Synthesis and Biological Evaluation of Cajaninstilbene Acid and Amorfrutins A and B as Inhibitors of the Pseudomonas aeruginosa Quorum Sensing System

Article abstract of DOI:10.1021/acs.jnatprod.8b00315

The quorum sensing (QS) system inhibitors of Pseudomonas aeruginosa are thought to attenuate bacterial pathogenicity and drug resistance by inhibiting biofilm formation and the production of virulence factors. In this study, a synthetic approach to the na

Full text of DOI:10.1021/acs.jnatprod.8b00315

Article
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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Synthesis and Biological Evaluation of Cajaninstilbene Acid and
Amorfrutins A and B as Inhibitors of the Pseudomonas aeruginosa
Quorum Sensing System
Xing-Jun Xu, Ting Zeng, Zhi-Xing Huang, Xiao-Fang Xu, Jing Lin,* and Wei-Min Chen*
College of Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China
S
* Supporting Information
ABSTRACT: The quorum sensing (QS) system inhibitors of Pseudomonas aeruginosa
are thought to attenuate bacterial pathogenicity and drug resistance by inhibiting
biofilm formation and the production of virulence factors. In this study, a synthetic
approach to the natural products cajaninstilbene acid (1) and amorfrutins A (2) and B
(3) has been developed and was characterized by the Heck reaction, which was used
to obtain the stilbene core and a Pinick oxidation to give the O-hydroxybenzoic acid.
The biological activities of these compounds against the P. aeruginosa quorum sensing
systems were evaluated. Amorfrutin B (3) showed promising antibiofilm activity
against P. aeruginosa PAO1 with a biofilm inhibition ratio of 50.3 2.7. Three lacZ
reporter strains were constructed to identify the effects of compound 3 on different
QS systems. Suppression efficacy of compound 3 on the expression of lasB-lacZ and
pqsA-lacZ as well as on the production of their corresponding virulence factors elastase
and pyocyanin was observed.
In view of the biological potential of cajaninstilbene acid (1)
and amorfrutins A (2) and B (3), several synthetic routes for
these compounds have been developed. These are focused on
manipulation of the double bond in the stilbene unit parent,
and various protocols have been developed, including use of
the (1) Wittig reaction,11 (2) benzylic metalation followed by
alkylation,12 and (3) Sonogashira coupling to link an
aryltriflate with a phenylacetylene.13 In a recent report of the
synthesis of cajaninstilbene acid (1) and amorfrutin A (2) and
their analogues, Julia olefination and monobenzylation
followed by desulfonylation with SmI2 were used as key
reactions.14 These methods all must use methyl 2-acetoxy-6-
(bromomethyl)-4-methoxybenzoate as the key intermediate,
prepared in a four- or five-step synthesis that can be quite
expensive. Thus, a concise efficient strategy to synthesize these
compounds in fewer steps and under mild conditions may
promote the development of such natural products.
In this study, a synthetic approach to the synthesis of
cajaninstilbene acid (1) and amorfrutins A (2) and B (3) was
developed. The activities of the final compounds and key
intermediates against the biofilm formation and quorum
sensing systems of P. aeruginosa were evaluated. Of these,
amorfrutin B (3) exhibited good antibiofilm activities. The
regulated effects of amorfrutin B on QS systems were
investigated using a lacZ-based bioreporter strain by
monitoring the gene expression of the las, rhl, and pqs QS
systems, and these effects were quantified by measurement of
the production of related virulence factors.
ultiple drug resistant (MDR) bacterial strains directly
Mcause the death of more than 16 million people
annually.1 At least 65% of these cases are linked to bacterial
communities that proliferate by forming biofilms.2 Bacterial
community activities, such as the production of numerous
extracellular proteases, biofilm maturation factors, and toxins,
are all associated with intercellular communication or “quorum
sensing” (QS).3,4 As an example, one of the most common of
the Gram-negative bacteria found in hospital-acquired
infections, Pseudomonas aeruginosa, has become increasingly
resistant to most current antibiotic therapies as a result of its
development of QS systems and its acquisition of antibiotic-
resistant genes by horizontal gene transfer. P. aeruginosa has a
relatively complex QS network that includes, at a minimum,
las, rhl, and pqs pathways, which can cause pathogenicity. It is
considered important to develop approaches to treat P.
aeruginosa infections by regulating these QS systems.5
Chemical strategies to inhibit QS in P. aeruginosa have
received significant recent attention.6 Interestingly, a long
lipophilic chain, the tail, accompanied by a polar protic moiety,
the head, appear to be common structural motifs of QS
inhibitors shared across multiple species.5 The natural
products cajaninstilbene acid (1) and its analogues amorfrutins
A (2) and B (3) (Figure 1), which have been identified with
many biological activities, including antibacterial,7 cytotoxic,8
PPARγ agonist,9 and anti-inflammatory effects,10 have a long
lipophilic isopentenyl or geranyl (tail) and O-hydroxybenzoic
acid core (head) and thus share the structural characteristics of
QS signaling molecules. Consequently, active natural products
of this sort are likely to be potential QS inhibitors.
Received: April 20, 2018
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.8b00315
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Figure 1. Structures of cajaninstilbene acid (1), amorfrutin A (2), and amorfrutin B (3).
Scheme 1. Synthesis Procedure of Cajaninstilbene Acid (1) with Low Yield
converted to the corresponding acetate 10 in 98% yield under
standard conditions. Pinnick oxidation of 10 to the
corresponding acid and hydrolysis of the acetate gave the
natural product 1, as shown in Scheme 2, for which the
structure was confirmed spectroscopically.
RESULTS AND DISCUSSION
■
Total Synthesis of Cajaninstilbene Acid (1). An initial
approach to the synthesis of cajaninstilbene acid (1) is shown
in Scheme 1. The benzaldehyde (5) was prepared starting from
1-bromo-3,5-dimethoxybenzene (4) by a Vilsmeyer formyla-
tion15 that proceeded with high yield (95%). A ligand-free
Heck reaction16 was reported by Yao’s group in 2003. Using
the same protocol, product 6 was obtained in a 90% yield.
Then, ortho-demethylation, giving 7 was achieved with the
Lewis acid boron trichloride. With intermediate 7 in hand, a C-
prenylation reaction was accomplished as the next step.
Compound 7 was treated with sodium hydride as a base and
prenyl bromide as an electrophile in toluene at 65 °C. This
generated the desired C-prenylated compound (8) with a yield
of 36%, the O-prenylated compound (9) in a 33% yield, and
recovered starting material 7 (28%). Although the yield of C-
prenylated compound (8) was acceptable, it was found that the
O-prenylated compound (9) can be converted to the C-
prenylated compound (8) in a 40% yield by a rearrangement
induced by Montmorillonite K10.14 Thus, the conversion of 7
to the C-prenylated compound (8) can be as high as 49% with
these two transformations. The next step was the oxidation of
the α-hydroxy benzylaldehyde to α-hydroxybenzoic acid.
