoisomer or a mixture of stereoisomers. Neither Kashman
3
4
Myrtaceae. Appendino et al. “rediscovered” myrtucommu-
lones in 2002 and confirmed the structure of MC A (1)
1
published by Kashman et al. In addition, semimyrtucommu-
5
lone (3) was also isolated. Later, Shaheen et al. isolated and
characterized the new myrtucommulones C, D, and E from
6
myrtle, and Appendino et al. found new galloylated
4
myrtucommulones from extracts of the same plant. During
their search for new natural products with TRH-receptor-2
binding affinity (TRH = thyrotropin releasing hormone),
and co-workers nor Appendino et al. reported specific
7
rotations for MC A. However, Quinn et al. recently published
an [α]1
7.6
D
value of +42.1 (c 0.066, MeOH). However, Quinn
7
Quinn et al. discovered new myrtucommulones F−I from
et al. offered two contradictory statements about the absolute
14
Corymbia scabrida, which also belongs to the Myrtaceae. The
latest members of the myrtucommulones, namely, myrtucom-
mulones J, K, and L, were isolated from Myrtus communis by
configuration of MC A. This confusion, together with our
observation of identical pharmacological activities of natural
and synthetic MC A (1), led us to explore naturally occurring 1
8
Cottiglia et al. Recently, Nicoletti et al. showed that the
endophytic fungus Neofusicoccum australe, isolated from myrtle,
also produces MC A.
Received: April 29, 2015
9
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00358
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
z = 667.3) ion as the only ion. The specific rotation of pure 1
7
20
D
under the conditions of Quinn et al. is [α]
= 0 (c 0.1,
MeOH), and the ECD spectrum recorded for compound 1
showed no Cotton effects. Thus, naturally occurring MC A is a
racemate or the meso form or a mixture of both forms.
Next, separation of MC A into racemate and meso form by
HPLC was investigated with different stationary phases, but
without success. Therefore, MC A (1) was cyclized and
dehydrated to the pentacyclic derivative 4 (Scheme 1)
1
according to the conditions of Kashman et al.
This cyclization and dehydration was previously used to
13
determine the ratio of racemate and meso form of synthetic 1.
The cyclization product 4 is a linear pentacyclic bispyrane
13
according to NMR analysis, and only traces of the angularly
cyclized isomer constitutionally identical with myrtucommu-
5
lone E reported by Shaheen et al. were present. Compound 4
is separable into its racemate 4a and its meso form 4b via
HPLC on a C18 column using 80:20 MeOH/MeCN as eluent
(
Figure 3). Thus, naturally occurring MC A is indeed a mixture
of the racemate and the meso form in the ratio of 52:48. As
1
3
Figure 1. 2D structures of MC A (1), MC B (2), and
semimyrtucommulone (3).
shown previously, the first eluting peak is the racemate 4a,
and the second is the meso form 4b. Additionally, ECD spectra
of 4, 4a, and 4b were recorded, and no Cotton effects were
observed. To prove that the first eluting peak of 4 is the
racemate, it was further separated into its enantiomers via
HPLC on a ChiralCel-OD-H column (Figure 3). The ECD
spectra of both enantiomers of 4a were recorded, and as
anticipated the compounds showed mirror-image Cotton
effects (Figure 3).
in more detail, especially regarding isomeric composition and
absolute configuration.
RESULTS AND DISCUSSION
■
For the isolation of 1 finely ground myrtle leaves (M.
communis) were extracted exhaustively with diethyl ether.
After evaporation of the solvent, the residual green gum was
subjected to flash chromatography on silica gel, yielding five
fractions. The third fraction contained 1 together with other
myrtucommulones and was further purified by preparative RP-
HPLC using 80:20 (v/v) (MeOH + 0.1% TFA)/(MeCN +
To determine the absolute configurations of the enantiomers
of 4a quantum-chemical ECD calculations were performed and
compared with the experimental results. A conformational
analysis using B97D/TZVP of PMCA (4) yielded 860 different
conformations within a small energy range, a number much too
high for subsequent TDDFT calculations. By several
simplifications and by using energies from B2GP-PLYP-D3/
def2-TZVPP single-point calculations (including solvent effects
with COSMO) of the aforementioned conformers, the number
of relevantly populated conformers could be reduced to 55,
giving about 80% of the population of PMC A in solution.
Details of this conformational analysis and the selection criteria
for the TDDFT calculations can be found in the Supporting
Information. Finally, TDBHLYP/def2-TZVP(-f) computations
gave an ECD curve for the R,R-enantiomer that corresponds
with the ECD spectrum of the faster eluting compound (+)-4a
= 70%, Figure 3). Conversely, the predicted ECD
spectrum of the S,S-configured compound matched that of the
slower eluting compound (−)-4a (Δ = 69%, Figure 3). In
the course of our enantioselective synthesis of MC A (1) the
S-Mosher ester of (+)-4a afforded crystals suitable for X-ray
diffraction analysis. The X-ray crystal structure additionally
verified the configurations established by the experimental and
computational ECD methods (Figure 4).
0
.1% TFA) on a C18 column. Owing to impurities, the isolate
was further purified to yield MC A (1) with a purity of 98−
00%, as shown in Figure 2.
MC A was obtained as a yellow powder (lit. mp: 185−186
1
1
7
°
C, crystallized from MeOH; lit.: yellow gum) and was further
characterized by H NMR spectroscopic and ESIMS data in the
1
1
negative ion mode. The H NMR data were identical to the
data of synthetic MC A, and ESIMS showed an [M − H] (m/
−
15
(
Δ
ESI
15
ESI
16
1
7
These results clearly show that natural MC A (1) consists of
16
the racemate and the meso form in a ca. 1:1 ratio. It is likely
that 1 is synthesized nonenzymatically in M. communis or in
Neofusicoccum australe like other natural compounds isolated
from various plants, bacteria, and even animals. If the
biosynthesis of MC A results from a double Michael addition of
isobutyrylphloroglucinol with isobutylidenesyncarpic acid
Figure 2. Analytical HPLC of the crude myrtle extract (gray),
analytical HPLC after first purification by preparative HPLC (green),
and analytical HPLC after second purification by preparative HPLC
18
(
red). Conditions for analytical HPLC: C18 ec (4.6 × 250, 5 μm,
M&N) + guard column C18 (4.0 × 5.0, 5 μm, M&N), 80:20 (MeOH +
.1% TFA)/(MeCN + 0.1% TFA); 1.5 mL/min, 24 °C. Conditions
0
(
possibly formed via a Knoevenagel condensation of syncarpic
for preparative HPLC: C18 ec (21 × 250, 5 μm, M&N) + guard
column C18 (20 × 30, 5 μm, Grom Saphir), 80:20 (MeOH + 0.1%
TFA)/(MeCN + 0.1% TFA); 16 mL/min, room temperature.
acid and isobutyraldehyde), then nor-semimyrtucommulone
(5) (NSMC) (Scheme 2) should be a precursor of 1 and
B
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Scheme 1. Cyclization of Naturally Occurring MC A (1) to the Pentacyclic Derivative 4 (PMCA)
Figure 3. Resolution of the racemate and the meso compound and subsequently of the enantiomers of 4a using HPLC on achiral and chiral phase
and determination of the absolute configurations of the enantiomers by comparison of the experimental and quantum-chemically calculated ECD
spectra. The resulting absolute configurations are consistent with the configuration found by X-ray structure determination of the S-Mosher
derivative of (+)-R,R-4a.
