N-Heterocyclic Carbene Boranes as Reactive Oxygen Species-Responsive Materials: Application to the Two-Photon Imaging of Hypochlorous Acid in Living Cells and Tissues

Article abstract of DOI:10.1002/anie.201711188

N-Heterocyclic carbene (NHC) boranes undergo oxidative hydrolysis to give imidazolium salts with excellent kinetic selectivity for HOCl over other reactive oxygen species (ROS), including peroxides and peroxynitrite. Selectivity for HOCl results from the

Full text of DOI:10.1002/anie.201711188

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
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Accepted Article
Title: N-Heterocyclic Carbene Boranes as Reactive Oxygen Species-
Responsive Materials: Application to the Two-Photon Imaging of
Hypochlorous Acid in Living Cells and Tissues
Authors: Yen Leng Pak, Sang Jun Park, Di Wu, BoHyun Cheon, Hwan
Myung Kim, Jean Bouffard, and Juyoung Yoon
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201711188
Angew. Chem. 10.1002/ange.201711188
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10.1002/anie.201711188
Angewandte Chemie International Edition
COMMUNICATION
N-Heterocyclic Carbene Boranes as Reactive Oxygen Species-
Responsive Materials: Application to the Two-Photon Imaging of
Hypochlorous Acid in Living Cells and Tissues
Yen Leng Pak,†[a] Sang Jun Park,†[b] Di Wu,†[a] BoHyun Cheon,[a] Hwan Myung Kim,*[b] Jean Bouffard,*[a]
and Juyoung Yoon*[a]
Abstract: N-Heterocyclic carbene (NHC) boranes undergo oxidative
hydrolysis to give imidazolium salts with excellent kinetic selectivity
for HOCl over other reactive oxygen species (ROS), including
peroxides and peroxynitrate. Selectivity for HOCl results from the
electrophilic oxidation mechanism of NHC boranes, which stands in
contrast to the nucleophilic oxidation mechanism of arylboronic acids
with ROS. The change in polarity that accompanies the conversion
of NHC boranes to imidazolium salts can control the formation of
emissive excimers, forming the basis for the design of the first
fluorescence probe for ROS based on the oxidation of B–H bonds.
Two-photon microscope (TPM) ratiometric imaging of HOCl in living
cells and tissues is demonstrated.
boranes[8] have emerged as attractive reagents that are stable to
air, column chromatography, Brønsted acids with a pKa > 2,[9] or
even recrystallization from boiling water.[10] Furthermore, Curran,
Lacôte and co-workers have demonstrated that NHC boranes
can perform as either radical[11] or ionic[9,12] reductants.[8]
• Previous Work: ROS Detection by C–B Oxidation
- Chang (2004)
(pin)B
O
B(pin)
HO
HO
HO
O
O
O
O
H2O2
O
COOH
O
O
- Yoon (2013)
(pin)B
O
B(pin)
O
OCl–
The oxidation of C–B bonds has played a prominent role in the
design of materials responsive to ROS. In particular, the borono-
Dakin oxidation of arylboronic acids and their esters has been
exploited in the development of fluorogenic probes for the
detection of hydrogen peroxide (H2O2) and endogenous
S
COOH
- Kalyanaraman (2010)
(HO)2B
ONOO–
O
O
O
hydroperoxides
(ROOH),[1]
hypochlorite
(OCl–),[2]
or
peroxynitrate (ONOO–).[3] Arylboronic acids and their esters are
oxidized to phenols with a kinetic selectivity of ca. 106 : 103-4 : 1
for peroxynitrate, hypochlorite, and peroxide, respectively.[3a]
Nevertheless, claims of selectivity for each of these ROS have
been reported in solution assays and cell imaging
experiments.[4,5] C–B bond oxidation of arylboronates by ROS
has also been widely used as a trigger in programmed
