Water-Soluble Polymeric Probes for the pH-Tunable Fluorometric Detection of Hydrogen Peroxide

Article abstract of DOI:10.1002/bkcs.12006

Hydrogen peroxide (H₂O₂) is an important reactive oxygen species (ROS) that plays a significant role in many biological systems. A new water-soluble polymeric probe appended with pyrene and boronic acid groups was designed and synthesized for the detection of H₂O₂. Glycidyl methacrylate (GMA) and N,N-dimethylacrylamide (DMA) were copolymerized by reversible addition-fragmentation chain-transfer (RAFT) polymerization to yield poly(glycidyl methacrylate-co-dimethylacrylamide) [p(GMA-co-DMA)](P1). The subsequent ring-opening reaction between the secondary amine of (3-((pyren-1-ylmethyl)amino)phenyl)boronic acid with the epoxide unit of P1 yielded a target polymer, P2. In the presence of H₂O₂, the phenyl boronic acid group of P2 transformed to a phenol group, which was accompanied by turn-off fluorescence. P2 also exhibited pH-tunable detection sensitivity. This polymeric probe is anticipated to promote the development of stimuli-responsive water-soluble polymers with fluorescence-sensing behaviors.

Full text of DOI:10.1002/bkcs.12006

Article
DOI: 10.1002/bkcs.12006
BULLETIN OF THE
T. N. Annisa and H.-i. Lee
KOREAN CHEMICAL SOCIETY
Water-Soluble Polymeric Probes for the pH-Tunable Fluorometric
Detection of Hydrogen Peroxide
*
Tiara Nur Annisa and Hyung-il Lee
Department of Chemistry, University of Ulsan, Ulsan 680-749 44610, Republic of Korea.
*E-mail: sims0904@ulsan.ac.kr
Received January 22, 2020, Accepted March 1, 2020
Hydrogen peroxide (H2O2) is an important reactive oxygen species (ROS) that plays a significant role in
many biological systems. A new water-soluble polymeric probe appended with pyrene and boronic acid
groups was designed and synthesized for the detection of H2O2. Glycidyl methacrylate (GMA) and N,N-
dimethylacrylamide (DMA) were copolymerized by reversible addition-fragmentation chain-transfer
(RAFT) polymerization to yield poly(glycidyl methacrylate-co-dimethylacrylamide) [p(GMA-co-DMA)]
(P1). The subsequent ring-opening reaction between the secondary amine of (3-((pyren-1-ylmethyl)amino)
phenyl)boronic acid with the epoxide unit of P1 yielded a target polymer, P2. In the presence of H2O2,
the phenyl boronic acid group of P2 transformed to a phenol group, which was accompanied by turn-off
fluorescence. P2 also exhibited pH-tunable detection sensitivity. This polymeric probe is anticipated to
promote the development of stimuli-responsive water-soluble polymers with fluorescence-sensing
behaviors.
Keywords: Water-soluble polymeric probe, Hydrogen peroxide, Fluorometric, Pyrene boronic acid
Introduction
investigated.16 The boron atom has a planar triangular con-
formation with sp2 hybridization in the neutral form of the
boronic acid group and a tetrahedral conformation with sp3
hybridization in the anionic form. This change can affect
the quench/enhance emission intensity of the fluorophore.
Boronic acid sensors are unique because of their covalent
bonds, instead of hydrogen bonds that form in molecular
recognition. As a result, the recognition of analytes with
boronic acid sensors can be carried out in aqueous
media.17,18
The fluorescence transduction mechanism is based on the
manipulation of electron transfer (ET), such as photoin-
duced electron transfer (PET), which is extremely useful in
the design and application of chemosensors.19,20 In general,
PET sensors, this type of fluorescent chemosensor consists
of an electron-donor nitrogen group and a pyrene fluo-
rophore as the electron acceptor of ET. In this case, a spe-
cific pH range is essential for optimizing the fluorescence
transduction of PET because the protonation of the nitrogen
group at low pH will enhance the background emission
intensity of the fluorophore. At normal or high pH, the
availability of the lone pair of electrons on the nitrogen
group tends to increase with increasing pH, leading to
quenching of the emission intensity. Therefore, a neutral or
high pH is required for most PET sensors to function effi-
ciently. Previous studies showed that these PET boronic
acid sensors do not work well in this system.21
Hydrogen peroxide (H2O2) is a reactive oxygen species
(ROS) that is used widely in many areas, including food
manufacturing applications, industrial chemical, clinical,
industrial wastewater and effluent treatment, textiles,
bleaching of wood pulp and paper, mining, and pharmaceu-
tical industry.1–4 In addition, H2O2 plays an essential role
in many biological systems, such as signal transduction,
immune-cell activation, cell differentiation, apoptosis, vas-
cular re-modeling, stomatal closure, and root growth.5–11
Therefore, the efficient detection of H2O2 is highly
desirable.
