Oxygenated N-Acyl Alanine Methyl Esters (NAMEs) from the Marine Bacterium Roseovarius tolerans EL-164

Article abstract of DOI:10.1021/acs.jnatprod.7b00757

The marine bacterium Roseovarius tolerans EL-164 (Rhodobacteraceae) can produce unique N-acylalanine methyl esters (NAMEs) besides strucutrally related N-acylhomoserine lactones (AHLs), bacterial signaling compounds widespread in the Rhodobacteraceae. The structures of two unprecedented NAMEs carrying a rare terminally oxidized acyl chain are reported here. The compounds (Z)-N-16-hydroxyhexadec-9-enoyl-l-alanine methyl ester (Z9-16-OH-C16:1-NAME, 3) and (Z)-N-15-carboxypentadec-9-enoyl-l-alanine methyl ester (16COOH-C16:1-NAME, 4) were isolated, and the structures were determined by NMR and MS experiments. Both compounds were synthesized to prove assignments and to test their biological activity. Finally, non-natural, structurally related Z9-3-OH-C16:1-NAME (18) was synthesized to investigate the mass spectroscopy of structurally related NAMEs. Compound 3 showed moderate antibacterial activity against microorganisms such as Bacillus, Streptococcus, Micrococcus, or Mucor strains. In contrast to AHLs, quorum-sensing or quorum-quenching activity was not observed.

Full text of DOI:10.1021/acs.jnatprod.7b00757

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Oxygenated N‑Acyl Alanine Methyl Esters (NAMEs) from the Marine
Bacterium Roseovarius tolerans EL-164
Hilke Bruns,† Jennifer Herrmann,‡ Rolf Muller,‡ Hui Wang,§ Irene Wagner Dobler,§
̈
̈
and Stefan Schulz*,†
†Institute of Organic Chemistry, Technische Universitat Braunschweig, Hagenring 30, 38106 Braunschweig, Germany
̈
‡Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Saarland
University, Campus E8.1, 66123 Saarbrucken, Germany
̈
§Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany
S
* Supporting Information
ABSTRACT: The marine bacterium Roseovarius tolerans EL-164
(Rhodobacteraceae) can produce unique N-acylalanine methyl esters
(NAMEs) besides strucutrally related N-acylhomoserine lactones
(AHLs), bacterial signaling compounds widespread in the Rhodo-
bacteraceae. The structures of two unprecedented NAMEs carrying a
rare terminally oxidized acyl chain are reported here. The compounds
(Z)-N-16-hydroxyhexadec-9-enoyl-L-alanine methyl ester (Z9-16-
OH-C16:1-NAME, 3) and (Z)-N-15-carboxypentadec-9-enoyl-L-
alanine methyl ester (16COOH-C16:1-NAME, 4) were isolated,
and the structures were determined by NMR and MS experiments.
Both compounds were synthesized to prove assignments and to test
their biological activity. Finally, non-natural, structurally related Z9-3-
OH-C16:1-NAME (18) was synthesized to investigate the mass spectroscopy of structurally related NAMEs. Compound 3
showed moderate antibacterial activity against microorganisms such as Bacillus, Streptococcus, Micrococcus, or Mucor strains. In
contrast to AHLs, quorum-sensing or quorum-quenching activity was not observed.
Roseovarius tolerans EL-164 (Rhodobacteraceae), isolated from
the hypersaline Ekho Lake (Antarctica),1 is a bacterium of the
Roseobacter group, an important group of marine bacteria.
Roseobacters occur in many different habitats such as the open
water column, algal or plankton surfaces, and algal blooms.2−4
This group of bacteria produces various secondary metabolites
such as the antibiotic tropodithietic acid (TDA),5 antialgal
roseobacticides,6 antioxidants such as methyl 3-hydroxybutyrate
oligomers,7,8 or volatile compounds.9 In addition, roseobacters
can produce a wide range of N-acylhomoserine lactones
(AHLs),7,10−12 which are involved in different quorum-sensing
regulated traits.5,13
Recently, we identified AHL-related compounds from R.
tolerans EL-164, N-acylalanine methyl esters (NAMEs).14
Despite their close structural similarity, differences occurred
in the acyl chains of AHLs and NAMEs in this bacterium. The
major compound was (Z)-N-9-hexadecenoyl-L-alanine methyl
ester (1), accompanied by smaller amounts of saturated and
unsaturated homologues.14 AHLs were also produced; the
major one was (Z)-N-7-tetradecenoyl-L-homoserine lactone
(2). In contrast to AHLs, NAMEs did not show any activity in
quorum-sensing assays,14 but proved to have antialgal and
antibacterial properties.7 During the investigation of the extract
of liquid cultures of this bacterium we observed new NAMEs
that carry additional oxygen atoms in the side chain, while
respective AHLs were absent. Here we report on the
identification and synthesis of these new NAMEs found in
culture medium extracts of R. tolerans EL-164.
RESULTS AND DISCUSSION
■
The secondary metabolites produced by liquid cultures of R.
tolerans EL-164 were collected by extraction via Amberlite
XAD-16 resin as described previously.14 These extracts were
investigated by HPLCMS (Figure 1). The known compounds
Z9-C16:1-NAME (1) and Z7-C14:1-AHL (2) were readily
identified by their ESI mass spectra with masses of m/z 340 and
m/z 310 [M + H]+ and typical ions at m/z 104 (alanine methyl
ester unit) or m/z 102 (homoserine lactone unit), respectively.
An HPLC/HR-MS2 investigation in ESI positive mode (Figure
2) and comparison with a synthetic sample confirmed the
assignment of 1. The mass spectrum is characterized by the loss
of H2O (−18 Da), MeOH (−32 Da), and MeOH/CO (−60
Da) from [M + H]+. The alanine methyl ester unit is indicated
by the ions m/z 104 and m/z 237 [M + H − 103]+, fragments
occurring analogously at m/z 102 and [M + H − 101]+ in
AHLs.15,16
Two additional compounds, A and B in Figure 1, showed [M
+ H]+ ions at m/z 356 and 370, respectively. The HR-MS data
Received: September 4, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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data. The absolute configuration of alanine in the NAMEs has
been previously shown to be L, and the same configuration was
tentatively assigned for A.14 The direction of the optical
rotation of the natural compound was not clearly assignable due
to the low specific rotation value. Based on these data, the
structure of A was therefore (Z)-N-16-hydroxyhexadec-9-enoyl-
L-alanine methyl ester (16OH-C16:1-NAME, 3), a terminally
oxidized analogue of NAME 1.
Compound B could not be isolated in sufficient quality for
NMR investigations. Nevertheless, the HRMS data of B (Figure
2C) showed the presence of a second additional oxygen
compared to 1, with the composition C20H26NO5. The ion m/z
104, characteristic for NAMES, was observed, while instead of
the ion [M + H − 103]+, m/z 249 [M + H − 103 − H2O]+
occurs. This indicated, together with the acyl ion m/z 231
[C16H23O]+, the presence of two double bonds and three O
atoms in the acyl chain. Ions of the NAME- and AHL-typical
loss of H2O and/or MeOH (m/z 352, 338, 320), and MeOH,
CO, and H2O (m/z 292) were also present. We therefore
concluded that this compound is likely (Z)-N-15-carboxypen-
tadec-9-enoyl-L-alanine methyl ester (16COOH-C16:1-NAME,
4), a further oxidized analogue of 1. The earlier retention times
on an RP-HPLC column are consistent with these assignments.
