Azulene-Derived Fluorescent Probe for Bioimaging: Detection of Reactive Oxygen and Nitrogen Species by Two-Photon Microscopy

Article abstract of DOI:10.1021/jacs.9b09813

Two-photon fluorescence microscopy has become an indispensable technique for cellular imaging. Whereas most two-photon fluorescent probes rely on well-known fluorophores, here we report a new fluorophore for bioimaging, namely azulene. A chemodosimeter, c

Full text of DOI:10.1021/jacs.9b09813

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Azulene-Derived Fluorescent Probe for Bioimaging: Detection of
Reactive Oxygen and Nitrogen Species by Two-Photon Microscopy
Lloyd C. Murfin,†,# Maria Weber,†,‡,# Sang Jun Park,§,# Won Tae Kim∥,§,# Carlos M. Lopez-Alled,†,‡
†
∥
Claire L. McMullin,† Fabienne Pradaux-Caggiano, Catherine L. Lyall, Gabriele Kociok-Kohn,
̈
Jannis Wenk,‡,⊥ Steven D. Bull,†,‡ Juyoung Yoon,∇ Hwan Myung Kim,*,§ Tony D. James,*,†,‡
and Simon E. Lewis*,†,‡
†Department of Chemistry, University of Bath, Bath BA2 7AY, United Kingdom
‡Center for Sustainable Circular Technologies, University of Bath, Bath BA2 7AY, United Kingdom
§Department of Energy Systems Research, Ajou University, Suwon 443-749, South Korea
∥Materials and Chemical Characterization (MC2), University of Bath, Bath BA2 7AY, United Kingdom
⊥Department of Chemical Engineering, University of Bath, Bath BA2 7AY, United Kingdom
∇Department of Chemistry and Nano Science, Ewha Woman’s University, Seoul 120-750, South Korea
S
* Supporting Information
ABSTRACT: Two-photon fluorescence microscopy has
become an indispensable technique for cellular imaging.
Whereas most two-photon fluorescent probes rely on well-
known fluorophores, here we report a new fluorophore for
bioimaging, namely azulene. A chemodosimeter, comprising a
boronate ester receptor motif conjugated to an appropriately substituted azulene, is shown to be an effective two-photon
fluorescent probe for reactive oxygen species, showing good cell penetration, high selectivity for peroxynitrite, no cytotoxicity,
and excellent photostability.
reaction-based probes). Probes of this second type often
have high analyte selectivity and are applicable even to those
ROS/RNS that have very short lifetimes.5 We and others have
reported many fluorescent probes for imaging ROS/RNS,6
INTRODUCTION
■
Reactive oxygen species (ROS) and reactive nitrogen species
(RNS) are important mediators in many physiological and
pathological processes.1 The one-electron reduction of O2 in
vivo leads to the formation of O2•− (superoxide), which in turn
undergoes disproportionation catalyzed by superoxide dis-
mutase to give O2 and H2O2 (hydrogen peroxide). Another
fate of O2•− is to react with endogenously produced NO (nitric
oxide) to form ONOO− (peroxynitrite) via a nonenzymatic
process. Ordinarily, the flux of H2O2 is tightly regulated;
aberrant H2O2 production or overexposure is implicated in the
pathogenesis of many diseases such as cancer and neuro-
degenerative conditions.2 Similarly, while ONOO− has roles in
signal transduction, its strongly oxidizing and nitrating
properties mean it can react in an uncontrolled manner with
various biomolecules.3 Elevated levels of ONOO− have been
linked to cardiovascular, neurodegenerative, and inflammatory
diseases as well as cancer.4 In view of this, there is a significant
need for tools and techniques to elucidate the roles of
ONOO−, H2O2, and other ROS/RNS in biological systems.