Scheme 2. 2D Structure of NSMC (5) and Cyclization to
MC B (2)
In fraction 4 of the first chromatographic separation of the
myrtle extract, semimyrtucommulone (3) together with 5 were
isolated. After two purification steps via preparative HPLC on a
Figure 4. ORTEP drawing of the S-Mosher ester of (+)-4a. The
C18 column [first elution with 80:20 (v/v) (MeOH + 0.1%
structure representation shows ellipsoids at 50% probability; hydrogen
TFA)/(MeCN + 0.1% TFA); second elution with 90:10 (v/v)
atoms were omitted for reasons of clarity.
(
MeOH + 0.1% TFA)/(H O + 0.1% TFA)] pure 5 was
2
1
obtained. The H NMR spectrum of compound 5 is of similar
complexity to that of compound 1 and is not helpful in
structure elucidation, but ESIMS showed only one peak at m/z
should likely also occur in myrtle extracts. Compound 5 should
also serve as the precursor of myrtucommulone B (2) (MC B).
−
= 431.2, which corresponds to [M − H] for 5. The specific
C
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Figure 5. Separation of the enantiomers of 2 and attribution of the absolute configurations of the enantiomers by comparison of experimental and
TDDFT-computed ECD spectra. The resultant absolute configuration is consistent with the configuration found by X-ray diffraction analysis.
rotation is [α]20 = 0 (c 0.1, MeOH), and the ECD spectrum
D
shows approximately a zero baseline, which implies that
compound 5 is also a racemate. To further confirm this and
to elucidate the structure of 5 unambiguously, NSMC (5) was
cyclized to the pyrane 2. The racemic mixture was separated on
a ChiralCel-OD H column as shown in Figure 5. Thus, 2 and,
therefore, also 5 are racemates.
By comparison of the experimental and calculated ECD
spectra, the absolute configuration of NSMC (5) and also of
MC B (2) could be deduced (Figure 5). The conformational
analysis of 2 using B2GP-PLYP-D3/def2-TZVPP//B97D/
TZVP (including solvent effects with COSMO) gave 12
conformations within an energy range of 3 kcal/mol. From
these conformations ECD spectra were calculated with
TDCAM-B3LYP/TZVP, and the resulting ECD spectra were
averaged using a Boltzmann statistical weighting. The
computed ECD curve of (S)-2 showed a good matching
Figure 6. ORTEP drawing of 2″,5-dibromo-(+)-2. The structure
representation shows ellipsoids at 50% probability; hydrogen atoms
were omitted for reasons of clarity.
in M. communis. This explains why pharmacological activities of
13
the synthesized compounds are similar to those of the
isolated natural products. This poses the question of whether
(
Δ
= 81%) with the experimental ECD spectrum of the more
ESI
5
,7,8
the other myrtucommulones
are enantiomerically pure and,
rapid eluting enantiomer (−)-2, while the calculated spectrum
of the R-enantiomer matches the Cotton effects of the slower
if so, how these compounds are biosynthesized. In view of the
9
19
results of Nicoletti et al., it seems that myrtle and its
eluting compound (+)-2 (Δ = 82%). Dibromination of
ESI
endophytic fungus both produce MC A and other myrtucom-
mulones. Our current research is directed to answer these
questions.
(
+)-2 yielded white needles of 2″,5-dibromo-2, which could be
used for X-ray analysis (Figure 6), revealing the absolute
2
0
configuration as R and confirming the results of the
calculations.
EXPERIMENTAL SECTION
■
Thus, MC A (1) isolated from the plant material is a mixture
General Experimental Procedures. All solvents were from
Merck, Darmstadt, Germany, in HPLC grade. Optical rotations were
measured in MeOH at 20 °C using a PerkinElmer 241 polarimeter
with a sodium lamp. ECD spectra were recorded on a Jasco J-715
spectropolarimeter in HPLC grade MeOH. NMR spectra (1D: H,
C, DEPT 135; 2D: H,H-COSY, HSQC, HMBC, NOESY) were
of racemate and meso form, and MC B (2) and NSMC (5) are
7
both racemates. Since Quinn et al. reported some optical
activity for 1, one possibility we have to rule out is that the
purchased material in our studies is not myrtle. Therefore, we
assessed the genetic identity of the dried leaves of M. communis
that were purchased from Alfred Galke, Gittelde, Germany.
After rigorous genetic analyses (see Experimental Section) the
plant material is without any doubt M. communis. Additionally,
the genetic analyses revealed that the plant material does not
contain any endophytic fungus. Thus, the isolated myrtucom-
mulones originate from myrtle and not from other organisms.
In summary, we unambiguously demonstrated that MC A
1
13
1
13
recorded on a Bruker AVANCE II 400 ( H NMR at 400 MHz,
C
NMR at 100 MHz). Chemical shifts are given in ppm, and coupling
constants in Hz. For processing the NMR spectra, MestReC 4.9.9.6
was used. ESIMS data were measured with a Shimadzu LC-MS 2020
from Shimadzu, Duisburg, Germany, and HRESIMS data were
measured on an LTQ Orbitrap XL from Thermo Scientific, Dreieich,
Germany.
TLC was carried out on silica glass plates (Si60 F254 from Merck)
and RP18 silica glass plates (RP18-F254 from Merck). For flash
(1) and the potential precursor NSMC (5) occur as racemates
D
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chromatography, silica gel with 40−63 μm particle size from Merck
was used. Analytical HPLC was performed on a SYKAM HPLC
GU984345, GU984370) and M. nivellei (JF304867, GU984339,
GU984338).
machine (SYKAM, Fu
mixer S8111, DAD S3210, Rheodyne valve 5210i and Jet-Stream II
̈
rstenfeldbruck, Germany, gradient pump S1122,
Maximum likelihood (ML) reconstructions were done using
RAxML. Internal branch support was estimated by heuristic
2
7
column oven). RP18ec Nucleodur columns (250 × 4.6 mm, 5 μm)
bootstrap (BS) searches with 10 000 replicates each. Bayesian analyses
2
8
from Macherey & Nagel, Du
̈
ren, Germany, were used as analytical
were performed with MrBayes v3.1.2, applying the GTR+Γ+I model.
