Targeting bacterial biofilms: Design of a terpenoid-like library as non-toxic anti-biofilm compounds

Article abstract of DOI:10.1016/j.bmcl.2010.12.134

A new class of anti-biofilm compounds possessing 1,4-disubstituted-(1H)-1, 2,3-triazolic cores was designed. Their efficient synthesis was performed by means of click chemistry through 1,3-dipolar cycloadditions. Two compounds were found to act as specific anti-biofilm agents against a gram negative species.

Full text of DOI:10.1016/j.bmcl.2010.12.134

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1493–1497
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Targeting bacterial biofilms: Design of a terpenoid-like library as non-toxic
anti-biofilm compounds
⇑
Cheikh Sall, Linda Dombrowsky, Olivier Bottzeck, Annie Praud-Tabariès, Yves Blache
Laboratoire MAPIEM, EA 4323, UFR Sciences et Techniques, Université du Sud Toulon-Var, 83957 La Garde cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of anti-biofilm compounds possessing 1,4-disubstituted-(1H)-1,2,3-triazolic cores was
designed. Their efficient synthesis was performed by means of click chemistry through 1,3-dipolar
cycloadditions. Two compounds were found to act as specific anti-biofilm agents against a gram negative
species.
Received 30 November 2010
Revised 24 December 2010
Accepted 31 December 2010
Available online 13 January 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Bacterial biofilm
Triazole
Click chemistry
Biofilms, which present problems in many different areas, result
from the adherence of bacteria to surfaces. Although bacteria grow
very poorly when floating in water or biological fluids, they grow
extremely well on surfaces. Bacteria produce these biofilms, in
part, to help them attach to surfaces and bind to one another.1
Bacterial biofilms cause problems in medical health care since they
colonize implants such as artificial joints or catheters,2 while in
marine environment, formation of biofilms on immersed substrata
leads to major economic problems which conducted to the use of
toxic biocides to eradicate these communities.3,4 Although eradica-
tion of planktonic bacteria communities have been largely con-
trolled, it has been estimated that bacteria within a biofilm can
display up to 1000-fold increased resistance to antibiotic or biocide
treatment. In this context, design of original compounds which can
limit formation of bacterial biofilms is of great need in the directive
of the rational use of antibiotics and/or biocides. In this way, we
initiated a programme aimed to allow the discovery of new poten-
tial leads from marine organisms which could be optimized by the
preparation of analogues. Linear diterpenes eleganolone and ele-
ganediol were identified as major compounds from the brown alga
Bifurcaria bifurcata which crude extracts exhibited antibacterial
activities5 while more recently, meroditerpenes (derivatives from
geranylgeranyltoluquinol) were isolated from brown alga Halidrys
siliquosa and showed antibacterial properties against different
strain of gram negative bacteria (Fig. 1).6 Furthermore, another ter-
pene namely 3-(40-geranyloxy-30-methoxyphenyl)-2-trans prope-
noic acid isolated from Acronychia baueri Schott was reported for
its capacity to inhibit the formation of Porphyromonas gingivalis
biofilm.7 For our purpose ‘click chemistry’ methodologies retained
our attention as an interesting process for the preparation of ana-
logues by mean of bioconjugations of a natural moiety (terpenoid)
and a synthetic part (related to the aromatic ring of meroditerp-
enes) through a triazole linker. A first polyprenyl-type library con-
taining 1,4-disubstituted triazoles was designed and reported to
exhibit anti-biofilm activities against a strain of Pseudoalteromonas
sp.8 However, since these compounds were obtained as Z/E mix-
tures from azido derivatives of terpenes, severe limitations of this
methodology resided in the difficulty to separate isomers and in
the final yield of pure E-isomers (27–60%). In continuation of our
investigations aimed to explore the chemical diversity around such
terpenic skeletons to probe anti-biofilm structure–activity rela-
tionships, we felt that the relatively simple structure of library A
invited the synthesis of new analogues. In this way, a new polypre-
nyl-type library B in which triazoles are obtained as pure E-isomers
was designed (Fig. 1).