To verify the structures of both A and B, compounds 3 and 4
were synthesized (Scheme 1). 1,7-Heptanediol (5) was
monoprotected with tert-butyldimethylsilyl chloride
(TBDMSCl) and oxidized via Parikh−Doering reaction to
give 6. The aldehyde was coupled with the Wittig salt 8,
obtained from 9-bromononanol (7). Wittig reaction furnished
monoprotected diol 9 in 76% yield with a Z-configured double
bond at C-9. After oxidation with tetrapropylammonium
perruthenate (TPAP) to the acid 10, coupling with L-alanine
methyl ester hydrochloride gave amide 11. Deprotection with
tetrabutylammonium fluoride (TBAF) furnished the desired
compound 3. Because direct oxidation proceeded only in poor
yield, a two-step oxidation process via Parikh−Doering and
subsequent Pinnick oxidations was performed, furnishing acid
4.
The synthetic material proved to be identical in all aspects to
the natural compounds A and B, e.g., in MS2 and MS3
experiments (Supporting Information). The absolute config-
uration of the alanine residue in NAMEs of R. tolerans EL-164
has been previously elucidated to be L.14 The new compounds
3 and 4 are obviously terminally oxidized analogues of the
already known NAME 1. Such oxidized acyl chains have not
been reported for the closely related AHLs that contain an
additional oxygen usually at C-3.10,18−20 Currently we are
analyzing a whole range of bacteria for similar analogues.
Therefore, it seemed plausible to also synthesize a 3-
hydroxyacyl-NAME (18), although this compound is not
known from nature. Knowledge of its mass spectrometric
properties would help in identification. Therefore, 3OH-C16:1-
NAME 18 was synthesized according to Scheme 2.
Figure 1. HPLCMS total ion chromatogram of an extract of R. tolerans
EL-164 showing C16:1-NAME (1) and C14:1-AHL (2), as well as the
unknown NAMEs A and B. The [M + H]+ ion is shown in
parentheses. Analysis was performed on an Agilent Eclipse Plus RP18
column (3.5 μm, 2.1 × 150 mm) with a gradient of H2O, MeCN, and
2% formic acid in ESI positive mode.
of A and its MS2 fragmentation pattern were similar to those of
NAME 1 (Figure 2B) and indicated this compound also to be
an acylated alanine methyl ester. The mass difference of 16 Da
compared to 1 and an additional loss of H2O in the mass
spectrum, indicated by ions m/z 306 [M + H − H2O −
CH3COOH]+, m/z 253 [M + H − 103]+, and m/z 235 [M + H
− 103 − H2O]+, revealed an additional HO group or a keto or
epoxide group instead of the double bond in the acyl side chain.
Compound A (0.8 mg) was then isolated by preparative
1
HPLC. NMR analysis was performed with the help of H, 13C,
DEPT, COSY, HSQC, HMBC, and NOESY experiments. As
1
expected, the H NMR spectrum showed signals of an alanine
methyl ester moiety: an ester methyl group at δ 3.69 (s), a
methine proton adjacent to a carbonyl and a methyl group (δ
4.54), the alanine methyl group (δ 1.33), and an amide proton
(δ 5.97) were observed (Table 1).
Additionally, an acyl chain with a Z-configured double bond
was present. A Δ9-double bond was evident from COSY
correlations along the chain between C-14 and C-20. The Z-
configuration was indicated by the chemical shift of the carbons
next to the double bond, δC 27.1.17 The connectivity of the
chain carbons was elucidated by COSY, HSQC, and HMBC
correlations (Figure 3). The CH2 group at δ 3.57 and 63.0 in
the 13C NMR indicated an additional HO group at the terminal
end of the chain, in accordance with the mass spectrometric
Ethyl-7-bromoheptanoate (12) was transformed into Wittig
salt 13. A Z-selective Wittig reaction with heptanal furnished
ethyl (Z)-tetradec-7-enoate (14). The free acid 15, obtained by
alkaline hydrolysis, was used to acylate Meldrum’s acid to
furnish 16. The crude product was directly coupled with L-
alanine methyl ester hydrochloride, furnishing (Z)-N-3-
oxohexadec-9-enoyl-L-alanine methyl ester (17). Final reduc-
tion with sodium borohydride delivered the target compound,
(Z)-N-3-hydroxyhexadec-9-enoyl-L-alanine methyl ester (3OH-
C16:1-NAME, 18).
B
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Figure 2. HR-MS/MS mass spectrum of C16:1-NAME (1) in ESI positive mode with m/z 340 as the precursor ion (a), of compound A, precursor
m/z 356 (b), compound B, precursor m/z 370 (c), and 3-OH-C16:1-NAME (18), precursor m/z 356 (d).
Table 1. NMR Data for Compound A in DMSO-d6
position
δC, type
δH, mult. (J in Hz)
1
52.5, CH3
173.8, C
3.69, s
2
Figure 3. Important COSY, HMBC, and NOESY correlations of
compound A.
3
47.8, CH
4.54, quin (7.2)
1.33, d (7.2)
4
18.6, CH3
172.7, C
5
The same ions were observed in the ESI mass spectra of 3
and 18. The only obvious difference was the intensity of the
ions m/z 324 and 338 in the HRESIMS2 spectra (Figure 2).
Low-resolution ESIMS2 spectra (Figure S3, Supporting
Information) also showed enhanced intensity of m/z 338,
explainable by the easier loss of H2O from C-3 compared to C-
16. Although 3 did show more fragmentation in MS3 compared
to 18 (Figure S3), it seems questionable whether the
differences observed are sufficient to differentiate these
compounds in complex samples. Compounds 3 and 18 can
also be analyzed by GCMS after trimethylsilylation. The
respective mass spectra are clearly different (Figure S4). The
derivative of 18 indicates the location of the OH substituent by
the ion m/z 246. No related ion is observable in the spectrum
of trimethylsilylated 3.
6
36.6, CH2
25.55, CH2
29.01, CH2
29.03, CH2
29.20, CH2
29.67, CH2
27.1, CH2
129.8, CH
129.9, CH
27.1, CH2
29.61, CH2
29.18, CH2
25.61, CH2
32.8, CH2
63.0, CH2
2.14, t (7.7)
1.59−1.54, m
1.24−1.21, m
1.30−1.25, m
1.24−1.21, m
1.30−1.25, m
1.97−1.92, m
5.30−5.25, m
5.30−5.25, m
1.97−1.92, m
1.30−1.25, m
1.24−1.21, m
1.30−1.25, m
1.53−1.48, m
3.57, t (6.7)
5.97, d (6.9)
7
8
9
10
11
12
13
14
15
16
17
18
19
20
NH
Moderate antibacterial activity against TolC-deficient E. coli
(minimum inhibitory concentration, MIC 8−16 μg/mL),
Micrococcus luteus DSM-1790 (MIC 32−64 μg/mL), Staph-
ylococcus aureus Newman (MIC 32−64 μg/mL), and Mucor
C
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a
Scheme 1. Synthesis of 3 and 4 Starting from 1,7-Heptanediol 5 and Bromo Alcohol 7
a
TBDMSCl: tert-butyldimethylsilyl chloride, EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, TBAF: tetrabutylammonium fluoride.
hiemalis DSM-2656 (MIC 32−64 μg/mL) was observed for 3.
