Oxazaborolidine derivatives inducing autoinducer-2 signal transduction in Vibrio harveyi

Article abstract of DOI:10.1016/j.bmc.2007.11.032

The bioluminescence of the marine bacterium Vibrio harveyi is controlled by quorum sensing. This effect is mediated by production, accumulation, and auto-detection of the species-specific autoinducer 1 (AI-1), autoinducer 2 (AI-2), and the V. cholerae autoinducer 1 (CAI-1). The V. harveyi AI-2 was recently identified as furanosyl borate diester. We synthesized several oxazaborolidine derivatives that chemically resemble the structure of AI-2. Five oxazaborolidine derivatives (BNO-1 to BNO-5) were tested, however only BNO-1 (3,4-dimethyl-2,5-diphenyl-1,3,2-oxazaborolidine), and BNO-5 (2-butyl-3,4-dimethyl-5-phenyl-1,3,2-oxazaborolidine) strongly induced V. harveyi bioluminescence in V. harveyi mutant (BB170) lacking sensor 1. A dose-dependent relationship between those oxazaborolidine derivatives and bioluminescence induction was observed with this V. harveyi strain (BB170). BNO-1 and BNO-5 did not affect V. harveyi BB886 lacking sensor 2. Using a mutant strain which produces neither AI-1 nor AI-2 (V. harveyi MM77) we showed that the presence of spent medium containing AI-2 is essential for BNO-1 and BNO-5 activity. This effect was similar when introducing the spent medium and the BNOs together or at a 3-h interval. A comparable induction of bioluminescence was observed when using synthetic DPD (pre-AI-2) in the presence of BNO-1 or BNO-5. The mode of action of BNO-1 and BNO-5 on bioluminescence of V. harveyi is of a co-agonist category. BNO-1 and BNO-5 enhanced AI-2 signal transduction only in the presence of AI-2 and only via sensor 2 cascade. BNO-1 and BNO-5 are the first oxazaborolidines reported to affect AI-2 activity. Those derivatives represent a new class of borates which may become prototypes of novel agonists of quorum sensing mediated by AI-2 in V. harveyi.

Full text of DOI:10.1016/j.bmc.2007.11.032

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1596–1604
Oxazaborolidine derivatives inducing autoinducer-2
signal transduction in Vibrio harveyi
R. Aharoni,a M. Bronstheyn,a A. Jabbour,a,b B. Zaks,a M. Srebnikb and D. Steinberga,*
aInstitute of Dental Sciences, Faculty of Dental Medicine, Hebrew University-Hadassah, Jerusalem 91120, Israel
bDepartment of Medicinal Chemistry and Natural Products, School of Pharmacy, Hebrew University, Jerusalem 91120, Israel
Received 25 September 2007; revised 12 November 2007; accepted 12 November 2007
Available online 17 November 2007
Abstract—The bioluminescence of the marine bacterium Vibrio harveyi is controlled by quorum sensing. This effect is mediated by
production, accumulation, and auto-detection of the species-specific autoinducer 1 (AI-1), autoinducer 2 (AI-2), and the V. cholerae
autoinducer 1 (CAI-1). The V. harveyi AI-2 was recently identified as furanosyl borate diester. We synthesized several oxazaboroli-
dine derivatives that chemically resemble the structure of AI-2. Five oxazaborolidine derivatives (BNO-1 to BNO-5) were tested,
however only BNO-1 (3,4-dimethyl-2,5-diphenyl-1,3,2-oxazaborolidine), and BNO-5 (2-butyl-3,4-dimethyl-5-phenyl-1,3,2-oxaz-
aborolidine) strongly induced V. harveyi bioluminescence in V. harveyi mutant (BB170) lacking sensor 1. A dose-dependent relation-
ship between those oxazaborolidine derivatives and bioluminescence induction was observed with this V. harveyi strain (BB170).
BNO-1 and BNO-5 did not affect V. harveyi BB886 lacking sensor 2. Using a mutant strain which produces neither AI-1 nor
AI-2 (V. harveyi MM77) we showed that the presence of spent medium containing AI-2 is essential for BNO-1 and BNO-5 activity.
