Sensitive and selective ratiometric fluorescent detection of monosaccharides in aqueous solutions at physiological pH using self-assembled peptides with different aromatic side chains

Article abstract of DOI:10.1039/c6nj03830a

The control of disassembly of supramolecular nanostructures of self-assembled peptides by specific stimuli has not been intensively investigated for the fluorescent detection of analytes in aqueous solutions. In the present study, we investigated the control of assembly and disassembly of self-assembled peptides by monosaccharides for ratiometric detection. A series of amphiphilic dipeptides (1-4) with a different aromatic side chain bearing a phenylboronic acid and pyrene fluorophore were synthesized. The dipeptides (1-3) spontaneously self-aggregated and formed nanoparticles in aqueous solutions, resulting in significant pyrene excimer emissions, whereas the dipeptide (4) did not aggregate, resulting in a little excimer emission. The addition of sugars induced the disassembly of the self-aggregates of 1-3, resulting in a decrease in excimer emission and an increase in monomer emission. All peptides (1-4) showed a high selectivity for fructose among monosaccharides and 3 showed the lowest detection limit for fructose among the peptides showed ratiometric responses. The addition of hydrophobic pinanediol stabilized the self-aggregates of 1-3, resulting in an increase in excimer emission and a decrease in monomer emission. Even though 4 did not self-aggregate in aqueous solution, the addition of pinanediol induced the assembly of nanoparticles, resulting in a significant increase of excimer emission and a decrease of monomer emissions. The results revealed that the assembly and disassembly of the self-assembled peptides could be controlled by covalent bonding with sugar, which was suitable for the sensitive ratiometric detection of monosaccharides in aqueous solution at physiological pH.

Full text of DOI:10.1039/c6nj03830a

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
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Sensitive and selective ratiometric fluorescent detection of
monosaccharides in aqueous solutions at physiological pH using
selfꢀassembled peptides with different aromatic side chains
Received 00th January 20xx,
Accepted 00th January 20xx
Lok Nath Neupane, Pramod Kumar Mehta, and KeunꢀHyeung Lee*
DOI: 10.1039/x0xx00000x
The control of disassembly of supramolecular nanostructures of the selfꢀassembled peptides by specific stimuli has not been
intensively investigated for the fluorescent detection of the analytes in aqueous solutions. In the present study, we
investigated the control of assembly and disassembly of selfꢀassembled peptides by monosaccharides for the ratiometric
detection. A series of amphiphilic dipeptides (1‒4) with a different aromatic side chain bearing a phenylboronic acid and
pyrene fluorphore were synthesized. The dipeptides (1‒3) spontaneously selfꢀaggregated and formed nanoparticles in
aqueous solutions, resulting in the significant pyrene excimer emissions, whereas the dipeptide (4) did not aggregate,
resulting in a little excimer emission. The addition of sugars induced the disassembly of the selfꢀaggregates of 1‒3, resulting
in a decrease in excimer emission and an increase in monomer emission. All peptides (1ꢀ4) showed a high selectivity for
fructose among monosaccharides and 3 showed the lowest detection limit for fructose among the peptides showed
ratiometric responses. The addition of a hydrophobic pinanediol stabilized the selfꢀaggregates of 1‒3, resulting in an
increase in excimer emission and a decrease in monomer emission. Even though 4 did not selfꢀaggregate in aqueous
solution, the addition of pinanediol induced the assembly of nanoparticles, resulting in a significant increase of excimer
emission and a decrease of monomer emissions. The results revealed that the assembly and disassembly of the selfꢀ
assembled peptides could be controlled by covalent bonding with sugar, which was suitable for the sensitive ratiometric
detection of monosaccharides in aqueous solutions at physiological pH.
www.rsc.org/
Monosaccharides are essential biomolecules because they
played significant roles in various biological processes such as
Introduction
Supramolecular structures of the selfꢀassembly of small
biomolecules played a critical role for the specific biological
functions in living systems. The selfꢀassembled peptides that
can form supramolecular structures have been extensively
studied for the potential use in diverse applications such as drug
delivery, tissue engineering, bioelectronics, and sensing due to
easy synthesis, high biocompatibility, and various
functionalities of peptides.1‒4 As the selfꢀassembled peptides
could form a various wellꢀdefined nanostructures such as
nanoparticles, nanofiber, nanorods, and nanotubes, control of
the primary structures of the selfꢀassembled peptides for the
specific nanostructures have been extensively investigated.1‒11
In recent years, triggers for the assembly of the selfꢀ
assembled peptides by external stimuli have been the focus of
much research. The selfꢀassembled peptides have been further
signal transduction, cell–cell interactions, bacteria–host
interactions, fertility, and development.21,22 Thus, the control of
disassembly and assembly of the selfꢀassembled peptides by
monosaccharides should be important for further applications
including sensing. However, it is rarely reported that the selfꢀ
assembled peptides was used a new sensing system for
monosaccharides in aqueous solutions.
Fluorescence has received great attention for the detection
of analytes due to inexpensive instrument, simple, rapid, and
high sensitivity.
A
variety of fluorescent probes for
monosaccharides has been reported.23‒36 However, most of the
probes showed turnꢀon responses to sugars, whereas a few of
the probes showed ratiometric responses to sugars and most of
the ratiometric probes required a tedious synthesis.23‒36 Turnꢀon
response was more preferred than turnꢀoff response because
turnꢀoff response could not be differentiated with the false
signals induced by the precipitation of the sensors or the
absorbance of impurities. However, turn on responses using one
emission band were affected by environmental effects such as
pH, polarity of the media, and temperature.37,38 Ratiometric
responses using two different emission bands were highly
recommended because the ratio between two emission
intensities could correct the environmental effects. However,
most of ratiometric probes for monosaccharides required a
difficult synthesis and did not show sensitive responses to sugar
in aqueous solutions at physiological pH. Furthermore,
considering the real applications of the fluorescent probe, a
relatively large emission change by target analytes was highly
designed with triggers for the assembly of
various
nanostructures by various stimuli such as solvent, pH,
temperature, salt concentration, metal ions, and enzymes.12‒20
Even though assembly as well as disassembly processes was
important for the various applications including drug delivery
and detection of analytes, the disassembly of supramolecular
nanostructures of the selfꢀassembled peptide by specific stimuli
has not been intensively investigated for the sensing systems.
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recommended. However, the recent reported ratiometric probe concomitant decrease of the excimer emissions at 470 nm.
showed a relatively small emission change and/or intensity ratio Among the peptideꢀbased probes ( ) showed ratiometric
change by monosaccharides at physiological pH. Thus, it is responses to monosaccharides, showed the lowest detection
1‒3
3
highly challenging for the design of fluorescent probes with limit for fructose and exhibited a selective ratiometric response
easy synthesis that show sensitive ratiometric responses to to fructose among the tested monosaccharides. The dipeptide
monosaccharides with significant emission intensity changes in
aqueous solutions at physiological pH.
