An unprecedented medium-chain diunsaturated n-acylhomoserine lactone from marine roseobacter group bacteria

Article abstract of DOI:10.3390/md17010020

N-acylhomoserine lactones (AHLs), bacterial signaling compounds involved in quorum-sensing, are a structurally diverse group of compounds. We describe here the identification, synthesis, occurrence and biological activity of a new AHL, N-((2E,5Z)-2,5-dodecadienoyl)homoserine lactone (11) and its isomer N-((3E,5Z)-3,5-dodecadienoyl)homoserine lactone (13), occurring in several Roseobacter group bacteria (Rhodobacteraceae). The analysis of 26 strains revealed the presence of 11 and 13 in six of them originating from the surface of the macroalgae Fucus spiralis or sediments from the North Sea. In addition, 18 other AHLs were detected in 12 strains. Compound identification was performed by GC/MS. Mass spectral analysis revealed a diunsaturated C₁₂ homoserine lactone as structural element of the new AHL. Synthesis of three likely candidate compounds, 11, 13 and N-((2E,4E)-2,4-dodecadienoyl)homoserine lactone (5), revealed the former to be the natural AHLs. Bioactivity test with quorum-sensing reporter strains showed high activity of all three compounds. Therefore, the configuration and stereochemistry of the double bonds in the acyl chain seemed to be unimportant for the activity, although the chains have largely different shapes, solely the chain length determining activity. In combination with previous results with other Roseobacter group bacteria, we could show that there is wide variance between AHL composition within the strains. Furthermore, no association of certain AHLs with different habitats like macroalgal surfaces or sediment could be detected.

Full text of DOI:10.3390/md17010020

marine drugs
Article
An Unprecedented Medium-Chain Diunsaturated
N-acylhomoserine Lactone from Marine Roseobacter
Group Bacteria
1
2
3
2
2
Lisa Ziesche , Laura Wolter , Hui Wang , Thorsten Brinkhoff , Marion Pohlner ,
2
3
1,
Bert Engelen , Irene Wagner-Döbler and Stefan Schulz *
1
Institute of Organic Chemistry, Technische Universität Braunschweig, Hagenring 30, 38106 Braunschweig,
Germany; l.ziesche@tu-braunschweig.de
Institute for Chemistry and Biology of the Marine Environment, University of Oldenburg,
2
Carl-von-Ossietzky-Straße 9-11, 26111 Oldenburg, Germany; laura.wolter@uni-oldenburg.de (L.W.);
thorsten.brinkhoff@icbm.de (T.B.); marion.pohlner@uni-oldenburg.de (M.P.);
bert.engelen@uni-oldenburg.de (B.E.)
3
Helmholtz Centre for Infection Research, Department of Medical Microbiology, Group Microbial
Communication, Inhoffenstr. 7, 38124 Braunschweig, Germany; Hui.Wang@helmholtz-hzi.de (H.W.);
Irene.Wagner-Doebler@helmholtz-hzi.de (I.W.-D.)
*
Correspondence: stefan.schulz@tu-braunschweig.de; Tel.: +49-531-391-5272
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 7 December 2018; Accepted: 21 December 2018; Published: 31 December 2018
Abstract: N-acylhomoserine lactones (AHLs), bacterial signaling compounds involved in
quorum-sensing, are a structurally diverse group of compounds. We describe here the identification,
synthesis, occurrence and biological activity of a new AHL, N-((2E,5Z)-2,5-dodecadienoyl)homoserine
lactone (11) and its isomer N-((3E,5Z)-3,5-dodecadienoyl)homoserine lactone (13), occurring in several
Roseobacter group bacteria (Rhodobacteraceae). The analysis of 26 strains revealed the presence of
11 and 13 in six of them originating from the surface of the macroalgae Fucus spiralis or sediments
from the North Sea. In addition, 18 other AHLs were detected in 12 strains. Compound identification
was performed by GC/MS. Mass spectral analysis revealed a diunsaturated C12 homoserine lactone
as structural element of the new AHL. Synthesis of three likely candidate compounds, 11
, 13 and
N-((2E,4E)-2,4-dodecadienoyl)homoserine lactone ( ), revealed the former to be the natural AHLs.
5
Bioactivity test with quorum-sensing reporter strains showed high activity of all three compounds.
Therefore, the configuration and stereochemistry of the double bonds in the acyl chain seemed to be
unimportant for the activity, although the chains have largely different shapes, solely the chain length
determining activity. In combination with previous results with other Roseobacter group bacteria, we
could show that there is wide variance between AHL composition within the strains. Furthermore,
no association of certain AHLs with different habitats like macroalgal surfaces or sediment could
be detected.
Keywords: quorum-sensing; structure elucidation; Rhodobacteraceae; autoinducers; bacterial
signaling; Heck coupling; mass spectrometry; gas chromatography; sediment; AHL
1
. Introduction
N-acylhomoserine lactones (AHLs) are well known signalling compounds used by Gram-negative
bacteria for quorum-sensing (QS)-driven cell-to-cell communication. QS is a cell density-dependant
mechanism to regulate physiological traits like antibiotic production, cell differentiation or biofilm
formation [1–7]. AHLs constitute a γ-lactone ring and an acyl side chain that is usually even-numbered
and unbranched, ranging in chain length from C –C
[3]. The stereochemistry of the lactone ring
4
18
Mar. Drugs 2019, 17, 20; doi:10.3390/md17010020
www.mdpi.com/journal/marinedrugs