The synthetic compounds 17 and 18 also showed moderate
antibacterial activity against Bacillus subtilis DSM10 (MIC 16
and 128 μg/mL), Staphylococcus carnosus DSM20501 (MIC 8
and 16 μg/mL), Micrococcus luteus DSM-1790 (MIC 16 and 8
μg/mL), and E. coli TolC (MIC 16 μg/mL for both). Similar to
the nonoxidized parent compound 1, NAME 3 did not induce
or suppress quorum sensing signaling in reporter strains for
long-chain and short-chain AHLs.14 If the NAMEs indeed
function as signaling compounds, it seems logical that no cross-
activity with AHLs is observable. The function of NAMEs is
currently being investigated by us. In summary, we identified
unique NAMEs carrying terminally oxidized acyl chains and
developed a straightforward approach to their synthesis.
The NAMEs 3 and 4 comprise new natural products. They
resemble structurally not only AHLs, important signaling
compounds, but also acylated amino acids (AAs) identified
from various bacteria.21−24 AAs are known from the amino
acids tyrosine, tryptophan, arginine, or phenylalanine and carry
in all cases nonoxidized saturated and unsaturated acyl chains.
Acyl chains oxidized at positions other than C-3 are not known
from either AHLs or AAs. Recently, AHLs have been detected
that obviously carry additional O along the chain, but their
exact structures were not determined.25 Terminally oxidized
fatty acids occur in plant materials, such as suberin26 or cutin,27
but are rare in microorganisms.28
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measuired with an Anton-Paar MD 150 instrument at 20 °C with a
100 mm path length. UV spectra were obtained with a Varian Cary
100 Bio spectrometer. IR spectra were obtained with a Bruker Tensor
27 ATR spectrometer. NMR spectra were obtained with Bruker DRX-
400, AV III-400, or AV II-600 spectrometers. The spectra were
1
referenced to internal tetramethylsilane (δH 0.00 ppm) for H NMR
and to CHCl3 (δ 77.01 ppm) for 13C NMR. Chemical shifts are given
in ppm; coupling constants J, in Hz.
For GC/MS an HP6890 GC system connected to an HP5973 MSD
(Hewlett-Packard) fitted with a BPX-5 fused silica cap was used:
column (25 m, 0.22 mm i.d., 0.25 mm film, SGE Inc.) or Zebron ZB-5
MSi column (30 m × 0.25 mm i.d., 0.25 μm film, Phenomenex) or
HP-5 MS fused silica capillary column (30 m, 0.25 mm i.d., 0.22 mm
film, Agilent); conditions: inlet pressure 97.0 kPa He, purge flow 45.5
mL/min; injection volume 1 μL; injector 250 °C, transfer line 300 °C,
D
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Antarctica.1 This strain has been sequenced with the GenBank
accession number LGVV0000000029 and is in the strain collection of
the Helmholtz Center for Infection Research, Braunschweig, Germany.
Precultures were routinely grown in marine broth medium (MB, Carl
Roth) in Erlenmeyer flasks at 28 °C on a rotary shaker at 150 rpm.
Erlenmeyer flasks (500 mL) containing 100 mL of MB were
inoculated with 2% preculture, and 2% of precleaned Amberlite
XAD-16 (Sigma-Aldrich) was added, which was activated before by
washing with 30 mL of MeOH and 3 × 30 mL of distilled H2O. After
growth of the culture for 3−5 days, the resin was filtered off and
extracted with 3 × 50 mL of CH2Cl2/H2O 3:1 (v/v). The two phases
were separated, the organic phase was dried (MgSO4), and the solvent
was evaporated under reduced pressure. The extract was concentrated
at 60 °C under N2 to a volume of ca. 500 μL. For HPLC analysis, 400
μL of the extract was evaporated to dryness and dissolved in 300 μL of
MeCN. For isolation and purification of oxygenated NAMEs 4 L of
culture was prepared as described above, which gave 91 mg of dry
extract.
a
Scheme 2. Synthesis of 3-OH-C16:1-NAME 18
HPLC/MS of Extract. The dry extract was dissolved in MeCN/
H2O (1:1) and filtered. An Agilent Eclipse Plus C18 column (3.5 μm,
2.1 × 150 mm) was used with a flow of 250 μL/min and injection
volume of 10 μL. Gradient elution was performed using 92.5% H2O
(B), 2.5% MeCN (C), and 5% MeCN containing 2% formic acid (D)
from 0 to 1.5 min, 2.5% B, 92.5% C, and 5% D from 8 to 15 min, and
reequilibration from 17 to 22 min to 92.5% B, 2.5% C, and 5% D.
Isolation and Purification. The extract was dissolved in H2O and
submitted to RP18 column chromatography with MeCN/H2O (2:1,
v/v) as eluents. Fractions were pooled after detection of compounds
via HPLC/MS. Subsequent evaporation of MeCN under reduced
pressure and lyophilization gave 1.5 mg of impure 16COOH-C16:1-
NAME (4) and 0.8 mg of pure 16OH-C16:1-NAME (3). The purity
was determined by LC/MS. Because the fraction of NAME 4 resisted
further attempts of purification, only the fraction containing NAME 3
was submitted to NMR spectroscopic analysis.
a
DMAP: 4-(N,N-dimethylamino)pyridine, DCC: dicyclohexylcarbo-
diimide, L-AME HCl: L-alanine methyl ester hydrochloride.
electron energy 70 eV. The gas chromatograph was programmed as
follows: 50 °C (5 min isothermal), increasing at 10 °C/min to 320 °C,
and operated in split mode (35:1), carrier gas (He) 1.2 mL/min.
Alternatively, an Agilent GC 7890A system connected to a 5975C
MSD (Agilent) fitted with an HP-5 MS fused silica capillary column
(30 m, 0.25 mm i.d., 0.22 mm film, Agilent) was used. Conditions:
inlet pressure 67.5 kPa He, purge flow 24.2 mL/min. The other data
were identical to the first system. Gas chromatographic retention
indices (I) were determined from a homologous series of n-alkanes
(C8−C33). Acids and alcohols were derivatized with N-methyl-N-
(trimethylsilyl)trifluoroacetamide (MSTFA) prior to injection. To a
60 μL sample in a glass vial dissolved in CH2Cl2 was added 20 μL of
MSTFA, and the vial was closed and heated to 60 °C for 1 h. The
solvent and excess MSTFA were evaporated in a stream of N2, and the
sample was taken up in CH2Cl2.
LC/MS data were acquired on a Thermo Fisher LTQ XL, equipped
with an Accela pump, autosampler, and PDA detector. Only LC/MS
grade eluents were used. Mass spectra were obtained in ESI positive
mode. The temperature of the ion source was 40 °C, and the capillary
temperature was 275 °C. The sheath and auxiliary gas flows were 15
and 10 mL/min, respectively. MS2 and MS3 analyses were carried out
in CID mode with a normalized collision energy of 35%. The
activation Q was 0.250 and activation time was 30 ms. Spectra were
analyzed with Thermo Xcalibur 2.2 software. HRESIMS were
performed with an LTQ Orbitrap Velos in MeOH acidified with 1%
formic acid. The spray voltage was set to 1.8 to 2.3 kV, and the flow
rate was 1 μL/min. HRMS/MS data were obtained via CID and a
collision energy of 25−30.