6
HPLC columns, together with an RP18 guard column (4.0 × 5.0 mm,
μm). Preparative HPLC was done on a SYKAM HPLC machine
SYKAM, Furstenfeldbruck, Germany, gradient pump S1521, DAD
S3210 and Rheodyne valve 5330). A ChiralCel-OD H HPLC column
250 × 4.6 mm, 5 μm) was purchased from VWR International,
Darmstadt, Germany. Preparative HPLC columns were from
Macherey & Nagel, Duren, Germany, with RP18ec Nucleodur 5 μm
Four runs with four chains (5 × 10 generations each) were run
5
simultaneously, with a chain sampling every 1000 generations.
2
9
(
̈
Tracer v1.4 was used to examine the log likelihoods to determine
the effective sampling size and stationary of the MCMC search.
Calculations of the consensus tree, including clade posterior
probability (PP), were performed based upon the trees sampled
after the chains converged (<generation 500 000). Consensus
topologies and support values from the different methodological
(
̈
as stationairy phase (250 × 21 mm) with Grom Saphir 110C18 5 μm
guard columns (30 × 20 mm).
̈
approaches were compiled and drawn using TreeGraph2 (Stover et
3
0
Plant Material. Dried leaves of Myrtus communis were purchased
from Alfred Galke, Gittelde, Germany. To verify the species identity of
the purchased plant material, a DNA-barcoding approach was chosen.
Owing to the wealth of sequence information available in GenBank,
two of the most prominent DNA markers used in molecular
phylogenetics were selected, namely, (1) the plastid trnL-F region
al. ).
Both marker systems unambiguously identified the plant material of
both purchased batches as M. communis. The plastid marker revealed
100% identity of batch 1 (ED993) with the published M. communis
2
3
haplotype LF1 (FJ611338; Migliore et al. ), whereas batch 2
(ED992) belongs to the haplotype LF2 (FJ611337). While the fast,
but in comparison to the ITS slow, evolving plastid trnL-F region
discriminates between only two haplotypes (LF1 and LF2), far more
ITS haplotypes have been reported for M. communis that even provide
21
(
compare Quandt et al. ), including the trnL
group I intron and
UAA
the trnL-trnF intergenic spacer (IGS), and (2) the nuclear ITS region,
including the internal transcribed spacers (ITS) 1 and 2 as well as the
2
2
23
5
.8S gene of the nuclear rDNA. Both markers have been successfully
phylobiogeographical information (compare Migliore et al. ).
used to describe distinct geographic haplotypes within the genus
Surprisingly, ITS sequences of batch 1 (ED993) revealed a new
undescribed nuclear M. communis haplotype with no ambiguous
character codings and close affinities to Western Mediterranean
samples (compare Figure 7), such as AM234149 or JF304838
(haplotype ITSF40). In contrast, ITS sequences of batch 2 (ED992)
were composed of a mixture of different published M. communis
haplotypes; that is, ambiguous characters in accordance with the
published haplotypes were recorded. As the identification of the plant
material was still unambiguous, we refrained from a cloning approach
that would have revealed the precise haplotypes and biogeographical
history.
Apart from the ambiguities that are in accordance with the different
nuclear M. communis haplotypes, no traces of foreign nuclear rDNA
were detected in the batches. As the amplification protocol and the
ITS primers are universal for eukaryotes, the presence of fungal DNA
would have obscured the PCR and sequencing results. Owing to its
shorter size, the fungal ITS region would have been amplified
preferably over the angiosperm ITS region. Thus, clear fungal
fragments would have been visible in the Agarose Gel Electrophoresis
(AGE) controls. However, neither have PCR amplicons of different
size been detected nor have unambiguous pherogram reads as a result
of fungal contaminations, etc., been observed. Thus, we found no
evidence of fungi present in both analyzed batches.
Myrtus and to address phylogeographic patterns (compare Migliore et
2
3
al. ).
Therefore, whole genomic DNA of random samples of the
purchased material was isolated using commercially available spin
columns (NucleoSpin Plant II, Macherey-Nagel, Germany) following
the manufacturer’s protocol. Prior to incubation with the lysis buffer
plant material was pulverized in 2 mL Eppendorf caps (round-bottom)
containing three glass beads (5 mm) with a Mixer Mill (Retsch
TissueLyser, Qiagen) at 30 Hz (two rounds of 1 min).
PCR amplifications (T3 Thermocycler, Biometra) were performed
in 50 μL reactions containing 1 U Taq-DNA polymerase (GoTaq,
Promega), 1 mM dNTP mix of each 0.25 mM, 1× buffer, 1.25−2.5
mM MgCl , and 20 pmol of each amplification primer. The ITS region
2
was amplified using the primers ITS4 and ITS5 designed by White et
2
4
al. with an amplification profile of 5 min at 94 °C, 40 cycles (1 min at
4 °C, 1 min at 48 °C with a time increment of +4 s/cycle, 45 s at 68
C), and a final extension of 7 min at 68 °C. PCR protocols for the
9
°
25
trnL-F region followed Borsch et al. Column cleaned PCR products
NucleoSpin Gel and PCR Clean-up, Macherey-Nagel, Germany)
(
were sequenced by Macrogen Inc., South Korea (www.macrogen.
com).
Sequences were edited and aligned using PhyDE version 0.996
26
(
Mu
̈
ller et al., available at http://www.phyde.de). Primer sequences
Extraction and Isolation. Dried leaves were frozen overnight at
were clipped off. Obtained trnL-F and ITS sequences of both
purchased batches were compared with existing records in GenBank
via BLAST. To track the geographic origin of the samples, we included
the obtained ITS sequences in the ITS data set provided by Migliore et
−30 °C and then powdered with a laboratory mixer. The powder (500
g) was extracted in a Soxhlet apparatus with 6 L of Et O as solvent for
2
11 h. The solvent was removed in vacuo at room temperature to yield
18 g of a dark green gum. This material was dissolved in 200 mL of
acetone and absorbed on 100 mL of silica gel (Merck Si80, 60−200
μm particle size). The acetone was removed in vacuo at room
temperature, and the green powder was applied on top of a
chromatography column (30 × 7.5 cm), packed with a slurry of 700
mL of silica gel (Merck Si60, 40−63 μm particle size) in ca. 1.2 L of
petroleum ether/acetone, 3:2 (v/v). The chromatography was
performed isocratically with petroleum ether/acetone, 3:2 (v/v), to
give four fractions, F1 to F4. The last fraction (F5) was obtained after
rinsing the column with MeOH. The solvent of each fraction was
removed in vacuo at room temperature, recovery factor 93%.