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is S. Functional groups like hydroxy- or carbonyl-group can be present at C-3 of the acyl chain.
The double bond of unsaturated AHLs is Z-configured and in the position -7, with few exceptions [ ].
ω
3
An additional double bond can occur at C-2 in E-configuration, a feature especially occurring in AHLs
produced by marine Roseobacter group bacteria (Rhodobacteraceae). Roseobacters are abundant in the
ocean, occurring in diverse habitats [
to biotic and abiotic surfaces as well as in symbiosis with higher organisms like algae [
of this bacterial group have saturated, unsaturated and sometimes oxygenated acyl chains, ranging
in length between C and C 12] with the exception of aromatic p-coumaroylhomoserine lactone
8], e. g. in open waters, shore environments, sediments, attached
9
–
11]. AHLs
[
18
8
produced by Ruegeria pomeroyi DSS-3 [13]. They are involved in various biological traits [14], e.g., in
the production of the antibiotic tropodithietic acid in Phaeobacter inhibens [5] or cell differentiation in
Dinoroseobacter shibae [4].
In a broader program, we currently look into the inventory of AHLs occurring in Roseobacter group
bacteria. A non-targeted analytical approach was developed using extraction of AHLs from bacterial
cultures by XAD-16 adsorption, solvent extraction, and direct analysis by GC/MS. This approach
combines high-sensitivity with unbiased analysis and allows structural proposals to be made basing
on the information rich EI-mass spectra obtained. We could successfully use this approach to
identify several previously unknown AHLs [12,15,16] as well as related N-acylalanine methyl esters
(
NAMEs) [17 18]. We have previously analyzed AHLs from roseobacters isolated from macroalgae
,
surfaces and found a high proportion of strains producing AHLs in various mixtures [12]. A specific
AHL signature of macroalgae associated strains was not observed. In an extension of this study we
investigated 16 strains obtained from one location of samples of the algae Fucus spiralis, collected
from a single location (Neuharlingersiel, German Wadden Sea), nine strains from Norwegian trench
sediments [19] and one sea water strain from the German/Danish coast to test for the occurrence of
specific AHL signatures. During this investigation we detected two previously unreported AHLs, their
identification, synthesis, and biological activities being reported.
2
. Results
2
.1. Occurrence of N-acylhomoserine Lactones in Roseobacter Group Bacteria of Fucus Spiralis and the Eastern
North Sea
Sixteen roseobacters originating from Fucus spiralis from the German Wadden Sea and 10 strains
from the eastern North Sea were cultivated in marine broth and analyzed for the presence of AHLs by
GC/MS as described previously [12 20]. AHLs were detected in eight of the isolates from F. spiralis
50%) and three of the sediment strains (33%) as well as in the open water strain (Table 1). The highest
numbers of individual compounds were detected on the extracts obtained from Octadecabacter sp.
Lw-22) and Loktanella sp. (D15 (40)), 12 and 13 AHLs being detected, exhibiting a distinct qualitative
,
(
(
profile. The sediment strain Phaeobacter sp. (SK040) contained nine different AHLs, while the other
sediment strains SK013 and SK032 had only one or two AHLs, comparable to the water column strain.
The AHL composition of other strains varied between single compounds and mixtures (Table 1).
Octadecenoylhomoserine lactone (C18:1-HSL) and C16:1-HSL were the most common compounds,
produced by six of the 12 bacteria. Roseovarius sp. D12-1.68 displayed a unique profile due to the
presence of C12:0 as major AHL with a relatively short chain length compared to most other major
AHLs of roseobacters [12,15,21]. Dinoroseobacter shibae MDLw-58 produced three different isomers of
the C18:2-HSL, consistent with previous observations of closely related strains [20]. While the major
component was 2E,11Z-C18:2-HSL, the location and configuration of the double bonds in the other
two isomers remains unknown. Other diunsaturated AHLs with shorter chain lengths, rarely observed
in strains taxonomically distant from roseobacters, occurred as well. These include C16:2-HSL in
Huaishuia sp. SK032, C14:2-HSL in Octadecabacter sp. Lw-22 and several strains containing C12:2-HSL.
Because this AHL was not previously reported and due to its abundant occurrence in the investigated
strains, we determined its structure of this new AHL, as reported in Section 2.2. Less abundant were

Mar. Drugs 2019, 17, 20
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the oxygenated AHLs, 3-OH-C10- and 3-OH-C14-HSL. The only odd numbered AHLs were C15:0-HSL,
C15:1-HSL and C17:1-HSL which occurred as minor components in several strains.
Table 1. AHL production of Roseobacter group isolates from Fucus spiralis (German Wadden Sea) and
sediments as well as open waters (Eastern North Sea) a.
Strain
Genus Affiliation
Fucus Spiralis
D12-1.68
D3
Lw-22
Roseovarius sp.
Loktanella sp.
Octadecabacter sp.
Octadecabacter sp.
Loktanella sp.
Loktanella sp.
Loktanella sp.
Dinoroseobacter sp.
Sediment
94.4
1.3
4.3
15.7 2.4 21.2 60.7
6.6 3.7 0.7
8.3 25.8 4.4
4.6 72.7 4.3 1.2 1.9 0.9 0.6
1.1 0.7
100
3.9 5.9 1.2 0.9 18.4 2.3 58.6
1.1 1.8
59.8
D4 (50)
Lw-26b
D15 (40)
Lw-55a
MDLw-58
0.5 0.8 0.6 6.1
0.5 0.3
100
5.4 3.9 2.5 88.2
SK013
SK032
SK040
Shimia sp.
Huaishuia sp.
Phaeobacter sp.
100
81.7 18.3
4.8 64.9
5.8 3.7 1.4 7.3 4.4
3.5
4.1
Water Column
Sulfitobacter sp.
SK038
a
100
relative amounts of AHLs for each strain in %. No AHLs detected in Puniceibacterium sp. Lw-III1a, Sulfitobacter
sp. B15 G2, Pseudooceanicola sp. Lw-13e, Roseobacter sp. B14, Loktanella sp. SK033, Phaeobacter sp. N05I, Sulfitobacter
sp. B14 27, A12, D4 55, SK012, Citreicella sp. Lw-41a, Roseovarius pelophilus G5II, Tateyamaria pelophila SAM4,
Pseudoruegeria sp. SK021.
2
.2. Identification and Synthesis of New Diunsaturated N-acylhomoserine Lactones from the Roseobacter Group
During these analyses two compounds,
were detected in five of the strains, whose mass spectra showed similarity to those of other AHLs.
The spectra of A (Figure 1b) and (Supplementary Figure S1) were very similar, although the quality
A as the major component and B in lower concentration,
B
of the spectra was often low due to overlapping peaks from other compounds. To elucidate their
structure, analysis of mass spectral data and total synthesis were performed.
Electron impact mass spectra of AHLs have a typical fragmentation pattern shown in Figure 1a.
+
AHLs are characterized by the fragment ions m/z 102, 143 and a small [M] [15
,22]. The ion
m/z 102 is formed by
α-cleavage of the homoserine lactone unit and transfer of two hydrogens,
while MCLAFFERTY-rearrangement forms the ion m/z 143. The intensity of m/z 102 is higher in
monounsaturated compared to diunsaturated AHLs and the intensities of m/z 102 and 143 decrease
+
with the acyl chain length [15]. Cleavage of the homoserine moiety explains the ion [M–101] , m/z 264,
in the spectrum of (Z)-11-octadecenoylhomoserine lactone (Z11-C18:1-HSL, Figure 1a).
+
Compound
A
and
B
showed both ions m/z 102 and 143 and a putative [M] at m/z 279, indicating
to be C12:2-homoserine lactones (C12:2-HSL). Additional ions at m/z 94 and 107, untypical for AHLs,
were present in high intensity. The gas chromatographic retention index of
388. High resolution ESI-MS of
A
was 2422 and that of
B
+
2
A
delivered an ion at m/z 280.19071 [M + H] , consistent with the
formula C H NO (calc. 280.19072) required for an diunsaturated AHL. The homoserine lactone unit
16
26
3
is indicated by the ion m/z 102.05496 (C H NO ) and the acyl chain by the ion m/z 179.14314 (C H O)
4
8
2
12 19
in the ESI spectrum [18]. The location of the double bonds could not be determined with dimethyl
disulfide derivatization [12 20] because of the low concentration of the compound. Nevertheless,
the usual -7 position of double bonds in AHLs and the previous detection of Z5-C12:1-HSL in
roseobacters [12] suggested to be 2E,5Z-C12:2-HSL, because the second double-bond in all known
,
ω
A
natural AHLs is located at C-2 with E-configuration. The close proximity of the double bonds might
also favor a double bond shift into conjugation during biosynthesis with concomitant double bond
isomerization, leading to 2E,4E-C12:2-HSL. Furthermore, deconjugation is a known process known
to occur during formation of
α,β-unsaturated amides under basic conditions, leading potentially to
3
E,5Z-C12:2-HSL [23 24]. Therefore, we decided to synthesize all three compounds to reveal insight
,