(
4
) that did not aggregate in aqueous solutions at physiological
pH, showed a turn on response to monosaccharide however
Since Gazit et al. reported that the selfꢀassembled dipeptide exhibited a ratiometric response to pinanediol by the increase of
consisting of PhePhe aggregated and formed a range of excimer emissions and decrease of monomer emissions. The
different nanostructures including nanoparticles, nanofibril, present study provides a new sensing system based on the selfꢀ
nanorods, nanotubular aggregates in different solvent assembled peptides for the sensitive ratiometric detections for
conditions,2,39 short selfꢀassembled peptides have received monosaccharides in aqueous solutions at physiological pH.
attentions as biomaterials for drug delivery systems,
bioelectronics, and tissue engineering.1‒4 However, the control
Experimental
of the assembly and disassembly of the selfꢀassembled peptides
Materials
by carbohydrates was rarely investigated.40‒42 In the previous
FmocꢀOrn(alloc)ꢀOH and FmocꢀNaIꢀOH were purchased from
Bachem. FmocꢀTrp(Boc)ꢀOH, FmocꢀPheꢀOH, FmocꢀHis(Trt)ꢀ
OH, Rink Amide MBHA resin, 1ꢀhydroxybenzotriazole (HOBt)
and N,N’’ꢀdiisopropylcarbodiimide (DIC) were purchased from
Bead Tech. 1ꢀpyreneacetic acid, 4ꢀCarboxyphenylboronic acid
research, we proposed a new ratiometric sensing system for
monosaccharides based on the fluorescent dipeptide bearing a
phenylboronic acid.43 Furthermore, unlike the chemical probes,
the sensitivity, the response type, and selectivity of the peptideꢀ
based probes for specific targets can be easily optimized by
further tuning of the amino acid sequences of the peptide part.
pinacol
dimethylformamide
ester,
trifluoroacetic
(DMF),
acid
(TFA),
N,N’ꢀ
(DCM),
Dichloromethane
palladium(0)
Tetrakis(triphenylphosphine)
Pd(PPh3)4,
Phenylsilane, (1S,2S,3R,5S)ꢀ(+)ꢀpinanediol, piperidine were
purchased from Sigma Aldrich.
Solid phase synthesis of compounds
The peptides were synthesized by solid phase synthesis with
Fmoc chemistry.44 Fmoc protected
LꢀTrp(Boc)ꢀOH, FmocꢀNaIꢀ
OH, FmocꢀPheꢀ OH and FmocꢀHis (Trt)ꢀOH (0.3 mmol of
each, 3 equiv) were separately assembled on Rink Amide
MBHA resin (0.1 mmol) for the synthesis of compound
1, 2, 3,
and , respectively. After deprotection of Fmoc group, Fmocꢀ
4
Orn(alloc)ꢀOH (0.3 mmol, 3 equiv) was coupled with the amino
acid on the resin (Scheme S1). After deprotection of Fmoc
group, coupling of 4ꢀcarboxyphenylboronic acid pinacol ester
was performed by the following procedure. 4ꢀ
carboxyphenylboronic acid pinacol ester (75 mg, 0.3 mmol),
HOBt (40 mg, 0.3 mmol) and DIC (47 ꢁL, 0.3 mmol) in DMF
(3mL) were added into the resin and the resulting solution
containing the resin was stirred for 4 h at room temperature.
The deprotection of alloc group of the side chain of ornithine
was carried out by the following literature procedure.45 1ꢀ
Pyreneacetic acid (78 mg, 0.3 mmol), HOBt (40 mg, 0.3 mmol)
and DIPC (47 ꢁL, 0.3 mmol) in DMF (3mL) were added into
the resin and the resulting solution was mixed for 4 h at room
temperature. Deprotection and cleavage of the compounds from
the resin was achieved by treatment with a mixture of
TFA/TIS/H2O (95:2.5:2.5, v/v) at room temperature for 5 h.
After filtration and washing of the resin by TFA, a gentle
stream of nitrogen was used to remove the excess TFA. The
crude was triturated with diethyl ether chilled at ꢀ20 °C and
then centrifuged at 3000 rpm for 10 min at ꢀ10 °C. The crude
product was purified by prepꢀHPLC with a Vydac C18 column
using water (0.1% TFA)ꢀacetonitrile (0.1% TFA) gradient to
give >79% yield. The successful synthesis was confirmed by
ESI mass spectrometry (Platform II, micromass, Manchester,
Scheme 1. Proposed binding mode of (a) 1‒3 and (b) 4 with sugars and pinanediol for
fluorescent change and aggregations.
In the present study, we synthesized several selfꢀassembled
dipeptides bearing a phenylboronic acid as a receptor and
investigated the control of assembly and disassembly of the
selfꢀassembled peptides by monosaccharides and a hydrophobic
diol compound for the ratiometric sensing (Scheme 1).
Dipeptides (1‒3) bearing a phenylboronic acid selfꢀaggregated
and formed nanoparticles in aqueous solutions at physiological
pH. Upon addition of hydrophilic monosaccharides, the
nanoparticles of the dipeptides (1‒3) were disassembled,
resulting in the increase of monomer emissions at 378 nm and
2 |New j. Chem.
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UK) and its homogeneity (>98%) was confirmed by reverse = 8.4 Hz, 1H), 7.85–7.79 (m, 5H), 7.32 (d,
J = 8.6 Hz, 2H),
phase analytical HPLC with a C18 column: 7.23 (s, 1H), 4.51–4.48 (m, 1H), 4.36–4.31 (m, 1H), 4.14 (s,
Compound 1. White solid, Yield : 79 %; tR = 44 min 2H), 3.09–3.06 (m, 3H), 2.95–2.90 (m, 1H), 1.75–1.69 (m, 2H),
1
(H2O/CH3CN); mp 260–262 ºC; H NMR (400 MHz, DMSOꢀ 1.58–1.40 (m, 2H). 13C NMR (50 MHz, DMSOꢀd6) δ: 171.8,
d6) δ: 10.8 (s, 1H), 8.43 (d,
1H), 8.25 (d, = 8.4 Hz, 1H), 8.23–8.19 (m, 3H), 8.17 (d,
8.4 Hz, 1H), 8.15–8.12 (m, 2H), 8.17 (d, = 8.4 Hz, 1H), 7.91 124.7, 124.0, 123.9, 123.8, 53.7, 51.2, 39.7, 39.5, 39.2, 28.5,
= 8.4 Hz, 1H), 7.87–7.85 (m, 2H), 7.82–7.80 (m, 2H), 7.56 26.0, ESIꢀMass (m/z) calculated for C36H35BN6O6 [M + H+]+,
= 8.5 Hz, 1H), 7.12 (brs, 1H), 7.05–6.99 (m, 2H), 6.92 (t, 659.27; found, 659.23; Elemental analysis (calcd, found for
J
= 8.4 Hz, 1H), 8.36 (d,
J
= 8.4 Hz, 171.7, 170.0, 167.0, 135.0, 133.8, 130.9, 130.7, 130.2, 129.6,
J
J =
128.9, 128.5, 127.3, 127.1, 126.7, 126.4, 126.1, 125.0, 124.8,
J
(d,
(d,
J
J
J
= 8.5 Hz, 1H), 4.5–4.4 (m, 2H), 4.2 (s, 2H), 3.19–3.0 (m, 4H), C36H35BN6O6): C (65.66, 56.26), H (5.36, 4.53), N (12.76,
1.8–1.7 (m, 2H). 13C NMR (50 MHz, DMSOꢀd6) δ: 173.9, 10.00).
172.1, 170.7, 167.4, 138.2, 136.6, 136.0, 134.5, 131.7, 131.4, General fluorescence measurements
131.0, 130.3, 129.6, 129.3, 128.1, 127.8, 127.4, 127.11, 126.8,
A stock solution of compounds at the concentration of 1.0 ×
125.7, 125.5, 125.4, 124.7, 124.7, 124.6, 124.1, 121.4, 119.1,
10−3 M were prepared in DMSO/water (1:1, v/v), and stored in
118.8, 111.8, 110.6, 54.1, 53.8, 39.5, 39.1, 29.7, 28.3, 26.7,
a cold and dark place. The concentration of stock solution was
ESIꢀMass (m/z) calculated for C41H38BN5O6 [M + Na+]+,
confirmed by UV absorbance at 342 nm for pyrene.
