Synthesis of a series of ethylene glycol modified water-soluble tetrameric TPE-amphiphiles with pyridinium polar heads: Towards applications as light-up bioprobes in protein and DNA assay, and wash-free imaging of bacteria

Article abstract of DOI:10.1016/j.tet.2019.05.044

Available online Development of water soluble AIE-active “light-up” bioprobes for the detection of biomacromolecules has drawn huge research interests in recent past. In this study, a series of ethylene glycol modified water soluble tetrameric tetraphenyl

Full text of DOI:10.1016/j.tet.2019.05.044

Accepted Manuscript
Synthesis of a series of ethylene glycol modified water-soluble tetrameric TPE-
amphiphiles with pyridinium polar heads: Towards applications as light-up bioprobes
in protein and DNA assay, and wash-free imaging of bacteria
Vikash Kumar, Viraj G. Naik, Avijit Das, Sourayan Basu Bal, Malabika Biswas, Nupur
Kumar, Anasuya Ganguly, Amrita Chatterjee, Mainak Banerjee
PII:
S0040-4020(19)30596-4
https://doi.org/10.1016/j.tet.2019.05.044
TET 30365
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 18 April 2019
Revised Date: 14 May 2019
Accepted Date: 21 May 2019
Please cite this article as: Kumar V, Naik VG, Das A, Bal SB, Biswas M, Kumar N, Ganguly A,
Chatterjee A, Banerjee M, Synthesis of a series of ethylene glycol modified water-soluble tetrameric
TPE-amphiphiles with pyridinium polar heads: Towards applications as light-up bioprobes in protein
and DNA assay, and wash-free imaging of bacteria, Tetrahedron (2019), doi: https://doi.org/10.1016/
j.tet.2019.05.044.
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Synthesis of a series of ethylene glycol
modified water-soluble tetrameric TPE-
amphiphiles with pyridinium polar heads:
towards applications as light-up bioprobes in
protein and DNA assay, and wash-free
imaging of bacteria
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Vikash Kumar,a Viraj G. Naik,a Avijit Das,b Sourayan Basu Bal,a Malabika Biswas,b Nupur Kumar,b Anasuya
Ganguly,b Amrita Chatterjee,*a and Mainak Banerjee*a
aDepartment of Chemistry, BITS, Pilani, Goa Campus, Goa-403726, INDIA.
bDepartment of Biological Sciences, BITS, Pilani, Goa Campus, Goa-403726, INDIA.

1
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Tetrahedron
journal homepage: www.elsevier.com
Synthesis of a series of ethylene glycol modified water-soluble tetrameric TPE-
amphiphiles with pyridinium polar heads: towards applications as light-up bioprobes
in protein and DNA assay, and wash-free imaging of bacteria
Vikash Kumar, †,a Viraj G. Naik, †,a Avijit Das,b Sourayan Basu Bal,a Malabika Biswas,b Nupur Kumar,b
Anasuya Ganguly,b Amrita Chatterjee,*a and Mainak Banerjee*a
aDepartment of Chemistry, BITS, Pilani, Goa Campus, Goa-403726, INDIA.
bDepartment of Biological Sciences, BITS, Pilani, Goa Campus, Goa-403726, INDIA.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Development of water soluble AIE-active “light-up” bioprobes for the detection of
biomacromolecules has drawn huge research interests in recent past. In this study, a series of
ethylene glycol modified water soluble tetrameric tetraphenylethylene amphiphiles with
pyridinium polar heads (TPE-xEG-Py, x = 3, 4, 6 or 1a-c) have been synthesized by varying the
ethylene glycol spacer. Their unique structure allows them to form vesicles and other
nanoaggregates in aqueous solutions. These amphiplihes were successfully utilized for
fluoremetric detection and quantitation of BSA and DNA based on the electrostatic interactions
to trigger AIE-emission from the TPE moiety. The electrostatic interaction was also proved very
effective in wash-free imaging of both Gram-negative (E. coli) and Gram-positive (S. aureus)
bacteria with up to 92 folds increase in fluorescence response within bacterial concentration 0–
12 x 108 CFU mL-1. The strategy is advantageous due to cost-effective and easy synthesis, high
water solubility, and fast response.
Available online
Keywords:
“Light-up” bioprobes
Tetraphenylethylene (TPE)
Aggregation-induced emission (AIE)
Water-soluble
Protein and DNA binding
Wash-free bacterial imaging
2009 Elsevier Ltd. All rights reserved
.
property of these probes is modulated by using an organic-water
mixed solvent system to display a strong fluorescence output. In
the last decade, a series of amphiphilic TPE-derivatives have
been reported mostly for the assay and imaging of
biomacromolecules.5b,9 As the assay is usually carried out in
water or buffer solutions, water solubility is one of the key
requirements of these bio-probes other than biocompatibility.
Tang and co-workers have designed a cationic TPE derivative
with ammonium polar heads which interacts with proteins and
DNAs.5a In subsequent years, Tang13 and Zhang’s group14 have
introduced anionic TPE-derivatives for the detection of specific
proteins and enzymes. Our group reported a TPE-derivative with
sulphonate functionality for selective detection of Gram-positive
bacteria.15 Neutral amphiphiles capable of interacting with
biomolecules are also reported. Hu et al. reported a TPE-based
glycoconjugate for detection of cholera toxin16 and another TPE-
oligosaccharide by Kato and co-workers is used for the detection
of influenza virus.17 In most of the cases the probe-biomolecule
interaction is based on electrostatic or hydrophobic in nature.
1. Introduction
Recently fluorescence (FL) detection and imaging of
biomacromolecules have attracted great research attention due to
requirements in health care and other biomedical applications.1
Because the FL-based technique offers high sensitivity and
selectivity, low background noises, and wide dynamic ranges.2 In
this context, aggregation-induced emission (AIE) based
fluorimetric assay has become the most popular approach due to
a unique photophysical phenomenon.3 The AIE-active molecules
are non-emissive in the monomeric form in the solution but
become highly emissive in the aggregated form due to the
restriction of intra-molecular rotations (RIR), which restrict the
energy dissipation through the nonradiative decay pathways.4
This could also address the long-lasting thorny problem of
aggregation-caused quenching (ACQ)5 and make a way to
develop efficient chemical and bioprobes.6
Tetraphenylethene (TPE), a propeller-shaped compound, is
probably the smartest AIE-luminogen reported so far because of
its cost-effective synthesis and easy functionalization for various
fluorimetric studies.7 The excellent utility of TPE has been
manifested by a vast amount of literature including sensing,8
bioimaging,9 light-harvesting materials,10 mechanochromism11
and so on.6 Multifunctional TPE-derivatives are even used for the
preparation of supramolecular assemblies.12 Generally, the AIE
Recently, growing interests have been shown in designing of
TPE-amphiphiles derived smart nanoaggregates (e.g. vesicles,
nanoparticles) because of their multi-targeting and better binding
abilities other than unique encapsulation capabilities for better
performance in delivery, bioimaging, diagnostics, and
theranostics. These amphiphiles can be classified into a) small-
molecule amphiphiles,18 b) amphiphilic polymers,19 and c)

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amphiphilic supramolecules/supramacromolecules.20 Of our
derivatives, 6a-c were quarternized by refluxing with excess
interest, among small-molecule amphiphiles Xia et al. reported a
rigid TPE-based bolaamphiphile which form flake-like
aggregates in water to emit strong green light.21 The assembly
was utilized for imaging of HeLa cells. Lu and co-workers
reported several TPE-based gemini or gemini like amphiphiles
with aza-crown ether as the polar head, which spontaneously
self-assemble to form micellar aggregates in water and used for
pyridine in chloroform to produce the desired cationic tetrameric
TPE-amphiphiles with pyridinium polar heads (1a-c) with iodide
as the counter anion in excellent yields.
