Color-Tunable Light-up Bioorthogonal Probes for In Vivo Two-Photon Fluorescence Imaging

Article abstract of DOI:10.1002/chem.201905183

Light-up bioorthogonal probes have attracted increasing attention recently due to their capability to directly image diverse biomolecules in living cells without washing steps. The development of bioorthogonal probes with excellent fluorescent properties

Full text of DOI:10.1002/chem.201905183

A Journal of
Accepted Article
Title: Color-Tunable Light-up Bioorthogonal Probes for in-vivo Two-
Photon Fluorescence Imaging
Authors: Yandong Dou, Yajun Wang, Yukun Duan, Bin Liu, Qinglian
Hu, Wei Shen, Hongyan Sun, and Qing Zhu
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Eur. J. 10.1002/chem.201905183
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Color-Tunable Light-up Bioorthogonal Probes for in-vivo Two-
Photon Fluorescence Imaging
Yandong Dou,+[a] Yajun Wang,+[a] Yukun Duan,[b] Bin Liu,*[b] Qinglian Hu,[a] Wei Shen, *[c] Hongyan
Sun*[d] and Qing Zhu*[a]
Abstract: Light-up bioorthogonal probes have attracted increasing
attention recently due to their capability of direct imaging of di-verse
biomolecules in living cells without washing steps. The development
of bioorthogonal probes with excellent fluorescent properties suitable
for in-vivo imaging, such as long excitation/emission wavelength, high
fluorescence turn-on ratio, and deep penetration has been rarely
reported. Herein, we design and synthesize a series of azide-based
light-up bioorthogonal probes with tunable colors based on a weak
fluorescent scaffold of 8-aminoquinoline (AQ). The azido quinoline
derivatives are able to induce large fluorescence enhancement (up to
1352-fold) after click reaction with alkynes. In addition, the probes
could be engineered to display excellent two-photon property (δ = 542
GM at 780 nm) after further introducing different styryl groups into the
AQ scaffold. Subsequent detailed bioimaging experiments
demonstrate that these probes are versatile, which have been
emerged, which attracts intense attention from scientific
community.4-6 These probes are usually consisted of two parts, a
fluorophore and a bioorthogonal group which also acts as a
quencher. Upon reacting with their bioorthogonal counterparts,
large fluorescence turn-on is induced which allows for real-time
imaging without washing steps. So far, bioorthogonal labeling has
been employed for in vitro imaging of various molecules, e.g.
small molecule drugs, natural products and proteins.7-9
During the past few years, considerable efforts have been
made on extracellular or intracellular turn-on bioimaging with
azide-/tetrazine fluorogenic probes in living cells.10-12 These
probes could undergo bioorthogonal reaction via copper
catalyzed (CuAAc) or “Diels–Alder” cycloaddition, respectively.13-
16
So far, azide-based light-up bioorthogonal probes have
attracted considerable attention due to their small size and high
successfully used for live cell/zebrafish imaging without washing steps. quenching efficiency.17 For example, Wong18 designed an azido-
Further in vivo two-photon imaging experiments demonstrated that
these newly developed light-up biorthogonal probe outperformed the
conventional fluorophores, e.g. high signal-to-noise ratio and deep
tissue penetration. The design strategy reported in this study
BODIPY based probe AzBOCEt (Az10), which can induce up to
52-fold fluorescence enhancement after click reaction (Fig. 1a).
Based on the principle of photo-induced electron transfer (PeT),
Bertozzi elegantly designed a panel of fluorogenic azide probes
that can be activated via click chemistry (Fig. 1a).19 Besides high
fluorescence turn-on efficiency, attempts have also been made to
develop fluorescent derivatives covering a wide range of emission
wavelengths based on monochromophoric design.20 Despite
many successful examples for in-vitro studying, in-vivo
experiments are less explored as they require long wavelength
excitation and large turn-on ratios to differentiate the signal from
auto-fluorescence of bio-substrates.
represents
a useful approach to realize a diversity of high-
performance biorthogonal light up probes for in vivo studying.
