1,4-Diazobicyclo(2,2,2)octane-modified multi-ammonium derivatives as ratiometric fluorescent sensors for lipopolysaccharide

Article abstract of DOI:10.1080/10610278.2012.732224

A series of pyrene derivatives bearing multi-quaternary ammonium 1,4-diazobicyclo(2,2,2)octane groups were synthesised as ratiometric fluorescent sensors for lipopolysaccharide (LPS) in aqueous solution. Ratiometric changes in fluorescence spectra of the

Full text of DOI:10.1080/10610278.2012.732224

Supramolecular Chemistry
Vol. 25, No. 2, February 2013, 69–78
1,4-Diazobicyclo(2,2,2)octane-modified multi-ammonium derivatives as ratiometric fluorescent
sensors for lipopolysaccharide
Wenwen Zhaoa,b, Jiasheng Wua, Weimin Liua and Pengfei Wanga*
aKey Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese
Academy of Sciences, Beijing 100190, P.R. China; Graduate School of Chinese Academy of Sciences, Beijing 100049, P.R. China
b
(Received 30 June 2012; final version received 16 September 2012)
A series of pyrene derivatives bearing multi-quaternary ammonium 1,4-diazobicyclo(2,2,2)octane groups were synthesised
as ratiometric fluorescent sensors for lipopolysaccharide (LPS) in aqueous solution. Ratiometric changes in fluorescence
spectra of the sensors were observed from the pyrene monomer–excimer conversion which is modulated by the
supramolecular self-assembly between sensors and LPS. These sensors show high selectivity towards LPS with the detection
limit down to tens of nanomolar levels. The interaction mechanism between the sensors and LPS was also discussed.
Keywords: lipopolysaccharide; pyrene; ratiometric; sensor; self-assembly
Introduction
the LPS toxic concentration (10212 M). Recently, we have
committed to the exploration of fluorescent sensors that
can trace LPS via the lipophilic pyrene derivatives (16),
cysteamine-modified gold nanoparticles (17) or co-
polythiophene derivatives (15). These sensors exhibit
high sensitivity with the detection limit at nanomolar or
even picomolar levels.
Lipopolysaccharide (LPS, also known as bacterial
endotoxin) is the main structural component in the outer
cell membranes of all Gram-negative bacteria (1, 2). It is
responsible for the integrity and low permeability of the
membrane, thereby protecting the bacteria from the
destruction by antibiotics (3). But massive existence of
LPS is an indication of bacteria contamination and can
result in sepsis and septic shocks (4, 5), which cause about
150,000 casualties annually in the USA (6). Due to its high
toxicity of LPS, considerable efforts have been devoted to
the development of specific quantification of LPS. Thus
far, some analytical methods for LPS have been
established including rabbit pyrogen test and enzymatic
limulus amebocyte lysate (LAL) assay (7). However, there
are some drawbacks for these methods, for instance, the
rabbit pyrogen test shows low sensitivity and its result
varies greatly with the individual differences in rabbits.
The LAL assay is a sensitive method, whereas it is highly
susceptible to changes in temperature and pH. Moreover,
carbohydrate derivatives other than LPS, such as b-glucan,
also respond positively in this assay (8). It is thus highly
desirable to develop specific, quantitative and sensitive
assays for detection of LPS.
LPS consists of a polysaccharide unit and a lipid A unit
(Scheme 1a). The two phosphorylated glucosamine sugars
in the lipid A and two 2-keto-3-deoxyoctanoate units, each
carrying carboxylic groups, make LPS highly negatively
charged. In addition, LPS is an amphiphilic molecule and
can be automatically arranged in order in aqueous solution
due to various long-chain fatty acid esters and amides
attached to the glucosamine disaccharide core. Along with
our continuing efforts in the exploration of fluorescent
sensors for trace detection of LPS, we herein report a series
of pyrene-based derivatives (C4-D-O-Py, C2-D-O-Py, Ph-
D-O-Py and C2-D-Py) bearing multi-ammonium 1,4-
diazobicyclo(2,2,2)octane (DABCO) groups as ratiometric
sensors for LPS in aqueous solution (Scheme 1b) via the
supramolecular interactions. The introduction of two
pyrene moieties into one molecular system is expected to
detect LPS ratiometrically, which can effectively eliminate
most or all interferences from the environment by built-in
correction of two emission bands. Moreover, the multi-
ammonium DABCO substituents in probes not only
improve their water solubility but also provide four
positive centres to bind with the negatively charged LPS
via the strong electrostatic, hydrophobic and p–p
stacking interactions. The most important differences
among these probes are the connector flexibility between
Chromo- and fluorogenic chemosensors for biological
species have attracted great attention due to their high
sensitivity, good selectivity, as well as on-line and real-
time analysis (9, 10). However, only a few such sensors for
LPS have been reported (11–15). Rangin and Basu (11)
first reported a colorimetric sensor for LPS based on
functionalised polydiacetylene liposome with the detec-
tion limit of about 100 mM, which is still much higher than
*Corresponding author. Email: wangpf@mail.ipc.ac.cn
ISSN 1061-0278 print/ISSN 1029-0478 online
q 2013 Taylor & Francis
http://dx.doi.org/10.1080/10610278.2012.732224
http://www.tandfonline.com