Syntheses. (9-Hydroxynonyl)triphenylphosphonium Bromide
(8). The reaction was performed under dry conditions. A solution of
9-bromononan-1-ol (7) (1.00 g, 4.48 mmol, 1.0 equiv) and Ph3P (1.29
g, 4.93 mmol, 1.1 equiv) in MeCN (8 mL) was heated to reflux for 48
h.32 After evaporation of MeCN under reduced pressure, the crude
product was dissolved in CH2Cl2 and purified by flash column
chromatography (SiO2, CH2Cl2/MeOH, 15:1). Product 8 was
obtained as a yellowish oil (2.13 g, 4.39 mmol, 98%): Rf (CH2Cl2/
1
MeOH, 15:1) 0.16; H NMR (CDCl3, 400 MHz) δ 7.85−7.80 (m, 9
arom. H), 7.75−7.71 (m, 6 arom. H), 3.68−3.62 (m, 2H, CH2), 3.57
(t, 2H, J = 6.6 Hz, CH2OH), 2.73 (s br, 1H, OH), 1.67−1.59 (m, 4H,
2 × CH2), 1.49 (quin, 2H, J = 6.9 Hz, CH2), 1.28−1.21 (m, 8H, 4 ×
CH2); 13C NMR (CDCl3, 100 MHz) δ 134.9 (d, J = 2.9, 3 Ar), 133.3
(d, J = 10.0 Hz, 6 Ar), 103.3 (d, J = 12.5 Hz, 6 Ar), 118.0 (d, J = 85.9
Hz, 3 Ar), 62.1 (CH2OH), 32.3 (CH2CH2OH), 30.0 (d, J = 15.6 Hz,
CH2), 28.7 (d, J = 10.2 Hz, CH2), 28.5 (CH2), 25.3 (CH2), 22.7
(CH2), 22.3 (CH2), 22.2 (CH2); 31P NMR (CDCl3, 162 MHz) 24.66
(s, PPh3).
7-((tert-Butyldimethylsilyl)oxy)heptan-1-ol. 1,7-Heptanediol (5)
(2.66 g, 20.10 mmol, 1 equiv) and imidazole (1.50 g, 22.11 mmol,
1.1 equiv) were dissolved in dry CH2Cl2 (140 mL), cooled to 0 °C,
and stirred for 15 min at 0 °C under a nitrogen atmosphere. Slowly
TBDMSCl (3.33 g, 22.11 mmol, 1.1 equiv) was added dropwise over a
period of 10 min. The solution was allowed to warm to rt and was
stirred for 3 days. H2O (80 mL) was added, the phases were separated,
and the H2O phase was extracted with CH2Cl2 (3 × 30 mL). The
combined organic layers were washed with saturated NaCl solution
and dried (MgSO4), and the solvent was evaporated under reduced
pressure.33,34 Column chromatography (SiO2, pentane/EtOAc, 4:1)
yielded the monoprotected alcohol (4.34 g, 17.61 mmol, 88%) as a
yellowish liquid: Rf (SiO2, pentane/EtOAc, 4:1) 0.29; GC (HP-5 MS)
I 1572; IR (ATR) νmax 3335 (br), 2929 (m), 2857 (m), 1468 (m),
1387 (w), 1361 (w), 1253 (m), 1096 (s), 1058 (m), 1006 (m), 833
(s), 773 (s), 771 (m), 661 (m) cm−1; 1H NMR (CDCl3, 400 MHz) δ
3.58 (t, 2H, J = 6.2 Hz, SiOCH2), 3.55 (t, 2H, J = 6.1 Hz, CH2OH),
1.81 (s br, 1H, OH), 1.55−1.44 (m, 8H, 4 × CH2), 1.35−1.26 (m, 2H,
For TLC, 0.20 mm Macherey-Nagel silica gel plates (Polygram SIL
G/UV254) were used. Column chromatography (CC): Merck silica
gel 60 (0.040−0.063 mm) using standard flash chromatographic
methods.
Chemicals were purchased from Sigma-Aldrich Chemie GmbH or
from Acros Organics and 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 N2 in vacuum-heated flasks with dried solvents.
Strains, Culture Conditions, and Extraction. Roseovarius
tolerans EL-164 was collected from hypersaline Ekho Lake in
E
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CH2), 0.85 (s, 9H, 3 × CH3), 0.00 (s, 6H, 2 × CH3); 13C NMR
(CDCl3, 100 MHz) δ 63.2 (CH2OH), 62.9 (SiOCH2), 32.8 (CH2),
32.7 (CH2), 29.2 (CH2), 26.0 (3 × CH3), 25.8 (CH2), 25.7 (CH2),
18.3 (C), −5.3 (2 × CH3); EIMS m/z 246 [M]+ (<1), 189 (5), 171
(6), 143 (13), 115 (23), 105 (48), 97 (65), 75 (98), 73 (39), 69 (29),
55 (100), 41 (32).
(w), 1361 (w), 1252 (m), 1099 (s),1005 (w), 937 (w), 834 (s), 775
1
(s), 723 (w), 662 (m) cm−1; H NMR (CDCl3, 400 MHz) δ 5.33−
5.25 (m, 2H, HCCH), 3.55 (t, 2H, J = 6.6 Hz, SiOCH2), 2.29 (t,
2H, J = 7.5 Hz, CH2COOH), 2.00−1.94 (m, 4H, CH2HCCHCH2),
1.58 (quin, 2H, J = 7.4 Hz, CH2CH2COOH), 1.50−1.43 (m, 2H,
CH2), 1.33−1.24 (m, 14H, 7 × CH2), 0.84 (s, 9H, 3 × CH3), 0.00 (s,
6H, 2 × CH3); 13C NMR (CDCl3, 100 MHz) δ 80.0 (C), 130.0
(HCCH), 129.8 (HCCH), 63.3 (SiOCH2), 34.1 (CH2), 32.8 (2
× CH2), 29.7 (CH2), 29.7 (CH2), 29.1 (3 × CH2), 29.0 (CH2), 27.1
(2 × CH2), 26.0 (3 × CH3), 24.7 (CH2), 18.4 (C), −5.3 (2 × CH3).