Introduction
Fluorescence imaging has been a powerful technique for
investigating biological molecules in living organisms with superb
spatiotemporal resolution.1-2 Traditional fluorophores for
fluorescent imaging usually display strong back-ground
fluorescence, which requires extensive washing steps.3 To
address this issue, a class of light-up bioorthogonal probes has
[a]
Y. Dou, Dr. Y. Wang, Dr. Q. Hu, Prof. Q. Zhu
College of Biotechnology and Bioengineering, Zhejiang University of
Technology, Hangzhou 310014, China E-mail: zhuq@zjut.edu.cn
These authors contributed equally to this work
Y. Duan, Prof. Dr B. Liu
[+]
[b]
Department of Chemical and Biomolecular Engineering, National
University of Singapore, 4 Science Drive 4 117585, Singapore
E-mail: cheliub@nus.edu.sg
[c]
[d]
W. Shen
Department of General Surgery, Jinhua Municipal Central Hospital,
Jinhua 321000, China，E-mail: zhuqshenw@hotmail.com
Prof. H. Sun
Fig. 1. (a) Classical light-up fluorophores based on PeT mechanism; (b) Our
strategy for designing light-up bioorthogonal probes based on ICT mechanism.
Department of Chemistry, City University of Hong Kong, 83 Tat
Chee Avenue, Kowloon, Hong Kong, China
E-mail: hongysun@cityu.edu.hk
Herein, we report a new class of color-tunable two-photon
light-up bioorthogonal probes based on a fluorescent scaffold of
8-aminoquinoline (AQ) (Fig. 1b). Specifically, we achieved site-
specific introduction of azido group at the C-5 position of AQ via
C-H/I activation. The azido-AQ derivatives showed low
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fluorescence background due to intramolecular charge transfer
(ICT) effect, whereas they could generate high fluorescence after
click reaction (~700-fold turn-on). The emission wavelength of the
clicked triazole fluorophores can be fine-tuned from 430 to 610
nm. Remarkably, higher quantum yields, larger fluorescence
enhancement (up to 1352-fold) and longer emission wavelength
(610 nm) could be achieved by introducing an aziridine ring and a
styryl group to AQ. The values of these probes have been further
demonstrated in two-photon imaging of living cells, and
zebrafishes without washing steps. The in vivo imaging of mice
demonstrated that these newly developed light-up biorthogonal
probe displayed much higher signal-to-noise ratio and deeper
penetration than the conventional fluorophores.
as catalyst and NaN3 as the azido source to give DQ, which was
hydrolyzed in potassium hydroxide to offer DA in 75% yield.
Subsequently, the click reactions between DA and different
alkynes, including aromatic and aliphatic substituted alkynes,
were carried out. The corresponding triazoles DZ-a~h were
obtained in more than 90% yields.
Results and Discussion
8-Aminoquinoline was chosen in our study due to its simple and
easily-modifiable structure. The introduction of azide group could
diminish the fluorescence background due to ICT quenching
effect.21-22 On the other hand, trans-formation of the azide to
triazole group will not only sup-press ICT effect, but also enlarge
the conjugation system of the fluorophore to yield tunable
emission.23 We envision that azido substituted 8-aminoquine
derivatives will serve as an excellent building block to construct a
new class of highly fluorogenic biorthogonal probes.
Fig. 2. Fluorescence enhancements of DQ, DAA, DAAS-1 and DAAS-2 after
copper-catalyzed click reactions. Reaction condition: CuSO4·5H2O (20 μM)
and sodium ascorbate (40 μM) were added to a mixture of ethynylbenzene (20
μM) and azido-fluorophore (10 μM), and fluorescence intensity was monitored
after 30 min at 25 °C in PBS solution (20 mM, pH 7.4, 10% DMSO).
With these compounds in hand, we firstly investigated the
fluorescent properties of DA and its triazole derivatives DZ-a~DZ-
h. Although DA has an absorption at 350 nm, it showed weak
fluorescence due to the quenching effect of the electron-rich azide
group via ICT mechanism (Fig. S1–S2).26 The triazole derivatives
displayed absorptions at around 365 nm with strong fluorescence
in 480-524 nm range (Fig. S2–S3) showing the turn-on ratios
ranging from 630 to 730-fold in H2O after click reaction (Table S1).