Pinnick oxidation conditions gave only 10% yield of an impure
product, and other oxidation systems, such as AgNO3/H2O2
and Ag2O/NaOH, all failed.17,18
Scheme 2. Optimized Total Synthesis Procedure of
Cajaninstilbene Acid (1)
Total Synthesis of Amorfrutins A (2) and B (3). The
synthesis protocol of amorfrutins A and B is shown in Scheme
3. Following the same multiple step strategy used in the
synthesis of cajaninstilbene acid (1), started from the
intermediate (6), for which the double bond was reduced
with hydrogen in the presence of 10% palladium on carbon to
give 12 (95%). The dimethyl product (13) was produced
quantitatively by following the procedure by which 7 was
produced from 6. The subsequent C-alkylation reaction with
3,3-dimethylallyl bromide and geranyl bromide as C-alkyation
reagents, but under the same conditions used for the
1
The H NMR spectrum of compound 8 was measured, and
it was found that there was an intramolecular hydrogen bond
occurring between the hydroxy group and the aldehyde, which
caused the signals from these groups to move downfield by
about 2 ppm. In addition, the phenolic hydroxy group could be
oxidized to the quinone, and, to avoid this, the O-hydroxy
group was protected before oxidation. The intermediate 8 was
B
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Scheme 3. Total Synthesis of Amorfrutins A (2) and B (3)
Table 1. Biofilm Inhibition Rates of Cajaninstilbene Acid
(1), Amorfrutins A (2) and B (3), and Key Intermediates
a
compound
biofilm inhibition ratio (%)
1
2
−41.0 5.9
−31.3 1.1
b
3
50.3 2.7***
8
9
42.4 0.4***
−14.3 1.6
−12.5 0.7
−24.4 1.2
26.9 2.6***
53.1 1.9***
−32.1 0.1
24.7 2.5***
36.9 0.5***
10
11
14a
14b
15a
15b
16a
16b
−32.9 2.1
b
a
Tested compounds were used at 50 μM. ***p < 0.005 indicates
statistically significant differences from the untreated control group.
Figure 2. Histogram of biofilm formation rates following treatment
with the target compounds cajaninstilbene acid (1), amorfrutins A (2)
and B (3), and key intermediates (8−11, 14a, 14b, 15a, 15b, 16a, and
16b). ***p < 0.005 indicates statistically significant differences from
the untreated control group.
conversion of 7 to 8, gave 14a (38%) and 14b (34%),
respectively. Compounds 14a and 14b were protected
separately by acetylation and Pinnick oxidation of the aldehyde
group to the acid, and deprotection with base gave amorfrutin
A (2) and amorfrutin B (3). The structures of the amorfrutins
were established by NMR and mass spectrometry.
compound 3 on the P. aeruginosa PAO1 biofilm formation was
further confirmed by concentration gradient experiments. As
shown in Figure 3A, biofilm formation was reduced 20−50%
by treatment with compound 3 in a concentration-dependent
manner. The toxicity of compound 3 on the growth of PAO1
at the same concentrations was also evaluated. As shown in
Figure 3B, the curve representing treatment with compound 3
corresponded well with the control curve. This suggested that
compound 3 at a range of concentrations between 1 and 50
μM had no influence on the growth of bacteria. These results
demonstrate that compound 3 can serve as a potential specific
biofilm formation inhibitor.
Involvement of Compound 3 in Quorum Sensing
Inhibition. Quorum sensing circuits, including the las, rhl, and
pqs pathways, are the core systems responsible for the
regulation of biofilm formation and the secretion of virulence
factors in P. aeruginosa. To explore the mechanism of
inhibition by compound 3, three reporter strains were used,
PAO1-mini-ctx-lasB-lacZ, PAO1-mini-ctx-rhlA-lacZ, and PAO1-
mini-ctx-pqsA-lacZ, in which the lacZ gene was integrated into
the PAO1 genome. Production of β-galactosidase in these
strains is known to reflect the activity of the promoters of the
lasB, rhlA, and pqsA genes19,20 and were constructed as
illustrated in Figure 4. In this assay, PAO1-mini-ctx-lacZ was
used as a control in the investigation of the influence of
Evaluation of the Antibiofilm Activity of Compounds
1−3 and Some Key Intermediates. A large number of
publications have shown that pathogenic bacteria such as P.
aeruginosa employed quorum sensing to regulate biofilm
development. It is becoming increasingly clear that quorum-
sensing regulation can control bacterial biofilm formation.
Consequently, the antibiofilm activities of all synthesized
compounds, including the final compounds 1−3 and some key
intermediates, which have many of the structural characteristics
of QS regulators, were evaluated initially in vitro with a crystal
violet staining assay using a PAO1 strain of P. aeruginosa. The
results of screening in Table 1 and Figure 2, showed that
compounds 3, 8, 14a, 14b, 15b, and 16a actively inhibited the
formation of biofilm. Compounds 3 and 14b showed
inhibition of more than 50%. It was found that the οrtho-
hydroxybenzoic acid or the οrtho-hydroxybenzaldehyde in the
structure of cajaninstilbene acid (1) and its analogues was key
to biofilm inhibition. If the carbon−carbon double bond
between the two benzene rings was reduced, the antibiofilm
activity was enhanced. Generally, similar compounds with a
geranyl substituent showed better antibiofilm activity than the
compounds with an isopentenyl group. Amorfrutin B (3) is a
natural product that exhibits good activity, and it was selected
for the further study of its antibiofilm activity. The effect of
C
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Figure 3. (A) Effects of different concentrations of compound 3 on biofilm formation. (B) Growth curve of P. aeruginosa PAO1 after treatment
with different concentrations of compound 3. ***p < 0.005 indicates statistically significant differences from the untreated control group.
Figure 4. Schematic diagram of reporter strain PAO1-mini-ctx-pqsA-lacZ and the mechanism of the effect of compound 3 on the pqs system.
compound 3 on the expression of the lacZ gene. As shown in
Figure 5A, no obvious impact of compound 3 on β-
galactosidase activity was observed, and consequently the
constructed lacZ reporter strain could be used to explore the
interaction of 3 with this gene. As expected, although
compound 3 had little effect on the rhl system since it did
not depress the expression of β-galactosidase except at 50 μM
(Figure 5C), the expression of both the las and pqs systems
was inhibited by compound 3 in a concentration-dependent
manner (Figure 5B and D). The amount of β-galactosidase
decreased after the addition of a 5 μM concentration of
compound 3. These findings confirm that compound 3 may
play a role in biofilm inhibition by interrupting the las and pqs
systems.
Effect of Compound 3 on Virulence Production. As
indicated by the lacZ reporter strain, it can be concluded that
compound 3 had a significant effect on the las and pqs systems.
To further verify the inhibitory effect of compound 3 on these
two systems, the production of the three virulence factors
elastase, rhamnolipid, and pyocyanin, which are directly
controlled by the las, rhl, and pqs system, respectively, was
measured. Consistent with the reporter strain assay results,
compound 3 at concentrations below 50 μM had no
discernible effects on the production of rhamnolipid that is
under the control of the rhl system. On the other hand,
compound 3 reduced the production of elastase and
pyocyanin, which are under the control of the las and pqs
systems, respectively. As shown in Figure 6A and C, compound
3 suppressed about 50% of the pyocyanin and elastase
expression at 10 μM. These results further demonstrate that
compound 3 serves as an inhibitor of the las and pqs pathways.