730.28; found, 730.16. Elemental analysis (calcd, found for
Fluorescence emission spectrum of the sample in a 10 mm path
C41H38BN5O6): C (69.59, 66.07), H (5.41, 5.11), N (9.90,
10.75).
length quartz cuvette was measured in 50 mM phosphate buffer
solution at pH 7.4 using
a PerkineElmer luminescence
Compound 2. White solid, Yield : 78 %; tR = 49 min
spectrophotometer (model LS 55). Emission spectra of the
compound in the presence of sugars and diol were measured by
excitation with 342 nm for pyrene.
1
(H2O/CH3CN); mp 269–270 ºC; H NMR (400 MHz, DMSOꢀ
d6) δ: 8.42 (d,
= 8.4 Hz, 1H), 8.24–8.19 (m, 3H), 8.15 (d,
8.13–8.11 (m, 4H), 8.08–8.0 (m, 1H), 7.99 (d,
7.99 (d,
1H), 7.55–7.50 (m, 2H), 7.38 (s, 1H), 7.29 (d,
J = 8.5 Hz, 1H), 8.35 (d, J = 8.4 Hz, 1H), 8.26 (d,
J
J
J
= 8.4 Hz, 1H),
= 8.5 Hz, 1H),
Determination of association constant
The association constant for the 1:1 complex was calculated
based on the titration curve of the fluorescent chemosensor with
J
= 8.4 Hz, 1H), 7.89–7.8 (m, 5H), 7.66 (d,
J = 8.4 Hz,
J
= 8.5 Hz, 1H),
sugar. Association constants
(Ka) was determined by a
7.28–7.20 (m, 1H), 7.13 (s, 1H), 4.60–1.52 (m, 1H), 4.39–4.30
(m, 1H), 4.16 (s, 2H), 3.60–3.58 (m, 1H), 3.30–3.21 (m, 1H),
3.10–2.90 (m, 1H), 1.70–1.60 (m, 2H), 1.45–1.30 (m, 2H). 13C
NMR (50 MHz, DMSOꢀd6) δ: 173.5, 172.1, 170.6, 167.4,
135.7, 134.3, 134.2, 133.8, 131.4, 131.3, 130.8, 130.2, 129.4,
129.1, 127.9, 127.7, 127.4, 127.3, 127.0, 126.7, 126.6, 126.0,
125.7, 125.6, 125.4, 125.2, 124.6, 124.4, 124.4, 124.2, 54.1,
53.7, 39.2, 38.9, 35.0, 29.2, 26.3, ESIꢀMass (m/z) calculated for
C43H39BN4O6 [M + H+]+, 719.30; found, 718.38; Elemental
analysis (calcd, found for C43H39BN4O6): C (71.87, 70.66), H
(5.47, 5.51), N (7.80, 7.37).
nonlinear least squares fitting of the data with the following
equation.35
Iꢀꢁꢂ + IꢀꢃꢄKꢃ[S]
I =
+ kꢅꢃ[s]
1 + Kꢃ[S]
Where Imin and Imax are the initial (no sugar) and final (plateau)
fluorescence intensities of the titration curves and ka’ is slope of
the linear equation for the fitting of the linearly increased
emission intensity as function of the concentration of sugar.
Measurement of the size of the aggregates of the compounds in
solutions
Compound 3. White solid, Yield : 81 %; tR = 43 min
1
(H2O/CH3CN); mp 265–266 ºC; H NMR (400 MHz, DMSOꢀ
The size distribution of the aggregates of compounds in
aqueous solution was characterized by using a particle size
analyzer (Brookhaven Instruments Corporation, New York,
USA, model 9863) equipped with HeꢀNe laser (633 nm). The
measurements of the aggregates in the solution were carried out
by 90º dynamic light scattering at 25ºC. Compound (30 µM)
was dissolved in 50 mM phosphate buffer solution containing
DMSO at pH 7.4 for the size measurement.
d6) δ: 8.45 (d,
(d, = 8.5 Hz, 1H), 8.21–8.20 (m, 3H), 8.18 (d,
1H), 8.15–8.13 (m, 4H), 8.10–8.05 (m, 1H), 8.01 (d,
J
= 8.5 Hz, 1H), 8.35 (d,
J
= 8.4 Hz, 1H), 8.25
= 8.4 Hz,
= 8.4 Hz,
J
J
J
1H), 8.95–8.80 (m, 5H), 7.39 (s, 1H), 7.0–7.05 (m, 4H), 4.55–
4.39 (m, 2H), 4.18 (s, 2H), 3.10–2.96 (m, 3H), 2.89–2.8 (m,
1H), 1.75–1.62 (m, 2H), 1.5–1.35 (m, 2H). 13C NMR (50 MHz,
DMSOꢀd6) δ: 172.9, 171.5, 170.1, 166.9, 137.9, 135.4, 134.0,
131.2, 130.9, 130.5, 129.8, 129.3, 129.1, 128.8, 128.1, 127.5,
127.3, 126.9, 126.6, 126.3, 125.2, 125.0, 124.9, 124.2, 124.2,
53.7, 53.6, 39.9, 39.7, 39.5, 29.0, 26.1, ESIꢀMass (m/z)
calculated for C39H37BN4O6 [M + H+]+, 669.28; found, 668.26;
Elemental analysis (calcd, found for C39H37BN4O6): C (70.07,
68.17), H (5.58, 5.53), N (8.38, 7.88).
Transmission Electron Microscopy (TEM) measurements
Transmission electron microscopy (TEM) was performed using
a Philips CM 200 operated at an acceleration voltage of 120
kV. The samples were prepared by dropping 5 ꢁL of
1 (0.5
mM) in aqueous buffered (50 mM phosphate, pH 7.4) solution
on a 300ꢀmesh copper grid coated with carbon followed by
staining with phosphotungstic acid (2 wt%). The TEM grid was
completely dried in the air before TEM measurements were
conducted.
Compound 4. White solid, Yield : 80 %; tR = 35 min
1
(H2O/CH3CN); mp 247–248 ºC; H NMR (400 MHz, DMSO–
d6) δ: 8.94 (s, 1H), 8.52 (d,
J = 8.5 Hz, 1H), 8.34 (d, J = 8.4,
Hz, 1H), 8.20 (d, = 8.6 Hz, 1H), 8.19–8.18 (m, 3H), 8.17 (d,
= 8.4 Hz, 1H), 8.14–8.10 (m, 4H), 8.05–8.0 (m, 1H), 7.95 (d,
J
J
J
Determination of detection limit
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The detection limit was calculated based on the fluorescence
titration. To determine the S/N ratio, the emission intensity of
dipeptides without Dꢀfructose was measured by ten times and
the standard deviation of blank measurements was determined.
Three independent duplication measurements of emission
intensity were performed in the presence of
Dꢀfructose and each
average value of the intensities was plotted as a concentration
of Dꢀfructose for determining the slope. The detection limit is
then calculated with the following equation.46
Detection limit = 3σ/m
Where, σ is the standard deviation of the emission intensity of
probe and m is the slope between the emission intensity vs.
concentration.
Results and Discussion
D-Fructose
D-Glucose
Pinanediol
D-Galactose
Design of the selfꢀassembled peptides bearing phenylboronic
acid
Figure 1. Structures of compound 1‒4, monosaccharides, and pinanediol.
