Structural Elucidation of Trace Components Combining GC/MS, GC/IR, DFT-Calculation and Synthesis—Salinilactones, Unprecedented Bicyclic Lactones from Salinispora Bacteria

Article abstract of DOI:10.1002/anie.201807923

The analysis of volatiles released by marine Salinispora bacteria uncovered a new class of natural compounds displaying an unusual bicyclic [3.1.0]-lactone skeleton. Although only sub-μg quantities of the compounds were available, the combination of analytical methods, computational spectroscopy, and synthesis allowed unambiguous structural identification of the compounds, called salinilactones, without the need for isolation. Orthogonal hyphenated methods, GC/MS and solid-phase GC/IR allowed to propose a small set of structures consistent with the data. A candidate structure was selected by comparison of DFT-calculated IR spectra and the experimental IR-spectrum. Synthesis confirmed the structure and absolute configuration of three bicyclic lactones, salinilactones A–C. The salinilactones are structurally closely related to the A-factor class of compounds, autoregulators from streptomycete bacteria. They exhibited inhibitory activity against Salinispora and Streptomyces strains.

Full text of DOI:10.1002/anie.201807923

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Accepted Article
Title: Structural Elucidation of Trace Components Combining GC/
MS, GC/IR, DFT-Calculation and Synthesis - Salinilactones,
Unprecedented Bicyclic Lactones from Salinispora Bacteria
Authors: Christian Schlawis, Simone Kern, Yuta Kudo, Jörg
Grunenberg, Bradley Moore, and Stefan Schulz
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201807923
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Structural Elucidation of Trace Components Combining
GC/MS, GC/IR, DFT-Calculation and Synthesis -
Salinilactones, Unprecedented Bicyclic Lactones from
Salinispora Bacteria
Christian Schlawis,[a] Simone Kern,[a] Yuta Kudo,[b] Jörg Grunenberg,[a] Bradley S. Moore,[b]
and Stefan Schulz*[a]
Abstract: The analysis of volatiles released by marine Salinispora
bacteria uncovered a new class of natural compounds displaying an
unusual bicyclic [3.1.0]-lactone skeleton. Although only sub-mg
quantities of the compounds were available, the combination of
analytical methods, computational spectroscopy and synthesis
allowed unambiguous structural identification of the compounds,
called salinilactones, without the need for isolation. Orthogonal
hyphenated methods, GC/MS and solid-phase GC/IR allowed to
propose a small set of structures consistent with the data. A candidate
structure was selected by comparison of DFT calculated IR spectra
and the experimental IR-spectrum. Synthesis confirmed structure and
absolute configuration of three bicyclic lactones, salinilactones A-C.
The salinilactones are structurally closely related to the A-factor class
of compounds, autoregulators from streptomycete bacteria. They
exhibited inhibitory activity against Salinispora and Streptomyces
strains.
are sometimes difficult to identify because the interpretation of
mass-spectra can become insufficient. At this point, IR offers
orthogonal information, e. g. on functional groups and double
bonds. Solid phase GC/IR techniques available nowadays allow
the use of infrared detectors integrating high sensitivity with high
chromatographic resolution comparable to GC/MS.[9] Finally,
structural proposals developed by these methods have to be
verified by synthesis, sometimes only successful by a trial- and
error method needing multiple syntheses to finally arrive at the
correct structure.
We add here a third type of information to these methods,
computational spectroscopy.[10] Combining solid phase GC/IR
analysis with DFT-simulations and MS data allows to select
effectively candidate compounds worth considering for synthetic
verification. This combination of methods led to the identification
of a hitherto unknown class of volatile bicyclic lactones emitted by
several Salinispora bacteria with minimal synthetic effort and
without the need of isolation of the natural compounds (Figure S1,
SI).
Marine actinomycete bacteria of the genus Salinispora are known
to produce a wide variety of natural products,[1-3] e. g. the
antimalarial salinipostins[4] or the proteasome inhibitor
salinosporamide A.[5,6] Salinispora bacteria also emit an extensive
range of volatiles.[7] In an effort to reveal the function of these
volatiles, we analyzed liquid cultures of Salinispora arenicola
CNQ748 by closed loop stripping analysis (CLSA).[8] These
experiments furnished complex samples of volatiles containing
many compounds in just ng amounts. Therefore, isolation and
standard NMR procedures for structure elucidation of unknowns
cannot be used. GC/EI-MS analyses are well suited for
identification of compounds in such samples because of the high
chromatographic resolution, sensitivity, and mass spectra
revealing skeletal and functional group information. In favorable
cases, mass spectrometric analysis and GC retention index data
can lead to a structural proposal for an unknown compound, a
method frequently used e. g. in chemical ecology, geochemistry
or metabolomics. Nonetheless, more complex target compounds
.