The structure of this new series of TPE-amphiphiles (TPE-xEG-
Py or 1a-c) was established by 1H NMR, 13C NMR, ESI-MS and
CHN analysis. The formation of cationic TPE- amphiphiles, 1a-c
from the corresponding iodides 6a-c was indicated by the
appearance of desired number of heteroaromatic protons at
gene
transfection
studies.22
TPE-based
cross-linked
autofluorescent vesicles (TPE-CV) have been prepared by Huang
et al. in aqueous media which can entrap rhodamine B (RhB) and
corresponding TPE-CV@ RhB was used for cell imaging and
drug delivery.23 However, most of these 2nd generation
bioprobes involve multistep, complex synthetic strategies and
therefore, they are not cost-effective. As one of our focused area
of research on TPE-based sensing and imaging systems,15,24 we
envisaged, simple design and cost-effective synthesis of TPE-
based amphiphiles, keeping high water solubility and
biocompatibility in mind, capable of forming vesicles or other
nanoaggregates for multipurpose use in binding assay, wash-free
imaging, as staining agent etc. will be a worthy pursuit. The
simple design involves incorporation of glycol units of variable
lengths in tetrahydroxy-TPE (3) for better water solubility and
pyridinium polar heads for better biocompatibility. The new
series of tetrameric TPE-based amphiphiles (TPE-xEG-Py, x =
3, 4, 6 or 1a-c) showed interesting results on spacer size
dependent binding with bio-macromolecules in protein and DNA
binding assay as well as detection of bacteria. The best of them
(1c) was used for wash-free bacterial imaging studies.
1
downfield within the range δ 8.71-8.25 ppm in H NMR, which
ensures attachment of pyridine ring for all the TPE-amphiphiles.
In addition, the appearance of two carbon (CH) signals at δ
~128.0-128.1 ppm, two carbon (CH) signals at δ ~144.7-144.8
ppm and one carbon (CH) signal at δ ~145.9-146.0 ppm for each
pyridinium ring in 13C NMR spectrum further supported the
formation of the desired products. The formation of these TPE-
amphiphiles 1a-c was re-confirmed by ESI-MS (positive ion)
mass spectroscopy. In each case, appearance of a base peak at
m/z = (1/4 x MW-4I) (as z = 4) confirmed the formation of
tetracationic TPE-amphiphiles. The peaks appeared at 293, 337,
and 425 for TPE-TREG-Py (i.e. x = 3, 1a), TPE-TEG-Py (i.e. x
= 4, 1b) and TPE-HEG-Py (i.e. x = 6, 1c), respectively. All the
spectral studies clearly established the formation of the desired
tetrameric TPE-amphiphiles with pyridinium polar heads, 1a-c.
2.2 Water solubility, CMC and morphologies of 1a-c
The tetrameric TPE-amphiphiles, 1a-c were tested for their
aqueous solubility and it was found that all three compounds
were highly soluble in water. For each case up to 10 mM aqueous
(or buffer) solution was prepared and nominal background
fluorescence was observed from up to 0.1 mM of the aqueous
solutions which makes them excellent light-up bioprobes.
Although background fluorescence increases a little at higher
concentrations the solution remains clear which can be seen by
naked eyes.
2. Results and discussion
2.1. 2.1 Synthesis and characterization of TPE-amphiphiles (1a-
c).
As shown in scheme 1, the TPE-derived bioprobes (1a-c)
were prepared by attaching four glycol units to tetrahydroxy-TPE
(3) and then carrying out simple transformations to convert
terminal alcoholic groups to corresponding iodides which was
finally converted to pyridinium salts by SN2 mechanism upon
heating with pyridine. The first-step involved the McMurry
coupling7a of 4,4’-dihydroxybenzophenone (2) to tetrahydroxy-
TPE (3). On the other hand, glycols were first monomesylated by
treatment with MsCl followed by treatment with sodium iodide
to afford glycols with a terminal iodide group (4a-c) in good
yields. Tetrahydroxy-TPE (3) on reaction with monoiodide
derivatives of different glycol units (4a-c) produced the TPE with
four glycol arms, 5a-c in good yields. These compounds undergo
mesylation to give the corresponding products in high yields,
which were found to be relatively unstable and were immediately
converted to iodide derivative, 6a-c by refluxing with sodium
iodide in high yields over two steps. In the final step, the iodide
However, the nominal fluorescence shown by these
amphiphiles can be beneficial to investigate the self-assembly
behaviour of 1a-c in aqueous solution. It may be assumed that the
monomeric units will start self-aggregating at and above a certain
concentration which results in increase in fluorescence output
from the aqueous solution.21,22 Accordingly, fluorescence of the
aqueous solutions of TPE-amphiphiles, 1a-c were examined at
different lower concentration levels (Fig. S1). The plots of the
fluorescence intensity versus corresponding concentrations of 1a-
c are shown in Fig. 1, wherein, inflection points can be located at
0.8 X 10-6 mol L-1, 0.77 X 10-6 mol L-1, and 0.75 X 10-6 mol L-1
for TPE-amphiphiles 1a, 1b, 1c, respectively. These inflection
points correspond to the critical micelle concentrations (CMCs),
where
the
fluorescence
intensity

3
H
I
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O
HO
HO
OH
HO
O
O
O
OH
m
m = 3, 4, 6
4(a-c)
O
O
O
O
TiCl4 / Zn
pyridine
O
n
n
n
K2CO3 / acetone (dry)
reflux, 18 h
THF (anhy.)
reflux, 12 h
72%
HO
OH
2
OH
HO
O
n
OH
88-92%
4,4'-Dihydroxybenzophenone
3
5(a-c)
n = 2, 3, 5
I
I
I
O
n
O
O
I
N
O
O
O
N
N
1. MsCl / THF (anhy.)
O
O
O
O
O
O
O
O
n
n
n
n
Et3N / 0 °C to rt
24 h
, CHCl3
N
reflux, 15 h
91-95%
2. NaI / acetone (dry)
I
I
reflux, 3 h
I
O
n
I
N
O
n
n
90-92%
1(a-c)
6(a-c)
n = 2, 3, 5
n = 2, 3, 5
H
OH
m
MsCl (0.3 equiv) / THF (anhy.)
