Modified N-acyl-homoserine lactones as chemical probes for the elucidation of plant-microbe interactions

Article abstract of DOI:10.1039/c3ob41215f

Gram-negative bacteria often use N-acyl-homoserine lactones (AHLs) as signal molecules to monitor their local population densities and to regulate gene-expression in a process called "Quorum Sensing" (QS). This cell-to-cell communication allows bacteria to adapt to environmental changes and to behave as multicellular communities. QS plays a key role in both bacterial virulence towards the host and symbiotic interactions with other organisms. Plants also perceive AHLs and respond to them with changes in gene expression or modifications in development. Herein, we report the synthesis of new AHL-derivatives for the investigation and identification of AHL-interacting proteins. We show that our new compounds are still recognised by different bacteria and that a novel biotin-tagged-AHL derivative interacts with a bacterial AHL receptor.

Full text of DOI:10.1039/c3ob41215f

Organic &
Biomolecular Chemistry
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Modified N-acyl-homoserine lactones as chemical
probes for the elucidation of plant–microbe
interactions†
Cite this: Org. Biomol. Chem., 2013, 11,
6994
Heike Thomanek,a Sebastian T. Schenk,b Elke Stein,b Karl-Heinz Kogel,b
Adam Schikora*b and Wolfgang Maison*a
Gram-negative bacteria often use N-acyl-homoserine lactones (AHLs) as signal molecules to monitor their
local population densities and to regulate gene-expression in a process called “Quorum Sensing” (QS).
This cell-to-cell communication allows bacteria to adapt to environmental changes and to behave as
multicellular communities. QS plays a key role in both bacterial virulence towards the host and symbiotic
interactions with other organisms. Plants also perceive AHLs and respond to them with changes in gene
expression or modifications in development. Herein, we report the synthesis of new AHL-derivatives for
the investigation and identification of AHL-interacting proteins. We show that our new compounds
are still recognised by different bacteria and that a novel biotin-tagged-AHL derivative interacts with a
bacterial AHL receptor.
Received 12th June 2013,
Accepted 21st August 2013
DOI: 10.1039/c3ob41215f
www.rsc.org/obc
significant effects on human health and crop plant
production.12–15 Gram-negative bacteria use N-acyl-L-homo-
Introduction
Bacteria produce signal molecules of low molecular weight to serine lactones (AHLs) as their primary QS signal
report information on their local population densities in order molecules.11,16–19 Bacteria produce different AHLs and one
to control and coordinate their behaviour. These signal mole- species may produce one or more autoinducers varying in con-
cules, also known as autoinducers, play a key role in a complex stitution and effects on the host. Here, we focus on AHLs of
cell-to-cell communication process.1 Fuqua et al. were the first plant-associated bacteria and their biological relevance for the
to describe and investigate bacterial communication and plant hosts.20
defined it as the cell density dependent alteration of gene
Recent studies showed that bacteria not only use AHLs for
expression.2 The signalling process also termed “Quorum their communication but that plants are also able to detect
Sensing” allows the bacteria to sense and respond to their bacterial signal molecules and respond to the autoinducers
local population density. The small, pheromone-like autoindu- with altered gene expression or modifications in development,
cers pass the cell membrane and, once a certain threshold con- which can be beneficial for the plant.21,22 von Rad et al. have
centration is reached, the signal molecules bind to a cognate shown that Arabidopsis, for example, takes up short-chained
receptor to form a complex which induces alteration of gene AHLs and allows their systemic distribution in the plant. On
expression.3,4 Examples of QS-regulated behaviours are biofilm the one hand, short-chained AHLs promote root growth and
production,5–9 induction of bioluminescence,10,11 antibiotic alter root hair development.23,24 On the other hand, some
production and virulence factor expression. QS plays a central AHL-producing bacteria have a beneficial effect on resistance
role in the interaction of bacteria with their hosts with often against microbial pathogens.25 Recently, oxo-C14-AHL has
been found to induce resistance in Arabidopsis and barley
plants towards biotrophic and hemibiotrophic pathogens.26
aPharmaceutical and Medicinal Chemistry, University of Hamburg, Bundesstr. 45,
Despite its importance for plant resistance and development,
20146 Hamburg, Germany. E-mail: maison@chemie.uni-hamburg.de;
the underlying molecular processes of AHL-perception by
Fax: (+)49 40 42838 3477; Tel: (+)49 40 42838 3497
bInstitute for Phytopathology and Applied Zoology, University of Giessen,
plants are widely unexplored and plant receptors for AHLs
have not been isolated so far. The identification and investi-
gation of these putative plant receptors will be crucial to
understand the plant response to bacterial pathogens. As a
critical step to elucidate the plants’ perception system for bac-
terial autoinducers, we present syntheses of modified oxo-C14-
Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany.
E-mail: adam.schikora@agrar.uni-giessen.de; Fax: (+)49 641 9937499;
Tel: (+)49 641 9937497
†Electronic supplementary information (ESI) available: Copies of NMR spectra
for new compounds, HPLC data for the separation of racemic 12c, experimental
details of biological evaluation. See DOI: 10.1039/c3ob41215f
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not give the desired target compound 5 and lead to complex
product mixtures.
Three additional functionalized AHL-derivatives 7–9 have
been prepared as depicted in Scheme 2. Azide 7 is a versatile
intermediate for copper catalyzed click-functionalization of
oxo-C14-AHL and was prepared from azidoacid 6. The two cate-
chol derivatives 8 and 9 were synthesized by NHS-ester coup-
ling of amine 3. The first catechol-AHL-derivatives have
recently been introduced by Gademann.42 The catechol group
is a surface anchor43,44 and may be used for the immobili-
zation of 8 and 9 on metal oxide surfaces (nanoparticles or
bulk materials). The resulting materials are anticipated to be
interesting for the modulation of plant root growth or as
stationary phases for the affinity purification of human, bac-
terial and putative plant AHL-receptors.
