Synthesis of 5-(bromomethylene)furan-2(5H)-ones and 3-(bromomethylene) isobenzofuran-1(3H)-ones as inhibitors of microbial quorum sensing

Article abstract of DOI:10.1039/b803926g

(E)- and (Z)-5-(Bromomethylene)furan-2(5H)-ones and (E)- and (Z)-3-(bromomethylene)isobenzofuran-1(3H)-ones have been prepared starting from commercially available maleic anhydrides and phthalic anhydrides, respectively. A debrominative decarboxylation or a bromodecarboxylation reaction is a key step in the synthesis. The furanones were investigated for their ability to interfere with microbial communication and biofilm formation by Staphylococcus epidermidis. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

Full text of DOI:10.1039/b803926g

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Synthesis of 5-(bromomethylene)furan-2(5H)-ones and
3-(bromomethylene)isobenzofuran-1(3H)-ones as inhibitors
of microbial quorum sensing
Tore Benneche,wa Zainab Hussain,a Anne Aamdal Scheieb and
¨
Jessica Lonn-Stensrud
b
Received (in Montpellier, France) 6th March 2008, Accepted 28th March 2008
First published as an Advance Article on the web 12th May 2008
DOI: 10.1039/b803926g
(E)- and (Z)-5-(Bromomethylene)furan-2(5H)-ones and (E)- and (Z)-3-
(bromomethylene)isobenzofuran-1(3H)-ones have been prepared starting from commercially
available maleic anhydrides and phthalic anhydrides, respectively. A debrominative
decarboxylation or a bromodecarboxylation reaction is a key step in the synthesis. The furanones
were investigated for their ability to interfere with microbial communication and biofilm
formation by Staphylococcus epidermidis.
furanones are thought to interfere with microbial communica-
tion, mainly in Gram-negative microorganisms.
Introduction
Many microorganisms communicate via chemical signal
molecules such as autoinducer-1 (AI-1) and autoinducer-2
(AI-2) to control gene expression in response to population
density.1 This phenomenon, called quorum sensing (QS),
regulates various virulence factors, for instance proteolytic
activity, carbohydrate metabolism and biofilm formation.2,3
Thus interference with QS, in principle, would reduce the
pathogenic potential of a bacterium. Since this occurs without
exerting a selective pressure on microbial viability, resistance
development is not likely.
The aim of this study was to synthesize 5-(bromomethyl-
ene)furan-2(5H)-ones (2 and 3, Scheme 1) and 3-(bromo-
methylene)isobenzofuran-1(3H)-ones (4 and 5, Scheme 2)
and to assess their ability to interfere with microbial commu-
nication and biofilm formation by S. epidermidis.
5-(Bromomethylene)furan-2(5H)-ones have previously
been prepared from levulinic acid11 or its derivatives,12
propenoates13 and allenic esters.14 3-(Bromomethylene)
isobenzofuran-1(3H)-ones have been prepared from 3-alkyli-
dene isobenzofuran-1(3H)-ones15 and by dehydration of bro-
mo keto acids.16
Most microorganisms in nature prefer a biofilm mode of
growth4 and biofilm formation is regulated by QS in a number
of microorganisms.5,6 Staphylococcus epidermidis is a Gram-
positive biofilm forming commensal bacterium on human skin
and mucous membranes, as well as a major nosocomial
pathogen associated with medical implant infections.7 The
virulence is related to S. epidermidis’s ability to form biofilm
on implanted devices. There are presently few effective means
to prevent medical implant infections,8 mainly due to in-
creased resistance of biofilm microorganisms to both antimi-
crobials and the human immune system, being up to 10–1000
times more tolerant to antimicrobials than their planktonic
counterparts.9 Thus antimicrobial treatment of implant infec-
tions often fails, necessitating removal of the implanted device.