Figure 1. Total ion chromatogram of a headspace extract of a liquid culture of
Salinispora arenicola CNQ748 obtained by CLSA. Compounds A-C are
indicated.
[a]
[b]
C. Schlawis, S. Kern, Prof. Dr. J. Grunenberg, Prof. Dr. S. Schulz,
Institut für Organische Chemie, TU Braunschweig
Hagenring 30, 38106 Braunschweig (Germany)
E-mail: stefan.schulz@tu-bs.de
Homepage: http://www.oc.tu-bs.de/schulz/index.html
Dr. Y. Kudo, Prof. Dr. B. S. Moore,
Scripps Institution of Oceanography, UC San Diego, 9500 Gilman
Drive, La Jolla, CA 92093-0204 (USA)
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These lactones are structurally related to the A-factor, a bacterial
autoregulator that triggers secondary metabolism and cellular
differentiation in Streptomyces griseus.[11,12] Several A-factor like
agreement between theory and experiment was sufficient for our
purpose. The visual comparison of the fingerprint areas of the
spectra revealed 8 as the most likely structure for B. A detailed
discussion of the spectra is given in Figures S10-S17 in the
autoregulators carrying
a monocyclic g-butyrolactone core
structure are already known from different Streptomycetes.[13-16]
The GC/MS analysis of volatile compounds extracted from liquid
cultures of Salinispora arenicola CNQ748 showed three unknown
compounds sharing the same characteristic ions m/z 140 and m/z
122 (Figure 1, A-C). Several other S. arenicola strains, CNS205,
CNB527, CNH646, CNH996A and CNQ748, produced the same
compounds. Structural analysis was initially performed for
compound B occurring in the highest concentration. A detailed
analysis of the mass spectrum (Figure 2 A) revealed the existence
of a C5-acyl side-chain from which the two characteristic
fragments arise by McLafferty-rearrangement (m/z 140) and
subsequent loss of water (m/z 122). Additional peaks resulting
from alkyl chain fragmentation at m/z 153 by loss of an ethyl group
from M+ at m/z 182 and at m/z 125 (a-carbonyl cleavage)
supported this assignment (Figure S3, SI).. GC/HR-MS delivered
the molecular formula C10H14O3 (found 182.0972, calc. 182.0943)
requiring four double-bond equivalents (DBE). Solid phase GC/IR
confirmed the presence of a ketone and an ester group,
recognizable by their C-O double-bond stretching bands at 1696
and 1769 cm-1, respectively (Figure 2B).[17]
supporting information. In order to quantify our results
a
computer-assisted comparison based on first derivative
a
correlation algorithm available through the GRAMS software
package was performed, also revealing 8 as the most similar
structure (Figure S9 and Table S5 in the SI).
Figure 2. A) Mass spectrum and B) infrared spectrum of natural compound B.
The high energy of the ester stretching vibration indicated ring
strain, excluding an open chain ester. Another important
information was the absence of a C-C double-bond stretch
vibration (1620-1680 cm-1). In order to reach the required number
of DBEs without a C-C double-bond we proposed a [3.1.0]-
bicyclic structure. Although
a good amount of structural
information was obtained, many candidate structures were still in
agreement with the data, among them e. g. compounds 1-8
(Figure 3). Synthesizing all possible isomers was therefore
considered to be ineffective.
Consequently, we decided to apply dispersion corrected DFT[18]
(density functional theory) calculations at the B3LYP level of
theory[19] in order to simulate the IR-spectra of the relevant lactone
isomers in question. The lowest energy conformer of each isomer
was determined using a hybrid Monte Carlo/Low Frequency Mode
algorithm[20] applying the MMFF[21] force field. In a second step,
each of the global force field minima was subject to a DFT-based
optimization and frequency calculation applying the B3LYP-D/6-
311G(p,d) level of theory (Figure 3). Though we are comparing
solid state IR-data with gas-phase computations,[22] the
Figure 3. Experimental infrared spectrum (bottom) and simulated IR-spectra of
the depicted structures based on lowest energy conformers. Conformational
analysis with MMFF and optimization/frequency calculation using B3LYP-D/6-
311G(p,d), scaling factor 0.975.