Fig. 1 Generalized AHL-structure and oxo-C14-AHL as
a functionalized
derivative.
AHLs and evaluate their biological activity in bacteria and
plants (Fig. 1).
Results and discussion
Substituents at the lactone ring are tolerated in a number
of known AHL-mimetics and the crystal structure of LasR from
P. aeruginosa with oxo-C12-AHL suggested to us that a cis-C5-
substitution pattern might be suitable for the introduction of
functional groups to our AHL-analogues.45 A number of
lactone ring modifications have been described in the litera-
ture. Most of them concentrate on the variation of the lactone
ring size or replacing the lactone scaffold with other moi-
eties.27,28,36,46 In 2002 Olsen et al. described the synthesis of
3- and 4-hydroxy substituted C6-AHL and some carbamate
derivatives starting from serine.47 Our approach is depicted in
Scheme 3. We planned to construct the modified lactone
moiety according to the Kazmaier method from N-protected
allylglycines 10 via a Claisen rearrangement of glycine allyl-
esters 11.48 Following the Kazmaier synthesis we started with
TFA-protected glycine 10a to synthesize glycine allyl ester 11a
(Scheme 3). The following Claisen rearrangement to 12a pro-
ceeded in varying yields up to 74%. In some cases we observed
Different acyl chain modified AHL-derivatives have been
synthesized.27–33 For the synthesis of acyl chain modified
oxo-C14-AHLs we chose the Meldrum’s acid approach.34–37
Following a route by Amara et al.38 we started with the Boc-
protected acid 1 which was converted to the novel oxo-C14-
AHL analogue 2 by treatment with Meldrum’s acid and
(S)-homoserine lactone. Boc-deprotection of 2 with TFA gave
the free amine 3 (Scheme 1). From this intermediate, different
functionalized AHL-mimetics were prepared. Acylation with
acetic anhydride gave the N-acetyl derivative 4, which was syn-
thesized as a simple and sterically non-demanding analogue
of oxo-C14-AHL. Most notably, the biotin-labeled analogue 5
was prepared, which is an interesting molecular probe for a
pull-down assay for putative plant AHL receptors. Only a few
biotin-tagged-AHLs can be found in literature and derivatives
of oxo-C14-AHL are unknown.39,40 We found NHS-ester coup-
ling of biotin to be the best method for conjugation.41
Standard peptide coupling conditions (e.g. HOBt, EDC) did
Scheme 1 Synthesis of amine 3 and its conversion to the N-acyl derivative 4 and biotin-labeled molecular probe 5.
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Scheme 2 Synthesis of azide 7 for click-functionalization and two catechol derivatives 8 and 9 for surface immobilisation.
Scheme 3 Synthesis of lactone ring modified AHL-derivatives (only one enantiomer of racemic mixtures is depicted).
deprotection of the TFA group after work up. In addition, the groups and prepared Cbz-protected glycine allyl ester 11b and
following iodolactonization to 13a was accomplished with only the Boc-protected analogue 11c. With both derivatives, the
37% yield. In consequence, we decided to switch protecting rearrangement worked fine and gave the expected allylglycines
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12b and 12c. Particularly Boc-allyl ester 11c gave consistently
high yields of Boc-allylglycine 12c. Unfortunately, as already
reported by Kazmaier for similar carbamates,49 the enantio-
selectivity of these conversions is low. For Boc- and Cbz-allyl-
glycine we obtained almost racemic mixtures. However, both
enantiomers of Boc-allylglycine 12c are easy to separate by
HPLC on a chiral phase (see ESI†) and are thus available in a
pure form if needed. For a first evaluation of our 4-substituted
AHL analogues, we used the racemic compounds 12b and 12c.
Iodolactonization worked well for the Cbz- and the Boc-pro-
tected compounds and gave iodolactones 13b and 13c in good
yields and with good diastereoselectivities for the cis-deriva-
tives (dr(cis:trans) = 10 : 1).50 Deprotection with TFA gave the free
amine 14 which was coupled to lauric acid in the presence of
Meldrum’s acid. The final AHL-analogue 15 was obtained in
43% yield over two steps. Alternatively, an azide moiety was
introduced to iodolactone 13 via nucleophilic displacement of
iodide with NaN3 to give the desired products 16b and 16c.
Deprotection of 16c to the free amine 17 followed by coupling
to lauric acid in the presence of Meldrum’s acid gave AHL-
analogue 18. Both derivatives 15 and 18 are versatile AHL
analogues because the iodide in 15 may be easily substituted
with various nucleophiles and the azide in 18 is an excellent
precursor for click functionalization or reduction to the amine
and subsequent functionalization via amide formation.
Fig. 2 Detection of AHL-derivatives with biosensor bacteria. Three different
bacterial strains were used to detect the derivatives of N-acyl-homoserine lac-
tones. Molecules were dissolved in acetone, or DMSO, and 5 µL were dropped
on bacterial lawns for
2 h. Green fluorescent protein (GFP) signals were
observed with fluorescent binocular using GFP filter Em: 505–550 nm.