Of the various techniques that have been employed for the
study of ROS/RNS, the use of fluorescent probes has several
advantages. They can enable realtime fluorescence imaging of
ROS/RNS with high sensitivity, are nondestructive, and are
usually operationally straightforward. Such fluorescent probes
may be categorized as chemosensors (those which reversibly
bind their analytes) and chemodosimeters (irreversible
7
including H2O2 and ONOO−.8 In the field of fluorescence
imaging, the use of two-photon excitation fluorescence
microscopy (TPM)9 brings particular advantages. Specifically,
TPM allows higher spatial resolution, and the use of excitation
wavelengths in the near-infrared (NIR) region allows much
greater tissue penetration, since minimal absorption by
hemoglobin and water occurs in the NIR window.10
Furthermore, excitation by lower energy photons means
photobleaching and damage to the sample are minimized,
and the autofluorescence of intrinsic fluorophores is avoided.11
The design and development of small-molecule based
fluorescent probes that can be used in TPM has been an
area of much recent research activity.12 Among these, various
two-photon fluorescent probes for ROS/RNS have been
exploited, since one-photon excitation with short wavelength
could generate artificial ROS/RNS.13 For H2O2, two-photon
fluorescent probes have been reported that utilize the reaction
with boronic acids or their esters, with nitroxide radicals, or
Baeyer−Villiger type rearrangements.14 For ONOO−, two-
photon fluorescent probes have been reported that also utilize
Received: September 10, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.9b09813
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the reaction with boronic acids or their esters, as well as ones
which function by oxidation of electron-rich arenes, α-
ketocarbonyls, selenides, or by nucleophilic addition to
electron-poor alkenes and arenes.15 These reported probes
incorporate a variety of fluorophores, including well-known
ones that have been extensively used in one-photon
fluorescence, such as naphthalimides, rhodamines, coumarins,
etc. In contrast, in the work presented here, we report a
fluorescent probe for ROS/RNS that incorporates a previously
unexploited class of fluorophore, namely an azulene.
Scheme 1. Initial Concept for Azulene-Based Fluorescent
Probe for ROS/RNS
Azulene is a nonalternant bicyclic aromatic hydrocarbon,
isomeric with naphthalene, yet with appreciably different
properties.16 The S0 − S1 energy gap is smaller than in
naphthalene, leading to an absorption in the visible region, and
hence the blue color for which azulene is named. The
nonradiative S1 → S0 transition is extremely fast,17 whereas the
nonradiative S2 → S1 transition is much slower.18 Con-
sequently, S2 → S0 fluorescence is the principal mode of
emission,19 in violation of Kasha’s rule.20 The predominance of
emission from higher excited states has also been noted for
many azulene derivatives.21 These properties have led to
azulenes being used extensively as both colorimetric22 and
fluorescent23 probes for various analytes. Azulene derivatives
have also been exploited as fluorescence sensitizers24 and
quenchers25 as well as in photoswitches26 and molecular logic
gates.27 In the context of bioimaging, 18F-labeled azulene
derivatives have been used for PET imaging,28 but to our
knowledge, azulenes have not previously been used for
fluorescence imaging. In the specific context of ROS/RNS
detection, no reports employing azulene-containing probes
have appeared to date. The two-photon fluorescence of
azulene29 and some derivatives30 has been studied, yet it has
never been utilized in any sensing context (although it has
been exploited for fluorescence lithography31 and applications
in optical data storage have been suggested32).
having reduced ICT character overall. On the other hand, the
effect of the hydroxyl group in the Az-6-OH system will
reinforce the inherent polarization of the fluorophore, and
hence lead to increased ICT character overall and enhanced
fluorescence.
The simplest embodiment of the design strategy described
above would be to synthesize and evaluate 6-azulenyl
pinacolborane itself as a fluorescent probe. However, the
reaction product, 6-hydroxyazulene, is reportedly unstable,
which may be due in part to the fact that it exists in tautomeric
equilibrium with its keto form.33 We therefore refined our
design strategy, introducing esters at the azulene 1- and 3-
positions, as this substitution pattern is known to stabilize 6-
hydroxyazulenic compounds.34 Finally, we introduced an
electron-donating group (amine) at the 2-position. The final
probe structure 1 and its oxidation product 2 are depicted in
Scheme 2a. The purpose of the amine substituent is to enhance
the contribution of the resonance effect that conflicts with the
inherent polarity of azulene, as shown in Scheme 2b, with an
additional ammonium-boronate resonance contribution com-
pared to Scheme 1c. Furthermore, the ester groups will not
only stabilize the hydroxyazulene product 2 of reaction with
ROS/RNS, they will also enhance the contribution of the
resonance effect that reinforces the inherent polarity of
azulene, as shown in Scheme 2c.