However, when the triazole is bearing an electron withdrawing
group (DZ-g), its fluorescent intensity could increase along with a
distinct blue shift of the emission. Interestingly, the emission of
DZ-h displays red-shift at 524 nm in DMSO and 500 nm in pure
water. This phenomenon could be attributed to that the electron-
donating group improves the energy of HOMO and LOMO level,
while the HOMO is improved more significantly, resulting in the
red-shift emission (Figure S6). Moreover, no significant influence
of the substituent at the triazole on the excitation wavelengths was
observed. These results demonstrate that our first generation of
light-up bio-orthogonal probe is capable of fluorescence turn on
after cycloaddition reaction.
To red-shift the absorption and emission of AQ based probes,
we introduced styrene at C-2, C-3, and C-6 of AQ to
systematically study the structure-property relationship. Firstly, 2-
styrylquinolin-8-amine was iodinated, followed by the substitution
with NaN3 in the presence of CuI, to afford DAS-1 (Scheme 1b,
Scheme S2). Following a similar route, C-3/-6 vinyl substituted
DAS-2 and DAS-3 were successfully obtained in good yields
(Scheme 1a, Scheme S2~S4). The click reaction with
ethynylbenzene gave the corresponding triazoles DZS-1~3. To
our delight that the introduction of vinyl group could red-shift the
absorption to around 380 nm and extend the emission wavelength
effectively from 486 nm to 540 nm (Figure S7~S8). It is noteworthy
that, while DZS-1~3 exhibit similar emission wavelength around
540 nm, the C-2 substitution gave the highest fluorescence
quantum yield (Φ= 0.374) and turn-on ratio (238-fold) (Table S2).
Therefore, C-2 substitution was selected for subsequent studies.
Scheme 1. The general synthetic pathway of the bioorthogonal probes and the
structures after click reaction. a) Synthetic pathway of DA bioorthogonal probes
and its triazole derivatives (DZ-a-h); b) Synthetic pathway of the second
generation of probes with long emission wavelength: styryl substituted 5-azido-
8-aminesquinolines (DAS) and their triazole derivatives (DZS-1-3); c) Synthetic
pathway for two-photon light-up probes DAAS by introducing an aziridine ring
and a styryl group to AQ.
To verify our design, 5-azido-8-aminoquinoline (DA) was firstly
synthesized via C-H azidation of AQ under mild condition
(Scheme 1a).24-25 Briefly, AQ was firstly treated with benzoyl
chloride in the presence of triethyl amine to afford N-benzoyl 8-
aminoquinoline. Then azidation was carried out utilizing Cu(OAc)2
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As the intramolecular hydrogen bond between the amino group
(C-8 position) and nitro atom (C-1 position) of 8-aminoquinoline is
highly prone to decrease the fluorescent signal because of the
excited-state proton transfer (ESPT) effect.27 To reduce the
impact of environmental factor and further enhance the
fluorescence quantum yield of the bio-orthogonal product,28 we
attempted to replace the amino group at C-8 position of the
quinoline with a dialkylated amino group (Scheme 1c, Scheme
S5), and to our delight, a four-membered azetidine displays the
higher quantum yield and its emission wavelength red-shits 10 nm
(Figure S9). Furthermore, various vinyl containing building blocks,
such as styryl groups with electron withdrawing (DZAS-2) and
donating groups (DZAS-3, 4), 2-vinyl indole and 2-(3-
vinylcyclohex-2-en-1-ylidene) malononitrile are introduced to AQ
scaffold to further red-shift the emission wavelength. To obtain 2-
styryl-8-(1-azetidinyl)-quinolines (DAAS), 5-iodo-2-methylquinolin-
8-amine (IntA-1) and 1,3-dibromopropane were incubated
together in a microwave oven to yield the four-membered
azetidine substituted qunioline IntA-2. Next, IntA-2 was
condensed with different aldehydes to give 2-styryl substituted
compounds IntA-3, which were subjected to Cu(I)-catalyzed C-I
azidation to afford various molecules DAA and DAAS-1~6
(Scheme 1c, Scheme S6). Subsequently, their triazoles DZA and
DZAS-1~6 derivatives could be obtained readily via click reaction
with ethynylbenzene. To our delight, the emission wave-length is
red-shifted to 610 nm when 4-methylbenzonitrile was conjugated
with quinoline (DZAS-2) (Table S3).
could be further easily modified to offer a series of new
fluorophores covering different colors with excellent fluorescence
turn-on properties.