70
W. Zhao et al.
(a) Lipopolysaccharides (LPS)
HO
OH
HO
HO
R
O
HO
O
O
–
OH
O
O
O
O
–
O
O
O
P
O
–
O
–
O
O
O
O
O
O
O
O
NH
O
HO
O
P
O
–
O
NH
O
–
O
O
O
HO
O
O
R=
HO
Lipid A
n
(b)
Sensors
O
O
O
O
+
+
+
+
+
N
N
+
N
O
+
+
N
+
N
–
N
–
4Br
–
4Br
O
+
N
4Br
N
+
N
+
N
+
N
–
+
N
4Br
N
+
N
N
+
N
O
O
O
O
O
O
Ph-D-O-Py
C2-D-O-Py
C4-D-O-Py
C2-D-Py
Scheme 1. Molecular structures of (a) LPS and (b) the sensors.
1
the two DABCO moieties and the alkyl chain length
between DABCO and pyrene. It is found that the more
flexible connector and the longer chain length of the sensor
can help to improve the sensitivity and selectivity of the
sensors to LPS.
compounds were verified by H, 13C NMR and MS or
element analysis. Details on the synthesis and character-
isation are described in ‘Experimental’ section.
Optical spectral studies with LPS
Figure 1 shows the results of absorption spectra of C4-D-O-
Py (5 mM) in 4-(2-hydroacidxyethyl)-1-piperazineethane-
sulfonic acid (HEPES) buffer (10.0 mM, pH 7.4,
H2O:DMSO ¼ 8:2, v/v) titrated with LPS. Without LPS,
C4-D-O-Pydisplaystheabsorptionbandscentredat275,327
and343 nm. UponadditionofLPSrangingfrom0to2.2 mM,
band broadening and a significant red shift are observed,
which can be attributed to the intermolecular p–p stacking
of two pyrene groups in their ground state (Figure 1(a)) (11).
Interestingly, the absorption intensity increases with a slight
blue shift when LPS exceeds 2.6 mM (Figure 1(b)).
Results and discussion
Synthesis and structural characterisation
The synthetic routes for fluorescent sensors are depicted in
Scheme 2. Alkylation of o-methyl phenol with 1,2-
dibromoethane or 1,4-dibromobutane afforded compounds
1 and 2, which were further brominated with N-
bromosuccinimide (NBS) to give dibromide derivatives 3
and 4, respectively. Compounds 5 and 7 were synthesised
according to the reported procedure, and then treated by
DABCO to yield 6 and 8. Finally, dibromide derivatives (3,
4 or m-xylylene dibromide) react with 6 or 8 to give the
desired sensors with moderate yields (50–70%). All
The excimer formation can be observed more directly
from the fluorescence spectra (Figure 2). In the absence of

Supramolecular Chemistry
71
OH
Br
O
O
NBS/AIBN
CCl4
Br
n
O
O
n
n
Br
Br
1: n = 1
2: n = 2
3: n = 1
4: n = 2
Br–
N+
CHO
CH2OH
CH2Br
N
N
PBr3
Toluene
N
NaBH4
CH3OH
3
C2-D-Py
CH3COOC2H5
CH3CN
5
6
Br–
N+
CH2OH
Br
Br
Br
Ph-D-O-Py
N
O
O
CH3CN
Br
N
Br
CTAB/NaOH
N
3
CH3COOC2H5
C2-D-O-Py
C4-D-O-Py
CH3CN
4
7
8
CH3CN
Scheme 2. The synthetic pathway of the sensors.
LPS, C4-D-O-Py exhibits the emission peaks at 375 and
395 nm and a broad band centred at 489 nm. The former
two are typical pyrene monomers and the last one
corresponds to fluorescence emission of the excimer. Upon
addition of 0–2.2 mM LPS, the fluorescence intensity of
the monomer emission markedly decreases while the
excimer emission intensity significantly increases with a
discernible isoemissive point at 418 nm (Figure 2(a)). By
plotting the emission intensity ratio (I485/I389) to the added
LPS, a good linearity (R 2 ¼ 0.992) was observed with the
LPS concentration ranging from 0 to 1.8 mM (Figure 2(b)),
indicating that the sensor C4-D-O-Py enables ratiometric
detection of LPS. The detection limits, defined as three
times the standard deviation of the background, were
calculated to be 18.5 nM, which is the lowest value of the
reported LPS probes based on small organic molecules.