In order to highlight analogies between the two series (A and B),
a molecular modeling study was performed with the 4-methoxy
derivatives (geranyl tail, R = OCH3) of each library.9 Results are
summarized in Figure 2. Although conformations of the two
compounds were similar, slight differences arised from these
calculations. Considering properties of library B versus library A,
the size of the triazolic linker between the terpenic unit and the
aromatic pharmacophore is longer of 1.76 Å, c Log(P) is lower and
an additional H-bonding site is located on the oxygen atom. Exam-
ination of electrostatic potential isosurface also highlighted
differences between the two series. In the two cases, two
electron-poor domains were highlighted: a large one constituted
of the terpenic chain and a smaller one constituted of the aromatic
ring, while an electron-rich domain is located of the triazole ring.
⇑
Corresponding author.
E-mail address: blache@univ-tln.fr (Y. Blache).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.134

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C. Sall et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1493–1497
OH
OH
3
3
OH
eleganolone
OH
elegandiol
OH
OCH3
O
2
Me
OH
3
CO2H
geranylgeranyltoluquinol
3-(4'-geranyloxy-3'-methoxyphenyl)
-2-trans propenoic acid
N
N
N
N
N
N
O
R
R
n
n
First generation of analogues (Z/E mixture)
(library A)
R = OCH3, OH
Targeted library B
(second generation of analogues)
R = OCH3, OH, CO2H
Figure 1. Structure of targeted library B, natural diterpenoids and previously studied anti-biofilm agents (library A).
Figure 2. Conformations and properties of the two samples of library A (left) and B (right): electrostatic potential surfaces (up), the values are color-coded onto the total
electron density surface, with colors toward red indicating electron rich regions of the molecule.
However, in the case of series B, this electron-poor area is extended
to the terpenic chain by overlapping the ether function. As these
elements should have their importance when considering
interactions with biological systems, we finally believe that design-
ing this new library should give some relevant informations in
terms of SAR in regards with anti-biofilm activities of such
compounds.
The practical interest of this second library resides in the facility
of preparation of aromatic azides from aromatic amines in situ, and
in the fact that resulting triazoles are obtained as pure E-isomers.
Synthesis of the targeted library was achieved by performing the
copper(I)-catalyzed 1,3-dipolar cycloaddition of organic azides
and alkynes resulting in the formation of 1,2,3-triazoles.10 In gen-
eral, these reactions usually proceed to completion in 6–36 h at
ambient temperature in water with a variety of organic co-sol-
vents, such as tert-butanol, ethanol, DMF, DMSO, THF, or CH3CN,
and this reaction is useful for large classes of azides and al-
kynes.11–15 Preparation of alkyne 1 and 2 was achieved as previ-
ously described.16,17 The reactions were investigated in a one pot
process without isolation of azides.18 In practice, all aromatic
azides were obtained from the corresponding amines by treatment
with sodium nitrite in acidic media for 2 h (HCl/H2O), followed by
addition of sodium azide (NaN3). Reactions were monitored by TLC
and after disappearance of the starting amine, a simple extraction
with dichloromethane led to the resulting azides to which a
solution of appropriate alkyne, CuSO4/sodium ascorbate in water/
ethanol mixture (50:50) was added. Choice of ethanol rather than
DMF, similar to our previous work, allowed an easier workup and a
better purity of products. All reactions were performed in good to
excellent yields at room temperature excepted for the 2-methoxy
derivatives 3, 12, which were obtained only in 5% and 7% respec-
tively. These poor yields can be explained by a probable steric
effect due to the methoxy group as well as by the nature of azides
which could be stabilized through
a conjugation with the
2-methoxy group (ortho effect).19 These two derivatives were final-
ly obtained in good yield at 40 °C (Scheme 1 and Table 1).20,21
Structures of all compounds were in good agreement with the
spectroscopic data.