Modifications of the molecular structure often lead to
altered activity of biologically active molecules. To assess
whether bacteria still recognize the modified AHL derivatives,
we used two reporter strains. These bacteria carry plasmids
with gene coding for an AHL receptor and the Green
Fluorescent Protein (GFP) gene under control of AHL-inducible
promoters: Pseudomonas putida strain F117 carrying the
pKR-C12 plasmid (PlasB-gfp(ASV)-Plac-lasR; Gmr),51 and Escheri-
chia coli strain MT102 carrying the pJBA89 plasmid (Apr;
pUC18Not-luxR-PluxI-RBSII-gfp(ASV)-T0-T1).52 These bacteria are
detecting nanomolar–micromolar concentrations of AHLs
from C6-AHL to C14-AHL.26 Both strains were treated with five
native AHL molecules (see Fig. S1 of the ESI†). AHLs were dis-
Fig. 3 Pull-down of the His-tagged oxo-C14-AHL receptor 6xHis-Sinme_0536
from S. meliloti. The biotinylated AHL-derivative 5 was immobilized on streptavi-
din beads, which were pretreated with BSA to minimize unspecific protein
binding. Protein bands were visualized with silver stain and 6xHis-Sinme_0536
solved in acetone and applied to lawns of reporter bacteria in was detected by a His-specific antibody (α-His). BSA: bovine serum albumin.
different concentrations. GFP signals were recorded 2 h after
application. Analysis of the obtained results revealed that
reporter bacteria slightly differ in their AHL perception. While purified as a 6xHis-tagged recombinant protein. Using strepta-
P. putida recognizes oxo-C10-AHL to oxo-C14-AHL, E. coli per- vidin-coated beads, we attempted to precipitate the protein in
ceives oxo-C8-AHL but also all other tested native AHLs. E. coli the presence of solely biotin (negative control, lane 1) or in the
and P. putida also recognize oxo-C14-AHL. Next, we addressed presence of 5 (lane 3). As an additional control we used a pull-
the question, whether the bacterial strains recognize the down setup without 6xHis-Sinme_0536 (lane 2). Proteins
modified oxo-C14-AHL derivatives. P. putida recognizes 2 in a pulled down in the presence of 5 were probed for the occur-
similar concentration range to oxo-C14-AHL as well as 3 and 4 rence of 6xHis-Sinme_0536 with a specific anti-His antibody.
at high concentration. E. coli, on the other hand, recognizes 3 As shown in Fig. 2, in the presence of 5 (lane 3) but not free
and 5 comparably to oxo-C14-AHL. E. coli also recognized 4 biotin (lane 1) 6xHis-Sinme_0536 was found in the precipitate
and 15, though only at high concentration (Fig. 2).
(αHis, lane 3).
In order to verify the potential of biotinylated 5, we per-
Recent reports suggest that AHLs produced by soil bacteria
formed an exploratory pull-down experiment with a member of can actively affect plant health and development.22,25,26,53–56
the LuxR AHL-receptor family (Sinme_0536) from Sinorhizo- An early plant response to pathogen attack is transcriptional
bium meliloti, a soil borne bacterium known to produce and activation of defense related genes. Among them are pathogen-
perceive oxo-C14-AHL (Fig. 3).29 Sinme_0536 was cloned and inducible WRKY transcription factors that are activated by
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different signaling pathways involved. It is likely that 5
binds to the cognate AHL receptor(s) preventing oxo-C14-AHL
binding and initiation of defense priming in the plant.
Whether 5 is a direct antagonist will be the subject of future
studies.
Conclusions
In summary, we have synthesized new AHL derivatives with
modified acyl chains and lactone moieties. The former are
attractive molecular probes for the elucidation of molecular
communication between plants and bacteria. The biotinylated
AHL-derivative 5 turned out to be particularly valuable. It is
still recognized by different bacteria as a signal molecule and
can be used for pull-down systems on streptavidin beads. This
was demonstrated with the specific pull-down of the LuxR-type
receptor Sinme_0536 from S. meliloti. In addition, we have
shown that 5 acts as an antagonist for native oxo-C14-AHL in
Arabidopsis seedlings.
In addition to the biotinylated derivative 5, the catechol
derivatives 8 and 9 are particularly attractive molecular probes
because they can easily be immobilized on metal surfaces and
may thus be used for affinity catching of putative AHL-interact-
ing proteins in plants.
Fig. 4 The biotinylated AHL derivative 5 acts as an antagonist for plant AHL-
induced resistance responses, measured by the transcriptional activation of
plant transcription factors WRKY22 and WRKY29 in the presence of the natural
autoinducer oxo-C14-AHL and the modified analogue 5. Error bars represent SD
from three independent experiments.
Experimental
Material and methods
TLC was performed on silica gel aluminum sheets. Reagents
used for developing plates include cerium stain (5 g molyb-
datophosphoric acid, 2.5 g cerium sulfate tetrahydrate, 25 mL
sulfuric acid and 225 mL water), potassium permanganate
(0.5% in 1 N NaOH w/v) and detection by UV light was used
when applicable. Flash column chromatography was per-
mitogen-activated protein kinases (MAPK) which are key
elements in early defense signalling. Consistent with this, acti-
vation (phosphorylation) of MPK6 by a combined treatment of
Arabidopsis thaliana with oxo-C14-AHL and the bacterial signal
flg22 (flagellin 22) resulted in transcriptional upregulation of
both WRKY22 and WRKY29.57–60 This scenario is thought to be
the molecular base for AHL-induced resistance.26 In order to
compare the activity of native oxo-C14-AHL and its derivatives,
we examined the impact of 5 on the relative expression levels
of WRKY22 and WRKY29 (Fig. 4). Two-week-old Arabidopsis
seedlings were pretreated with 6 μM oxo-C14-AHL (positive
control), AHL derivatives or combinations of both and sub-
sequently treated with 100 nM flg22. Total RNA was extracted
and transcript levels of WRKYs normalized to the expression
of UBQ4 (At5g25760). Pretreatment with the AHL followed
by treatment with 100 nM flg22 resulted in upregulation of
WRKY22 and WRKY29 (Fig. 4). In contrast, pretreatment with
modified AHL derivative 5 had no impact on WRKYs expression
pattern. Notably, however, when 5 was added in addition to
oxo-C14-AHL the observed AHL effect was abolished. We
suspect that 5 is an antagonist to oxo-C14-AHL in plants.