Control of biofilm formation by for instance S. epidermidis
represents a novel, non-antimicrobial approach of implant
infection prevention. Thus identification of compounds that
inhibit QS has become an area of intense research.2 The macro
algae Delisea pulchra is known to prevent microbial colonisa-
tion of its surface by producing brominated furanones.10 Such
5-(Alkylidene)furan-2(5H)-ones, without the bromo substi-
tuent in the alkylidene group, have been synthesized from
maleic anhydrides in a Wittig reaction using stabilized phos-
phorus ylides.17 We wanted to use this method in our synthesis
since a number of maleic and phthalic anhydrides are com-
mercially available. The necessity to use stabilized phosphorus
ylides in the Wittig reaction indicates a bromodecarboxylation
step of an a,b-unsaturated acid in order to prepare the 5-
bromomethylene substituent. A number of methods have been
used in the bromodecarboxylation of a,b-unsaturated acids
having a b-aryl substituent.18 Without such a substituent the
methods are much more limited.19
Results and discussion
Synthesis of furanones
The unsaturated esters 1 (Scheme 1) were prepared in good
yields from commercially available maleic anhydrides in the
Wittig reaction and the esters were transformed into the 5-
(bromomethylene)furan-2(5H)-ones (2 and 3) in three ways
(Method A, B and C, Table 1). Methods A and B have three
steps, while Method C has two. All methods are one-pot
procedures—with only solvent evaporation between the steps.
In Method A the tert-butyl ester of 1 is first cleaved by TFA
in dichloromethane and then brominated before the resulting
a Department of Chemistry, University of Oslo, Norway
b Department of Oral Biology, Faculty of Dentistry, University of
Oslo, Norway. E-mail: tore.benneche@kjemi.uio.no;
Fax: + 47 22 85 55 07
w Address for correspondence: Department of Chemistry, PO Box
1033 Blindern, 0315 Oslo, Norway.
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Scheme 1
Scheme 2
a,b-dibromo acid is debromodecarboxylated with triethyl-
amine in DMF. Method B is similar to Method A except that
the two first steps are switched. The yields in Method A are
somewhat better than the yields in Method B, but the reaction
times in Method A are much longer than in Method B. The
bromination of the unsaturated acids takes 3–4 days
with a large excess of bromine, while the bromination of
the unsaturated esters is over in a couple of hours with
a slight excess of bromine. The product distribution of
the E- and Z-isomer of the 5-(bromomethylene)furan-2(5H)-
one (2 and 3) is almost the same in these two methods
(Table 1). The Z-isomer is always the major isomer except in
one case (R1 and R2 = Me). The identity of the E- and
Z-isomers were determined by NOE experiments and/or by 1H
NMR chemical shift of the methine proton and any ring
protons.20
The product ratios in Table 1 are probably not equal to the
thermodynamic ratios of the products, since treatment of
either pure 2c or pure 3c with iodine in CDCl3 gives the same
mixture of 2c and 3c (29 : 71) which is significantly different
from the ratio in Table 1 (12 : 88)
Phthalic anhydrides can also be used as starting material in
the synthesis discussed above. Thus the 3-(bromomethylene)-
isobenzofuran-1(3H)-ones 4a and 5a (R = H, Scheme 2) were
formed as a 1 : 2 mixture in 53% yield while the nitro
compounds 4b and 5b (R = NO2, Scheme 2) were formed as
a 1 : 1 mixture in 27% yield. We could separate the E- and
Z-isomers 4a and 5a by flash chromatography but not the
nitro compounds 4b and 5b.
Bromination of the unsaturated ester 1 (Method B, step 1)
or the corresponding unsaturated acid (Method A, step 2) is
completely regioselective: only the exocyclic carbon–carbon
double bond is brominated at room temperature even if an
excess of bromine is used.
Bromination of ordinary alkenes is stereospecific.22 The
bromination of the unsaturated ester 1 (R1 = R2 = Me) or
the corresponding unsaturated acid is, however, not stereo-
specific because the pure E-isomer and the pure Z-isomer of 1
give the same mixture of dibrominated ester or acid (Scheme 3).
The isomerization occurs only in the bromination step,
since no isomerization was observed when a pure dibromide
In Method C the unsaturated esters are cleaved to the acids
and then bromodecarboxylated using bis(2,4,6-trimethyl-
pyridine)bromine(I) hexafluorophosphate in dichloromethane.
The yield in this reaction is low to moderate, 10–58% over two
steps (Table 1). The reaction shows some degree of stereo-
specificity since treatment of the pure E-isomer of the corres-
ponding acid of 1 (R1 = R2 = Me) gives mainly the
E-isomer (E : Z ratio 71 : 29) of the bromodecarboxylated
product. The pure Z-isomer gives only the Z-isomer.