(Z)-(16-((tert-Butyldimethylsilyl)oxy)hexadec-9-enoyl)-L-alanine
Methyl Ester (11). Acid 10 (0.20 g, 0.52 mmol, 1 equiv) was dissolved
in dry CH2Cl2 (10 mL) under a nitrogen atmosphere. After addition of
4-(N,N-dimethylamino)pyridine (DMAP, 0.06 g, 0.52 mmol, 1 equiv),
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.1 g, 0.68
mmol, 1.3 equiv) was added at 0 °C, and the solution was stirred for 1
h at 0 °C, allowed to warm to rt, and stirred for an additional 1 h. A
solution of L-alanine methyl ester·HCl (0.09 g, 0.68 mmol, 1.3 equiv)
in dry CH2Cl2 (10 mL) was prepared, and NEt3 (0.07 g, 0.68 mmol,
1.3 equiv) was added. This solution was added dropwise to the
solution containing 10 and was stirred overnight at rt. The solution
was washed with 1 M HCl (10 mL), NaHCO3 (10 mL), and H2O (10
mL), the phases were separated, and the organic phase was dried with
MgSO4.39−41 Product 11 (0.12 g, 0.26 mmol, 49%) was obtained by
column chromatography (SiO2, pentane/EtOAc, 4:1) as a colorless
liquid: Rf (pentane/EtOAc, 4:1) 0.4; GC (HP-5 MS) I 3087; [α]D20
−0.98 0.05 (c 0.0215, CH2Cl2); IR (ATR) νmax 3292 (br), 3002 (w),
2928 (s), 2855 (s), 1748 (m), 1649 (s), 1539 (m), 1459 (m), 1382
(w), 1360 (w), 1253 (m), 1207 (m), 1162 (m), 1098 (s), 1058 (m),
1005 (w), 990(w), 937 (w), 919 (w), 835 (s), 775 (s),732 (m), 661
7-((tert-Butyldimethylsilyl)oxy)heptanal (6). The reaction was
performed under a nitrogen atmosphere. 7-((tert-Butyldimethylsilyl)-
oxy)heptan-1-ol (3.68 g, 14.81 mmol, 1 equiv) was dissolved in dry
CH2Cl2 (120 mL), and NEt3 (20.53 mL, 148.10 mmol, 10 equiv) was
added. A solution of pyridine·SO3 (7.07 g, 44.43 mmol, 3 equiv),
which was dissolved in DMSO (45 mL) and stirred for 15 min at rt,
was then added to the alcohol at 0 °C. The solution was stirred for 30
min at rt, and H2O (100 mL) was added. The phases were separated,
and the H2O phase was extracted with CH2Cl2 (3 × 30 mL). The
combined organic layers were washed with saturated NaCl solution
and dried with MgSO4.35 After evaporation of the solvents under
reduced pressure column chromatography (SiO2, pentane/EtOAc,
17.1) gave aldehyde 6 (3.51 g, 14.35 mmol, 97%) as a yellowish liquid:
Rf (SiO2, pentane/EtOAc, 17:1) 0.36; GC (HP-5 MS) I 1506; UV/vis
(CH2Cl2) λmax (log ε) 227 (2.29), 222 (2.08) nm; IR (ATR) νmax 2930
(s), 2857 (m), 1710 (s), 1467 (m), 1411 (w), 1389 (w), 1361 (w),
1253 (s), 1097 (s), 1005 (w), 937 (m), 833 (s), 774 (s), 661 (m)
1
cm−1; H NMR (CDCl3, 400 MHz) δ 9.72 (t, 1H, J = 1.8 Hz, HC
O), 3.55 (t, J = 6.5 Hz, 2H, SiOCH2), 2.38 (dt, 2H, J = 7.4 Hz, 1.9 Hz,
CH2HCO), 1.63−1.56 (m, 4H, 2 × CH2), 1.51−1.44 (m, 2H,
CH2), 1.32−1.29 (m, 2H, CH2), 0.85 (s, 9H, 3 × CH3), 0.00 (s, 6H, 2
× CH3); 13C NMR (CDCl3, 100 MHz) δ 202.7 (HCO), 63.0
(CH2), 43.8 (CH2), 32.6 (CH2), 28.9 (CH2), 25.9 (3 × CH3), 25.6
(CH2), 22.0 (CH2), 18.3 (C), −5.3 (2 × CH3); MS (70 eV, EI m/z
244 (<1, [M]+), 187 (5), 173 (4), 157 (12), 131 (19), 115 (17), 105
(27), 101 (22), 95 (48), 75 (100), 73 (32), 67 (14), 59 (19), 57 (17),
44 (20), 41 (29).
1
(m), 539 (w) cm−1; H NMR (CDCl3, 400 MHz) δ 5.97 (d, 1H, J =
6.8 Hz, NH), 5.31−5.28 (m, 2H, HCCH), 4.56 (quin, 1H, J = 7.2
Hz, CHCH3), 3.71 (s, 3H, OCH3), 3.55 (t, 2H, J = 6.6 Hz, SiOCH2),
2.16 (t, 2H, J = 7.6 Hz, CH2CO), 2.00−1.93 (m, 4H, CH2HC
CHCH2), 1.62−1.55 (m, 2H, CH2), 1.51−1.43 (m, 2H, CH2), 1.36 (d,
3H, J = 7.2 Hz, CHCH3), 1.31−1.24 (m, 14H, 7 × CH2), 0.85 (s, 9H,
3 × CH3), 0.00 (s, 6H, 2 × CH3); 13C NMR (CDCl3, 100 MHz) δ
173.7 (C), 172.6 (C), 129.9 (HCCH), 129.8 (HCCH), 63.3
(SiOCH2), 52.4 (OCH3), 47.8 (CHCH3), 36.5 (CH2), 32.8 (CH2),
29.73 (CH2), 29.68 (CH2), 29.23 (CH2), 29.18 (CH2), 29.11 (CH2),
29.09 (CH2), 27.2 (CH2HCCHCH2), 26.0 (3 × CH3), 25.7 (CH2),
25.5 (CH2), 18.6 (C), 18.4 (CH3), −5.3 (2 × CH3); EIMS m/z 469
[M]+ (6), 454 (4), 413 (37), 412 (100), 352 (6), 308 (5), 272 (2),
158 (2), 145 (4), 104 (14), 75 (21), 55 (7), 44 (17).
(Z)-16-((tert-Butyldimethylsilyl)oxy)hexadec-9-en-1-ol (9). Wittig
salt 8 (5.30 g, 10.91 mmol, 1.05 equiv) was dissolved in dry THF (120
mL), and NaHDMS was added dropwise (21.82 mL, 1 M in THF,
21.82 mmol, 2.1 equiv) at 0 °C under a nitrogen atmosphere. The
solution was stirred for 45 min at rt, and a strong orange color evolved.
The solution was cooled to −78 °C, and aldehyde 6 (2.54 g, 10.39
mmol, 1.0 equiv) was added slowly. After stirring for 1 h at −78 °C the
temperature was allowed to rise to rt and ice cold pentane (300 mL)
was added.36,37 The formed Ph3PO was filtered off, and after
evaporation of two-thirds of the solvent SiO2 was added to form a
slurry. Column chromatography with gradient elution (SiO2, pentane/
EtOAc, 10:1, 5:1) furnished 9 (2.93 g, 7.90 mmol, 76%) as a colorless
oil: Rf (pentane/EtOAc, 10:1) 0.4; GC (HP-5 MS) I 2463; IR (ATR)
νmax 3339 (br), 3005 (w), 2927 (s), 2855 (s), 1463 (m), 1438 (w),
1387 (w), 1361 (w), 1253 (m), 1185 (m), 1098 (s), 1006 (m), 938
(Z)-(16-Hydroxyhexadec-9-enoyl)-L-alanine Methyl Ester, 16OH-
C16:1-NAME (3). The educt 11 (0.17 g, 0.35 mmol, 1 equiv) was
dissolved in dry THF (20 mL) and cooled to 0 °C. TBAF was added
slowly (1 M in THF, 0.53 mL, 1.5 equiv), and the solution was stirred
for 3 h at rt. Saturated NH4Cl solution (20 mL) was added, the phases
were separated, and the H2O phase was extracted with CH2Cl2 (3 ×
20 mL). The combined organic layers were washed with saturated
NaCl solution and dried with MgSO4, and the solvent was evaporated
under reduced pressure.42 Column chromatography (RP18, MeCN/
H2O, 3:1) afforded product 3 (0.07 g, 0.21 mmol, 61%) as a colorless
1
(w), 834 (s), 774 (s), 722 (m), 695 (m), 662 (m), 541 (s) cm−1; H
NMR (CDCl3, 400 MHz) δ 5.34−5.25 (m, 2H, HCCH), 3.59 (t,
2H, J = 6.7 Hz, SiOCH2), 3.55 (t, 2H, J = 6.6 Hz, CH2OH), 2.02−1.94
(m, 4H, CH2HCCHCH2), 1.55−1.43 (m, 4H, 2 × CH2), 1.33−1.23
(m, 16H, 8 × CH2), 0.85 (s, 9H, 3 × CH3), 0.00 (s, 6H, 2 × CH3);
13C NMR (CDCl3, 100 MHz) δ 129.9 (CH), 129.8 (CH), 63.3
(CH2), 63.1 (CH2), 32.9 (CH2), 32.8 (CH2), 29.7 (CH2), 29.7 (CH2),
29.5 (CH2), 29.4 (CH2), 29.2 (CH2), 29.1 (CH2), 27.2 (CH2), 27.2
(CH2), 26.0 (3 × CH3), 25.7 (CH2), 25.7 (CH2), 18.4 (C), −5.3 (2 ×
CH3); EIMS m/z 370 [M]+ (<1), 313 (10), 295 (2), 221 (2), 151 (6),
137 (14), 123 (29), 109 (54), 95 (79), 83 (54), 81 (77), 75 (100), 69
(58), 67 (60), 55 (61), 41 (34).