H
I
m
H
OMs NaI / acetone (dry)
O
O
O
m
Et3N / 0 °C to rt
12 h
reflux, 15 h
4(a-c)
m = 3, 4, 6
Scheme 1. Synthetic route to the tetrameric TPE-amphiphiles, 1a-c.
starts increasing due to AIE effect by the formation of
nanoaggregates. Similarly, UV-Vis studies were also carried out
to reconfirm the CMC values. At lower concentrations, below
the CMC values, the absorption spectrum showed two small
peaks at 223 and 259 nm but as the concentration was increased,
a new peak appeared at 311 nm for all three cases and became
stronger with increase in probe concentration (Fig. S1 of SI). The
absorbance of other peaks was increased as well. It may be
assumed that the small absorption peaks correspond to monomer
and the new peak at 311 nm should be attributed to the formation
of aggregates in the solution. UV-Vis data gave similar CMC
values as that of fluorescence measurements. All the CMC values
were in micromolar range and it can be observed that elongation
of the chain length in the glycol unit does not have any additional
effect on CMC.
It is expected that the four amphiphilic arms of the TPE-
amphiphiles, 1a-c with a hydrophobic TPE moiety at the center
would provide a right hydrophobic-hydrophilic balance in the
molecules in order to form unique aggregate morphologies in the
aqueous solution. The aggregation morphologies of 1a-c in
contact with water were initially investigated under optical
micrograph with fluorescence attachment and further confirmed
by FE-SEM and DLS analysis (Fig. 2 and Fig. S2, SI). FE-SEM
images revealed that all three TPE-amphiphiles spontaneously
Fig 2. Microscopic images of 1c in aqueous solution (10-5 M): (A) and (C) are
the optical micrographs showing formation of fluorescent vesicles and tubes,
after aging the solution for 30 min and 1 day, respectively and (B) and (D) are
the FE-SEM images at the same condition.
Fig 1. Plots of fluorescence intensity vs concentration of (A) 1a, (B) 1b and
(C) 1c, respectively in aqueous solution. The inflection points correspond to
CMCs.

4
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Fig 3. Schematic representation of vesicle formation by tetrameric TPE-amphiphiles, 1a-c.
form autofluorescent spherical vesicles of variable sizes above
their CMCs (10-5 M) after incubation of their aqueous solutions
for just 30 min (Fig. 2A and Fig. S2A,E, SI). The formation of
vesicles was found in FE-SEM analysis as well (Fig. 2B and Fig.
S2B,F, SI). Both optical micrograph and FE-SEM further
revealed that the spherical vesicles self-aggregate with time to
form various unique nano- to micro-sized morphologies. Upon
aging the aqueous solutions of 1a (10-5 M) for 1 day formation of
rings and short tubular morphologies were observed (Fig. S2C,D,
SI). On the other hand, co-existence of vesicles and long tubules
were seen for 1b and long tubes for 1c upon aging the solutions
for just 1 day (Fig. 2C,D and Fig. S2G,H, SI). In a separate study,
the particle size of these TPE-amphiphiles 1a-c in aqueous
solution (10-5 M) was estimated by DLS analysis after incubation
of the samples for 30 min and the results were in coherence with
optical micrograph and FE-SEM analysis to re-establish the
coexistence of variable sized assemblies. In all cases, narrow
ranges of particle size distributions of smaller aggregates as well
as bigger aggregates were observed in the similar ranges for these
TPE-amphiphiles (Fig. S3, SI). The smaller assemblies found to
have average sizes of 60 nm, 59 nm and 69 nm for TPE-
amphiphiles 1a, 1b, 1c, respectively; whereas, bigger assemblies
of the same appeared at an average diameters of 282 nm, 279 nm
and 260 nm indicating spontaneous formation of vesicles. In
addition, for 1b and 1c a lesser distribution of particles of around
1 µM size were observed in DLS analysis which indicate
initiation of the formation of tubular assemblies in the solution.
Formation of vesicles is schematically represented in Fig. 3.
Presumably, TPE-core with two of the glycolic arms at both sides
forms a definite array of lipid monolayer which in turn gets
elongated to form monolayer membrane (MLM) vesicles. Quick
formation of vesicles and other bigger morphologies is
advantageous for better interaction with biomacromolecules.
Low toxicity of fluorescent bioprobes towards animal cell
lines is an essential parameter to test biocompatibility.
Cytotoxicity of these TPE-amphiphiles 1a-c was assessed on
HeLa cell line using MTT assay. Live HeLa cells were treated
with these amphiphiles at a concentration range 10-100 µM. The
percentages of viable HeLa cells were quantified and it was
found that cell viability does not alter much for any of the three
candidates. The cell viability was more than 90% after 24 h even
when the concentration of TPE-amphiphiles was as high as 100
µM in culture medium (Fig. 4). These results also demonstrated
that these new TPE-amphiphiles do not cause toxicity to the
living human cells.
Fig 4. Cytotoxicity study of TPE-amphiphiles 1a-c on Hela cells by MTT
assay.
2.4 Protein binding assay
Application of TPE-amphiphiles 1a-c as fluorescent bioprobes
was explored by analyzing their interactions with bio
macromolecules like proteins and DNA. Complexation of the
water-soluble TPE based AIE compounds with Bovine serum
2.3 Cytotoxicity

5
albumin (BSA) was investigated by spectrofluorimetric titration
in aqueous phosphate buffer (pH = 7.0) at 25 °C (Fig. 5). The
experiment was triplicated and similar results were found each
time. It was found that the TPE-amphiphiles 1a-c in buffer
solutions (5 µM) in the absence of BSA are almost non-
fluorescent, whereas, they show a fluorescence enhancement up
to 6-7 folds by the addition of BSA (Fig. 5). The intensity of the
probe solution increases up to 2 mg/mL of BSA. A hypsochromic
shift from 464 nm (λmax of native probe molecules in phosphate
buffer) to 442 nm was observed upon interaction with BSA. The
plots display good linear ranges with R2 values of 0.9959, 0.9976,
0.9959, for 1a, 1b, 1c, respectively. The linear range of I-I0/ I0 vs.
concentration of BSA plot for 1a is 0.2 - ~1.2 mg/mL, that of 1b
is 0.4 - ~1.2 mg/mL, and that of 1c is 0.2 - ~1.2 mg/mL. The
studies revealed that all the probes were equally efficient in
detecting and quantifying BSA. Notably, the highest
enhancement in fluorescence intensity was observed for the
probe with six glycol units i.e. TPE-HEG-Py (1c). As known,
the isoelectric point of BSA is at pH 4.5–5.0, therefore, the
protein is negatively charged at neutral pH. The fluorescence
enhancement may be attributed to the fact that the native folded
structures of BSA possess negatively charged regions on the
surface and in the pockets, and the positively charged TPE-
amphiphiles in the monomeric or aggregated forms bind with
them by simple electrostatic interactions, wherein the rotation of
the molecules are seized, hence inducing this complex to emit
after aggregation.5a,13 So, AIE property makes the TPE-
amphiphiles 1a-c as efficient probes for protein detection and
quantitation.
native probe solution at 478 nm got marginally red shifted to
ACCEPTED MANUSCRIPT
484 nm upon addition of DNA solution. The change of intensity
(I-I0/I0) versus concentration of plasmid DNA can be easily fitted
to the Boltzman function as shown in (Fig. 6-inset). The plot
displays a good linear range from 0.1-1.0 µg with an R2 value of
0.9987 for 1c. Thus, TPE-amphiphile 1c showed good binding
efficiency with about 8 folds fluorescence enhancement with
plasmid DNA (pET-28a). Similarly for other two amphiphiles viz
TPE-TREG-Py (1a) and TPE-TEG-Py (1b) enhancement in
fluorescence intensity has been observed upon their interaction
with p-DNA. However, in both cases the enhancement is not as
much as for 1c. Particularly for 1a the initial increment is little
slow, a saturation point comes after addition of 1.8 µg of DNA.