Table 1 Yields and product distribution in the synthesis of 2 and 3
E : Z Starting
E : Z
Product 2 : 3
R1
R2
material 1
Methoda
Product
Yieldb 2
Yieldb 3
Total yieldb
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
0 : 100
0 : 100
0 : 100
100 : 0
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
C
2a/3ac
2a/3ac
2a/3ac
2b/3b
2b/3b
2b/3b
2c/3c
2c/3c
2c/3c
2d/3d
2d/3d
2d/3d
2e/3e
2e/3e
2e/3e
2e/3e
—
2
22
10
7
32
7
6
—
44
0
77
46
6
53
45
4
50
48
4
61
46
22
87
53
38
60
51
58
56
49
10
75
66
—
48
5 : 85
4 : 96
100 : 0
11 : 89
13 : 87
84 : 16
12 : 88
12 : 88
93 : 7
Me
Me
Me
Ph
Ph
Ph
Br
Br
Br
Me
Me
Me
Me
a
54
6
1
11 : 89
2 : 98
6
60 : 40
88 : 12
79 : 21
0 : 100d
71 : 29
66
52
—
34
9
14
—
14
Method A: 1. TFA, CH2Cl2, rt, 2 h; 2. Br2, CDCl3, rt, 2–3 d; 3. N(Et)3, DMF, rt, 2 h; Method B: 1. Br2, CDCl3, rt, 2–3 h; 2. TFA, CH2Cl2, 2 h; 3.
N(Et)3, DMF, rt, 2 h; Method C: 1. TFA, CH2Cl2, rt, 2 h; 2. Br+(coll)2PF6ꢁ, CH2Cl2, rt, 2 h. Isolated yield. Ref. 21. From the 1H NMR of
b
c
d
the crude product.
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Scheme 3
Fig. 2 Bioluminescence response in the reporter strain V. harveyi BB
170 induced by V. harveyi BB152 supernatant and repressed by 6.0 mM
of furanones 2e, 3b–e, 4a, 5a or the reference F202. The results are
mean values and standard errors from three independent experiments
with three parallels. * Significantly different from control (ctr) without
furanone (P o 0.01).
Scheme 4
(6, R = t-Bu) was carried through to the end product. The
5-bromomethylenefuran-2(5H)-ones will, however, isomerize
if the compounds are kept in a polar solvent like CDCl3 for a
couple of weeks. This tendency to isomerize can be utilized in
a synthesis of the natural product bovolide23 (7, Scheme 4). A
mixture of 2e and 3e was isomerized to pure 3e with iodine in
CH2Cl2 and coupled with butylzinc chloride in THF at room
temperature in a Pd(0)-catalyzed reaction.
The brominated furanone 3d (Table 1) has proven to be a
valuable intermediate in the synthesis of another g-alkylidene
butenolide: lissoclinolide.24
Interference with microbial communication and biofilm
formation
All synthesized furanones (2e, 3b–e, 4a, 5a) and the reference
F20221 (Fig. 1) at 6.0 mM reduced bioluminescence in V.
harveyi BB170 significantly (P o 0.01 compared to control
without furanone), with 5a being slightly more effective than
the F202 reference (Fig. 2).
Fig. 3 24 h Biofilm formation by S. epidermidis on discs coated with
furanone 5a and the reference F202. The results are mean values and
standard errors from three independent experiments done in triplicate.*
Significantly different from control (ctr) without furanone (P o 0.01).
V. harveyi BB170 lacks the receptor for AI-1 and thus
responds only to intermicrobial communication via the AI-2
QS molecule. We assume therefore that the tested furanones
interfered with AI-2 QS communication. This is in line with
previous data in both Gram-negative25 and Gram-positive
microorganisms.26 More recently, furanones were found to
structurally alter LuxR in V. harveyi thus preventing binding
to promoter sequences.27 Further studies are needed to verify
the mechanism of action against S. epidermidis.
