N-Acylated amino acid methyl esters from marine Roseobacter group bacteria

Article abstract of DOI:10.3762/bjoc.14.276

Bacteria of the Roseobacter group (Rhodobacteraceae) are important members of many marine ecosystems. Similar to other Gram-negative bacteria many roseobacters produce N-acylhomoserine lactones (AHLs) for communication by quorum sensing systems. AHLs regulate different traits like cell differentiation or antibiotic production. Related N-acylalanine methyl esters (NAMEs) have been reported as well, but so far only from Roseovarius tolerans EL-164. While screening various roseobacters isolated from macroalgae we encountered four strains, Roseovarius sp. D12_1.68, Loktanella sp. F13, F14 and D3 that produced new derivatives and analogs of NAMEs, namely N-acyl-2-aminobutyric acid methyl esters (NABME), N-acylglycine methyl esters (NAGME), N-acylvaline methyl esters (NAVME), as well as for the first time a methyl-branched NAME, N-(13-methyltetradecanoyl)alanine methyl ester. These compounds were detected by GC–MS analysis, and structural proposals were derived from the mass spectra and by derivatization. Verification of compound structures was performed by synthesis. NABMEs, NAVMEs and NAGMEs are produced in low amounts only, making mass spectrometry the method of choice for their detection. The analysis of both EI and ESI mass spectra revealed fragmentation patterns helpful for the detection of similar compounds derived from other amino acids. Some of these compounds showed antimicrobial activity. The structural similarity of N-acylated amino acid methyl esters and similar lipophilicity to AHLs might indicate a yet unknown function as signalling compounds in the ecology of these bacteria, although their singular occurrence is in strong contrast to the common occurrence of AHLs. Obviously the structural motif is not restricted to Roseovarius spp. and occurs also in other genera.

Full text of DOI:10.3762/bjoc.14.276

N-Acylated amino acid methyl esters from marine
Roseobacter group bacteria
Hilke Bruns1,§, Lisa Ziesche1,§, Nargis Khakin Taniwal1, Laura Wolter2,
Thorsten Brinkhoff2, Jennifer Herrmann3, Rolf Müller3 and Stefan Schulz*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2018, 14, 2964–2973.
1Institute of Organic Chemistry, Technische Universität
Braunschweig, Hagenring 30, 38106 Braunschweig, Germany,
2Institute for Chemistry and Biology of the Marine Environment,
University of Oldenburg, Carl-von-Ossietzky-Straße 9–11, 26111
Oldenburg, Germany and 3Helmholtz Institute for Pharmaceutical
Research Saarland (HIPS), Helmholtz Center for Infection Research
(HZI), Saarland University, Campus E8.1, 66123 Saarbrücken,
Germany
doi:10.3762/bjoc.14.276
Received: 13 September 2018
Accepted: 15 November 2018
Published: 03 December 2018
Associate Editor: J. S. Dickschat
© 2018 Bruns et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
Stefan Schulz* - stefan.schulz@tu-braunschweig.de
* Corresponding author
§ Both authors added equally to this work and are therefore both first
authors.
Keywords:
amino acid derivatives; 2-aminobutyric acid; homoserine lactones;
natural products; quorum sensing
Abstract
Bacteria of the Roseobacter group (Rhodobacteraceae) are important members of many marine ecosystems. Similar to other Gram-
negative bacteria many roseobacters produce N-acylhomoserine lactones (AHLs) for communication by quorum sensing systems.