The assembly and/or disassembly of the selfꢀassembled
peptides (1‒4) triggered by sugar were synthesized, as shown in
Figure 1. Arylboronic acid as a receptor was conjugated with
the peptides because arylꢀboronic acid formed reversible
Fluorescence emission spectra and the size and shape analysis of
the peptides in aqueous buffered solutions containing DMSO
We measured the fluorescence emission spectra of the peptides
(1‒4) in aqueous buffered solution (50 mM phosphate pH 7.4)
covalent
adducts
with
diol
compounds
including
monosaccharides in aqueous solutions and several artificial
receptors using arylboronic acid based on various organic
compounds, polymers, proteins, and DNA were reported for the
recognition of various analytes including saccharides and
glycosylated biomolecules.23‒36,47‒49 A pyrene fluorophore was
incorporated into the dipeptides for the ratiometric detection
because the pyrene fluorophore has interesting photophysical
properties such as high fluorescence quantum yield and dual
fluorescence emission (monomer emission at 376 nm and
excimer at 470 nm) depending on the proximity between two
pyrene fluorophores.50,51
containing different volume percentage of DMSO because
amphiphilic peptides dissolved well in DMSO. As shown in
Figure 2,
1 displayed a relatively strong excimer emission at
470 nm and weak monomer emissions at 377 and 396 nm in
aqueous solution containing 1 % (v/v) DMSO. As the volume
percent of DMSO increased from 1 to 10 % (v/v), the excimer
emission significantly decreased and the monomer emission
considerably increased. The strong pyrene excimer emissions
indicated that
1 might aggregate in aqueous solution containing
low percentages of DMSO. However, the low excimer emission
in aqueous solutions containing high percentages of DMSO
suggests that the peptides did not aggregate in this solvent
condition.
As shown in Figure 1, we synthesized the dipeptides (1‒4)
bearing a phenylboronic acid in which the second amino acid
was Trp, (3ꢀ(Naphthyl)ꢀLꢀalanine), Phe, and His among
aromatic amino acids, to have different selfꢀassembled
properties by the change of the hydrophobicity and the structure
2
showed significant monomer emissions at 377 and 396
nm and a relatively weak excimer emission in aqueous solution
at physiological pH containing 1% DMSO. As the volume
percent of DMSO increased from 1 to 20 % in the solution
of the 2nd amino acid. When the peptides bearing
a
phenylboronic acid might interact with the hydrophilic sugars
or hydrophobic diols and form covalent adducts, the peptides
might assemble or disassemble depending on the selfꢀassembly
property of the covalent adducts and exhibit the change of
monomer emissions and excimer emissions depending on the
proximity between the pyrene fluorophores.
containing 2, the monomer emission significantly increased and
the excimer emission slightly decreased (Figure 2). About 15 %
DMSO was enough for the complete disappearance of excimer
emissions in aqueous solution. As the volume percent of
DMSO increased from 1 to 15%, the monomer emission of
3
significantly increased and the excimer emission decreased
(Figure S21). About 3 % DMSO was required for the complete
The peptides (1‒4) with high purity were easily synthesized
in solid phase synthesis with high yields.44 The detailed
procedure for the synthesis and characterization of the pyrene
labelled peptides were described in the experimental section
(Figure S1‒S20). The high purity (> 98%) of the peptide was
confirmed by analytical HPLC with a C18 column and ESI mass
spectrometer.
disappearance of excimer emission of
3 in aqueous solution. 4
in which the second amino acid was His, exhibited only
monomer emission in 100 % aqueous solution at pH 7.4 and
aqueous solution containing high percentages of DMSO (Figure
S21).
4 did not aggregate even in 100% aqueous solutions,
resulting in only monomer emissions because hydrophilic
might not aggregate.
4
4 |New j. Chem.
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100
80
60
40
20
0
400
1 % DMSO
1.5 % DMSO
2 % DMSO
1 % DMSO
(a)
2 % DMSO
5 % DMSO
10 % DMSO
12 % DMSO
15 % DMSO
20 % DMSO
(b)
3 % DMSO
300
5 % DMSO
10 % DMSO
15 % DMSO
200
100
0
360
420
540
600
Wavele4n8g0th (nm)
360
420
480
540
600
Wavelength (nm)
100
80
60
40
20
0
100
80
60
40
20
0
(c)
(d)
100 ± 30 nm
170 ± 60 nm
0
100
200
Size (nm)
300
400
500
Size (nm6)00
0
200
400
800
1000
Figure 2. Fluorescence spectra of (a) 1 (10 ꢁM) and (b) 2 (10 ꢁM) in aqueous buffered solution (50 mM phosphate, pH 7.4) containing different percentage of DMSO (λex = 342 nm,
slit = 12/2.5 nm). Size distribution of DLS measurements of (c) 1 (30 ꢁM) and (d) 2 (30 ꢁM) in aqueous buffered solution containing 3 % and 10 % DMSO, respectively.
We investigated the size of the selfꢀaggregates of the considerable increase of absorbance at 343 nm was observed
peptides in aqueous buffered solution containing DMSO using (Figure S23). As the peptide selfꢀaggregated in solutions, the
a dynamic light scattering (DLS) measurement. As shown in pyrene fluorophore might be overlapped and the absorbance
Figure 2,
solutions formed aggregates with an average diameter of ca. of DMSO increased, the selfꢀaggregates did not form, resulting
100 nm and 170 nm, respectively. Similarly, that exhibited in the increase of the absorbance of the pyrene. UVꢀvisible
excimer emissions in solutions also formed nanoꢀaggregates spectra of and indicated that the absorbance at 343 nm
with an average diameter of ca 90 nm in aqueous buffered increased as the volume percentage of DMSO increased. This
solution (Figure S22). DLS result indicates that which did not indicates that did not aggregate as the percentage of DMSO
show excimer emissions in 100% aqueous solutions did not in aqueous solution increased. UVꢀvisible spectra of revealed
form aggregates in this solvent condition (Figure S22). that the absorbance at 343 nm was not considerably changed as
1 and 2 that showed a significant excimer emission in corresponding to the pyrene might decrease. As the percentage
3
2
3
4
1‒3
4
The overall result suggests that the hydrophobicity of the the volume percentage of DMSO in the solution increased
second amino acid in the peptides play a critical role for the (Figure S23). This indicated that as relatively hydrophilic
selfꢀaggregations and excimer emissions in aqueous solution. peptide (4) did not aggregate in aqueous solutions containing
The retention time on C18 column might correlate well with the different volumes of DMSO, the absorbance of the pyrene at
hydrophobicity.52 According to the retention time of the 343 nm did not change considerably.
peptides, the order of hydrophobicity was
hydrophobic peptides required more DMSO in aqueous assembled aggregates of
solutions for the observation of a little excimer emission. The (TEM). As shown in Figure 3,
2
>
1
>
3
>
4
. More
Furthermore, we investigated the structures of the selfꢀ
by transmission electron microscopy
selfꢀaggregated and formed
1
1
maximum excimer intensity of the peptides may correlate with vesicleꢀlike structures in aqueous buffered solution. The size of
the hydrophobicity of the peptides; the maximum excimer vesicle ranged from 50 nm to 80 nm, which was smaller than
emission intensity at 470 nm was 23 for
1
, 50 for
2
, and 20 for that measured by DLS maybe due to the dehydration process.
Interestingly, when the concentration of was increased to 2
1 in aqueous solutions containing mM, the formation of nanofibrils with the length of 100~200
3, respectively.
1
UVꢀvisible spectra of
different percentage of DMSO showed that as the volume nm was observed (Figure 3b). This suggests that if the
percentage of DMSO in aqueous solutions increased,
a
concentration of the peptide reaches to certain high
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concentration, the formation of nanofibrils might be band at 470 nm were observed with a clear isoemission point at
predominant. This result was consistent with the fact that the 432 nm. The intensity ratio (I377/I470) increased from 0.71 to 16
selfꢀassembled
dipeptide
(PhePhe)
formed
different as the concentration of
Dꢀfructose increased and about 20 mM
nanostructures such as nanoparticles, nanofibril, nanotubular of
D
ꢀfructose was required for the complete disappearance of
aggregates in different solvent conditions.2,39
the excimer emissions.