68%. Notably, this reduction could not be ascribed to an
antimicrobial effect since for both compounds, total growth
was unaffected (Fig. 3). These findings are interesting in view of
the increased use of implanted medical devices and the con-
comitant implant infections. We suggest that synthetic com-
pounds like 5a and F202, immobilised on implant surfaces, may
prevent implant infection. F202 was more effective than 5a in
reducing biofilm formation by S. epidermidis. F202 has pre-
viously been reported to interfere with colonization by the
Gram-negative Pseudomonas aeruginosa in lungs of experimen-
tally infected mice.28 Thus F202 appear to be able to interfere
with both AI-1 and AI-2 communication. The present and other
studies confirm the possibility of interfering with microbial
virulence without inhibiting microbial growth.
The two most effective bioluminescence reducers (5a and
F202) were subsequently tested for effect on biofilm formation
by S. epidermidis. S. epidermidis carries the AI-2 synthase gene,
although its role in biofilm formation is not clearly defined. The
biofilm assay clearly showed the biofilm inhibitory potential of
both furanone 5a and F202 (P o 0.01 compared to control
without furanone). Furanone 5a reduced S. epidermidis biofilm
by 57%, while the reference F202 reduced biofilm formation by
Conclusion
We have shown that 5-(bromomethylene)furan-2(5H)-ones
and 3-(bromomethylene)isobenzofuran-1(3H)-ones can be
Fig. 1 Structure of compound F202, (Z)-5-(bromomethylene)furan-
2(5H)-one.21
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easily synthesized in 3 or 4 steps from commercially available
maleic anhydrides and phthalic anhydrides, respectively. This
study also shows that furanones may be potential inhibitors of
microbial communication. The most effective furanones, 5a
and F202, also reduced biofilm formation by S. epidermidis
without affecting the growth. The bioluminescence assay
indicates that the efficacy may be structurally dependent. We
see a potential for furanones and isobenzofuranones in pre-
venting implant infections.
(E)-5-(Bromomethylene)-3-methylfuran-2(5H)-one (2b). Elu-
ent EtOAc–hexane 1 : 6 Rf 0.30; mp 52–56 1C; dH (300 MHz;
CDCl3) 2.01 (3H, Me), 6.33 (1H, s, CHBr), 7.37 (1H, m, H4);
dC (75 MHz; CDCl3) 11.03, 91.96, 133.42, 134.52, 150.93,
170.02; m/z (EI) 190 (M+ + 2, 63%), 188 (M+, 64), 125 (65),
122 (40), 120 (42), 97 (42), 68 (49), 39 (100); HRMS (EI) calcd.
for C6H5O2Br: 187.9472, found 187.9467.
(Z)-5-(Bromomethylene)-3-methylfuran-2(5H)-one (3b). Elu-
ent EtOAc–hexane 1 : 6 Rf 0.13; mp 91–94 1C; dH (200 MHz;
CDCl3) 1.95 (3H, Me), 5.91 (1H, s, CHBr), 7.03 (1H, m, H4);
dC (75 MHz; CDCl3) 10.75, 89.57, 131.24, 135.81, 151.18,
169.52; m/z (EI): 190 (M+ + 2, 99%), 188 (M+, 100), 162
(16), 160 (16), 122 (55), 120 (57), 53 (76); HRMS (EI) calcd. for
C6H5O2Br: 187.9472, found 187.9469.
Experimental
The 1H NMR and the 13C NMR spectra were recorded on
Bruker Avance DPX instruments. Mass spectra, under elec-
tron impact conditions, were recorded at 70 eV ionizing energy
on a Fision ProSpec instrument.
(E)-5-(Bromomethylene)-3-phenylfuran-2(H)-one (2c). Elu-
ent EtOAc–hexane 1 : 3 Rf 0.48; dH (200 MHz; CDCl3) 6.48
(1H, s, QCHBr), 7.41–7.47 (3H, m, Ph), 7.82 (1H, s, H4),
7.91–7.96 (2H, m, Ph); dC (75 MHz; CDCl3) 93.89, 124.50,
128.78, 128.95, 130.43, 131.12 , 133.15, 150.75, 167.74; m/z
(M+) 252 (M+ + 2, 59%), 250 (M+, 60), 171 (100), 115 (74),
102 (88), 57 (20); HRMS (EI) calcd. for C11H7O2Br: 249.9629,
found 249.9626.