(Z)-16-((tert-Butyldimethylsilyl)oxy)hexadec-9-enoic Acid (10).
To a solution of alcohol 9 (2.93 g, 7.90 mmol, 1.0 equiv), N-
methylmorpholin-N-oxide (NMO, 9.23 g, 79.0 mmol, 10.0 equiv), and
H2O (1.44 mL, 79.0 mmol, 10 equiv) in MeCN (100 mL) was added
TPAP (0.28 g, 0.79 mmol, 0.1 equiv). The solution was stirred
overnight at rt, and the solvent was evaporated under reduced
pressure.38 Acid 10 (2.16 g, 5.61 mmol, 71%) was obtained after
column chromatography (SiO2, pentane/EtOAc + 1% AcOH, 10:1) as
a colorless liquid: Rf (pentane/EtOAc + 1% AcOH, 10:1) 0.36; IR
(ATR) νmax 3005 (w), 2927 (s), 2855 (s), 1710 (s), 1463 (m), 1412
liquid: Rf (MeCN/H2O, 3:1) 0.36; [α]2D0 −1.15
0.39 (c 0.0026,
CH2Cl2); IR (ATR) νmax 3282 (br), 3002 (w), 2927 (s), 2855 (s),
1746 (s), 1651 (s), 1545 (m), 1456 (m), 1379 (w), 1263 (m), 1209
(m), 1163 (m), 1057 (m), 724 (w), 553 (w), 532 (w) cm−1; 1H NMR
(CDCl3, 400 MHz) δ 6.07 (d, 1H, J = 5.3 Hz, NH), 5.35−5.33 (m,
2H, HCCH), 4.61 (quin, 1H, J = 7.2 Hz, CHCH3), 3.75 (s, 3H,
OCH3), 3.64 (t, 2H, J = 6.7 Hz, CH2OH), 2.21 (t, 2H, J = 7.6 Hz,
CH2CO), 2.04−1.99 (m, 4H, CH2HCCHCH2), 1.66−1.61 (m,
2H, CH2), 1.60−1.55 (m, 2H, CH2), 1.40 (d, 3H, J = 7.2 Hz,
CHCH3), 1.38−1.29 (m, 14H, 7 × CH2); 13C NMR (CDCl3, 100
MHz) δ 173.8 (CO), 172.7 (CO), 129.9 (HCCH), 129.8
(HCCH), 63.0 (CH2OH), 52.5 (OCH3), 47.8 (CHCH3), 36.5
(CH2), 32.8 (CH2), 29.64 (CH2), 29.59 (CH2), 29.18 (CH2), 29.16
(CH2), 29.01 (CH2), 28.99 (CH2), 27.1 (CH2HCCHCH2), 25.6
(CH2), 25.5 (CH2), 18.6 (CH3); HRESIMS m/z 356.27967 (calcd for
C20H38NO4, 356.27954).
F
DOI: 10.1021/acs.jnatprod.7b00757
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(Z)-(15-Carboxypentadec-9-enoyl)-L-alanine Methyl Ester,
16COOH-C16:1-NAME (4). The oxidation of the alcohol 3 (17 mg,
48 mmol, 1 equiv) to the aldehyde was performed as described for 6.
The aldehyde (16 mg, 51 mmol, 1 equiv) obtained was directly used in
the following reaction. The aldehyde was dissolved in a t-BuOH/THF
mixture (1.2 mL/1 mL), and 2-methyl-2-butene was added. A solution
of NaClO2 (32 mg, 356 mmol, 7 equiv) and NaH2PO4 (63 mg, 458
mmol, 9 equiv) in H2O (0.4 mL) was added dropwise, resulting in a
yellowish color of the solution. After stirring at rt for 3 h, a saturated
NaCl solution was added and the phases were separated. The H2O
phase was extracted with CH2Cl2 (3 × 2 mL), and the combined
organic layers were washed with a saturated NaCl solution. After
drying with MgSO4, the solvent was evaporated. A saturated NaHCO3
solution was added to adjust the pH to 9, and the solution was
extracted with diethyl ether (2 × 10 mL). After phase separation, 2 N
HCl was added to lower the pH to 2 and the mixture extracted with
diethyl ether (4 × 6 mL) again.43−45 The organic phase was dried with
MgSO4, and the solvent was evaporated under reduced pressure.
Product 4 (7.2 mg, 19 mmol) was obtained in 40% yield: [α]2D0 −4.843
0.32 (c 0.0031, CH2Cl2); IR (ATR) νmax 3290 (br), 3002 (w), 2928
(s), 2856 (s), 1735 (s), 1645 (s), 1545 (m), 1457 (m), 1351 (w), 1213
(m), 1162 (m), 1060 (w), 985 (w), 729 (w), 566 (w), 542 (w) cm−1;
1H NMR (CDCl3, 300 MHz) δ 6.34 (d, 1H, J = 7.1 Hz, NH), 5.40−
5.28 (m, 2H, HCCH), 4.64 (quin, 1H, J = 7.2 Hz, CHCH3), 3.76 (s,
3H, OCH3), 2.34 (t, 2H, J = 7.3 Hz, CH2COOH), 2.22 (t, 2H, J = 7.7
Hz, CH2CO), 2.07−1.97 (m, 4H, CH2HCCHCH2), 1.69−1.58
(m, 6H, 3 × CH2), 1.34 (d, 3H, J = 7.2 Hz, CHCH3), 1.31−1.12 (m,
12H, 6 × CH2); 13C NMR (CDCl3, 75 MHz) δ 177.9 (COOH), 174.1
(CO), 173.3 (CO), 130.1 (HCCH), 129.6 (HCCH), 52.5
(OCH3), 47.9 (CHCH3), 36.4 (CH2), 33.9 (CH2), 29.5 (CH2), 29.2
(CH2), 29.1 (CH2), 29.0 (CH2), 28.8 (CH2), 28.5 (CH2), 27.0
(CH2HCCHCH2), 26.8 (CH2HCCHCH2), 25.6 (CH2), 24.6
(CH2), 18.5 (CH3); HRESIMS m/z 370.25921 (calcd for C20H36NO5,
370.25880).