AHLs regulate different traits like cell differentiation or antibiotic production. Related N-acylalanine methyl esters (NAMEs) have
been reported as well, but so far only from Roseovarius tolerans EL-164. While screening various roseobacters isolated from
macroalgae we encountered four strains, Roseovarius sp. D12_1.68, Loktanella sp. F13, F14 and D3 that produced new derivatives
and analogs of NAMEs, namely N-acyl-2-aminobutyric acid methyl esters (NABME), N-acylglycine methyl esters (NAGME),
N-acylvaline methyl esters (NAVME), as well as for the first time a methyl-branched NAME, N-(13-methyltetradecanoyl)alanine
methyl ester. These compounds were detected by GC–MS analysis, and structural proposals were derived from the mass spectra and
by derivatization. Verification of compound structures was performed by synthesis. NABMEs, NAVMEs and NAGMEs are pro-
duced in low amounts only, making mass spectrometry the method of choice for their detection. The analysis of both EI and ESI
mass spectra revealed fragmentation patterns helpful for the detection of similar compounds derived from other amino acids. Some
of these compounds showed antimicrobial activity. The structural similarity of N-acylated amino acid methyl esters and similar
lipophilicity to AHLs might indicate a yet unknown function as signalling compounds in the ecology of these bacteria, although
their singular occurrence is in strong contrast to the common occurrence of AHLs. Obviously the structural motif is not restricted to
Roseovarius spp. and occurs also in other genera.
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Beilstein J. Org. Chem. 2018, 14, 2964–2973.
Introduction
The identification and structural elucidation of naturally occur- though NAMEs are structurally similar to AHLs by an acyl
ring compounds traditionally requires isolation and NMR inves- chain linked to an amino acid derivative via an amide bond,
tigation as key method to detect novel compounds and new they do not activate AHL receptors in roseobacters [21].
structural classes. Although the advent of NMR spectrometers Instead, they show moderate antialgal activity [21]. In contrast
with high frequencies and cryoprobes with small diameters to AHLs, the acyl chain can also be terminally oxidized [24].
enables experiments to be performed in the μg scale with pure During our analyses of different Roseobacter isolates, we en-
compound, the isolation of the pure material from complex countered several compounds, which mass spectra show simi-
samples as well as the access to the expensive equipment still larities to known NAMEs. These compounds proved to be
pose a considerable challenge to find new compounds. The either new NAMEs or constitute new classes of acylated amino
ongoing quest for new structures also increasingly addresses acid methyl esters, derived from valine (NAVME), glycine
minor components, requiring larger amounts of the producing (NAGME), or 2-aminobutyric acid (NABME). The identifica-
organism, not always readily accessible, to isolate a targeted tion of these compounds will be discussed based on the outlined
compound [1-3].
approach including GC/MS analysis, interpretation of mass
spectra, and verification by synthesis.
An alternative methodology can avoid the laborious isolation
procedure. Direct analysis by mass-spectrometric methods of
natural materials, e.g., extracts, may give enough information to
infer the structure of an unknown compound that is finally
proven by synthesis and comparison with natural material. The
use of GC/EI-MS is especially advantageous because such mass
spectra often reveal key structural features. Furthermore, the
availability of large cross-platform databases useful for derepli-
cation allows focussing on new compounds.
We are interested in natural compounds from Roseobacter
group bacteria, an abundant class of marine bacteria occurring
in diverse habitats with a broad metabolic potential [4-7]. Espe-
cially attached-living roseobacters produce diverse secondary
metabolites, e.g., N-acylhomoserine lactones (AHLs) that the
bacteria use for communication by quorum sensing [8-10].
AHLs are extensively investigated because of the broad know-
ledge on their biosynthesis, the underlying gene organization, as
well as their function in many bacteria [11-13]. In the
Roseobacter group, AHLs are involved, e.g., in antibiotic pro-
duction [9] or cell differentiation [10]. Although many other
bacterial signalling compounds must exist, only few of them
Figure 1: N-Acylhomoserine lactones 1 (Z7-C14:1-AHL) and
N-acylalanine methyl esters 2 (Z9-C16:1-NAME) occurring in
Roseobacter group bacteria.