Probe molecule 1 and its oxidation product 2 were modeled
using DFT as well as azulene itself. NBOs (natural bonding
orbitals) were computed, and selected molecular orbitals
(HOMO, LUMO, and LUMO+1) for each compound are
shown in Figure 1.
The probe molecule 1 may be accessed in three steps from
commercial materials (see ESI).35 We have termed this
substance “AzuFluor 483-Bpin” (to indicate the nature of the
receptor motif and the emission maximum of its turn-on
response). AzuFluor 1 was then reacted with H2O2 (THF, 24
h, 95%) and the novel product isolated and fully characterized
as the stable 6-hydroxyazulene 2. Crystals of 2 suitable for X-
ray diffraction were grown from THF/hexane and the solid
state structure of 2 is shown in Figure 2.
RESULTS AND DISCUSSION
■
Design Principles. Our design strategy for an azulene
chemodosimeter imaging probe is centered on the concept of
an inversion of internal charge transfer (ICT) directionality
upon reaction with the analyte. This exploits the inherent
dipole moment of azulene of 1.08 D, unusually high for a
hydrocarbon. Thus, the charge distribution in azulene is as
shown in Scheme 1a, with the smaller ring having the higher
electron density as a result of the contribution of the resonance
structure shown, in which both rings (tropylium and
cyclopentadienide) are discrete 6π aromatic systems. Our
approach involves the use of a pinacol boronate ester as a
receptor motif, whose reaction with ROS/RNS leads to the
desired reversal of the direction of ICT. In many cases,
boronate-based probes function by the carbon−boron bond
undergoing oxidation by the ROS/RNS to give a carbon−
oxygen bond, which in turn leads to the cleavage of a self-
immolative linker and a change in fluorescence response. In the
present case, we reasoned that a boronate group appended
directly to the azulene seven-membered ring could effect the
desired perturbation of the fluorophore upon reaction with
ROS/RNS (Scheme 1b). This approach exploits the fact that
the boronic ester is electron-withdrawing (Scheme 1c),
whereas the hydroxyl group is electron-donating by resonance
(Scheme 1d). Our strategy anticipates that the electronic
influence of the boronic ester will attenuate the inherent
polarization of the azulene, leading to the Az-6-Bpin system
Probe Characterization. The AzuFluor probe 1 itself
exhibits near-negligible fluorescence, whereas the oxidation
B
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Scheme 2. Final Sensor Design for Azulene-Based
Fluorescent Probe for ROS/RNS
Figure 2. Solid state structure of 2. Ellipsoids are represented at 50%
probability. H atoms are shown as spheres of arbitrary radius.
CCDC 1899490.
Figure 3. Comparison of one-photon fluorescence emission spectrum
of 1 with 2. Emission spectrum of 2 (500 nM) and 1 (500 nM) with
and without H2O2 (200 μM) in PBS buffer 52% H2O: MeOH, pH 8.2
at 25 °C. Fluorescence intensities were measured with λex = 350
(bandwidth: 20) nm on a BMG Labtech CLARIOstar plate reader.
observed in the absorption spectrum of 2; thus, the oxidation
product displays an appreciable Stokes shift of 148 nm.
Fluorescence emission of 2 was also examined in various
solvents (Figure S5), and we observed the solvatofluorochrom-
ism expected for an ICT fluorophore; the observed λem value
underwent a bathochromic shift from 480 to 491 nm with
increasing solvent polarity.