Elastase can degrade the elastin, collagen, and other matrix
proteins, and consequently, it can delay the healing of
wounds,21,22 while pyocyanin can easily penetrate biological
membranes and has crucial roles in P. aeruginosa infection. A
considerable amount of research has indicated that pyocyanin
can interfere with multiple cellular functions in vitro. This can
lead to a wide spectrum of cellular damage and can induce
neutrophil apoptosis and impair neutrophil-mediated host
defenses in vivo.23,24 More significantly, chronic exposure to
pyocyanin can induce destruction of the alveolar airspace in
mice, and pyocyanin is closely related to the aggravation of
cystic fibrosis.25 Thus, reduction of the virulence factor itself
D
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Figure 5. Dose-dependent inhibition curves of compound 3 incubated with the QS monitors PAO1-mini-ctx-lasB-lacZ, rhlA-lacZ, and pqsA-lacZ.
PAO1-mini-ctx-lacZ was selected as a control to explore the effect on the expression of the lacZ gene after treatment with compound 3. ***p <
0.005, **p < 0.01, *p < 0.05 indicate statistically significant differences from the untreated control group. The experiments were done in triplicate.
Figure 6. Effects of compound 3 on virulence production. (A) Elastase activity. (B) Rhamnolipid production. (C) Pyocyanin production.
PAO1ΔlasIΔrhlI was used as a blank, and the control used DMSO only, rather than a test compound under the same conditions. Error bars are
means SDs. ***p < 0.005 indicate statistically significant differences from the untreated control group.
Figure 7. Dose-dependent inhibition histogram of compound 3 incubated with different concentrations of ODdHL and PQS, respectively. ***p <
0.005, **p < 0.01, *p < 0.05 indicate statistically significant differences from the DMSO control group. The experiments were performed in
triplicate.
and attenuating bacterial toxicity and infectivity has great
clinical significance.
To further confirm that compound 3 can competitively bind
ODdHL and PQS, competitive assays were conducted. The
reporter strains PAO1-mini-ctx-lasB-lacZ and PAO1-mini-ctx-
pqsA-lacZ were treated with compound 3 (50 μM) together
with different concentrations of autoinducers. As shown in
the receptors of native AHLs from P. aeruginosa such as
E
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concentrated to dryness under vacuum. The crude product was
purified by flash chromatography on silica (2:1 hexane−EtOAc).
Deprotection of the Hydroxy Group, General Procedure E.
K2CO3 (2.0 equiv) was added to a stirred solution of substrate (1.0
equiv) in MeOH (0.20 M) at rt. The mixture was stirred at rt for 8 h.
The solvent was removed under vacuum, and 2.0 M HCl was added
until the pH was 6. The aqueous phase was extracted with CH2Cl2 (3
× 15 mL). The combined organic extracts were washed with brine,
dried over anhydrous Na2SO4, and concentrated to dryness under
vacuum. The crude product was purified by flash chromatography on
silica (3:1 hexane−EtOAc).
2-Bromo-4,6-dimethoxybenzaldehyde (5). POCl3 (6.4 mL, 69.0
mmol) was added dropwise to a stirred solution of 3,5-
dimethoxybromobenzene (5.00 g, 23.0 mmol) in DMF (10.8 mL,
138.0 mmol) at 0 °C. The resulting mixture was stirred at rt for 0.5 h,
then heated to 100 °C, and stirring was continued for 4 h. The
reaction mixture was poured onto crushed ice (100 mL), warmed to
rt, left at rt for 16 h, and then filtered. The filter cake was dissolved in
ether, and insoluble materials were filtered off. The filtrate was
concentrated, and the resulting crude product was purified by flash
chromatography on silica (5:1 hexane−acetone) to give pure product
5 as a white solid: mp 94.8−95.7 °C; 1H NMR (300 MHz, CDCl3) δ
10.29 (1H, s), 6.77 (1H, d, J = 2.3 Hz), 6.42 (1H, d, J = 2.3 Hz), 3.88
(3H, s), 3.85 (3H, s); 13C NMR (75 MHz, CDCl3) δ 189.3, 164.6,
163.7, 127.5, 117.0, 111.7, 98.3, 56.2, 56.0; ESIMS m/z 245.0 [M +
H]+; HRMS m/z 244.9811 [M + H]+, calcd for C9H979BrO3,
244.9808. The spectroscopic data were in agreement with those
reported in the literature.15
Figure 7, compound 3 did not inhibit Las and Pqs pathways
with increasing concentrations of ODdHL and PQS. These
results again support the conclusion that compound 3 inhibits
the QS system, which involves competition with the receptors
of native AHLs from P. aeruginosa.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All reagents and solvents
were purchased from commercial sources and used without further
purification. CH2Cl2, dimethylacetamide (DMA) and dimethylforma-
mide (DMF) were distilled from CaH2; toluene was distilled from Na.
Thin-layer chromatography (TLC) analysis was conducted on
Qingdao Haiyang GF254 silica gel plates. Flash chromatography was
performed using Qingdao Haiyang 200−300 mesh silica gel. Melting
points were measured with an Optimelt automated melting point
system and are uncorrected. Unless otherwise specified, 1H NMR and
13C NMR spectra were recorded at room temperature in base-filtered
CDCl3 on a Varian spectrometer operating at 300 MHz for protons
and 75 MHz for carbon nuclei. The signal due to residual CHCl3
appearing at δH 7.26 and the central resonance of the CDCl3 triplet
appearing at δC 77.16 were used to reference 1H and 13C NMR
spectra, respectively. 1H NMR data were recorded as follows:
chemical shift (δ) [multiplicity, coupling constant(s) J (Hz), relative
integral], where multiplicity is defined as s = singlet; d = doublet; t =
triplet; q = quartet; m = multiplet or combinations of the above. Low-
resolution ESIMS were recorded on a single quadrupole liquid
chromatograph−mass spectrometer, while high-resolution measure-
ments were conducted on a time-of-flight instrument.
Demethylation by BCl3, General Procedure A. A 1.0 M
solution of BCl3 in CH2Cl2 (1.5 equiv) was added dropwise at 0 °C to
a solution of substrate (1.0 equiv) in CH2Cl2 (0.20 M). After being
stirred at the same temperature for 5 min, the mixture was warmed to
rt and stirred for 1 h. The reaction mixture was then poured into cold
water and extracted with CH2Cl2. The combined organic layer was
washed successively with saturated NaHCO3 and brine and then dried
over anhydrous Na2SO4. The solvent was evaporated under vacuum,
and the crude product was purified by column chromatography on
silica gel and eluted with a mixture of hexane and EtOAc.