When the concentration of LPS is further increased (2.6–
25.2 mM), the fluorescence change reverses. This indicates
that the monomer emission intensity increases dramati-
cally, accompanied by a decrease in the excimer emission
(Figure 2(c)). And the plot of I485/I389 ratio to LPS
concentration decreases gradually (Figure 2(d)).
Similar results were obtained with the addition of LPS
to the solution of Ph-D-O-Py or C2-D-O-Py. It can be seen
that the band broadening and red shift in absorption spectra,
and a significant excimer/monomer ratio enhancement in
emission spectra were observed at low LPS concentration,
whereas the optical spectra reverse at high LPS concen-
tration (Figure S1 and S2 of the Supporting Information,
available online, respectively). To clarify the effect of
connector flexibility between two DABCO moieties on LPS
sensing, we compared the responses of the three sensors
(a)
(b)
0
25.2
23.2
21.2
19.2
17.2
15.2
13.2
11.2
0.4
0.2
(µΜ)
0.4
(µΜ)
0.6
0.3
0.3
0.8
1.0
1.2
0.2
9.2
0.2
1.4
8.2
7.2
6.2
5.2
4.2
3.2
2.6
1.6
1.8
0.1
0.1
0.0
2.0
2.2
2.4
0.0
270
300
330
360
390
300
320
340
360
380
400
Wavelength (nm)
Wavelength (nm)
Figure 1. Absorption spectra of C4-D-O-Py (5 mM) in the presence of different concentrations of LPS in a buffered (10 mM HEPES, pH
7.4, H2O:DMSO ¼ 8:2, v/v) solution: (a) 0–2.4 mM; (b) 2.6–25.2 mM.

72
W. Zhao et al.
(a)
(b)
8
y = a + b*x
No Weighti
0.19658
Equation
Weight
Residual
sum of
7
6
4000
3000
2000
1000
0
(µM)
Squares
0.99236
Adj. R-Squa
Value Standard Error
2.4
Intercept
Slope
1.7120
2.9519
0.09213
B
B
0.086029
5
I
4
I
0
3
2
1
0.0
0.5
1.0
1.5
400
450
500
550
600 650
Wavelength (nm)
Concentration (µm)
(c)
(d)
8
7
6
5
4
3
2
1
0
(µM)
4000
3000
2000
1000
0
2.6
I
27.2
I
0
5
10
15
20
25
400
450
500
550
600
650
Wavelength (nm)
Concentration (µm)
Figure 2. Fluorescence emission spectra (lex ¼ 360 nm) of C4-D-O-Py (5 mM) recorded with different concentrations of LPS: (a) 0–
2.4 mM; (c) 2.6–25.2 mM in a buffered (10 mM HEPES, pH 7.4, H2O:DMSO ¼ 8:2, v/v) solution; (b) and (d) fluorescence intensity ratio
(I485/I389) against the LPS concentrations.
above to LPS, and the results are summarised in Figure 3
and Table 1. It can be seen that all of the three sensors can
detect LPS by utilising the ratiometric signal of the
excimer/monomer emission with tens of nanomolar
detection limit, suggesting that the connector between the
two DABCO moieties had a little effect on the spectral
performance of the sensors. However, compared with Ph-
D-O-Py and C2-D-O-Py, C4-D-O-Py with the maximal
slope in the I485/I389 ratio exhibits the lowest detection limit
and the widest linear range. These results indicate that the
more flexible of the connector between the two DABCO
moieties, the better responses of the sensor towards LPS.
Thus, the sensor C4-D-O-Py was chosen for further
discussion on pH effect and selectivity measurement.
For the sensor C2-D-Py in which the alkyl chain length
between the DABCO and pyrene is shorter than that of the
probes discussed above (Ph-D-O-Py, C2-D-O-Py and C4-
D-O-Py), a distinctly different fluorescence response
towards LPS was observed. As shown in Figure 4, the
addition of LPS at even a higher concentration can give
8
C4-D-O-Py
C2-D-O-Py
Ph-D-O-Py
7
6
5
C2-D-Py
I
4
I
3
Table 1. Summarised performance of the probes to LPS.
2
1
0
The LPS
concentrations
corresponding
to the increased
485/I389 ratio
(mM)
The linear
range of LPS
(mM)
0
5
10
15
20
I
Detection
limit (nM)
Concentration (µM)
Compounds
Figure 3. The fluorescence intensity in the I485/I389 ratio of four
different probes (5 mM) against the LPS concentrations in a
buffered (10 mM HEPES, pH 7.4, H2O:DMSO ¼ 8:2, v/v)
solution.