Given the fact that 5 induced GFP expression in bacterial
reporter strains (agonistic action) this antagonistic effect in
plants is remarkable and might be the consequence of the two
1
formed on silica gel (60–200 μm). H chemical shifts are refer-
enced to residual non-deuterated solvent (CDCl3, δH
=
7.26 ppm; DMSO-d6, δH = 2.50 ppm; CD3OD, δH = 3.31 ppm).
13C chemical shifts are referenced to the solvent signal (CDCl3,
δC = 77.16 ppm; DMSO-d6, δC = 128.06 ppm; CD3OD, δH
=
49.00 ppm). NMR spectra were recorded using 400 (100) MHz
instruments. ESI mass spectra were recorded using a TOF
instrument operated in positive mode (Bruker MicrOTOF Q).
Samples were dissolved in MeOH or H2O–MeCN-mixtures and
directly injected via a syringe. Analytical HPLC analysis was
recorded using a VWR HITACHI ELITE LaChrom L-2130 HPLC
(RI Detector: L2490). The following chiral column was used:
CHIRALPAK IA (DAICEL Chemical Industries; particle size:
5 µm; dimensions: 4.6 mm ϕ × 150 mm). Solvents were dried
by distillation from sodium under a nitrogen atmosphere prior
to application.
The following compounds were prepared according to lit-
erature procedures: Boc-protected acid 1,49 biotin-NHS ester,39
azido acid 6,38 TFA-glycine allylester 11a,48 TFA-allyl glycine
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12a,48 TFA-iodolactone 13a,48 Boc-protected iodolactone 13c,50 was washed with brine, dried over Na2SO4 and filtered. The
and Boc-protected lactone 16c.50
solvent was evaporated in vacuo and the crude product was
purified by flash chromatography.
General procedure 1: coupling with Meldrum’s acid
General procedure 6: preparation of azido lactones
1 eq. of the appropriate fatty acid was dissolved in CH2Cl2 and
1 eq. DMAP, 1 eq. DCC and 1 eq. of Meldrum’s acid were To a solution of the iodide in DMF, NaN3 was added and the
added to the mixture. The solution was stirred overnight at mixture was stirred at 40 °C for 24 h. The solvent was distilled
room temperature and then filtered to remove the N,N-dicyclo- off in vacuo and the residue was dissolved in EtOAc. The
hexyl urea formed in the reaction. The filtrate was concen- organic layer was washed with water and brine. The organic
trated in vacuo and the resulting residue was dissolved in DMF. phase was then dried over Na2SO4, filtered and the solvent was
α-Amino-γ-butyrolactone hydrobromide was added and the evaporated in vacuo. The crude product was purified by flash
mixture was stirred at room temperature for 1 h and additional chromatography.
4 h at 60 °C. The solvent was distilled off in vacuo and the
residue was dissolved in EtOAc. The organic phase was washed
General procedure 7: NHS-ester coupling
three times with saturated sodium bicarbonate solution, 1 M 1 eq. AHL 3, 1 eq. NHS-ester and 1.3 eq. Et3N were dissolved in
sodium hydrogen sulfate solution and brine. Afterwards, the DMSO. After stirring at room temperature for 24 h the solvent
organic phase was dried over Na2SO4, filtered and then the was distilled off in vacuo and the crude product was purified
solvent was distilled off. The crude product was purified by by flash chromatography if necessary.
flash chromatography if necessary.
AHL-derivative (2)
General procedure 2: Boc deprotection
According to general procedure 1, the title compound 2
The Boc protected amine was dissolved in TFA–CH2Cl2 (1 : 1; (278 mg, quant.) was obtained from carboxylic acid 1 (200 mg,
5 mL per 0.1 mmol educt) and stirred at room temperature for 0.6 mmol), DMAP (77.5 mg, 0.6 mmol), DCC (131 mg,
3 h. The solvent was distilled off in vacuo. The crude product 0.6 mmol), Meldrum’s acid (91 mg, 0.6 mmol) and α-amino-
was purified by flash chromatography if necessary.
γ-butyrolactone hydrobromide (115 mg, 0.6 mmol) as a colour-
less solid. δH (CDCl3, 400 MHz) 4.44–4.62 (m, 2H), 4.23–4.30
(m, 1H), 3.46 (s, 2H), 3.05–3.11 (m, 2H), 2.70–2.79 (m, 1H),
General procedure 3: preparation of glycine allyl esters
1 eq. N-protected glycine was dissolved in CH2Cl2 and cooled 2.51 (t, 1H, J = 9.4 Hz), 2.12–2.29 (m, 1H), 1.90–1.94 (m, 1H),
to 0 °C. 1 eq. of allylic alcohol was added and the solution was 1.64–1.73 (m, 2H), 1.53–1.64 (m, 2H), 1.43 (s, 9H), 1.18–1.20
cooled to −15 °C. A solution of 1 eq. DCC and 0.1 eq. DMAP in (m, 14H); δC (CDCl3, 100 MHz) 206.5, 174.8, 166.4, 156.1, 78.9,
CH2Cl2 was added and the reaction was stirred at room temp- 65.8, 48.9, 48.1, 43.9, 40.6, 33.8, 30.0, 29.8, 29.4, 29.2, 28.9,
erature for 12 h. The precipitated N,N-dicyclohexyl urea was 28.4, 26.8, 25.6, 24.9, 23.3; HRMS (ESI) m/z calcd for
filtered off and washed with CH2Cl2. The organic layer was C23H40N2NaO6 [M + Na]+ 463.2779, found 463.2784.
washed with 1 M HCl and saturated NaHCO3 solution. The
AHL-derivative (3)
organic phase was dried over Na2SO4, filtered and the solvent
was evaporated in vacuo.