Compared to other methods, the fluorometric detection
method has several advantages, including high selectivity,11
good sensitivity,5 invasiveness,9 speed,12 operational
simplicity,8 and useful applications in chemistry, biology,
and medicine.13 The design strategies are based on a spe-
cific reaction between recognition groups, such as boronate
groups (boronic acid or boronate ester), and H2O2, which
give products with different photophysical properties.8
Among the various fluorophores reported for the detection
ROS, such as pyrene, rhodamine, bodipy, and
naphthalimide, pyrene has a large stokes shift for the detec-
tion of H2O2 in aqueous media and is available in many
easily manipulated derivatives.13,14
A phenyl boronic acid group (PBA) had attracted consid-
erable interest for its ability to bind covalently to H2O2.8,15
Recently, a polymeric probe containing a pyrene boronic
acid sensor, P2, showed a donor-PET (d-PET) effect (fluo-
rophore as the donor of the ET). Surprisingly, with this
The difference between the structure of the neutral and
−
anionic forms [–B(OH)3
] of the PBA group was
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system, the emission intensity was quenched at low pH and
enhanced gradually at neutral and high pH.22 This type of
d-PET sensor is rarely reported.23
protons (peak d) confirmed the successful deprotection of
PyTDA to PyPBA.
11B NMR was used to compare the boron position
between PyTDA and PyPBA after being deprotected
(Figure S3). The B-OH coupling after deprotection at δ–
26.45 ppm was similar to that previously reported.24
Another type of NMR spectroscopy, 19F NMR, was used to
confirm the intermediate product of compound [3a]
(Figure S4). The phenyltrifluoroborate anion gives rise to
B F coupling at δ–139.53 ppm, which was similar to that
reported previously.25
This paper reports the results of d-PET sensors as a
water-soluble polymeric probe containing pyrene boronic
acid groups in the side chains for the detection of H2O2
within a specific pH range. The lone pair of electrons in the
tertiary amine unit in the probe acts as an electron donor
that can transfer the electron to pyrene, leading to
quenching of the emission intensity of the pyrene fluo-
rophore. This emission intensity of the probe was con-
trolled by varying the pH of the aqueous solutions.
The next step, GMA and DMA, were copolymerized by
RAFT polymerization to yield [p(GMA-co-DMA)]
(P1) using AIBN as the initiator and DMP as the chain
transfer agent (CTA). The initial feed ratio of GMA and
DMA for this random copolymerization was 5:95. DMA
and PyPBA were introduced in this system to provide P2
with water solubility and fluorometric detection capabilities
because it contains pyrene units as the fluorophore and
boronic acid as a binding site for the fluorometric sensing
of H2O2. The successful synthesis of P1 and P2 was con-
Results and Discussion
Scheme 1 outlines the synthesis of the designed polymer,
P2, for the detection of H2O2. A random copolymer con-
taining pyrene units in the side chains were prepared in sev-
eral steps. In the first step, 3-aminophenyl boronic acid
hemisulfate was used as a starting material in the presence
of pinacol in DCM to obtain compound 1 (Figure S1).