Results and Discussion
The secondary metabolites released by liquid cultures of various
roseobacters were collected by extraction via Amberlite XAD-
16 resin and analysed by GC/MS. Four of these strains, Roseo-
varius sp. D12_1.68 and Loktanella sp. F13, F14 and D3,
contained low amounts of compounds with similar mass spec-
tra to those of NAMEs [23].
have been characterized so far [14-18]. Such signalling com- Roseovarius sp. D12_1.68
pounds as well as many other unknown metabolites often occur The investigation of an extract by GC/MS (Figure 2) revealed
in small amounts, which renders trace detecting methods like the presence of several NAMEs and AHLs due to their charac-
GC/MS a suitable approach for their detection and structure teristic ions at m/z 104, 145, and 158 and m/z 102, 143, and 156,
elucidation, provided their polarity falls into the analytical respectively [21,23]. Some of these compounds, E and L in
window of the method.
Figure 2, were readily identified by their mass spectra and gas
chromatographic retention indices Inat as known AHLs, contain-
A wide variety of AHLs, e.g., widespread (Z)-N-(tetradec-7- ing saturated C12 and C14 acyl chains (Table 1).
enoyl)homoserine lactone (1, Z7-C-14:1-AHL, Figure 1), have
been identified in roseobacters by these methods [19-22]. A Similarly, compounds C, F, H, I, and M were identified as the
related group of compounds occurring in Roseovarius only, are already known C14:0-, C15:0-, 9-C16:1-, C16:0-, and 9-C17:1-
N-acylalanine methyl esters (NAMEs), e.g., (Z)-N-(hexadec-9- NAMEs. Compounds A, B, and O proved to be not previously
enoyl)alanine methyl ester (2, Z9-C16:1-NAME), the major reported C13:0-, C14:1- and C18:1-NAMEs, assignable by their
NAME produced by Roseovarius tolerans EL 164 [23]. Al- mass spectra and gas chromatographic retention indices. These
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Beilstein J. Org. Chem. 2018, 14, 2964–2973.
Figure 2: Total ion chromatogram (TIC) of an XAD extract of Roseovarius sp. D12_1.68. AHLs, NAMEs and related compounds are assigned by bold
letters (Table 1).
Table 1: Composition of the extracellular metabolites of Roseovarius sp. D12_1.68. [M]+, m/z: molecular and characteristic ions in EI mass spectrum.
Inat: gas chromatographic retention index of natural compounds. HRMS: HR-mass spectral data of [M + H]+ obtained by HPLC/HR–ESI+–MS.
peak
compound
[M]+
m/z
Inat
HRMS [M + H]+
A
B
C
D
E
F
C13:0-NAME
C14:1-NAME
C14:0-NAME
iso-C15:0-NAME
C12:0-AHL
299
311
313
327
283
327
339
339
341
353
355
311
353
367
367
104, 145, 158
104, 145, 158
104, 145, 158
104, 145, 158
102, 143, 156
104, 145, 158
104, 145, 158
104, 145, 158
104, 145, 158
118, 159, 172
118, 159, 172
102, 143, 156
104, 145, 158
132, 173, 181
104, 145, 158
2181
2265
2289
2354
2367
2392
2457
2473
2497
2548
2569
2570
2570
2588
2677
C18H34NO3
C18H36NO3
C19H38NO3
C15:0-NAME
C16:1-NAME
Z9-C16:1-NAME
C16:0-NAME
9-C16:1-NABME
C16:0-NABME
C14:0-AHL
C19H38NO3
C20H38NO3
C20H38NO3
C20H40NO3
C21H40NO3
C21H42NO3
G
H
I
J
K
L
M
N
O
9-C17:1-NAME
9-C16:1-NAVME
11-C18:1-NAME
C21H40NO3
C22H42NO3
C22H42NO3
conclusions were supported by HRMS data obtained by bond location in unsaturated NAMEs seems to be fixed at the
HPLC/MS (Table 1). Localization of the double bond was ω-7 position [8,25].