C-Alkylation, General Procedure B. A solution of the substrate
(1.0 equiv) in anhydrous toluene (0.20 M) was added dropwise at 0
°C to a stirred solution of NaH (1.2 equiv) in anhydrous toluene
(0.20 M). The combined mixture was stirred for 0.5 h, the C-
alkylation reagent (1.3 equiv) was added by syringe, and then the
solution was heated to 65 °C for 3 h. After cooling to rt, the solution
was quenched with saturated NH4Cl solution and extracted with
EtOAc (3 × 50 mL). The combined organic extracts were washed
with brine, dried over anhydrous Na2SO4, and concentrated to
dryness under vacuum. The crude product was purified by flash
chromatography on silica (30:1 hexane−EtOAc).
(E)-2,4-Dimethoxy-6-styrylbenzaldehyde (6). An oven-dried 100
mL Schlenk flask was charged under Ar with K3PO4 (5.77 g, 27.2
mmol) and compound 5 (5.20 g, 19.4 mmol) in dimethylacetamide
(DMA) (25 mL). Styrene (2.60 mL, 23.3 mmol) was added by
syringe. Pd(OAc)2 (0.061 g, 0.27 mmol) was then added, and the
Schlenk tube was sealed under Ar and placed in an oil bath preheated
to 120 °C; the reaction mixture was stirred for 5 h at 120 °C. After
being cooled to rt, the reaction mixture was poured into water (150
mL) and extracted with EtOAc (3 × 100 mL). The combined organic
extracts were washed with brine, dried over anhydrous Na2SO4, and
concentrated to dryness under vacuum. The crude product was
purified by flash chromatography on silica (5:1 hexane−EtOAc) to
give pure trans-stilbene (6, 4.68 g, 90%) as white solid: mp 95.1−96.5
1
°C; H NMR (300 MHz, CDCl3) δ 10.53 (1H, s), 8.17 (1H, d, J =
16.2 Hz), 7.61−7.52 (2H, m), 7.42−7.32 (2H, m), 7.33−7.22 (1H,
m), 7.00 (1H, d, J = 16.2 Hz), 6.75 (1H, d, J = 2.2 Hz), 6.40 (1H, d, J
= 2.2 Hz), 3.91 (3H, s), 3.88 (3H, s); 13C NMR (75 MHz, CDCl3) δ
190.6, 165.1, 164.7, 142.9, 137.3, 132.5, 128.7, 128.1, 127.9, 127.1,
116.3, 103.8, 97.3, 56.0, 55.7; ESIMS m/z 269.1 [M + H]+; HRMS
m/z 269.1184 [M + H]+, calcd for C17H16O3, 269.1172.
(E)-2-Hydroxy-4-methoxy-6-styrylbenzaldehyde (7). Following
procedure A, the substrate, compound 6 (1.50 g, 5.60 mmol), gave
1
compound 7 (1.39 g, 98%) as a white solid: H NMR (300 MHz,
Protection of Hydroxy Groups by Acetylation, General
Procedure C. Triethylamine (1.2 equiv) and Ac2O (1.1 equiv) were
added to a stirred solution of substrate (1.0 equiv) in CH2Cl2 (0.20
M) at 0 °C; then dimethylaminopyridine (DMAP) (0.05 equiv) was
added. The mixture was warmed to rt and stirred for 1 h. It was
quenched with a saturated NH4Cl solution and extracted with EtOAc
(3 × 50 mL). The combined organic extracts were washed with brine,
dried over anhydrous Na2SO4, and concentrated to dryness under
vacuum. The crude product was purified by flash chromatography on
silica (10:1 hexane−EtOAc).
Pinnick Oxidation, General Procedure D. Tetrahydrofuran
(1.00 mL/mmol) and H2O (1.00 mL/mmol) at rt, NaH2PO4·2H2O
(2.0 equiv), and 2-methyl-2-butene (6.0 equiv) were added to a
stirred solution of substrate (1.0 equiv) in t-BuOH (5.00 mL/mmol).
The mixture was stirred for 5 min, and then NaClO2 (2.0 equiv) was
added. The reaction mixture was stirred at rt for 4 h. Then the solvent
was removed under vacuum, water was added, and the aqueous phase
was extracted with EtOAc (3 × 15 mL). The combined organic
extracts were washed with brine, dried over anhydrous Na2SO4, and
CDCl3) δ 12.49 (1H, s), 10.20 (1H, s), 7.56−7.45 (3H, m), 7.45−
7.28 (3H, m), 6.98 (1H, d, J = 15.9 Hz), 6.61 (1H, d, J = 2.3 Hz),
6.37 (1H, d, J = 2.3 Hz), 3.87 (3H, s); 13C NMR (75 MHz, CDCl3) δ
193.0, 166.7, 166.3, 144.4, 136.4, 135.7, 129.0, 128.8, 127.0, 122.9,
112.5, 107.1, 100.0, 55.8; ESIMS m/z 255.1 [M + H]+.; HRMS m/z
255.1019 [M + H]+, calcd for C16H14O3, 255.1016.
(E)-2-Hydroxy-4-methoxy-3-(3-methylbut-2-en-1-yl)-6-styrylben-
zaldehyde (8) and (E)-4-Methoxy-2-((3-methylbut-2-en-1-yl)oxy)-6-
styrylbenzaldehyde (9). Following procedure B, the substrate,
compound 7 (1.39 g, 5.50 mmol), and 3,3-dimethylallyl bromide as
a C-alkylation reagent gave the product 8 (0.63 g, 36%) as yellow
1
solid: mp 139.2−141.7 °C; H NMR (300 MHz, CDCl3) δ 12.41
(1H, s), 10.23 (1H, s), 7.60−7.48 (3H, m), 7.45−7.29 (3H, m), 6.95
(1H, d, J = 15.9 Hz), 6.61 (1H, s), 5.21 (1H, tdt, J = 5.7, 2.8, 1.4 Hz),
3.96 (3H, s), 3.36 (2H, d, J = 7.2 Hz), 1.80 (3H, d, J = 1.4 Hz), 1.69
(3H, d, J = 1.4 Hz); 13C NMR (75 MHz, CDCl3) δ 193.3, 163.8,
162.4, 142.5, 136.5, 135.2, 132.3, 129.0, 128.7, 126.9, 123.6, 121.8,
116.9, 112.9, 102.0, 56.0, 26.0, 21.6, 17.9; ESIMS m/z 323.2 [M +
H]+; HRMS m/z 323.1650 [M + H]+, calcd for C21H22O3, 323.1642.
F
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The O-alkylation compound 9 (0.58 g, 33%) was prepared as yellow
solid: mp 75.9−77.3 °C; H NMR (300 MHz, CDCl3) δ 10.56 (1H,
55.9, 55.5, 37.6, 37.3; ESIMS m/z 271.0 [M + H]+; HRMS m/z
271.1335 [M + H]+, calcd for C17H18O3, 271.1329.