Preparation of 5-(bromomethylene)furan-2(5H)-ones and 3-
(bromomethylene)isobenzofuran-2(3H)-ones
Method A. The a,b-unsaturated ester (1.0 mmol) was dis-
solved in a mixture of CH2Cl2 (1 mL) and TFA (1 mL). The
reaction mixture was stirred at room temperature for 2 h,
evaporated and redissolved in a mixture of CDCl3 (2 mL) and
TFA (0.1 mL). Bromine (2 mL, 2 M in CCl4) was added and
(Z)-5-(Bromomethylene)-3-phenylfuran-2(H)-one (3c). Elu-
ent EtOAc–hexane 1 : 3 Rf 0.32; mp 123–125 1C; dH
(200 MHz; CDCl3) 6.10 (1H, s, QCHBr), 7.40–7.44 (3H, m,
Ph), 7.47 (1H, s, H4), 7.85–7.90 (2H, m, Ph); dC (75 MHz;
CDCl3) 91.58, 127.15, 128.69, 128.91, 130.13, 131.30, 132.50,
151.03, 167.1; m/z (EI): 252 (M+ + 2, 68%), 250 (M+, 69),
172 (14), 171 (100), 116 (10), 115 (93), 102 (100), 76 (17);
HRMS (EI) calcd. for C11H7O2Br: 249.9629, found 249.9623.
1
the mixture was stirred at room temperature until H NMR
showed that all starting material had been consumed (2–3 d).
The solvents were evaporated off and the residue was dissolved
in DMF (2 mL). Triethylamine (0.15 mL, 1.08 mmol) was
added at 0 1C and the mixture stirred at room temperature for
45 min before water was added. The product was extracted
into Et2O, washed with brine (3 ꢂ 10 mL), dried (MgSO4) and
evaporated. The E- and Z-isomers were separated by flash
chromatography on silica gel.
(E)-3-Bromo-5-(bromomethylene)furan-2(5H)-one (2d). Elu-
ent EtOAc–hexane 1 : 6 Rf 0.23; mp 52–55 1C; dH (200 MHz;
CDCl3) 6.53 (1H, s, QCHBr), 7.83 (1H, s, H4); m/z (EI) 256
(M+ + 4, 49%), 254 (M+ + 2, 100), 252 (M+, 52), 228 (9),
226 (19), 224 (10), 175 (11), 173 (11), 149 (15), 147 15), 145
(15), 122 (18), 120 (19) 119 (17), 117 (17).
Method B. Bromine (0.55 mL, 1.10 mmol, 2 M in CCl4) was
added to a solution of the a,b-unsaturated ester (1.0 mmol) in
CH2Cl2 (2 mL). The mixture was stirred at room temperature
for 2 h before the solvent was evaporated and the residue
dissolved in a mixture of CH2Cl2 (1 mL) and TFA (1 mL). The
mixture was stirred at room temperature for 2 h before the
solvent was evaporated off. The residue was dissolved in DMF
(2 mL) and triethylamine (0.15 mL, 1.08 mmol) was added at
0 1C. The mixture was stirred at room temperature for 45 min
before water was added and the product was extracted into
Et2O, washed with brine (3 ꢂ 10 mL), dried (MgSO4) and
evaporated. The E- and Z-isomers were separated by flash
chromatography on silica gel.
(Z)-3-Bromo-5-(bromomethylene)furan-2(5H)-one (3d).24 Eluent
EtOAc–hexane 1 : 6 Rf 0.15; mp 73–76 1C; dH (300 MHz; CDCl3)
6.19 (1H, s, QCHBr), 7.49 (1H, s, H4); dC (75 MHz; CDCl3)
93.27, 114.42, 139.42, 150.80, 164.01; m/z (EI) 256 (M+ + 4,
49%), 254 (M+ + 2, 100), 252 (M+, 51), 228 (8), 226 (16), 224 (8),
145 (16), 147 (15), 53 (37). HRMS (EI) calcd. for C5H2O2Br2:
251.8421, found 251.8422.