A good linear range of R2 = 0.9983 was observed from 0.1-1.6
µg. The less response can be attributed to the fact that the
nanoaggregates (or the monomers) derived from TPE with
shorter glycol units may not have right charge balance or
effective length to interact as efficiently as it’s congener with six
glycol units (i.e 1c). However, a moderately long TPE-TEG-Py
(1b) offers relatively better binding than 1a and a linear range
was observed from 0.2-1.4 (R2 = 0.9984) In this case as well, the
binding is mainly electrostatic between positively charged TPE
derivative and negatively charged DNA. Nonetheless, from the
results it may be postulated that a chain length of six glycol units
could be the ideal length to interact with DNA better than its
congeners. While, the exact reason for this behavior is yet to be
established firmly, similar chain length dependent binding
affinities for DNA was observed by others.21 The detection range
for DNA is much lower than BSA binding which may be
attributed to the fact that the negative charge density on DNA is
much higher than BSA to effectively go for stronger electrostatic
interactions with the bioprobes.
2.2. 2.5 DNA binding assay
As TPE-amphiphile 1c showed excellent results for DNA
binding assay, in a demonstration study, it was used as an
autofluorescent staining agent for nucleic acids in agarose gel
As a part of preliminary investigations on the efficacy of these
systems as potential fluorescent probes for DNA and gene
delivery, we studied the interaction of these cationic TPE-
amphiphiles 1a-c with plasmid DNA (Fig. 6). pET-28a was used
as model plasmid DNA for the spectrometric titration. A dilute
solution of TPE-amphiphiles 1a-c (5 µM) in Tris-EDTA buffer
was non-fluorescent. Addition of a small amount of plasmid
DNA to the aqueous solution of TPE-amphiphiles (5 µM) turned
on its emission. As observed in Fig. 6, increase of the DNA
concentrations further increased the fluorescent intensity. Here,
the best results were observed for the one with six glycol units
(TPE-HEG-Py, 1c) and for other two TPE-amphiphiles the
response was relatively lower. The fluorescence intensity of the
electrophoresis experiment as
a potential replacement of
conventional dyes. It is worthy to mention that some of the
common conventional small-molecule staining agents (e.g.
ethidium bromide) are carcinogenic in nature while few others
(e.g. nile red) are not adequately photostable. This instigates the
search for new staining agents. Gel electrophoresis experiment
was carried out in agarose gel (1%) using pET-28a plasmid
DNA. It was found that TPE-amphiphile 1c effectively function
as autofluorescent staining agent for pDNA. As shown in (Fig.
6), after binding with the TPE-amphiphile 1c, DNA bands
Fig 5. Fluorescence spectra of 1a-c (5 µM) (A: 1a; B: 1b; C: 1c) upon addition of BSA in an aqueous phosphate buffer (pH = 7.0). Inset: plots of corresponding
fluorescence intensities at 442 nm vs BSA concentration.

6
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Fig 6. Fluorescence spectra of 1a-c (5 µM) (A: 1a; B: 1b; C: 1c) upon addition of plasmid DNA (pET-28a) in an aqueous Tris-EDTA buffer. Inset: plots of
corresponding fluorescence intensities at 484 nm vs DNA concentration.
become visible under UV illumination after
a
certain
bacteria would undergo electrostatic interaction with the
positively charged probe molecules to exhibit strong fluorescence
from the surface in the same manner as demonstrated by Tang
and co-workers.25 At 10 µM concentration these probes
themselves showed little or negligible fluorescence. However,
upon incubation of these probes with increasing bacterial
concentrations (E. coli, S. aureus) it led to a gradual increase in
fluorescence intensities (Fig. 8 and 9). Bacterial concentration
ranges from 2.25 X 106 to 12 X 108 CFU mL-1 for E. coli and 3.5
X 106 to 12 X 108 CFU mL-1 for S. aureus were screened. The
initial bacterial colony formation was estimated from OD of the
sample by McFarland standard and the other sample solutions
were prepared from it by half dilution method. The interaction of
probe molecules with the bacterial surface is very fast as the
fluorescence spectra were recorded instantaneously after each
addition. It was observed that even after incubation of probe with
bacterial solution for 30 min there is hardly any change in the
fluorescence output. Although all the probes were capable of
interacting with these bacterial surfaces to transmit a strong
fluorimetric response with almost equal efficiency, the highest
enhancement of fluorescence was observed for probe 1c. Again,
the trend is like the longer the length of the glycol units the better
is the interaction with the bacteria. The detection limit 1c, which
is considered here as the lowest concentration at which a
fluorescence response is obtained, was estimated to be
approximately 9.37 X 106 CFU mL-1 for E. coli (Fig. 8C, inset)
and 18.75 X 106 CFU mL-1 for S. aureus (Fig. 9C, inset) with 92
and 38 folds increase in fluorescence intensity from its initial
value. Similarly, the detection limits for 1a and 1b for E. coli and
S. aureus are estimated to be the same as that of 1c. Notably,
highest enhancement in fluorescence upon interaction with
concentration of DNA and get intensified with increasing
concentration of DNA. It showed fairly good sensitivity as 0.25
µg of plasmid DNA by showing a strong enough band in gel
electrophoresis. As expected, positively charged pyridinium units
and negatively charged phosphates of DNA strands are mainly
involved in the electrostatic interaction leading to the formation
of DNA-TPE-amphiphiles conjugates. As the bioprobes, 1c gets
a solid surface in the form of DNA, it light’s-up by AIE-effect.
Fig 7. Agarose gel (1%) electrophoresis gel pattern of TPE-amphiphile 1c (5
µM) – pDNA complex at different concentrations of pET-28a plasmid DNA
(0, 0.125, 0.25, 0.5, 1, 5, 10 µg).
2.6 Bacterial detection and wash-free imaging
These TPE-amphiphiles, 1a-c were also examined as light-up
bioprobes for the detection and imaging of various bacterial
species. Accordingly, fluorimetric studies were carried out on
three different classes of bacteria viz. Gram-negative E. coli,
Gram-positive S. aureus and acid-fast M. smegmatis keeping the
sensing condition same as in the case of BSA binding and pure
water was used as the media. It is presumed that the different
negatively charged functionalities available on the cell-walls of
bacterial surface has been observed for TPE-HEG-Py, 1c.
A
large hypsochromic shift of 76 nm from 476 to 400 nm was
observed for E. coli in all cases. Similarly a relatively less
hypsochromic shift from 476 nm to 422 nm was observed for S.
aureus. However, no change in fluorescence intensities was
observed for M. smegmatis (acid-fast) for all three probes. As
seen in Fig.