1
s), 8.19 (1H, d, J = 16.2 Hz), 7.64−7.51 (2H, m), 7.41−7.32 (2H,
m), 7.27 (1H, tt, J = 6.4, 1.4 Hz), 7.00 (1H, d, J = 16.2 Hz), 6.76 (1H,
d, J = 2.2 Hz), 6.42 (1H, d, J = 2.3 Hz), 5.49 (1H, tdd, J = 6.4, 2.9, 1.4
Hz), 4.60 (2H, d, J = 6.7 Hz), 3.91 (3H, d, J = 0.8 Hz), 1.81 (3H, d, J
= 1.4 Hz), 1.76 (3H, d, J = 1.3 Hz); 13C NMR (75 MHz, CDCl3) δ
191.0, 164.6, 142.8, 138.9, 137.4, 132.4, 128.7, 128.1, 127.2, 119.0,
116.7, 103.8, 98.5, 65.9, 55.7, 25.9, 18.4; ESIMS m/z 323.2 [M + H]+;
HRMS m/z 323.1642 [M + H]+, calcd for C21H22O3, 323.1642.
(E)-2-Hydroxy-4-methoxy-3-(3-methylbut-2-en-1-yl)-6-styrylben-
zaldehyde (8). Montmorillonite K10 (0.59 g) was added to a solution
of compound 9 (0.58 g, 1.80 mmol) in anhydrous CH2Cl2 (10 mL),
and the reaction mixture stirred at rt for 8 h. The progress of the
reaction was monitored by TLC. After complete consumption of
starting material, the reaction mixture was filtered through a Celite
bed and the filtrate obtained was concentrated under vacuum. The
crude compound was purified by silica gel column chromatography to
obtain the title compound 8 (0.23 g, 40%) as a yellow solid together
with the deprenylated compound 7 (0.29 g, 50%) as the major side
product.
2-Hydroxy-4-methoxy-6-phenethylbenzaldehyde (13). Following
procedure A, compound 12 (2.70 g, 10.00 mmol) as the substrate
gave 2.51 g (98%) of compound 13 as a white solid: mp 131.1−134.0
1
°C; H NMR (300 MHz, CDCl3) δ 12.53 (1H, s), 10.01 (1H, s),
7.41−7.23 (5H, m), 7.22−7.09 (3H, m), 6.43−6.05 (3H, m), 3.84
(4H, s), 3.43- 3.06 (2H, m), 3.05−2.89 (2H, m); 13C NMR (75 MHz,
CDCl3) δ 192.5, 166.8, 166.7, 147.8, 140.5, 128.7, 128.5, 126.6, 112.6,
109.9, 99.0, 55.7, 38.7, 33.9; ESIMS m/z 257.1 [M + H]+; HRMS m/
z 257.1183 [M + H]+, calcd for C16H16O3, 257.1172.
2-Hydroxy-4-methoxy-3-(3-methylbut-2-en-1-yl)-6-phenethyl-
benzaldehyde (14a). Following procedure B, compound 13 (1.00 g,
3.90 mmol) as the substrate and 3,3-dimethylallyl bromide as a C-
alkylation reagent gave product 14a (0.48 g, 38%) as a yellow oil: 1H
NMR (300 MHz, CDCl3) δ 12.43 (1H, s), 10.06 (1H, s), 7.37−7.23
(3H, m), 7.19−7.09 (2H, m), 6.22 (1H, s), 5.20 (1H, dddd, J = 8.5,
5.7, 2.8, 1.4 Hz), 3.83 (3H, s), 3.40−3.27 (2H, m), 3.16 (2H, dd, J =
9.2, 6.5 Hz), 2.94 (2H, dd, J = 9.2, 6.5 Hz), 1.79 (3H, d, J = 1.3 Hz),
1.69 (3H, d, J = 1.4 Hz); 13C NMR (75 MHz, CDCl3) δ 192.8, 163.9,
162.8, 145.9, 140.6, 132.0, 128.7, 128.6, 126.5, 122.0, 115.5, 112.9,
104.9, 55.8, 39.2, 34.2, 25.9, 21.4, 17.9; ESIMS m/z 325.2 [M + H]+;
HRMS m/z 325.1808 [M + H]+, calcd for C21H24O3 325.1798.
(E)-3-(3,7-Dimethylocta-2,6-dien-1-yl)-2-hydroxy-4-methoxy-6-
phenethylbenzaldehyde (14b). Following procedure B, compound
13 (1.00 g, 3.90 mmol) as the substrate and geranyl bromide as a C-
alkylation reagent gave product 14b as a yellow oil (0.52 g, 34%): 1H
NMR (300 MHz, CDCl3) δ 12.47 (1H, s), 10.09 (1H, s), 7.37−7.25
(4H, m), 7.20−7.14 (2H, m), 6.25 (1H, s), 5.23 (1H, q, J = 1.3 Hz),
5.11 (1H, tt, J = 5.5, 3.1 Hz), 3.86 (3H, s), 3.36 (2H, d, J = 7.1 Hz),
3.24−3.15 (2H, m), 2.98 (2H, d, J = 8.6 Hz), 2.14−2.05 (3H, m),
2.05−1.96 (3H, m), 1.82 (3H, d, J = 1.3 Hz), 1.69 (3H, d, J = 1.3
Hz), 1.62 (3H, d, J = 1.3 Hz); 13C NMR (75 MHz, CDCl3) δ 192.7,
163.9, 162.8, 145.8, 140.6, 135.4, 131.2, 128.6, 128.5, 126.5, 124.5,
121.8, 115.5, 112.8, 104.8, 55.8, 39.9, 39.2, 34.2, 26.8, 25.8, 21.3, 17.8,
16.2; ESIMS m/z 393.2 [M + H]+; HRMS m/z 393.233 [M + H]+,
calcd for C26H32O3 393.2424.
(E)-2-Formyl-5-methoxy-6-(3-methylbut-2-en-1-yl)-3-styrylphen-
yl Acetate (10). Following procedure C, compound 8 (0.50 g, 1.55
mmol) as the substrate gave product 10 (0.55 g, 98%) as a white
1
solid: mp 97.2−99.6 °C; H NMR (300 MHz, CDCl3) δ 10.23 (1H,
s), 7.83 (1H, d, J = 16.1 Hz), 7.59−7.50 (2H, m), 7.43−7.34 (2H,
m), 7.34−7.27 (1H, m), 6.93 (1H, s), 6.95 (2H, d, J = 16.1), 5.10
(1H, tt, J = 7.1, 1.4 Hz), 3.97 (3H, s), 3.28 (2H, d, J = 7.0 Hz), 2.40
(3H, s), 1.77 (3H, d, J = 1.4 Hz), 1.69 (3H, d, J = 1.4 Hz); 13C NMR
(75 MHz, CDCl3) δ 188.6, 169.5, 162.2, 151.2, 142.0, 136.7, 134.6,
132.5, 128.9, 128.5, 127.1, 125.5, 123.4, 121.0, 119.2, 107.0, 56.1,
25.8, 23.0, 20.9, 17.9; ESIMS m/z 365.2 [M + H]+; HRMS m/z
365.1759 [M + H]+, calcd for C23H24O4, 365.1747.