This effect was similar when introducing the spent medium and the BNOs together or at a 3-h interval. A comparable induction of
bioluminescence was observed when using synthetic DPD (pre-AI-2) in the presence of BNO-1 or BNO-5. The mode of action of
BNO-1 and BNO-5 on bioluminescence of V. harveyi is of a co-agonist category. BNO-1 and BNO-5 enhanced AI-2 signal trans-
duction only in the presence of AI-2 and only via sensor 2 cascade. BNO-1 and BNO-5 are the first oxazaborolidines reported to
affect AI-2 activity. Those derivatives represent a new class of borates which may become prototypes of novel agonists of quorum
sensing mediated by AI-2 in V. harveyi.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
membranal or intracellular receptors which control
numerous physiological pathways of the bacteria.36
Regulation of bacterial properties by population density
is a manifestation of bacterial cell–cell communication
(quorum sensing) and is mediated by signal molecules
known as autoinducers (AIs).6,29,36 Quorum sensing is
associated with genetic competence, bacterial coloniza-
tion, biofilm formation,24,30 virulence,5,37,40 and other
bacterial properties, by regulating the expression of the
corresponding genes.29
Although AI-2 cascade has been found in many spe-
cies,35 the free living marine bacterium Vibrio harveyi
has been proposed as a model for investigating quorum
sensing related to AI-1, AI-22,3 and CAI.16 V. harveyi
produces three types of AIs that regulate its biolumines-
cence: species-specific AHL designated AI-1,3,7 AI-
2,2,3,34,39,41 and the recently described CAI-1 (V. chol-
erae AI).16 Both AI-1 and AI-2 are perceived by two-
component signal transduction systems: Sensor 1, the
membrane-bound histidine kinase LuxN, binds AI-
1,3,13 while the AI-2 cognate sensor 2 includes the peri-
plasmic binding protein LuxP and the membrane-bound
histidine protein kinase LuxQ.28 In the absence of AI-1
and AI-2, the kinase activity of the sensors results in
activation (phosphorylation) of the central biolumines-
cence repressor LuxO. The phosphorylated active
repressor, in conjugation with the transcription factor
r54, activates transcription of five regulatory small
RNAs which interact with a RNA chaperon, called
Typically, oligopeptides play the role of AIs in Gram-
positive bacteria,19,22 Acylated homoserine lactones
(AHL) act as AIs in Gram-negative bacteria,14 whereas
AI-2s are considered signal molecules, affecting a broad
spectrum of bacteria.6,36,41 The AIs are recognized by
Keywords: Quorum sensing; Autoinducers; Vibrio harveyi; Lumines-
cence; Oxazaborolidines.
*
Corresponding author. Tel.: +972
6758561; e-mail: dorons@cc.huji.ac.il
2 6758633; fax: +972 2
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.032

R. Aharoni et al. / Bioorg. Med. Chem. 16 (2008) 1596–1604
1597
Hfq, and destabilized the mRNA encoding the tran-
scriptional activator LuxR which is required to activate
transcription of the luciferase operon luxCDABE. In the
presence of AIs, the phosphatase activity of the sensors
causes inactivation (dephosphorylation) of the central
repressor LuxO, LuxR is activated, promoting tran-
scription of the luciferase operon, and allows biolumi-
nescence of the bacteria.4,11,12,36,39
Therefore, it appeared to us that those molecules might
be suitable candidates for studying quorum sensing via
the AI-2 receptors of V. harveyi. We reasoned, based
on the chemical structure of oxazaborolidines and the
biological activity displayed by other boron compounds
with B–N bonds, that oxazaborolidines might selectively
bind to a receptor like sensor-2 (AI-2 receptor of V. har-
veyi) thereby triggering an agonistic response.
AI-2 is an unstable molecule which was recently crystal-
lized in a complex with LuxP and was identified as a
furanosyl borate diester in which the boron plays a piv-
otal role in the structure of the AI-2 molecule of V. har-
veyi.8,9,26 AI-2 is synthesized by V. harveyi in a series of
enzymatic reactions.32 In the final steps, LuxS converts
the ribose moiety of S-ribosylhomocysteine into 4,5-
dihydroxy-2,3-pentanedione (DPD).25,31,34,38 In V. har-
veyi, DPD reacts in its cyclic form with boric acid to
form AI-2.9 However, not all AI-2 are boron containing
compounds as was identified for the AI-2 of Salmonella
typhimurium.27
2. Materials and methods
2.1. Synthesis of oxazaborolidines
The oxazaborolidines were synthesized as described by
Jabbour et al.18 Briefly, BNO-1 to BNO-5 (Fig. 1a) were
synthesized by reacting an amino alcohol with a boronic
acid. The synthesis of BNO-1 and BNO-5 and their hy-
drated forms is illustrated in Figure 2. The purity of the
oxazaborolidines was determined by 1H, 13C, 11B NMR,
microanalysis and mass-spectral analysis.18 The maxi-
mal solubility of oxazaborolidines in double distilled
water (saturated solution) at 37 ꢁC (mM): BNO-1, 71;
BNO-2, 486; BNO-3, 358; BNO-4, 121; BNO-5, 123.
The luxS gene, encoding the LuxS enzyme synthesizing
DPD, plays a crucial role in quorum sensing of V. har-
veyi.10,36,41 Clearly, DPD (pre-AI-2) and the furanosyl
borate diester (AI-2) are potential prototypes for analog
designs due to their pivotal role in the quorum sensing
cascade.
2.2. Bacterial strains and growth conditions
All V. harveyi strains and mutants (Table 1), kindly pro-
vided by B. Bassler (Princeton University), were grown
aerobically at 30 ꢁC with constant shaking in AB
medium.15
Oxazaborolidines are five-membered heterocyclic boron
compounds containing oxygen and nitrogen atoms. No
information on the biological activity of oxazaboroli-
dines was available until our recent study on the effect
of several oxazaborolidines on the viability of S.
mutans.17,18
2.3. Determination of oxazaborolidines, MIC for V.
harveyi
Minimal inhibitory concentration (MIC) was tested
according to the CLSI (formerly NCCLS) guidelines.