established via DMDS derivatization as described previously
[23]. Due to the low amounts no double bond position could be Compound D (C19H37NO3) showed a mass spectrum identical
established for C14:1-NAME, while the double bond of C18:1 to C15:0-NAME, albeit the retention index deviated by
NAME was located at C-11. Similar as in AHLs, the double 38 units. A methyl branch at iso- or anteiso-position seemed
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Beilstein J. Org. Chem. 2018, 14, 2964–2973.
likely. Therefore, the theoretical retention index Ic were calcu-
lated for methyl branched C15:0-NAMEs using an empirical
model established in our work group [26] that had successfully
been used for the detection of methyl branched AHLs [27]. The
retention indices were calculated using the formula
with N indicating the number of n carbons in the chain times
hundred, FG as the functional group increment depending on
and Mei as an increment for the methyl branching in different
positions. The increments for Mei are known [26]. The func-
tional group increment was calculated to be 836 + 4 · n using
the retention indices of C14:0, C15:0, and C16:0-NAME.
Therefore, the calculated retention index for iso-C15:0 and
Figure 3: Mass spectrum of natural compound D, N-(13-methyltetra-
decanoyl)alanine methyl ester (iso-C15:0-NAME, 6).
anteiso-C15:0-NAME are Ic = 2352 and Ic = 2365, respectively, zation of the compound that could either be methyl-branched in
while all other methyl branch locations had a lower value. The the acyl chain or might show a different double bond position or
close similarity of Inat = 2354 and Ic = 2352 suggested the configuration.
methyl branch to be located in the iso-position. Consequently,
iso-C15:0-NAME (6) was synthesized as shown in Scheme 1 to The remaining three compounds J, K, and N with the molecu-
verify the structural proposal.
lar composition C21H42NO3, C21H40NO3, and C22H42NO3 de-
termined by HRMS showed related mass spectra with a charac-
11-Bromoundecan-1-ol (3) was converted into the alcohol teristic mass shift compared to NAMEs. Ions m/z 44, 104, 145,
13-methyltetradecan-1-ol (4) with isobutylmagnesium bromide and 158 where shifted, however, to m/z 58, 118, 159, and 172 in
under Li2CuCl4 catalysis according to Mori et al. [28]. After the spectra of J and K (Figure 4a,b).
Jones oxidation, 13-methyltetradecanoic acid (5) was coupled
with -alanine methyl ester hydrochloride to deliver the desired These ions can be explained by an additional CH2 group in the
product 6. The mass spectra and retention indices of the natural alanine part, leading to a 2-aminobutyric acid fragment in these
and synthetic samples were identical, proving the proposed compounds. The later eluting compound K with a molecular ion
structure (Figure 3). This compound is the first natural NAME at m/z 355 was therefore proposed to be N-(hexadecanoyl)-2-
featuring a methyl branched acyl chain.
aminobutyric acid methyl ester (7), while the earlier eluting J
with m/z 353 compound was likely N-[(Z)-hexadec-9-enoyl]-2-
Compound G showed a mass spectrum identical to C16:1- aminobutyric acid methyl ester (8). The double bond position
NAME and a retention index with I = 2457, eluting earlier than was determined by DMDS derivatization. The structures of both
compound H, Z9-C16:1-NAME with I = 2473. The low amount K and J were verified by synthesis according to Scheme 2.
of the material produced excluded further structural characteri- Palmitoleic acid was synthesized in g-scale by standard proce-
Scheme 1: Synthesis of iso-C15:0-NAME (6). DMAP: 4-dimethylaminopyridine, EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,
L-AME: -alanine methyl ester.
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Beilstein J. Org. Chem. 2018, 14, 2964–2973.
Compound N showed a mass spectrum (Figure 4c) featuring
characteristic ions at m/z 72, 132, and 173, indicating a further
carbon in the amino acid part. Together with the molecular ion
at m/z 367 (C22H41NO3) the data indicate the presence of valine
in the compound. DMDS derivatization proved the unknown
compound to also carry a double bond at C-9 of the alkenoyl
chain. The proposed structure N-[(Z)-hexadec-9-enoyl]valine
methyl ester (9) was proven by synthesis starting from valine
methyl ester as described (Scheme 2). The gas chromatograph-
ic retention indices of the synthetic material and the natural
compound matched perfectly. We propose to call the N-acylated
valine methyl ester 9 Z9-C16:1-NAVME.