8 and Fig. 9, the probes interact better
Fig. 8. Fluorescence response of aqueous solution of TPE-amphiphiles 1a-c (10 µM) [A: 1a; B: 1b; C: 1c] upon addition of increasing concentration of E. coli.
[λex 311 nm; λem 400 nm]. Inset: plots of corresponding fluorescence intensities against increasing bacterial concentration at λmax 400 nm.

7
ACCEPTED MANUSCRIPT
Fig. 9. Fluorescence response of aqueous solution of TPE-amphiphiles 1a-c (10 µM) [A: 1a; B: 1b; C: 1c] upon addition of increasing concentration of S. aureus
[λex 311 nm; λem 422 nm]. Inset: : plots of corresponding fluorescence intensities against increasing bacterial concentration at λmax 422 nm.
with E. coli than S. aureus giving rise to a better fluorescence
output. Similarly, the UV-Vis spectra of each solution were
recorded and it was observed that all the major bands at around
223, 259 and 311 nm were intensified upon interaction with both
bacterial species (see SI, Fig. S4). These experiments
unambiguously established the utility of these tetrameric TPE-
based probes with pyridine as polar head (1a-c) for the detection
of both Gram-positive and Gram-negative bacteria. It may be
presumed that the bacteria with negatively charged functionality
on the cell surface can be efficiently detected by probes 1a-c.
both Gram-positive and Gram-negative bacteria. Notably,
imaging of E. coli by probe 1c is relatively faster than that of S.
aureus.
Based on the above results a plausible mechanism for bacterial
detection and imaging may be proposed (Fig. 11). The cell
surface of E. coli possesses LPS (lipopolysaccharides), a
characteristic of Gram-negative bacteria, with several carboxylate
residues at the outer layer as well as phosphate groups in the
backbone. These negatively charged functionalities are easily
accessible to the positively charged probe molecules for strong
electrostatic interactions. Presumably, the monomers of probe 1c
mostly interact with the cell surface of E. coli to form a
definitrray of TPE molecules resulting in turn-on type
fluorescence response by AIE effect (Fig. 11). Similarly, the
deep-seated negatively charged phosphate groups of LTA
(lipoteichoic acid) on the cell-wall of the Gram-positive bacteria
(viz. S. aureus) undergo electrostatic interactions with probe 1c
to exhibit turn-on fluorescence by AIE effect in the same manner
like a Gram-negative bacterium (viz. E. coli). On the contrary,
M. smegmatis with mycolic acid, a charge-neutral long chain
aliphatic acid, on the cell wall do not effectively interact with the
probe molecules and fail to produce any fluorescence response
from the cell surface.
TPE-amphiphile, 1c was considered as the best candidate of
this series to act as light-up bioprobe for the imaging of various
bacterial species. Again for imaging studies the same set of
bacteria of three different classes namely S. aureus, E. coli and
M. smegmatis were chosen. The higher aqueous solubility with
negligible background fluorescence makes 1c an efficient
candidate for wash-free imaging of bacteria. The images were
captured under an inverted microscope with fluorescence
attachment. Both phase contrast and fluorescence images of E.
coli were taken by incubating probe 1c with the bacterial culture
for 15 min before capturing images. Similarly, for S. aureus 30
min of incubation time was given to intensify the fluorescence
output from their cell surface and for M. smegmatis images were
captured after 1 h. The study revealed that probe 1c can
efficiently interact with the cell-walls of both E. coli and S.
aureus but not with M. smegmatis (Fig. 10). For the first two
cases i.e. E. coli and S. aureus an intense blue fluorescence was
observed from the bacterial surface, on the contrary, the other
bacteria M. smegmatis do not show any fluorescence from the
cell surface. As there is no requirement of washing of media
before imaging, the process remains simple with minimum loss
of bacteria. This study once again indicates that probe 1c can be
used as an efficient light-up bioprobe for wash-free imaging of
Fig. 11. A schematic representation of the sensing mechanism of bioprobes
(1a-c) with E. coli. The bioprobes undergo simple electrostatic interaction
with carboxylates and phosphates groups of LPS seated on the cell-wall of E.
coli and produce blue fluorescence by AIE-effect.
3. Conclusion
Fig. 10. Phase contrast (A-C) and fluorescence (D-F) images of, E. coli, S.
aureus and M. smegmatis, respectively treated with probe 1c (10 µM). Scale
bar for all the images are 100 µm.
In the present study, a new series of ethylene glycol modified
water-soluble tetrameric TPE-amphiphiles with pyridinium polar
heads have been synthesized in good overall yields and used for

8
Tetrahedron
ACCEPTED MANUSCRIPT
protein and DNA binding assay, wash-free bacterial imaging,
transilluminator (BIO-RAD, USA). Fluorescence studies were
and as staining agents for nucleic acids. They are non-fluorescent
in aqueous solution and their CMC values were estimated to be
just below micromolar range (0.75-0.8 µM). Their self-
aggregation behavior was studied under fluorescence microscope
and FE-SEM. They spontaneously form vesicles and other
nanoaggregates in aqueous solutions. DLS studies were also
conducted to check particle size distribution of the
nanoaggregates, which are in well agreement with the
microscopic results. MTT assay on HeLa cells revealed that 1a-c
are fairly non-toxic in nature. It was observed that these TPE-
amphiphiles 1a-c bind well with BSA and pDNA (pET-28a)
through electrostatic interactions, which turns on fluorescence
emission by AIE. Among them TPE-amphiphile with longest
glycolic arms, 1c showed best results. These TPE-amphiphiles,
1a-c were effectively utilized for wash-free imaging of both
Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria.
Presumably, these cationic amphiphiles interact with
lipopolysaccharide (LPS) situated at the cell-walls of Gram-
negative bacteria (E. coli) and lipotiechoic acid (LTA) of Gram-
positive (S. aureus), and efficiently illuminate them by sitting on
their surface. Up to 92 and 38 folds increase in fluorescence was
observed for 1c against E. coli and S. aureus, respectively, within
bacterial concentration 0–12 x 108 CFU mL-1. From the results it
may be concluded that this new series of TPE-based amphiphiles
can be used as multipurpose “light-up” bioprobes for electrostatic
interaction based detection and quantitation studies. While the
current work focuses on the simple design strategy and cost-
effective synthesis of the tetrameric TPE-amphiphile based new
bioprobes, and their preliminary applications the advanced
applications in drug- and gene-delivery can be considered as
future scope of this project.
carried out on a JASCO FP6300 fluorescence spectrophotometer
(JASCO Corp., Japan); the slit width was 2.5 nm for both
excitation and emission. UV-Visible spectra of probe molecules
were taken on a JASCO V-550, the bandwidth and data pitch
were set as 1 nm or 5 nm whenever required. Absorbance studies
were also carried out on a SHIMADZU UV–2450 UV-visible
spectrophotometer (SHIMADZU Corp., Japan) for cytotoxicity
assay and for finding optical density of bacterial sample. Optical
micrographs were carried out on inverted microscope, IX-51
(Olympus) with fluorescence attachment. Beckman Coulter Delsa
Nano particle size analyzer was used for DLS study.
4.3 Materials and methods
4.3.1 General procedure
THF was dried over sodium and freshly distilled before use.