(E)-5-(Bromomethylene)-3,4-dimethylfuran-2(5H)-one (2e). Elu-
ent EtOAc–hexane 1 : 4 Rf 0.40; mp 48–50 1C; dH (200 MHz,
CDCl3) 1.87 (3H, m, 4-Me), 2.35 (3H, m, 3-Me), 6.38 (1H, s,
QCHBr); dC (75 MHz, CDCl3) 8.8, 13.7, 91.3, 129.0, 146.2,
150.8, 169.0; m/z (EI) 204 (M+ + 2, 100%), 202 (M+, 100),
191(9), 189(9), 139(10), 127 (17), 122 (26), 120 (26); HRMS (M+)
calcd. for C7H7O2Br: 201.9629, found 201.9631.
Method C. The a,b-unsaturated ester (1.0 mmol) was dis-
solved in a mixture of CH2Cl2 (1 mL) and TFA (1 mL). The
reaction mixture was stirred at room temperature for 2 h,
evaporated and redissolved in CH2Cl2 (4 mL). Bis(2,4,6-
trimethylpyridine)bromine(I) hexafluorophosphate29 (700 mg,
1.50 mmol) was added at 0 1C and the mixture was stirred at
0 1C for 15 min and at room temperature for 2 h before Et2O
was added. The Et2O was washed with 1M HCl, saturated
NaHCO3 and brine before it was dried (MgSO4) and evapo-
rated. The E- and Z-isomers were separated by flash chroma-
tography on silica gel.
(Z)-5-(Bromomethylene)-3,4-dimethylfuran-2(5H)-one (3e).
Eluent EtOAc–hexane 1 : 4 Rf 0.26; mp 113–116 1C; dH (200
MHz; CDCl3) 1.92 (3H, m, 4-Me), 2.10 (3H, m, 3-Me), 5.98
(1H, s, QCHBr); dC (75 MHz; CDCl3) 8.5, 9.6, 86.5, 125.7,
145.7, 152.7, 169.0; m/z (EI) 204 (M+ + 2, 100%), 202 (M+,
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100), 191(10), 189 (10), 176 (6), 174 (6), 122 (33), 120 (33), 111
(17), 67 (68); HRMS (M+) calcd. for C7H7O2Br 201.9629,
found 201.9634.
at ꢁ70 1C until use.31 S. epidermidis was grown on Brain Heart
Infusion agar plates (BHI, Difco Laboratories, Detroit, MI,
USA) for 24 h at 37 1C in an aerobic atmosphere before
inoculation into Brain Heart Infusion medium (BHI, Difco
Laboratories, USA) for biofilm formation and planktonic
growth.
(E)-3-(Bromomethylene)isobenzofuran-1(3H)-one (4a). Elu-
ent EtOAc–hexane 1 : 6 Rf 0.23; mp 80–85 1C; dH (200 MHz;
CDCl3) 6.56 (1H, s, QCHBr), 7.62–8.46 (Ar); dC (75 MHz;
CDCl3) 91.05, 124.56,125.73, 125.97, 131.05, 134.70, 137.23,
147.49, 165.65; m/z (EI) 226 (M+ + 2, 98%), 224 (M+, 100),
170 (11), 168 (11), 104 (48), 89 (82), 76 (52); HRMS (M+)
calcd. for C9H5O2Br: 223.9472 found 223.9475.
A slightly modified bioluminescence assay was performed as
previously described6,26,30 to assess the ability of the various
furanones (2e, 3b–e, 4a, 5a) to interfere with QS communica-
tion. Furanone F202 was included as a reference. Briefly, cell-
free supernatants prepared from V. harveyi BB152 were added
at final concentration of 10% to BB170. The various fura-
nones were added to a final concentration of 6.0 mmol Lꢁ1
where upon bioluminescence induction was followed during
the next six hours in a Synergy HT Multi-Detection Micro-
plate Reader (Biotek, VT, USA).
(Z)-3-(Bromomethylene)isobenzofuran-1(3H)-one (5a).15 Elu-
ent EtOAc–hexane 1 : 6 Rf 0.15; mp 128–131 1C; dH (200
MHz; CDCl3) 6.33 (1H, s, QCHBr), 7.57–7.91 (Ar); dC (75
MHz; CDCl3) 85.72, 119.96, 124.47, 125.84, 130.56, 134.87,
137.90, 148.72, 165.54; m/z (EI) 226 (M+ + 2, 98%), 224
(M+, 100), 170 (10), 168 (10), 104 (36), 89 (83), 76 (41); HRMS
(EI) calcd. for C9H5O2Br: 223.9472, found 223.9476.