(6-Carboethoxyheptyl)triphenylphosphonium Bromide (13). The
Wittig salt was prepared as described for 8 from ethyl 7-
bromoheptanoate (2.47 mL, 12.65 mmol, 1.05 equiv) and PPh3
(3.15 g, 12.01 mmol, 1.05 equiv). The product 13 (5.56 g, 11.13
mmol, 88%) was obtained as a colorless oil: Rf (CH2Cl2/MeOH, 25:1)
0.56; 1H NMR (CDCl3, 200 MHz) δ 7.84−763 (m, 15H, Ar), 4.00 (q,
2H, J = 7.1 Hz, CH2CH3), 3.72−3.58 (m, 2H, PCH2), 2.18 (t, 2H, J =
7.2 Hz, CH2COOEt), 1.71−1.54 (m, 4H, 2 × CH2), 1.52−1.41 (m,
2H, CH2), 1.35−1.22 (m, 2H, CH2), 1.15 (t, 3H, J = 7.1 Hz, CH3);
13C NMR (CDCl3, 50 MHz) δ 172.6 (CO), 134.2 (d, 3C, J = 2.9
Hz, 3 × CH Ar), 132.7 (d, 6C, J = 9.9 Hz, 6 × CH Ar), 129.7 (d, 6C, J
= 12.5 Hz, 6 × CH Ar), 117.2 (d, 3C, J = 85.9 Hz, 3 × C Ar), 59.2
(OCH2CH3), 33.1 (CH2), 29.2 (CH2), 28.9 (CH2), 27.5 (CH2), 23.4
(CH2), 22.2 (CH2), 13.1 (OCH2CH3).
After acidification with 2 N HCl to pH 2.0, the solution was extracted
with EtOAc (3 × 50 mL). The organic layers were combined, dried
with MgSO4, and concentrated in vacuo.46 Column chromatography
(SiO2, pentane/EtOAc, 10:1) gave the product 15 (0.99 g, 4.36 mmol,
66%) as a yellowish oil: Rf (SiO2, pentane/EtOAc, 10:1) 0.20; GC
(ZB-5 MSi, MSTFA derivative) I 1857; IR (ATR) νmax 3005 (m),
2924 (s), 2855 (s), 2671 (w), 1707 (s), 1460 (m), 1413 (m), 1278
(m), 1227 (m), 935 (m), 725 (m) cm−1; 1H NMR (CDCl3, 400 MHz)
δ 7.19 (s, 1H, OH), 5.22−5.33 (m, 2H, HCCH), 2.28 (t, 2H, J = 7.7
Hz, CH2COOH), 1.92−1.99 (m, 4H, H2CHCCHCH2), 1.54−1.61
(m, 2H, CH2CH3), 1.13−1.37 (m, 12H, 6 × CH2), 0.81 (t, 3H, J = 7.2
Hz, CH3); 13C NMR (CDCl3, 100 MHz) δ 180.3 (CO), 129.8
(HCCH), 34.1 (CH2COOH), 31.8 (CH2), 29.5 (2 × CH2), 28.9 (2
× CH2), 27.1 (H2CHCCHCH2), 24.6 (CH2), 22.6 (CH2CH3), 14.1
(CH3); EIMS m/z (MSTFA derivative) 298 [M]+ (21), 283 [M+-
CH3] (96), 255 (5), 227 (5), 199 (29), 183 (11), 166 (48), 145 (54),
117 (100), 96 (44), 73 (93), 55 (58), 39 (10).
(Z)-2,2-Dimethyl-5-(tetradec-7-enoyl)-1,3-dioxan-4,6-dione (16).
The acid 15 (0.69 g, 3.05 mmol, 1.00 equiv), DCC (3.35 mL, 1 M in
CH2Cl2, 0.69 mg, 3.53 mmol, 1.1 equiv), Meldrum’s acid (2,2-
dimethyl-1,3-dioxane-4,6-dione, 0.44 g, 3.05 mmol, 1.0 equiv), and
DMAP (0.39 g, 3.20 mmol, 1.05 equiv) were dissolved in 30 mL of dry
CH2Cl2 and stirred at rt overnight in a nitrogen atmosphere. The
mixture was filtered, the solvent was evaporated under reduced
pressure, and the residue was redissolved in EtOAc (30 mL). After
shaking with 2 N HCl (5 × 15 mL) the organic phase was dried with
MgSO4 and concentrated in vacuo.47 The product 16 (0.56 g, 1.59
mmol, 52%) was obtained as a dark yellowish oil and used in the next
reaction without further purification: Rf (SiO2, pentane/EtOAc, 1:1)
0.46.
(Z)-N-(3-Oxohexadec-9-enoyl)alanine Methyl Ester, 3-Oxo-C16:1-
NAME (17). The ester 16 (0.56 g, 1.56 mmol, 1.00 equiv),
triethylamine (0.35 mL, 2.54 mmol, 1.60 equiv), and L-alanine methyl
ester·HCl (0.22 g, 1.59 mmol, 1.0 equiv) were dissolved in 30 mL of
MeCN. The solution was stirred for 2 h at rt and then heated to reflux
for 4 h. The formed precipitate was filtered off, the solvent evaporated
under reduced pressure, and the residue redissolved in EtOAc (30
mL).47 After washing with 2 M HCl (3 × 10 mL) the organic phase
was dried with MgSO4 and dried in vacuo. Column chromatography
(RP18, MeCN/H2O, 5:1) gave 3-Oxo-C16:1-NAME 17 (0.16 g, 0.47
mmol, 30%) as a yellowish oil: Rf (RP18, MeCN/H2O, 5:1) 0.32;
[α]2D0 −0.02
0.07 (c 0.0153, CH2Cl2); IR (ATR) νmax 3309 (w),
3003 (w), 2926 (m), 2855 (m), 1748 (m), 1721 (m), 1648 (m), 1540
(m), 1455 (m), 1376 (w), 3151 (w), 1211 (m), 1163 (m), 1057 (w),
1
987 (w),852 (w), 724 (w), 567 (w) cm−1; H NMR (CDCl3, 400
MHz) δ 7.43 (d, 1H, J = 5.7 Hz, NH), 5.39−5.29 (m, 2H, HCCH),
4.59 (quin, 1H, J = 7.2 Hz, CHCH3), 3.75 (s, 3H, OCH3), 3.41 (s, 2H,
OCCH2CO), 2.53 (t, 2H, CH2CO), 1.97−2.05 (m, 4H,
H2CHCCHCH2), 1.54−1.65 (m, 2H, CH2), 1.43 (d, 3H, J = 7.3
Hz, CHCH3), 1.39−1.22 (m, 12H, 6 × CH2), 0.88 (t, 3H, J = 6.8 Hz,
CH2CH3); 13C NMR (CDCl3, 100 MHz) δ 206.4 (CO), 173.0
(CO), 165.2 (CO), 129.8 (HCCH), 52.5 (OCH3), 48.6 (O
CCH2CO), 48.1 (CHCH3), 43.8 (CH2CO), 31.8 (CH2), 29.6 (2
× CH2), 28.8 (2 × CH2), 27.1 (H2CHCCHCH2), 22.9 (2 × CH2),
18.2 (CHCH3), 14.1 (CH3).
