Ratiometric fluorescence sensing of sugars via a reversible disassembly and assembly of the peptide aggregates mediated by sugars

Article abstract of DOI:10.1039/c4cc01439a

An amphiphilic dipeptide (1) bearing pyrene and phenylboronic acid was demonstrated as a unique example of a ratiometric sensing system for sugars by reversibly converting the peptide aggregates into the monomer form of the complex with sugars in aqueous solutions. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc01439a

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Ratiometric fluorescence sensing of sugars via a
reversible disassembly and assembly of the
peptide aggregates mediated by sugars†
Cite this: Chem. Commun., 2014,
50, 5854
Received 25th February 2014,
Accepted 3rd April 2014
Lok Nath Neupane, Song Yee Han and Keun-Hyeung Lee*
DOI: 10.1039/c4cc01439a
www.rsc.org/chemcomm
An amphiphilic dipeptide (1) bearing pyrene and phenylboronic acid the excimer emissions without a change in monomer emissions.
was demonstrated as a unique example of a ratiometric sensing system In general, a ratiometric response using two different emission
for sugars by reversibly converting the peptide aggregates into the bands is more ideal because a ratiometric response could measure
monomer form of the complex with sugars in aqueous solutions.
analytes more accurately with a correction of environmental effects
such as pH, temperature, and solvent media.7
Carbohydrates are the major energy sources for living organisms
In the present study, we synthesized a new ratiometric
and play an important role in a wide range of biological processes sensing system based on peptides for sugars including glucose
such as signal transduction, inflammation, cell–cell interactions, capable of controlling the pyrene monomer and excimer emissions
bacteria–host interactions, fertility, and development.1 Thus, new upon sugar binding. Amphiphilic dipeptide 1 containing phenyl-
methods of monitoring carbohydrates have received much attention boronic acid as a receptor was aggregated in the absence of sugars
in the field of biology and medicinal chemistry. As fluorescence in aqueous solutions at pH 7.4, resulting in a considerable excimer
is one of the most powerful and simple ways for detecting low emission. The covalent bonding of the phenylboronic acid of 1
concentrations of analytes, fluorescent chemosensors for sugars with glucose converted the aggregates into a monomer form of the
have received growing interest.
complex, resulting in a decrease of excimer emission and a
Fluorescent chemosensors for sugars have been used mainly concomitant increase of monomer emission intensity. To the best
with arylboronic acids as receptors because arylboronic acids of our knowledge, this is a unique ratiometric sensing system for
rapidly form reversible covalent bonding with several diol sugars by changing the aggregate form into the monomer form in
compounds including sugars in aqueous solutions.2,3 As it aqueous solutions after sugar binding.
was believed that the covalent interactions of arylboronic acids
As peptides are biologically compatible and highly water
with carbohydrates did not considerably affect the fluorescence soluble and act as ligands for specific analytes,8 the amphiphilic
of the neighboring fluorophore, most of the chemosensors dipeptide (1) was designed to contain pyrene as a fluorophore, and
based on arylboronic acids detected carbohydrates by a turn-on phenylboronic acid as a receptor. A Trp amino acid was selected
response using a photoinduced electron transfer (PET) process.2,3 because Trp is reported to act as a ligand for several analytes
Among various fluorophores, pyrene has received much attention including sugars (Scheme 1).9 1 was easily synthesized in 79% yield
in the design of the chemosensors because the unique monomer by a solid phase synthesis method. Details of the synthesis and
and excimer emissions with a distinctive different wavelength characterization of 1 are described in the ESI† (Fig. S1–S5).
varied depending on the proximity between the pyrene fluoro-
We investigated the fluorescence response of 1 to glucose in
phores.4 Until now, various pyrene systems capable of controlling an aqueous solution containing 1% DMSO. As shown in Fig. 1,
the pyrene monomer and excimer emissions by external stimuli
such as metal ions, anions, and pH have been reported.5 Even
though some sugar sensing systems involving a pyrene fluoro-
phore have been reported,6 they detected sugars by a change in
Department of Chemistry, Bioorganic Chemistry Lab, Inha University,
253 Yunghyun-dong, Nam-Gu, Incheon city, 402-751, South Korea.
E-mail: leekh@inha.ac.kr; Fax: +82-32-8675604; Tel: +82-32-8607674
† Electronic supplementary information (ESI) available: Synthesis scheme,
additional fluorescence spectra, and 1H NMR and ESI-MS data. See DOI: 10.1039/
Scheme 1 Structures of 1 and 2.
c4cc01439a
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Fig. 1 Fluorescence emission spectra of 1 (10 mM) upon addition of
D-glucose (0–0.056 M) (lex = 342 nm) in 50 mM phosphate buffer solution
containing 1% DMSO at pH 7.4.
