Synthesis of near-infrared fluorescent rhodamines via an SNArH reaction and their biological applications

Article abstract of DOI:10.1039/c8ob01701h

Near-infrared (NIR) dyes are of great interest in biomedicine due to diminished interfering absorption and fluorescence from biological samples, reduced scattering, and enhanced tissue penetration depth. In this context, we report the synthesis of rectili

Full text of DOI:10.1039/c8ob01701h

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
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H
Synthesis of Near-Infrared Fluorescent Rhodamines via an S Ar
N
Reaction and Their Biological Applications
Received 00th January 20xx,
Accepted 00th January 20xx
ab
a
b
a
a
a
b
Qing Wang, Kun Huang, Songtao Cai, Chang Liu, Xiaojie Jiao, Song He, Liancheng Zhao, and
ab
Xianshun Zeng*
DOI: 10.1039/x0xx00000x
Near-infrared (NIR) dyes are of great interest in biomedicine due to diminished interfering absorption and fluorescence
from biological samples, reduced scattering, and enhanced tissue penetration depth. In this context, we report the
synthesis of rectilinearly π-extended rhodamine dyes using a unique intramolecular nucleophilic substitution of aromatic
www.rsc.org/
H
H
hydrogen (S
N
Ar ) strategy. The strategy makes use of an S
N
Ar reaction between a preorganized aromatic amino nitrogen
H
and an electron-deficient carbon in the xanthylium ion. The S
N
Ar reaction presented herein can be performed under mild
conditions without a transition metal catalyst and can be expected to enable the preparation of a wide variety of π-
extended near-infrared fluorescent rhodamine dyes. Using this strategy, seven rectilinearly π-extended rhodamines (RE1–
RE7) that had fluorescence emission wavelengths in the near-infrared region were synthesized. RE1, RE3, and RE4 were
lysosome targetable and showed good photostabilities. In addition, using dye RE1 as a precursor, we constructed a novel
2
+
NIR fluorescent turn-on probe (RE1-Cu), which can be used for detecting Cu in living cells, demonstrating the value of our
NIR functional fluorescent dyes.
6
have emission wavelengths in the NIR region (λem > 660 nm).
Introduction
The development of these probes was an important milestone
in NIR fluorescence bioimaging. Si-rhodamines possess all the
Considerable effort has been expended towards the
development of fluorescent-probe-based imaging techniques,
which are powerful, noninvasive tools for visualization of
biological processes and which have potential clinical
favorable characteristics of traditional rhodamines, and a
number of excellent rationally designed Si-rhodamine probes
7
have been synthesized for NIR imaging. Extending the π-
1
conjugation of the xanthene moiety by adding aromatic rings
has also been shown to be a versatile strategy for red-shifting
the excitation and emission bands of xanthene dyes. For
example, Strongin et al. synthesized ring-expanded
seminaphthofluorones that exhibit clearly red-shifted
applications. However, the performance of the probes
depends strongly on their photophysical properties. Various
2
fluorescent dyes have been used for designing probes. It has
been reported that the use of near-infrared (NIR) fluorescence
imaging (λem > 650 nm) techniques can minimize photodamage
to living samples, increase tissue penetration depth, and
8
absorption and emission maxima. Other angular π-expanded
9
xanthenes, such as seminaphthorhodamine, also show
3
minimize interference due to autofluorescence. The most
fluorescence emission that is significantly red-shifted (into NIR
region).
widely used NIR dyes for such purposes are cyanine dyes, such
4
as Cy5 and Cy7. However, these dyes have some drawbacks
Herein, we reported the synthesis of rectilinearly π-
for biological imaging; for example, they often suffer from
photobleaching, low quantum yields, and narrow Stokes
extended NIR fluorescent rhodamines via an intramolecular
H
Ar ) reaction
5
nucleophilic substitution of aromatic hydrogen (S
0
N
shifts.
1
under mild metal-free conditions (Scheme 1a). Most of the
widely used methods for constructing C–N bond involve
Recently, Qian, Nagano, and co-workers synthesized Si-
rhodamines, in which the bridging oxygen atom in the
xanthene moiety is replaced with a silicon atom and which
1
1
transition metal catalysis. However, these methods generally
require harsh reaction conditions or generate metal waste,
11d
and these features limit their applications. Metal-free S
H
N
Ar
reactions offer an potential alternative approach for
10
constructing C–N bonds, but reported examples of metal-
H
free S
conducted under mild conditions are limited in number. In
this study, we synthesized seven dyes RE1 RE7 (Scheme 1b) by
