Long-Chain Alkyl Cyanides: Unprecedented Volatile Compounds Released by Pseudomonas and Micromonospora Bacteria

Article abstract of DOI:10.1002/anie.201611940

The analysis of volatiles from bacterial cultures revealed long-chain aliphatic nitriles, a new class of natural products. Such nitriles are produced by both Gram-positive Micromonospora echinospora and Gram-negative Pseudomonas veronii bacteria, although the structures differ. A variable sequence of chain elongation and dehydration in the fatty acid biosynthesis leads to either unbranched saturated or unsaturated nitriles with an ω?7 double bond, such as (Z)-11-octadecenenitrile, or methyl-branched unsaturated nitriles with the double bond located at C-3, such as (Z)-13-methyltetradec-3-enenitrile. The nitrile biosynthesis starts from fatty acids, which are converted into their amides and finally dehydrated. The structures and biosyntheses of the 19 naturally occurring compounds were elucidated by mass spectrometry, synthesis, and feeding experiments with deuterium-labeled precursors. Some of the nitriles showed antimicrobial activity, for example, against multiresistant Staphylococcus aureus strains.

Full text of DOI:10.1002/anie.201611940

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201611940
German Edition: DOI: 10.1002/ange.201611940
Natural Products
Hot Paper
Long-Chain Alkyl Cyanides: Unprecedented Volatile Compounds
Released by Pseudomonas and Micromonospora Bacteria
Diogo Montes Vidal+, Anna-Lena von Rymon-Lipinski+, Srinivasa Ravella, Ulrike Groenhagen,
Jennifer Herrmann, Nestor Zaburannyi, Paulo H. G. Zarbin, Adithi R. Varadarajan,
Christian H. Ahrens, Laure Weisskopf, Rolf Mꢀller, and Stefan Schulz*
Abstract: The analysis of volatiles from bacterial cultures
revealed long-chain aliphatic nitriles, a new class of natural
products. Such nitriles are produced by both Gram-positive
Micromonospora echinospora and Gram-negative Pseudomo-
nas veronii bacteria, although the structures differ. A variable
sequence of chain elongation and dehydration in the fatty acid
biosynthesis leads to either unbranched saturated or unsatu-
rated nitriles with an wÀ7 double bond, such as (Z)-11-
octadecenenitrile, or methyl-branched unsaturated nitriles with
the double bond located at C-3, such as (Z)-13-methyltetradec-
3-enenitrile. The nitrile biosynthesis starts from fatty acids,
which are converted into their amides and finally dehydrated.
The structures and biosyntheses of the 19 naturally occurring
compounds were elucidated by mass spectrometry, synthesis,
and feeding experiments with deuterium-labeled precursors.
Some of the nitriles showed antimicrobial activity, for example,
against multiresistant Staphylococcus aureus strains.
bacteria cover a broad structural range, including metabolites
of the fatty acid biosynthetic pathway.[2,3] These are short-
chain compounds that are unspecifically produced by many
bacteria[2] and more specific compounds with longer aliphatic
chains, for example, methyl ketones or methyl esters.[4] Such
a compound, namely 1-undecene, which is produced by
Pseudomonas strains,[5,6] inhibits the growth and sporulation
of one of the most harmful phytopathogenic oomycetes,
Phytophtera infestans.[5] We have now studied the volatile
compounds released by 1-undecene-producing Pseudomonas
strains in more detail, and found that P. veronii R02 (NCBI
acc. number CP018420) emits long-chain aliphatic nitriles.
This compound class has not been reported before to occur in
nature, and nitriles in general are rarely reported as natural
products.[7]
We herein describe the identification, synthesis, and first
insight into the biosynthesis of these nitriles, which are
released by the proteobacterium P. veronii R02 but also by
the actinomycete Micromonospora echinospora DSMZ
43816.