The fluorescence emission spectra of
3 in aqueous buffered
solution (50 mM phosphate, pH 7.4) containing 0.5% (v/v)
DMSO, displayed weak excimer emission at 470 nm. Upon
addition of Dꢀfructose to the solution of 3, a significant increase
of monomer intensity at 376 nm and 396 nm and slightly
decrease of excimer emission intensity were observed (Figure
4). The intensity ratio (I376/I470) between the monomer and
excimer emission increased from 2.2 to 26 and 20 mM of
fructose was enough for the complete change of the intensity
ratio. The results suggest that the covalent bonding with
fructose might induce disassembly of supramolecular
nanostructures of the peptides ( 3) in aqueous solutions.
We also investigated the fluorescence responses of
to ꢀglucose and ꢀgalactose, respectively (Figure S24‒S26).
Although they required a high concentration of ꢀglucose and
ꢀgalactose for the fluorescent emission changes, the addition
of ꢀglucose and ꢀgalactose at physiological pH resulted in the
Dꢀ
D
ꢀ
Figure 3. TEM images of 1 (a) (0.5 mM) and (b) (2 mM) in aqueous buffered solution
(50 mM phosphate buffer, pH 7.4). The samples were negatively stained with 2%
phosphotungstic acid. The bar represents 200 nm (a) and 100 nm (b).
1
‒
1, 2, and
3
D
D
D
Fluorescence emission spectra of 1‒4 in the presence of
monosaccharide
D
D
D
The fluorescence emission responses of
were investigated in aqueous buffered solution at pH 7.4
(Figure 4). displayed a strong excimer emission band at 470
1ꢀ3 to monosaccharides
increase of monomer emission intensity and decrease of the
excimer emission, respectively. This suggests that the
1
disassembly of the selfꢀassembled peptide aggregates (
triggered by monosaccharides in aqueous solution. As
1
‒
3
4
) was
was
nm and weak monomer emissions at 377 and 396 nm in
aqueous buffered solution (50 mM phosphate, pH 7.4)
dissolved well in distilled water, the fluorescence spectrum was
measured in 100 % aqueous solution (50 mM phosphate, pH
containing 1% (v/v) DMSO. Upon addition of
Dꢀfructose, a
significant decrease of pyrene excimer emission at 470 nm and
a concomitant increase of pyrene monomer emissions at 377
and 396 nm were observed. The intensity ratio (I377/I470) was
7.4) without organic solvent. As shown in Figure 4,
4 displayed
strong emission intensities at 376 and 396 nm, corresponding to
the typical monomer emission bands of the pyrene, which
significantly changed from 0.60 to 15 as the concentration of
fructose increased from 0 to 7.5 mM. Approximately 7.5 mM
of ꢀfructose was necessary for the complete disappearance of
the pyrene excimer. Similarly, also showed a strong excimer
Dꢀ
indicated that
Upon addition of
396 nm was gradually increased. About 6 mM
required to completely change the emission intensity in aqueous
solution. Similar monomer emission enhancement of was
ꢀgalactose,
4
might not aggregate in 100% aqueous solutions.
ꢀfructose, the monomer emission at 376 and
ꢀfructose was
D
D
D
2
emission at 470 nm and weak monomer emissions at 376 and
396 nm in aqueous buffered solution (50 mM phosphate, pH
4
observed in the presence of
respectively (Figure S27).
Dꢀglucose and D
7.4) containing 5% (v/v) DMSO. Upon addition of
Dꢀfructose,
a significant increase of the monomer emissions at 377 nm and
396 nm and considerable decrease of the excimer emission
6 |New j. Chem.
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100
80
60
40
20
0
300
16
12
8
15
12
9
6
3
(a)
(b)
250
200
150
100
50
4
0
0
0
2
4
6
8
0
5
10 15 20
[D-Fructose] mM
[D-Fructose] mM
0
360
420
480
540
600
360
540
600
4W20avele4n8g0th (nm)
Wavelength (nm)
250
600
25
20
15
10
5
600
500
400
300
200
100
0
(c)
(d)
500
400
300
200
200
150
100
50
0
0
5 10 15 20 25 30
0
2
4
6
8 10
[D-Fructose] mM
[D-Fructose] mM
0
360
420
480
540
600
360
420
480
540
600
Wavelength (nm)
Wavelength (nm)
Figure 4. Fluorescence spectra of (a) 1 (b) 2 (c) 3, and (d) 4 upon the gradual addition of Dꢀfructose in aqueous buffered solution (50 mM phosphate, pH 7.4) containing DMSO
solvent (1 %, 5 %, 0.5 % and 0 %, respectively). The concentration of peptides is 10 ꢁM.
UV−visible titration experiment was carried out (Figure When the peptides formed a covalent bonding with hydrophilic
S28). In the UV−visible absorption titration of with sugars, the nanoparticles of the peptide disassembled, resulting
1
Dꢀ
fructose, a considerable increase in absorbance at 343 nm in the decrease of the excimer emission and increase of
corresponding to the pyrene fluorophore was observed as the monomer emissions. Thus, we investigated whether the binding
concentrations of
showed a considerable increase of the absorbance at 343 nm in emission spectrum and the formation of the nanoparticles.
the presence of ꢀfructose (Figure S28). The results indicated As shown in Figure 5, the addition of pinanediol to the
that after the peptides ( ) were complexed with ꢀfructose, solution containing induced a significant increase of the
the aggregates might disassemble, resulting in the increase of excimer emission at 470 nm and a slight decrease of the
the absorbance of pyrene. However, the absorbance spectra of monomer emissions at 376 nm. The intensity ratio I470/I376
were not changed in the presence of ꢀfructose. As did not increased from 1.8 to 31 and just 80 ꢁM of pinanediol was
Dꢀfructose increased. Similarly, 2 and 3 also of a hydrophobic diol compound would affect the fluorescence
D
1
‒3
D
1
4
D
4
form selfꢀaggregates in aqueous solution even in the absence of enough for the complete change of both monomer and excimer
sugar, disassembly could not be triggered by covalent bonding emission intensities. Upon addition of increasing concentration
with
D
ꢀfructose.
of pinanediol to the solution of 2, the increase of excimer
We also investigated whether the nanoparticles of the selfꢀ emission and the decrease of monomer emissions were
assembled peptide disassembled in the presence of hydrophilic observed (Figure S30). About 60 ꢁM of pinanediol was
sugars or not. Interestingly, DLS measurements of the peptide required for the saturation of the intensity ratio change. Upon
in the presence of
fructose induced the disassembling of the nanoparticles of
(Figure S29). Furthermore, upon addition of ꢀfructose, the significant decrease in the pyrene monomer emissions were
vesicleꢀlike nanostructures disappeared in TEM, which observed (Figure 5). The intensity ratio (I470/I376) between the
suggests that the supramolecular nanostructures of were excimer and monomer emissions changed from 0.2 to 7. The
disassembled by covalent bonding with hydrophilic ꢀfructose. results indicate that the covalent bonding of pinanediol with
may stabilize the nanoparticles in aqueous solutions, resulting
D
ꢀfructose revealed that the addition of
D
ꢀ
addition of pinanediol into the solution of
3, a significant
1‒3.
increase in the pyrene excimer emission at 470 nm and
D
1
D
1‒
3
Fluorescence emission spectra and the size distributions of
the peptides of 1‒4 in the presence of a hydrophobic diol
in the enhanced excimer emission and a concomitant decrease
of the monomer emission.