Mar. Drugs 2019, 17, 20
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into the MS and GC behavior of the isomers and also allowing to perform bioassays to investigate
whether slight changes in double bond location and geometry have an influence on the activity in
AHL reporter assays.
Figure 1.
(
a) Mass spectrum and fragmentation pattern of N-((Z)-11-octadecenoyl)homoserine lactone
(
Z11-C18:1-HSL); (
b
) mass spectrum of the unknown AHL ; ( ) total ion chromatogram of the natural
A c
extract of Octadecabacter D4 (50) and characteristic ion traces m/z 102 and 143, indicating potential
presence of AHLs.

Mar. Drugs 2019, 17, 20
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The synthesis of 2E,4E-C12:2-HSL (
of triethyl (E)-4-phosphonocrotonate (
) (Scheme 1). After saponification with lithium hydroxide,
lactone hydrobromide (HSL
hydrochloride (EDC
homoserine lactone (5).
5
1
) started with a homologous Horner-Wadsworth-Emmons reaction
) with octanal ( ) to furnish ethyl (2E,4E)-2,4-dodecadienoate
2
(3
4
was coupled with L-homoserine
0
·
HBr) in the presence of N-(3-dimethylaminopropyl)-N - ethylcarbodiimide
HCl) and triethylamine, yielding the desired AHL N-((2E,4E)-2,4-dodecadienoyl)
·
O
P
O
O
N
H
O
O
+
O
O
O
Li
6
4%
1
2
3
THF:MeOH:H2O (2:2:1)
LiOH,
O
O
O
HSL·HBr, Et N, EDC·HCl
3
OH
N
9
2%
99%
H
O
4
5
Scheme 1. Synthesis of N-((2E,4E)-2,4-dodecadienoyl)homoserine lactone (2E,4E-C12:2-HSL,
5
).
L-homoserine lactone hydrobromide (HSL
hydrochloride (EDC·HCl).
·
HBr), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide
The isomers 2E,5Z-C12:2-HSL (11) and 3E,5Z-C12:2-HSL (13) were synthesized from octyne
(
6
) (Scheme 2). The addition of paraformaldehyde furnished non-2-yn-1-ol (
7
). The allylic alcohol
0
(Z)-non-2-en-1-ol (
8) was obtained after hydrogenation with Lindlar s catalysts. A Heck reaction
according to Tsukuda et al. [25] using methyl acrylate instead of butyl acrylate yielded in excellent
yield the desired diastereomerically pure methyl (2E,5Z)-2,5-dodecadienoate ( ) that was saponified
with lithium hydroxide. The basic conditions lead to a rearrangement of the double bond from C-2
to C-3, forming acid 12. This acid was coupled with HSL HBr in the presence of EDC HCl and
triethylamine to form N-((3E,5Z)-3,5-dodecadienoyl)homoserine lactone (13). The saponification
of ester can also be performed under milder conditions, thus preventing the rearrangement.
First, ester was transesterified to the trimethylsilyl ester with trimethylsilyl iodide (TMSI) in
CCl . Aqueous hydrolysis delivers acid 10 without rearrangement [26]. Finally, acid 10 was
9
·
·
9
9
4
coupled with HSL
·
HBr in the presence of EDC HCl and p-dimethylaminopyridine (DMAP) to furnish
·
N-((2E,5Z)-2,5-dodecadienoyl)homoserine lactone (11). The isomerization of the double bond, leading
again to 13, can also be induced by the use of triethylamine in this step instead of the weaker base
DMAP [24]. AHL 11 is not stable for prolonged storage under room temperature, leading to the
isomerized compound 13 and other stereoisomers.
Scheme 2. Synthesis of N-((2E,5Z)-2,5-dodecadienoyl)homoserine lactone (2E,5Z-C12:2-HSL, 11) and
N-((3E,5Z)-3,5-dodecadienoyl)homoserine lactone (3E,5Z-C12:2-HSL, 13). Tricyclohexylphosphine (Cy P),
3
tris(dibenzylideneacetone)dipalladium (Pd (dba) ), L-homoserine lactone hydrobromide (HSL
·
HBr),
2
3
N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC·HCl), p-dimethylaminopyridine
(DMAP).

Mar. Drugs 2019, 17, 20
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Comparison of both mass spectra (Figure 2) and gas chromatographic retention indices of the
synthesized AHLs with those of the natural compounds indicated the unknown major AHL to be
E,5Z-C12:2-HSL (11), while the minor component is its rearrangement product, 3E,5Z-C12:2-HSL
13). The mass spectrum of differs from those of 11 and 13, showing an ion at m/z 180 instead of m/z
78. The ions m/z 94, 102, 107 and 143 are present, but the intensity of these fragment ions is different
A
2
B
(
5
1
to those of the natural compounds, as is the retention index of 2510. HR-MS data showed that the ions
m/z 107 and 94, not observed in high intensity in other diunsaturated AHLs with longer acyl chains,
are composed of C H and C H O, respectively. They seem to be formed by acyl cleavage followed
8
11
6
6
by allylic chain cleavage with or without previous elimination of water.
Figure 2. Mass spectra of synthetic AHLs: (
E,5Z-C12:2-HSL (13).
a) 2E,5Z-C12:2-HSL (11); (b) 2E,4E-C12:2-HSL (5); (c)
3
2
.3. Activity of N-acylhomoserine Lactones in AHL Reporter Assays
The three C12:2-HSLs 11, and 13 as well as 2E,11Z-C18:2-HSL, a major AHL of the Roseobacter
group model strain Dinoroseobacter shibae DFL12 [ 20] were tested for quorum sensing activity with
5,
4
,
two sensor strains (Table 2). Escherichia coli MT102 (pJBA132) [27] responds primarily to short chain
AHLs while Pseudomonas putida F117 (pRK-C12) [28] prefers long chain AHLs. Both strains do not
produce AHLs but are able to respond to them through the expression of a LuxR-controlled promoter
fused to a gene coding for an easily detectable output signal, fluorescence [15]. In addition, C6:0-, C8:0-
and 3-oxo-C8:0-HSL for E.coli MT102 and Z9-C16:1-, 3-oxo-C8:0- and 3-oxo-C12:0-HSL for P. putida
F117 were tested as references. The maximum fold induction with sensor strain E.coli MT102 was
low for all target AHLs,
as expected.
5, 11, 13 as well as 2E,11Z-C18:2-HSL, but C6 and C8-HSLs showed activity
Table 2. Activity of synthetic AHLs in quorum sensing experiments with the sensor strains E.coli
MT102 (pJBA132) and P. putida F117 (pRK-C12). Maximum fold induction.
AHLa
MT102
F117
2
E,4E-C12:2 (5)
1.00 ± 0.01
1.01 ± 0.01
1.03 ± 0
81.22 ± 1.26
78.03 ± 2.05
73.64 ± 2.34
1.32 ± 0.04
30.46 ± 2.79
2E,5Z-C12:2 (11)
3E,5Z-C12:2 (13)
2
E,11Z-C18:2
1.02 ± 0.01
13.41± 0.49
17.09 ± 1.04
10.24 ± 0.55
3
-oxo-C8:0
C6:0
C8:0
3
-oxo-C12:0
Z9-C16:1
77.75 ± 2.51
72.37 ± 2.47
a
The final concentration of each tested compound was 10 µM.