A pH titration showed that the fluorescence intensity is
greatest at pH 7−8 (Figure 4; see also Figures S6−S8), which
Figure 4. Effect of various pH on fluorescence intensity of 2 (500
nM) in 52% MeOH: 48% 0.1 M NaCl with λmax = 483 nm.
corresponds to the pH range at which a probe for cellular
imaging would need to function in most cases. Fluorescence
intensity decreases markedly at lower pH, and is also
attenuated above pH 8. An NMR study of 2 at different pH
values (Figures S9−S11) shows it to be stable upon prolonged
standing in solution across a range of pH values, albeit with a
downfield shift of the aryl resonances at low pH. Thus, we do
not believe the decrease in fluorescence at extremes of pH is
due to decomposition of 2. Rather, we ascribe these
phenomena to changes in the ionization state of 2. Thus, at
Figure 1. Selected molecular orbitals for azulene (left), 1 (center) and
2 (right). DFT calculations used the BP86 functional and 6-31G**
basis set; see ESI for full details.
product 2 exhibits fluorescence emission at λem = 483 nm,
upon excitation at 350 nm, in a mixed aqueous buffer/
methanol system, as shown in Figure 3. Fluorescence excitation
spectra were also acquired (Figures S1−S3), as well as UV−vis
absorption spectra (Figure S4). A value of λmax = 335 nm was
C
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low pH the amino group in 2 would be protonated; in [2+H]+
the ICT interaction between the amine and the esters would
be abolished. Conversely, at high pH, deprotonation of the aryl
hydroxyl group to give [2-H]− would also affect the
fluorescence response. The computed pKa values for the
amine and hydroxyl groups in 2 are 3.60 and 10.95,
respectively (Figures S12−S13), which supports the above
explanation.
weight, such as dansyl hydrazine, Lucifer yellow, Cascade blue,
or Indo-1 (see SI, Table S3 for a tabulated comparison). The
fluorescence quantum yield of 2 was determined to be 0.010
(Figures S34−S36). Thus, at 810 nm the two-photon
absorption cross-section of 2 (Φδ÷Φ) is 320 GM.
Probe Evaluation in Cell Studies. In vitro studies were
performed to validate the ability of 1 in aqueous solution to
detect endogenous and exogenous H2O2 and ONOO− by
TPM. MTT assays confirmed that 1 is not cytotoxic under the
identified imaging conditions (Figure S28). Two-photon
excited fluorescence intensities were determined by staining
HeLa cells with 1 at a concentration of 5 μM (by adding 5 μL
of a 1 mM DMSO stock solution of 1 per 1 mL of total volume
of medium) in the presence and then absence of ONOO−
(100 μM), to find the optimal two-photon excitation
It is well established in the literature14,15 that boronate
receptors respond to both ONOO− and H2O2, although the
greater nucleophilicity of ONOO− leads to a reaction rate
∼106 greater than for H2O2.36 We therefore evaluated 1 as a
probe for both potential analytes, with the expectation of a
faster and more sensitive response to peroxynitrite. Fluo-
rescence titration experiments (Figure S14) with 1 and H2O2
established the limit of detection to be 1.72 μM. A time drive
experiment (Figure S15) showed the fluorescence response of
1 (500 nM) to H2O2 to be slow, with fluorescence intensity
continuing to increase after 30 min even at [H2O2] = 500 μM.
In contrast, 1 was much more sensitive to ONOO− as
expected, with fluorescence titration experiments (Figure S16)
establishing the limit of detection to be 21.7 nM. The
selectivity of 1 for various ROS was then examined, and 1 was
found to be highly selective for ONOO− and H2O2 over
HOCl, HO•, O2•−, ROO•, and 1O2 (Figure S17). Furthermore,
with equimolar concentrations of all ROS, 1 exhibited
pronounced selectivity for ONOO− over H2O2 at both 5 and
30 min time points (Figures 5, S18−S19). The UV−vis
spectroscopic response of 1 to ONOO− and H2O2 was also
studied (Figures S20−S25; Tables S1−S2).
wavelength (Figure S27a). This was determined to be λex
=
800 nm, at which wavelength the mean fluorescence intensity
was nearly 4-fold higher after treatment with ONOO− (Figure
S27b). Probe 1 showed high photostability, without any decay
of fluorescence intensity even after irradiation for 1 h at 2 s
intervals, both with and without pretreatment with ONOO−
(Figure S29). Co-localization experiments were performed
with 1 and representative organelle markers (ER, mitochon-
dria, and lysosomes). The Pearson correlation coefficients with
the markers for the ER, mitochondria and lysosomes are 0.79,
0.70 and 0.41, respectively (Figure S30). This indicates that 1
is distributed through the cytosol, including in these organelles,
rather than any specific localization.