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100
300
250
200
150
100
50
30
25
20
15
10
5
0
6
4
2
0
(b)
(a)
80
60
0
20 40 60 80 100
[Pinanediol]
0
20 40 60 80
µ
M
[Pinanediol]
µM
40
20
0
0
360
420
480
540
600
360
480
600
42W0 avelength (n5m40)
Wavelength (nm)
100
80
60
40
20
0
100
80
60
40
20
0
(c)
170 ± 80 nm
240 ± 40 nm
(d)
0
200
400
800
1000
0
200
400
600
800
1000
Size (n6m00)
Size (nm)
Figure 5. Fluorescence spectra of (a) 1 (10 ꢁM) and (b) 3 (10 ꢁM) upon the gradual addition of pinanediol in aqueous buffered solution (50 mM phosphate, pH 7.4) containing 1%
and 0.5 % DMSO, respectively (λex = 342 nm, slit = 12/2.5 nm). Particle size distribution of (c) 1 (30 ꢁM) and (d) 3 (30 ꢁM) in aqueous buffered solution containing 3% DMSO.
UV−vis titration of
(Figure S31). Upon addition of pinanediol to the solution of
, a significant decrease in the absorbance at 343 nm assembled peptides formed covalent adducts with the
1‒
3
with pinanediol was carried out average size of the nanoparticles was consistent well with the
1‒
increase of the excimer emissions (Figure S33). When the selfꢀ
3
corresponding to the pyrene and red shift were observed, which hydrophobic diol, the size of the resulting nanoparticles
indicated the overlap of the pyrene fluorophores was induced increased and the overlapped pyrene fluorophore increased,
by the covalent bonding of pinanediol with
1
‒
3 50,51
.
resulting in the increase of the excimer emissions.
Even though did not show excimer emission in aqueous
4
The effect of hydrophobic pinanediol on the assembly of 4
solution, the addition of pinanediol induced the significant was much significant.
increase of the excimer emission and the decrease of monomer 100% aqueous solution without organic cosolvent. However,
emissions (Figure S32). The relatively hydrophilic peptide ( upon addition of pinanediol, the nanoparticles with the average
4 did not form nanoparticles even in
4
)
required about 200 ꢁM of pinanediol for the complete change size of 130 nm were observed with the significant excimer
of the excimer emission and monomer emissions. During the emission (Figure S33). The result suggested that the assembly
UVꢀvisible titration of
of the absorbance at 343 nm and a red shift of the absorption addition of the hydrophobic diol compound.
spectra were observed (Figure S32). This suggests that after Furthermore, we investigated the nanostructure of the selfꢀ
complexation of with pinanediol, the pyrene fluorophores assembled peptide ( ) in the presence of pinanediol by using a
overlapped, resulting in the decrease of the absorbance at 343 transmission electron microscopy (TEM). Upon addition of
nm and an increase in excimer emission. hydrophobic pinanediol, the vesicleꢀlike structures in the
We investigated whether the addition of pinanediol induced absence of pinanediol were converted to the fibrils (Figure 6).
the change of the nanoparticles of or not. DLS experiments The average size of the fibrils was much larger than those
indicated that the size of the nanoparticles increased in the measured by DLS. This is because small fibrils induced by
presence of pinanediol. The selfꢀassembled peptides ( , and covalent bonding with the hydrophobic pinanediol might
) formed nanoparticles with the average size of 100 nm, 170 convert to larger fibrils during the dehydration process or larger
nm, and 90 nm in the absence of diol compounds, respectively. fibrils could not be well detected by DLS measurements due to
Upon addition of pinanediol to the solution of , the average the precipitations.
size of the nanoparticles increased from 100 nm to 240 nm We also investigated whether
(Figure 5). Similarly, the size of nanoparticles of and presence of pinanediol or not. Upon addition pinanediol into the
increased in the presence of pinanediol. The increase of the solution, the nanoparticle structures were clearly observed in
4 with pinanediol, a significant decrease of the peptide for the nanoparticles could be triggered by the
4
1
1‒4
1, 2
3
1
4
formed nanoparticles in the
2
3
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TEM images (Figure 6b). This suggests that the covalent with the order of Ka values of the reported fluorescent probes
bonding of the hydrophilic peptide ( which indicates that the
) with a hydrophobic diol using phenylboronic acid23,24,27
induce the selfꢀaggregation and formation of nanoparticles.
4
,
boronic ester formation between the phenylboronic acid moiety
of the peptides and the diol compounds induced the change of
the fluorescent spectra and nanostructures. The binding affinity
of the boronic acid for sugar was determined by the orientation
and relative position of hydroxyl groups of the sugar. In general,
(a)
(b)
the binding affinity for Dꢀfructose was most potent because Dꢀ
fructose existed as a furanose form with a relative high
percentage in aqueous solutions that contained a synꢀperiplanar
pair of hydroxyl groups.27 The association constants of
1‒4 for
pinanediol were calculated based on the titration curve (Figure
S37). The binding affinity of the peptides for pinanediol is
much higher than those for the monosaccharides. The high Ka
100 nnmm
50 nmm
Figure 6. TEM images of (a) 1 (0.5 mM) and (b) 4 (0.5 mM) in the presence of
pinanediol in aqueous buffered solutions (50 mM phosphate, pH 7.4) containing
CH3CN. Samples were negatively stained with 2% phosphotungstic acid. The bar values for pinanediol may be due to the position and
represents 100 nm (a) and 50 nm (b).
configuration of the diol of pinanediol that formed a stable
boronic ester form.23,24,47
Binding affinity, detection limit, and selectivity for the diol
compounds
Considering the applications for the detection of fructose,
we measured the detection limits and the linear response ranges
The binding stoichiometry was investigated by ESI mass
spectrometry. After adding ꢀfructose to the solution of , a
new peak at 850.31 (m/z) appeared. This peak corresponded to
ꢀfructose ꢀ 2H2O ꢀ H+]ꢀ (Figure S34), which confirmed that
formed a 1:1 complex with ꢀfructose. ESI mass spectrum for
the sample containing and ꢀfructose showed that the peak at
862.42 (m/z) corresponding to [
ꢀfructose ꢀ 2H2O ꢀ H+]‒ was
observed (Figure S35). This result supports that also formed a
1:1 complex with ꢀfructose. In general, one phenylboronic
of the changes in intensity ratio by fructose. As shown in Fig.
S38, showed sensitive linear ratiometric responses to subꢀ
molar concentrations (100~800 ꢁM) of ꢀfructose in aqueous
buffered solution. The detection limit was calculated based on
the linear relationships between the intensity ratio (I378/I475) and
D
1
1ꢀ3
D
[1·D
1
D
2
D
the concentration of
Dꢀfructose. Even though 3 did not have the
2·D
highest association constant for fructose among the peptides (
1ꢀ
2
3
3
) that showed a ratiometric response to sugar, the sensitivity of
was the greatest among the peptides so had the lowest
ꢀfructose. had some advantages for the
ꢀfructose because aggregated in aqueous
solutions without organic cosolvent and showed the most
D
3
acid of the receptor formed a 1:1 complex with various diol
compounds.28‒36
detection limit for
detection of
D
3
D
3
Assuming the formation of a 1:1 complex, the association
constant (Ka) of 1‒4 for sugar and pinanediol were calculated
sensitive ratiometric response to
As shown in Figure 7,
ratiometric response to
the concentration of fructose increased, the intensity ratio
(I376/I470) significantly increased from 2.2 to 26 and the
intensity at 376 nm increased from 40 to 240. The large
enhancement of the emission by fructose and sensitive
ratiometric response to fructose provided a selective detection
of fructose among monosaccharides in aqueous solutions.