It was also found that the Cu-catalyzed alkyne-azide
cycloaddition could achieve 85% conversion within 30 min (Fig.
S10, S11), which was fast enough for most applications.29 A fast
reaction kinetic (k2) of DAA was obtained as 11.5 M−1 s−1 in PBS
by treatment with ethynylbenzene (Fig. S12). These results
indicate that our bioorthogonal probes can generate light-up
fluorescent signal effectively for biomedical application.
To understand the low background signal and the fluorescence
turn-on, we further carried out time-dependent density functional
theory (TD-DFT) calculations (CAM-B3LYP/6-31G*).30 As shown
in Fig. 3, the main orbitals contributing to the S0-S1 transition of
azide-based probes is an π→π* type transition. Based on the
lowest first singlet excited state of azide-based probes, the
nonbonding orbitals largely contribute to the π→π* transition.
Meanwhile, the optically inactive π→π* transition at the lowest
lying state disappears after click reaction with extended aromatic
systems, but the n→π* transition becomes the most important
transition between the S0 and S1 states in all triazole-structures
(Table S4, Table S5).31 Moreover, f values for the S0-S1 transition
in triazole-structures (from 0.14 to 0.15) are significantly higher
than that of the S0−S1 transition in azide structures (from 0.0095
to 0.015). The high f values of triazole-structures indicate that the
excited fluorophores could lose radiative energy after click
reaction. Taken together, computational calculation demonstrates
that azide moiety in the molecule causes the optically inactive
π→π* transition and low f value of the major S0−S1 transition, and
thus result in quenching the fluorescence of the chromophores.
Fig. 3. Molecular orbital distribution vertical transition energy and oscillator
strength values (f) .a) DQ, DA, DAS-1 from left to right by TD-DFT calculation
(CAM-B3LYP/6-31G*) of the corresponding first excited-state optimized
structures. H and L stand for HOMO and LUMO, respectively. b) after click
reaction obtained by TD-DFT calculation (CAM-B3LYP/6-31G*) of the
corresponding first excited-state optimized structures. H and L stand for HOMO
and LUMO, respectively.
Fig. 4. No-wash living cell two-photon fluorescence imaging of
lysosome/mitochondria-targeting molecules. a, b, c, d): Cells incubated with
DAAS-1 (5 μM); e, f, g, h): Cells incubated with DAAS-1 (5 μM) THPTA (10 μM),
CuSO4·5H2O (10.0 μM), sodium ascorbate (20 μM) N, N-dimethylprop-2-yn-1-
amine (DPA)(10 μM) and DND-99 (100 nM) as the lysosomes tracker; I, j, k, l)
DAAS-1 (5.0 μM) THPTA(10 μM), CuSO4·5H2O (10 μM), sodium ascorbate (20
μM), triphenyl phosphine-alkyene(TPA) (10 μM) and Mito-Red-FM (500 nM) as
the mitochondria tracker; b ,c, f, j) Ex = 780 nm, Em = 520 nm-580 nm; g) Ex =
1040 nm, Em = 560-600 nm; k) Ex = 1040 nm, Em = 620-650 nm); a, e, i) Bright
light; d, h, l) Overlay. Scale bar: 20 μm.
Next, we studied the fluorescence turn-on response of the
probes before and after click reactions. As shown in Table S1-S3,
all of these compounds display excellent turn-on ratios.
Specifically, DQ, DAA, DAAS-1, DAAS-2 were treated with
ethynylbenzene, CuSO4·5H2O and sodium ascorbate for 30 min
Compared to one-photon imaging, two-photon (TP) imaging
possesses distinct advantages, including increased penetration
depth and reduced specimen photodamage due to its longer
excitation wavelength. Specifically, the two-photon increased
to generate the corresponding fluorophores of DZ-B, DZA, DZAS
-
1, DZAS-2. Their emission wavelengths cover from blue to red
(430 to 610 nm), and the fluorescence turn-on ratios were
penetration depth and reduced specimen photodamage due to its
33
longer excitation wavelength.32,
As DZAS-1~6 possess the
determined as 216 (DQ to DZ-B), 1352 (DAA to DZA), 738 (DAAS
-
typical D-π-A structure of two-photon probes, the two-photon
absorption cross-sections (δ) of DZAS were determined by two-
1 to DZAS-1), and 352 (DAAS-2 to DZAS-1-2) folds, respectively (Fig.