2
3
al., plus additional data available from GenBank, and subjected the
data set to phylogenetic analyses. As outgroups Xanthostemon
chrysanthus (EF041515), Stockwellia spec. (AF390445), Angophora
costata (DQ499112), Callistemon viminalis (JX856429), Eugenia
tropophylla (AY487303), Ugni selkirkii (JN660934), U. molinae
(
JN660933), Archirhodomyrtus paitensis (HQ225436), A. baladensis
(
HQ225434), and A. turbinata (HQ225437) were chosen. Sequences
with ambiguous character codings were omitted from the ITS data set;
thus only the following sequences were included: Myrtus communis
(
AM234149, JF304834, JF304838, GU984375, GU984340,
GU984364, GU984363, GU984373, GU984372, GU984349,
JQ740194, GU984365, GU984348, GU984367, JF304823,
GU984360, GU984342, GU984362, GU984351, GU984352,
GU984356, GU984341, GU984359, GU984350, GU984361,
GU984374, GU984368, GU984366, GU984358, GU984357,
GU984355, GU984354, GU984353, GU984376, GU984347,
GU984371, GU984369, GU984346, GU984344, GU984343,
MC A (1). Fraction F3 (60 mg) was dissolved in 20 mL of MeOH
and applied in portions of 4 mL to preparative HPLC using 80:20
(MeOH + 0.1% TFA)/(MeCN + 0.1% TFA). Two fractions were
obtained (F3.1 and F3.2), and F3.1 contained MC A (purity ca. 70%).
F3.1 was purified a second time under the same conditions to give two
fractions, F3.1.1 and F3.1.2. F3.1.1 is pure MC A (1) (purity 98−
100%). In total, from 60 mg of F3, 16 mg of pure MC A was obtained:
E
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Journal of Natural Products
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Cyclization of MC A (1) to PMCA (4). MC A (10 mg, 15 μmol) was
dissolved in dry benzene (10 mL). p-Toluenesulfonic acid (15.6 mg,
9
0 μmol) was added, and the mixture was refluxed for 1 h. After
cooling to room temperature, the mixture was washed with saturated
NaHCO solution (20 mL), dried with MgSO , and evaporated to
3
4
dryness to yield 10 mg of crude PMCA (4), which was dissolved in
MeOH (3 mL) and purified by preparative HPLC (MeOH/H O,
2
2
0
D
9
0:10). Yield: 4.2 mg of racemic 4a as a white powder; [α] 0 (c 0.1,
MeOH); H NMR (400 MHz, CDCl ) δ 0.78 (d, J = 6.8 Hz, 6 H, H-
1
3
1
0′ and H-10‴), 0.82 (d, J = 6.8 Hz, 6 H, H-9′ and H-9‴), 1.23 (d, J =
7
.1 Hz, 3 H, H-4″), 1.25 (d, J = 7.1 Hz, 3 H, H-3″), 1.40 (s, 6 H, H-14′
and H-13‴), 1.42 (s, 6 H, H-11′ and H-12‴), 1.43 (s, 6 H, H-13′ and
H-14‴), 1.54 (s, 6 H, H-12′ and H-11‴), 1.94 (m, 2 H, H-8′ and H-
8
‴), 3.16 (sept, J = 7.0 Hz, 1 H, H-2″), 4.32 (d, J = 3.4 Hz, 2 H, H-1′
13
and H-1‴), 5.88 (br s, 1 H, OH); C NMR (100 MHz, CDCl ) δ
3
1
7.98 (CH , C-3″ and C-4″), 18.60 (CH , C-9′ and C-9‴), 19.24
3
3
(
CH , C-10′ and C-10‴), 24.40 (CH , C-14′ and C-13‴), 24.81 (CH ,
3
3
3
C-11′ and C-12‴ and C-13′ and C-14‴), 25.19 (CH , C-12′ and C-
3
1
1‴), 32.29 (CH, C-1′ and C-1‴), 35.10 (CH, C-8′ and C-8‴), 42.67
(
CH, C-2″), 47.54 (C, C-4′ and C-4‴), 55.84 (C, C-6′ and C-6‴),
1
5
3
08.70 (C, C-2′ and C-2‴), 110.21 (C, C-1), 110.88 (C, C-3 and C-
), 147.55 (C, C-2 and C-6), 152.03 (C, C-4), 168.82 (C, C-3′ and C-
‴), 198.72 (C, C-7′ and C-7‴), 204.30 (C, C-1″), 211.78 (C, C-5′
and C-5‴); HRESIMS m/z 655.3240 (calcd for C H O Na
3
8
48
8
6
55.3241).
Yield: 3.8 mg of meso 4b as a white powder; [α]2 0 (c 0.1,
0
D
1
MeOH); H NMR (400 MHz, CDCl ) δ 0.78 (d, J = 6.9 Hz, 6 H, H-
3
1
7
6
0′ and H-10‴), 0.82 (d, J = 6.9 Hz, 6 H, H-9′ and H-9‴), 1.29 (d, J =
.0 Hz, 6 H, H-3″ and H-4″), 1.35 (s, 6 H, H-14′ and H-14‴), 1.38 (s,
H, H-11′ and H-11‴), 1.42 (s, 6 H, H-13′ and H-13‴), 1.52 (s, 6 H,
H-12′ and H-12‴), 1.97 (m, 2 H, H-8′ and H-8‴), 3.19 (sept, J = 7.0
Hz, 1 H, H-2″), 4.38 (d, J = 3.8 Hz, 2 H, H-1′ and H-1‴), 6.18 (br s, 1
H, OH); 13C NMR (100 MHz, CDCl ) δ 17.73 (CH , C-3″ and C-
3
3
4
2
″), 18.77 (CH , C-9′ and C-9‴), 19.12 (CH , C-10′ and C-10‴),
3
3
4.27 (CH , C-13′ and C-13‴), 24.76 (CH , C-11′ and C-11‴), 24.78
3
3
(
CH , C-14′ and C-14‴), 25.19 (CH , C-12′ and C-12‴), 32.47 (CH,
3
3
C-1′ and C-1‴), 35.11 (CH, C-8′ and C-8‴), 43.26 (CH, C-2″), 47.52
(C, C-4′ and C-4‴), 55.97 (C, C-6′ and C-6‴), 108.48 (C, C-2′ and
C-2‴), 110.50 (C, C-1), 111.27 (C, C-3 and C-5), 147.49 (C, C-2 and
C-6), 152.31 (C, C-4), 169.08 (C, C-3′ and C-3‴), 198.58 (C, C-7′
and C-7‴), 205.30 (C, C-1″), 211.76 (C, C-5′ and C-5‴); HRESIMS
m/z 655.3240 (calcd for C H O Na 655.3241).