The extract of Roseovarius sp. D12_1.68 was also investigated
by HPLC/ESI+–MS to detect more polar compounds compared
to GC. The NAMEs, NABMEs and NAVMEs reported here
were detected by MS2 analyses based on their characteristic
fragmentation (see below). The only oxygenated derivative
present was 16OH-C16:1-NAME, which has been described
before from Roseovarius tolerans EL-164 [24].
Loktanella sp. D3, F13 and F14
Investigation of extracts of the three isolates F14, F13, and D3
by GC–MS indicated the presence of compounds whose mass
spectra were again similar to those of NAMEs (Figure 5). The
spectra show characteristic ions at m/z 90, 131, and 144
(Figure 6), a loss of one methylene group compared to ions
m/z 104, 145, and 158 of NAMEs. The lack of an analogous ion
to m/z 44 (m/z 30 is outside the mass range of the spectrometer
used) pointed this time to glycine as the core amino acid.
Molecular formulas were obtained via HPLC/HRMS measure-
ments and supported the N-acylglycine methyl ester (NAGME)
structure proposed for these compounds.
Saturated and unsaturated NAGMEs can be distinguished by the
intensity of the ions m/z 90 and 131. Similar to NAMEs satu-
rated NAGMEs show a high intensity of m/z 131 whereas unsat-
urated NAGMEs show higher intensity of m/z 90 (Figure 6).
Figure 4: Mass spectra of natural compounds a) K (7, C16:0-NABME),
b) J (8, C16:1-NABME), and c) N (9, C16:1-NAVME).
dures as shown in the Supporting Information File 1, Scheme The low amounts available did not allow to determine the posi-
S1. This acid and palmitic acid were converted into the respec- tion of the double bond in unsaturated NAGMEs. Nevertheless,
tive chlorides and standard acylation delivered 2-aminobutyric the predominance of the (Z)-9-hexadecenoyl side chain in all
acid derivatives 7 and 8 (Scheme 2) that proved to be identical NAME family compounds suggested compound R to be N-[(Z)-
with the natural products. The absolute configuration of the hexadec-9-enoyl]glycine methyl ester (11, Z9-C16:1-NAGME),
amino acid could not be determined due to the low amount of while S is its saturated analogue. Therefore, both compounds
material present. Nevertheless, because NAMEs showed the were synthesized as described before from glycine methyl ester
common -configuration [23], this configuration also seems and the respective acid (Scheme 2) and their identity confirmed.
likely for the other amino acid derivatives reported here. We The other components P, Q and T–W were also NAGMEs.
suggest the term NABME (N-acylated 2-aminobutyric acid
methyl esters) for the new compounds that can thus be assigned Their chain length was established using EI mass spectra and
as C16:0-NABME (7) and Z9-C16:1-NABME (8).
the gas chromatographic retention indices Inat of the com-
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Beilstein J. Org. Chem. 2018, 14, 2964–2973.
Scheme 2: Synthesis of N-acylated amino acid methyl esters 7–11. AAME: amino acid methyl ester.
Figure 5: TIC of an XAD extract of Loktanella sp. F14. Compound S is a minor component within the broad peak.
pounds (Table 2). Overall, six saturated and unsaturated com- Analysis by HPLC/MS revealed no additional NAGME.
pounds with a chain length between C14 and C19 were detected. Furthermore, no NAMEs, NABMEs, or NAVMEs were ob-
The roughly 100 retention index units between the members of served in the three strains.
this homologous series indicated that the acyl chains are
unbranched. Additionally, two unsaturated glycine derivatives Mass spectrometry
were present, 9-C16:1-NAGME (11) and C18:1-NAGME The analysis of the mass spectra of NAMEs, NABMEs,
(Table 2).