C4-D-O-Py
C2-D-O-Py
Ph-D-O-Py
0–2.2
0–1.6
0–1.3
0–1.8
0–1.3
0–1.2
18.5
25.2
36.9

Supramolecular Chemistry
73
(b)
(a)
14.0
12.0
1.0
10.0
5000
4000
3000
2000
1000
0
8.0
6.0
4.0
3.0
2.2
1.8
1.4
1.0
0.8
0.6
0.4
0.2
0
(µM)
0.8
I
0.6
I
0.4
0.2
0
4
8
12
16
20
400
450
500
550
600
650
Wavelength (nm)
Concentration (µM)
Figure 4. (a) Changes in fluorescent spectra (lex ¼ 360 nm) of C2-D-Py (5 mM) upon addition of LPS in HEPES:DMSO (8:2, v/v)
buffer solution (10 mM, pH 7.4). (b) Fluorescence intensity ratio (I485/I389) against the LPS concentration.
rise to a small change in monomer/excimer emission ratio,
replaced by a significant increase in monomer emission.
This could be attributed to the rigid conformation of
DABCO groups which hinders the two pyrene groups
close to form the excimer. This means that the sensors just
with a flexible enough alkyl chain between DABCO and
pyrene can detect LPS with a ratiometric change.
To further demonstrate the supramolecular self-
assembly mechanism, the controlled experiments using
C4-D-O-Py treated with LPS in different solvents were
carried out and the results are shown in Figure 5. Only in
aqueous solution, C4-D-O-Py exhibits a distinct spectral
response to LPS (Figures 1 and 2), whereas in absolute
DMSO, it cannot give rise to any spectral changes even at a
higher LPS concentration (8.0 mM) (Figure 5), indicating
that supramolecular self-assembly indeed plays an
important role for LPS sensing.
The proposed schematic illustration of the sensing
mechanism for the probes to LPS is depicted in Scheme 3a.
Without LPS, some molecules of Ph-D-O-Py, C2-D-O-Py
or C4-D-O-Py, containing six bonds between DABCO and
pyrene, exist in the monomer form in aqueous solution due
to the relative rigid conformation of DABCO and the
intramolecular electrostatic repulsion. Upon addition of
low concentration of LPS, the strong electrostatic,
hydrophobic and p–p stacking interactions could drive
the probes and LPS to form supramolecular self-assembly,
which weakens the intramolecular electrostatic repulsion
and induces two pyrene moieties close to each other, thus
leading to a significant increase in excimer/monomer
emission ratio. Subsequently, with increasing concen-
tration of LPS to a certain extent, the highly ordered
phospholipids bilayers of LPS formed in aqueous solution
may give rise to solubility enhancement, so that most
pyrene moieties incorporate into micelle in monomer
form. Moreover, the lower polarity inside LPS micelles
reduces the possibility of an excited-state pyrene to
encounter another ground-state pyrene. Those might be
interpreted as the main reasons for the obvious decrease of
excimer/monomer emission ratio at high LPS
concentrations.
pH effect
The effect of pH on the sensing performance to LPS was
investigated. Taking C4-D-O-Py for example, the
fluorescence intensity I485/I389 ratios of free sensor are
relatively stable in a wide pH range from 3 to 11. However,
the I485/I389 ratios show an obvious pH dependency upon
addition of LPS at a constant concentration of 2.5 mM. As
shown in Figure 6, at acidic conditions, the I485/I389 ratio
drops a lot with decreasing pH. This phenomenon can be
attributed to the carboxylate and phosphate (Pi) moieties
of LPS may be partially protonated, which decreases the
electrostatic interactions between the sensor and LPS.
When pH ranges from 7 to 10, C4-D-O-Py shows the
highest fluorescence response towards LPS and the
I
485/I389 ratios vary slightly with the pH changes.
Therefore, a physiological condition of pH 7.4 (10 mM
HEPES buffer) was chosen as the optimum determining
medium for LPS sensing.
In contrast, although C2-D-Py can also bind with LPS
via strong electrostatic and hydrophobic interactions to
form supramolecular self-assembly, no obvious excimer
formations were found because the too short connector
between DABCO and pyrene moiety cannot overcome the
hindrance from rigid conformation of DABCO groups to
form the excimer via the supramolecular interactions
(Scheme 3b).
