Rational Design of Fluorescent Phthalazinone Derivatives for One- and Two-Photon Imaging

Article abstract of DOI:10.1002/chem.201601499

Phthalazinone derivatives were designed as optical probes for one- and two-photon fluorescence microscopy imaging. The design strategy involves stepwise extension and modification of pyridazinone by 1) expansion of pyridazinone to phthalazinone, a larger conjugated system, as the electron acceptor, 2) coupling of electron-donating aromatic groups such as N,N-diethylaminophenyl, thienyl, naphthyl, and quinolyl to the phthalazinone, and 3) anchoring of an alkyl chain to the phthalazinone with various terminal substituents such as triphenylphosphonio, morpholino, triethylammonio, N-methylimidazolio, pyrrolidino, and piperidino. Theoretical calculations were utilized to verify the initial design. The desired fluorescent probes were synthesized by two different routes in considerable yields. Twenty-two phthalazinone derivatives were synthesized and their photophysical properties were measured. Selected compounds were applied in cell imaging, and valuable information was obtained. Furthermore, the designed compounds showed excellent performance in two-photon microscopic imaging of mouse brain slices.

Full text of DOI:10.1002/chem.201601499

DOI: 10.1002/chem.201601499
Full Paper
&
Fluorescent Probes
Rational Design of Fluorescent Phthalazinone Derivatives for One-
and Two-Photon Imaging
+
[a, c]
+ [b]
[b]
[a]
[d]
Lingfei Yang ,
Yuanjun Zhu , Mengyang Shui, Tongliang Zhou, Yuanbo Cai,
[a]
[a]
[a]
[a]
[d]
[a]
Wei Wang, Fengrong Xu, Yan Niu, Chao Wang, Jun-Long Zhang, Ping Xu,*
Lan Yuan,* and Lei Liang*
[c]
[a]
Abstract: Phthalazinone derivatives were designed as optical
probes for one- and two-photon fluorescence microscopy
imaging. The design strategy involves stepwise extension
and modification of pyridazinone by 1) expansion of pyrida-
zinone to phthalazinone, a larger conjugated system, as the
electron acceptor, 2) coupling of electron-donating aromatic
groups such as N,N-diethylaminophenyl, thienyl, naphthyl,
and quinolyl to the phthalazinone, and 3) anchoring of an
alkyl chain to the phthalazinone with various terminal sub-
stituents such as triphenylphosphonio, morpholino, triethy-
lammonio, N-methylimidazolio, pyrrolidino, and piperidino.
Theoretical calculations were utilized to verify the initial
design. The desired fluorescent probes were synthesized by
two different routes in considerable yields. Twenty-two
phthalazinone derivatives were synthesized and their photo-
physical properties were measured. Selected compounds
were applied in cell imaging, and valuable information was
obtained. Furthermore, the designed compounds showed
excellent performance in two-photon microscopic imaging
of mouse brain slices.
Introduction
to decipher local information on intracellular and extensive re-
[3]
gions of tissues is required.
Fluorescence imaging for observing life processes in single
molecules, living cells, and deep tissues attracts attention in
fundamental medicine, clinical diagnostics, and drug discov-
On the subcellular level, there has been broad interest in de-
veloping novel imaging techniques and dyes for cellular organ-
ization, structures, and molecular events during biological pro-
cesses. Many existing fluorophores, such as fluorescein, rhoda-
mine, coumarin, cyanine, and BODIPY, have exhibited good ap-
[
1]
ery. As chemobiological tools, fluorescent probes have been
developed with extraordinary luminescence, accurate localiza-
tion, excellent spatial resolution, and high sensitivity through
continuous efforts to improve their structures and related
[4]
plicability and promising prospects. Furthermore, to improve
the physical and chemical properties and enhance the per-
formance, the development of novel fluorescent probes with
appropriate solubility, high luminescence, low toxicity, high sta-
bility, and large Stokes shift is still required. Besides, efficient
synthesis and easy modification are also in demand for build-
ing large libraries of compounds.