depolymerization, sensing amplification, drug delivery, and
theragnostics.[4b,6]
In view of the need for ROS-responsive materials that are
not merely selective but instead specific, it is remarkable that
designs relying on the oxidation of B–H bonds have so far been
overlooked. Although hydroboranes and borohydrides have
been among the most popular and selective reducing agents
since the 1950’s, their sensitivity to aerobic oxidation or
hydrolysis has constrained their applications as ROS-responsive
materials. The reactivity of hydroboranes is tamed by the
formation of adducts with a Lewis base.[7] In particular, NHC
• Previous Work: NHC-Boranes as Hydrolytically Stable, Selective Reductants
- Curran & Lacôte (2009-2013)
Br
OTf
H
N
H
Ts
N
N
N
N
R
N
R
R
R
(tBuO)2
Ts
N
H
B
H
H
H
B
H
H
Ts
H– Transfer
H· Transfer
N
Ts
O
O
Ph
Me
Ph
H
HOAc
or SiO2
p-BrC6H4NH2,
OH
Me
NHAr
HOAc
Ph
Ph
H– Transfer
• This Work: ROS Detection & Two-Photon Imaging by B–H Oxidation
HOCl
N
N
N
Me
N
Me
H
B
H
H
H
1·BH3
1·H+
Herein, we report the first fluorescence imaging probe for
ROS based on the selective oxidation of a B–H bond. This probe
(1·BH3) is composed of a NHC borane as the ROS reaction site,
and of a pyrene as fluorescence reporter. Because of its low
polarity, 1·BH3 reversibly forms colloidal aggregates, facilitating
pyrene excimer emission.[13,14] Selective oxidative hydrolysis of
the B–H bonds of 1·BH3 by aq. HOCl results in the formation of
the imidazolium salt 1·H+, which exclusively emits as a distinct
monomeric species. 1·BH3 enables the ratiometric imaging of
biologically relevant HOCl levels in living cells and tissues using
TPM.[15]
[a]
[b]
[†]
Y. L. Pak, Dr. D. Wu, B. Cheon, Prof. J. Bouffard, Prof. J. Yoon
Department of Chemistry and Nano Science (BK 21 Plus)
Ewha Womans University, Seoul 03760, Korea
bouffard@ewha.ac.kr, jyoon@ewha.ac.kr
Sang Jun Park, Prof. H. M. Kim
Department of Chemistry and Energy Systems Research
Ajou University, Suwon 443-749, Korea
kimhm@ajou.ac.kr
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document
This article is protected by copyright. All rights reserved.

10.1002/anie.201711188
Angewandte Chemie International Edition
COMMUNICATION
The absorption and emission spectra of 1·HBr (10 µM) in
phosphate-buffered saline solution (PBS) (10 mM, 99:1
H2O:CH3CN, pH 7.4, 25˚C) reflect those of an isolated pyrene
chromophore. By contrast, chromophore stacking is
unmistakable in the emission spectra of 1·BH3, which features
the characteristically broad pyrene excimer band centered near
477 nm (Figure S1). Addition of NaOCl to 1·BH3 results in its
immediate conversion to a species indistinguishable from 1·HBr
(Figures 1 and S1). Analysis of the reaction mixture by ESI-MS
confirmed the disappearance of 1·BH3, and its conversion to the
imidazolium salt 1·H+ (Figure S2).
Figure 2. Relative fluorescence responses of 1·BH3 (10 µM) to various ROS
t
and other possible interferences: 1. Blank, 2. NaOCl, 3. H2O2, 4. BuOOH, 5.
ROO•, 6. ONOO-, 7. HO•, 8. •NO, 9. HCHO, 10. Me2CO, 11. GSSG (10 equiv,
100 µM) in PBS (10 mM, pH 7.4) after 1 min at room temperature.
In dry CDCl3, the oxidation of 2·BH3 to 2·BCl3 is instantaneous
(< 30 s), and likely proceeds through the intermediacy of
2·BH2Cl and 2·BHCl2. Chlorinated products are not seen when
the oxidation is carried out in buffered CD3CN-D2O. Instead, the
disappearance of 2·BH3 is accompanied by the growth of 2·H+,
t
following a 2:1 stoichiometry of BuOCl to 2·BH3 (Figure S13).
Boric acid was identified as the ultimate B-containing by-product.