Selectivity
The selectivity of the sensors for LPS was evaluated by
)
monitoring the fluorescence intensity ratio (I485/I389
response in the presence of various anions and biological
species. We still take C4-D-O-Py as an example, and the
results are illustrated in Figure 7. It reveals that only the
addition of LPS could result in a prominent increase of the

74
W. Zhao et al.
Scheme 3. The proposed schematic illustration of LPS sensing. (a) Ph-D-O-Py, C2-D-O-Py and C4-D-O-Py; (b) C2-D-Py.
I485/I389 ratio. In contrast, negligible variations of the
I485/I389 ratio were observed in the presence of Pi,
pyrophosphate, sodium citrate, adenosine triphosphate
(ATP), adenosine diphosphate (ADP) or adenosine mono-
posphate (AMP). This finding indicates that C4-D-O-Py
shows a sound selectivity for LPS over other simple anionic
phosphates. Molecules with two carboxyl groups, including
malic acid, aspartic acid and glutamic acid, cannot induce

Supramolecular Chemistry
75
(a)
(µM)
(b)
(µM)
2000
1600
1200
800
400
0
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0
0
LPS
LPS
1.6
8
1.6
8
400
450
500
550
600
650
270
300
330
360
390
Wavelength (nm)
Wavelength (nm)
Figure 5. Spectral responses of C4-D-O-Py (5.0 mM) to LPS in absolute DMSO.
any significant fluorescence changes in the I485/I389 ratio.
For some ordinary fatty acid salts such as sodium dodecyl
sulphate and lysophosphatidic acid (LPA), although they
have long alkyl chain and negatively charged end groups,
they cannot induce enhancements in the I485/I389 ratio.
Moreover, C4-D-O-Py shows no responses to some
coexisting biomolecules in practical samples, such as
glucose, albumin from bovine serum (BSA) and lecithin. In
addition, C4-D-O-Py exhibits an obvious enhancements of
the I485/I389 ratio to LPS even in the presence of other
coexisting species, indicating that C4-D-O-Py as a LPS-
selective sensor is not affected by common competing
species. These above results suggest evidently that C4-D-O-
Py shows highly selective fluorescence response for LPS in
aqueous solution. As we know, LPS is highly negatively
charged and more hydrophobic than other biomolecules,
such as ATP, ADP, AMP, BSA and LPA. The good
selectivity could be attributed to the stronger electrostatic,
hydrophobic and p–p stacking interactions between the
sensor and LPS, enabling them to form the supramolecular
self-assembly. This selectivity results are well consistent
with the sensing mechanism proposed above.
9
8
7
6
5
4
3
2
I
Sensor
I
Sensor + LPS
2
4
6
8
10
pH
Figure 6. Effect of pH on the fluorescence intensity ratio
(I485/I389) of free C4-D-O-Py (5 mM) in the absence and presence
of LPS (2.5 mM) at room temperature.
10
8
Conclusions
In summary, we have successfully developed a series of
bis-pyrene derivatives bearing multi-ammonium DABCO
groups as ratiometric fluorescent sensors for quantitative
detection of LPS. These sensors show excellent selectivity
towards LPS with tens of nanomolar sensitivity, which is
the lowest value of the reported small molecule-based
probes for LPS. The sensing mechanism could be
suggested that the strong electrostatic and hydrophobic
interactions, as well as p–p stacking of pyrene moiety
induce the supramolecular self-assembly between the
sensor and LPS in aqueous solution, then, if the alkyl chain
between DABCO and pyrene is flexible enough to
overcome the rigid conformation of DABCO groups, the
overlap between the pyrene could be regulated effectively,
showing a good fluorescence ratio of pyrene excimer over
monomer in emission.
6
I
I
4
2
0
Figure 7. The selectivity of C4-D-O-Py (5 mM) to LPS over
competing anions or biological molecules. Grey bar: Profiles of
the I485/I389 ratio changes in a single analyte at 2.5 mM (BSA was
50 mg l21). Black bar: Profiles of normal fluorescence intensity
of a mixture of competing species (2.5 mM) and LPS (2.5 mM).
lex ¼ 360 nm.

76
W. Zhao et al.
precipitate was filtered out and dissolved in water
Experimental section
(40 ml). The solution was extracted with CH2Cl2
(3 £ 25 ml). The combined organic layer was dried over
MgSO4 and concentrated to produce the crude product,
which was further purified by recrystallisation from
acetonitrile to yield 1.72 g (80%) of 2 as white powder.
General procedures
All commercial chemicals were used without further
purification. Carbon tetrachloride (CCl4) and toluene were
dried over P2O5 and Na, respectively. And other reagents
were used as reagent grade without further purification. 1H
and 13C NMR spectra were obtained on a Bruker Avance
400 NMR spectrometer using tetramethylsilane as an
internal reference. EI-MS spectra were recorded on a
Waters GCT Premier mass spectrometer, whereas ESI-MS
on a Shimadzu LC-MS 2010 instrument. UV–vis and
fluorescence spectra were measured with a Hitachi U-3010
and FL-4600 spectrometer, respectively. Fluorescence
lifetime was conducted on an Edinburgh Instruments Xe-
920 spectrometer. The pH measurements were carried out
on a Mettler-Toledo Delta 320 pH meter.