V. harveyi BB170, MM77, and the BB886 strains (ad-
justed to 0.5 Macfarlane standard suspension) were
grown at 30 ꢁC, in the presence of BNO-1, BNO-2,
BNO-3, BNO-4, and BNO-5 at concentrations ranging
A structural relationship between V. harveyi AI-2 and
oxazaborolidines is evident (Fig. 1). Both are heterocy-
clic hydrated complexes, containing negatively charged
tetra-coordinated boron atom that has the ability to
form hydrogen bonds.
a
CH3
CH3
NH
CH3
O
N CH3
O
NH
O
NH
O
O
N CH3
B
B
B
B
B
n-Bu
n-Bu
BNO1
BNO2
BNO3
BNO4
BNO5
HO OH
B
b
O
O
HO
HO
CH3
O
AI-2
Figure 1. (a) Structure of BNO-1 to BNO-5. (b) Structure of AI-2.

1598
R. Aharoni et al. / Bioorg. Med. Chem. 16 (2008) 1596–1604
a
b
CH3
CH3
+
RB(OH)2
O
N
HO HN
CH3
CH3
B
R
2H2O
H2O
CH3
CH3
CH3
H
-
O
N
O
N
CH3
+
B
R
B
HO
R
Figure 2. (a) Synthesis of BNO-1 (R = Phenyl) and BNO-5 (R = Butyl) by reacting (À)-ephedrine with either phenyl or butyl boronic acid with the
azeotropic removal of water. (b) Hydrated complex formation of BNO-1 (R = Phenyl) and BNO-5 (R = Butyl).
Table 1. V. harveyi strains
V. harveyi strain
Relevant genotype
Relevant phenotype
BB170
BB886
MM77
BB152
MM30
(BB7) luxN:: Tn5
sensor -1À, sensor-2+; AI-1+, AI-2+
sensor-1+, sensor-2À; AI-1+, AI-2+
sensor-1+, sensor-2+; AI-1À, AI-2À
sensor-1+, sensor-2+; AI-1À, AI-2+
sensor-1+, sensor-2+; AI-1+, AI-2À
(BB7) luxP:: Tn5
(BB7 or BB120) luxLM:: Tn5, luxS:: Cmr
(BB7 or BB120) luxLM::Tn5
(BB120) luxS:: Tn5
from 0.5 mM to 150 mM, in 96-well transparent plates
(NUNC, Denmark). After 18-h of incubation, bacterial
growth was determined by absorbance at OD595 (GEN-
ios reader TECAN, Austria). Each experiment was con-
ducted in triplicate and repeated three times.
by V. harveyi BB170 or BB886 at minimum lumines-
cence time point after 3–4 h of incubation.2,30,34 For
the above rapid screening, the induction was determined
only according to the luminescence. The screening as-
says were performed at concentration of 200 lM of each
tested agent. This concentration was chosen as it is
about 100 times less than the MIC and no significant ef-
fect was recorded on bacterial growth at this concentra-
tion (data not shown). Based on those screening results
we have further investigated agents which have demon-
strated a potential effect on quorum sensing (BNO-1,
BNO-5).
2.4. Rapid screening of oxazaborolidines
We screened five different oxazaborolidine derivatives
(BNO-1, BNO-2, BNO-3, BNO-4, and BNO-5) for their
effect on bioluminescence of V. harveyi strains. The mu-
tant V. harveyi strains (BB170, BB886) were grown to
cell density of 1.5–2.5 · 109 CFU/ml (15–16 h) in AB
medium at 30 ꢁC with constant shaking. The fresh cul-
tures were then diluted 5000-fold in fresh AB medium
containing the tested compound at a concentration of
200 lM. Each compound was exposed to spent medium
of V. harveyi MM30 (AI-1+, AI-2À) containing AI-1 (in
the case of strain BB886) or to spent medium of V. har-
veyi BB152 (AI-1À, AI-2+) containing AI-2 (in the case
of strain BB170) to examine possible effect on biolumi-
nescence of the tested compound. Each sample was
tested also without the spent medium in order to exam-
ine potential self-induction. The negative control con-
tained bacteria in fresh AB medium and the positive
control contained bacteria, fresh AB medium, and
10% v/v spent medium with the appropriate autoinducer
(V. harveyi BB152 or MM30 spent medium). The test
was conducted in white 96-well plates (NUNC). Lumi-
nescence was measured automatically by a GENios
reader (TECAN) every 30 min. The end point of the
bacterial density was determined (OD595). Each sample
was tested in three separate biological experiments, each
conducted in triplicate. Fold induction was determined
above the endogenous level of luminescence expressed
2.5. Induction of V. harveyi bioluminescence by BNO-1
and BNO-5
Due to the fact that in our screening assay, BNO-1 and
also BNO-5 have been found to be the most effective in
inducing bioluminescence of V. harveyi, their induction
activity was further explored. The tested V. harveyi
strains (BB170 and BB886) were grown to cell density
of 1.5–2.5 · 109 CFU/ml (15–16 h). Next the suspension
was diluted (1:5000 or 1:10,000) in fresh AB medium
supplemented with BNO-1 or BNO-5 at various concen-
trations below the MIC. Positive and negative controls
were used as described before.