C. Sall et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1493–1497
1495
NH2
OH
with R = 2-OCH3, 3-OCH3, 4-OCH3,
3,5-(OCH3)2, 3,4,5-(OCH3)3, 3-OH,
4-OH, 3-CO2H, 4-CO2H
n
R
n=1 (geraniol)
n=2 (farnesol)
1)NaNO2/ H2O,HCl, 5°C
2)NaN3, 70°C
1) NaH, THF
2) propargyl bromide
N3
O
R
n
1 n=1
2 n=2
CuSO4, Na ascorbate, EtOH/H2O
N
N
N
O
R
n
3-20
Scheme 1. Synthesis of compounds 3–20.
methoxy derivatives in the geranyl series (n = 1) as well as in the
farnesyl series (n = 2). Replacement of the methoxy group by a hy-
droxy group decreases the efficiency, and activities of the carbox-
ylic acids are modulated by the length of the terpenic chain.
When comparing the effect of derivatives of the previously de-
scribed library A and those of the present library B, further consid-
erations of structure–activity relationships can be highlighted.
Although it is difficult to affirm that the modification of the linker
enhance the activity, we can reasonably consider that in the case of
geranyl derivatives, the structure of the linker has an impact on the
activities of the methoxy derivatives since the second generation of
compounds are generally more active (except for the 4-methoxy
and 3-hydroxy derivatives), while in the case of the farnesyl
derivatives, the impact is moderate except for the 3,5-dimethoxy
derivative, which was the most potent compound of library A
Table 1
Selected 1,2,3-triazolesa and biological screening against Pseudoalteromonas sp. D41
biofilm
Compounds
n
R
Yield
(%)b
(%) of adhesiond
(brackets)e
EC50
M)
(
l
3
4
5
6
7
1
1
1
1
1
2-OCH3
3-OCH3
4-OCH3
3,5-(OCH3)2
3,4,5-
80c
72
92
63
96
29
19
20
76
36
9
6
8
1
5
(>90)
(>90)
(3 8)
(>90)
—
166 14
103 10
276 132
—
—
(OCH3)3
3-OH
4-OH
3-CO2H
4-CO2H
2-OCH3
3-OCH3
4-OCH3
3,5-(OCH3)2
3,4,5-
8
9
1
1
1
1
2
2
2
2
2
95
75
87
82
75c
67
81
57
91
55 24 (30 6)
—
—
74
—
>90
32
39
35
25
24
76
—
—
—
10
11
12
13
14
15
16
5
9
7
4
6
5
8
(25 7)
(15 39) 198 33
(46 7)
(0 7)
—
—
(EC50 of E-isomer = 71 lM).
117 13
—
—
The more potent compounds 3, 4, 5, 10, 13, 14, as well as ele-
ganolone and elegandiol were selected to evaluate their EC50 (ex-
pressed as the effective concentration to inhibit 50% of the
bacterial adhesion). This evaluation showed a dose-dependent re-
sponse, since the most potent candidate at 500 lM was not the
best candidate of the selection. Three compounds 3, 5, 13 and
50 28
(OCH3)3
3-OH
4-OH
3-CO2H
4-CO2H
—
17
18
19
20
2
2
2
2
3
3
90
70
92
76
—
>90
73 29
>90
>90
17 11
(34 2)
—
—
—
—
—
—
—
—
—
Eleganolone
Eleganediol
174 56
224 65
the two natural compounds were shown be finally inactive with
—
—
14
6
EC50 >150
tively 103 and 117
concentration of 74
active derivative of library
3,5-dimethoxy substituents on aromatic ring.
l
M. Two of them 4, 14 exhibited mild activities (respec-
M), and finally compound 10 was active at a
M. These value is in the range of the most
bearing farnesyl tail and
a
l
l
All experiments were achieved in the same conditions of concentrations of
reactants, catalyst, and volume of solvents.
b
Yield calculated from crude azides.
Yield obtained at 40 °C.
Percentage of adhesion at a concentration of 500 lM (% of adhesion).