(E)-2-Acetoxy-4-methoxy-3-(3-methylbut-2-en-1-yl)-6-styrylben-
zoic Acid (11). Following procedure D, compound 10 (0.55 g, 1.51
mmol) as the substrate gave oxidation product 10 (0.50 g, 88%) as
1
white solid: mp 135.3−138.1 °C; H NMR (300 MHz, CDCl3) δ
10.76 (1H, s), 7.62 (1H, d, J = 16.1 Hz), 7.53 (2H, d, J = 7.5 Hz),
7.36 (2H, t, J = 7.4 Hz), 7.29 (1H, d, J = 7.2 Hz), 7.07 (1H, s), 7.01
(1H, d, J = 16.1 Hz), 5.13 (1H, t, J = 7.0 Hz), 3.97 (3H, s), 3.28 (2H,
d, J = 7.0 Hz), 2.31 (3H, s), 1.77 (3H, s), 1.70 (3H, s); 13C NMR (75
MHz, CDCl3) δ 172.0, 169.5, 160.4, 149.1, 138.6, 137.0, 132.3, 131.8,
128.8, 128.2, 127.0, 126.9, 123.5, 121.2, 116.1, 106.2, 56.0, 25.8, 23.5,
20.9, 17.9; ESIMS m/z 379.1 [M − H]−; HRMS m/z 379.1549 [M −
H]−, calcd for C23H24O5, 379.1551.
2-Formyl-5-methoxy-6-(3-methylbut-2-en-1-yl)-3-phenethyl-
phenyl Acetate (15a). Following procedure C, compound 14a (0.48
g, 1.48 mmol) as the substrate gave the product 15a (0.52 g, 96%) as
a colorless oil: H NMR (300 MHz, CDCl3) δ 10.18 (1H, s), 7.35−
7.26 (2H, m), 7.25−7.17 (3H, m), 6.50 (1H, s), 5.07 (1H, tt, J = 7.0,
1.5 Hz), 3.82 (3H, s), 3.33−3.18 (4H, m), 2.91 (2H, dd, J = 9.3, 6.6
Hz), 2.39 (3H, s), 1.75 (3H, d, J = 1.3 Hz), 1.68 (3H, d, J = 1.5 Hz);
13C NMR (75 MHz, CDCl3) δ 188.3, 169.5, 162.1, 152.0, 146.0,
1
(E)-2-Hydroxy-4-methoxy-3-(3-methylbut-2-en-1-yl)-6-styrylben-
zoic Acid (1). Following procedure E, compound 11 (0.50 g, 1.32
mmol) as the substrate gave product 1 (0.42 g, 95%) as a white solid:
mp 170.7−173.8 °C; H NMR (300 MHz, acetone-d6) δ 12.27 (1H,
141.2, 132.3, 128.7, 128.5, 126.2, 122.0, 121.2, 119.3, 110.8, 56.0,
38.3, 35.9, 25.8, 22.9, 20.9, 17.9; ESIMS m/z 367.2 [M + H]+; HRMS
m/z 367.1914 [M + H]+, calcd for C23H26O4, 367.1904.
1
(E)-2-(3,7-Dimethylocta-2,6-dien-1-yl)-6-formyl-3-methoxy-5-
phenethylphenyl Acetate (15b). Following procedure C, compound
14b (0.52 g, 1.32 mmol) as the substrate gave product 15b (0.54 g,
93%) as a colorless oil: 1H NMR (300 MHz, CDCl3) δ 10.19 (1H, s),
7.35−7.25 (2H, m), 7.22 (3H, d, J = 7.0 Hz), 6.50 (1H, s), 5.08 (2H,
ddd, J = 6.9, 5.4, 2.9 Hz), 3.81 (3H, s), 3.33−3.19 (4H, m), 2.92 (2H,
dd, J = 9.3, 6.6 Hz), 2.38 (3H, s), 2.07 (2H, q, J = 7.6, 6.4 Hz), 2.01−
1.92 (2H, m), 1.75 (3H, d, J = 1.4 Hz), 1.66 (3H, d, J = 1.4 Hz), 1.59
(3H, d, J = 1.3 Hz); 13C NMR (75 MHz, CDCl3) δ 188.3, 169.5,
162.1, 152.1, 145.9, 141.2, 135.9, 131.4, 128.7, 128.5, 126.2, 124.3,
122.1, 121.2, 119.3, 110.8, 55.9, 39.7, 38.2, 35.8, 26.7, 25.8, 22.8, 20.8,
17.8, 16.2; ESIMS m/z 435.3 [M + H]+; HRMS m/z 435.2536 [M +
H]+, calcd for C28H34O4 435.2535.
2-Acetoxy-4-methoxy-3-(3-methylbut-2-en-1-yl)-6-phenethyl-
benzoic Acid (16a). Following procedure D, compound 15a (0.52 g,
1.42 mmol) as the substrate gave the oxidation product 16a (0.48 g,
89%) as a yellow oil: 1H NMR (300 MHz, CDCl3) δ 7.30−7.22 (2H,
m), 7.19 (3H, dt, J = 8.0, 2.0 Hz), 6.52 (1H, s), 5.09 (1H, tt, J = 5.6,
1.8 Hz), 3.78 (3H, s), 3.22 (2H, d, J = 7.1 Hz), 3.17−3.07 (2H, m),
2.95 (2H, dd, J = 9.9, 6.0 Hz), 2.28 (3H, s), 1.73 (3H, d, J = 1.3 Hz),
1.67 (3H, d, J = 1.4 Hz); 13C NMR (75 MHz, CDCl3) δ 172.0, 169.5,
160.1, 149.0, 142.6, 141.7, 132.1, 128.7, 128.5, 126.1, 121.9, 121.5,
s), 8.08−7.91 (1H, m), 7.69−7.51 (2H, m), 7.38 (2H, ddd, J = 7.9,
6.5, 1.6 Hz), 7.28 (1H, td, J = 7.1, 1.5 Hz), 7.01 (1H, dd, J = 16.1, 1.6
Hz), 6.87 (1H, d, J = 1.5 Hz), 5.22 (1H, tt, J = 5.8, 2.9 Hz), 3.98 (3H,
d, J = 1.6 Hz), 3.36 (2H, d, J = 7.3 Hz), 1.77 (3H, s), 1.64 (3H, s);
13C NMR (75 MHz, acetone-d6) δ 174.2, 163.0, 162.4, 141.8, 138.7,
131.5, 131.2, 131.2, 129.5, 128.5, 127.5, 123.2, 116.9, 104.8, 103.4,
56.2, 25.9, 22.7, 17.9; ESIMS m/z 337.0 [M − H]−; HRMS m/z
337.1441 [M − H]−, calcd for C21H22O4, 337.1445.