According to general procedure 2, the title compound 3
(51 mg, 61%) was obtained as a brown oil from AHL 2
(108 mg, 0.2 mmol). δH (CD3OD, 400 MHz, COCH2CO signal is
General procedure 4: Claisen rearrangement
LHMDS solution was prepared by adding 1 eq. 1.6 M n-BuLi in hidden under solvent peak) 4.61 (t, 1H, J = 10.0 Hz), 4.44 (t,
hexane at room temperature under an argon atmosphere to 1H, J = 2.3 Hz, J = 8.8 Hz), 4.27–4.33 (m, 1H), 2.91 (t, 2H, J =
1.2 eq. hexamethyldisilazane in abs. THF. The solution was 8.0 Hz), 2.58 (t, 2H, J = 5.3 Hz), 2.24–2.35 (m, 1H), 1.52–1.68
stirred for 20 min. In a second flask 0.2 eq. of the N-protected (m, 5H), 1.28–1.42 (m, 14H); δC (DMSO-d6, 100 MHz) 204.6,
glycine ester, 0.2 eq. Al(OPr-i)3 and 0.5 eq. quinidine were dis- 174.8, 166.4, 65.3, 50.0, 48.1, 42.6, 41.9, 34.0, 33.3, 28.8, 28.7,
solved in abs. THF under an argon atmosphere and cooled to 28.4, 28.3, 28.1, 26.9, 25.7, 22.8; HRMS (ESI) m/z calcd for
−78 °C. The LHMDS solution was added slowly and the reac- C18H33N2O4 [M + H]+ 341.2435, found 341.2440.
tion was stirred at room temperature for 12 h. The mixture was
AHL-derivative (4)
treated with 1 M KHSO4 and the organic layer was dried over
Na2SO4. After filtration the solvent was distilled off in vacuo AHL-derivative 3 (95 mg, 0.3 mmol), acetic anhydride (0.1 mL,
and the crude product was purified by flash chromatography if 5 eq., 1.4 mmol) and Et3N (0.4 mL, 10 eq., 2.8 mmol) were dis-
necessary.
solved in CH2Cl2 (30 mL) and stirred for 12 h at room tempera-
ture. The solvent was evaporated in vacuo and the crude
residue was purified by flash chromatography (EtOAc–MeOH,
General procedure 5: iodolactonization
The Claisen product was dissolved in THF at 0 °C and 1 eq. I2 9 : 1, Rf = 0.3) to give the title compound 4 (52 mg; 49%) as a
was added. The reaction was stirred for 12 h at room tempera- brown oil. δH (CD3OD, 400 MHz) 4.58–4.64 (m, 1H), 4.41–4.47
ture and then diluted with EtOAc. Afterwards the mixture was (m, 1H), 4.27–4.33 (m, 1H), 3.28 (s, 2H), 3.13 (t, 2H, J = 8.2 Hz),
quenched with saturated Na2S2O4 solution. The organic layer 2.57 (t, 3H, J = 8.2 Hz), 2.25–2.35 (m, 1H), 1.91 (s, 3H),
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1.44–1.60 (m, 4H), 1.26–1.29 (m, 14H); δC (CD3OD, 100 MHz) (20 mL) was added. DCU was filtered off and the solvent was
213.4, 206.5, 177.0, 173.4, 169.1, 67.1, 64.7, 50.2, 43.9, 40.7, evaporated in vacuo to give the crude product as a colourless
30.6, 30.5, 30.4, 30.3, 30.2, 30.0, 29.6, 28.0, 24.4, 22.5; HRMS oil, which was used without further purification. MS (ESI) m/z
(ESI) m/z calcd for C20H34N2NaO5 [M + Na]+ 405.2360, found 274.1 (M + Na+, 100%).
405.2382.