[
2]
properties. Apart from optimization of the basic optical prop-
erties, improved applicability with the aim of visualizing specif-
ic subcellular organelles and large-scale tissues is a critical
issue. Hence, the development of suitable fluorescent probes
+
[
a] L. Yang, T. Zhou, W. Wang, F. Xu, Y. Niu, C. Wang, Prof. Dr. P. Xu,
Both one-photon microscopy (OPM) and two-photon micros-
copy (TPM), which utilizes two near-infrared (NIR) photons as
the excitation light source, have emerged as powerful imaging
tools for biological and medicinal research. These two tech-
niques have their own advantages and limitations, and thus
they are often utilized complementarily depending on the spe-
Prof. Dr. L. Liang
Department of Medicinal Chemistry, School of Pharmaceutical Sciences
Peking University Health Science Center, Beijing (P. R. China)
E-mail: pingxu@bjmu.edu.cn
leiliang@bjmu.edu.cn
+
[
b] Dr. Y. Zhu, M. Shui
Department of Molecular and Cellular Pharmacology
School of Pharmaceutical Sciences, Peking University, Beijing (P. R. China)
[5]
cific application. For example, OPM is capable of imaging
fixed thin samples with high resolution, especially for cell
imaging, and TPM offers advantages such as longer observa-
tion duration, deep penetration into tissues, and low phototox-
icity. Therefore, a variety of two-photon probes have been de-
veloped, and their utility in bioimaging applications has been
+
[
c] L. Yang, Dr. L. Yuan
Medical and Healthy Analysis Center, Peking University, Beijing (P. R. China)
E-mail: yuan_lan@bjmu.edu.cn
[
d] Y. Cai, Prof. Dr. J.-L. Zhang
Beijing National Laboratory for Molecular Science
State Key Laboratory of Rare Earth Materials Chemistry and Applications
College of Chemistry and Molecular Engineering
[6]
demonstrated. To meet the requirements of both OPM and
TPM imaging techniques, herein we designed a novel type of
fluorescent probes by rational design and theoretical calcula-
tions.
Peking University, Beijing (P. R.China)
+
[
] These authors contributed equally to this work.
Supporting information for this article, including all experimental details,
can be found under http://dx.doi.org/10.1002/chem.201401499.
Chem. Eur. J. 2016, 22, 1 – 9
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Pyridazinone, a six-membered aromatic ring which is not
naturally synthesized in living organisms, has been synthetical-
Then coupling with different electron-donating aromatic
groups such as N,N-diethylaminophenyl, thienyl, naphthyl, and
quinolyl at the 2-NH group endows the whole molecule with
tunable luminescent properties (Figure 1b). Furthermore, to
achieve tailor-made specificity in intracellular localization, an
anchoring group on the skeleton is necessary, so that a 4-phe-
noxyl group was added to the phthalazinone followed by in-
troduction of a bromo alkyl chain at the phenolic hydroxyl
group. Finally, various carriers with lipophilic and cationic prop-
erties were installed at the alkyl chain to enhance the affinity
to particular organelles. For example, an n-butyl chain was
anchored to the phenolic hydroxyl group with a terminal posi-
tively charged triphenylphosphonio group, which was expect-
ed to act as a mitochondria-directing group, and morpholino
was expected to behave as a lysosome-directing group.
[7]
ly prepared and applied as a pharmacophore in drug design.
Pyridazinones have been developed as phosphodiesterase III
inhibitors, a1-adrenoreceptor antagonists, antiplatelet agents,
anti-inflammatory agents, HIV-1 reverse-transcriptase inhibitors,
[
8]
GPCR antagonists, and c-Met inhibitors. Previously, we de-
signed a series of full-color tunable fluorescent dyes based on
a pyridazinone scaffold. Preliminary application of these pyrida-
zinone derivatives in cell imaging revealed ideal cell permeabil-
[
9]
ity and high stability. To meet the requirements of biological
research, we have now employed phthalazinone, an analogue
of pyridazinone, to construct a novel fluorophore. To the best
of our knowledge, phthalazinone has never been used in the
development of fluorescent probes.