During GC/MS analysis of the volatile compounds
emitted by liquid cultures of P. veronii R02, we detected
a range of compounds with mass spectra similar to those
reported for long-chain aliphatic nitriles, such as (Z)-9-
octadecenenitrile (1–8; Figure 1A).[8]
E
mission of volatile compounds is a widespread trait found
in many bacteria. Only recently it became clear that such
compounds may influence the physiology and/or behavior of
other organisms.[1] The compounds that are emitted by
[*] Dr. D. Montes Vidal,[+] Dr. A.-L. von Rymon-Lipinski,[+] S. Ravella,
Dr. U. Groenhagen, Prof. Dr. S. Schulz
Institut fꢀr Organische Chemie, TU Braunschweig
Hagenring 30, 38106 Braunschweig (Germany)
E-mail: stefan.schulz@tu-bs.de
The mass spectra (Figure 2) revealed the presence of
saturated as well as unsaturated nitriles with chain lengths
ranging from C12 to C18. As oleonitrile and hexadecanenitrile
are sometimes present as trace contaminants in samples
Homepage: http://www.oc.tu-bs.de/schulz/index.html
Dr. D. Montes Vidal,[+] Prof. Dr. P. H. G. Zarbin
Laboratꢁrio de Semioquꢂmicos
Departamento de Quꢂmica—UFPR
Curitiba PR (Brazil)
Dr. J. Herrmann, Dr. N. Zaburannyi, Prof. Dr. R. Mꢀller
Microbial Natural Products
Helmholtz Institute for Pharmaceutical Research Saarland
Helmholtz Centre for Infection Research
Saarland University, Saarbrꢀcken (Germany)
Prof. Dr. L. Weisskopf
Institute for Viticulture and Oenology
University of Applied Sciences and Arts Western Switzerland
(Switzerland)
Prof. Dr. L. Weisskopf
Agroscope (Switzerland)
A. R. Varadarajan, Dr. C. H. Ahrens
SIB Swiss Institute of Bioinformatics (Switzerland)
[+] These authors contributed equally to this work.
Figure 1. Total-ion chromatograms of the headspace extracts of liquid
cultures of Pseudomonas veronii R02 (A) and Micromonospora echino-
spora DSMZ 43816 (B). 1–19: aliphatic nitriles, U=1-undecene (see
Table S1). Slightly different chromatography conditions were used so
that the retention times are not comparable.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201611940.
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These nitriles were then synthesized to confirm their
structures and to elucidate the double-bond configurations.
Nitriles 6 and 8 were obtained from palmitoleic and vaccenic
acid through reactions with chlorosulfonyl isocyanate and
acetonitrile, respectively.[10] Compound 3 was synthesized
from 7-octynoic acid (20). Chain elongation provided acid 21,
which afforded (Z)-7-tetradecenoic acid (22), the precursor of
nitrile 3, by Lindlar hydrogenation (Scheme 1). The natural
compounds were all shown to be Z-configured, as determined
by GC analysis. The saturated nitriles with chain lengths
ranging from C14 to C16 were similarly synthesized from the
respective acids, establishing their release by the bacteria (see
the Supporting Information).
Surprisingly, Micromonospora echinospora DSMZ 43816
produced eleven compounds with mass spectra similar to
those of nitriles 1–9 (9–19; Figure 1B) together with a ple-
thora of other components, although the release rate was
much lower than in P. veronii. In most cases, these mostly
unsaturated nitriles had different gas-chromatographic reten-
tion indices (I) than the compounds released by P. veronii.
The three isomers 9, 10, and 17 with I = 1638, 1652, and 1675
are isomers of 3 (I = 1675). The DMDS adducts revealed that
the double bond is consistently located at the C3 carbon atom
because of the strong ion M+À100 (Figure 2G).
The I value indicated that compound 17 was likely to be
3-tetradecenenitrile whereas 9 and 10 were obviously methyl-
branched derivatives. An iso-methyl group leads to an I value
that is 40 units lower[11] than that of the unbranched isomer.
This is in good agreement with the value for compound 9,
suggesting it to be 12-methyltridec-3-enenitrile. The structure
was confirmed by synthesis as shown in Scheme 2.
Tosylate 24 was coupled with isopropylmagnesium bro-
mide 23 to yield alkene 25, which was then transformed by
cross-metathesis with allyl cyanide (26) using Grubbsꢀ second-
generation catalyst[12] into a 10:1 mixture of (E)- and (Z)-12-
methyltridec-3-enenitrile (10/9). Comparison with the natural
products showed that both the E and Z isomers occur
naturally. Compounds 11–19 were synthesized (see the
Supporting Information) and identified in a similar fashion
(Scheme 1). (Z)-13-Methyltetradec-3-enenitrile (13) was the
nitrile most abundantly produced by M. echinospora.
The nitriles synthesized by the two strains are structurally
very different. Whereas P. veronii produces saturated and
unsaturated nitriles with an wÀ7 double bond, M. echino-
spora emits predominately D3-unsaturated, methyl-branched
nitriles.
Figure 2. Mass spectra of naturally occurring nitriles and their deriva-
tives. A) (Z)-7-Tetradecenenitrile (3). B) Tetradecanenitrile (4).