2,4-Dimethoxy-6-phenethylbenzaldehyde (12). Pd/C (0.3 g, 10%
by wt) was added to a solution of 6 (3.0 g, 11.18 mmol) in EtOAc (60
mL). The suspension was sealed with a septum under an atmosphere
of H2 supplied via a balloon. The reaction was stirred vigorously for 4
h. Following complete hydrogenation, the suspension was filtered
through Celite and the filtrate was concentrated in vacuo. The crude
product was purified by flash chromatography on silica (15:1 hexane−
EtOAc) to give 12 (2.96 g, 98%) as white solid: mp 81.3−83.3 °C; 1H
NMR (300 MHz, CDCl3) δ 10.53 (1H, d, J = 1.0 Hz), 7.40−7.23
(4H, m), 7.24−7.15 (1H, m), 6.35 (1H, d, J = 2.3 Hz), 6.23 (1H, d, J
= 2.3 Hz), 3.89 (3H, s), 3.80 (3H, s), 3.36- 3.19 (2H, t, J = 7.5 Hz),
2.92- 2.81 (2H, t, J = 7.5 Hz); 13C NMR (75 MHz, CDCl3) δ 190.3,
165.6, 164.6, 148.3, 142.2, 128.8, 128.3, 125.9, 116.9, 108.4, 96.3,
G
DOI: 10.1021/acs.jnatprod.8b00315
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
116.7, 110.5, 55.9, 38.1, 37.3, 25.8, 23.4, 21.0, 17.9; ESIMS m/z 381.0
[M − H]−; HRMS m/z 381.1697 [M − H]−, calcd for C23H26O5,
381.1707.
according to previous documents.28,29 First, the promoters of lasB,
rhlA, and pqsA were amplified by following specific primers. lasB-F:
5′-GGGGTACCGCGGCCAGGAAAGCGTGCAA-3′, lasB-R: 5′-
CGGGATCCTTGTTCAGTTCTCCTGGT-3′. rhlA-F: 5′-
GGGGTACCTCCAATACCACCAACCTGCC-3′, rhlA-R: 5′-
CGGGATCCTTCACACCTCCCAAAAATTTTCG-3′. pqsA-F: 5′-
GGGGTACCATGCCGTCGCCCCCTTGGA-3′, pqsA-R: 5′-
AACTGCAGCATGACAGAACGTTCCCTCTT-3′. T4 ligase was
used to link the plasmid mini-ctx-lacZ and promoter sequences,
which were cut by restriction enzymes (KpnI and PstI for PqsA, KpnI
and BamH1 for lasB and rhlA). The recombinant plasmids were
transferred to DH5α by heat shock and then conjugated to the PAO1
with the helper plasmid RK600 (a kind gift from Singapore Centre for
Environmental Life Sciences Engineering, Nanyang Technological
University). A control strain, PAO1-mini-ctx-lacZ, was constructed
using the same technique. These recombined strains were used to
evaluate the effect of synthetic compounds on different QS systems by
measuring the activity of β-galactosidase. Briefly, a 2 mL bacterial
culture in ABTGC medium (OD600 = 0.1) was shaken for 6 h at 37
°C with different concentrations of compound 3. Then, the amount of
β-galactosidase was measured using a published method.30
Elastase Assay. Overnight cultures of P. aeruginosa wild-type
PAO1 were diluted in ABTGC medium to a final optical density at
600 nm of 0.01. Cultures were grown for 24 h at 37 °C with shaking
(200 rpm). The cultures were centrifuged, and the amount of elastase
in the supernatants was measured by using the EnzChek elastase assay
kit (Invitrogen).
Pyocyanin Quantification Assay. Pyocyanin activity was
estimated by measuring OD at 695 nm according to the protocol
of O’Loughlin with some modifications.31 The overnight-cultured P.
aeruginosa PAO1 was diluted to OD600 = 0.01 with LB medium, and
then 20 mL was inoculated into the 50 mL conical flask with shaking
at 37 °C. After 20 h of incubation, the culture was centrifuged at
1500g for 10 min, and the absorbance at 695 nm of the supernatant
(E)-2-Acetoxy-3-(3,7-dimethylocta-2,6-dien-1-yl)-4-methoxy-6-
phenethylbenzoic Acid (16b). Following procedure D, compound
15b (0.54 g, 1.24 mmol) as the substrate gave the oxidation product
1
16b (0.46 g, 82%) as yellow oil: H NMR (300 MHz, CDCl3) δ
7.31−7.24 (2H, m), 7.23−7.15 (3H, m), 6.54 (1H, s), 5.17−5.02
(2H, m), 3.79 (3H, s), 3.25 (2H, d, J = 6.9 Hz), 3.14 (2H, dd, J = 9.5,
5.7 Hz), 2.97 (2H, dd, J = 9.9, 6.1 Hz), 2.29 (3H, s), 2.06 (2H, s),
1.99 (2H, d, J = 7.2 Hz), 1.74 (3H, d, J = 1.3 Hz), 1.69−1.63 (3H,
m), 1.60 (3H, d, J = 1.3 Hz); 13C NMR (75 MHz, CDCl3) δ 172.1,
169.5, 160.1, 149.1, 142.5, 141.7, 135.6, 131.4, 128.7, 128.5, 126.1,
124.4, 122.0, 121.4, 116.7, 110.4, 55.8, 39.8, 38.1, 37.3, 26.8, 25.8,
23.2, 20.9, 17.8, 16.2; ESIMS m/z 449.1 [M − H]−; HRMS m/z
449.2324 [M + H]−, calcd for C28H34O5, 449.2333.
2-Hydroxy-4-methoxy-3-(3-methylbut-2-en-1-yl)-6-phenethyl-
benzoic Acid (2). Following procedure E, compound 16a (0.48 g,
1.26 mmol) as the substrate gave a deprotected product (2, 0.41 g,
1
96%) as a white solid: mp 139.2−141.2 °C; H NMR (300 MHz,
CDCl3) δ 11.60 (1H, s), 7.38−7.28 (2H, m), 7.26−7.19 (3H, m),
6.24 (1H, s), 5.30−5.16 (1H, m), 3.81 (3H, s), 3.37 (2H, d, J = 7.1
Hz), 3.34−3.23 (2H, m), 2.95 (2H, dd, J = 9.6, 6.3 Hz), 1.81 (3H, d, J
= 1.3 Hz), 1.71 (3H, d, J = 1.5 Hz); 13C NMR (75 MHz, CDCl3) δ
176.4, 163.0, 162.3, 146.0, 142.0, 131.9, 128.7, 128.5, 126.1, 122.3,
115.5, 106.7, 103.8, 55.7, 39.4, 38.3, 26.0, 22.1, 17.9; ESIMS m/z
339.0 [M − H]−; HRMS m/z 339.1605 [M − H]−, calcd for
C21H24O4 339.1602.