To gain more insight into our design of phthalazinone deriv-
atives, calculations were performed on the thermodynamic
properties and electronic transitions by geometrical optimiza-
Results and Discussion
tion of their structures at the B3LYP/6-31G** level of density
Design and computational verification
[10]
functional theory.
The vertical excitation energy from the
To fulfil our aim of utilizing phthalazin-1(2H)-one, we chose 6-
phenylpyridazinone as the initial electron-withdrawing moiety
to build the fluorescent core skeleton. Conjugation with a ben-
zene ring at the 4,5-positions of pyridazinone yields phthalazi-
none with a larger p-system as electron acceptor (Figure 1a).
ground state to the lowest-lying excited state in the singlet
[11]
manifold was also calculated.
To simplify the calculation, the modified alkyl chain linked at
the phenolic hydroxyl group was omitted and replaced by
a simple methyl group without influencing the results. As
shown in Tables 1 and 2, as well as Table S2 of the Supporting
Information, the corresponding molecules were abbreviated as
PY for pyridazinone analogues and PH for phthalazinone ana-
logues. The structurally analyzed molecular framework was
composed of three aromatic rings: pyridazinone/phthalazi-
none, anisole, and N-substituent. In the optimized structures of
PY1, PY2, and PY3, the dihedral angle between the anisole
and pyridazinone rings is about 228, but it increased to about
508 between the anisole and phthalazinone rings because of
the steric effect between 2’-H of anisole and 5-H of phthalazi-
none. The dihedral angles between pyridazinone/phthalazi-
none and N,N-diethylaminophenyl rings range from 37.8 to
408. However, this angle is only 2.68 when N,N-diethylamino-
phenyl is replaced by thienyl. The significant difference in dihe-
dral angles is determined by the interaction between the car-
bonyl O atom and the nearest H atom of the N,N-diethylami-
nophenyl or thienyl group. Moreover, alkyl substitution on the
amino group of aniline had no effect on the structure of the
whole molecule (Supporting Information, Table S2).
Figure 1. a) Structure of pyridazine-3(2H)-one and phthalazin-1(2H)-one.
b) Design strategy of the phthalazinone derivatives as fluorescent probes
based on the pyridazinone scaffold.
For all these compounds, the S !S transition mainly con-
0
1
sists of HOMO!LUMO. All the aniline-containing compounds
[
a]
Table 1. Calculated parameters (S
0
!S
1
) of different pyridazinone and phthalazinone derivatives.
[
b]
[c]
Compound
HOMO [eV]
LUMO [eV]
Excitation energy [eV]
f
Composition
Dihedral angles
PY1
PH1
PH6
ꢀ4.91
ꢀ4.84
ꢀ5.41
ꢀ1.61
2.88
3.06
3.27
0.3235
0.2451
0.2493
HOMO!LUMO
22.1/37.8
50.2/39.6
48.9/2.6
ꢀ1.32
HOMO!LUMO
LUMO: ꢀ1.69
LUMO+1: ꢀ1.33
HOMO!LUMO (94%)
HOMO!LUMO+1 (6%)
[a] Only selected excited states were considered. [b] Oscillator strength. [c] The former is the dihedral angle between anisole and pyridazinone/phthalazi-
none moieties, and the latter is that between the N-substituent and the pyridazinone/phthalazinone ring.
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uted to the methoxyl group lowering the electron-donating
ability of anisole, which reduces the energy of LUMO. Although
PH6 shows greater planarity, the five-membered thienyl ring
reduces the repulsion between the carbonyl O atom and 5’-H
of the thienyl substituent, and its weaker electron-donating
ability leads to indistinct charge-transfer characteristics in
a higher-energy electronic transition.
Table 2. DFT calculated HOMOs and LUMOs of pyridazinone and phthala-
zinone derivatives.
Model
HOMO
LUMO
PY1
PH1
Synthesis and characterization
With the results of our calculations in hand, we started our
synthesis of the phthalazinone derivatives. To prepare the
target molecules, two efficient synthetic routes were explored
depending on the substituents of choice.
LUMO:
As shown in Scheme 1, Method A was established with 2,3-
dihydrophthalazine-1,4-dione (1) as starting material. By treat-
ment with POBr and acetic acid, 2 was obtained in 86% yield.