Bacterial cultures were incubated at 30 ꢁC for approxi-
mately 7 h, and the luminescence emitted during growth
was measured with a Lumac/3M Biocounter M2010
(Lumac BV, Schaesberg, Netherlands) or GENios read-
er (TECAN). The relative intensity of light emission was
calculated in proportion to cell density, represented by
number of CFU or relatively to the sample OD595. Fold
induction at each compound’s concentration was deter-

R. Aharoni et al. / Bioorg. Med. Chem. 16 (2008) 1596–1604
1599
mined at minimum luminescence time point after 3–4 h,
above the endogenous level of luminescence expressed
by V. harveyi BB170 or BB886, as was conducted by
Bassler et al.2
were recorded every 30 min parallel with absorbance
measurement (OD595). The value of each reading (bacte-
ria in various conditions) was divided by the absorbance
values to normalize the luminescence value of each sam-
ple to its cell density and avoid dissimilarities caused by
differences in growth rates.
2.6. The combined effect of BNO-1 and BNO-5 on
bioluminescence
Fold induction, above non specific luminescence back-
ground, was determined at the end of bacterial growth
after approximately 7 h of growth. We used this time
period because in our experimental system the most sig-
nificant inductive effect occurred then.
The potential combined effect of spent medium contain-
ing AI-2, spent medium containing AI-1 or synthetic
DPD (pre-AI-2)25 (The Scripps Research Institute,
USA) together with BNO-1 or with BNO-5 on lumines-
cence induction was determined using V. harveyi MM77
(sensor 1+, sensor 2+; AI-1À, AI-2À). Bacteria were
grown as described above. Response curves were re-
corded for 0.4% (v/v) spent medium of V. harveyi
BB152 (AI-1À, AI-2+), 10% (v/v) spent medium of
V.harveyi MM30 (AI-1+, AI-2À) or synthetic DPD at
various concentrations, ranging from 0.0002 to
200 nM, with BNO-1 or with BNO-5 at constant con-
centration of 800 lM. The effects of those combinations
on luminescence induction were examined when the bac-
teria were exposed to the tested compounds and spent
media or DPD, immediately upon the beginning of bac-
terial growth period. Further examination was per-
formed on V. harveyi BB152 spent medium and BNO-
1 or BNO-5 which were added together 3 h after the
beginning of bacterial growth or when one of the tested
agents was introduced to the bacteria upon the begin-
ning of bacterial growth and the other agent 3 h later.
The readings were taken in triplicates in 96-well white
plates with an optic bottom (NUNC) in a GENios read-
er (TECAN) at 30 ꢁC. Luminescence measurements
3. Results
3.1. Aqueous stability of oxazaborolidines
The B–N bond of the oxazaborolidines is susceptible to
nucleophilic attack and upon the addition of 1 mole of
H2O immediately forms a hydrated complex with H2O
(11B d ꢀ 7 ppm) (Fig. 2). We have investigated the hy-
drated complexes of tested oxazaborolidines and found
them to be very stable to hydrolysis in the pH range
5–13, meaning that no free boronate (boronic acid) is re-
leased from these compounds as was determined by 11B
NMR.18
3.2. Induction of V. harveyi bioluminescence by BNOs
Of the five oxazaborolidine derivatives we tested, only
BNO-1 and BNO-5 showed the strongest effect on V.
harveyi bioluminescence (Fig. 3). The effective concen-
10000
1000
100
10
V. harveyi BB170 (sensor1-, sensor2+)
V. harveyi BB886 (sensor1+, sensor2-)
1
V. harvey i BB152/MM30
spent medium (10%
Vol.)
BNO-1 200
µ
M
BNO-2 200
µ
M
BNO-3 200
µ
M
BNO-4 200
µ
M
BNO-5 200 µM
Figure 3. Screening of bioluminescence effect of BNO-1 to BNO-5 on V. harveyi BB170 (sensor-1À, sensor-2+) and V. harveyi BB886 (sensor-1+,
sensor-2À). Each screened compound was added at the beginning of bacterial growth at a concentration of 200 lM. Sample without any compound
served as negative control. Fold induction above the endogenous level of luminescence expressed by V. harveyi BB170 or BB886 was determined at
the minimum luminescence time point of the negative control (after 3–4 h). Sample with 10% v/v spent medium of V. harveyi BB152 (AI-1À, AI-2+)
served as positive control for V. harveyi BB170 and sample with 10% v/v spent medium of V. harveyi MM30 (AI-1+, AI-2À) served as positive control
for V. harveyi BB886.

1600
R. Aharoni et al. / Bioorg. Med. Chem. 16 (2008) 1596–1604
trations were below the MIC (Table 2). Therefore, we
continued to explore the influence of these two com-
pounds on the bioluminescence of V. harveyi. The induc-
tion effect of BNO-1 on the bioluminescence of V.
harveyi BB170 (sensor-1À, sensor-2+) was positively
dependent on BNO-1 concentrations in the range of 0–
600 lM. At higher concentrations, the bioluminescence
response was stable (Fig. 4). Under the same conditions,
the effect of BNO-1 on V. harveyi strain BB886 (sensor-
1+, sensor-2À) was much lower. BNO-5 was less effective
than BNO-1 in induction of bioluminescence in V. har-
veyi BB170. Similar to BNO-1 also BNO-5 had rela-
tively little effect on the mutant strain lacking sensor 2
(Fig. 5).