AHL-derivative (8)
Biotin-labeled-AHL (5)
According to general procedure 7, the title compound 8
Biotin-NHS ester (109 mg, 0.3 mmol) was dissolved in DMSO (180 mg, 88% over 2 steps) was obtained as a colourless oil
(50 mL). 3 (103 mg, 0.3 mmol) and Et3N (0.06 mL, 1.3 eq., from dihydroxybenzoic acid NHS-ester (147 mg, 0.6 mmol), 3
0.4 mmol) were added to the solution and the mixture was (200 mg, 0.6 mmol) and Et3N (0.1 mL, 0.8 mmol). δH (CD3OD,
stirred at room temperature for 24 h. The solvent was distilled 400 MHz) 7.20–7.48 (m, 2H), 6.83 (d, 1H, J = 7.4 Hz), 4.62
off in vacuo to give the title compound 5 (97 mg, 54%) as a (t, 1H, J = 10.0 Hz), 4.47 (t, 1H, J = 11.0 Hz), 4.28–4.35 (m, 1H)
brown oil. δH (DMSO-d6, 400 MHz) 6.36 (d, 2H, J = 12.3 Hz), 3.17–3.22 (m, 2H), 2.93 (t, 1H, J = 11.0 Hz), 2.71 (s, 2H),
4.25–4.30 (m, 1H), 4.07–4.13 (m, 1H), 3.02–3.09 (m, 3H), 2.57–2.61 (m, 2H), 2.26–2.40 (m, 1H), 1.53–1.72 (m, 4H),
2.93–2.96 (m, 1H), 2.65–2.76 (m, 4H), 2.54–2.60 (m, 2H), 1.26–1.43 (m, 14H). δC (CD3OD, 100 MHz) 206.7, 177.2, 175.1,
2.45–2.47 (m, 6H), 1.24–1.64 (m, 12H), 1.10–1.21 (m, 12H); δC 170.5, 151.4, 150.0, 146.0, 124.0, 117.6, 115.7, 67.1, 50.2, 47.9,
(DMSO-d6, 100 MHz) 204.7, 172.8, 170.2, 170.0, 162.8, 65.4, 40.8, 30.6, 30.5, 30.4, 30.2, 30.1, 29.6, 28.6, 27.4, 26.3, 24.5,
61.1, 59.3, 55.2, 45.7, 42.7, 40.3, 30.0, 29.6, 29.1, 28.7, 28.5, 24.4; HRMS (ESI) m/z calcd for C25H36N2NaO7 [M + Na]+
28.4, 28.2, 28.0, 27.97, 27.9, 27.5, 26.4, 25.4, 25.3, 25.25, 25.2; 499.2415, found 499.2396.
HRMS (ESI) m/z calcd for C28H46N4NaO6S [M + Na]+ 589.3030,
found 589.3038.
Dimethoxybenzoic acid NHS-ester
2.3-Dimethoxybenzoic acid (300 mg, 2.6 mmol), N-hydroxy-
succinimide (303 mg, 2.6 mmol) and EDC*HCl (503 mg,
Azido acid (6)38
12-Bromododecanoic acid (0.50 g, 1.8 mmol) was dissolved in 2.6 mmol) were dissolved in DMF (50 mL). After stirring for
CH2Cl2–DMF (100 mL, 6 : 4) and NaN3 (1.17 g, 18 mmol) was 24 h at room temperature the solvent was distilled off in vacuo
added to the mixture. After stirring for 24 h at 60 °C the and EtOAc (20 mL) was added. The organic phase was washed
solvent was evaporated in vacuo and CH2Cl2 (60 mL) was three times with 20 mL 1 M KHSO4 solution, saturated
added. The organic phase was washed three times with 1 M NaHCO3 solution and saturated NaCl solution. The organic
HCl (30 mL) and saturated NaCl solution (30 mL). The organic layer was then dried over Na2SO4, filtered and the solvent was
layer was then dried over Na2SO4, filtered and the solvent was evaporated in vacuo to give the title compound (373 mg, 81%)
distilled off in vacuo to give the title compound 6 (0.41 g, 94%) as a colourless solid. δH (CDCl3, 400 MHz) 7.53 (d, 1H, J =
as a colourless oil. δH (CDCl3, 400 MHz) 11.23 (b, 1H), 3.23 (t, 4.7 Hz), 7.11–7.19 (m, 2H), 3.93 (s, 3H), 3.89 (s, 3H), 2.88 (s,
2H, J = 6.6 Hz), 2.32 (t, 2H, J = 7.4 Hz), 1.53–1.64 (m, 4H), 4H). δC (CDCl3, 100 MHz) 169.2, 161.0, 153.8, 150.7, 124.2,
1.23–1.35 (m, 14H); MS (ESI) m/z 264.2 (M + Na+, 100%).
122.9, 120.4, 118.1, 61.9, 56.4, 25.9; HRMS (ESI) m/z calcd for
C13H13NNaO6 [M + Na]+ 302.0635, found 302.0639.
AHL-derivative (7)
AHL-derivative (9)
According to general procedure 1, the title compound 7
(480 mg, 77%) was obtained from carboxylic acid 6 (408 mg, According to general procedure 7, the title compound 9
1.7 mmol), DMAP (207 mg, 1.7 mmol), DCC (349 mg, (127 mg, 39%) was obtained as a colourless oil from dimethoxy-
1.7 mmol), Meldrum’s acid (244 mg, 1.7 mmol) and α-amino- benzoic acid NHS-ester (178 mg, 0.6 mmol), 3 (217 mg,
γ-butyrolactone hydrobromide (308 mg, 1.7 mmol) as a colour- 0.6 mmol) and Et3N (0.1 mL, 0.8 mmol). The crude product
less solid after flash chromatography (EtOAc–MeOH 9 : 1, Rf = was purified by flash chromatography (EtOAc 100%, Rf = 0.2).