Biofilm assay. Biofilm by S. epidermidis was allowed to form
during 24 h at 37 1C in an aerobic atmosphere on polystyrene
discs (Nunc) coated with furanone. Coating with furanone was
performed by adding 1 ml 60 mmol Lꢁ1 furanone dissolved in
ethanol to each well. After 24 h, the solvent had evaporated.
Discs similarly treated but without furanone were included as
controls with no inhibitory effect on biofilm.
(E)- and (Z)-3-(Bromomethylene)-4-nitroisobenzofuran-1(3H)-
one (4b and 5b). Eluent EtOAc–hexane 2 : 3 Rf 0.35 gave a 1 : 1
mixture of 4b and 5b; dH (200 MHz; CDCl3) 6.95 (1H, s,
QCHBr), 7.59 (1H, s, QCHBr), 7.80–8.50 (2 ꢂ 3H, m, Ph);
m/z (EI): 271 (M+ + 2, 23%), 269 (M+, 23), 218 (34), 190 (49),
161 (55), 104 (60), 75 (100), 57 (59).
Biofilm mass formed was quantified as follows; the discs
were rinsed twice in distilled water and stained for 10 min with
a 0.1% solution of safranin in new wells. The discs were rinsed
again, and bound safranin was released from stained cells
using 30% glacial acetic acid. OD measurements at 530 nm
were compared to the non-furanone coated control and the
reference F202.
(Z)-3,4-Dimethyl-5-pentylidenefuran-2(5H)-one (7).23 Zinc
chloride (3.0 mL, 1.5 mmol, 0.50 M in THF) was added
dropwise to a solution of n-butyllithium (0.15 mL, 1.5 mmol,
10 M) at ꢁ78 1C under N2. After 1 h a solution of (Z)-5-
(bromomethylene)-3,4-dimetylfuran-2(5H)-one (3e) (0.102 g,
0.5 mmol) in dry THF (5 mL) was added, followed by
tetrakis(triphenylphosphine)palladium [generated in situ from
tris(dibenzylideneacetone)dipalladium chloroform adduct
(0.026 g, 0.025 mmol) and triphenylphosphine (0.026 g,
0.1 mmol)] in dry THF (4 mL). The mixture was stirred at
room temperature for 18 h before the solvent was evaporated
off. The residue was dissolved in diethyl ether, washed with a
saturated solution of NH4Cl (10 mL) and brine. The solution
was dried (MgSO4), evaporated and the crude product purified
by flash chromatography on silica gel. Eluent 0–15% EtOAc
in hexane; 0.61 g (68%) yellow oil; dH (200 MHz; CDCl3) 0.88
(3H, t, J = 7.0 Hz, –CH3), 1.30–1.42 (2 ꢂ 2H, m, 2 ꢂ CH2),
1.86 (3H, s, 4-Me), 1.99 (3H, s, 3-Me), 2.32 (2H, q, J = 7.3 Hz,
ꢁCH2), 5 .17 (1H, t, J = 7.8 Hz, CHnBu); dC (75 MHz;
CDCl3) 8.48, 9.77, 13.74, 22.28, 25.60, 31.26, 110.86, 123.89,
146.83, 149.93, 170.93; m/z (EI): 180 (M+, 38%), 137 (98), 138
(16), 125 (27), 124 (100), 110 (16), 82 (17), 55 (65). HRMS (EI)
calcd. for C11H16O2: 180.1150, found 180.1147.
To exclude an antimicrobial effect of the furanones, total
growth, including both scraped biofilm and planktonic cells,
was measured. After vigorous shaking to evenly disperse the
cells, total growth was quantified by measuring optical density
at 600 nm.
Statistics. Each assay was performed in triplicate in three
independent experiments. One-way ANOVA on Ranks fol-
lowed by Student–Newman–Keuls method for normal distri-
bution was used for multiple comparisons on biofilm
formation and growth and for comparisons of biolumines-
cence induction. The level of significance was set at P r 0.01.
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