D
ꢀfructose.
showed a highly selective
based on the titration curve by nonlinear least square fitting
(Figure S36).35 The fitting of titration curves provided less than
5% errors, which also confirmed the 1:1 binding stoichiometry
of the peptide. Table 1 summarized Ka values and detection
3
D
ꢀfructose among monosaccharide, As
limits of
values revealed that all peptides showed a high selectivity for
fructose among sugars. The order of Ka values ( ꢀfructose>
galactose> ꢀglucose) of for the sugar is similar to the order
of the association constants of phenylboronic acid for the
sugars.53 The order of
a values of for the sugars correlated
1‒4 for monosaccharides and pinanediol. The Ka
D
Dꢀ
D
1‒4
K
1‒4
Table 1. Apparent association constants (
K
a) and detection limits of
a [Mꢀ1]
1‒
4
for diol compounds.
Detection limit for Detection limit for
Peptides
K
D
ꢀfructose [M]
pinanediol [M]
D
ꢀFructose
D
ꢀGalactose
D
ꢀGlucose
Pinanediol
61.4 × 103
34.7 × 103
73.2 × 103
1
2
3
1.15 × 103
0.14 × 103
0.22 × 103
1.01 × 102
0.24 × 102
0.45 × 102
41
14
21
9.74 × 10ꢀ5
9.55 × 10ꢀ5
8.66 × 10ꢀ5
2.13 × 10ꢀ7
9.96 × 10ꢀ7
3.14 × 10ꢀ7
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4
1.32 × 103
1.40 × 102
46
21.3 × 103
2.31 × 10ꢀ5
4.20 × 10ꢀ6
excimer emissions which might slowly induce the fibril
formation.
50
40
30
20
10
0
D
-Fructose
-Glucose
-Galactose
-(+)Pinanediol
3
2
1
0
D
D
As shown in Scheme 1, the peptide (
aggregate in aqueous solution. When the hydrophobic
pinanediol covalently bonded with the peptide ( ), the resulting
4) did not selfꢀ
4
0.00 0.02 0.04 0.06 0.08
covalent adduct with pinanediol selfꢀaggregated and formed
nanoparticles, resulting in the increase of excimer emission and
decrease of monomer emissions. As the hydrophilic sugar
[Diols] mM
covalently bonded with the peptide (4), the monomer emission
was significantly increased without the formation of
nanoparticles. This enhancement can be explained by the strong
quenching effect of phenylboronic acid to the adjacent pyrene
fluorophore because phenylboronic acid acted as a quencher for
pyrene fluorophore and the tetrahedral boronate form showed a
much weaker quenching effect rather than the corresponding
boronic acid form.32
0
5 10 15 20 25 30 35 40 45 50 55
[Diols] mM
Figure. 7. Emission intensity ratio of 3 (10 ꢁM) upon the addition of
galactose (▲), ꢀglucose (●), and –(+)–pinanediol (▼) in 50 mM phosphate buffer
solution containing 0.5% DMSO at pH 7.4.
Dꢀfructose (■), Dꢀ
D
Herein, we report the discovery of a selective and highly
sensitive fluorescent probes based on the selfꢀassembled
peptides for fructose in aqueous solutions. In last two decades,
the worldwide consumption of fructose and highꢀfructose corn
syrup has increased significantly and the high administration of
fructose highly related to the increase of the obesity, diabetes,
hypertension, and kidney disease.54 Thus, the simple and easy
detection of fructose in food is important to control of the
administration of fructose. Even though several ratiometric
fluorescent probes for fructose have been reported, most of
them suffered from one of the limitations such as the difficulty
of the synthesis, poor solubility in water, low sensitivity
(enhancement), and low emission intensity change by
fructose.23ꢀ26 The fluorescent probes developed in this work are
easy to synthesize and selectively and sensitively detected
fructose in aqueous solutions by significant emission intensity
changes. Thus, the probes will provide an efficient method for
the detection of fructose in aqueous solutions.
Figure 8 presents a visible emission color images of
presence of sugars and pinanediol under UV light (λem = 365
nm) with a UV lamp. displayed a blue color in the absence of
sugars and pinanediol whereas displayed a violet color in the
presence of ꢀfructose and a brighter cyan color in the presence
of pinanediol. This result indicates that the ratiometric probe (
3 in the
3
3
D
3
)
made it possible for the selective detection of fructose among
monosaccharides by the naked eye with a UV lamp.
Figure 8. Visible emission color change of
monosaccharides (20 mM) and pinanediol (0.1 mM) under UV light (λem = 365 nm).
3 (10 ꢁM) in the presence of
Conclusion
We synthesized selfꢀassembled peptides in aqueous solution at
physiological pH and demonstrated the disassembly and
assembly of supramolecular nanoparticles of the selfꢀassembled
peptides controlled by the diol compounds for the ratiometric
detection. The amphiphilic peptides (1‒3) selfꢀaggregated and
formed vesicleꢀlike nanostructures, resulting in the considerable
excimer emission. The covalent bonding of the phenylboronic
We proposed how the assembly and disassembly of the selfꢀ
assembled peptides were controlled by diols compounds. As
shown in Scheme 1,
1‒3 selfꢀaggregated and formed
nanoparticles in aqueous solution at physiological pH in which
the pyrene fluorophores were close each other, resulting in the
excimer emissions. When the hydrophilic sugars such as
fructose, ꢀglucose, and ꢀgalactose covalently bonded with the
phenylboronic acid of , the hydrophilicity of the complex
Dꢀ
acid of the peptides (1‒3) with hydrophilic sugars induced
D
D
disassembly of the nanoparticles, resulted in a decrease of
excimer emissions and a concomitant increase of monomer
emissions. The covalent bonding of hydrophobic pinanediol
1‒3
increased and the selfꢀaggregates disassembled, resulting in the
increase of monomer emissions and a concomitant decrease of
excimer emissions. When the hydrophobic pinanediol
covalently bonded with the selfꢀassembled peptides 1‒3, the
hydrophobicity of the complex increased, resulting in the
stabilization of nanoparticle structures with the enhancement of
with
in the significant enhancement of excimer emissions and a
decrease of monomer emissions. The dipeptide ( ) did not selfꢀ
aggregate in aqueous solution and only showed monomer
emissions. The assembly of to the nanoparticles which
1‒3 stabilized the formation of the nanoparticles, resulting
4
4
10 |New j. Chem.
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DOI: 10.1039/C6NJ03830A
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Sakurai, J. Zhao and T. D. James, Acc. Chem. Res., 2013, 46,
312–326.
24 R. Nishiyabu, Y. Kubo, T. D. James and J. S. Fossey, Chem.
Commun., 2011, 47, 1106–1123.
25 Z. Guo, I. Shin and J. Yoon, Chem. Commun., 2012, 5956–
5967.
26 H. Fang, G. Kaur and B. Wang, J. Fluoresc., 2004, 14, 481–
exhibited a considerable excimer emission could be triggered
by pinanediol. showed selective ratiometric responses to
fructose among sugars and showed the lowest detection limit
for fructose. exhibited a selective ratiometric response to
pinanediol by the enhancement of excimer emissions at 475 nm
and exhibited turn on responses to sugars by the enhancement
of monomer emissions at 376 nm. This study revealed that the
selfꢀassembled peptides triggered by sugars will be a valuable
tool for the sensitive ratiometric detection for sugar in aqueous
solutions.
1ꢀ3
3
4
489.
27 X. Wu, Z. Li, X.ꢀX. Chen, J. S. Fossey, T. D. James and Y.ꢀ
B. Jiang, Chem. Soc. Rev., 2013, 42, 8032–8048.
28 X. Sun, B. Zhu, D.ꢀK. Ji, Q. Chen, X.ꢀP. He, G.ꢀR. Chen, T.
D. James, ACS Appl. Mater. Interfaces, 2014, 6, 10078–
10082.