0.3). δH (CD3OD, 400 MHz) 4.61 (t, 1H, J = 10.3 Hz), 4.44 (dt, δH (CD3OD, 400 MHz, COCH2CO signal is hidden under
1H, J = 3.0 Hz, J = 8.1 Hz), 4.27–4.33 (m, 1H), 3.27 (t, 2H, J = solvent peak) 7.32 (dd, 1H, J = 2.5 Hz, J = 4.0 Hz), 7.11–7.17 (m,
5.9 Hz), 2.57 (t, 2H, J = 8.0 Hz), 2.23–2.34 (m, 1H), 1.67–1.73 2H), 4.61 (t, 1H, J = 10.0 Hz), 4.44 (dt, 1H, J = 2.0 Hz, J =
(m, 1H), 1.53–1.63 (m, 6H), 1.28–1.38 (m, 14H); δC (CD3OD, 9.0 Hz), 4.26–4.43 (m, 1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.38 (t,
100 MHz) 206.5, 171.2, 169.4, 67.1, 52.5, 50.0, 43.9, 34.8, 30.9, 2H, J = 6.8 Hz), 2.57 (t, 2H, J = 7.8 Hz), 2.27–2.34 (m, 1H),
30.7, 30.6, 30.5, 30.2, 30.0, 29.7, 27.9, 26.8, 24.5; HRMS (ESI) 1.51–1.74 (m, 5H), 1.60–1.45 (m, 14H). δC (CD3OD, 100 MHz)
m/z calcd for C18H30N4NaO4 [M + Na]+ 389.2160, found 205.2, 175.8, 173.6, 166.9, 153.1, 147.0, 128.1, 123.9, 120.9,
389.2160.
115.2, 65.8, 60.3, 55.1, 48.7, 42.3, 42.3, 33.3, 29.2, 29.1, 29.0,
28.9, 28.6, 28.2, 26.6, 23.0; HRMS (ESI) m/z calcd for
C27H40N2NaO7 [M + Na]+ 527.2728, found 527.2735.
Dihydroxybenzoic acid-NHS ester
3,4-Dihydroxybenzoic acid (350 mg, 2.3 mmol), N-hydroxy-
succinimide (261 mg, 2.3 mmol) and DCC (468 mg, 2.3 mmol)
Cbz-glycine allylester (11b)
were dissolved in DMF (40 mL). After stirring for 48 h at room According to general procedure 3, the title compound 11b
temperature the solvent was distilled off in vacuo and H2O (458 mg, 64%) was obtained from Cbz-glycine (600 mg,
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2.9 mmol), allylic alcohol (0.2 mL, 2.9 mmol), DMAP (35 mg, Cbz-protected azide (16b)
2.9 mmol) and DCC (603 mg, 2.9 mmol). The crude product
According to general procedure 6, the title compound 16b
(296 mg, 93%) was obtained from lactone 13b (400 mg,
1.1 mmol) and NaN3 (347 mg, 5.3 mmol). The crude product
was purified by flash chromatography PE–EtOAc (1 : 1, Rf = 0.3)
to give 16b as a brown oil. δH (DMSO-d6, 400 MHz) 7.29–7.37
(m, 5H), 5.03 (s, 2H), 4.41–4.65 (m, 2H), 3.61–3.80 (m, 2H),
2.40–2.48 (m, 1H), 1.93–2.07 (m, 1H); δC (DMSO-d6, 100 MHz)
174.5, 155.5, 136.8, 128.4, 128.35, 127.9, 127.8, 127.7, 75.4,
65.7, 52.9, 50.5, 30.4; HRMS (ESI) m/z calcd for C13H14N4O4
[M + Na]+ 313.0907, found 313.0907.
was purified by flash chromatography CH2Cl2–MeOH (10 : 0.2,
Rf = 0.6) to give the product as a colourless oil. δH (CDCl3,
400 MHz) 7.05–7.12 (m, 5H), 5.60–5.70 (m, 1H), 4.99–5.09 (m,
2H), 4.87 (s, 2H), 4.39 (d, 2H, J = 6.0 Hz), 3.75 (d, 2H, J =
6.0 Hz); δC (CDCl3, 100 MHz) 169.7, 156.3, 136.2, 131.54,
128.54, 128.51, 128.21, 128.15, 128.1, 119.0, 67.1, 66.0, 42.8.
Boc-glycine allylester (11c)
According to general procedure 3, the title compound 11c
(893 mg, 73%) was obtained as a colourless oil from Boc-
glycine (1.0 g, 5.7 mmol), allylic alcohol (0.4 mL, 5.7 mmol),
DMAP (70 mg, 5.7 mmol) and DCC (1.2 g, 5.7 mmol). δH
(CDCl3, 400 MHz) 5.70–5.89 (m, 1H), 5.17–5.28 (m, 3H, NH,
–CH2), 4.57 (d, 2H, J = 6.7 Hz), 3.86 (d, 2H, J = 9.0 Hz), 1.39 (s,
9H); δC (CDCl3, 100 MHz) 170.5, 155.7, 131.7, 118.6, 79.9, 65.8,
42.2, 28.2.
Iodolactone (14)
Deprotection of the Boc-group was performed according to
general procedure 2. Boc-protected iodolactone 13c (209 mg,
0.6 mmol) was dissolved in TFA–CH2Cl2 (30 mL, 1 : 1) to
give the crude product which was used in the next step
without further purification. HRMS (ESI) m/z calcd for
C5H8INO2 [M + H]+ 241.9672, found 241.9668.
Cbz-protected allyl glycine (12b)
According to general procedure 4, the title compound 12b
(263 mg, 90%) was obtained as a brown oil. LHMDS solution
was prepared freshly from hexamethyldisilazane (1.5 mL,
7 mmol) in abs. THF (5 mL) with n-BuLi (5 mL, 6 mmol). Cbz-
glycine allylester 11b (300 mg, 1.2 mmol), Al(OPr-i)3 (269 mg,
1.3 mmol) and quinidine (972 mg, 3 mmol) were dissolved in
abs. THF (50 mL). The LHMDS solution was added slowly to
the reaction mixture. δH (CDCl3, 400 MHz) 7.29–7.38 (m, 5H),
5.67–5.78 (m, 1H), 5.09–5.18 (m, 4H, 7-H), 4.46–4.51 (m, 1H),
2.52–2.67 (m, 2H); δC (CDCl3, 100 MHz) 175.0, 154.9, 135.2,
130.9, 127.5, 127.4, 127.3, 127.2, 127.1, 118.5, 65.9, 52.3, 35.3.