BNO-5 on V. harveyi MM77 (sensor-1+, sensor-2+; AI-
1À, AI-2À).
A strong effect was evident when 800 lM of BNO-1 or
BNO-5 and 0.4% v/v of V. harveyi BB152 spent medium
containing AI-2 were used (Fig. 6). There was no effect
of the same concentrations of BNO-1 or BNO-5 with
10% of V. harveyi MM30 spent medium containing
AI-1. There was also no effect of these concentra-
tions of BNO-1, BNO-5 or these spent media alone on
bioluminescence, and these fold induction values were
considered to be in the background area of the biolumi-
nescence assay (fold induction of 10 and below).
Similar results of BNO-1 and BNO-5 with V. harveyi
AI-2 were obtained when the same combinations were
added together to V. harveyi MM77, 3 h after the begin-
ning of growth, although the fold induction values were
smaller (Fig. 7). To determine whether there was a car-
ryover or mutual effect of those agents on each other, we
have also added the second agent 3 h after the first
(Fig. 7). The strongest effect was observed using
800 lM of BNO and 0.4% v/v of spent media containing
AI-2. The effect was similar whether both agents were
added at the beginning of growth period, 3 h after begin-
ning of growth or when one agent was added after the
other (Figs. 6 and 7).
3.3. The combined effect of BNO-1 and BNO-5 on
bioluminescence
To examine the mutual effect of V. harveyi autoinducers
and the BNOs, we tested the combined influence of
spent media containing AI-1 or AI-2 and BNO-1 or
Table 2. Minimal inhibitory concentration (MIC) of the different
oxazaborolidines for V. harveyi BB170, BB886 and MM77
V. harveyi
V. harveyi
V. harveyi
BB170
BB886
MM77
We have also tested the mutual effect of BNO-1 or
BNO-5 at concentration of 800 lM together with syn-
thetic and chemically defined DPD at various concentra-
tions ranging from 0.0002 to 200 nM. A strong positive
dose response effect was also obtained after 7 h of
growth, by BNO-1 or BNO-5 with DPD, at concentra-
BNO-1
BNO-2
BNO-3
BNO-4
BNO-5
20–40
150
20–40
5–10
20–40
10–20
120–150
10–25
10–30
120–150
10–25
150
120–150
10–25
10–30
Values are in mM.
1000
V. harveyi BB 170 (sensor 1-, sensor 2+)
V. harveyi BB 886 (sensor 1+,sensor 2-)
100
10
1
0
200
400
600
800
1000
1200
1400
1600
BNO-1 concentration (µM)
Figure 4. Effect of BNO-1 on induced bioluminescence of V. harveyi BB170 (sensor-1À, sensor-2+) and V. harveyi BB886 (sensor-1+, sensor-2À).
BNO-1 was added at the beginning of bacterial growth to various concentrations, ranging from 0 to 1500 lM. Sample without any compound served
as negative control. Fold induction above the endogenous level of luminescence expressed by V. harveyi BB170 or BB886 was determined at the
minimum luminescence time point of the negative control (after 3–4 h). Sample with 10% v/v spent medium of V. harveyi BB152 (AI-1À, AI-2+)
served as positive control for V. harveyi BB170 and sample with 10% v/v spent medium of V. harveyi MM30 (AI-1+, AI-2À) served as positive control
for V. harveyi BB886.

R. Aharoni et al. / Bioorg. Med. Chem. 16 (2008) 1596–1604
1601
1000
100
10
V. harveyi BB170 (sensor 1-, sensor 2+)
V. harveyi BB886 (sensor 1+, sensor 2-)
1
0
200
400
600
800
1000
1200
1400
1600
BNO-5 concentration (µM)
Figure 5. Effect of BNO-5 on induced bioluminescence of V. harveyi BB170 (sensor-1À, sensor-2+) and V. harveyi BB886 (sensor-1+, sensor-2À).
BNO-5 was added at the beginning of bacterial growth in various concentrations, ranging from 0 to 1500 lM. Sample without any compound served
as negative control. Fold induction above the endogenous level of luminescence expressed by V. harveyi BB170 or BB886 was determined at the
minimum luminescence time point of the negative control (after 3–4 h). Sample with 10% v/v spent medium of V. harveyi BB152 (AI-1À, AI-2+)
served as positive control for V. harveyi BB170 and sample with 10% v/v spent medium of V. harveyi MM30 (AI-1+, AI-2À) served as positive control
for V. harveyi BB886.
10000
BNO-1
BNO-5
1000
0.4%AI-2
10% AI-1
100
10
1
800µM BNO
0.4% AI-2+800µM BNO
10% AI-1+800µM BNO
0.4% AI-2
10% AI-1
Figure 6. Mutual effect of BNO-1, BNO-5 and spent medium containing AI-1 or AI-2 on induced bioluminescence of V. harveyi MM77 (AI-1À, AI-
2À). Bacteria were grown for approximately 7 h. V. harveyi BB152 (AI-1À, AI-2+) or V. harveyi MM30 (AI-1+, AI-2À) spent medium and BNO-1 or
BNO-5 were added to the reaction medium immediately at the beginning of the bacterial growth. Sample without any compound or spent medium
served as negative control. Fold induction above non specific luminescence background was determined at the end of bacterial growth. Constant
oxazaborolidine concentration of 800 lM was used, while spent media (represented in graph as ‘AI-1’ or ‘AI-2’) are presented as percentage from
each sample volume (200 ll).