3
8
48
8
3
1
Esterification of (+)-PMCA (4) with (S)-Mosher Acid. To
(
+)-PMCA (4a) (50 mg, 0.08 mmol) in a 4 mL vial were added 1.1
mL of a 0.09 M solution of (S)-Mosher acid in CH Cl (23.5 mg, 0.1
2
2
mmol), 1.5 mL of a 0.07 M solution of N,N-Dicyclohexylcarbodiimide
DCC) in CH Cl (21 mg, 0.1 mmol), and several crystals of 4-(N,N-
(
2
2
Dimenthylamino)-pyridine (DMAP). After 24 h, additional (S)-
Mosher acid (13 mg, 0.06 mmol) and DCC (12 mg, 0.06 mmol)
were added. After 4 days, the reaction was judged complete by TLC. A
Figure 7. Bayesian inferences with posterior probabilities and
maximum likelihood bootstrap support values (bold) are shown on
the tree branches. Values below 0.85 or 85, respectively, were
eliminated. Statistical significance was considered at >0.95 for PP and
saturated aqueous solution of NH Cl was added, and the mixture was
4
extracted with Et O, dried over MgSO , and concentrated. The residue
2
4
>90 for BS.
was purified by flash chromatography on silica gel with acetone/
petroleum ether (1:15) to yield 46 mg (0.05 mmol, 68%) of the
yellow powder; [α]20 0 (c 0.1, MeOH); H NMR spectrum due to a
1
Mosher ester as a white foam, which was crystallized from CH
Cl
39.5 (c 1.0,
); the NMR spectra show two conformers; only the data for the
3
to
D
2
2
20
mixture of rotamers and keto−enol tautomers was complicated; see
Supporting Information; HRESIMS m/z 669.3639 [M + H] (calcd
afford thin white needles, mp 224−230 °C; [α]
CHCl
major conformer are given; H NMR (400 MHz, CDCl
= 7.8 Hz, 2 H, HAr), 7.66−7.60 (m, 2 H, HAr), 7.56−7.50 (m, 1 H,
D
+
1
3
for C H O 669.3633).
3
) δ 7.82 (d, J
38
53 10
Nor-semimyrtucommulon (5). Fraction F4 (72 mg) was dissolved
in 24 mL of MeOH and applied in portions of 4 mL to preparative
HPLC using 80:20 (MeOH + 0.1% TFA)/(MeCN + 0.1% TFA). Two
fractions were obtained, F4.1 and F4.2, the former containing NSMC
5). This fraction (26 mg) was dissolved in 9 mL of MeOH and
applied in portions of 3 mL to a second preparative HPLC, now using
0:10 (MeOH + 0.1% TFA)/(H O + 0.1% TFA) as eluent. Collecting
the peak with t = 12.3 min and evaporating the solvent in vacuo gave
6 mg of pure NSMC (5), as a light yellow powder: [α] 0 (c 0.1,
MeOH); H NMR spectrum due to mixture of rotamers and keto−
enol tautomers was complicated; see Supporting Information;
3
H
Ar), 4.19 (br s, 2 H, H-1′, H-1‴), 3.86 (s, 3 H, OMe), 3.12 (sept, J =
7.0 Hz, 1 H, H-2″), 1.73−1.65 (m, 2 H, H-8′ and H-8‴), 1.53 (s, 6 H,
H-12′, H-11‴), 1.43−1.33 (m, 18 H, H-11′, H-12‴, H-13′, H-13‴, H-
3
3
(
14′, H-14‴), 1.27 (d, J = 7.0 Hz, 3 H, H-3″), 1.26 (d, J = 7.3 Hz, 3
13
H, H-4″), 0.70−0.49 (m, 12 H, H-10′, H-10‴, H-9′, H-9‴); C NMR
9
(100 MHz, CDCl ) δ 211.71, 211.67 (C-5′, C-5‴), 203.6 (C-1″),
2
3
197.2, 197.0 (C-7′, C-7‴), 167.87, 167.82 (C-3′, C-3‴), 165.1
R
20
1
(−C(O)O−), 147.33 (C-2, C-6), 144.8 (C-4), 130.1 (p-C ), 129.15
D
Ar
1
(C ), 127.8 (C ), 124.5, 121.6 (CF , other signals of the quartet to
Ar
Ar
3
small, JC−F = 289 Hz), 115.9, 115.5 (C ), 110.5, 110.2 (C-3, C-5),
Ar
+
HRESIMS m/z 433.2224 [M + H] (calcd for C H O 433.2221).
110.0 (C-1), 109.6 (C-2′, C-2‴), 85.1, 85.1, 84.9, 84.6 (C-CF , J
=
C−F
24
33
7
3
F
DOI: 10.1021/acs.jnatprod.5b00358
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
2
4
2
1
8 Hz), 56.1, 55.9 (C-6′, C-6‴), 56.0 (O-CH ), 47.5 (C-4′, C-4‴),
Crystallographic data (CIF)
NMR and ESIMS spectra of compounds 1, 2, 4, and 5
PDF)
3
2.7 (C-2″), 35.3 (C-8′, C-8‴), 32.7 (C-1′, C-1‴), 25.7, 25.3, 24.7,
3.7 (C-12′, C-11‴, C-13′, C-14‴, C-11′, C-12‴, C-14′, C-13‴), 20.8,
(
9.8, 19.3, 19.1 (C-10′, C-10‴, C-9′, C-9‴), 17.9, 17.50 (C-3″, C-4″);
+
HRESIMS m/z 849.3830 (calcd for C H F O 849.3825).
48
56
3
10
AUTHOR INFORMATION
Corresponding Authors
Tel: +49 (0) 681 302 64 301. Fax: +49 (0) 681 302 64 150. E-
mail: j.jauch@mx.uni-saaarland.de (isolation, purification, struc-
ture elucidation, enantioseparation, and derivatization of
myrtucommulones).