29 Y. Liu, J. Zhu, Y. Xu, Y. Qin and D. Jiang, ACS Appl.
Mater. Interfaces, 2015, 7, 11141–11145.
Acknowledgements
30 M. Elstner, K. Weisshart, K. Mullen and A. Schiller, J. Am.
This work was supported by Inha University Research Grant.
Chem. Soc., 2012, 134, 8098–8100.
31 T. L. Halo, J. Appelbaum, E. M. Hobert, D. M. Balkin and A.
Schepartz, J. Am. Chem. Soc., 2009, 131, 438–439.
32 L. N. Neupane, C. R. Lohani, J. Kim and K.ꢀH. Lee,
Tetrahedron, 2013, 69, 11057–11063.
Notes and references
1
2
3
I. W. Hamley, Soft Matter, 2011, 7, 4122–4138.
E. Gazit, Chem. Soc. Rev., 2007, 36, 1263–1269.
S. Fleming and R. V. Ulijn, Chem. Soc. Rev., 2014, 43, 8150–
8177.
33 Y.ꢀJ. Huang, W.ꢀJ. Ouyang, X. Wu and Z. Li, J. Am. Chem.
Soc., 2013, 135, 1700–1703.
34 Y. Liu, C. Deng, L. Tang, A. Qin, R. Hu, J. Z. Sun and B. Z.
Tang, J. Am. Chem. Soc., 2011, 133, 660–663.
35 N. D. Cesare and J. R. Lakowicz, J. Photochem. Photobiol. A,
2001, 143, 39–47.
4
5
6
7
8
9
H. Hosseinkhani, P. Hong and D. Yu, Chem. Rev., 2013, 113,
4837−4861.
Y.ꢀb. Lim, K.ꢀS. Moon and M. Lee, Chem. Soc. Rev., 2009,
38, 925–934.
H. Wang, Z. Yang and D. J. Adams, Mater. Today, 2012, 15,
36 H. S. Mader and O. S. Wolfbeis, Microchim. Acta., 2008,
162, 1–34.
500–507.
37 B. Valeur, Molecular Fluorescence: Principles and
Applications, WileyꢀVCH, Weinheim, 2002.
38 J. R. Lakowicz, Topics in Fluorescence Spectroscopy: Probe
Design and Chemical Sensing, vol. 4, Plenum Press, New
York, 1994.
A. Lakshmanan, S. Zhang and C. A. Hauser, Trends
Biotechnol., 2012, 30, 155–165.
D. Mandal, A. N. Shirazi and K. Parang, Org. Biomol. Chem.,
2014, 12, 3544–3561.
H. Cui, M. J. Webber and S. I. Stupp, Biopolymers, 2010, 94,
39 M. Reches and E. Gazit, Nat. Nanotechnol., 2006, 1, 195‒
200.
1–18.
10 X. Zhao, F. Pan, H. Xu, M. Yaseen, H. Shan, C. A. Hauser, S.
Zhang and J. R. Lu, Chem. Soc. Rev., 2010, 39, 3480‒3498.
11 P.ꢀP. Yang, X.ꢀX. Zhao, A.ꢀP. Xu, L. Wang and Hao Wang,
J. Mater. Chem. B, 2016, 4, 2662‒2668.
40 M. Mandal, S. K. Mandal, M. Ghosh and P. K. Das, Chem.
Eur. J., 2015, 21, 12042–12052.
41 T. HoegꢀJensen, S. Havelund, P. K. Nielsen and J.
Markussen, J. Am. Chem. Soc., 2005, 127, 6158–6159.
42 B. H. Jones, A. M. Martinez, J. S. Wheeler, B. B. McKenzie,
L. L. Miller, D. R. Wheeler and E. D. Spoerke, Chem.
Commun., 2015, 51, 14532–14535.
43 L. N. Neupane, S. Y. Han and K.ꢀH. Lee, Chem. Commun.,
2014, 50, 5854–5857.
44 G. B. Fields and R. L. Nobel, Int. J. Pept. Protein Res., 1990,
12 R. Zou, Q. Wang, J. Wu, J. Wu, C. Schmuck and H. Tian,
Chem. Soc. Rev., 2015, 44, 5200–5219.
13 W. She, K. Luo, C. Zhang, G. Wang, Y. Geng, L. Li, B. He,
Z. Gu, Biomaterials, 2013, 34, 1613–1623.
14 M. R. Dreher, A. J. Simnick, K. Fischer, R. J. Smith, A.
Patel, M. Schmidt and A. Chilkoti, J. Am. Chem. Soc., 2008,
130, 687–694.
35, 161–214.
15 M. J. Webber, C. J. Newcomb, R. I. Bitton and S. I. Stupp,
Soft Matter, 2011, 7, 9665–9672.
45 N. Thieriet, J. Alsina, E. Giralt, F. Guibe and F. Albericio,
Tetrahedron Lett., 1997, 38, 7275–7278.
16 A. N. Rissanou, E. Georgilis, E. Kasotakis, A. Mitraki and V.
Harmandaris, Phys. Chem. B, 2013, 117, 3962−3975.
17 M. Ikeda, T. Tanida, T. Yoshii, K. Kurotani, S. Onogi, K.
Urayama and I. Hamachi, Nat. Chem., 2014, 6, 511–518.
18 A. Ghosh, M. Haverick, K. Stump, X. Yang, M. F. Tweedle
and J. E. Goldberger, J. Am. Chem. Soc., 2012, 134, 3647–
3650.
46 G. L. Long and J. D. Winefordner, Anal. Chem. 1983. 55,
713ꢀ724.
47 D. G. Hall, Structure, Properties, and Preparation of Boronic
Acid Derivatives. Overview of Their Reactions and
Applications. Wiley, Weinheim, 2005.
48 W. L. A. Brooks and B. S. Sumerlin, Chem. Rev., 2016, 116,
1375–1397.
19 C. J. Bowerman and B. L. A. Nilsson, J. Am. Chem. Soc.,
2010, 132, 9526–952.
49 N. Y. Edwards, T. W. Sager, J. T. McDevitt, E. V. Anslyn, J.
Am. Chem. Soc., 2007, 129, 13575–13583.
20 H. Xu, A. K. Das, M. Horie, M. S. Shaik, A. M. Smith, Y.
Luo, X. Lu, R. Collins, S. Y. Liem, A. Song, P. L. A.
Popelier, M. L. Turner, P. Xiao, I. A. Kinloch and R. V.
Ulijn, Nanoscale, 2010, 2, 960–966.
21 H. J. Allen and E. C. Kisailus, Glycoconjugates:
Composition, Structure and Function, CRC Press, 1992.
22 C. R. Bertozzi and L. L. Kiessling, Science, 2001, 291, 2357–
2364.
50 F. M. Winnik, Chem. Rev., 1993, 93, 587–614.
51 C. Yao, H.ꢀB. Kraatz and R. P. Steer, Photochem. Photobiol
Sci., 2005, 4, 191–199.
52 R. A. Houghten and S. T. Degraw, J. Chromatogr. A, 1987,
386, 223–228.
53 J. Yan, G. Springsteen, S. Deeter and B. Wang, Tetrahedron,
2004, 60, 11205–11209.
54 R. J. Johnson, M. S. Segal and Y. Sautin, Am. J. Clin. Nutr.,
2007, 86, 899–906.
.
23 S. D. Bull, M. G. Davidson, J. M. H. Vanden Elsen, J. S.
Fossey, A. T. Jenkins, Y. B. Jiang, Y. Kubo, F. Marken, K.
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