1602
R. Aharoni et al. / Bioorg. Med. Chem. 16 (2008) 1596–1604
1000
100
10
BNO-1
BNO-5
AI-2
1
0.4% AI-2 after 3 800µM BNO after 800µM BNO and
0.2% AI-2
400µM BNO
800µM BNO
0.4% AI-2
hrs
3 hrs
0.4% AI-2 after 3
hrs
immediatly and
immediatly and
immediatly and
immediatly and
0.2% AI-2 after 3 400µM BNO after 0.4% AI-2 after 3 800µM BNO after
hrs 3 hrs hrs 3 hrs
Figure 7. Mutual effect of BNO-1 or BNO-5 and AI-2 on induced bioluminescence of V. harveyi MM77 (AI-1À, AI-2À). Bacteria were grown for
approximately 7 h. V. harveyi BB152 (AI-1À, AI-2+) spent medium and BNO-1 or BNO-5 were added together to the reaction medium 3 h after the
beginning of the bacterial growth or one agent immediately upon the beginning of the bacterial growth and the other agent 3 h later. Sample without
any compound or spent medium served as negative control. Fold induction above non specific luminescence background was determined at the end
of bacterial growth. Constant oxazaborolidine concentration of 800 lM was used, while V. harveyi BB152 spent medium (represented in graph as
‘AI-2’) is presented as percentage from each sample volume (200 ll).
tions of 0.02–20 nM on V. harveyi MM77, lacking AI-1
and AI-2 (Fig. 8).
essential for triggering maximal bioluminescence by
both BNO-1 and BNO-5 (Figs. 3–5). Supplementing
spent medium containing AI-1 together with the BNOs
to V. harveyi MM77, lacking AI-1 and AI-2 also did
not evoke bioluminescence as did with AI-2. The find-
ings that neither BNO-1 nor BNO-5 enhanced or inhib-
ited bioluminescence induced by AI-1 in a V. harveyi
mutant lacking sensor 2 (BB886) further indicates the
specificity of the reaction, demonstrating that BNOs
act via the AI-2, and not by the AI-1 receptors (Figs.
3–5). In addition, the inability of BNOs solely to induce
bioluminescence in V. harveyi MM77 (Sensor1+, sen-
sor2+, AI-1À, AI-2À), indicates that BNO1 or BNO-5
cannot fully substitute AI-2 (Figs. 6–8). The effect of
the BNOs is therefore a co-agonistic effect where the
biological outcome is dependent on the presence of
BNO-1 or BNO-5 and AI-2 or pre-AI-2 (DPD) and sen-
sor 2.
4. Discussion
Quorum sensing has been studied by several means, such
as genes involved in quorum sensing, enzymatic inhibi-
tion in the metabolic pathway of AI synthesis or at var-
ious points in the signal transduction pathway .1,21 Only
after identification of the AI-2 molecule by Chen et al.,8
an alternative approach, enabling evaluation of the
structural requirements of AI-2, was available. Clearly,
DPD in its cyclic form and furanosyl borate diester
(AI-2) are potential prototypes for analog design due
to their pivotal role as the final products of the AI-2 syn-
thesis cascade.
The atom boron is considered a pivotal functional part
of the AI-2 molecule.8,9 We synthesized oxazaborolidine
derivatives which chemically resemble the structure of
AI-2 (Fig. 1). Five oxazaborolidine derivatives were
tested on the bioluminescence of V. harveyi. The oxaz-
aborolidine derivatives, BNO-1 and BNO-5 most
strongly induced the bioluminescence of the reporter
strain V. harveyi BB170 (Fig. 3). This finding prompted
us to further investigate a potential mechanism of action
of this induction.
BNOs, in the presence of exogenous AI-2, evoked a sig-
nificant level of bioluminescence. A similar trend was
observed when the agents were introduced in the begin-
ning of incubation period of the bacteria or 3 h later or
when the sequence of introducing BNOs and AI-2 was
altered. These indicate that the effect is non-time depen-
dent and the sequence of exposure of the bacteria to
either BNOs or AI-2 is not relevant to the induction
of bioluminescence.
Using a mutant strain of V. harveyi, lacking sensor 1
(BB170), we found that the presence of sensor 1 is not
The use of structural bacterial AI analogs provides a un-
ique means of manipulating and studying quorum sens-

R. Aharoni et al. / Bioorg. Med. Chem. 16 (2008) 1596–1604
1603
100000
10000
1000
100
BNO-1 (800µM)
BNO-5 (800µM)
control
10
1
0
0.0002
0.002
0.02
0.2
2
20
200
DPD concentration (nM)
Figure 8. Mutual effect of BNO-1 or BNO-5 and synthetic DPD (pre-AI-2) at different concentrations on induced bioluminescence of V. harveyi
MM77 (AI-1À, AI-2À). Bacteria were grown for approximately 7 h. Synthetic DPD was added, at various concentrations, to the reaction medium
immediately upon the beginning of the bacterial growth together with BNO-1 or BNO-5. Sample without any compound served as negative control.