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In contrast, sensor P. putida F117, most sensitive to C12:0-HSL [16], showed high induction
upon exposure to AHLs 11, and 13, as well as 3-oxo-C12:0-HSL and Z9-C16:1-HSL, a lower
5
,
activity for 3-oxo-C8:0-HSL being noted. The almost identical values exhibited by the four C12
compounds evidence a certain degree of selectivity dependent on chain length, but the reporter strain
is insensitive to position and configuration of the double bonds, as well as presence or absence of a
3
-oxo functional group.
3
. Discussion
The unknown AHL
A was identified to be 2E,5Z-C12:2-HSL (11). This new AHL is the smallest
AHL with two double-bonds identified so far and occurs in several roseobacters. The minor
E,5Z-C12:2-HSL (13) seems to be a rearrangement product of 11. Although we cannot exclude that the
3
process took place during work-up, the observed chemical instability of 11 may point to the formation
of 13 as a bacterial metabolic product. Different roseobacters are able to produce a bishomologous
series of diunsaturated AHLs with double-bonds at C-2 and the
ω-7 position [12,15,16,20]. AHL 11
extends this series to a shorter C12 chain length. It is also within the preferred chain-length of major
AHLs of roseobacters that ranges from C12 to C , with the exception of 3-OH-C10-AHL. Although
18
we analyzed many roseobacters for AHL presence using GC/MS as well as HPLC/MS methods, we
never found evidence for the presence of p-coumaroyl-HSL reported from Ruegeria pomeroyi DSS-3 [13
]
in any of these strains.
Although the AHLs
The two (E)-configured double bonds of 2E,4E-C12:2-HSL lead to a straight aliphatic chain, while the
Z)-configured double-bonds in 2E,5Z-C12:2-HSL and 3E,5Z-C12:2-HSL induce a bend in the chain.
-oxo-C12:0-HSL even has a bend chain due to H-bonding towards the amide carbonyl group [29].
5, 11 and 13 have the same chain length, the shape of the side chain differs.
(
3
Obviously, there is no influence of the chain configuration on the activity of the reporter strain.
Therefore, the configuration of the side chain does not seem to be recognized by the receptor, although
the chain-length obviously is. Whether this is also true for the cognate receptors in the roseobacters is
unknown. Nevertheless, the (2E)-double bond is not common in fatty acids of roseobacters [30] and
thus the prominent occurrence of this structural motif and of diunsaturated AHLs in general indicate
the importance of the double bonds for their function as signaling compounds of these bacteria.
No general association of specific AHLs or their mixtures with certain habitats was observed.
This includes strains originating from the same host organism as F. spiralis reported here, but also
strains from the sediment or the water phase [12,15]. In general, surface-associated strains seem to
produce AHLs more often under laboratory conditions compared to those obtained from sediments or
the water column.
4
. Materials and Methods
4
.1. General Experimental Procedures
General conditions: Chemicals were obtained from Sigma-Aldrich (Taufkirchen, Germany),
Carl Roth (Karlsruhe, Germany) or from Acros Organics (Schwerte, Germany) and were used
without further purification. Solvents were purified by distillation and dried according to standard
procedures. Moisture- and/or oxygen-sensitive reactions were carried out under a nitrogen atmosphere
in vacuum-heated flasks with dried solvents. Thin-layer chromatography (SiO , TLC) was performed
2
on 0.20 mm Macherey-Nagel silica gel plates (Polygram SIL G/UV254), and column chromatography
was performed with Merck silica gel 60 (0.040–0.063 mm) by using standard flash chromatographic
methods. NMR spectra were recorded on DRX-400 (400 MHz), AV III-400 (400 MHz) or AV II-600
(
600 MHz) spectrometers (Bruker: Bremen, Germany), and were referenced against TMS (
δ
= 0.00 ppm),
1
= 77.01 ppm) for 13C-NMR experiments. GC/MS
CDCl3 (
δ
= 7.26 ppm) for H-NMR and CDCl (
δ
3
analyses of extracts were carried out on an GC 7890A gas chromatograph connected to a 5975C
mass-selective detector (Agilent; Waldbronn, Germany). Synthetic samples were analyzed on an HP