C) DMDS adduct of 6. D) DMDS adduct of 6 after feeding of
perdeuterated palmitic acid. The boxed ion contains 14 deuterium
atoms. E) (Z)-13-Methyltetradec-3-enenitrile (13). F) (Z)-Pentadec-3-
enenitrile. G) DMDS adduct of 13. H) 13-Methyltetradecanenitrile after
feeding of [D6]-13-methyltetradecanoic acid amide. The boxed ion
contains six deuterium atoms.
obtained from the headspace of bacteria, a contamination
could initially not be completely excluded. However, the
presence of nitriles in only one of several different Pseudo-
monas strains, their relatively high production rates, and their
absence in control samples of the medium excluded this
possibility.
The positions of the double bonds in the unsaturated
nitriles were determined by derivatization of a headspace
sample with dimethyl disulfide (DMDS).[9] Mass-spectromet-
ric cleavage of these derivatives occurs preferentially between
the two thiomethyl groups (Figure 2C). The mass spectrum of
the adduct of hexadecenenitrile (6) showed characteristic ions
at m/z 145 ([C7H14SCH3]+) and m/z 184 ([NCC8H15SCH3]+),
locating the double bond at the C-9 carbon atom. Similarly,
the other unsaturated nitriles were identified as (Z)-7-
tetradecenenitrile (3) and (Z)-11-octadecenenitrile (8). All
three compounds carry the double bond at the wÀ7 position,
which is typical for many bacterial fatty acid derivatives.
Therefore, we became interested in investigating the
biosynthetic pathways leading to these nitriles, which are most
likely formed from the corresponding fatty acids. Perdeuter-
ated palmitic acid (27) was fed to P. veronii. Surprisingly,
whereas labeled saturated and unsaturated nitriles were
formed, the deuterium content was lower than expected.
Analysis of their mass spectra and that of the DMDS adduct
of unsaturated labeled 6 (Figure 2D) revealed labeling only at
the terminal seven carbon atoms. Obviously, palmitic acid is
degraded by the bacterium until octanoyl-CoA is formed,
which then serves as the starting material for de novo
biosynthesis of the required fatty acids. While the saturated
fatty acids are formed by conventional fatty acid biosynthesis
2
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beled nitriles were not
observed, as was the case
after feeding labeled nitrile
3. These results rule out the
possibility that the C-3
double bond is introduced
by
a
specific desaturase
acting on the saturated acid
or the nitrile.
The final step in the bio-
synthesis is the transforma-
tion of the acyl group into
the nitrile. Nitriles in natural
products are often formed
via an oxime from an alde-
hyde, followed by dehydra-
tion.[7,15] It has recently been
shown that an amide can also
serve as a precursor, which
forms the nitrile upon dehy-
dration.[16] One of these two
possibilities seemed to be
plausible in our case whereas
Scheme 1. Nitriles naturally occurring in P. veronii R02 (1–8) and M. echinospora DSMZ 43816 (9–19) and
synthesis of (Z)-7-tetradecenenitrile (3).
Scheme 2. Synthesis of (E)- and (Z)-12-methyltridec-3-enenitrile (10
and 9).
with FabZ enzymes, the wÀ7-unsaturated acids are produced
in Pseudomonas by isomerization of (E)-2-decenoyl-ACP
into (Z)-3-decenoyl-ACP using a FabA enzyme,[13] followed
Scheme 3. Feeding deuterated precursors (black dots indicate deute-
rium labeling) to P. veronii R02 or M. echinospora DSMZ 43816.
by chain elongation (ACP = acyl carrier protein). During this
process, one deuterium of the perdeuterated octanoic acid
precursor should be lost. Indeed, both saturated [D15]nitriles
and unsaturated [D14]nitriles were formed, such as com-
pounds 28 and 29. Interestingly, the major volatile compound
1-undecene was obtained as its D15-labeled variant, formed
from an octanoyl-CoA starter unit as well. Elongation to
dodecanoic acid and oxidative decarboxylation then lead to
1-undecene.[14] Feeding experiments with perdeuterated dec-
anoic acid or its N-acetylcysteamine (SNAC) ester led to
identical results.
To investigate the biosynthesis in M. echinospora,
[2,2,3,3,4,4-D6]-13-methyltetradecanoic acid (32), its amide
31, and [3,3,4,4-D4]-13-methyltetradecanenitrile (33) were
synthesized (see the Supporting Information, Scheme S1).