2). Therefore, those probes constructed from 8-aminequinoline
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photon induced fluorescence measurement technique (Fig. S13).
As shown in Table S3 and Figure S22, DZAS-1~6 display δ as high
as 542 GM, and high photostability, implying that the DZAS could
serve as two-photon fluorescent probes for bioimaging.
the tracker dyes, whose overlap coefficient is 0.921 (for lysosome)
and 0.902 (for mitochondria) respectively. These experiments
demonstrated that these bioorthogonal light-up probes could
distribute and turn on fluorescence in at specific location in living
cells.
To evaluate their performance in cell imaging experiments, DQ,
DZA, DAAS-1 and DAAS-2 were chosen as they display different
colors. MTT assays were firstly performed and more than 85% of
A549 cells survived after incubation with the probes (5.0 μM) for
24 h, indicating low cytotoxicity of the probes (Fig. S14~S17). The
further time-dependent imaging experiments indicated that the
cell could show the obviously signal after 30 min incubation
(Figure S23). Subsequently, A549 cells were incubated with
ethynylbenzene and treated with DQ, DZA, DAAS-1 and DAAS-2
and CuSO4·5H2O, sodium ascorbate, THPTA for 30 min. The
turn-on fluorescence of various colors is clearly observed in living
cells (Fig. S24). These results indicate that our newly developed
light-up bio-orthogonal probes are suitable for living cell imaging
study with multiple emission channels. In addition, DAA was also
demonstrated to be capable of imaging cell surface glycans
successfully utilizing metabolic engineering approach (Fig. S25).
These experiments showed that light-up bioorthogonal probes are
capable of wash-free cell imaging study.
Encouraged by the above results, we further performed
zebrafish imaging experiments using DAAS-1. As shown in Fig.
S28, using two-photon excitation microscopy, a substantial two-
photon fluorescence signal could be detected in zebrafish treated
with N, N-dimethylprop-2-yn-1-amine, CuSO4·5H2O, sodium
ascorbate, THPTA and DAAS-1. In contrast, no two-photon
fluorescence signal was observed when the zebrafish was treated
solely with DAAS-1. These results indicated that the two-photon
signal is attributed to the successful cycloaddition of the alkynyl
group and DAAS-1.
Fig. 6. In situ two-photon imaging of mice with tumor xenograft. A: a)
biorthogonal light-up imaging with DAAS-1 at a depth of 400 μm; b) conventional
fluorescent imaging strategy with RB at a depth of 400 μm; B: Relative
fluorescent intensity at a depth of 400 μm of tumor xenograft and no tumor
xenograft. Mice was intravenously injected with DBCO-(CH2)4CONH(CH2)2NH-
Biotin (100 mM, 100 μL) followed by subsequent intravenous injection (0.5 h
later) of DAAS-1 (10 mM, 100 μL) in PBS.
In order to further expand the biological application scope of
the probe, we performed in-vivo imaging experiments with DAAS
-
1 in mice. Biotin unit, as an effective tumor-targeting ligand for
cancer imaging, has been successfully applied for tracking cancer
cells and targeted drug delivery.34-36 Consequently we selected
the DBCO-(CH2)4CONH(CH2)2NH-Biotin as the tumor-targeting
ligand for the subsequent biorthogonal imaging study of tumors
(Fig. S30). To avoid the usage of catalyst copper ion which may
be toxic to the mice, dibenzocyclooctyne was used for the copper-
Fig. 5. In situ two-photon imaging of mice with tumor xenograft. a) Mice
intravenous injected with DBCO-(CH2)4CONH(CH2)2NH-Biotin (100 mM, 100
μL)with subsequent intravenous injection (0.5 h later) of DAAS-1 (10 mM, 100
μL) in PBS. b) Two-photon imaging of mice with tumor xenograft after 2 h
incubation. Control I: without tumor xenografts; control II: without addition of
DBCO-conjugated biotin. c) 3D image of xenograft tumors in a living mouse.