The reactions were monitored by thin layer chromatography
(TLC) carried out on 0.25 mm silica gel plates (60F-254) using
UV light (254 or 365 nm) for visualization. All the stock
solutions of TPE-amphiphiles (1a-c) were prepared by using
Tris-EDTA buffer solution or phosphate-buffered saline (PBS)
(pH = 7.0) or deionized water (Milli-Q, 18 Mꢀ) as per
requirements for dilution purpose.
4.3.2 Procedure for agarose gel electrophoresis
and cytotoxicity assay
For agarose gel electrophoresis, 0.125, 0.25, 0.5, 1.0, 5.0 and
10.0 µg of plasmid DNA (pET-28a) and 5 µM solution of TPE-
amphiphiles (1a-c) were mixed so as to make 20 µL volumes and
incubated. Then each reaction mixture was loaded on a 1%
agarose gel made in tris-acetate-EDTA buffer. After completion
of the assay, the gel was viewed under UV transilluminator and
photographed.
4. Experimental Section
4.1 General information
Cytotoxicity was performed using MTT assay. HeLa cell lines
were grown in standard medium as per protocol and they were
seeded at a density of 5 x 104 cells per well. After 24 h of
incubation, the cells were supplemented with TPE-amphiphiles
(1a-c) at different concentrations. After incubation for 24 h, MTT
was added in each well and incubated for 4 h. Finally absorbance
was measured after following standard protocol at 570 nm and
cell viability was expressed as relative absorbance (%) of the
sample vs control cells. The experiments were triplicated and the
average data were presented.
4,4ʹ-dihydroxybenzopheneone, various ethylene glycols were
purchased from Sigma-Aldrich and used without further
purification. TiCl4 was purchased from Spectrochem Pvt. Ltd.
Mumbai (India) and Albumin fraction V (from bovine serum)
was procured from LOBA Chemie (India). Dulbecco’s Modified
Eagle’s Media (DMEM), Phosphate Buffer Saline (PBS) and
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) were purchased from Himedia Laboratories, Mumbai.
Plasmid DNA (pET-28a) was purchased from Bangalore GeNei,
Bangalore, India. Other common reagents were procured either
from SD Fine - Chem Limited, Mumbai, India or from Merck,
India and were used without further purification. All ultrapure
water used was collected from Millipore water system and
purged with N2 for 15 min prior to use. All the stock solutions of
TPE-amphiphiles were prepared by using Tris-EDTA buffer
solution or phosphate-buffered saline (PBS) (pH = 7.0) or Milli-
Q (18 Mꢀ) water unless otherwise stated.
4.3.3 Fluorimetric titration of BSA and p-DNA with
TPE- amphiphiles (1a-c):
For fluorescence measurements 1 mM of stock solutions of
1a-c were prepared in phosphate buffer (pH 7) or deionized
water. BSA was dissolved in a pH 7.0 phosphate buffer solution
(10.0 mg/mL). DNA was dissolved in Tris-EDTA buffer (1.0
mg/mL) and filtered through a 0.45 ꢁm filter.
Fluorimetric titration for BSA was carried out by adding
aliquots of BSA solution in the aqueous phoshate buffer to 10 µL
of a 1.0 mM stock solution of TPE-amphiphiles 1a, 1b and 1c
followed by adding a measured volume of autoclaved aqueous
phosphate buffer (pH 7) to acquire a 2 mL solution. The mixture
was stirred for 10 min and then incubated for 10 min at room
temperature prior to recording the fluorescence spectrum. All
observations were expressed by intensity vs. wavelength plots.
4.2 Instruments and measurements
NMR spectra were recorded on BRUKER NMR 300 MHz or
400 MHz systems using tetramethylsilane as an internal standard.
Mass spectra were recorded on Waters Q-TOF micro mass
spectrometer or Brucker Esquire 3000 mass spectrometer using
ESI as the ion source. CHN data were recorded using Vario
elementar CHNS analyzer. Agarose gel was viewed under UV
Fluorimetric titration for plasmid DNA was carried out by
adding aliquots of p-DNA solution in Tris-EDTA buffer to 10 µL
of a 1.0 mM stock solution of TPE-amphiphiles 1a, 1b and 1c in

9
1.5 mL of Tris-EDTA buffer followed by adding adequate
amount of Tris-EDTA buffer to acquire a 2 mL solution (pH 8).
The mixture was subjected to vortex for few seconds and then
incubated for 10 min at room temperature prior to recording the
fluorescence spectrum. All observations were expressed by
intensity vs. wavelength plots.
addition of 10% aqueous K2CO3 solution and worked up with
ACCEPTED MANUSCRIPT
CHCl3. The organic layer was collected and concentrated. The
crude product was purified by column chromatography using
100-200 silica gel and 2:5 ethyl acetate in petroleum ether (60-
80) as an eluent to afford the desired product (3). Off-white solid,
yield: 72%; 1H NMR (300 MHz, DMSO-d6): δ (ppm) 6.48 (d, J =
8.4 Hz, 8H), 6.70 (d, J = 8.4 Hz, 8H), 9.22 (s, 4H, exchangeable);
13C NMR (75 MHz, DMSO-d6): δ (ppm) 114.9, 132.4, 135.5,
138.2, 155.8; ESI-MS: m/z 419 [M + Na]+; Anal. Calcd. for
C26H20O4: C, 78.77; H, 5.09; Found: C, 78.92; H, 5.11.
4.3.4 Bacterial sampling
The cultures of different classes of bacteria, namely E. coli
DH5α, S. aureus RN4220 and M. smegmatis MC2 155 were
selected for the study. These cultures were routinely grown on
Luria Bertani broth (Hi-Media, India) for E. coli, Tryptic Soy
Broth (Becton-Dickinson and Company, USA) for S. aureus, and
Middlebrook 7H9 broth base (Hi-Media, India) for M.
smegmatis, respectively. Agar was added as per requirement of
routine culture maintenance. At OD600 ~0.5 of the grown cells
were harvested. OD of bacterial cultures were measured by UV-
Visible spectrophotometer and the colony-forming unit (CFU)
was obtained by McFarland standards (Table S1, SI).26 The
sample 1 was half-diluted to obtain bacterial samples of lower
concentrations.
4.4.2. General procedure for the preparation of amphiphilic
TPE-glycols (5a-c):
The general procedure is illustrated by the preparation of 5a.
To a solution of tetra(p-hydroxyphenyl)ethylene (3, 0.7 g, 1.8
mmol) in dry acetone (7 mL) was added K2CO3 (4.88 g, 30.4
mmol) and the reaction mixture was then refluxed for 2 h. Next,
monoiodide derivative of triethylene glycol (4a) (2.76 g, 10.6
mmol) in dry acetone (10 mL) was added drop by drop in the
reaction mixture. The mixture was again refluxed for another 18
h. Then the reaction mixture was filtered and the filtrate was
evaporated under vacuum and then the corresponding residue
was chromatographed over silica gel (100-200) using 10%
MeOH in chloroform to afford the desired compound (5a).