With ideal imaging conditions established, the ability of 1 to
detect ROS in living cells was then demonstrated. Thus, the
mean fluorescence intensity was measured for TPM images of
1 in RAW 264.7 macrophages pretreated with H2O2 (2 mM)
and ONOO− (100 μM). The fluorescence intensity of cells
pretreated with ROS was shown to increase nearly 3-fold
(Figure 6b-c), compared to negative control (Figure 6a, cells
stained with probe 1 only). Next, RAW 264.7 macrophages
were pretreated with endogenous ROS-inducing reagents to
observe whether probe 1 could respond to the endogenous
ROS of macrophages. Phorbol myristate acetate (PMA),38 and
lipopolysaccharide/gamma interferon (LPS/IFN-γ)39 are
known to stimulate macrophages to release ROS such as
NO•, O2•−, H2O2, and ONOO−, whereas 3-morpholinosydno-
nimine (SIN-1)40 is employed as a positive control (exogenous
source of ONOO−). Each cell was individually incubated with
either PMA (1 μg mL−1), LPS (1 μg mL−1) + IFN-γ (50 ng
mL−1), or SIN-1 (50 μM) and then stained with probe 1. As a
result, 1 showed strong fluorescence intensity, increasing by
approximately 2-fold, as compared with the control group
(Figure 6d,e,g). ROS-induced fluorescence intensity did not
rise above basal levels upon cotreatment with ebselen (50
μM), a commonly used scavenger of superoxide (the precursor
of ONOO− and H2O2;41 Figure 6f,h). Overall, the obtained
outcomes showed that 1 can directly detect the presence of
H2O2 and ONOO− in biological systems.
Figure 5. Selectivity data for 1 (500 nM) in the presence of ONOO−,
•−
H2O2, HOCl, OH•, O2
,
1O2, and ROO• (each 500 nM). Data
acquired 5 min after mixing, in PBS buffer 52% H2O: MeOH, pH 8.2
at 25 °C, at λmax = 483 nm. Fluorescence intensities were measured
with λex = 350 (bandwidth 20) nm, on a BMG Labtech CLARIOstar
plate reader.
Two-photon action spectra (Φδ, where Φ is the fluorescence
quantum yield and δ is the two-photon absorption cross-
section) of probe 1 in the presence and absence of H2O2 or
ONOO− were investigated (Figure S26) to determine the
maximum optical brightness. Upon addition of ROS, the
spectroscopic maximum (Φδmax) was red-shifted from 700 to
810 nm, along with an increase of the Φδ value (at 810 nm)
from 1.2 to 3.2 GM. A similar result was observed with two-
photon microscopy imaging (Figure S27). The measured
maximum two-photon optical brightness of Φδmax = 3.2 GM
compares favorably with the reported values37 for other
commonly used fluorophores of similar or greater molecular
Finally, we investigated the ability of probe 1 to visualize
ONOO− and H2O2 in living tissues. A fresh slice of rat
hippocampus was stained with 1 at a concentration of 50 μM
(by adding 5 μL of a 10 mM DMSO stock solution of 1 per 1
mL total volume) for 1 h at 37 °C. The TPM images obtained
in the CA1 regions displayed only weak signals, as we observed
in cultured cells (Figure 7a,e). However, bright fluorescence
was observed upon pretreatment with PMA (10 μg mL−1) and
SIN-1 (200 μM), increased by approximately 2 times
D
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has been shown to possess high selectivity for peroxynitrite and
hydrogen peroxide over other reactive oxygen species.