Images were acquired using 780 nm TP excitation with Olympus FVMPE-RS.
TP fluorescence emission windows: 520−580 nm. Scale bar = 50 μm.
free click reaction.37 Firstly, tumor xenografts were generated by
39
standard protocol.38,
Then, the tumor-bearing mice were
intravenously injected with DBCO-(CH2)4CONH(CH2)2NH-Biotin
(100 mM, 100 μL), followed by injection of the DAAS-1 (10 mM,
100 μL, Fig. 5) after 0.5 h. The same experiments were carried
out with two additional control groups concurrently. The first
control group was the mice without tumor xenografts, and the
other control group utilized mouse tumor xenograft without
treatment of DBCO-conjugated biotin (Control I and II, Fig. 5).
After 2 h incubation, imaging of the tumor was carried out with
two-photon excitation microscopy. As shown in Fig. 5b, a strong
two-photon signal could be observed in the tumor tissue of the
mice. In contrast, there were no signal observed in the control
groups. Furthermore, the penetrating depth could reach deeper
than 500 µm, which demonstrates that our probes are superior to
all of the existing one-photon light-up probes (Figure S30- S31).
In addition, 3D image of xenograft tumors in a living mouse was
successfully constructed, which is difficult to acquire from one-
photon microscopy. These results demonstrated that our probes
Next, we examined whether our probes can be used for two-
photon in vivo imaging experiments. DAAS-1 was chosen in our
study due to its good two-photon properties and relatively long-
wavelength emission. N, N-dimethylprop-2-yn-1-amine and
triphenyl phosphine-alkyne were used as targeting groups for
lysosomes and mitochondria. Live A549 cells were firstly
incubated with N, N-dimethylprop-2-yn-1-amine (mitochondrion-
targeting group) and triphenyl phosphine-alkyne (lysosome-
targeting group) respectively (Fig. S26-S27). Subsequently,
DAAS-1 and click reaction cocktail were added, and after
incubating at 37 ℃ for 2 h, and live cells were imaged by two-
photon microscopy without washing steps. In addition, DND-99
and Mito-Red-FM were used as the tracker of lysosomes and
mitochondria for comparison purpose. As shown in Fig. 4, cells
treated with DAAS-1 after click reactions displayed enhanced
fluorescent signals and the fluorescent images overlay well with
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A novel class of light-up bioorthogonal probes with color
tunable from blue to orange was successfully developed.
Generally, azido derivatives of AQ can produce large
fluorescence after cycloaddition reaction. Remarkably, the
replacement of amine with four-membered azetidine ring in AQ
scaffold leads to significant fluorescence enhancement up to
1352-fold by effectively suppressing ICT effect. Interestingly, the
substitution of styryl group at C-2 position exhibits long emission
wavelength (up to 610 nm) and large two-photon absorption cross
section (δ up to 542 GM). Subsequent bioimaging experiments
demonstrate that these probes are versatile, which have been
successfully used for live cell imaging without washing steps, and
in-vivo two-photon imaging of live zebrafish and mice.
Remarkably, these light-up biorthogonal probes with two-photon
property displayed better resolution and higher signal to noise
ratio for in vivo tumor imaging than conventional fluorophores
without light-up property. It is also noteworthy that, the design
strategy reported in this study represents a unique approach to
realize a diversity of high-performance light-up biorthogonal
probes for bioimaging. We envison that the bioorthognal probes
developed in this study will provide useful tools for cancer
diagnosis or fluorescence-guided therapy.
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[a]
Yandong Dou, [a] Yajun Wang,
Yukun Duan,[b] Bin Liu,*[b] Qinglian Hu,[a]
Wei Shen,*[c] Hongyan Sun*[d] and Qing
Zhu*[a]
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Color-Tunable Light-up
Bioorthogonal Probes for in-vivo
Two-Photon Fluorescence Imaging
Text for Table of Contents
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