TPE-triethylene glycol 5a:27 Colourless sticky thick liquid,
yield: 88%; H NMR (300 MHz, DMSO-d6): δ (ppm) 3.48-3.68
(m, 40 H), 3.99 (brs, 8H), 6.69 (d, J = 8.4 Hz, 8H), 6.83 (d, J =
8.4 Hz, 8H); 13C NMR (75 MHz, DMSO-d6): δ (ppm) 60.3, 67.0,
69.0, 69.8, 72.4, 113.7, 132.1, 136.4, 138.0, 156.8; ESI-MS: m/z
925 [M + H]+; Anal. Calcd. for C50H68O16: C, 64.92; H, 7.41;
Found: C, 65.02; H, 7.44.
4.3.5 Procedure for fluorescence and UV-Vis
titrations with bacterial stains
1
mM stock solutions of probes were used for the
experiments. The grown bacterial cells were harvested by
centrifuging at 4 °C, washed twice with sterilized phosphate-
buffered saline (1X PBS, pH 7.4) solution and used for
fluorescence and UV-Vis measurements. The bacterial cells were
re-suspended in 0.9% NaCl solution and the same solution was
used for any dilution purpose. The spectra were recorded
immediately after mixing probes with bacteria. For fluorescence
measurements the excitation wavelength was set to 311 nm and
emission spectra were recorded in the range of 312 to 600 nm for
1a-c. UV-Vis spectra were recorded from 200-800 nm. All the
experiments were performed at room temperature.
1
TPE-tetraethylene glycol 5b:27 Colourless sticky thick liquid,
yield: 89%; H NMR (300 MHz, DMSO-d6): δ (ppm) 3.41-3.54
1
(m, 48 H), 3.68 (brs, 8H), 4.00 (brs, 8H), 6.69 (d, J = 8.7 Hz,
8H), 6.83 (d, J = 8.7 Hz, 8H); 13C NMR (75 MHz, DMSO-d6): δ
(ppm) 60.7, 67.2, 67.3, 69.4, 70.2, 72.8, 114.1, 132.5, 136.7,
138.4, 157.2; ESI-MS: m/z 1123 [M + Na]+; Anal. Calcd. for
C58H84O20: C, 63.26; H, 7.69; Found: C, 63.38; H, 7.71.
4.3.6 Wash-free bacterial imaging with phase-
contrast and fluorescence microscopy
Cells from overnight grown culture were used to inoculate the
secondary culture media and finally the young grown cells were
harvested. At OD600 ~0.5 of the grown cells were harvested by
centrifugation at 4 °C and washed twice with sterilized PBS
solution. The washed cells were suspended in an appropriate
volume of sterile PBS. Different bacterial sets were prepared
with and without probe 1c, incubated at room temperature for 15
min, 30 min or 1 h as per requirement and directly viewed under
an Olympus IX51 inverted microscope, combining the phase-
contrast system and the fluorescence system.
TPE-hexaethylene glycol 5c:27 Colourless sticky thick liquid,
1
yield: 92%; H NMR (300 MHz, DMSO-d6): δ (ppm) 3.49-3.68
(m, 88 H), 3.99 (brs, 8H), 6.69 (d, J = 8.4 Hz, 8H), 6.83 (d, J =
8.4 Hz, 8H); 13C NMR (75 MHz, DMSO-d6): δ (ppm) 60.3, 67.0,
69.0, 70.0, 72.4, 113.8, 132.1, 136.4, 138.0, 156.8; ESI-MS: m/z
1476 [M + Na]+; Anal. Calcd. for C74H116O28: C, 61.14; H, 8.04;
Found: C, 61.30; H, 8.07.
4.4.3. General procedure for the preparation of iodide
derivatives (6a-c) of amphiphilic TPE-glycols (5a-c):
4.4 Synthetic procedures and spectral data of (1a-c)
4.4.1. Tetra(p-hydroxyphenyl)ethylene 3:7 a
The general procedure is illustrated by the preparation of 6a.
To a solution of 5a (0.7 g, 0.76 mmol) in dry THF (7 mL) was
added triethylamine (0.92 g, 9.1 mmol) at 0 °C under nitrogen
atmosphere. To it mesyl chloride (0.52 g, 4.55 mmol) was added
and the mixture was stirred at room temperature for 24 h till the
completion of the reaction. Then THF was removed under vacuo
and the crude product was diluted with CHCl3 (20 mL) and
washed with 5% HCl to remove excess triethylamine. Then the
organic extracts were washed with brine, dried over Na2SO4 and
concentrated. Column chromatogtaphy of the crude product over
silica gel (100-200 mesh) eluting with 2% MeOH in CHCl3
afforded the mesylated compound in high yield. The mesylated
A three-necked flask equipped with a magnetic stirrer was
charged with zinc powder (3.1 g, 47 mmol) and 30 mL
anhydrous THF under nitrogen atmosphere. The mixture was
cooled to 0 to -5 °C and TiCl4 (2.6 mL, 23.5 mmol) was slowly
added by a syringe. The suspension was warmed to room
temperature and stirred for 30 min, then heated at reflux for 2.5 h.
The mixture was again cooled to 0 to -5 °C, charged with
pyridine (0.9 mL, 11.3 mmol) and stirred for 10 min. The
solution of 4,4ʹ-dihydroxybezophenone (1 g, 4.7 mmol) in 10 mL
of THF was added slowly. After addition, the reaction mixture
was heated at reflux for 8 h. The reaction was quenched by

10
Tetrahedron
compound is relatively unstable and is immediately subjected
C7 H84I4N4O12: C, 50.01; H, 5.04; N, 3.33; Found: C, 50.15; H,
ACCEPTED MANUSCRIPT
0
to iodide formation.
5.06; N, 3.34.
To a solution of mesylated compound of 5a (0.32 g, 0.26
mmol) in dry acetone (3 mL) was added sodium iodide (0.47 g,
3.10 mmol) under nitrogen atmosphere. The mixture was then
refluxed for 3 h. Then the reaction mixture was concentrated in
vacuo to get crude residue, which was extracted with ethyl
acetate (2 x 10 mL). The combined extracts were first washed
with 10% Na2S2O3 then with brine, dried over Na2SO4. Column
chromatogtaphy of the crude product over silica gel (100-200
mesh) eluting with 1% MeOH in CHCl3 afforded the desired
iodide derivative (6a).
Bioprobe TPE-TEG-Py (1b): Light brown sticky liquid, yield:
92%; H NMR (400 MHz, D2O): δ (ppm) 3.41-3.48 (m, 40H),
1
3.61 (brs, 8H), 3.82-3.85 (m, 16H), 6.47 (d, J = 8.6 Hz, 8H), 6.80
(d, J = 8.6 Hz, 8H), 7.82 (t, J = 7.2 Hz, 8H), 8.27 (t, J = 7.9 Hz,
4H), 8.65 (d, J = 6.0 Hz, 8H); 13C NMR (100 MHz, D2O): δ
(ppm) 61.0, 67.1, 68.7, 68.9, 68.6, 69.5, 69.8, 113.9, 128.0,
132.4, 136.9, 138.7, 144.7, 145.9, 156.4; ESI-MS: m/z 337 (z =
4); Anal. Calcd. for C78H100I4N4O16: C, 50.44; H, 5.43; N, 3.02;
Found: C, 50.61; H, 5.45; N, 3.04.