Furthermore, 1 is cell-permeable, photostable, and non-
cytotoxic in vitro on the time scale of the two-photon
fluorescence microscopy experiments. In view of the
susceptibility of the azulene fluorophore to perturbation by
attached substituents, the wide array of known receptor motifs
that could be attached, and the availability of synthetic
methodology to attach them, we anticipate that azulene
fluorophores based on the design principles we have described
here will find many further applications in two-photon
fluorescence imaging.
EXPERIMENTAL SECTION
■
Preparation of Diethyl 2-amino-6-hydroxyazulene-1,3-di-
carboxylate (2). Diethyl 2-amino-6-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)azulene-1,3-dicarboxylate 135 (100 mg, 0.24 mmol, 1.00
equiv) was dissolved in THF (2.5 mL) to which 30% aq. H2O2
solution (50 μL, 0.48 mmol, 2.0 equiv) was added. The bright-orange
solution was left to stir in air for 24 h, affording a pale-yellow solution,
after which water (10 mL) was added. The mixture was extracted with
CH2Cl2 (2 × 20 mL), and the combined organic extracts were dried
over MgSO4 and then filtered. The filtrate was concentrated under
reduced pressure, then the crude material was purified via silica
column chromatography, eluting with EtOAc/Petrol (1:4 → 2:3) to
afford 2 as an orange powder (70 mg, 95%). Rf = 0.16 (EtOAc/Petrol
1:4); m. pt. 117 °C (dec.); δH (500 MHz, (CD3)2SO) 11.02 (1H, s,
OH), 8.99 (2H, dt, J 11.6, 1.4 Hz, H4, H8), 7.41 (2H, s, NH2), 7.28
(2H, dt, J 11.6, 1.4 Hz, H5, H7), 4.35 (4H, q, J 7.1 Hz, CH2), 1.37
(6H, t, J 7.1 Hz, CH3) ppm; δC (125 MHz, (CD3)2SO) 165.6, 163.8,
158.6, 139.9, 131.9, 121.4, 98.6, 59.2, 14.5 ppm; IR νmax (film) 3499,
3353, 3243, 2959, 2925, 1660, 1636, 1580, 1536, 1508, 1491, 1416,
1383, 1354, 1327, 1262, 1197, 1166, 1100, 1025, 955, 908, 848, 803,
790, 745, 718, 692 cm−1; HRMS (ESI+) m/z calcd for
(C16H17NO5+Na)+, 326.0999; found 326.0996.
Figure 6. TPM images of RAW 264.7 macrophages stained with 1 (5
μM, 30 min). (a) Control image. (b−h) Cells were pretreated with
(b) exogenous added H2O2 (2 mM, 30 min), (c) exogenous added
ONOO− (100 μM, 30 min), (d) PMA (1 μg mL−1, 30 min), (e) LPS
(1 μg mL−1, 4 h) and IFN-γ (50 ng mL−1, 1 h), (f) LPS, IFN-γ and
ebselen (50 μM, 30 min), (g) SIN-1 (50 μM, 30 min), and (h) SIN-1
and ebselen. (i) Average fluorescence intensity in the corresponding
TPM images. Images were obtained using 800 nm as the excitation
wavelength and 400−600 nm emission windows. Scale bars = 20 μm.
Asterisks represent statistical significance (***p < 0.001) and n is the
number of counted cells.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.9b09813.
Synthetic procedures for 1; procedures for preparation
of ROS in Figures 4, 6, and 7; computational data for 1
and 2; spectroscopic and crystallographic data for 2;
procedures for two-photon fluorescence imaging;
supplemental figures and tables (PDF)
Figure 7. TPM images of rat hippocampal slices stained with 1 (50
μM, 1 h). (a) Control image. (b−d) Tissues were pretreated with (b)
PMA (10 μg mL−1, 30 min), (c) SIN-1 (200 μM, 30 min), and (d)
SIN-1 and ebselen (each 200 μM, 30 min). (e) Bright-field image of
the CA1 and CA3 regions of rat hippocampal slices. (f) Average
fluorescence intensity in the corresponding TPM images. Images were
obtained in the CA1 layer using 800 nm as the excitation wavelength
and 400−600 nm emission windows. Scale bars = 25 (a−d) and 250
μm (e). Asterisks represent statistical significance (***p < 0.001) and
n is the number of ROI from three samples for each imaging
condition.
