Enhancing the Anti-Solvatochromic Two-Photon Fluorescence for Cirrhosis Imaging by Forming a Hydrogen-Bond Network

Article abstract of DOI:10.1002/anie.201800293

Two-photon imaging is an emerging tool for biomedical research and clinical diagnostics. Electron donor–acceptor (D–A) type molecules are the most widely employed two-photon scaffolds. However, current D–A type fluorophores suffer from solvatochromic quenching in aqueous biological samples. To address this issue, we devised a novel class of D–A type green fluorescent protein (GFP) chromophore analogues that form a hydrogen-bond network in water to improve the two-photon efficiency. Our design results in two-photon chalcone (TPC) dyes with 0.80 quantum yield and large two-photon action cross section (210 GM) in water. This strategy to form hydrogen bonds can be generalized to design two-photon materials with anti-solvatochromic fluorescence. To demonstrate the improved in vivo imaging, we designed a sulfide probe based on TPC dyes and monitored endogenous H₂S generation and scavenging in the cirrhotic rat liver for the first time.

Full text of DOI:10.1002/anie.201800293

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Accepted Article
Title: Harvesting Hydrogen Bond Network: Enhance the Anti-
Solvatochromic Two-Photon Fluorescence for Cirrhosis Imaging
Authors: Tianbing Ren, Wang Xu, Qianling Zhang, Xingxing Zhang,
Siyu Wen, Haibo Yi, Lin Yuan, and Xiaobing Zhang
This manuscript has been accepted after peer review and appears as an
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using the Digital Object Identifier (DOI) given below. The VoR will be
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201800293
Angew. Chem. 10.1002/ange.201800293
Link to VoR: http://dx.doi.org/10.1002/anie.201800293
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10.1002/anie.201800293
Angewandte Chemie International Edition
COMMUNICATION
Harvesting Hydrogen Bond Network: Enhance the Anti-
Solvatochromic Two-Photon Fluorescence for Cirrhosis Imaging
Tian-Bing Ren, Wang Xu, Qian-Ling Zhang, Xing-Xing Zhang, Si-Yu Wen, Hai-Bo Yi, Lin Yuan* and
Xiao-Bing Zhang
Abstract: Two-photon imaging is an emerging tool for biomedical
research and clinical diagnostics. Electron donor–acceptor (D–A)
type molecules are the most widely employed two-photon scaffolds.
However, current D–A type fluorophores suffer from solvatochromic
quenching in aqueous biological samples. To address this issue, we
devised a novel class of D-A type GFP chromophore analogues that
form hydrogen bond network in water to improve the two-photon
efficiency. Our design results in TPC dyes with 0.80 quantum yield
and large two-photon cross section (210 GM) in water. This strategy
to harvest hydrogen bonds can be generalized to design two-photon
materials with anti-solvatochromic fluorescence. To demonstrate the
improved in vivo imaging, we designed a sulfide probe based on
TPC dyes and monitored endogenous H2S generation and
scavenging in the cirrhotic rat liver for the first time.
(ICT) for bright emission.[12] The fluorescence properties might
be jeopardized if the D-A group were overly truncated. Given all
the complexities, a thorough evaluation is necessary to design
new D-A type fluorophores.
Molecular imaging has revolutionized biomedical research and
clinical diagnostics.[1] Among the great diversity of imaging
techniques, two-photon fluorescence imaging stands out due to
its non-invasiveness and deep tissue penetration.[2] Particularly,
electron donor–acceptor (D–A) type molecules are the most
commonly adopted two-photon scaffolds due to their large
absorbance coefficients and redshifted emission.[3] However, in
protic polar solvents, D-A type molecules usually suffer from
fluorescence quenching with bathochromic shifts, known as
solvatochromism.[3b,4] Although this phenomenon was proven
useful in polarity sensing,[5] the poor quantum yields of D-A type
molecules under physiological condition inevitably hamper their
applications in vivo.[6] There remains an urgent need to design
D-A type two-photon fluorophores with minimized quenching in
water and robust performance in biological research.[4a]
Physically, solvatochromism is manifested as emission
redshift and fluorescence quenching, both originate from the
polar interactions between the electronic structure and the
solvent.[7] Redshift is due to matching alignment between the
molecules and the solvent that lowers the HOMO-LUMO gap.[8]
Quenching is more complicated and influenced by many factors.