Values reported in brackets are those obtained for the corresponding analogues
A
a
c
d
e
The further step was to investigate if these compounds exhib-
ited a specific anti-biofilm activity or if this observation was simply
related to a general toxic effect on the bacteria. In this way, the two
more active compounds 4 and 10 were tested for their capacity to
inhibit the growth of Pseudoalteromonas sp. (D41) using the antibi-
otic norfloxacin as a positive antibacterial reference.24,25 Experi-
ments were performed at the concentrations of 1, 2.5, 5, 10, 25,
of library A.8
In order to assess anti-biofilm activity of these compounds as
well as the natural leads eleganolone and eleganediol against a
gram negative bacterial biofilm, a Pseudoalteromonas sp., strain
was chosen.22,23 All compounds were first screened at a concentra-
80, 100 l
M for each compound.26 Results showed that when com-
tion of 500
l
M for their capacity to inhibit biofilm formation. At
pared to norfloxacin, the two compounds 4, 10 exhibit no effect on
the bacterial growth even at high concentrations, indicating that
the anti-biofilm activity was not connected to antibacterial effect
(Fig. 3).
this concentration, eleganolone and eleganediol showed the best
effect. Concerning the substituent effects on the aromatic ring,
mono-methoxy derivatives exhibit better activities than di or tri-

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C. Sall et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1493–1497
0.3
0.25
0.2
0.3
cpd 4
cpd 4
0.25
Norfloxacin
Control
cpd 10
Norfloxacin
0.2
Control
0.15
cpd 10
0.1
0.15
0.1
0.05
0
0.05
0
0
100
200
300
400
500
0
100
200
300
400
500
Time (min)
Time (min)
Figure 3. Effect of compounds 4, 10 and norfloxacin on bacterial growth at concentrations of 10
lM (left) and 100
lM (right).
10. Rodrıguez-Borges, J.-E.; Goncalves, S.; Vale, M. L.; Garcia-Mera, X.; Coelho, A.;
Sotelo, E. J. Comb. Chem. 2008, 10, 372.
11. Zhang, J.; Chen, H. N.; Chiang, F. I.; Takemoto, J. Y.; Bensaci, M.; Chang, C. W. T. J.
Comb. Chem. 2007, 9, 17.
12. Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.
13. Meldal, M.; Tornoe, C. W. Chem. Rev. 2008, 108, 2952.
14. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596.
15. Huisgen, R. 1, 3-Dipolar Cycloaddition Chemistry; Wiley: New York, 1984.
pp 1–176.
16. Olagnier, D.; Costes, P.; Berry, A.; Linas, M. D.; Urrutigoity, M.; Dechy-Cabaretb,
O.; Benoit-Vical, F. Bioorg. Med. Chem. Lett. 2007, 17, 6075.
17. Wilkinson, B. L.; Long, H.; Sim, E.; Fairbanks, A. J. Bioorg. Med. Chem. Lett. 2008,
18, 6265.
18. Formation of azide was monitored by TLC. Trying to purify azides by
chromatography did not improve yields of the following steps, and in
addition some of them were obtained after with low yields purification
(probably due to their degradation under the conditions of purification).
19. Dyall, L. K.; L’abbé, G.; Dehaen, W. J. Chem. Soc. 1997, 2, 971.
20. Typical method for preparation of compounds 3-20: To a stirred solution of
aniline (1 g) in H2O/HCl (50/50, 15 mL/15 mL) was added NaNO2 (0.8 g,
11.55 mmol). The whole was stirred at 0 °C for 2 h, and then NaN3 was
added (0.78 g, 11.55 mmol). The resulting solution was refluxed for 3 h. After
extraction with dichloromethane, the resulting crude azide (100 mg) was
In summary, from natural diterpenic frameworks, we have de-
signed biological active analogues in an efficient way allowing fur-
ther developments. Through the generation of this library, the
principal several relevant point when concerning SAR data in the
field of specific anti-biofilm activity is that, as hypothesized from
semi-empirical calculations which showed the similarities of the
two series, the nature of the linker modulates the activity but does
not generate fundamental changes in the biological response.