3
Then a palladium-catalyzed Suzuki coupling reaction was used
to form a CꢀC bond between 2 and 4-hydroxyphenylboronic
acid to give 3, which is suitable for construction of an optional
substituted hydroxyl group at the 4-phenyl position of phtha-
lazinone, in moderate to high yields. 4-(4-Hydroxyphenyl)ph-
thalazin-1(2H)-one (3) is the core skeleton of our target com-
pounds. Extension of 3 by coupling of an electron-donating
substituent to the 2-NH group expands the conjugation and
PH6
LUMO+1:
[12]
give a donor–acceptor system. Moreover, modification of the
hydroxyl group allows the functionality to be increased. Selec-
tive modification of 3 with different electron-donating aromat-
ic halides by copper(I) iodide catalyzed coupling reactions pro-
ceeded with retention of the hydroxyl group. For example,
after coupling with 4-bromo-N,N-diethylaniline, fluorescent
product 4 was obtained in 72% yield. 2-Iodothiophene could
also be used to couple with 3, and the corresponding product
was obtained in 68% yield. Then the hydroxyl group was sub-
stituted with different dibromo alkanes such as 1,4-dibromobu-
tane and 1,6-dibromohexane in order to investigate the role of
the length of the alkyl chain, which showed little effect on the
structure and photophysical properties of the luminescent
core. Finally, compounds 6 were obtained through terminal
bromo substitution with different nucleophiles such as triphe-
nylphosphine, morpholine, pyrrolidine, and piperidine. The
total yields ranged from 18 to 32%, depending on the nucleo-
phile. The advantage of this route lies in the 4-substituent of
the phthalazinone, which could be varied by means of differ-
have HOMO!LUMO S !S transitions. For compound PH6,
0
1
9
4% HOMO!LUMO and 6% HOMO!LUMO+1 was obtained
due to the thienyl group. The distribution of the molecular
HOMO and LUMO reveals that S !S transition is basically an
0
1
intramolecular charge transfer from aniline to pyridazinone/
phthalazinone. Alkyl substitution of the amino group enhances
the HOMO energy by increasing the electron density, but it
has no influence on the energy of the LUMO because the
LUMO does is not located on the aniline moiety. With increas-
ing the substituent on the amino group of the aniline moiety,
the fluorescence spectrum shows a redshift and the HOMO–
LUMO energy gap decreases. Compared with phthalazinone,
pyridazinone showed a 25 nm redshift and increased oscillator
strength due to its greater planarity. Moving the methoxyl
group to the meta position had no effect on the planarity or
electronic transition of the whole molecule (Supporting Infor-
mation, Table S3). The slight spectroscopic redshift was attrib-
Scheme 1. Method A. Reagents and conditions: i) 1,2-Dichloroethane, POBr
3
, reflux, 12 h; acetic acid, reflux, 2 h; ii) 4-Hydroxyphenylboronic acid, [Pd(PPh
3 4
) ],
Na CO , DMF, H O, 1208C, 24 h; iii) 4-Bromo-N,N-diethylaminophenyl, CuI, Cs
2
3
2
2
CO , DMF, 1208C, 12 h; iv) 1,4-Dibromobutane, K CO , CH CN, reflux 4 h; v) PPh
3
2
3
3
3
3
or other nucleophiles, CH CN, reflux, 24 h.
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Scheme 2. Method B. Reagents and conditions: i) NaOH, hydroxylamine hydrochloride, H
zene, dimethylacetamide, reflux, 2 h; iii) 1-Iodo-4-nitrobenzene, CuI, Cs CO
other nucleophiles, CH CN, reflux, 16 h; vi) Hydrazine hydrate, FeCl ·6H O, activated carbon, 1,4-dioxane, reflux 4 h; vii) Iodoethane, Cs
6 h.