Fold induction above non specific luminescence background was determined at the end of bacterial growth. Constant oxazaborolidine concentration
of 800 lM was added. DPD concentrations are in nM.
ing.5,21,33 The gene luxS is found among many types of
bacteria and products of LuxS are homologs and are
tightly related to bacterial AIs.36,41 AI-2 agonists and
antagonists can affect quorum sensing of a wide range
of bacteria, although AI-2-like signals, from various
bacterial species, are not identical.27
factor in activation of quorum sensing in V. harveyi and
that the butyl or phenyl substitutes on the boron are of
less importance.
While BNO-1 and BNO-5 share the same amino alcohol
moiety, different groups are attached to boron. In BNO-
1, the aromatic phenyl group is attached to boron. In
BNO-5, the alkyl n-butyl group is attached. The fact
that the two compounds substantially differ in activity
may, therefore, indicate that the aromatic group must
interact favorably at the active site, affecting the AI-2
cascade.
The unexpected finding by Chen et al.8 that V. harveyi
AI-2 is a furanosyl borate diester has highlighted boron
as a cardinal structural element of AI-2,9 although it was
recently shown that boron is not essential in all bacterial
quorum sensing.23,27 The oxazaborolidines and V. har-
veyi AI-2 are hydrated complexes of heterocyclic boron.
Both contain a negatively charged tetra-coordinated
boron atom that has the ability to form hydrogen bonds.
However, the boron atom in the BNOs is a part of the
oxazaborolidine ring, whereas in V. harveyi AI-2 it is
part of a dioxaborolane ring that does not contain nitro-
gen and has no aromatic substitute. Both chelation of
boron and the position of the hydroxyl moieties in V.
harveyi AI-2 are critical for activation of sensor-2.20,32
In addition, BNO-1 and BNO-5 activity indicates that
the presence of a five-membered heterocyclic ring con-
taining tetrahedral boron bearing a hydroxyl group is
sufficient for specific interaction with V. harveyi AI-2
signal transduction, despite the fact that this structure
is only slightly reminiscent of V. harveyi AI-2. The find-
ing that BNO-1 and BNO-5 induce bioluminescence in
V. harveyi, but BNO-2, BNO-3, BNO-4 do not, may
indicate that the methyl on the nitrogen is an important
We have investigated the hydrated complexes of tested
oxazaborolidines and found them to be very stable to
hydrolysis in the pH range 5–13, meaning that no free
boronate (boronic acid) is released from these com-
pounds as was determined by 11B NMR.18 In a separate
study (data not shown), we demonstrated that BNO-1
does not exchange with B(OH)3. This indicates that an
exchange with V. harveyi AI-2 is also highly unlikely,
and that under the conditions of the above experiment,
BNO-1 remains intact. This is reasonable since boro-
nates are stronger Lewis acids than boric acid.
The use of synthetic bacterial AI analogs allows better
insight into the quorum sensing cascade. We propose
that derivatives of oxazaborolidines may mimic V. har-
veyi AI-2 activity. Based on this study, a full scale
screening of more oxazaborolidine derivatives should re-

1604
R. Aharoni et al. / Bioorg. Med. Chem. 16 (2008) 1596–1604
19. Kleerebezem, M.; Quadri, L. E.; Kuipers, O. P.; de Vos,
W. M. Mol. Microbiol. 1997, 24, 895–904.
20. Lowery, C. A.; McKenzie, K. M.; Qi, L.; Meijler, M. M.;
Janda, K. D. Bioorg. Med. Chem. Lett. 2005, 15, 2395–
2398.
veal substances with stronger agonistic activity, opening
up new possibilities for studies on the structure–function
relationships of V. harveyi AI-2.
21. Lyon, G. J.; Muir, T. W. Chem. Biol. 2003, 10, 1007–
1021.
Acknowledgments
22. Lyon, G. J.; Novick, R. P. Peptides 2004, 25, 1389–
1403.
23. McKenzie, K. M.; Meijler, M. M.; Lowery, C. A.; Boldt,
G. E.; Janda, K. D. Chem. Commun. (Camb.) 2005, 4863–
4865.
24. McNab, R.; Ford, S. K.; El-Sabaeny, A.; Barbieri, B.;
Cook, G. S.; Lamont, R. J. J. Bacteriol. 2003, 185, 274–
284.
25. Meijler, M. M.; Hom, L. G.; Kaufmann, G. F.; McKen-
zie, K. M.; Sun, C.; Moss, J. A.; Matsushita, M.; Janda,
K. D. Angew. Chem. Int. Ed. Engl. 2004, 43, 2106–2108.
26. Miller, M. B.; Bassler, B. L. Annu. Rev. Microbiol. 2001,
55, 165–199.
This work is part of the M.Sc. thesis of R. Aharoni, and
the Ph.D. thesis of M. Bronstheyn and A. Jabbour.
This work was supported in part by the Israeli Science
Foundation (ISF).
We thank Prof. B.L. Bassler for providing the Vibrio
harveyi strains and Prof. K.D. Janda for providing the
synthetic DPD.