However, further studies are needed in order to highlight precisely
the effect of the terpenic chain. Finally, the low toxicity of the
derivatives allows us to focuses our interest in the development
of these molecules as non-toxic anti-biofilm compounds for poten-
tial use as non toxic co-biocides or co-antibiotics in view of rational
eradication of persistent biofilms. In this way, further studies are
actually in course in order to optimize the nature of the linker as
well as to define the mechanism of action of this class of
compounds.
dissolved in
a solution of H2O/EtOH (50/50 1.5 mL/1.5 mL) containing
Acknowledgements
CuSO4ꢀ5H2O (0.3 equiv), alkyne (1.5 equiv) and sodium ascorbate (0.4 equiv).
The resulting mixture was stirred 12 h at room temperature. A saturated
solution of Na2CO3 was added and the resulting solution extracted three times
with ethyl acetate. Organic layers were then dried over Na2SO4 and evaporated
to give the crude triazoles which were purified by flash chromatography eluted
with hexane/ethyl acetate.
The Pseudoalteromonas sp. strain was gracefully supplied by
IFREMER; Service Interfaces et Capteurs, IFREMER Brest, France.
21. Spectral data for selected compounds: 4-[((E)-3,7-dimethylocta-2,6-dienyl-
oxy)methyl]-1-(2-methoxyphenyl)-1H-1,2,3-triazole (3); (ESI, m/z) 364.35
(M+Na+), 342.38 (M+H+). 1H NMR (CDCl3, 400 MHz) d 1.29 (s, 3H, CH3), 1.37
(s, 3H), 1.38 (s, 3H), 1.73-1.84 (m, 4H), 3.50 (s, 3H), 3.83 (d, 2H, J = 6.7 Hz), 4.39
(s, 2H), 4.88 (t, 1H, J = 6.8 Hz), 5.11 (t, 1H, J = 6.7 Hz), 6.37 (m, 2H), 7;05 (dt, 1H,
J = 8.3 and 1.6 Hz), 7.43 (dd, 1H, J = 7.7 and 1.3 Hz), 7,95 (s, 1H). 13C NMR
(CDCl3, 100 MHz) d 15.7, 16.8, 24.9, 25.6, 38.8, 55.0, 62.4, 66.0, 111.5, 119.9,
120.2, 120.4, 123.2, 124.3, 124.9, 125.5, 129.3, 130.6, 139.7, 150.2. 4-[((E)-3,7-
dimethylocta-2,6-dienyloxy)methyl]-1-(3-methoxyphenyl)-1H-1,2,3-triazole
(4); (ESI, m/z) 364.33 (M+Na+), 342.37 (M+H+). 1H NMR (CDCl3, 400 MHz) d 1.26
(s, 1H), 1.33 (s, 1H), 1.35 (s, 1H), 1.69–1.80 (m, 4H), 3.50 (s, 3H), 3.82 (d, 2H,
J = 7.1 Hz,), 4.35 (s, 2H), 4.77 (t, 1H, J = 5.5 Hz), 5.07 (t, 1H, J = 6.7 Hz), 6,60 (m,
1H), 6.96 (m, 1H), 7.10–7.14(m, 2H), 7.90 (s, 1H). 13C NMR (CDCl3, 100 MHz) d
16.3, 17.5, 25.5, 26.3, 39.5, 55.3, 60.2, 66.9, 106.0, 112.1, 114.2, 120.5, 123.9,
124.0, 130.3, 130.4, 131.3, 138.0, 140.5, 160.5, 170.7. 4-[((E)-3,7-dimethylocta-
2,6-dienyloxy)methyl]-1-(4-methoxyphenyl)-1H-1,2,3-triazole (5); (ESI, m/z)
364.25 (M+Na+), 342.28 (M+H+), 1H NMR (CDCl3, 400 MHz) d 1.59 (s, 3H); 1.66
(brs, 6H); 2.02–2.09 (m, 4H), 3.86 (s, 3H), 4.08 (d, 2H, J = 7.0 Hz’), 4.12 (s, 2H),
5.08 (t, 1H, J = 5.6 Hz), 5.32 (t, 1H, J = 6 Hz), 7.01 (d, 2H, J = 7.0 Hz), 7.61 (d, 2H,