For instance, twisted intramolecular charge transfer (TICT)
dissipates the excitation energy through bond rotation (Figure
1A).[9] "Energy gap law" releases energy through electron
transition from excited states to the empty orbitals.[10] Excited
state proton transfer mostly occurs in protic polar solvents,
where protons migrate from solvents to the fluorophores and
quench the excited electrons (Figure 1A).[11] On the other hand,
D-A type fluorophores rely on intramolecular charge transfer
Figure 1. (A) The quenching mechanisms of traditional D-A type fluorophores
(dimethylamino acedan). (B) General design strategy of new D-A type TPC
(two-photon chalcone) dyes.
Our design is inspired by a natural D-A type molecule, the
GFP chromophore,[13] which shows three features: 1) reduced
aggregation and non-radiative decay from the small aromatic
scaffold;[14]
2)
attenuated
quenching
by
restrained
donor/acceptor; 3) most remarkably, a hydrogen bond network
(HBN) that modulates the emission profile. Inside the protein
pocket, GFP chromophore is surrounded by HBN that stabilizes
the electronic states, minimizes the energy dissipation and
strengthens ICT (Figure 1B).[15] It is noteworthy that most
fluorophores are quenched by hydrogen bonds, with GFP
chromophore being an exception only in the protein pocket.[11]
Our design aims at incorporating all the three features of GFP
chromophore with an emphasis on harvesting hydrogen bonds
in the open solvent environment.
By replacing the dihydroimidazole ring of GFP chromophore
with cyclopentanone appended on dimethylaminobenzene, we
developed a simple and facile scheme to produce TPC dyes by
one-step nucleophilic substitution with indanones and
benzaldehydes under mild conditions (Scheme S1). As a proof-
of-concept, we synthesized three TPC dyes by altering the
substituents on the 3-position of ring A (Figure 2A). Their
emission spectra in different solvents were measured together
with three known D-A type two-photon dyes, Acedan-NH2, Ans
and Naph-NH2 (Figures 2B, S1). The known D-A type
fluorophores exhibit substantial solvatochromism, quenching in
protic polar media (Figures 2B, S2a-2c, S2g-h). In comparison,
all TPC dyes exhibit anti-solvatochromic fluorescence emission.
TPC1 displays gradual quantum yield increment with solvent
polarity, changing from Φf = 0.16 in DCM (λmax 482 nm) to 0.23
in MeCN (λmax 506 nm) and 0.30 in ethanol (λmax 508 nm)
[*]
T.-B. Ren,∇ Dr. W. Xu,∇ Q.-L. Zhang, X.-X. Zhang, S.-Y. Wen, Dr.
H.-B. Yi, Prof. Dr. L. Yuan, Prof. Dr. X.- B. Zhang
State Key Laboratory of Chemo/Biosensing and Chemometrics,
College of Chemistry and Chemical Engineering
Hunan University
Changsha 410082 (PR China)
E-mail: lyuan@hnu.edu.cn
∇These authors contributed equally
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201800293
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(Table S1). However, in PBS buffer the quantum yield drops to
less than 0.02. The dimethylamino group is likely too
hydrophobic and electron-donating, thus not only reduces the
water solubility of TPC1 (Figure S3), but also counters our
design to reduce TICT. By altering the dimethylamino group, we
produced more hydrophilic TPC2 and TPC3, both emitting
stronger in PBS buffer (Figure S2e-2f). TPC3 exhibits the most
remarkable anti-solvatochromic fluorescence (quantum yields in
PBS (0.80) > EtOH (0.53) > MeCN (0.12) > DCM (0.02)), 113-
time stronger emission in PBS than in DCM (Figure S2f). Under
UV irradiation, TPC3 exhibits clear fluorescence turn-on with
increasing PBS ratio (Figures 2C, S4a), contrasting the
quenching of Acedan-NH2 (Figures 2D, S4b). Fluorescence
lifetime also exhibit anti-solvatochromic pattern: as the solvent
polarity increases, the lifetime of TPC3 significantly extends
(Table S2 & Figures S5).