NAVMEs, and NAGMEs revealed the typical fragmentation of
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Beilstein J. Org. Chem. 2018, 14, 2964–2973.
sity. In addition to these features, the molecular ion is visible, as
is the loss of the carbomethoxy group [M − 59]+.
Figure 7: EI mass spectrometric fragmentation of N-acylated amino
acid derivatives. The ions w–z allow identification of the respective
amino acid residue.
The MS2 spectrum of the [M + H]+-ion obtained in ESI posi-
tive mode shows loss of water, an intense ion due to loss of
methanol, and loss of the carbomethoxy group [24]. Often the
amino acid ion can also be observed. These features indicate the
presence of an amino acid methyl ester.
Biological activity
Several of the synthesized compounds were tested in a broader
screening program for their antimicrobial activity (Table 3).
While the valine derivative 9 was virtually inactive, the other
compounds showed some activity. Glycine compound 11
showed good activity against the Gram-positive bacteria
Figure 6: Mass spectra of natural compounds
a) R (11, C16:1-NAGME) and b) S (10, C16:0-NAGME).
N-acylated amino acid methyl esters under both EI (Figure 7) Bacillus subtilis, Staphylococcus aureus, and Micrococcus
and ESI ionization (Figure 8). Detailed structural information luteus, and against the filamentous fungus Mucor hiemalis. In
can be obtained by EI-MS. A dominant peak in the mass spec- addition, 11 displayed moderate active on Mycobacterium
trum is the McLafferty rearrangement ion y, if the acyl chain is smegmatis and the efflux-deficient Escherichia coli TolC strain.
saturated. Together with prominent ion w [NH–CH2–R]+, often The 2-aminobutyric acid derivative 7 was active against
the base peak, and x it defines the amino acid, while z is usually M. hiemalis, M. luteus and S. aureus, while the unsaturated ana-
of low abundance. Formation of x requires transfer of two H logue 8 was mainly active against E. coli TolC, and it was the
atoms to this fragment. In compounds carrying an unsaturated only compound that showed moderate cytotoxicity on a human
acyl chain the intensity of y is reduced, and x increases in inten- cancer cell line.
Table 2: NAGMEs produced by Loktanella related isolates F14, F13 and D3. Inat: gas chromatographic retention index on a HP-5 phase;
[M]+: molecular mass; m/z: characteristic ions in EI mass spectra.
peak
compound
Inat
[M]+
m/z
Loktanella isolate
F14
F13
D3
P
Q
R
S
C14:0-NAGME
C15:0-NAGME
C16:1-NAGME
C16:0-NAGME
C17:0-NAGME
C18:1-NAGME
C18:0-NAGME
C19:0-NAGME
2312
2415
2495
2515
2618
2702
2720
2832
299
313
325
327
341
353
355
369
131 > 90
131 > 90
90 < 131
131 > 90
131 > 90
90 < 131
131 > 90
131 > 90
x
x
x
x
x
x
x
x
x
x
x
x
T
U
V
x
x
x
W
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Beilstein J. Org. Chem. 2018, 14, 2964–2973.
Figure 8: MS2 spectra in ESI positive mode of a) Z9-16OH-C16:1-NAME with [M + H − H2O]+ 356, b) Z9-C16:1-NABME (8) with [M + H]+ 354,
c) Z9-C16:1-NAVME (9) with [M + H]+ 368, and d) Z9-C16:1-NAGME (11) with [M + H]+ 326.