AUTHOR INFORMATION
■
Corresponding Authors
*kimhm@ajou.ac.kr
*t.d.james@bath.ac.uk
*s.e.lewis@bath.ac.uk
ORCID
Maria Weber: 0000-0002-7492-1035
Carlos M. Lopez-Alled: 0000-0002-8117-4971
Claire L. McMullin: 0000-0002-4924-2890
compared to negative control (Figure 7b,c) whereas fluo-
rescence did not increase above basal levels upon cotreatment
with SIN-1 and ebselen (200 μM) (Figure 7d,f). This confirms
that our probe can directly detect endogenous ROS in living
tissues with TPM.
̈
Gabriele Kociok-Kohn: 0000-0002-7186-1399
Jannis Wenk: 0000-0001-9182-0407
Steven D. Bull: 0000-0001-8244-5123
Juyoung Yoon: 0000-0002-1728-3970
Hwan Myung Kim: 0000-0002-4112-9009
Tony D. James: 0000-0002-4095-2191
Simon E. Lewis: 0000-0003-4555-4907
CONCLUSIONS
■
We have synthesized and evaluated the first two-photon
fluorescent bioimaging probe containing an azulene fluoro-
phore. The rationally designed probe, AzuFluor 483-Bpin (1),
E
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(5) Chan, J.; Dodani, S. C.; Chang, C. J. Reaction-based small-
molecule fluorescent probes for chemoselective bioimaging. Nat.
Chem. 2012, 4, 973−984.
Author Contributions
#L.C.M., M.W., S.J.P., and W.T.K. contributed equally.
Notes
(6) For reviews, see: (a) Kwon, N.; Hu, Y.; Yoon, J. Fluorescent
chemosensors for various analytes including reactive oxygen species,
biothiol, metal ions, and toxic gases. ACS Omega 2018, 3, 13731−
13751. (b) Chen, X.; Wang, F.; Hyun, J.; Wei, Y. T.; Qiang, J.; Ren,
X.; Shin, I.; Yoon, J. Recent progress in the development of
fluorescent, luminescent and colorimetric probes for detection of
reactive oxygen and nitrogen species. Chem. Soc. Rev. 2016, 45, 2976−
3016. (c) Chen, X.; Tian, X.; Shin, I.; Yoon, J. Fluorescent and
luminescent probes for detection of reactive oxygen and nitrogen
species. Chem. Soc. Rev. 2011, 40, 4783−4804. (d) Dickinson, B. C.;
Srikun, D.; Chang, C. J. Mitochondrial-targeted fluorescent probes for
reactive oxygen species. Curr. Opin. Chem. Biol. 2010, 14, 50−56.
(e) Nagano, T. Bioimaging of nitric oxide. Chem. Rev. 2002, 102,
1235−1269.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for Ph.D. funding to C.M.L.-A. from the EU
Horizon 2020 research and innovation program under Grant
Agreement H2020-MSCA-CO-FUND, No. 665992. We thank
the Center for Doctoral Training in Sustainable Chemical
Technologies for Ph.D. funding to M.W. under EPSRC Grant
EP/L016354/1. We also thank EPSRC for DTP Ph.D. funding
to L.C.M. H.M.K. acknowledges a grant from the National
Leading Research Lab Program of the National Research
Foundation of Korea (NRF), funded by the Korean govern-
ment (MSIP; No. 2016R1E1A1A02920873). In addition,
T.D.J. wishes to thank the Royal Society for a Wolfson
Research Merit Award and for funding to F.P.-C. under Grant
CHG\R1\170010. This work was supported by a seed corn
grant from the CR@B (Cancer Research at Bath) network.
The British-Spanish Society and Plastic Energy are thanked for
a 2017 Scholarship to C. M. L.-A. This research made use of
the Balena High Performance Computing (HPC) Service at
the University of Bath. NMR, X-ray crystallography, and MS
facilities were provided through the Materials and Chemical
Characterisation Facility (MC2) at the University of Bath. We
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