fluorescence (Figure S7 and Table 1). We hence confirm that by
modifying ring B with acceptors at less sterically hindered
positions, slight emission tuning can be achieved without
compromising the anti-solvatochromic fluorescence. TPC9 and
TPC10 replaced the benzene ring B with electron deficient
pyridine. Both dyes showed substantial redshift, further
confirming our hypothesis. TPC11 and TPC12 changed the
cyclopentanone linker between ring
A
and ring
B
to
dihydrofuranone and cyclohexanone, affecting the planar
conjugation and reducing their quantum yields (Table 1). As
revealed by DFT calculations, ring B of TPC12 forms an angle of
149.24° at cyclohexanone (Figure S11). C3 has flexible
connection between ring A and B, hence showing negligible
emission in all solvents (Table 1). In total, structure tuning on
ring B resulted in emission wavelengths adjustment.
Table 1. Chemical structures and optical properties of TPC dyes.
O
O
O
O
1
9
2
8
A
(
X
)
B
)
(
N
3
H2N
5
R2
7
=
O
CH2CH2
:
:
4
H2N TP
X
X
H2N
C11
TPC12
TPC9
O
C
1
H2N
N
6
=
O
-
-
-
-
=
=
=
=
:
:
:
:
e
TPC4 R2 5 OM
O
TPC5 R2 5 COOH
e
TPC6 R2 7 OM
TPC7 R2 7 COOH
N
-
H2N
=
N
H2N
C2
C3
:
TPC10
TPC8 R2 7 CN
λAbs
/nm
375
381
374
388
382
388
381
383
363
378
426
445
344
λEm
/nm
498
501
503
493
518
535
522
535
505
528
572
524
/
Stokes
shift/nm
123
120
129
105
136
147
141
152
142
150
146
79
Dye
ε(M−1cm-1)
Φ[a]
TPC3
TPC4
TPC5
TPC6
TPC7
TPC8
TPC9
TPC10
TPC11
TPC12
C1
43400
34400
8010
10200
6200
0.80
0.34
0.39
0.20
0.49
0.17
0.52
0.29
0.13
0.072
0.004
/
9800
36000
37200
19000
18200
9020
Figure 2. (A) Structures of TPC1-3. (B) Normalized fluorescence intensities of
Acedan-NH2, Ans, Naph-NH2, and TPC1-3 (5 µM) in different solvents. (C)
and (D) Fluorescent photographs of TPC3 and Acedan-NH2 (5 µM) in MeCN-
PBS buffer mixtures under 365 nm excitation.
C2
C3
7440
14400
/
/
[a]The quantum yields were determined using fluorescein as reference (Φf
=
0.95 in 0.1 M NaOH solution).
DFT calculations indicate that HOMO distribute on ring A of
TPC3, whereas LUMO cover ring B (Figure S6). We hypothesize
that HOMO determines the solvatochromism whereas
To elaborate the functions of HBN in improving the
fluorescence of TPC dyes, we conducted a series of simulations.
We hypothesize that the HBN functions in three ways: 1) it
restrains the internal rotation, thus reducing the non-radiative
dissipation; 2) it helps dissolve the molecules in protic polar
solvents; 3) it facilitates and stabilizes the ICT states that lead to
redshifts. These assumptions were confirmed by energy
comparisons between solvated and non-solvated TPC3. In
Figure 3A, S1-min and S1-max are the lowest and highest
energy excited states of TPC3. S1-min has a nearly planar
conformation with 0-degree dihedral angle between ring A and B,
whereas S1-max exhibits 90-degree dihedral angle (Figure
S12a). The excited state energy increases with the dihedral
angle and solvent polarity (Figure S12b). The calculated
modifications on ring
B lead to adjustment of emission
wavelengths. The calculations suggest that 5-, 7-, and 9-
positions of TPC3 have denser LUMO electrons. Thus we
synthesized a series of TPC dyes with their 5- and 7-positions
modified by electron donors or acceptors. Fluorescence spectra
comparison between TPC4-TPC5 and TPC6-TPC7 indicates
that both weak electron donors and acceptors redshift the
emission maxima (Figure S7A-D & Table 1). However, TPC4
and TPC6 are not completely anti-solvatochromic because the
strong electron donor - methoxy group pushes HOMO to ring B
(Figure S6). To confirm the assumption, we synthesized C1 with
7-dimethylamino and compared its spectra with TPC6. On TPC6, oscillator strengths reveal that in polar solvents, TPC3 presents
the HOMO still mainly rests on ring A, whereas in C1 the HOMO
shifts to ring B (Figure S8), thus becoming solvatochromic
(Figure S9a-10a). Similar observation was seen on C2 (Figures
S8, S9b-S10b). The HOMO migrates further to ring B, making
C2 even more solvatochromic. Thus weak electron acceptors on
ring B can redshift the emission of TPC dyes while maintaining
their anti-solvatochromic fluorescence.