Only 2·BH2OD is observed as plausible intermediate, indicating
that its direct hydrolysis leads to 2·H+, or that further oxidation
yields species too hydrolytically labile to accumulate (2·BH(OD)2
or 2·B(OD)3). These results are consistent with an oxidative
hydrolysis mechanism that involves the electrophilic attack of the
tetracoordinate NHC borane by HOCl/tBuOCl.[9,11b,12e] This
stands in contrast with the borono-Dakin oxidation of arylboronic
acids and their esters, which proceeds through the nucleophilic
Figure 1. Fluorescence titration of 1·BH3 (10 µM) upon addition of NaOCl (0-
100 µM) in PBS (10 mM, pH 7.4) after 1 min at room temperature, with
excitation at 350 nm. Inset: Photographs of 1·BH3 before (left) and after (right)
the addition 10 equiv. of NaOCl under UV irradiation (365 nm).
The conversion of 1·BH3 to 1·H+ was followed by plotting
the ratio of the fluorescence emission intensities in the green
(477 nm) and blue (374 nm) color regions (F477/F374), which
decreases ca. 540-fold in less than one minute upon the addition
of 100 µM NaOCl (Figure S3; t1/2 = 1.5 s for [1·BH3]·[HOCl/OCl–]
= 1x10-9 M2). The detection limit for HOCl/OCl– in this ratiometric
fluorescence assay (3 µM, Figures S4-S5) is far below the
physiological concentrations generated by neutrophils (20-400
µM).[16] Probe 1·BH3 is highly selective for HOCl/OCl– over other
ROS and relevant interferences. As shown in Figure 2, only the
addition of NaOCl gave rise to a significant change of the
F477/F374 fluorescence ratio. Other species including hydrogen
peroxide, tert-butyl hydroperoxide, peroxynitrite, nitric oxide
(·NO), hydroxyl (HO·), peroxyl radicals (ROO·), formaldehyde,
acetone, or glutathione disulfide (GSSG) only resulted in
comparably minor changes in the fluorescence ratio (Figures
S6-S11). Furthermore, the stability of the probe 1·BH3, and the
magnitude of the response to HOCl/OCl– were unchanged in a
physiologically relevant range from pH 2 to 10 (Figure S12).
To gain insight into the conversion of 1·BH3 to 1·H+ and its
selectivity for HOCl over other ROS, NMR titration experiments
–
attack of OCl– (or HO2 , ONOO–) on the Lewis acidic
tricoordinate boron center. NMR kinetics experiments carried out
t
with 2·BH3 and BuOOH or H2O2 reveal kinetic selectivities for
hypochlorites over peroxides as high as 108 : 1, resulting from
the electrophilic oxidation mechanism of NHC boranes (Figures
S20-S26).
The bio-imaging capability of 1·BH3 using TPM was then
investigated. For the in situ detection of ROS, TPM is an
attractive method because it uses lower energy light (>700 nm)
as the excitation source, limiting photodamage, cellular
autofluorescence, and artificial ROS generation.[17] In addition,
ratiometric imaging using dual detection windows can rule out
experimental artifacts, such as a heterogeneous distribution of
the probe, and instrumental variability. After incubation of 1·BH3
for 30 min in live HeLa cells, TPM images were collected with
various excitation sources (690-740 nm). Images obtained from
excitation at 710 nm were the brightest (Figure S27). They
displayed bright emission throughout the cell, except in the
nucleus region.
After NaOCl treatment of the cells, spectral changes that
parallel those seen in solution occur, allowing for ratiometric
image analysis (Fgreen/Fblue) with 380–420 nm (Fblue) and 480–
600 nm (Fgreen). Clearer images were obtained using this ratio
(Fgreen/Fblue) than with its inverse (Fblue/Fgreen); therefore the
former was chosen for the analyses.
t
were carried out between 2·BH3 and BuOCl as surrogates for
1·BH3 and HOCl, respectively (Scheme 1, and Figures S13-S19).
This article is protected by copyright. All rights reserved.

10.1002/anie.201711188
Angewandte Chemie International Edition
COMMUNICATION
N
N
N
N
N
N
anh.