1
m.p. ,778C. H NMR (CDCl3, 400 MHz, d): 7.16–7.12
(m, 4H), 6.87–6.81 (m, 4H), 4.05 (t, 4H), 2.23 (s, 6H),
2.03 (m, 4H). 13C NMR (CDCl3, 100 MHz, d): 157.2,
130.7, 126.9, 126.8, 120.3, 111.0, 67.5, 26.4, 16.3. MS (EI)
calcd for C18H22O2 [M] 270.1; found: 270.1.
Synthesis of compounds 3 and 4
Compounds 3 and 4 were synthesised according to
reported procedures (19). Under a nitrogen atmosphere, a
mixture of compounds 1 or 2 (1.24 mmol), N-bromosucci-
nimide (482 mg, 2.72 mmol) and azobisisobutyronitrile
(60 mg) in CCl4 (20 ml) was heated under refluxing for l h.
After cooling to the room temperature, the reaction
mixture was filtered and concentrated by evaporation. The
residue was dissolved in 40 ml of CH2Cl2 and washed with
aqueous saturated Na2S2O3 (30 ml). The combined organic
layer was dried over MgSO4. The resulting crude product
was further purified by column chromatography on silica
gel (CH2Cl2:petroleum ether ¼ 1:3, v/v) to give the
dibromide derivatives as white powder.
For the spectral measurements, a stock solution
(1 £ 1023 M) of sensors was prepared by dissolving
10 mmol sensors into 10 ml of DMSO. The spectro-
photometic determinations of the probes, including the
UV–vis spectra, fluorescence titration and selectivity,
were conducted in a 10 mM HEPES buffer solution (pH
7.4; water:DMSO ¼ 8:2, v/v).
Synthesis of 1,2-bis(o-tolyloxy)ethane (1)
Compound 1 was synthesised according to the literature
with a minor modification (18). Sodium hydroxide (1.6 g,
40 mmol) was added into a solution of o-methyl phenol
(2.16 g, 20 mmol) in THF (20 ml). After the mixture was
refluxed for 0.5 h, 1,2-dibromoethane (1.3 g, 7 mmol) was
added. The mixture was refluxed for another 48 h and
concentrated by evaporation. The residue was poured into
40 ml of distilled water under stirring and then extracted
with 3 £ 15 ml of CH2Cl2. The combined organic layer
was washed with saturated brine and dried over MgSO4.
The crude product was further purified by column
chromatography on silica gel (ethyl acetate:petroleum
ether ¼ 5:100, v/v) to give 1 (338 mg, 20% yield) as white
powder. m.p. ,878C. 1H NMR (CDCl3, 400 MHz, d):
7.17–7.15 (m, 4H), 6.90–6.97 (m, 4H), 4.35 (s, 4H), 2.23
(s, 6H). 13C NMR (CDCl3, 100 MHz, d): 157.0, 130.9,
127.3, 126.8, 120.9, 111.5, 67.0, 16.3. HR-MS (EI) calcd
for C16H18O2 [M] 242.1307; found: 242.1302.
1,2-Bis(2,bromomethylphenoxy)ethane (3)
1
Yield 24%, 120 mg, m.p. 122–1258C. H NMR (CDCl3,
400 MHz, d): 7.35–7.29 (m, 4H), 6.99–6.94 (m, 4H), 4.56
(s, 4H), 4.47 (s, 4H). MS (EI) calcd for C16H16Br2O2 [Mþ]
397.9; found: 397.9.
1,4-Bis(2-(bromomethyl)phenoxy)butane (4)
Yield 24%, 197 mg, m.p. ,1448C. 1H NMR (CDCl3,
400 MHz, d): 7.34–7.26 (m, 4H), 6.93–6.88 (q, 4H), 4.58
(s, 4H), 4.15 (t, 4H), 2.13 (m, 4H). 13C NMR (CDCl3,
100 MHz, d): 157.0, 130.9, 130.3, 126.3, 1207, 111.8,
67.6, 29.3, 26.2. HR-MS (EI) calcd for C18H20Br2O2 [M]
425.9830; found: 425.9824.
Synthesis of compounds 5 and 7
Compounds 5 and 7 were synthesised according to the
previously reported procedures (20, 21).