The close similarity of NAMEs, NAVMEs, and NAGMEs to costatum while Loktanella sp. D3 showed no antagonistic activ-
AHLs might indicate a function as signalling compounds, al- ity [29]. This observation fits well with the detection of antimi-
though experiments with NAMEs and AHL reporter assays did crobial or cytotoxic NAVME 9 and NABMEs 7 and 8 only in
not reveal any activity on the LuxR type receptors for AHLs in Roseovarius sp. D12_1.68.
bacteria [23,24]. Other potential functions are antimicrobial or
cytotoxic activity of the tested derivatives. Roseovarius sp. Bacteria are known to produce acylated amino acids, although
D12_1.68 showed antimicrobial activity against a Rhodobacter- the number reported so far is small, including tyrosine, trypto-
aceae sp. TL and antialgal activity against Skeletonema phan, arginine, or phenylalanine [30-32]. They all carry long
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Beilstein J. Org. Chem. 2018, 14, 2964–2973.
Table 3: Antimicrobial activity (minimum inhibitory concentration, MIC, in µg/mL) and cytotoxicity (minimum inhibitory concentration, MIC, in µg/mL) of
selected NAVME, NABME and NAGME derivatives. Minimal inhibitory concentration and IC50 values for cytotoxicity in μg/mL.
strain
11
9
8
7
Chromobacterium violaceum DSM-30191
Escherichia coli DSM-1116
>128
>128
128
>128
4–8
4
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
nd
nd
>128
>128
>128
>128
>128
16
Escherichia coli (TolC-deficient)
Pseudomonas aeruginosa PA14
Bacillus subtilis DSM-10
16
nd
>128
64–128
nd
Micrococcus luteus DSM-1790
Staphylococcus aureus Newman
Mycobacterium smegmatis mc2155
Mucor hiemalis DSM-2656
16
8–16
>128
32
64
nd
8–16
>128
>128
nd
Pichia anomala DSM-6766
nd
>128
>128
Candida albicans DSM-1665
nd
cytotoxicity
HCT-116 (human colon carcinoma)
>67
>67
15.8
>67
chain saturated or unsaturated acyl chains similar to those re-
ported here. Recently, derivatives of the hydrophobic amino
acids valine, leucine and isoleucine were also reported [33].
These compounds are produced by a family of acyl amino acid
synthases structurally related to AHL synthases, further
suggesting a function as bacterial signalling compounds [34]. In
contrast to the reported compounds carrying a free acid group,
all derivatives reported here are native methyl esters because no
methanol was used during sample preparation
Supporting Information
Supporting Information File 1
Experimental synthetic procedures, biological tests and
NMR spectra.
[https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-14-276-S1.pdf]
Acknowledgements
We thank the Deutsche Forschungsgemeinschaft (DFG) for
supporting our work through the Transregional Collaborative
Research Center “Roseobacter” (SFB TRR 51/3)
Conclusion
We have identified here new classes of acylated amino acid de-
rivatives including previously unknown glycine and 2-aminobu-
tyric acid derived compounds. The combination of GC/MS,
HPLC/MS, retention indices and synthesis proved to be espe-
cially suited to structurally identify minor components of com-
plex extracellular metabolite mixtures. The reported com-
pounds are specific for Roseobacter group bacteria of the
genera Roseovarius and Loktanella, in contrast to broadly distri-
buted AHLs. Although their function as signalling compounds
is not proven, the occurrence of 2-aminobutyric acid might indi-
cate some similarity to homoserine in AHLs because both are
non-proteinogenic amino acids. This similarity might also be
functional, because the structures of NAMEs, NABMEs,
NAGMEs and NAVMEs are similar to other bacterial signalling
compounds, often carrying a lipophilic side chain and a medi-
um polar core structure [8,16]. Nevertheless, the ecological
function of NAMEs and its derivatives could also be antago-
nistic activity against concurrent biofilm microorganisms, sug-
gested by the bioactivity of some of the compounds as ob-
served in this study and the antimicrobial and antialgal activity
of the producing organism [29].
ORCID® iDs
Laura Wolter - https://orcid.org/0000-0002-1238-2232
Thorsten Brinkhoff - https://orcid.org/0000-0003-3917-6882
Rolf Müller - https://orcid.org/0000-0002-1042-5665
Stefan Schulz - https://orcid.org/0000-0002-4810-324X
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