J = 7.0 Hz), 7.91 (s, 1H). 13C NMR (CDCl3, 100 MHz) d 16.4, 17.8, 25.8, 26.5, 39.7,
55.7, 63.5, 67.2, 114.9, 120.4, 122.3, 123.6 (2C), 124.1 (2C), 130.6, 131.8, 139.4,
141.2, 159.9. 3-(4-(((E)-3,7-dimethylocta-2,6-dienyloxy)methyl)-1H-1,2,3-
triazol-1-yl)benzoic acid (10); (ESI, m/z) 377.94 (M+Na+). 1H NMR (CDCl3,
400 MHz) d 1.49 (s, 3H), 1.55 (s, 3H), 1.60 (s, 3H), 1.90–2.10 (m, 4H), 4.07 (d,
J = 6.8 Hz, 2H), 4.67 (s, 2H), 4.99 (m, 1H), 5.31 (t, J = 6.7 Hz, 1H), 7.55 (d, 2H,
J = 7.8 Hz), 7.98 (d, 1H, J = 8.0 Hz), 8,06 (m, 2H), 8.32 (s, 1H). 13C NMR (CDCl3,
100 MHz) d 16.8, 17.9, 25.9, 26.5, 39.8, 63.4, 67.4, 120.1, 120.6, 121.8, 124.1,
125.7, 130.3, 130.5, 131.4, 132.0, 137.4, 141.6, 146.8, 170.7. 4-[((2E,6E)-3,7,11-
trimethyldodeca-2,6,10-trienyloxy)methyl]-1-(3-methoxyphenyl)-1H-1,2,3-
triazole (13); (ESI, m/z) 410.42 (M+H+). 1H NMR (CDCl3, 400 MHz) d 1.40 (s, 3H),
1.41 (s, 3H), 1.47 (s, 3H), 1.50 (s, 3H), 1.77–1.97 (m, 8H), 3.67 (s, 3H), 3.95 (d,
2H, J = 6.8 Hz), 4.51 (s, 2H), 4.91 (m, 2H), 5.22 (t, 1H, J = 5.6 Hz), 6.74 (m, 1H),
7.05 (m, 1H), 7.13–7.19 (m, 2H), 7.84 (s, 1H). 13C NMR (CDCl3, 100 MHz) d 16.1,
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9. Computational methods: The geometry of a selected molecule was optimized to
an rms (root mean square) gradient of 0.01 in vacuo (Polak–Ribière method). A
periodic box, 15–15–15 Å around the drug was then set up, containing 112
water molecules. The system was optimized in MM+ using switched cut-offs
(outer 10 and inner 14 Å) to an rms gradient of 0.5. Then a molecular dynamics
program was run for 1 ps, with 0.001 ps steps, relaxation time 0.1 ps, to a 30
simulation temperature of 300 K. This was followed by MM+ geometry
optimization to an rms gradient of 0.2. The molecular dynamics run was
repeated and a further MM+ protocol was carried out to a gradient of rms 0.2
on the selected drug. Finally, the geometries were optimized using the
semiempirical AM1 programme in singly excited configuration interaction to
a
gradient of rms 0.01. (RHF [Restricted Hartree–Fock], charge 0, spin
multiplicity 1, lowest state, orbital criterion, five occupied and five
unoccupied orbitals.). Properties (c Log P, hydratation energies) were
obtained from these semiempirical calculations with the QSAR package
implemented in HyperChem Release 8.05 pro for Windows (Hypercube Inc.
Gainesville, Florida.)

C. Sall et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1493–1497
1497
16.6, 17.8, 25.8, 26.4, 26.8, 39.7, 39.8, 55.6, 63.3, 67.2, 106.2, 112.3, 114.5,
120.5, 121.0, 123.9, 124.4, 130.5, 130.6, 131.2, 135.3, 138.1, 141.0, 160.6.