Synthesis of 1,4-bis(o-tolyloxy)butane (2)
The procedure for synthesis of compound 3 was similar to
the process of compound 1. Sodium hydroxide (1.6 g,
40 mmol) was added into a solution of o-methyl phenol
(2.16 g, 20 mmol) in acetonitrile (20 ml). After the mixture
was refluxed for 0.5 h, 1,4-dibromobutane (1.49 g, 7 mmol)
was added. The mixture was refluxed for another 10 h.
After cooling to the room temperature, the formed
1-(Bromomethyl)pyrene (5)
Yield 24%. m.p. ,1388C. 1H NMR (CDCl3, 400 MHz, d):
8.38(d, 1H), 8.25–8.21 (m, 3H), 8.12–8.01 (m, 5H), 5.26
(s, 2H).

Supramolecular Chemistry
77
1-((4-Bromobutoxy)methyl)pyrene (7)
1
4H), 8.08 (t, 4H), 7.77–7.70 (m, 4H), 5.15 (s, 4H), 4.95 (s,
4H), 4.01 (br, 12H), 3.92 (br, 12H), 3.60–3.54 (m, 8H),
1.77 (br, 4H), 1.60 (t, 4H). 13C NMR (DMSO, 100 MHz,
d): 136.7, 135.6, 131.8, 130.7, 130.5, 130.2, 130.1, 128.5,
127.6, 127.3, 127.2, 126.9, 126.2, 125.3, 125.2, 124.5,
123.9, 123.8, 123.4, 70.2, 68.8, 65.6, 63.5, 50.5, 50.1, 25.9,
18.7. Element analysis: calcd for C62H70Br4N4O2·2H2O:
C, 57.51; H, 6.07; N, 4.33; found: C, 57.01; H, 5.88; N,
4.24%.
m.p. ,2158C. H NMR (CDCl3, 400 MHz, d): 8.36 (d,
1H), 8.14–8.22 (m, 4H), 8.00–8.07 (m, 4H), 5.22 (s, 2H),
3.64 (t, 2H), 3.41 (t, 2H), 1.98 (m, 2H), 1.82 (m, 2H).
Synthesis of compounds 6 and 8
Compounds 6 and 8 were synthesised using the same
procedure. In the typical process, compound 5 (400 mg,
1.36 mmol) or 7 (497 mg, 1.36 mmol) and DABCO
(182 mg, 1.50 mmol) were dissolved into 30 ml of ethyl
acetate. The mixture was stirred at room temperature for
12 h and then the products were obtained by filtration.
4,40-(2,20-(Ethane-1,2-diylbis(oxy))bis(2,1-phenylene))
bis(methylene)bis(1-(4-(pyren-1-ylmethoxy)butyl)-1,4-
diazonia-bicyclo[2.2.2]octane) (C2-D-O-Py)
Yield 60%, 125 mg. m.p. ,2208C. 1H NMR (DMSO,
400 MHz, d): 8.37 (d, 2H), 8.33–8.27 (m, 8H), 8.18–8.07
(m, 8H), 7.60–7.52 (q, 4H), 7.29 (d, 2H), 7.12 (t, 2H), 5.20
(s, 4H), 4.76 (s, 4H), 4.52 (s, 4H), 3.94 (br, 12H), 3.83 (br,
12H), 3.65 (t, 4H),3.56 (m, 4H), 1.76 (br, 4H), 1.63 (t, 4H).
13C NMR (DMSO, 100 MHz, d): 157.7, 134.9, 132.9,
131.9, 130.7, 130.5, 130.2, 128.6, 127.6, 127.3, 127.2,
126.9, 126.2, 125.3, 125.2, 124.6, 123.9, 123.8, 123.5,
121.1, 114.7, 113.2, 70.3, 68.8, 66.8, 63.1, 61.3, 50.5, 50.4,
25.9, 18.6. Element analysis: calcd for C70H78Br4N4O4·2-
H2O: C, 60.27; H, 5.92; N, 4.02; found: C, 59.17; H, 5.80;
N, 4.01%.
1-(Pyren-1-ylmethyl)-4-aza-1-azonia-bicyclo
[2.2.2]octane (6)
White solid (276 mg, 50%). 1H NMR (CH3OD, 400 MHz,
d): 8.55 (d, 2H), 8.36–8.33 (m, 4H), 8.26–8.11 (m, 4H),
5.30 (s, 2H), 3.56 (t, 6H), 3.14 (t, 6H). 13C NMR (CH3OD,
100 MHz, d): 134.5, 133.2, 133.0, 132.4, 131.5, 130.6,
130.2, 128.1, 127.7, 127.5, 127.2, 126.0, 125.8, 125.2,
123.7, 120.5, 66.0, 53.6, 46.2. MS (ESI) calcd for
C23H23Nþ2 [Mþ] 327.1; found: 327.3.