Regioisomeric comparison was conducted between TPC5
and TPC7. Both molecules exhibit anti-solvatochromic
fluorescence, but the bulky 5-COOH on TPC5 hampers the
overall conjugation, making it less redshifted than TPC7. TPC8,
equipped with 7-cyanide, clearly emits anti-solvatochromic
higher chance to absorb or emit light than in non-polar solvent
(Figure S12c).
On TPC3 structure, there are two exposed heteroatoms that
can form hydrogen bonds with solvents: the carbonyl group of
the cyclopentanone and the amino group on ring A. In the
simulated environment of MeCN-water mixture, we tested
hydrogen bonding on both positions (Figure 3A and S13A).
Apparently, the change of rotation barrier and oscillator strength
is associated with the change of hydrogen bonds (Figure 3B and
S13B). The ability of molecules undergoing electronic state
transition is manifested by the difference of oscillator strengths
at 0 degree and 90 degree dihedral angles. More hydrogen
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Figure 4. Two-photon imaging of TPC-N3 (10 μM) in liver tissue of cirrhotic
mice: (A) Normal mice liver tissue only; (B) Normal mice liver tissue with TPC-
N3, (C)-(I) mice with CCl4 treatment after 1 to 7 weeks, liver tissue extracted
then incubated with TPC-N3. Excitation: 810 nm. Emission: 500-560 nm.
Scale bar: 150 μm. (J) Quantification of cystathionine γ lyase (CSE) levels in
normal and cirrhosis liver tissue by ELISA. (K) Serum ALT levels in normal
and cirrhosis mice.
bonds prompt smaller depth, hence the molecule is easier to
absorb and emit energy (Figure 3B). In addition, we postulate
that the HBN shapes the molecule in a maximally fluorescent
state by limiting vibrational and rotational energy loss. This
phenomenon can be named hydrogen bond induced enhanced
emission (HIEE). In addition, dynamic light scattering indicates
that TPC3 remains solvated in the water-MeCN mixture. Hence
its quantum yield increase originates from the interactions with
water molecules, not through aggregation-induced emission
(Figure S14).
To build a reliable cirrhosis model, mice were subcutaneously
injected with CCl4 to induce hepatotoxicity on a daily level for
over 7 weeks. The liver tissue was harvested at different time
points and stained with TPC-N3. Two-photon imaging clearly
indicated a sharp purge of H2S in liver upon first week of
treatment (Figure 4A-I and S27a). Comparing to normal liver, the
fluorescence intensity increased over three-fold (Figure S27a).
Reports showed that to antagonize acute liver injury,
endogenous H2S was abundantly produced and consumed as
an internal gasotransmitter.[17] In addition, quantitation of key
enzymes expressed by hepatocytes, such as aspartate
transaminase (AST) and alanine transaminase (ALT) saw their
increased expression levels (Figure 4K and S27b), indicating
liver self-remediation.[18] Interestingly, with the progression of
liver injury, H2S level significantly dropped (Figure 4A-I, Figure
S27a). Immunoassay reveals that CSE level in liver tissue
initially decreased, followed by increase and a final purge at
week 7 (Figure 4J). This indicates that liver CSE is heavily
expressed to compensate for H2S depletion. The ALT and AST
levels also stepped down after the initial increase, reaching
normal levels (Figure 4K and S27b). This proved that liver injury
has entered late stage and hepatocytes can no longer support
the liver self-repairing, resulting in cirrhosis (Figure S26 and
S27).[19] Hematoxylin and eosin stain clearly showed fibrotic
bands and distortion of the liver tissue (Figure S27c). This is the
first direct visualization of H2S generation and consumption in a
liver cirrhosis model. The probe provides a sensitive and
powerful tool to study chronic liver diseases.