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A more sophisticated calculation with additional optimization
steps and Boltzmann averaging of several conformers further
emphasized the degree of similarity (Figure S18, SI). The close
resemblance between this simulation and the spectrum of the
natural product justified the synthesis of compound 8.
temperature, yet the yield dropped significantly. The absolute
configuration of the major enantiomer suggested by the proposed
transition state from Nakada et al. was proven by electronic
circular dichroism measurements, DFT-simulations and literature
comparison of the specific rotation of structurally related
compounds (Figures S19-S20, SI). Chiral GC/MS investigations
disclosed the absolute configuration of the natural compound B
using the synthesized scalemic mixture (Figure S21, SI). The
natural compound B occurred in a 97:3 (1R,5S)/(1S,5R)-mixture.
Both the natural compounds A and C were also submitted to chiral
GC analyses and compared to the corresponding racemic
mixtures as references. In each case the natural product was
identical with the later eluting enantiomer (Figure S12, SI),
indicating the same absolute configuration as found in B.
The racemic synthesis started from commercially available ester
9 (Scheme 1). Saponification and subsequent esterification with
allyl alcohol gave the enyne ester 16 that was converted into the
target compound 8 under oxidizing conditions and Pd catalysis
according to Sanford et al.[23] Structural proposals for compounds
A and C, 18 and 19, were made with respect to the gas
chromatographic retention index of each compound. The
synthesis of 18 and 19 started from the terminal alkynes 10 and
11 which were transformed into acids 12 and 14 with CO2 and
further developed as outlined in Scheme 1. Mass and IR spectra
as well as gas chromatographic retention indices of compounds
8, 18 and 19 matched those of the natural products A-C, thus
confirming their identity (Figure S2, SI).
Scheme 2. Asymmetric synthesis of 8 via intramolecular cyclopropanation.[25]
ligand*:
2,2-bBis((4S)-(-)-4-isopropyloxazoline)propane,
p-ABSA:
4-
acetamidobenzene sulfonyl azide.
The biosynthesis of the A-factor (30), related to the salinilactones,
has been investigated in detail (Scheme 3).[27] Horinouchi et al.
showed that the enzyme A-factor synthase AfsA synthesizes
ester 25 by coupling an acyl carrier protein (ACP) bound b-
ketoacyl unit (24) and dihydroxyacetone phosphate (23).[28] The
phosphorylated ester 25 is then converted into lactone 26,
followed by reduction to lactone 27 that only needs a phosphatase
to release 30. We hypothesized that the salinilactones might be
biosynthesized by the same pathway.
We recently reported the creation of a mutant of the AfsA-like
gene called spt9 in the related isolate S. arenicola CNS205
associated with salinipostin biosynthesis.[29] In contrast to the
wildtype, no salinilactones were detected in the mutant. Therefore,
the biosynthetic routes of g-butyrolactone signaling compounds
and salinilactones seemed to be closely connected. Especially
phosphate 27 seems to be a suitable precursor for salinilactone
biosynthesis, offering a leaving group for the construction of the
three membered ring. We propose that compound 27 is
enzymatically deprotonated to form enolate 28. This enolate can
then form the cyclopropane unit of the salinilactones by C-
alkylation via nucleophilic displacement of the phosphate group.
We suspect that the phosphate 27 also gives rise to the recently
discovered salinipostins (29), potent anti-malaria compounds also
isolated from Salinispora,[4] as the spt9 knockout mutant also
abolishes its biosynthesis.[29] The oxygen of enolate 28 likely
Scheme 1. Racemic synthesis of bicyclic lactones 8, 18 and 19.
In order to determine the absolute configuration of B, an
asymmetric synthesis was performed. Valeroyl chloride (20) was
converted into the b-keto ester 21 utilizing meldrums acid and allyl
alcohol.[24] The carbene precursor 22 was synthesized by diazo
transfer under basic conditions.[25] The key step was the
asymmetric copper catalyzed intramolecular cyclopropanation
described by Nakada and coworkers (Scheme 2).[26]
The mediocre enantioselectivity of 52% ee at standard conditions
could be slightly enhanced to 58%ee by lowering the reaction
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attacks alternatively the phosphate group, or a derivative thereof,
leading finally to 29.
Preliminary experiments were performed to test the biological
effects of these compounds. Agar diffusion assays of the
salinilactones A-C showed inhibition of growth of the test strain S.
arenicola CNH996A that also can produce these metabolites.