2
O, 808C; sulfuric acid, H
, DMF, 1208C, 12 h; iv) 1,4-Dibromobutane, K
2
O, 1008C; ii) Hydrazine hydrate, chloroben-
2
3
2
CO
3
, CH
3
CN, reflux, 4 h; v) PPh
3
or
3
3
2
2
CO , CH CN, reflux,
3
3
1
ent aromatic boronic acids to expand the scope of our com-
pound library.
light of 6a versus 6 f, 6c versus 6g, and 6d versus 6o, and the
pyridazinone derivatives showed obvious redshifts. The batho-
chromic shift of the absorption peak may arise from the in-
creasing electron-withdrawing ability of the pyridazinone
moiety. By comparing 6 f, 6j–6o, 6t, and 6u, all of which have
the same fluorophore but different terminal modification of
the alkyl chain, we found that the appended alkyl moiety has
a negligible effect on the photophysical properties of these
compounds (Supporting Information, Figures S1–S3).
To simplify the synthetic route with the aim of rapid synthe-
sis of 3 with low cost and on a large scale, we explored an op-
timized method. As shown in Scheme 2, by treating phenolph-
thalein with NaOH, hydroxylamine hydrochloride, and then sul-
furic acid in hot water, 2-(4-hydroxybenzoyl)benzoic acid (8)
[
13]
was obtained in quantitative yield. Then the core skeleton 3
was obtained in quantitative yield by condensation of 8 with
[
14]
hydrazine hydrate in 2 h. In another adjustment, 4-bromo-
N,N-diethylaminophenyl was replaced by p-nitrobenzene in the
copper(I) iodide catalyzed reaction at the 2-NH group of
phthalazinone. To investigate the electron-donating effect of
different amino groups and avoid the simultaneous substitu-
tion at both amino and hydroxyl groups, compound 9 was
prepared. Its phenol group could be fully modified with reten-
According to our previous report, the hydrophobicity and
charge of the N-substituents of the pyridazinone strongly
[9]
affect the photophysical properties. In this work, electron-do-
nating aromatic groups such as N-alkylated aminophenyl,
thienyl, naphthyl, and quinolyl were used for coupling with the
pyridazinone and phthalazinone moiety. The results obtained
for 6 f–6i and 6o–6s indicated that the rigidity and electron-
donating ability of the N-substituents affect the electronic
structures and the absorptions of the fluorophores more signif-
icantly than variation of the pyridazinone/phthalazinone moiet-
ies.
tion of the nitro group. Then, we used FeCl and hydrazine to
3
[
15]
reduce the nitro to an amino group in 94% yield. The amino
group could be substituted with one or two alkyl groups by
a controlled procedure to give the desired products 6 in con-
siderable total yields. The optimized route also expands the
scope of our target compounds due to the variety of tailor-
made substituents on the amino group. Besides, 2-iodothio-
phene, 2-bromonaphthalene, and 3-bromoquinoline were also
employed in this route with moderate to good yields.
Pyridazinone and phthalazinone derivatives with similar N-
substituents were found to exhibit similar vibronically struc-
tured emission bands. For instance, phthalazinone 6 f with a p-
N,N-dialkylated aminophenyl N-substituent showed maximum
emission at 612 nm, which is blueshifted by about 24 nm com-
pared to the similar pyridazinone analogue 6a (636 nm).
To probe how the N-substituents affect the emissions of the
compounds, we chose 6o–6s for comparison. As shown in
Table 3, they emit with a maximum at 610, 607, 497, 465, and
426 nm, respectively. Their fluorescence spectra show progres-
sive blueshifts with decreasing electron-donating ability, as
was observed in the absorption spectra.
With the optimized synthetic route, 22 compounds were
prepared in moderate to good yields. Before biological evalua-
tion, their photophysical properties were measured. Absorp-
tion data for all the compounds are compiled in Table 3. In
DMSO at 298 K, they generally show an intense band around
3
1
13–392 nm with molar absorptivity e in the range of 0.49ꢁ
4
4
ꢀ1
ꢀ1
1
0 to 1.68ꢁ10 m cm , corresponding to the p–p* transi-
tion of the basic fluorophores. To examine how the pyridazi-
none/phthalazinone moiety influences the photophysical prop-
erties, these two series of compounds were compared in the
Fluorescent quantum yields F were measured in DMSO by
fl
16]
[
using fluorescein as reference.