In conclusion, a new series of D-A type GFP chromophore
analogues (TPC dyes) that can harvest hydrogen bonds in water
to induce strong anti-solvatochromic fluorescence was devised.
Such a feature makes TPC dyes especially attractive for two-
photon molecular imaging in the biological specimens.
Mechanism and structure-activity studies elaborated that TPC
dyes bind to water molecules through multiple hydrogen bonds,
achieving better solubility and higher stability of the ICT excited
states. Such an unprecedented design strategy utilizing HBN
can potentially be generalized to the pursuit of two-photon
fluorophores with strong fluorescence in aqueous environment.
To demonstrate their biological applications, we synthesized a
H2S selective probe based on TPC scaffold and for the first time
visualized H2S generation and consumption in the cirrhotic liver
tissue.
(B)
1.4
1.2
Non
1.0
0.8
One-1
One-2
Two-1
Four
Two-2
0
40 80 120 160
Dihedral angel (°)
Figure 3. (A) S1-min and S1-max conformations of TPC3 in the first excited
state with hydrogen bonds attached to either carbonyl or amino group. (B) The
oscillator strength of S1 excited state of TPC3 with different numbers of
hydrogen bonds (None to four). Both metrics were plotted with increasing twist
angles (φ) at the bridging bond of ring B. The simulation is conducted in the
mixture of MeCN and water.
We next conducted a series of studies on the selected
molecule TPC3 and proved that its excellent two-photon
performance, chemical stability and photo-stability is suitable for
biological studies (Figure S15-20). By engineering the 3-amino
group of TPC3 with azide, we altered its electronic structure and
generated TPC-N3 (Figure 4). TPC-N3 itself exhibits negligible
fluorescence emission under UV; upon reaction with hydrogen
sulfide (H2S), the azide is reduced to amino, yielding TPC3 and
recovering the emission (Scheme S2). TPC-N3 can thus serve
as a potent two-photon fluorescent probe for sulfide in the
biological systems.
Endogenous H2S plays an essential role in antagonizing
chronic liver injury or cirrhosis by mediating vasodilation and
increasing hepatic arterial buffer responses.[16] Internally, H2S
level is modulated by cystathionine γ lyase (CSE). However,
there is no direct evaluation of the H2S dynamics during
cirrhosis due to the lack of potent sensing tools. To tackle this
issue, we first proved that TPC-N3 could selectively monitor
endogenous sulfide generation both in vitro and in vivo (Figure
S21-24). The penetration depth of TPC-N3 can reach 100 μm in
the liver tissue (Figure S25).
(J)
400
O
350
300
N3
Acknowledgements
-
TPC N3
250
l
This work is supported by NSFC (21622504, 21735001,
21521063), the Science and Technology Project of Hunan
Province (2017RS3019). We are grateful to Prof. Peng Wu from
Sichuan University for measuring the fluorescence lifetime data.
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Keywords: fluorescent dyes • hydrogen bond network • anti-
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solvatochromism • two-photon • cirrhosis
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Entry for the Table of Contents (Please choose one layout)
COMMUNICATION
Tian-Bing Ren, ∇ Wang Xu, ∇ Qian-
Ling Zhang, Xing-Xing Zhang, Si-Yu
Wen, Hai-Bo Yi, Lin Yuan, * Xiao-
Bing Zhang
Turn bane into a boon: A unique
class of two-photon dyes with anti-
solvatochromic
fluorescence,
optically tunable groups, high
fluorescence quantum yields and
large two-photon action cross
sections in water were developed.
They are expected to be useful
Page No. – Page No.
Harvesting Hydrogen Bond
Network: Enhance the Anti-
Solvatochromic Two-Photon
Fluorescence for Cirrhosis
Imaging
fluorophore
scaffolds
in
the
development of new two-photon
probes for bioimaging.
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