Compound 1 was coupled with 1-pyrenecarboxaldehyde in
the presence of NaBH4 in methanol to afford PyTDA (com-
pound 2). PyTDA was deprotected to the corresponding
PyPBA (compound 3b) via potassium pyrene phe-
nyltrifluoroborate (compound 3a) by a treatment with an
aqueous potassium hydrogen fluoride solution. The
resulting crude compound 3a was recrystallized from ace-
tone or diethyl ether to afford pure compound 3a. The
hydrolysis of compound 3a was then investigated with
aqueous alkali metal hydroxides (lithium hydroxide) to
obtain pure compound 3b free of pinacol. The successful
1
firmed by H NMR spectroscopy (Figure S5). The appear-
ance of the characteristic diastereotopic –OCH2– protons
(peaks a and a’) of GMA and the dimethyl unit (peak b) of
DMA confirmed the successful formation of P1 (Figure S5
(a)). To calculate the final incorporation ratio, the integral
area of the diastereotopic protons of GMA units at 4.33 and
3.76 ppm was compared with that of the dimethyl protons
of the DMA unit. The final incorporation ratio of GMA and
DMA was calculated to be 6:94, which is similar to the
actual feed ratio (Figure S5(a)). The number averaged
molecular weight (Mn) of P1 was determined to be 15 730
with a low polydispersity (Mw/Mn = 1.1) (Figure S6).
To obtain the final polymer, P2, the epoxide ring of P1
was opened by the secondary amine of PyPBA. The suc-
cessful post-modification reaction was monitored by 1H
NMR spectroscopy, in which peak a’ of the diastereotopic
proton of GMA shifted; new b and c peaks appeared at the
5.1–4.7 ppm region along with the pyrene and aromatic
protons at 8.3–8.1 ppm (Figure S5(b)). GPC analysis rev-
ealed P2 to have a number average molecular weight (Mn)
of 18 740 with (Mw/Mn = 1.1) (Figure S6).
1
deprotection of compound 2 to 3b was confirmed by H
NMR and 11B NMR spectroscopy (Figure S2 and S3). In
Figure S2, the disappearance of the characteristic –CH3–
The photophysical responses of P2 (0.05 mM; assuming
the 6% incorporation of pyrene units in the P2 backbone)
were examined by emission spectroscopy in various aqueous
HEPES buffer solutions (pH 3–11) (Figure 1(a)). The emis-
sion intensity of the pyrene peak at 397 nm increased gradu-
ally with increasing pH of the medium, leading to an
increasing color intensity of the solution under UV light irra-
diation (Figure 1(b)). The amino phenylboronic acid (N-
PBA) groups of P2 can be protonated at low pH to form
quaternary ammonium cations, which quenches the emission
intensity by the d-PET process from pyrene to NH+-PBA.
At high pH, the boronic acid group is present in the anionic
−
form [–B(OH)3 ], resulting in a change in the configuration
Scheme 1. Synthesis of small molecule [1, 2], [3b], and polymeric
probe P2.
of the boron atom from sp2 hybridization (triangular planar)
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Figure 1. (a) Fluorescence spectra of P2 alone (0.05 mM of
pyrene units) and (b) photograph of P2 under UV lamp
(λmax = 365 nm), in water at various HEPES buffer solution
(pH 3–11).
Figure 2. (a) Emission spectral changes in P2 upon the gradual
addition of H2O2 in aqueous media at pH 11), (inset: Photograph
of P2 in the absence and presence of H2O2 under UV lamp) and
(b) selectivity of P2 with different analytes in water pH 7 (where
I0 and I indicate the fluorescence emission of P2 in the absence
and presence of H2O2, respectively), (inset: Photograph of P2
under UV lamp with other analytes).
Figure 3. (a) Extent of emission quenching upon the addition of
H2O2 with various HEPES buffer solutions (inset: Photograph of
P2 before and after the addition of H2O2 under UV lamp), and
emission intensity of P2 upon the gradual addition of H2O2
according to pH (b) pH 3, (c) pH 5, (d) pH 7, (e) pH 9, and (f)
pH 11 (inset: Photograph of P2 before and after the addition of
H2O2 under UV lamp).
to sp3 hybridization (tetrahedral boronate). This change
induced an increase in emission intensity via the suppression
of the d-PET because of the loss of the electron-withdrawing
properties for the anionic forms of the boronic acid
group.21,22,26,27 These pH-responsive properties allow P2 to
behave as a fluorescent pH indicator.