Cyclization of NSMC to MC B (2). NSMC (17.0 mg, 39 μmol) was
■
dissolved in dry benzene (8.5 mL). p-Toluenesulfonic acid (20.3 mg,
1
17 μmol) was added, and the mixture was refluxed for 1 h. After
*
cooling to room temperature, the mixture was washed with a saturated
aqueous NaHCO3 solution (20 mL), dried with MgSO , and
evaporated to dryness to yield 15.9 mg of crude MC B, which was
dissolved in MeOH (5 mL) and purified by preparative HPLC
4
*
Tel: +49 (0) 931 31 85323. Fax: +49 (0) 931 31 84755. E-
(
MeOH/H O, 90:10). Yield: 11.0 mg of racemic MC B as a white
2
1
mail: bringmann@chemie.uni-wuerzburg.de (calculations of
ECD spectra and determination of absolute configuration).
*Tel. +49 (0) 118 73 3315. Fax: +49 (0) 228 73 3120. E-mail:
quandt@uni-bonn.de (genetic analysis of myrtle).
powder; H NMR (400 MHz, CDCl ) δ 0.78 (d, J = 6.9 Hz, 3 H, H-
3
1
1
1
0′), 0.83 (d, J = 6.9 Hz, 3 H, H-9′), 1.24 (d, J = 6.9 Hz, 3 H, H-3″),
.26 (d, J = 6.9 Hz, 3 H, H-4″), 1.39 (s, 3 H, H-13′), 1.43 (s, 3 H, H-
1′), 1.45 (s, 3 H, H-14′), 1.62 (s, 3 H, 12′), 1.91 (m, 1 H, H-8′), 3.90
(
sept, J = 6.7 Hz, 1 H, H-2″), 4.32 (d, J = 3.6 Hz, 1 H, H-1′), 6.29 (s, 1
Notes
13
H, H-5), 6.59 (br s, 1 H, OH at C-4), 13.32 (s, 1 H, OH at C-6);
C
The authors declare no competing financial interest.
NMR (100 MHz, CDCl ) δ 17.68 (CH , C-3″ and C-4″), 18.71 (CH ,
3
3
3
C-10′), 18.84 (CH , C-9′), 24.16 (CH , C-13′), 24.86 (CH , C-11′),
3
3
3
ACKNOWLEDGMENTS
The authors are grateful to the Saarland University, to the
2
4.96, (CH , C-14′), 25.12 (CH , C-12′), 31.53 (CH, C-1′), 34.80
■
3
3
(
CH, C-8′), 39.68 (CH, C-2″), 47.28 (C, C-2′), 56.41 (C, C-6′),
1
2
3
00.62 (CH, C-7), 103.60 (C, C-1), 103.94 (C, C-3), 112.07 (C, C-
′), 153.48 (C, C-2), 159.31 (C, C-6), 164.71 (C, C-4), 167.84 (C, C-
′), 198.20 (C, C-7′), 208.92 (C, C-1″), 211.79 (C, C-5′); HRESIMS
University of Wurzburg, and to DFG (Ja 616-5/1) for financial
̈
support and the German Barcode of Life Initiative: Barcoding
the German Flora (BMBF GBoL5b (01LI1101F)). We thank
Prof. Dr. R. Bernhardt, A. Schifrin, and Dr. F. Hannemann
m/z 415.2115 (calcd for C H O 415.2115).
24
31
6
3
2
Bromination of MC B (2). (+)-MC B (121 mg, 0.29 mmol) was
(
Chair of Biochemistry at Saarland University) for using their
ECD spectropolarimeter, Prof. Dr. R. Muller and T. Hoffmann
Chair of Pharmaceutical Biotechnology at Saarland Univer-
dissolved in HOAc (3 mL), and 0.4 mL of a 1.7 M solution of Br in
HOAc (0.69 mmol, 2.4 equiv) was slowly added to the mixture. After
2
̈
(
3
0 min of vigorous stirring, the reaction mixture was poured into a
saturated aqueous Na S O solution and stirred for an additional 5
sity), and Prof. Dr. D. Volmer and T. Dier (Chair of Analytical
Chemistry at Saarland University) for the ESIMS spectra.
2
2
3
min. NaHCO was then added until the gas formation had ceased, and
3
the product was extracted with Et O, dried over MgSO , and
2
4
concentrated. The residue was purified by flash chromatography on
silica gel with acetone/petroleum ether (1:5) to yield 152 mg (0.27
mmol, 92%) of (+)-(R)-dibromo-MC B as a white foam, which was
crystallized from CH Cl to afford thin white needles: mp 179−181
REFERENCES
■
(
9
1) (a) Kashman, Y.; Rotstein, A.; Lifshitz, A. Tetrahedron 1974, 30,
91−997. (b) Rotstein, A.; Lifshitz, A.; Kashman, Y. Antimicrob. Agents
2
2
Chemother. 1974, 6, 539−542.
C; [α]20 40.1 (c 1.0, CHCl ); H NMR (400 MHz, CDCl ) δ 5.91
1
°
D
3
3
(
2) The structure of isolated myrtucommulone B is not yet finally
(
s, 1 H, C-6-OH), 5.77 (s, 1 H, C-4-OH), 4.36 (d, J = 3.5 Hz, 1 H, H-
settled. Whereas ref 1 assigns structure 2 to MC B, ref 4 takes into
account that it could be an isolation artifact and ref 5 assigns structure
′ on the basis of an X-ray analysis.
1
6
3
′), 2.064, 2.059 (2s, 6 H, H-3″, H-4″), 1.98 (dsept, J = 3.5 Hz and J =
.8 Hz, 1 H, H-8′), 1.55 (s, 3 H, H-12′), 1.41 (s, 3 H, H-14′), 1.36 (s,
H, H-11′), 1.35 (s, 3 H, H-13′), 0.84 (d, J = 7.0 Hz, 3 H, H-9′) 0.79
2
1
3
(
d, J = 7.0 Hz, 3 H, H-10′); C NMR (100 MHz, CDCl ) δ 211.9 (C-
3
5
6
6
3
1
′), 198.9 (C-7′), 197.7 (C-1″), 168.4 (C-3′), 151.0 (C-4), 149.0 (C-
), 147.3 (C-2), 111.3 (C-2′), 108.5 (C-3), 105.6 (C-1), 95.5 (C-5),
5.2 (C-2″), 56.1 (C-6′), 47.5 (C-4′), 34.8 (C-8′), 33.1 (C-1′), 30.54,
0.51 (C-3″, C-4″), 25.2, 24.7, 24.5 24.3 (C-14′, C-12′, C-11′, C-13′),
9.0 (C-9′), 18.9 (C-10′); HRESIMS m/z 571.0312 (calcd for
+
C H Br O 571.0331).