Fig. 2 (a) Fluorescence response of 1 (10 mM) in the absence of carbo-
hydrates at different pH values and (b) the emission intensity at 378 nm as a
function of pH in the presence (K) and absence (’) of glucose (0.05 M) in
a 50 mM phosphate buffer solution containing 1% DMSO.
in the absence of glucose, significant excimer emission at
470 nm with weak typical monomer emission bands at 378
and 395 nm were observed. This indicated that 1 might form
self-assembled aggregates and two pyrene fluorophores were
stacked even in the absence of glucose. Interestingly, upon the
addition of glucose, a significant decrease of the excimer
emission and a concomitant increase of the monomer emission
with an isoemissive point at 417 nm were observed. 1 exhibited a
ratiometric response to glucose in an aqueous solution at neutral
pH. The intensity ratio (I378/I470) at 378 and 470 nm changed
significantly from 0 to 7 upon adding glucose. About 0.05 M
glucose was enough for the saturation of the emission intensity
change. During the UV-visible titration of 1 with glucose, an
increase of the absorbance at 343 nm was observed (Fig. S6,
ESI†), which indicated a decrease of pyrene–pyrene interactions
in the presence of glucose. The covalent bonding between 1 and
glucose was analyzed by ESI mass spectroscopy (Fig. S7, ESI†).
A new peak corresponding to [1Áglucose – 2H2O – H+]À appeared
at 850.46 (m/z), which reveals that the covalent complex between
1 and glucose was formed by a boronate ester group and 1
formed a 1 : 1 complex with glucose. The covalent bonding of 1
with glucose might induce the formation of the monomer form
from the aggregates of 1 in aqueous solutions, resulting in a
decrease of excimer emission and a concomitant increase of
monomer emission intensity.
To investigate the role of phenylboronic acid in the binding
mode, the fluorescence spectra of 1 were measured at different
pH values. Fig. 2a shows the fluorescence spectra of 1 in the
absence of glucose at different pH. As the neutral pH of the
solution was changed to an acidic pH, the excimer emission
increased and a small decrease of monomer emission was
observed. However, as the neutral pH was increased to a basic
pH, the excimer emission significantly decreased and the monomer
emission significantly increased, which indicates that the two
pyrene fluorophores were less overlapped at basic pH values. As
shown in Fig. 2b, the monomer emission intensity, depending on
the pH in the absence of sugar, indicated that the pKa value of
phenylboronic acid played a critical role in the ratio between
excimer and monomer emissions. As the pH is higher than the
pKa value of phenylboronic acid, the boronic acid with sp2 hybrid-
ization was converted into a tetrahedral boronate form with sp3
hybridization. The anionic boronate form of 1 preferred the
monomer form rather than the aggregates, which may be due to
the charge–charge repulsion and an increase of hydrophilicity,
which induced the increase of monomer emission intensity and
a decrease of excimer emission. UV-visible titration of 1 at different
pH values showed that as the pH was increased, a considerable
increase of the absorbance at 343 nm was observed (Fig. S8, ESI†),
which revealed that pyrene–pyrene interactions decreased as the
pH was increased.
Fig. 2b shows the emission intensity at 378 nm as a function
of pH in the presence of glucose in aqueous buffer solutions.
When glucose interacted with the phenylboronic acid of 1 to
form a boronate-ester, the pKa value of the phenylboronic acid
decreased from 8.36 to 7.62. As a result, 1 was converted into an
anionic boronate form at neutral pH and this complex preferred
the monomer form rather than the aggregates, resulting in a
decrease of excimer emission and a concomitant increase of
monomer emissions. The shift in the pKa value upon binding of
glucose was in agreement with the previously reported results.4a–c,10
We also investigated the fluorescence response of 1 to
D-fructose, D-galactose, and D-mannose (Fig. S9, ESI†). 1 showed
a ratiometric response to the sugars at a neutral pH by increasing
monomer emission intensity and decreasing excimer emission.
Assuming a 1 : 1 complex formation based on the ESI mass
spectrum, the association constants of 1 for the sugars were
calculated by fitting the emission intensity at 378 nm versus
concentrations of the sugars (Fig. 3).11 The association constants
for D-fructose, D-galactose, D-mannose, and D-glucose were calcu-
lated to be 1199.72 MÀ1, 105.08 MÀ1, 90.13 MÀ1 and 42.91 MÀ1
,
respectively. The order of the binding affinity of 1 for sugars was
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Fig. 4 The proposed binding mode of 1 with sugars.
hydrophobicity of 1 is likely to be a major driving force for the
aggregation of 1 in aqueous solutions.