27. Miller, S. T.; Xavier, K. B.; Campagna, S. R.; Taga, M.
E.; Semmelhack, M. F.; Bassler, B. L.; Hughson, F. M.
Mol. Cell 2004, 15, 677–687.
28. Neiditch, M. B.; Federle, M. J.; Miller, S. T.; Bassler, B.
L.; Hughson, F. M. Mol. Cell 2005, 18, 507–518.
29. Reading, N. C.; Sperandio, V. FEMS Microbiol. Lett.
2006, 254, 1–11.
30. Rickard, A. H.; Palmer, R. J., Jr.; Blehert, D. S.;
Campagna, S. R.; Semmelhack, M. F.; Egland, P. G.;
Bassler, B. L.; Kolenbrander, P. E. Mol. Microbiol. 2006,
60, 1446–1456.
31. Schauder, S.; Shokat, K.; Surette, M. G.; Bassler, B. L.
Mol. Microbiol. 2001, 41, 463–476.
32. Semmelhack, M. F.; Campagna, S. R.; Hwa, C.; Federle,
M. J.; Bassler, B. L. Org. Lett. 2004, 6, 2635–2637.
33. Suga, H.; Smith, K. M. Curr. Opin. Chem. Biol. 2003, 7,
586–591.
34. Surette, M. G.; Miller, M. B.; Bassler, B. L. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 1639–1644.
35. Vendeville, A.; Winzer, K.; Heurlier, K.; Tang, C.
M.; Hardie, K. R. Nat. Rev. Microbiol. 2005, 3, 383–
396.
36. Waters, C. M.; Bassler, B. L. Annu. Rev. Cell Dev. Biol.
2005, 21, 319–346.
37. Williams, P.; Camara, M.; Hardman, A.; Swift, S.; Milton,
D.; Hope, V. J.; Winzer, K.; Middleton, B.; Pritchard, D.
I.; Bycroft, B. W. Philos Trans. R. Soc. Lond. B. Biol. Sci.
2000, 355, 667–680.
38. Winzer, K.; Hardie, K. R.; Burgess, N.; Doherty, N.;
Kirke, D.; Holden, M. T.; Linforth, R.; Cornell, K. A.;
Taylor, A. J.; Hill, P. J.; Williams, P. Microbiology 2002,
148, 909–922.
39. Winzer, K.; Hardie, K. R.; Williams, P. Adv. Appl.
Microbiol. 2003, 53, 291–396.
40. Winzer, K.; Williams, P. Int. J. Med. Microbiol. 2001, 291,
131–143.
References and notes
1. Bassler, B. L.; Greenberg, E. P.; Stevens, A. M. J.
Bacteriol. 1997, 179, 4043–4045.
2. Bassler, B. L.; Wright, M.; Showalter, R. E.; Silverman,
M. R. Mol. Microbiol. 1993, 9, 773–786.
3. Bassler, B. L.; Wright, M.; Silverman, M. R. Mol.
Microbiol. 1994, 13, 273–286.
4. Bassler, B. L.; Wright, M.; Silverman, M. R. Mol.
Microbiol. 1994, 12, 403–412.
5. Camara, M.; Williams, P.; Hardman, A. Lancet Infect.
Dis. 2002, 2, 667–676.
6. Camilli, A.; Bassler, B. L. Science 2006, 311, 1113–1116.
7. Cao, J. G.; Meighen, E. A. J. Biol. Chem. 1989, 264,
21670–21676.
8. Chen, X.; Schauder, S.; Potier, N.; Van Dorsselaer, A.;
Pelczer, I.; Bassler, B. L.; Hughson, F. M. Nature 2002,
415, 545–549.
9. Coulthurst, S. J.; Whitehead, N. A.; Welch, M.; Salmond,
G. P. Trends Biochem. Sci. 2002, 27, 217–219.
10. De Keersmaecker, S. C.; Sonck, K.; Vanderleyden, J.
Trends Microbiol. 2006, 14, 114–119.
11. Freeman, J. A.; Bassler, B. L. J. Bacteriol. 1999, 181, 899–906.
12. Freeman, J. A.; Bassler, B. L. Mol. Microbiol. 1999, 31,
665–677.
13. Freeman, J. A.; Lilley, B. N.; Bassler, B. L. Mol.
Microbiol. 2000, 35, 139–149.
14. Fuqua, C.; Parsek, M. R.; Greenberg, E. P. Annu. Rev.
Genet. 2001, 35, 439–468.
15. Greenberg, E. P.; Hastings, J. W.; Ulitzur, S. Arch.
Microbiol. 1979, 120, 87–91.
16. Henke, J. M.; Bassler, B. L. J. Bacteriol. 2004, 186, 6902–
6914.
17. Jabbour, A.; Srebnik, M.; Zaks, B.; Dembitsky, V.; Stein-
berg, D. Int. J. Antimicrob. Agents 2005, 26, 491–496.
18. Jabbour, A.; Steinberg, D.; Dembitsky, V. M.; Moussaieff,
A.; Zaks, B.; Srebnik, M. J. Med. Chem. 2004, 47, 2409–
2410.
41. Xavier, K. B.; Bassler, B. L. Curr. Opin. Microbiol. 2003,
6, 191–197.