Mar. Drugs 2019, 17, 20
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GC 6890 system connected to an HP 5973 mass selective detector fitted with a HP-5 MS fused-silica
capillary column (30 m 0.25 mm i.d., 0.22 m film; (Agilent; Waldbronn, Germany). Conditions
were as follows: carrier gas (He): 1.2 mL min ; injection volume: 1 mL; injector: 250 C; transfer
×
µ
−
1
◦
◦
◦
line: 300 C, EI 70 eV. The gas chromatograph was programmed as follows: 50 C (5 min isothermal),
◦
−1
◦
◦
increasing with 5 C min to 320 C, and operated in splitless mode for XAD extracts and 50 C (5 min
◦
−1
◦
isothermal), increasing with 10 C min to 320 C in split mode (20:1) for synthetic compounds. Gas
chromatographic retention indices, I, were determined from a homologous series of n-alkanes. Acids
were transformed into volatile trimethylsilyl esters with N-methyl-N-(trimethylsilyl)trifluoroacetamide
(MSTFA) for analysis by GC/MS [31]. HRMS analyses were carried out on a Thermo Fisher linear
iontrap (Thermo Fisher: Bremen, Germany) coupled with an LTQ-Orbitrap mass spectrometer (Thermo
Fisaher: Bremen, Germany) using ESI positive mode. ESI measurements were performed by direct
infusion mode using a custom-made micro-spray device mounted on a Proxeon nano-spray ion source.
All solvents used were of LC/MS grade.
4
.2. Strains and Culture Conditions
The bacterial strains were collected at various occasions in the North Sea [19]. Information on
bacterial strains investigated is shown in Table 3.
Table 3. Bacterial isolates with GenBank accession number, genus affiliation and origin.
Strain
GenBank Acc No (16S)
Genus Affiliation
Strain Origin/Host
Location
Lw-III1a
B15 G2
Lw-13e
D12-1.68
B14
B14 27
A12
D3
D4 55
Lw-22
D4 50
Lw-26b
D15 40
Lw-55a
MDLw-58
Lw-41a
N05I
KM268064
KM268068
KM268063
KM268065
KM268066
KM268072
KM268070
KC731427
KM268071
KM268073
KM268074
KM268054
KM268056
KM268057
KM268059
KM268061
AJ968647
Puniceibacterium sp.
Sulfitobacter sp.
Pseudooceanicola sp.
Roseovarius sp.
Roseobacter sp.
Sulfitobacter sp.
Sulfitobacter sp.
Loktanella sp.
Sulfitobacter sp.
Octadecabacter sp.
Octadecabacter sp.
Loktanella sp.
Fucus spiralis
Fucus spiralis
Fucus spiralis
Fucus spiralis
Fucus spiralis
Fucus spiralis
Fucus spiralis
Fucus spiralis
Fucus spiralis
Fucus spiralis
Fucus spiralis
Fucus spiralis
Fucus spiralis
Fucus spiralis
Fucus spiralis
Fucus spiralis
Neuharlingersiel
Neuharlingersiel
Neuharlingersiel
Neuharlingersiel
Neuharlingersiel
Neuharlingersiel
Neuharlingersiel
Neuharlingersiel
Neuharlingersiel
Neuharlingersiel
Neuharlingersiel
Neuharlingersiel
Neuharlingersiel
Neuharlingersiel
Neuharlingersiel
Neuharlingersiel
Loktanella sp.
Loktanella sp.
Dinoroseobacter sp.
Citreicella sp.
Phaeobacter sp.
Roseovarius pelophilus
German North Sea Coast Tidal-flat sediment
German North Sea Coast Tidal-flat sediment
German North Sea Coast Tidal-flat sediment
Danish North Sea
Norwegian North Sea
Norwegian North Sea
German North Sea
Norwegian North Sea
Danish North Sea
G5II
AJ968650
AJ968651
a
SAM4
SK012
SK013
SK021
SK032
SK033
SK040
SK038
Tateyamaria pelophila
HG423260
LAJH00000000
HG423263
HG423269
HG423270
HG423272
HG423271
Sulfitobacter sp.
sediment
sediment
sediment
sediment
sediment
sediment
seawater
Shimia sp. b
Pseudoruegeria sp. b
Huaishuia sp.
Loktanella sp.
Phaeobacter sp.
Sulfitobacter sp.
a
German North Sea
[
32]; b [33,34].
4
.3. Bacteria Cultivation and XAD Extraction
Bacterial strains were inoculated from marine broth (MB) medium agar plates into a preculture
◦
(
50 mL, MB medium) and were cultivated for one to three days at 20/28 C and 160 rpm. Bacterial
◦
cultures (100 mL) were grown from precultures for three to five days in MB medium at 20/28 C and
1
60 rpm containing Amberlite XAD-16 (2 g). The XAD-16 resin was cleaned using Soxhlet extraction
with acetonitrile, methanol and finally diethyl ether. The adsorbent was separated from the culture
by filtration and extracted three times with CH Cl /H O (10:1). The combined organic phases were
2
2
2

Mar. Drugs 2019, 17, 20
9 of 14
dried with MgSO , and the solvent was removed under reduced pressure. The extract was dissolved
4
in CH Cl (50 µL) and analyzed by GC/MS [20].
2
2
4
.4. AHL Reporter Assays
The sensor strain Pseudomonas putida pRK-C12 [27] was inoculated from plates into a preculture
◦
which was grown on LB medium (20 mL with 20 mg/mL gentamycin) at 30 C with shaking (160 rpm)
overnight. The next day fresh medium was added, and the culture was grown on a shaking platform
for 1–2 h until an OD620 value of 1.0 was reached. For the test, LB medium (99
µL) and 1 µL of
the test compound (final concentration 10 M, stock solution 1 mg/mL in dichlormethane) were
µ
pipetted into 96-well microtiter plates, and the sensor strain (100
were incubated at 30 C and shaken. Fluorescence was determined in a Victor 1420 Multilabel Counter
µ
L) was added. Microtiter plates
◦
(
Perkin Elmer; Rodgau, Germany) at an excitation wavelength of 485 nm and a detection wavelength
of 535 nm every 60 min for the first 6 h, and finally after 24 h of incubation. The OD620 value was also
measured. Dichlormethane was used as negative control, and synthetic 3-oxo-C12:0-HSL was used as
positive control. Fold induction of fluorescence was calculated by dividing the specific fluorescence
(gfp535/OD620) of the test sample by the specific fluorescence of the negative control. Mean and
standard deviation of three biological replicas after 6 h were determined, because fluorescence
decreased slightly in the 24 h time point. The sensor strain Escherichia coli MT102 [29] was used
as described [15] and the highest values obtained after 24 h in this case are reported in Table 2.
4
.5. Synthetic Procedures
4
.5.1. L-Homoserine Lactone Hydrobromide
A solution of bromoacetic acid (5.12 g, 36.9 mmol) and L-methionine (5.00 g, 33.5 mmol) in
H O/2-propanol/AcOH (5:5:2, 48.3 mL) was heated to reflux for 8 h. The solvent of the cooled mixture
2
◦
was evaporated. The orange solid was dissolved in dioxane/HCl (2:1, 20.1 mL), heated 10 min at 50 C
and stirred for 5 h at room temperature. The reaction mixture was placed in a fridge over night to
evoke precipitation. L-Homoserine lactone hydrobromide was obtained by filtration and washed with
cold isopropanol (3.19 g, 17.5 mmol, 52%) [35].
1
H-NMR (400 MHz, DMSO-d6):
δ = 4.44 (t, J = 8.8 Hz, 1H), 4.36–4.25 (m, 2H), 2.60–2.51 (m, 1H),
2
4
.42–2.31 (m, 1H); 13C-NMR (100 MHz, DMSO-d6):
3 (100), 57 (90.5), 56 (62.4), 42 (40.1), 44 (16.0), 41 (8.3), 101 (5.0) [M ], 54 (4.8), 39 (4.3), 73 (4.0).
δ
= 173.4, 66.3, 47.8, 27.0; MS (70 eV, EI): m/z (%):
+
4
.5.2. Ethyl (2E,4E)-2,4-Dodecadienoate (3)
◦
78 C.
N,N-Diisopropylamine (1.64 mL, 11.7 mmol) was dissolved in THF (25 mL) and cooled to
−
n-Butyl lithium in hexane (1.6 M, 4.88 mL, 7.8 mmol) was added slowly to the mixture. The solution
◦
◦
was stirred for 10 min at
−
78 C, followed by 15 min at 0 C. (E)-Triethyl-4-phosphonocrotonate (
1,
◦
1
3
.30 mL, 5.8 mmol) was added slowly at
−
78 C under stirring and after 15 min octanal (
2
, 0.61 mL,
.9 mmol) was added at the same temperature, again stirring continued for 15 min. The reaction
mixture was allowed to warm up to room temperature and stirred for 2.5 h. Sat. NH Cl solution was
4
added and the mixture was extracted three times with ethyl acetate. The combined organic phases
were dried with MgSO , filtered and the solvent was evaporated in vacuo. The crude product was
4
purified by flash chromatography on silica [pentane/EtOAc (30:1)] to receive the desired product as a
clear oil (280 mg, 1.25 mmol, 64%) [36].
1
R = 0.4 (pentane/EtOAc 30:1); H-NMR (400 MHz, CDCl ):
δ = 7.29–7.22 (m, 1H), 6.20–6.08 (m,
f
3
2
H), 5.78 (d, J = 15.4 Hz, 1H), 4.19 (q, J = 7.2 Hz, 2H), 2.16 (q, J = 7.0 Hz, 2H), 1.43 (quin, J = 7.3 Hz, 2H),
1
1
.32–1.24 (m, 11H), 0.88 (t, J = 7.1 Hz, 3H); 13C-NMR (100 MHz, CDCl3):
δ
= 167.3, 145.1, 144.8, 128.3,
19.1, 60.1, 33.0, 31.8, 29.1, 29.1, 28.7, 22.6, 14.3, 14.1; MS (70 eV, EI): m/z (%): 125 (100), 97 (58.4), 81
+
(51.7), 67 (39.6), 179 (34.9), 98 (30.6), 79 (30.5), 95 (25.3), 127 (22.8), 99 (22.4), 224 (21.0) [M ].