As shown in Table 3, the
phthalazinone derivatives 6 f, 6j–6o, 6t, and 6u have consid-
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[
a]
Table 3. Structures and photophysical properties of 6.
[
b]
1
max
fl
max
[c]
[d]
fl
2
max
Compound
A
R
1
R
2
e
max
l
(ex)/l (em)
F
l
(ex)
+
6
6
6
6
6
6
6
6
a
b
c
1
1
1
1
1
2
2
2
PPh
3
3
3
0.81
0.78
0.93
0.93
0.72
0.84
0.59
1.05
392/636
380/640
383/633
391/635
382/645
351/612
330/615
355/479
0.098
0.090
0.084
0.077
0.074
0.244
0.120
0.215
820
820
800
820
820
820
800
800
+
+
PPh
PPh
d
e
f
+
+
+
PPh
PPh
PPh
3
3
3
g
h
+
+
[e]
6
i
2
PPh
3
1.23
351/479
0.027
n.d.
6
6
6
j
2
2
2
P(Cy)
3
0.60
0.49
0.75
350/613
352/611
350/611
0.213
0.198
0.204
820
n.d.
820
k
l
PPh
2
6
m
2
0.72
354/613
0.185
820
6
6
6
6
n
o
p
q
2
2
2
2
0.68
0.97
0.85
1.06
351/611
351/610
342/607
353/497
0.177
0.196
0.133
0.209
820
820
800
800
6
6
r
2
2
1.39
313/465
314/426
0.022
0.018
n.d.
n.d.
s
1.68
0.70
6
6
t
2
2
349/607
352/610
0.188
0.224
820
820
u
0.56
4
ꢀ1
ꢀ1
[
a] All data were measured in DMSO at 298 K. [b] Extinction coefficient in 10 m cm . [c] lmax values of the one-photon absorption and emission spectra
in nanometers. [d] Fluorescence quantum yield, with fluorescein as standard (F=92% in 0.1 N NaOH). [e] Not determined.
erable fluorescence quantum yields (0.177–0.244), which are
pounds 6 by the two-photon induced fluorescence method
higher than those of pyridazinone derivatives 6a (0.098) and
referenced to fluorescein (Supporting Information, Figure S5).
The compounds exhibit reasonable 2PA cross sections d across
the 740–860 nm spectral window with 6 f showing a maximum
value of d=221 GM at 820 nm.
6
d (0.077). The electron-donating ability of 6a bearing an N,N-
diethylaminophenyl group exhibited higher F than 6b with
fl
an N,N-dimethylaminophenyl moiety. Singly alkylated 6c
showed lower F than dialkylated analogues such as 6a and
In our previous study, the viability, stability against photo-
bleaching, and cytotoxicity of a series of pyridazinone com-
pounds were measured and compared with those of some
commonly used fluorescent dyes such as fluorescein. Herein,
commercial trackers were used for comparison to test the
phthalazinone derivatives. We found that 6a and 6 f showed
obviously better properties than MitoTracker, and 6o showed
fl
6
b. The same trend was also observed for phthalazinone deriv-
atives.
Phthalazinone derivatives have never been used in two-
photon fluorescence spectroscopy. To study their potential and
the relationship between the 2PA cross section and the elec-
tronic structure, we measured the 2PA cross sections of com-
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better performance than LysoTracker (Supporting Information,
Figures S6–S8). To reveal the structural relationship between
the photophysical properties and the biological behaviors, the
cell viabilities of the compounds were assessed by CCK-8 assay
physical properties. As negative control, an electroneutral di-
phenylphosphanyl group was used in 6k, and poor colocaliza-
tion with mitochondria was observed. For 6l, 6m, and 6n, pyr-
idinio, N-methylimidazolio, and triethylammonio were used as
positively charged nitrogen cores in the mitochondrion-target-
ing component, but lower colocalization with mitochondria
was verified by the confocal micrographs and Pearson’s coeffi-
cients (Supporting Information, Tables S4 and S6). Compounds
6b and 6c based on a pyridazinone scaffold also exhibited
high luminescence and colocalization, which indicated alkyl
substitution on the amino group has an obvious effect on cell
imaging. When triphenylphosphonio was replaced by tricyclo-
hexylphosphonio, 6j still showed good colocalization per-
formance, but the distribution in the mitochondria was not
even as good as that of 6 f. Besides, phthalazinone analogue
6v with an n-hexyl chain showed similar features to 6 f when
applied in OPM (Supporting Information, Tables S4 and S6).