Similarly, growth of Streptomyces violaceoruber A3(2), producer
of the g-butyrolactone SCB1 closely related to the A-factor was
inhibited.[13] No inhibition was observed with loadings below 50 µg
(see SI). The analysis of S. violaceoruber A3(2) showed no
production of salinilactones. The self-inhibitory activity of the
salinilactones may hint to a signaling function, but further
experiments are needed to clarify the function of these
compounds in the biology of Salinispora.
g-Butyrolactones like A-factor are generally produced in small
amounts in Streptomyces. To determine the amount of
salinilactones produced by Salinispora, we opted for an isotope
dilution method. Therefore, the deuterated analog d2-8 was
synthesized and spiked to bacterial cultures. The following
headspace analysis of liquid cultures, performed at different time
points during the culture growth allowed determination of the
amount of 8 produced and its time course.
Figure 4. Production of salinilactones A-C in a liquid culture of S. arenicola
CNS205 within a six day period. square: salinilactone A; circle: salinilactone B;
triangle: salinilactone C.
The salinilactones have an interesting structural feature, the
reactive cyclopropane ring. An analysis of the relaxed DFT force
constants revealed kinetically labile C-C bonds in the three-
membered ring of 8 indicated by a deviation from the Badger rule
by more than 20%, not far from the value for fluxional
molecules.[32] The b-keto functionality on one side and the alkyl
group on the other side are structural features of push-pull
cyclopropanes, which show enhanced reactivity against
nucleophiles and/or electrophiles.[33] Several synthetic strategies
using this concept have been developed. Therefore, multiple
activities of salinilactones in biological systems can be assumed.
The bicyclic fused cyclopropabutyrolactone motif has not been
reported before as a natural product.
Scheme 3. Proposed biosynthesis of salinilactones basing on A-factor
biosynthesis in Streptomyces griseus,[4,27] The key intermediate, enolate 28,
gives salinilactones via C-alkylation, while O-phosphorylation leads to
salinipostins (29).
Reduction of acryloyl chloride gave crude 1,1-d2-allyl alcohol[30]
used for the synthesis of ester d2-16 that was converted to the
deuterated bicyclic lactone d2-8 as described (Scheme S1, SI). S.
arenicola CNS205, the strain with the highest production rate of
salinilactones A-C was used for the spiking experiments (Figure
4). A well grown liquid culture of strain CNS205 yielded about
500 µg/L of salinilactone B. Quantification of salinilactones A and
In summary, we were able to combine solid phase GC/IR and
DFT-based simulation of infrared spectra to complement GC/MS
analysis, thus enabling identification of natural compounds
present in trace amounts without NMR spectroscopy. This
approach led to the discovery of a new class of A-factor related
g-butyrolactones produced by Salinispora bacteria. Research on
naturally occurring derivatives and the biological function of these
unique fused bicyclic lactones is currently ongoing.
C
was possible by referencing characteristic ions to the
deuterated internal standard (Figure 4). The amounts of
salinilactones produced by S. arenicola CNS205 are one order of
magnitude higher than that of A-factor in Streptomyces griseus
that produces 25-30 µg/L.[31]
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This work was supported by NIH grant GM085770 to B.S.M. and
a JSPS Overseas Research Fellowships postdoctoral fellowship
from the Japan Society for Promotion of Science to Y.K. The
authors thank Prof. Paul Jensen, Scripps Institution of
Oceanography in La Jolla, for the Salinispora strains and
cultivation advice and Prof. Joachim Wink, Helmholtz Centre for
Infection Research in Braunschweig, for advice regarding the
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COMMUNICATION
The combination of orthogonal
hyphenated methods, GC/MS, gas
Christian
Schlawis,[a]
Simone Kern,[a]
Yuta Kudo,[b]
Jörg
chromatography/solid-phase infrared
spectroscopy (GC/IR), computational
spectroscopy and synthesis lead to the
discovery of three volatile bicyclic lactones
emitted by marine Salinispora bacteria,
salinilactones A-C. These unprecedented
bicyclic [3.1.0]-lactones are closely related
to the A-factor class of g-butyrolactone
autoregulators from certain actinomycetes.
Grunenberg,[a]
Bradley
Moore,[b] and
Stefan Schulz*[
Page No. –
Page No.
Structural
Elucidation of
Trace
Components
Combining
GC/MS, GC/IR,
DFT-
Calculation and
Synthesis -
Salinilactones,
Unprecedented
Bicyclic
Lactones from
Salinispora
Bacteria
Text for Table of Contents
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