Mar. Drugs 2019, 17, 20
10 of 14
4
.5.3. (2E,4E)-2,4-Dodecadienoic Acid (4)
Lithium hydroxide (106.8 mg, 4.46 mmol) was added to a stirred solution of ester
3
(50 mg,
0
.22 mmol) in THF/MeOH/H O (2:2:1) and stirred for 12 h under reflux. The mixture was acidified
2
with 2 M sulfuric acid and extracted three times with dichloromethane. The combined organic phases
were dried with Na SO , filtered and concentrated in vacuo to get the desired product after purification
2
4
by flash chromatography on silica [pentane/EtOAc (2:1)] as a white solid (39.9 mg, 0.20 mmol, 92%).
1
R = 0.3 (pentane/EtOAc 2:1); H-NMR (400 MHz, CDCl ):
δ = 7.35 (dd, J = 15.3, 10.0 Hz, 1H),
f
3
6
.24–6.16 (m, 2H), 5.78 (d, J = 15.3 Hz, 1H), 2.18 (q, J = 7.3 Hz, 2H), 1.43 (quin, J = 7.3 Hz, 2H), 1.33–1.20
13
(
m, 8H), 0.88 (t, J = 7.0 Hz, 3H); C-NMR (100 MHz, CDCl3):
δ = 172.8, 147.5, 146.3, 128.2, 118.2, 33.1,
3
1.8, 29.1, 29.1, 28.6, 22.6, 14.1; MS (70 eV, EI, trimethylsilyl ester): m/z (%): 169 (100), 253 (64.0), 73
+
(31.9), 155 (30.1), 170 (20.5), 75 (17.5), 254 (15.7), 268 (13.8) [M ], 81 (13.4), 171 (9.2).
4
.5.4. N-((2E,4E)-2,4-Dodecadienoyl)homoserine Lactone (5)
L-Homoserine lactone hydrobromide (53 mg, 0.29 mmol) was dissolved in dry dichloromethane.
Triethylamine (0.04 mL, 0.29 mmol) was added to the solution, followed by the addition of acid
57 mg, 2.29 mmol) and EDC HCl (56 mg, 0.29 mmol). The reaction mixture was stirred for 12 h at
room temperature and washed with H O, sat. NaHCO solution, and brine. The organic layers were
4
(
·
2
3
extracted three times with dichloromethane, dried with Na SO , filtered and concentrated. The crude
2
4
product was purified by flash chromatography on silica [pentane/EtOAc (1:1)] to obtain pure AHL
5
(
80.6 mg, 0.28 mmol, 99%).
1
R = 0.3 (pentane/EtOAc 1:1); H-NMR (600 MHz, CDCl ):
f
δ
= 7.23 (dd, J = 15.0, 9.7 Hz, 1H),
3
6.25 (br s, 1H), 6.17–6.08 (m, 2H), 5.81 (d, J = 15.1 Hz, 1H), 4.66 (ddd, J = 11.6, 8.6, 6.0 Hz, 1H), 4.48
(td, J = 9.0, 1.1 Hz, 1H), 4.31 (ddd, J = 11.3, 9.3, 5.9 Hz, 1H), 2.87 (dddd, J = 12.5, 8.5, 5.9, 1.2 Hz, 1H),
2
.22–2.18 (m, 1H), 2.15 (q, J = 7.0 Hz, 2H), 1.41 (quin, J = 7.0 Hz, 2H), 1.33–1.26 (m, 8H), 0.88 (t, J = 7.2
13
Hz, 3H); C-NMR (150 MHz, CDCl3):
3
calculated for C H NO Na 302.1727 [M + Na] .
δ
= 175.5, 166.6, 144.4, 142.7, 127.8, 119.9, 66.0, 49.2, 32.8, 31.5,
+
0.4, 28.9, 28.9, 28.5, 22.4, 14.0 (Supplementary Figure S4); HRMS (ESI+) m/z: 302.1728 [M + Na] ,
+
16
25
3
4
.5.5. Non-2-yn-1-ol (7)
n-Butyl lithium in hexane (1.6 M, 31.25 mL, 50 mmol) was added at
-octyne ( , 5 g, 45 mmol) in dry diethyl ether and stirred for 30 min. Paraformaldehyde (2.77 g, 90 mmol)
◦
78 C to a stirred solution of
−
1
6
was added and the mixture was allowed to warm up to room temperature. Sat. NH Cl solution was
4
added, the phases were separated and extracted three times with diethyl ether. The combined organic
phases were washed with brine, dried with Na SO , filtered and the solvent was evaporated. The crude
2
4
product was purified by flash chromatography on silica [pentane/EtOAc (7:1)] to furnish the desired
product as a clear oil (6.2 g, 40 mmol, 98%) [37].
1
R = 0.4 (pentane/EtOAc 7:1); H-NMR (400 MHz, CDCl ):
δ = 4.25 (dt, J = 5.8, 2.1 Hz, 2H), 2.21
f
3
13
(
tt, J = 7.1, 2.2 Hz, 2H), 1.51 (quin, J = 7.3 Hz, 2H), 1.41–1.24 (m, 6H), 0.89 (t, J = 6.8 Hz, 3H); C-NMR
(
100 MHz, CDCl3):
δ
= 86.7, 78.3, 51.4, 31.3, 28.6, 28.5, 22.5, 18.7, 14.0; MS (70 eV, EI): m/z (%): 67 (100), 55
+
(97.2), 41 (95.0), 93 (73.4), 70 (72.5), 39 (68.8), 43 (64.0), 69 (58.0), 79 (57.4), 83 (56.5), 122 (10.0) [M -H O].
2
4
.5.6. Non-2-en-1-ol (8)
Lindlar’s catalyst (50 mg) was added to a solution of ynol 7 (500 mg, 3.57 mmol) and methanol
(
10 mL). The mixture was stirred for 20 min at room temperature under a H atmosphere. The catalyst
2
was filtered through a short pad of silica and the solvent was evaporated. The product was received
after purification by flash chromatography on silica [pentane/EtOAc (10:1)] as a colorless oil (454 mg,
3
.19 mmol, 90%).
R = 0.3 (pentane/EtOAc 10:1); H-NMR (400 MHz, CDCl ):
1
δ
= 5.63–5.51 (m, 2H), 4.2 (d, J = 6.0
f
3
13
Hz, 2H), 2.07 (q, J = 6.7 Hz, 2H), 1.40–1.23 (m, 8H), 0.88 (t, J = 7.0 Hz, 3H); C-NMR (100 MHz, CDCl3):