CDCl3
Me
R
Me
R
Me
R
tBuOCl
tBuOCl
D2O
BH2Cl
2·BH2Cl
BHCl2
2·BHCl2
BCl3
k1
k2
k3
2·BCl3
11B δ = -18.7
(t, J = 110 Hz)
11B δ = -6.9
(d, J = 130 Hz)
11B δ = +2.0 (s)
k3 > k2 > k1
N
N
R t
BuOCl
N
N
Me
+ B(OD)
R
Me
D2O
D2O
D2O
3
11B δ = +19.7 (s)
BH3
D
NMR: not seen
tBuOCl
D2O
1·BH3 [R = CH2(pyrenyl)]
2·BH3 [R = Me]
2·D+
tBuOCl
D2O
N
N
N
N
N
N
Me
R
Me
R
Me
R
CD3CN-
D2O
D2O
11B δ = -37.5
(q, J = 86 Hz)
BH2OD
2·BH2OD
11B δ = -13.7
(t, J = 84 Hz)
BH(OD)2
2·BH(OD)2
B(OD)3
2·B(OD)3
PBS
Scheme 1. Mechanistic insight from the NMR titration of 2·BH3 with tBuOCl as surrogates for 1·BH3 and HOCl, respectively. Plain arrows represent the reaction
pathway most consistent with the observed stoichiometry, and dashed arrows alternate plausible pathways.
The average emission ratios (Fgreen/Fblue) of 1·BH3 labeled HeLa
cells gradually change with NaOCl treatment in dose-
reactive oxygen species. Oxidative hydrolysis of 1·BH3 occurs
selectively for HOCl over other ROS, through an electrophilic
mechanism that is distinct from the nucleophilic mechanism for
the oxidation of aryl C–B bonds by ROS. The probe 1·BH3
features a ca. 100 nm blue-shift in emission that occur within
seconds, is cell permeable, and shows low cytotoxicity. These
advantageous properties made 1·BH3 suitable for the ratiometric
visualization by TPM of both exogenous and endogenous HOCl,
in living cells and tissues. Forthcoming extensions of this design
to fluorescent reporters that are less sensitive to aggregation
effects than pyrene excimer formation will be required to
improve sensitivity in extended assays, Moreover, foreseeable
applications of this B–H bond oxidation trigger extend beyond
luminescent probes, and to other families of ROS-responsive
materials that so far relied on arylboronic acid or ester C–B bond
a
dependent manner (Figure S28). Standard MTT assay indicates
that 1·BH3 has no marked cytotoxicity to cells at the low
micromolar concentrations used in TPM imaging experiments
(Figure S29). The probe is highly photostable under the imaging
conditions (Figure S30). Moreover, the values of the Fgreen/Fblue
ratios for 1·BH3 labeled cells remained nearly constant for 2 h,
which is likely due to the fast response of the probe to HOCl in
living cells (Figure S31). These outcomes indicated that 1·BH3 is
able to detect HOCl in live cells using ratiometric TPM imaging,
with minimal interference of cytotoxicity and photobleaching.
A second TPM imaging study was carried out with RAW
264.7
macrophages
activated
by
incubation
with
lipopolysaccharides (LPS), and then by interferon gamma
(IFN-γ). Phorbol myristate acetate (PMA) was used to produce
H2O2, which is then transformed to HOCl/OCl– by
myeloperoxidase (MPO). When the cells were pretreated with
NaOCl, the average emission ratios (Fgreen/Fblue) were reduced
from 5.13 to 2.50. (Figure 3a and b). Similar results were
obtained in the TPM analysis after addition of the stimulants
(100 ng mL-1 LPS, 50 ng mL-1 IFN-γ) followed by 10 nM PMA
(Figure 3c). Furthermore, the decrease of average emission
ratios (Fgreen/Fblue) was moderated when the MPO inhibitors 4-
aminobenzoic acid hydrazide (4-ABAH) and flufenamic acid
(FAA) were present (Figure 3d and e). These results show that
TPM images of 1·BH3 directly reflect the presence of HOCl, as
expected from solution assays.
oxidation.