Bioprobe TPE-HEG-Py (1c): Light brown sticky liquid, yield:
1
TPE-triethylene glycol iodide 6a: Colourless sticky thick
95%; H NMR (400 MHz, D2O): δ (ppm) 3.47-3.57 (m, 72H),
1
liquid, yield: 90%; H NMR (400 MHz, CDCl3): δ (ppm) 3.24-
3.69 (brs, 8H), 3.90-3.91 (m, 16H), 6.54 (d, J = 8.0 Hz, 8H), 6.81
(d, J = 8.0 Hz, 8H), 7.95-7.99 (m, 8H), 8.44-8.48 (m, 4H), 8.76
(d, J = 6.1 Hz, 8H); 13C NMR (100 MHz, D2O): δ (ppm) 61.1,
67.2, 68.7, 69.0, 69.5, 69.6, 69.8, 114.0, 128.1, 132.4, 136.9,
138.7, 144.8, 146.0, 156.5; ESI-MS: m/z 425 (z = 4); Anal.
Calcd. for C94H132I4N4O24: C, 51.09; H, 6.02; N, 2.54; Found: C,
51.23; H, 6.04; N, 2.55.
3.27 (m, 8H), 3.66-3.77 (m, 24H), 3.82-3.84 (m, 8H), 4.05-4.07
(m, 8H), 6.63 (d, J = 8.8 Hz, 8H), 6.89 (d, J = 8.8 Hz, 8H); 13C
NMR (100 MHz, CDCl3): δ (ppm) 3.0, 67.2, 69.9, 70.2, 70.3,
70.6, 70.7, 70.8, 72.0, 113.7, 132.5, 137.0, 138.4, 156.9; ESI-MS:
m/z 1387 [M + Na]+; Anal. Calcd. for C50H64I4O12: C, 44.01; H,
4.73; Found: C, 43.89; H, 4.75.
TPE-tetraethylene glycol iodide 6b: Colourless sticky thick
liquid, yield: 91%; 1H NMR (300 MHz, CDCl3): δ (ppm) 3.25 (t,
J = 6.9 Hz, 8H), 3.67-3.78 (m, 40 H), 3.83 (t, J = 4.5 Hz, 8H),
4.04-4.08 (m, 8H), 6.64 (d, J = 8.4 Hz, 8H), 6.90 (d, J = 8.4 Hz,
8H); 13C NMR (100 MHz, CDCl3): δ (ppm) 2.9, 67.2, 69.8, 70.2,
70.5, 70.6, 70.7, 70.8, 72.0, 113.7, 132.5, 137.0, 138.4, 156.9;
ESI-MS: m/z 1563 [M + Na]+; Anal. Calcd. for C58H80I4O16: C,
45.21; H, 5.23; Found: C, 45.07; H, 5.26.
Acknowledgments
M.B.
thanks
DST-SERB,
India
(project
no.
EMR/2016/002253) and A. C. thanks DST-SERB, India (project
no. EMR/2016/001416) for the research fund. V.K. is thankful to
CSIR, India for SRFship and V.G.N. thanks DST-SERB for
JRFship. The authors also acknowledge central instrumentation
facility of BITS-Pilani, Goa campus for FE-SEM. The authors
thank Prof. N. N. Ghosh of the same department for the help in
DLS measurements.
TPE-triethylene glycol iodide 6c: Colourless sticky thick
1
liquid, yield: 92%; H NMR (400 MHz, CDCl3): δ (ppm) 3.22-
3.26 (m, 8H), 3.64-3.76 (m, 72 H), 3.79-3.82 (m, 8H), 4.02-4.05
(m, 8H), 6.61 (d, J = 8.8 Hz, 8H), 6.87 (d, J = 8.4 Hz, 8H); 13C
NMR (100 MHz, CDCl3): δ (ppm) 3.0, 67.1, 69.8, 70.2, 70.6,
70.6, 70.7, 70.8, 71.9, 113.6, 132.5, 137.0, 138.3, 156.9; ESI-MS:
m/z 1893 [M + H]+; Anal. Calcd. for C74H112I4O24: C, 46.94; H,
5.96; Found: C, 47.07; H, 5.98.
Conflicts of interest
The authors declare no personal, financial or organizational
conflict of interests
.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
Notes
4.4.4. General procedure for the preparation of cationic
pyridinium amphiphile of TPE (1a-c):
The general procedure is illustrated by the preparation of 1a.
To a solution of 6a (0.2 g, 0.15 mmol) in dry pyridine (1.0
mL) was added dry CHCl3 (2 mL) under nitrogen atmosphere. It
was then refluxed for 12 h till the completion of reaction. The
reaction mixture was then dried under vacuo to remove CHCl3
and excess pyridine. The residue was re-dissolved in CHCl3 (2
mL) and diethyl ether (10 mL) was added into it to get a light
brown coloured crude product as thick oil. The supernatant liquid
was decanted and the oily product was subsequently triturated
with cold ethyl acetate (2 x 2 mL) followed by cold acetone (2 x
2 mL) to remove any trace of unreacted starting material and
pyridine. The sufficiently pure final product was subjected to
drying under vacuo for several hours to afford tetrameric TPE-
amphiphile 1a.
Corresponding author 1. MB. Tel.: +91-832-2580-347; fax:
+91-832--2557-031; Email: mainak@goa.bits-pilani.ac.in
Corresponding author 2. AC. Tel.: +91-832-2580-320; fax:
+91-832--2557-031; Email: amrita@goa.bits-pilani.ac.in
† These authors contributed equally to this work.
References
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A Guide to Fluorescent Probes and Labeling Technologies, 10th
ed.; Haugland, R. P., Ed.; Molecular Probes: Eugene, 2005.
2. (a) Chemosensors: principles, strategies and applications, Eds.
Wang, B.; Anslyn, E. V. John Wiley & Sons, Inc., 2011. (b)
Valeur, B.; Berberan-Santos, M. N. Molecular Fluorescence:
Principles and Applications, 2nd ed., John Wiley & Sons: New
York, 2012.
Bioprobe TPE-TREG-Py (1a): Light brown sticky liquid,
yield: 91%; 1H NMR (400 MHz, D2O): δ (ppm) 3.49-3.58 (m, 28
H), 3.79-3.92 (m, 20 H), 6.52 (d, J = 8.4 Hz, 8H), 6.89 (d, J = 8.4
Hz, 8H), 7.84 (t, J = 7.0 Hz, 8H), 8.29 ((t, J = 7.0 Hz, 4H), 8.71
(d, J = 6.0 Hz, 8H); 13C NMR (100 MHz, D2O): δ (ppm) 61.1,
67.2, 68.7, 68.9, 69.6, 69.9, 114.1, 128.1, 132.4, 137.0, 138.9,
144.7, 146.0, 156.5; ESI-MS: m/z 293 (z = 4); Anal. Calcd. for
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Highlights
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A series of ethylene glycol modified TPE with pyridinium polar heads is synthesized
The AIE-active “light-up” probes are water-soluble and nonfluorescent in solution
They spontaneously form vesicles and other nanoaggregates in aqueous solution
The bioprobes are biocompatible and used for protein and DNA binding
assay
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They were effectively used in wash-free imaging of bacteria and as staining agent