2
4
29
2
6
Computational Details. The conformational optimizations of
3
3
compounds 2 and 4 were done with B97D/TZVP using
34
Gaussian09. Energies for the Boltzmann statistical weightings were
35
36
computed with ORCA and the B2GP-PLYP-D3/def2-TZVPP
(3) Lounasmaa, M.; Puri, H.-S.; Widen, C.-J. Phytochemistry 1977, 16,
851−1852.
37
method (including COSMO, MeOH) taking advantage of the chain-
1
(
38
39
of-spheres approximation. TDCAM-B3LYP/TZVP (without sol-
4) Appendino, G.; Bianchi, F.; Minassi, A.; Sterner, O.; Ballero, M.;
vent effects) calculations were performed with Gaussian09, while
40
Gibbons, S. J. Nat. Prod. 2002, 65, 334−338.
5) Shaheen, F.; Ahmad, M.; Khan, S. N.; Hussain, S. S.; Anjum, S.;
TDBHLYP/def2-TZVP(-f) (plus COSMO) was done in ORCA,
again in combination with the RIJCOSX keyword. For further
(
15,41
Tashkhodjaev, B.; Turgunov, K.; Sultankhdzhaev, M. N.; Choudhary,
processing of the computed spectra SpecDis
was applied (all data
are from the length formalism): 2: σ = 0.25 eV, UV shift of 20 nm; 4: σ
M. I.; Atta-ur-Rhaman. Eur. J. Org. Chem. 2006, 2006, 2371−2377.
(6) Appendino, G.; Maxia, L.; Bettoni, P.; Locatelli, M.; Valdivia, C.;
=
0.3 eV, UV shift of 35 nm.
Ballero, M.; Stavri, M.; Gibbons, S.; Sterner, O. J. Nat. Prod. 2006, 69,
ASSOCIATED CONTENT
Supporting Information
■
251−254.
*
S
(7) Carroll, A. R.; Lamb, J.; Moni, R.; Guymer, G. P.; Forster, P. I.;
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00358.
Quinn, R. J. J. Nat. Prod. 2008, 71, 1564−1568.
(8) Cottiglia, F.; Casu, L.; Leonti, V.; Caboni, P.; Floris, C.;
Busonera, B.; Farci, P.; Ouhtit, A.; Sanna, G. J. Nat. Prod. 2012, 75,
225−229.
Crystallographic data (CIF)
G
DOI: 10.1021/acs.jnatprod.5b00358
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(
9) Nicoletti, R.; Ferranti, P.; Caira, S.; Misso, G.; Castellano, M.; Di
Lorenzo, G.; Caraglia, M. World J. Microbiol. Biotechnol. 2014, 30,
047−1052.
10) (a) Feißt, C.; Franke, L.; Appendino, G.; Werz, O. J. Pharmacol.
group P2 2 2 with a unit cell of a = 9.7566(2) Å, b = 19.1985(4) Å, c
= 27.6028(5) Å. The structures were solved by direct methods,
showing the positions of all non-hydrogen atoms. Subsequent
1
1 1
42
1
(
refinement revealed the position of hydrogen atoms in difference
2
Exp. Ther. 2005, 315, 389−396. (b) Koeberle, A.; Pollastro, F.;
Fourier maps. Full-matrix least-squares refinement against Fo with 646
Northoff, H.; Werz, O. Br. J. Pharmacol. 2009, 156, 952−961.
anisotropic thermal parameters resulted in R1 = 0.044 and wR2 =
(
c) Gerbeth, K.; Meins, J.; Werz, O.; Schubert-Zsilavecz, M.; Abdel-
0.127 with I > 2σ . Further details are available from the Cambridge
I
Tawab, M. Planta Med. 2011, 77, 450−454. (d) Fiorini-Puybaret, C.;
Aries, M.-F.; Fabre, B.; Mamatas, S.; Luc, J.; Degouy, A.; Ambonati,
M.; Mejean, C.; Poli, F. Planta Med. 2011, 77, 1582−1589.
Crystallographic Data Center, on quoting the depository number
CCDC 1038052.
(21) Borsch, T.; Quandt, D. Plant Syst. Evol. 2009, 282, 169−199.
(
11) (a) Tretiakova, I.; Blaesius, D.; Maxia, L.; Wesselborg, S.;
̃
(22) Wicke, S.; Costa, A.; Munoz, J.; Quandt, D. Mol. Phylogenet.
Schulze-Osthoff, K.; Cinatl, J., Jr.; Michaelis, M.; Werz, O. Apoptosis
008, 13, 119−131. (b) See ref 8.
12) (a) Rosa, A.; Deiana, M.; Casu, V.; Corona, G.; Appendino, G.;
Bianchi, F.; Ballero, M.; Dessi, M. A. Free Radical Res. 2003, 37, 1013−
019. (b) Rosa, A.; Melis, M. P.; Deiana, M.; Atzeri, A.; Appendino,
G.; Corona, G.; Incani, A.; Loru, D.; Dessi, M. A. Chem. Phys. Lipids
008, 155, 16−23.
13) (a) Muller, H.; Paul, M.; Hartmann, D.; Huch, V.; Blaesius, D.;
Evol. 2011, 61, 321−332.
(23) Migliore, J.; Baumel, A.; Juin, M.; Med
39, 942−956.
2
(
́
ail, F. J. Biogeogr. 2012,
(24) White, T. J.; Bruns, T.; Lee, S.; Taylor, J. PCR Protocols: a Guide
to Methods and Applications; Innis, M. A., Gelfand, D. H., Sninsky, J. J.,
White, T. J., Eds.; Academic Press: San Diego, 1990; pp 315−322.
(25) Borsch, T.; Hilu, K. W.; Quandt, D.; Wilde, V.; Neinhuis, C.;
Barthlott, W. J. Evol. Biol. 2003, 16, 558−576.
1
2
(
̈
Koeberle, A.; Werz, O.; Jauch, J. Angew. Chem. 2010, 122, 2089−2093;
̈ ̈
(26) Muller, K., Quandt, D., Muller, J., Neinhuis, C. PhyDe v0.996;
Angew. Chem., Int. Ed. 2010, 49, 2045−2049. (b) Jauch, J. Ger. Offen.
Phylogenetic Data Editor; 2005, available online from www.phyde.de.
(27) Stamatakis, A. Bioinformatics 2006, 22, 2688−2690.