With appropriate nucleophilicity and alkalinity, morpholino
tails are effective in carrying the parent compounds into lyso-
(Supporting Information, Figures S9 and S10). The results indi-
cated that all these compounds have low cytotoxicity, even
when the cell were incubated with 20 mm of the compounds
for 24 h. These newly developed fluorescent dyes showed
better photobleaching resistance and pH tolerance in different
buffers, and thus may have potential in biological applications.
Cell imaging
Live-cell imaging experiments were performed to explore the
relation between the structure of compounds 6, their photo-
physical properties, and the visualization of specific cellular be-
haviors.
Due to the specific charge condition of the membrane of
mitochondria, positively charged groups including P-/N-con-
taining groups have been widely employed for mitochon-
[18]
somes. The morpholino moieties of 6d, 6e, and 6o–6s are
responsible for their similar lysosomal localizations. As shown
in Figure 3 and Table S5 (Supporting Information), 6d, 6o, and
[
17]
dria. Herein, triphenylphosphonio was used as mitochondri-
on-directing group with a commercially available mitochondri-
al tracker as control, and excellent results were obtained.
Images of SH-SY5Y cells incubated with 2 mm of 6a, 6 f, and
6
h are shown in Figure 2. All of them exhibited high selectivity
Figure 3. One-photon fluorescence colocalization images of RD cells incu-
bated with 2 mm probes and commercial dyes. a) Image of 6. b) image of Ly-
soTracker Red. c) Merged images of a) and b). d) Bright-field image. Images
were acquired in two channels. For channel 1 with 405 nm excitation and
fluorescence emission windows: green=420–550 nm (6d, 6o), Green=450–
Figure 2. One-photon fluorescence colocalization images of SH-SY5Y cells in-
cubated with 2 mm probes and commercial dyes. a) Image of 6. b) Image of
MitoTracker Deep Red FM. c) Merged images of a) and b). d) Bright-field
image. Images were acquired in two channels. For channel 1 with 405 nm
excitation and fluorescent emission windows: green=450–570 nm (6a, 6 f),
green=420–550 nm (6h), and for channel 2 with 633 nm excitation and
fluorescence emission windows: red=643–700 nm.
570 nm (6q), and for channel 2 with 561 nm excitation and fluorescence
emission windows: red=575–650 nm.
6q showed excellent colocalization with LysoTracker Red in
both RD cells and SH-SY5Y cells. Their Pearson’s coefficients
are around 0.90. Since tertiary amines such as morpholine, pyr-
rolidine, and piperidine are widely used as leading substances
for lysosomes, ,6t and 6u bearing pyrrolidino and piperidino
moieties, respectively, were applied, and good colocalization
with commercial LysoTracker Red was observed due to the ap-
propriate pendant groups (Figure 4).
for mitochondria, and the details are even better revealed than
with MitoTracker. Their Pearson’s coefficients, which are all
greater than 0.90, indicate excellent colocalization with the mi-
tochondria. Apart from aminophenyl and thienyl, neither naph-
thyl nor quinolyl as electron-donating group in 6i, 6r, or 6s
was suitable for one-photon microscopy due the poor photo-
Therefore, variation of the basic skeleton modulates the in-
tracellular emission without disturbance of the subcellular lo-
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Figure 6. TPM image of a fresh rat brain slice labeled with a) 6 f (20 mm,
30 min) and b) fluorescein (20 mm, 30 min). c) Merged image of a) and b).
d) Bright-field image. Images were acquired in two channels. For channel 1
with 720 nm excitation and fluorescence emission windows: green=450–
Figure 4. One-photon fluorescence colocalization images of SH-SY5Y cells in-
cubated with 2 mm probes and commercial dyes. a) Image of 6. b) Image of
LysoTracker Red. c) Merged images of a) and b). d) Bright-field image.