Mar. Drugs 2019, 17, 20
11 of 14
δ
= 133.3, 128.3, 58.6, 31.7, 29.6, 28.9, 27.4, 22.6, 14.1; MS (70 eV, EI): m/z (%): 57 (100), 41 (69.6), 55 (50.7),
+
+
4
3 (49.0), 54 (41.0), 67 (39.4), 82 (38.7), 81 (36.0), 68 (34.6), 95 (33.7), 124 (22.7) [M -H O], 142 (0.3) [M ].
2
4
.5.7. Methyl (2E,5Z)-2,5-Dodecadienoate (9)
The allylic alcohol
8 (454 mg, 3.19 mmol) was added to a mixture of tris(dibenzylideneacetone)
dipalladium (83 mg, 0.08 mmol), tricyclohexylphosphine (45 mg, 0.16 mmol) and p-toluenesulfonic
◦
anhydride (1.25 g, 3.8 mmol) in methyl acrylate (16 mL) and stirred for 12 h at 80 C in a special
high-pressure vial under argon. The catalyst was filtered through a short silica column and washed
with diethyl ether. The solvent was evaporated in vacuo and the crude product was purified by flash
chromatography on silica [pentane/EtOAc (30:1)] to the desired product (599.7 mg, 2.85 mmol, 89%) [25].
1
R = 0.4 (pentane/EtOAc 30:1); H-NMR (400 MHz, CDCl ):
δ = 6.97 (dt, J = 15.7, 6.4 Hz, 1H), 5.82
f
3
(
2
dt, J = 15.7, 1.8 Hz, 1H), 5.56-5.34 (m, 2H), 3.73 (s, 3H), 2.88 (dd, J = 6.9, 6.3 Hz, 2H), 2.01 (q, J = 6.6 Hz,
H), 1.38–1.26 (m, 8H), 0.88 (t, J = 7.1 Hz, 3H); 13C-NMR (100 MHz, CDCl3):
δ
= 167.1, 148.1, 133.8,
1
24.9, 121.1, 51.4, 35.1, 32.6, 31.7, 29.3, 28.8, 22.6, 14.1; MS (70 eV, EI): m/z (%): 79 (100), 111 (90.3), 81
+
(70.2), 67 (63.6), 41 (60.0), 210 (52.8) [M ], 55 (50.0), 95 (47.4), 80 (47.3), 100 (45.9).
4
.5.8. (2E,5Z)-2,5-Dodecadienoic Acid (10)
Trimethylsilyl iodide (0.038 mL, 0.268 mmol) was added to a stirred solution of ester
9
(30 mg,
◦
0
.134 mmol) in 0.5 mL CCl and warmed to 50 C for one hour. The mixture was washed with
4
sat. NH Cl solution followed by sat. sodium thiosulfate solution and extracted three times with
4
dichloromethane. The combined organic phases were dried with MgSO , filtered and concentrated in
4
vacuo to get the desired product after purification by flash chromatography on silica [pentane/Et O
2
(
5:1)] as a white solid (15 mg, 0.077 mmol, 58%).
1
R = 0.2 (pentane/EtOAc 5:1); H-NMR (400 MHz, CDCl ):
f
δ = 7.08–7.01(m, 1H), 5.81 (d, J = 15.6
3
Hz, 1H), 5.42–5.26 (m, 2H), 2.94 (dd, J = 6.9, 6.3 Hz, 2H), 2.01 (q, J = 6.9 Hz, 2H), 1.35–1.24 (m, 8H), 0.81
t, J = 7.0 Hz, 3H); 13C-NMR (100 MHz, CDCl3):
= 172.8, 148.8, 133.4, 124.4, 121.5, 33.0, 28.9, 28.8, 28.7,
7.8, 22.6, 14.0; MS (70 eV, EI, trimethylsilyl ester): m/z (%): 169 (100), 253 (42.4), 73 (40.2), 155 (21.9), 75
(
δ
2
+
(19.9), 170 (15.1), 81 (13.5), 254 (8.8), 43 (8.7), 268 (8.0) [M ].
4
.5.9. N-((2E,5Z)-2,5-Dodecadienoyl)homoserine Lactone (11)
L-Homoserine lactone hydrobromide (22 mg, 0.1224 mmol) was dissolved in dry dichloromethane,
followed by the addition of p-dimethylaminopyridine (DMAP) (15 mg, 0.1224 mmol) and acid 10
◦
(
5
24 mg, 0.1224 mmol). EDC
·
HCl (23.5 mg, 0.1224 mmol) was added at 0 C, the solution was stirred for
◦
min at 0 C and for 12 h at room temperature. The reaction mixture was washed one time with H O,
2
sat. NaHCO solution and brine. The organic layers were extracted three times with dichloromethane,
3
dried with Na SO , filtered and the solvent was evaporated. The crude product was purified by flash
2
4
chromatography on silica [pentane/EtOAc (1:1)] to the desired AHL (15 mg, 0.054 mmol, 44%).
1
R = 0.2 (pentane/EtOAc 1:1); H-NMR (600 MHz, CDCl ):
δ = 6.91 (dt, J = 15.4, 6.4 Hz, 1H), 6.06
f
3
(
(
8
6
2
br s, 1H), 5.83 (dt, J = 15.3, 1.7 Hz, 1H), 5.53–5.36 (m, 2H), 4.62 (ddd, J = 11.6, 8.6, 5.6 Hz, 1H), 4.50–4.47
m, 1H), 4.33–4.28 (m, 1H), 2.92–2.87 (m, 3H), 2.20–2.12 (m, 1H), 2.01 (q, J = 7.0 Hz, 2H), 1.39–1.25 (m,
H), 0.88 (t, J = 7.0 Hz, 3H); 13C-NMR (150 MHz, CDCl3):
δ
= 175.5, 166.3, 145.1, 133.8, 125.0, 122.5,
6.2, 49.4, 34.9, 32.6, 31.7, 30.7, 29.3, 28.9, 22.6, 14.1 (Supplementary Figure S2); HRMS (ESI+) m/z:
+
+
+
80.1909 [M + H] , calculated for C H NO 280.1907 [M + H] ; 302.1729 [M + Na] , calculated for
16
26
3
+
C H NO Na 302.1727 [M + Na] .
16
25
3
4
.5.10. (3E,5Z)-3,5-Dodecadienoic Acid (12)
Lithium hydroxide (0.238 mL, 1.5 M) was added to a stirred solution of ester
in THF/MeOH (1:1) and stirred for 3 h at 0 C. The mixture was acidified with 1 M HCl and extracted
9
(50 mg, 0.238 mmol)
◦
three times with dichloromethane. The combined organic phases were dried with Na SO , filtered and
2
4