Azide lactone (17)
Deprotection of the Boc-group was performed according to
general procedure 2. Boc-protected azide 16c (129 mg,
0.5 mmol) was dissolved in TFA–CH2Cl2 (30 mL, 1 : 1). The
product was not isolated and used in the next step without
further purification.
AHL-derivative (15)
According to general procedure 1, the title compound 15
(120 mg, 43% over 2 steps) was obtained from lauric acid
(119 mg, 0.6 mmol), DMAP (72 mg, 0.6 mmol), DCC (122 mg,
0.6 mmol), Meldrum’s acid (85 mg, 0.6 mmol) and iodolactone
14 (143 mg, 0.6 mmol). The crude product was purified by
flash chromatography (PE–EtOAc 9 : 1, Rf = 0.2). δH (CDCl3,
400 MHz) 4.46–4.80 (m, 2H), 3.44–3.49 (m, 2H), 3.30–3.38 (m,
1H), 2.93–3.00 (m, 1H), 2.44–2.54 (m, 2H), 1.89–1.98 (m, 1H),
1.55–1.71 (m, 3H), 1.19–1.35 (m, 16H), 0.87 (t, 3H, J = 8.1 Hz).
δC (CDCl3, 100 MHz) 206.8, 173.6, 166.3, 76.3, 50.7, 48.2, 44.1,
36.1, 34.1, 32.1, 29.6, 29.4, 29.3, 29.0, 25.5, 23.36, 22.65, 14.1,
7.9; HRMS (ESI) m/z calcd C19H32INO4 [M + Na]+ 488.1268,
found 488.1256.
Boc-protected allyl glycine (12c)
According to general procedure 4, the title compound 12c
(300 mg, quant.) was obtained as a brown oil. LHMDS solution
was prepared freshly from hexamethyldisilazane (1.7 mL,
8.1 mmol) in abs. THF (5 mL) with n-BuLi (7 mL, 7 mmol).
Boc-glycine allylester 12b (300 mg, 1.4 mmol), Al(OPr-i)3
(314 mg, 1.5 mmol) and quinidine (1.13 g, 3.5 mmol) were dis-
solved in abs. THF (50 mL). The LHMDS solution was added
slowly to the reaction mixture. δH (CDCl3, 400 MHz) 5.67–5.78
(m, 1H), 5.02–5.18 (m, 2H), 4.94 (1H, NH), 4.32–4.41 (m, 1H),
2.41–2.62 (m, 2H), 1.42 (s, 9H); δC (CDCl3, 100 MHz) 179.2,
155.7, 132.0, 119.5, 86.0, 53.0, 36.3, 28.4.
AHL-derivative (18)
Cbz-protected iodolactone (13b)
According to general procedure 1, the title compound 18
According to general procedure 5, the title compound 13b (106 mg, 35% over 2 steps) was obtained from lauric acid
(330 mg, 83%) was obtained from Cbz-protected allyl glycine (163 mg, 0.8 mmol), DMAP (99 mg, 0.8 mmol), DCC (167 mg,
12b (264 mg, 1.0 mmol) and I2 (536 mg, 2.0 mmol). The crude 0.8 mmol), Meldrum’s acid (117 mg, 0.8 mmol) and azide 17
product was purified by flash chromatography PE–EtOAc (1 : 1, (127 mg, 0.8 mmol). The crude product was purified by flash
Rf = 0.4) to give 13b as a brown oil. δH (DMSO-d6, 400 MHz) chromatography (PE–EtOAc 1 : 1, Rf
= 0.2). δH (CDCl3,
7.32–7.41 (m, 5H), 5.06 (s, 2H), 4.56–4.67 (m, 1H), 4.44–4.45 400 MHz) 4.55–4.76 (m, 2H), 3.43–3.67 (m, 2H), 2.55–2.65 (m,
(m, 1H), 3.24–3.55 (m, 2H), 2.56–2.63 (m, 1H), 1.77–1.92 (1H); 2H), 2.06–2.15 (m, 1H), 1.82–1.88 (m, 1H), 1.67–1.74 (m, 1H),
δC (DMSO-d6, 100 MHz) 174.2, 155.9, 136.9, 128.4, 128.3, 1.53–1.64 (m, 3H), 1.24–1.36 (m, 16H), 0.89 (t, 3H, J = 8.1 Hz);
127.9, 127.8, 127.6, 75.4, 65.8, 51.4, 34.7, 7.8; HRMS (ESI) m/z δC (CDCl3, 100 MHz) 206.4, 175.7, 169.7, 79.3, 77.7, 54.8, 51.0,
calcd for C13H14INNaO4 [M + Na]+ 397.9860, found 397.9861.
43.6, 34.9, 33.1, 32.1, 30.8, 30.5, 30.2, 26.9, 26.1, 24.6, 23.9,
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14.6; HRMS (ESI) m/z C19H32N4O4 [M + Na]+ 403.2316, found: 21 C. Götz, A. Fekete, I. Gebefuegi, S. Forczek, K. Fuksová,
403.2329.
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Acknowledgements
The authors thank Prof. Dr Anton Hartmann, Helmholtz
Centre Munich, for the kind gift of biosensor bacteria used in
this study. The work of STS and AS was supported by the
Bundesanstalt für Landwirtschaft und Ernährung (BLE) grant
no. 2811NA033.
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