4-[((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyloxy)methyl]-1-(4-methoxy-
phenyl)-1H-1,2,3-triazole (14); (ESI, m/z) 432.34 (M+Na+), 410.34 (M+H+). 1H
NMR (CDCl3, 400 MHz) d 1.60 (s, 6H), 1.66 (d, 3H), 1.96–2.10 (m, 8H), 3.86 (s,
3H), 4.08 (d, 2H, J = 7.2 Hz), 4.12 (s, 2H), 5.07 (m, 2H), 5.33 (t, 1H, J = 5.6 Hz),
7.01 (d, 2H, J = 6.4 Hz), 7.61 (d, 2H, J = 7,0 Hz); 7,98 (s, 1H). 13C NMR (CDCl3,
100 MHz) d 16.1, 16.6, 17.8, 25.8, 26.4, 26.8, 39.7, 39.9, 55.7, 63.5, 67.9, 114.9,
120.0, 120.4, 122.2, 123.9, 123.9, 124.4, 131.4,135.4, 141.1, 141.8, 159.9.
22. Leroy, C.; Delbarre-Ladrat, C.; Ghillebaert, F.; Rochet, M. J.; Compère, C.;
Combes, D. Lett. Appl. Microbiol. 2007, 44, 372.
by three hand washings (36 g/L NaCl solution). The DAPI was then solubilized
in 200 L of a 95% ethanol solution. Fluorescence was measured (kexc = 380 nm,
l
kem = 495 nm) using an Infinit 200 micro-plate fluorescence reader (TECAN,
Lyon, France). The dose-response curves fitting and the determination of the
EC50 for each molecule were achieved using GraphPad Prism software.
24. Biagi, G.; Calderone, V.; Giorgi, I.; Livi, O.; Martinotti, E.; Martelli, A.; Nardi, A.
Farmaco 2004, 59, 397.
25. Cruickshank, R.; Duguid, J.-P.; Marion, B.-P.; Swain, R. H., 12th ed. In Medicinal
Microbiology; Churchil Livingstone: London, 1975; vol. II, pp 196–202.
26. Typical procedure for antibacterial assays: The antibacterial assay was
performed on Pseudoalteromonas sp. strain D41. The strains were grown on
VNSS at 20 °C under shaking (120 rpm) and sampled in the exponential phase.
23. Typical procedure for bacterial adhesion assays (adapted from Leroy et al.13).
Pseudoalteromonas sp. D41 was grown on VNSS (Vaatanen Nine Salt solution)
at 20 °C and sampled in the stationary phase. After centrifugation, cells were
suspended in sterile artificial sea water (ASW) until an optical density of
The culture was subculture at an OD600 of 0.1 in VNSS. 200 lL of VNSS was
inoculated on border-rowwells of the 96-well PS microtiter plates
(steril,Greiner Bio-one CellstarÒ). The 6th column was inoculated with
0.2–0.4 at 600 nm was achieved. 200
row wells of the 96-well microtiter plates (sterile black polystyrene NUNC),
and 100 L of the bacterial suspension on other wells using an eight-channel
pipette. 100 L of diluted standard biocide (Seanine) and purified molecules
were added in the latter wells. All the concentrations were tested in triplicates.
100 L of ASW was added in six wells to constitute the bacterial adhesion
control. After 15 h at 20 °C, the non-adhered bacteria were eliminated by three
successive hand washings (36 g/L of sterile NaCl solution). The adhered
bacteria were fixed for 90 min at 4 °C with sterile NaCl solution (36 g/L)
l
L of ASW was inoculated on the border-
180
The 10 wells of lines A and G were filed with 180
Norfloxacin (standard biocides) or natural products at varied concentrations.
The others wells were filled with 160 L of VNSS, 20 L of the bacterial
subculture and 20 L of Norfloxacin or tested products (4 and 10) at varied
concentrations were added. All the concentrations were tested in triplicates.
The maximum percentage of solvent (dimethylsulfoxide or methanol) used for
the dilution of biocides was also tested as additional control in triplicate.
The plate was incubated at 20 °C under shaking (120 rpm). The OD600 was
taken all the hour by using an Infinit 200 microplate reader (TECAN, Lyon,
France).
l
L of VNSS and 20
l
L of the bacterial subculture as a growth control.
l
L of VNSS and 20 L of
l
l
l
l
l
l
l
containing 2% formaldehyde, then bacteria were stained by adding 200
lL of
4
l
g/mL DAPI (40,6-diamidino-2-phenylindole). The excess stain was removed