To investigate the relationship between the fluorescence and
the aggregates, we investigated the aggregates by dynamic light
scattering (DLS) and by fluorescence spectroscopy. As shown in
Fig. S17 (ESI†), the amphiphilic dipeptide 1 formed the aggregates of
diameters ca. 100 nm and resulted in excimer emissions in aqueous
solutions. The addition of glucose to the solution induced the
disassembly of the aggregates with increasing monomer emissions
and a concomitant decrease of the excimer emissions. At pH 4 10,
when the phenylboronic acid was converted into the boronate anion,
neither aggregates nor excimer emissions were observed, whereas at
pH r 7.4, the aggregates of diameters ca. 100 nm and excimer
emission were observed. These results indicated that the excimer
emission was due to the formation of the aggregates of 1. DLS
measurements of 2 indicated that 2 did not form aggregates in
aqueous solutions.
The binding mode of 1 with sugars as proposed is shown in
Fig. 4. When the sugars covalently interacted with the phenyl-
boronic acid of 1 to form boronate esters, the pKa value of the
boronic acid of the complex decreased. The resulting anionic
boronate form of the complex with sugars preferred the mono-
mer form rather than the aggregates due to charge–charge
repulsions and an increase of the hydrophilicity of the complex.
The sugar-induced disassembly of the aggregates of 1 resulted
in an increase of monomer emissions and a concomitant
decrease of excimer emissions.
In summary, we report a unique ratiometric sensing system
based on a peptide containing phenylboronic acid for sugars by
changing the aggregates into the monomer form in aqueous
solutions after sugar binding. The covalent bonding of the
phenylboronic acid of 1 with sugars induced disassembly of
the aggregates into the monomer form of the complex and
resulted in a decrease of excimer emissions and a concomitant
increase of monomer emissions in aqueous solutions. To the
best of our knowledge, this may be the first example of ratio-
metric sugar monitoring via a reversible disassembly of the
aggregates into the monomer form in aqueous solutions by
sugar binding.
Fig. 3 Emission intensity of 1 (10 mM) upon addition of D-glucose (K),
D-galactose (m), D-mannose (.) and D-fructose (’) in 50 mM phosphate
buffer solution containing 1% DMSO at pH 7.4.
consistent with that of the reported sugar sensor using phenyl-
boronic acid.4a–c,10,12
Interestingly, the excimer emission was significantly decreased
by adding the sugar, whereas the enhancement of monomer
emission intensity depended on the kind of the binding sugar.
The enhancement of monomer emission intensity of 1 correlates
well with the association constants for the sugar.
To investigate the solvent effect on the response to sugars,
we measured fluorescence of 1 in an aqueous solution containing
different volumes of DMSO (Fig. S10, ESI†). As the percent of
DMSO in the solvent increased from 1 to 10%, excimer emission
significantly decreased and few excimer emissions were observed,
whereas the monomer emission was enhanced less than 2 times.
This indicates that the increase of the volume percent of the
hydrophobic solvent, DMSO in solution may weaken the inter-
actions between the compounds and stabilize the monomer form
of 1. Interestingly, the enhancement of the monomer emission
intensity of 1 upon binding with glucose in aqueous solution
containing 1% DMSO was much higher than that measured in
aqueous solution containing 10% DMSO without glucose. This may
be due to the quenching effect of phenylboronic acid on the
fluorescence of pyrene.13 It was believed that the covalent inter-
actions of the arylboronic acid with the sugars did not considerably
change the fluorescence of the neighboring fluorophore. However,
we found that the phenylboronic acid with sp2 hybridization
showed a more potent quenching effect on the fluorescence of
pyrene than the tetrahedral boronate form with sp3 hybridization.13
Even though the aggregates of 1 were disassembled in aqueous
solution by increasing the percentage of DMSO, the phenylboronic
acid still acted as a quencher for the fluorescence of the pyrene to
induce a weak monomer emission. The solvent effect on the
fluorescence of 1 suggested that the hydrophobic interactions
between the compounds play an important role in the aggregation
in an aqueous solution. To confirm this, we synthesized compound
2 in which the hydrophobic Trp amino acid was replaced with Gly
(Fig. S11–S15, ESI†). 2 showed only weak monomer emission in the
absence of sugars in aqueous solutions containing 1% DMSO and
showed a turn-on response to glucose by the enhancement of
monomer emissions (Fig. S16, ESI†). This result confirms that the
This work was supported by a grant (2012R1A1B3000574)
from the National Research Foundation and a grant from Inha
University.
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