(E)-3-(3,7-Dimethylocta-2,6-dien-1-yl)-2-hydroxy-4-methoxy-6-
phenethylbenzoic Acid (3). Following procedure E, compound 16b
(0.46 g, 1.02 mmol) as the substrate gave deprotection product 3
1
(0.38 g, 92%) as a white solid: mp 80.2−83.1 °C; H NMR (300
MHz, CDCl3) δ 11.60 (1H, s), 7.31 (2H, dd, J = 7.9, 6.8 Hz), 7.23
(3H, d, J = 7.0 Hz), 6.24 (1H, s), 5.22 (1H, tt, J = 5.7, 3.2 Hz), 5.09
(1H, tt, J = 5.5, 2.6 Hz), 3.80 (3H, s), 3.38 (2H, d, J = 7.1 Hz), 3.33−
3.22 (2H, m), 2.95 (2H, dd, J = 9.6, 6.3 Hz), 2.07 (2H, t, J = 7.4 Hz),
1.99 (3H, dd, J = 9.6, 5.0 Hz), 1.81 (3H, d, J = 1.3 Hz), 1.66 (3H, d, J
= 1.3 Hz), 1.59 (3H, d, J = 1.3 Hz); 13C NMR (75 MHz, CDCl3) δ
176.4, 163.0, 162.3, 146.0, 142.0, 135.3, 131.3, 128.7, 128.5, 126.1,
124.6, 122.1, 115.6, 106.7, 103.8, 55.7, 40.0, 39.4, 38.3, 26.9, 25.8,
22.0, 17.8, 16.3; ESIMS m/z 407.1 [M − H]−; HRMS m/z 407.2216
[M − H]−, calcd for C26H32O4, 407.2228.
was read before obtaining the final cell density by reading the OD600
Data were normalized by dividing this absorbance value by the final
OD600
.
.
Rhamnolipid Quantification Assay. Rhamnolipid quantification
was carried out using orcinol according to an original method
established by Koch and associates.32 A fresh minimal medium (49.3
mM Na2HPO4, 5 mM KH2PO4, 4.8 mM MgSO4, 7.6 mM
(NH4)2SO4, 0.6 mM CaCl2, 25 μM FeSO4, 0.162 μM
(NH4)6Mo7O24, 38 μM ZnSO4, 14 μM MnCl2, 1.6 μM CuSO4,
0.86 μM CoCl2, 1.9 μM boric acid, 5.5 μM NiCl2, 6.72 μM EDTA,
0.6% glycerol in 18 MΩ deionized water) was prepared as described
previously.33 The overnight cultured P. aeruginosa PAO1 was diluted
with minimal medium, and then 5 mL was inoculated into the 15 mL
glass tube with different concentrations of compound under shaking
at 37 °C for 20 h. DMSO was selected as a control to measure
whether the solvent affects the production of rhamnolipid. The
culture was then centrifuged at 12000g at 4 °C for 5 min. Then
supernatant (2 mL) was extracted with Et2O (6 mL) with complete
mixing. The organic extract (1 mL) was transferred to a 1.5 mL tube
and placed in the fume cupboard while evaporating the solvent. Crude
rhamnolipid was redissolved by deionized water (40 μL) and orcinal
solution (360 μL, 0.19% in 53% H2SO4) to measure the production.
The tube was mixed thoroughly, kept in an 80 °C heating block for 30
min, and then cooled to room temperature. Data were normalized by
Biofilm Formation Inhibition Assay by Crystal Violet
Staining. The steps to measure biofilm inhibition used the method
developed earlier26 with some modifications. Thus, P. aeruginosa
PAO1 cultured overnight in LB broth was diluted in ABTGC
medium27 (B-medium (0.1% MgCl2, 0.1% CaCl2, 0.1% FeCl3)
supplemented with 10% A10, 0.2% glucose, and 0.2% casamino
acids). Test compounds were mixed with ABTGC medium, and 100
μL was added to each well. An equal amount of the bacterial
suspension was added to the wells to give a final concentration of 50
μM. DMSO and medium served as a negative control and a blank,
respectively. The plates were sealed with preservative film and then
incubated at 37 °C for 24 h without agitation. After incubation, the
supernatant was removed, and each well was washed gently with
phosphate-buffered saline (PBS) three times before adding MeOH
(200 μL) for 20 min. The MeOH was discarded, and 200 μL of 0.1%
w/v crystal violet solution, previously filtered, was loaded into each
well for staining lasting 20 min. The staining solution was removed
after 20 min, and the plate was washed gently with PBS three times.
Finally, 33% glacial AcOH solution was used to dissolve the crystal
violet stain absorbed into the biofilm matrix. The OD570 was
determined using a microplate reader, and the biofilm inhibitory effect
was calculated and compared with a negative control. The experiment
was repeated at least three times.
dividing the absorbance value at 421 nm by the final OD600
.
Competition Assays. Overnight cultures of reporter strains
PAO1-mini-ctx-lasB-lacZ and PAO1-mini-ctx-pqsA-lacZ were diluted
with ABTGC medium to a final OD600 of 0.01. Then, compound 3
(50 μM) was added to the dilute culture together with ODdHL or
PQS, respectively, to give final concentrations of 50, 20, 10, and 0 μM.
The amount of β-galactosidase was measured when the OD600
reached 1.0.
Construction of lacZ Reporter Strains and β-Galactosidase
Measurement. Mini-ctx-lacZ plasmid was a kind gift from Singapore
Centre for Environmental Life Sciences Engineering of Nanyang
Technology University. PAO1-mini-ctx-lasB-lacZ, PAO1-mini-ctx-rhlA-
lacZ, and PAO1-mini-ctx-pqsA-lacZ reporter strains were constructed
H
DOI: 10.1021/acs.jnatprod.8b00315
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.8b00315.
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NMR and mass spectra of compounds 1−3, 5−13, 14a−
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +86 20 8522 1367. Fax: +86 20 8522 4766. E-mail:
linjing_jnu@163.com (J. Lin).
*Tel: +86 20 8522 4497. Fax: +86 20 8522 4766. E-mail:
twmchen@jnu.edu.cn (W.-M. Chen).
ORCID
Jing Lin: 0000-0001-7707-8066
Wei-Min Chen: 0000-0002-0292-7841
Notes
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(29) Brouwer, S.; Pustelny, C.; Ritter, C.; Klinkert, B.; Narberhaus,
F.; Haussler, S. J. Bacteriol. 2014, 196, 4163−4171.
(30) Patriquin, G. M.; Banin, E.; Gilmour, C.; Tuchman, R.;
Greenberg, E. P.; Poole, K. J. Bacteriol. 2008, 190, 662−671.
(31) O’Loughlin, C. T.; Miller, L. C.; Siryaporn, A.; Drescher, K.;
Semmelhack, M. F.; Bassler, B. L. Proc. Natl. Acad. Sci. U. S. A. 2013,
110, 17981−17986.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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This work was supported by the National Natural Science
Foundation of China (81673336), Pearl River S&T Nova
Program of Guangzhou (201806010116), and the Fundamen-
tal Research Funds for the Central University (21617478).
̈
(32) Koch, A. K.; Kappeli, O.; Fiechter, A.; Reiser, J. J. Bacteriol.
1991, 173, 4212.
(33) Welsh, M. A.; Eibergen, N. R.; Moore, J. D.; Blackwell, H. E. J.
Am. Chem. Soc. 2015, 137, 1510−1519.
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DOI: 10.1021/acs.jnatprod.8b00315
J. Nat. Prod. XXXX, XXX, XXX−XXX