1-(4-(Pyren-1-ylmethoxy)butyl)-4-aza-1-azonia-
bicyclo[2.2.2]octane (8)
4,40-(2,20-(Butane-1,4-diylbis(oxy))bis(2,1-phenylene))
bis(methylene)bis(1-(4-(pyren-1-ylmethoxy)butyl)-1,4-
diazonia-bicyclo[2.2.2]octane) (C4-D-O-Py)
White solid (325 mg, 50%). m.p. ,1208C. 1H NMR
(CH3OD, 400 MHz, d): 8.44 (d, 2H), 8.27–8.19 (m, 4H),
8.11–8.04 (m, 4H), 5.22 (s, 2H), 3.67 (t, 2H), 2.97–2.83
(m, 14H), 1.63 (t, 2H). 13C NMR (CH3OD, 100 MHz, d):
133.0, 132.8, 132.6, 132.0, 130.7, 128.9, 128.8, 128.6,
128.5, 127.4, 126.5, 126.4, 126.0, 125.7, 125.6, 124.9,
72.3, 69.8, 65.2, 65.1, 65.1, 53.1, 53.1, 53.1, 45.7, 27.4,
19.9. MS (MALDI) calcd for C27H31N2Oþ [Mþ] 399.2;
found: 399.5.
Yield 60%, 127 mg. m.p. ,1888C. 1H NMR (DMSO,
400 MHz, d): 8.37 (d, 2H), 8.33–8.26 (m, 8H), 8.16–8.06
(m, 8H), 7.57–7.52 (m, 4H), 7.21 (d, 2H), 7.09 (t, 2H),
5.18 (s, 4H), 4.85 (s, 4H), 4.14 (s, 4H), 3.92-3.82 (m, 24H),
3.63 (t, 4H), 3.35 (t, 4H), 2.01 (s, 4H), 1.78 (br, 4H), 1.62
(t, 4H). 13C NMR (DMSO, 100 MHz, d): 158.1, 134.8,
132.9, 131.8, 130.6, 130.5, 130.2, 128.5, 127.5, 127.3,
127.2, 126.9, 126.2, 125.3, 125.2, 124.5, 123.9, 123.8,
123.4, 120.6, 114.5, 112.9, 70.3, 68.8, 68.1, 63.1, 61.5,
50.5, 25.9, 25.3, 18.6. Element analysis: calcd for
C72H82Br4N4O4·2H2O: C, 60.77; H, 6.09; N, 3.94;
found: C, 60.04; H, 6.06; N, 3.90%.
General procedure for synthesis of the sensors
A mixture of dibromide derivatives (3, 4 or m-xylylene
dibromide, 0.15 mmol) and the intermediates 6 or 8
(0.34 mmol) in appropriate amount of acetonitrile was
stirred at room temperature for 12 h. The formed
precipitate was filtered, washed with acetonitrile and
dried under vacuum to give the desired products as pale
yellow solid with moderate yield (50–70%).
4,40-(2,20-(Ethane-1,2-diylbis(oxy))bis(2,1-phenylene))
bis(methylene)bis(1-(pyren-1-ylmethyl)-1,4-diazonia-
bicyclo[2.2.2]octane) (C2-D-Py)
1
Yield 50%, 94 mg. H NMR (DMSO, 400 MHz, d): 8.73
4,40-(1,3-Phenylenebis(methylene))bis(1-(4-(pyren-1-
ylmethoxy)butyl)-1,4-diazonia-bicyclo[2.2.2]octane) (Ph-
D-O-Py)
Yield 70%, 131 mg. m.p. ,1948C. 1H NMR (DMSO,
400 MHz, d): 8.34 (d, 2H), 8.31–8.25 (m, 8H), 8.16 (s,
(d, 2H), 8.47–8.43 (m, 8H), 8.36 (d, 2H), 8.30–8.17 (m,
6H), 7.45 (q, 4H), 7.13 (d, 2H), 7.01 (t, 2H), 5.65 (s, 4H),
4.69 (s, 4H), 4.37 (s, 4H), 3.98 (br, 12H), 3.86 (br, 12H).
13C NMR (DMSO, 100 MHz, d): 158.2, 135.6, 134.2,
133.8, 132.9, 132.3, 131.4 130.6, 130.3, 128.0, 128.0,

78
W. Zhao et al.
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127.6, 127.2, 125.9, 125.0, 124.1, 123.2, 122.1, 119.4,
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Supporting information
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Changes of absorption and fluorescence spectra of Ph-D-O-
Py (Figure S1) and C2-D-O-Py (Figure S2) towards LPS.
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Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (Grant Nos 21173244 and
21073213) and the Main Direction Program of Knowledge
Innovation of Chinese Academy of Sciences.
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