(28) Huelsenbeck, J. P.; Ronquist, F. Bioinformatics 2001, 17, 754−
755.
2
010, DE102008044993A1 20100318.
(
14) First, the authors state that the relative configuration is R*; later
they explain that the MC A is the meso form and that the observed
optical activity could come from some kind of atropisomers.
(29) Rambaut, A., Drummond, A. J. Tracer; 2007, available online at
http://tree.bio.ed.ac.uk/software/tracer.
(
15) Bruhn, T.; Schaumlo
Chirality 2013, 25, 243−249.
16) Recently, we reported enantioselective syntheses of (+)-1 and
−)-5. Structure elucidation of the synthetic compounds was carried
̈
ffel, A.; Hemberger, Y.; Bringmann, G.
̈
̈
(30) Stover, B. C.; Muller, K. F. BMC Bioinf. 2010, 11, 7.
(
(31) Romaine, I. M.; Hempel, J. E.; Shanmugam, G.; Hori, H.;
Igarashi, Y.; Polavarapu, P. L.; Sulikowski, G. A. Org. Lett. 2011, 13,
4538−4541.
(
out after cyclization and dehydration in the same way as for natural 1
and 5. Thus, during isolation of synthetic (+)-1 and (−)-5 from the
respective reaction mixtures and acid-catalyzed cyclization no
racemization occurs; see Charpentier, M.; Hans, M.; Jauch, J. Eur. J.
Org. Chem. 2013, 2013, 4078−4084. If the enantiomerically enriched
synthetic 1 and 5 do not racemize, the myrtucommulones must be
regarded as configurationally stable, and therefore, natural 1 and 5
must occur in the plant as mixtures of stereoisomers. Furthermore, a
sample of (+)-1 with 70% ee and 18% de was treated with pTsOH in
refluxing benzene for 1, 2, 3, and 6 h, and the mixture was analyzed by
HPLC on a ChiralCel OD-H column. The ee- and de-values obtained
were identical within ±1%, so no racemization or epimerization could
be observed.
(32) Cechinel-Filho, V.; Vaz, Z.; Zunino, L.; Calixto, J.; Yunes, R.
Eur. J. Med. Chem. 1996, 31, 833−839.
(33) (a) Grimme, S. J. Comput. Chem. 2006, 27, 1787−1799.
̈
(b) Schafer, A.; Huber, C.; Ahlrichs, R. J. Chem. Phys. 1994, 100,
5829−5835.
(34) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.;
Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin,
K. N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.;
Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.;
Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.;
Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.;
Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.;
Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador,
P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.;
Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian09,
revision B.01; Gaussian, Inc.: Wallingford, CT, 2010.
(
17) Thin needle shaped colorless crystals for structure determi-
nation were grown from CH Cl . The data sets were gathered at 123 K
2
2
on an X8-Apex BrukerAXS diffractometer (Mo Kα radiation). In the θ
range from 2° to 27° collected reflections yielded 15 089 independent
reflections (Rint = 0.08). We determined the monoclinic space group
P2 with a = 6.448(1) Å, b = 40.961(5) Å, c = 17.888(3) Å, β =
1
4
2
9
1.687(8)°. The structures were solved by direct methods, showing
the positions of all non-hydrogen atoms. Subsequent refinement
revealed the position of hydrogen atoms in difference Fourier maps.
Full-matrix least-squares refinement against Fo2 with 1149 anisotropic
thermal parameters resulted in R1 = 0.091 and wR2 = 0.199 with I >
(35) (a) Neese, F. WIREs Comput. Mol. Sci. 2012, 2, 73−78.
(b) Neese, F.; Becker, U.; Ganyushin, D.; Hansen, A.; Liakos, D.;
Kollmar, C.; Koßmann, S.; Petrenko, T.; Reimann, C.; Riplinger, C.;
Sivalingam, K.; Wezisla, B.; Wennmohs, F. ORCA, version 2.9.1; Max
Planck Institute for Bioinorganic Chemistry: Germany, 2012.
(36) (a) Tarnopolsky, A.; Karton, A.; Sertchook, R.; Vuzman, D.;
Martin, J. M. L. J. Phys. Chem. A 2008, 112, 12868−12886.
(b) Weigend, F.; Ahlrichs, R. Phys. Chem. Chem. Phys. 2005, 7,
3297−3305.
2
σI. Further details are available from the Cambridge Crystallographic
Data Center, on quoting the depository number CCDC 1038053.
18) (a) Finefield, J. M.; Sherman, D. H.; Kreitman, M.; Williams, R.
M. Angew. Chem. 2012, 124, 4886−4920; Angew. Chem., Int. Ed. 2012,
(
5
1, 4802−4836. (b) Batista, J. M., Jr.; Batista, A. N. L.; Rinaldo, D.;
Vilegas, W.; Ambrosio, D. L.; Cicarelli, R. M. B.; Bolzani, V. S.; Kato,
M. J.; Nafie, L. A.; Lopez, S. N.; Furlan, M. J. Nat. Prod. 2011, 74,
1
154−1160. (c) Usami, Y.; Okada, Y.; Yamada, T. Chirality 2011, 23,
E7−E11.
19) Cechinel-Filho, V.; Vaz, Z.; Zunino, L.; Calixto, J.; Yunes, R.
Eur. J. Med. Chem. 1996, 31, 833−839.
20) Thin needle-shaped colorless crystals for structure determi-
nation were grown from CH Cl . The data sets were gathered at 123 K
(37) Klamt, A. WIREs Comput. Mol. Sci. 2011, 1, 699−709.
(38) Izsak, R.; Neese, F. J. Chem. Phys. 2011, 135, 144105/1−
144105/11.
(
(39) Yanai, T.; Tew, D. P.; Handy, N. C. Chem. Phys. Lett. 2004, 393,
(
51−57.
(40) Becke, A. D. J. Chem. Phys. 1993, 98, 1372−1377.
2
2
on an X8-Apex BrukerAXS diffractometer (Mo Kα radiation). In the θ
range from 2° to 27° collected reflections yielded 10 541 independent
reflections (Rint = 0.03). We determined the orthorhombic space
̈
(41) Bruhn, T.; Schaumloffel, A.; Hemberger, Y.; Bringmann, G.
SpecDis, version 1.62 ed.; University of Wu
Germany, 2014.
̈ ̈
rzburg: Wurzburg,
H
DOI: 10.1021/acs.jnatprod.5b00358
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(
42) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Crystallogr.
2
008, A64, 112−122.
I
DOI: 10.1021/acs.jnatprod.5b00358
J. Nat. Prod. XXXX, XXX, XXX−XXX