6.0
a
b
c
4.5
3.0
1.5
0.0
d
e
f
6
5
4
3
2
1
0
Finally, an investigation was conducted to evaluate the
C ontrol NaO C l IF N-
γ
IF N-
γ
IF N-
γ
usefulness of 1·BH3 in monitoring HOCl in
a fresh rat
+
L P S
+
L P S
+ L P S
+
P MA+ 4-AB AH + F AA
hippocampal slice. The average emission ratios (Fgreen/Fblue) of a
slice that had been incubated with 1·BH3 (100 µM) for 1.5 h were
1.57 and 1.63 in the CA1 and CA3 regions, respectively (Figure
4a and b). When tissue slices were pretreated with PMA (10 ng
mL-1), the average emission ratios (Fgreen/Fblue) dropped to 1.12
and 1.23 in the CA1 and CA3 regions, respectively (Figure 4d
and e).
This work demonstrates the potential of B–H bond
oxidation-based approaches, in particular those of NHC boranes,
in the design of materials that respond to oxidative stress and
Figure 3. Pseudocolored ratiometric TPM images of RAW 264.7 cells
incubated with 1·BH3 (5 µM). (a) Control image. (b) Cells pretreated with
NaOCl (200 µM) for 30 min and then incubated with 1·BH3. (c) Cells
pretreated with LPS (100 ng mL-1) for 16 h, IFN- γ (50 ng mL-1) for 4h, PMA
(10 nM) for 30 min and then incubated with 1·BH3 for 30 min. (d) Cells
pretreated with LPS for 16 h, IFN-γ for 4h, PMA (10 nM) + 4-ABAH (50 µM) for
4h and then 1·BH3 for 30 min. (e) Cells pretreated with LPS for 16 h, IFN-γ for
4h, PMA (10 nM) + FFA (50 µM) for 4h and then 1·BH3 for 30 min. (f) Average
Fgreen/Fblue intensity ratios in the TPM images. Images were acquired using 710
nm excitation and emission windows of 380–420 nm (blue) and 480–600 nm
(green). Scale bars = 20 µm.
This article is protected by copyright. All rights reserved.

10.1002/anie.201711188
Angewandte Chemie International Edition
COMMUNICATION
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a
d
b
e
c
CA1
1.5
1.0
0.5
0.0
CA3
C ontrol
P MA (10 ng mL
f
2.0
1.5
1.0
0.5
0.0
[5]
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-1
)
C A1
C A3
Figure 4. Pseudocolored ratiometric TPM images of a rat hippocampal slice
stained with 1·BH3 (100 µM). TPM images of a rat hippocampal slice stained
with (a, b) 1·BH3 for 1.5 h and (d, e) pretreated with PMA (10 ng mL-1) for 30
min before labeling with 1·BH3. TPM images were taken at 40x magnification
in the neuron layer of the (a, d) CA1 and (b, e) CA3 regions. (c) Bright-field
image of the CA1 and CA3 regions at 10x magnification. (f) Average Fgreen/Fblue
intensity ratios in the TPM images. Images were acquired using 710 nm
excitation and emission windows of 380–420 nm (blue) and 480–600 nm
(green). Scale bars = 50 µm.
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(2012R1A3A2048814
to H.
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Keywords: boron • N-heterocyclic carbenes • reactive oxygen
species • fluorescence probes • two-photon microscopy
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10.1002/anie.201711188
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
Bleached Boranes: NHC boranes
Yen Leng Pak,† Sang Jun Park,† Di Wu,†
BoHyun Cheon, Hwan Myung Kim,*
Jean Bouffard,* and Juyoung Yoon*
are
oxidatively
hydrolyzed
to
imidazolium salts with high kinetic
selectivity for HOCl over other reactive
oxygen species. This reactivity is
Page No. – Page No.
harnessed
in
the
two-photon
N-Heterocyclic Carbene Boranes as
Reactive Oxygen Species-Responsive
Materials: Application to the Two-
Photon Imaging of Hypochlorous
Acid in Living Cells and Tissues
fluoresence imaging probe 1·BH3, the
first ROS-responsive material that rely
on a B–H bond oxidation as triggering
event.
This article is protected by copyright. All rights reserved.