Images were acquired in two channels. For channel 1 with 405 nm excitation
and fluorescence emission windows: green=450–570 nm, and for channel 2
with 561 nm excitation and fluorescence emission windows: red=575–
570 nm, and for channel 2 with 488 nm excitation and fluorescence emission
windows: red=500–550 nm.
stronger luminescence and more homogeneous distribution in
the brain than fluorescein (Figure 6).
650 nm.
calization. The terminal pendants of the alkyl chain determine
the lipophilicity, with different partition coefficients, and the
subcellular localizations with different affinity to specific sub-
cellular organelles, while having negligible effect on the emis-
sion.
Conclusion
By rational design and theoretical calculations, we constructed
a pyridazinone/phthalazinone library of one- and two- photon
emissive probes for use in live-cell and brain-tissue imaging.
The structural relationship between the photophysical proper-
ties and the biological behaviors was elucidated. More impor-
tantly, the electronic states of the N-substituents of pyridazi-
none/phthalazinone moieties can effectively modulate the
photophysical properties of the fluorescent probes, while the
terminal pendants on the alkyl chain determine the lipophilici-
ty and subcellular organelle selectivity. These novel fluorescent
probes were used successfully in both one-photon cell imagin-
ing with high luminescence and subcellular organelle selectivi-
ty, and two-photon brain-tissue imaging with excellent per-
formance exceeding those of some commercial probes, which
provides an important insight into modular design of lumines-
cent probes and further expands the repertoire of small-mole-
cule fluorophores.
Two-photon imaging of brain tissue
To further establish the utility of compounds 6 for TPM bio-
imaging, the probes were employed to visualize mouse brain
tissue. The brain slices were isolated from two-month-old
mice, and each slice was incubated with 20 mm 6 f for 30 min
at 378C. Because the structure of the brain tissue is heteroge-
neous, we acquired eight TPM images at the depths of 100–
200 mm to investigate the overall distribution of the fluores-
cence. As shown in Figure 5, 6 f is capable of imaging in live
Acknowledgements
This work was supported by the National Natural Science
Foundation
of
China
(21202003/21172012/81302763/
91330103), the National Basic Research Program of China
Figure 5. a) TPM image of a fresh mouse brain slice labeled with 6 f (20 mm,
3
0 min). b) Bright field image. c) Merged images of a) and b). Images were
(2012CB518000), and the Specialized Research Fund for the
Doctoral Program of Higher Education of China
acquired with 720 nm excitation and fluorescent emission windows:
green=450–570 nm.
(
20120001110010).
Keywords: density functional calculations
·
fluorescence
tissue at depths ranging from 100–200 mm by TPM. Moreover,
the entire brain slices, including the cerebral cortex and central
core, were observed with strong fluorescence. The fluores-
cence intensity in the central core was not only stronger but
also more stable than that in the cortex, and indicated differ-
ent cell permeabilities of 6 f.
microscopy · fluorescent probes · imaging agents · synthesis
design
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Published online on && &&, 0000
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FULL PAPER
&
Fluorescent Probes
L. Yang, Y. Zhu, M. Shui, T. Zhou, Y. Cai,
W. Wang, F. Xu, Y. Niu, C. Wang,
J.-L. Zhang, P. Xu,* L. Yuan,* L. Liang*
&& – &&
Better probes by design: A pyridazi-
none/phthalazinone library of one- and
two-photon emissive probes for use in
live-cell and brain-tissue imaging (see
figure) was constructed. The electronic
states of the N-substituents of the pyri-
dazinone/phthalazinone moieties effec-
tively modulate the photophysical prop-
erties of the fluorescent probes, and the
terminal groups on the alkyl chain de-
termine the lipophilicity and subcellular
organelle selectivity.
Rational Design of Fluorescent
Phthalazinone Derivatives for One-
and Two-Photon Imaging
Chem. Eur. J. 2016, 22, 1 – 9
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9
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers! ÞÞ