Ethyl (Z)-Tetradec-7-enoate (14). Ester 14 was obtained as
described for the synthesis of 9 from Wittig salt 13 (6.00 g, 12.01
mmol, 1.05 equiv), NaHDMS (12.58 mL, 1 M in THF, 12.58 mmol,
1.1 equiv), and freshly distilled heptanal (1.6 mL, 1.30 g, 11.44 mmol,
1.00 equiv). Column chromatography (SiO2, pentane/EtOAc, 20:1)
furnished pure 14 (2.71 g, 10.64 mmol, 93%) as a yellowish oil: Rf
(pentane/EtOAc, 20:1) 0.40, GC (ZB-5 MSi) I 1800; IR (ATR) νmax
2926 (m), 2856 (m), 1738 (s), 1462 (w), 1373 (w), 1249 (w), 1179
(Z)-N-(3-Hydroxyhexadec-9-enoyl)alanine Methyl Ester, 3-OH-
C16:1-NAME (18). NAME 17 (92 mg, 0.26 mmol, 1.00 equiv) was
dissolved in MeOH (3.1 mL). The ice-cooled solution was adjusted
with 2 M HCl to pH 3, and subsequently NaBH4 (13.2 mg, 0.35
mmol, 1.4 equiv) was added in portions. After adding 2 M HCl to pH
3 the solution was stirred for 30 min at rt.47 The solvent was
evaporated under reduced pressure, and column chromatography
(RP18, MeCN/H2O, 8:1) afforded the diastereomeric product 18
(ratio 49:51) (29 mg, 0.08 mmol, 32%) as a bright yellowish oil: Rf
(RP18, MeCN/H2O, 8:1) 0.32; GC (HP-5 MS, MSTFA derivative) I
1
(m), 1033 (w), 726 (w) cm−1; H NMR (CDCl3, 400 MHz) δ 5.23
(m, 2H, HCCH), 3.99 (q, 2H, J = 7.7 Hz, CH2CH3), 2.17 (t, 2H, J
= 7.7 Hz, CH2COOEt), 1.86−1.93 (m, 4H, H2CHCCHCH2),
1.47−1.56 (m, 2H, CH2CH3), 1.15−1.29 (m, 12H, 6 × CH2), 1.13 (t,
3H, J = 7.2 Hz, CH3), 0.76 (t, 3H, J = 6.9 Hz, CH3); 13C NMR
(CDCl3, 100 MHz) δ 178.7 (CO), 129.8 (HCCH), 60.1
(CH2CH3), 34.3 (CH2COOEt), 31.7 (CH2), 29.5 (2 × CH2), 28.9 (2
× CH2), 27.1 (H2CHCCHCH2), 24.8 (CH2), 22.6 (CH2CH3), 14.1
(2 × CH3); EIMS m/z 254 [M]+ (37), 225 (4), 208 (74), 191 (16),
166 [M+ − C4H8O2] (88), 151 (35), 124 (71), 109 (62), 88
[C4H8O2]+ (94), 70 (56), 55 (100), 39 (28).
2604, 2611; [α]20 −2.72 0.10 (c 0.0104, CH2Cl2); IR (ATR) νmax
D
3312 (m, br), 3082 (w), 3003 (m), 2925 (s), 2854 (m), 1747 (m),
1730 (m), 1641 (s), 1540 (m), 1455 (m), 1374 (w), 1345 (w), 1212
(m), 1161 (m), 1091 (w), 1059 (w), 986 (w), 871 (w), 851 (w), 671
(Z)-Tetradec-7-enoic Acid (15). The ester 14 (1.68 g, 6.60 mmol, 1
equiv) was dissolved in EtOH (25 mL), and KOH (0.57 g, 10.21
mmol, 1.55 equiv) was added. The solution was stirred overnight at rt.
1
(m), 536 (m) cm−1; H NMR (CDCl3, 400 MHz) δ 6.40 (d, 1H, J =
G
DOI: 10.1021/acs.jnatprod.7b00757
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6.6 Hz, NH), 5.39−5.30 (m, 2H, HCCH), 4.60 (dquin, 1H, J = 7.2
Hz, 2.3 Hz, CHCH3), 4.01−3.94 (m, 1H, CHOH), 3.76 (s, 3H,
OCH3), 2.44−2.25 (m, 2H, CH2CO), 2.04−1.99 (m, 4H,
CH2HCCHCH2), 1.73−145 (m, 4H, 2 × CH2), 1.42 (dd, 3H, J
= 7.2 Hz, 1.0 Hz, CHCH3), 1.38−1.26 (m, 12H, 6 × CH2), 0.88 (t,
3H, J = 6.9 Hz, CH3); 13C NMR (CDCl3, 100 MHz) δ 173.7, 173.5
(CO), 172.2, 171.9 (CO), 130.0 (HCCH), 129.6 (HCCH),
68.7, 68.5 (CHOH), 52.6, 52.5 (OCH3), 48.0, 47.9 (CHCH3), 42.9,
42.4 (CH2CO), 36.9, 36.7 (CH2CHOH), 31.7 (CH2), 29.70 (CH2),
29.66 (CH2), 29.2 (CH2), 29.0 (CH2), 27.2 (CH2HCCHCH2),
27.1 (CH2HCCHCH2), 25.40, 25.39 (CH2), 22.6 (CH2), 18.3, 18.1
(CHCH3), 14.1 (CH3); EIMS m/z 355 [M]+ (8), 296 (9), 187 (6),
174 (21), 145 (25), 114 (11), 104 (100), 102 (19), 81 (12), 67 (10),
55 (16), 44 (60); HRESIMS m/z 356.27949 (calcd for C20H38NO4,
356.27954).
Bioassays. All compounds were dissolved in DMSO and were
tested in standard microbroth dilution assays as described earlier.30
Minimum inhibitory concentrations (MICs) were determined in
standard microdilution assays. Cultures of Bacillus subtilis DSM-10,
Staphylococcus carnosus DSM-20501, Micrococcus luteus DSM-1790,
TolC-deficient Escherichia coli, Staphylococcus aureus Newman, and
Mucor hiemalis DSM-2656 in mid log phase were diluted to achieve a
final inoculum of ca. 5 × 105 to 5 × 106 cfu/mL in Mueller-Hinton
broth (1.75% casein hydrolysate, 0.2% beef infusion, 0.15% starch; pH
7.4; used for all bacteria) or Myc medium (1% phytone peptone, 1%
glucose, 50 mM HEPES, pH 7.0; Mucor hiemalis). Serial dilutions of
samples were prepared from THF stock solutions (2 mg/mL) in
sterile 96-well plates, the cell suspension was added, and micro-
organisms were grown for 16−20 h at their optimal growth
temperature at either 30 or 37 °C. Given MIC values are the lowest
concentration of antibiotic at which there was no visible growth.
Quorum quenching and quorum sensing assays were performed as
described previously.14 Briefly, the sensor strain Pseudomonas putida
F117 (pKRC12)31 was grown overnight in Luria−Bertani medium
with addition of gentamycin (20 μg/mL) at 30 °C with shaking (160
rpm), and compound 3 and 4 were added at different concentrations.
The full assay is described in detail by Bruns et al.14
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ASSOCIATED CONTENT
* Supporting Information
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prod.7b00757.
ChemBioChem 2009, 10, 1861−1868.
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HPLC/MS TIC, mass spectra, and NMR spectra of
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(PDF)
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AUTHOR INFORMATION
Corresponding Author
*Phone: +495313917353. Fax: +495313915272. E-mail: stefan.
schulz@tu-bs.de.
■
(26) Company-Arumí, D.; Figueras, M.; Salvado,
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ORCID
Rolf Muller: 0000-0002-1042-5665
̈
Stefan Schulz: 0000-0002-4810-324X
(29) Voget, S.; Bruns, H.; Wagner-Dobler, I.; Schulz, S.; Daniel, R.
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Notes
Genome Announc. 2015, 3, 15.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank the Deutsche Forschungsgemeinschaft for funding
through the Transregional Collaborative Research Centre
“Roseobacter”, TRR51/2 C02.
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