Feeding of 32 to M. echinospora led to the formation of [D6]-
13-methyltetradecanenitrile (34; Scheme 3). Unsaturated la-
a third naturally occurring alternative, namely the addition of
cyanide to a double bond, is unlikely.[17] When labeled amide
31 was fed to M. echinospora, clean formation of 34 was
observed, thus excluding the oxime pathway. Nitrile forma-
tion via an amide has been proposed to occur over several
steps in a process catalyzed by the ToyM enzyme.[16] Although
two homologues of genes belonging to the QueC/ToyM
family are present in the genome of P. veronii R02 (see the
Supporting Information), they are not closely associated with
the known fatty acid biosynthesis gene clusters. Therefore,
these genes are not considered as strong and unambiguous
candidate biosynthetic genes for the nitriles.
The results of the labeling experiments are summarized in
a general biosynthetic scheme that highlights the similarities
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4 mgmLÀ1). (E)-12-Methyltridec-
3-enenitrile (10), on the other
hand, was active against the
fungus
Mucor hiemalis
(see
Table S2).
In summary, long-chain ali-
phatic nitriles represent a new
class of natural products produced
by Gram-negative proteobacteria
and Gram-positive actinomycetes.
It seems likely that long-chain
aliphatic nitriles are common sec-
ondary metabolites of many other
bacteria as well. There are subtle
differences in the biosynthetic
pathways of the two bacteria
investigated. Their biological
activities make the nitriles worth-
while targets for further analysis
of their biosynthetic machinery on
the genetic level and of their
ecological function for the bacte-
ria.
Scheme 4. Biosynthesis of the nitriles in P. veronii R02 and M. echinospora DSMZ 43816.
Acknowledgements
and differences of the two bacteria (Scheme 4). The nitriles of
We are grateful for a fellowship to
P. veronii are formed by conventional fatty acid biosynthesis.
The key intermediate 38 is either reduced to 39 and addi-
tionally extended to the required chain length, leading to
saturated nitriles, or it is isomerized to 40. The following chain
elongation leads to wÀ7-unsaturated nitriles. In M. echino-
spora, the isomerization from 38 to 40 is performed one
elongation step later, with substrates with chain lengths
between C13 and C16. No additional chain elongation occurs
after the isomerization; instead, the isomerized products 40
are directly transformed into the nitriles so that the double
bond is fixed at the C-3 position. Both E and Z isomers are
formed, in contrast to the pure Z isomers in the case of 48.
Feeding of [D7]-11-methyldodecanoic acid led directly to
nitrile 13 without the loss of any deuterium labels, supporting
our proposal (see Figure S23). The double bond is clearly
introduced during the final attachment of the last building
block of the carbon chain. The methyl groups of the nitriles
seem to originate from isobutyryl- as well as 2- and
3-methylbutyryl-CoA starter units, which are derived from
the amino acids valine, leucine, and isoleucine, respec-
tively.[18,19] The odd-numbered unbranched acids may either
be formed from a propionyl-CoA starter unit as shown in
Scheme 4 or by a-oxidation of fatty acids.[18,19] Finally,
dehydration of the amides might first require isomerization
to the imidate, which in turn loses water to form the cyanide
group.
D.M.V. from CiÞncia sem Fronteiras (CNPq/206529/2014-7).
Conflict of interest
The authors declare no conflict of interest.
Keywords: antibiotics · biosynthesis · fatty acids ·
isotopic labeling · nitriles
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Final Article published: && &&, &&&&
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ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
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Angewandte
Communications
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Communications
Natural Products
A new class of natural products featuring
long-chain aliphatic nitriles is produced
by Gram-positive and Gram-negative
bacteria. Variable chain lengths and
degrees of hydration in the fatty acid
biosynthesis lead to either unbranched or
methyl-branched unsaturated nitriles
with an wÀ7 or C-3 double bond. Some of
these nitriles showed antimicrobial activ-
ity, for example, against multiresistant
Staphylococcus aureus strains.
D. Montes Vidal,
A.-L. von Rymon-Lipinski, S. Ravella,
U. Groenhagen, J. Herrmann,
N. Zaburannyi, P. H. G. Zarbin,
A. R. Varadarajan, C. H. Ahrens,
L. Weisskopf, R. Mꢁller,
S. Schulz*
&&&& — &&&&
Long-Chain Alkyl Cyanides:
Unprecedented Volatile Compounds
Released by Pseudomonas and
Micromonospora Bacteria
6
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
These are not the final page numbers!