The sensing ability was examined in aqueous media with
the addition of various concentrations of H2O2 in a HEPES
buffer solution at pH 11, where the probe showed the
highest intensity. Marked quenching (approximately
14-fold) in fluorescence at 397 nm was observed as H2O2
was added gradually to the solution of P2 (0.05 mM)
(Figure 2(a)). The apparent emission color changes from
blue to green could be observed by the naked eye under a
UV lamp (see inset in Figure 2(a)). In an aqueous solution,
H2O2 dissociates immediately to H+ and hydroperoxide
(OH-O−). The tertiary N-PBA attached to the pyrene unit
in P2 converted to the tetrahedral boronate in the presence
of H2O2, leading to fluorescence quenching of the pyrene
monomer. To evaluate the selectivity of P2, other ROS,
such as singlet oxygen (1O2), cation (Na+), and anion
(ClO−), were tested in aqueous media at pH 7 (Figure S7).
None of the other analytes showed significant quenching,
suggesting the excellent selectivity of P2 to H2O2 (Figure 2
Figure 4. Schematic diagram for the mechanism of H2O2 sensing
according to pH in aqueous media.
(b)). A good linear relationship was observed between the
(I0 / I) and concentration of H2O2; the detection limit of P2
to H2O2 was calculated to be 0.18 M (Figure S8).15,20,28–30
Due to the bulky polymer probes, the sensitivity of this sys-
tem is not good as reported previously.1–6
After examining the blue to green fluorescence detection
of H2O2, the sensing behaviors according to a specific pH
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range were studied. The relative emission intensity at
397 nm was measured before and after the addition of
H2O2 in various HEPES buffer solutions (pH 3, 5, 7, 9, and
11). The extent of fluorescence quenching was maximized
at high pH because the emission intensity was highest. The
detection sensitivity decreased with decreasing pH of the
solution (Figure 3). The pH range difference in sensitivity
could be observed clearly under a UV lamp (Figure 3
(a) inset).
intensity by the d-PET process from pyrene to the NH+-
PBA. At high pH, the boronic acid group changes to the
anionic form, leading to an increase in emission intensity
via the suppression of d-PET. The H2O2 detection abilities
within a specific pH range under the optimal conditions
were observed at the highest pH (pH 11), showing up to
14-fold fluorescence quenching and color changes from
blue to green under UV light irradiation. P2 also showed
the highest selectivity toward H2O2 over the other analytes.
Therefore, this polymeric probe (P2) has the potential to
detect other ROS by the d-PET process.
The fluorescence response of the sensor is in contrast to
normal a-PET chemosensors. From previous studies, this
system showed the d-PET effect (fluorophore as the donor
of the electron transfer) because of the presence of boronic
acid.21,22 The d-PET effect was characterized by a lower
background fluorescence at low pH than at high pH. In this
system, the fluorescence emission intensity was quenched
at low pH compared to that at high pH (Figure 4). Quater-
nary ammonium cations were formed at low pH because of
the protonated N-PBA groups of P2. The boronic acid
group acts as a Lewis acid. This is because boron has an
empty p orbital, which is an electron acceptor that can
induce quenching of the emission intensity by the d-PET
process from the pyrene fluorophore to the NH+-PBA.
At high pH, the configuration of the boron atom changed
from sp2 hybridization (triangular planar) to sp3 hybridiza-
tion (tetrahedral boronate), which transformed the boronic
acid group to the anionic form [–B(OH)3−]. This anionic
form of the boronic acid group is no longer an electron
acceptor; instead, [–B(OH)3−] is an electron donor. Under
this condition, P2 also has a nitrogen group with an avail-
able lone pair of electrons, which served as an electron
donor. As a result, the emission intensity in the pyrene fluo-
rophore was enhanced by the suppression of the d-PET pro-
cess. After the addition of H2O2 at high pH, the role of [–B
(OH)3−] as an electron donor decreased. The anionic forms
of the boronic acid group cannot act as an electron donor,
whereas the lone pair of electrons in the nitrogen group can
act as an electron donor that can transfer an electron to
pyrene. These phenomena led to quenching of the emission
intensity of the pyrene fluorophore.21,22,26,27
Acknowledgments
This work was supported by University of Ulsan Research
Fund of 2019.
Supporting Information. Additional supporting informa-
tion may be found online in the Supporting Information
section at the end of the article.
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