Mar. Drugs 2019, 17, 20
12 of 14
concentrated in vacuo to get the desired product after purification by flash chromatography on silica
[
pentane/EtOAc (5:1)] as a white solid (25 mg, 0.128 mmol, 54%).
1
R = 0.2 (pentane/EtOAc 5:1); H-NMR (400 MHz, CDCl ):
δ = 6.19–5.98 (m, 2H), 5.72 (dt, J = 15.1,
f
3
7
.1 Hz, 1H), 5.39 (dt, J = 10.8, 7.6 Hz, 1H), 3.10 (d, J = 7.2 Hz, 2H), 2.09 (q, J = 7.6, 7.2 Hz, 2H), 1.43–1.26
13
(
m, 8H), 0.86 (t, J = 6.9 Hz, 3H); C-NMR (100 MHz, CDCl3):
δ = 178.2, 135.4, 134.7, 129.3, 121.3, 37.7,
3
1.7, 29.2, 28.9, 27.8, 22.6, 14.1; MS (70 eV, EI, trimethylsilyl ester): m/z (%): 73 (100), 75 (23.5), 253
+
(15.2), 74 (14.4), 268 (12.2) [M ], 79 (10.9), 150 (10.9), 41 (6.4), 67 (6.4), 224 (6.4).
4
.5.11. N-((3E,5Z)-3,5-Dodecadienoyl)homoserine Lactone (13)
L-Homoserine lactone hydrobromide (21.8 mg, 0.12 mmol) was dissolved in dry dichloromethane.
Triethylamine (0.02 mL, 0.12 mmol) was added to the solution, followed by the addition of acid 12
24.4 mg, 0.12 mmol) and EDC HCl (23 mg, 0.12 mmol). The reaction mixture was stirred for 12 h at
room temperature and washed successively with H O, sat. NaHCO solution and brine. The organic
(
·
2
3
layers were extracted three times with dichloromethane, dried with Na SO , filtered and the solvent
2
4
was evaporated. The crude product was purified by flash chromatography on silica [pentane/EtOAc
(
1:1)] to obtain the desired AHL 13 (32 mg, 0.115 mmol, 92%).
1
R = 0.2 (pentane/EtOAc 1:1); H-NMR (400 MHz, CDCl ):
f
δ = 6.34 (br s, 1H), 6.12–5.93 (m, 2H),
3
5.66–5.51 (m, 2H), 4.51 (ddd, J = 11.5, 8.5, 6.5 Hz, 1H), 4.41–4.36 (m, 1H), 4.2 (ddd, J = 11.0, 9.3, 6.0 Hz,
1
8
4
+
3
H), 3.00 (d, J = 7.3 Hz, 2H), 2.74–2.68 (m, 1H), 2.15–2.04 (m, 1H), 2.00 (q, J = 7.2 Hz, 2H), 1.32–1.20 (m,
H), 0.81 (t, J = 7.0 Hz, 3H); 13C-NMR (100 MHz, CDCl3):
= 175.4, 171.6, 135.8, 135.7, 129.2, 122.0, 66.0,
9.2, 39.9, 32.6, 31.7, 30.1, 29.1, 28.8, 22.5, 14.0 (Supplementary Figure S3); HRMS (ESIs1/z: 280.1909 [M
δ
+
+
+
H] , calculated for C H NO 280.1907 [M + H] ; 302.1729 [M + Na] , calculated for C H NO Na
16 26 3 16 25 3
+
02.1727 [M + Na] .
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/17/1/20/s1,
1
13
Figure S1: Mass spectrum of natural compound B. Figure S2: H NMR and C NMR spectrum of 2E,5Z-C12:2-HSL
1
13
1
13
(
11). Figure S3: H NMR and C NMR spectrum of 3E,5Z-C12:2-HSL (13). Figure S4: H NMR and C NMR
spectrum of 2E,4E-C12:2-HSL (5).
Author Contributions: Conceptualization, L.Z. and S.S.; Data curation, L.Z.; Methodology, L.Z.; Validation, S.S.
and I.W.-D.; Investigation, L.Z. and H.W.; Resources, S.S., L.W., M.P., T.B., B.E. and I.W.-D.; Writing-Original Draft
Preparation, L.Z.; Writing-Review & Editing, S.S.; Visualization, S.S.; Supervision, S.S.; Project Administration,
S.S.; Funding Acquisition, S.S.
Funding: This research was funded by The Deutsche Forschungsgemeinschaft (DFG) in the framework of
TRR/SFB 51 “Roseobacter”.
Acknowledgments: We thank the DFG for funding in the framework of TRR/SFB 51 “Roseobacter”.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to
publish the results.
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