fusing an aromatic ring directly to the xanthene scaffold of
N
Ar reactions for constructing C–N bond that can be
1
2
–
H
N
rhodamine via a metal-free intramolecular S Ar reaction. The
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1
0
emission maxima of RE1
–
RE7 were significantly red-shifted (to Both computational and experimental studies
have
the NIR region) compared to the corresponding emission demonstrated that the critical factor for the success of an
H
maxima of traditional rhodamine dyes. Dyes RE1
,
RE3 and RE4
N
S Ar reaction is that the aromatic carbon center attacked by
are highly lysosome targetable and exhibit clear fluorescence the nucleophile must be sufficiently electron deficient to favor
H
signals in living cells without any background interferences. In the formation of a transient σ adduct, which undergoes
order to demonstrate the value of our dyes for probe design, a subsequent oxidation to yield the C–N linking product.
2
probe, RE1-Cu for Cu was synthesized and applied for Therefore, we began by synthesizing xanthylium ion
+
2
+
imaging of Cu in living cells.
intermediate
6, which has an electron-deficient carbon at the
7
-position of the xanthene moiety and which we expected
H
would efficiently form the transient σ adduct necessary for
X
N
Ar
the synthesis of RE1 (Scheme 2b). Specifically, an S
reaction of 2-chloronitrobenzene ( ) and 4-methoxylphenol (
afforded intermediate (Scheme 2a). Condensation of with
-(4-(N,N-diethylamino)-2-hydroxybenzoyl)benzoic acid in
CH SO H afforded rhodamine analogue through
Then was
reduced with SnCl ·2H O in ethanol to afford amine , which
was refluxed in methanol in the presence of concentrated
H2SO4 to afford the key xanthylium ion ( ). Finally, the
rectilinearly π-extended rhodamine dye RE1 was obtained in
1
2)
3
3
2
4
3
3
2
f, 26
Friedel−Craꢀs type reacꢁons (Scheme S10).
4
5
2
2
6
H
5
2% yield by intramolecular S Ar reaction of 6 under basic
N
conditions (Na CO ) at 50 °C (Schemes 2a and S1). It is
2
3
noteworthy that
a solution of 6 in DMF underwent
spontaneously conversion to RE1 at room temperature within
Scheme 1 a) Retrosynthetic analysis of rectilinearly π-extended rhodamines RE1–RE6
and b) structures of RE1–RE7.
a few days in the absence of any base, which indicated that the
H
S Ar reaction could occur under mild conditions.
N
H
A proposed mechanism for the critical intramolecular S Ar
N
annulation of
to the ammonium ion form, compound
resonance structures: oxonium ion and transient carbocation
. The oxonium ion and carbocation structures are favorable
6 to form RE1 is shown in Scheme 2b. In addition
2
c
6
has two other
6
6
'
H
for the intramolecular S Ar reaction at the highly electron-
N
H
deficient C-7. From
6, the S Ar reaction can proceed via a
N
Michael-type addition of the amino nitrogen atom to C-7 to
H
form a six-membered-ring transient σ adduct RE1 . From 6'
H
,
H
the S Ar reaction can proceed via addition of the nitrogen
N
atom to the transient carbocation at the 7-position to form
H
RE1 . Then RE1 is converted to RE1 via spontaneous oxidation
H
1
5
to yield the C–N linking product. It is noteworthy that amine
did not undergo the annulation reaction even at elevated
temperature (110 °C) in toluene, mainly because exists in a
5
5
spirolactone structure under neutral and basic conditions in
organic solvents. This result implies that the intramolecular
H
SNAr annulation reaction of
6 was driven primarily by
electronic effects, that is, by the electron deficiency at C-7.
H
To evaluate the feasibility of this S
N
Ar annulation strategy,
H
we considered a number of factors that might influence the
H
Scheme 2 a) Synthesis of RE1. b) Proposed mechanism of the S
form RE1. Reagents and conditions: (i) DMF, Na CO
diethylamino)-2-hydroxybenzoyl)benzoic acid, CH SO H, 85 °C. (iii) SnCl
iv) MeOH, H SO (98 wt%), reflux. (v) Na CO , EtOAc, 50 °C. (vi) Na CO
C.
N
Ar reaction of 6 to
2
3
N
, 100 °C. (ii) 2-(4-(N,N- S Ar annulation reaction, such as electronic effects, as well as
3
3
2
, EtOH, 85 °C.
replacement of the benzene ring with a heterocyclic ring or
replacement of the bridging oxygen atom with a sulfur atom or
(
2
4
2
3
2
3
, toluene, 110
°
a benzo ring. Specifically, we synthesized rectilinearly π-
H
extended rhodamines RE2–RE7 via the intramolecular S Ar
N
reaction (Schemes 1 and S2–S7). The synthetic strategy mainly
includes two critical steps: the synthesis of 2'-aminoaryl linked
Results and discussion
H
N
Design and synthesis of π-extended rhodamine dyes RE1-RE7
xanthylium and the intramolecular S Ar reaction to form the
rectilinearly π-extended NIR rhodamine dyes. Followed the
similar synthesis procedures for the preparation of dye RE1
,
2
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dyes RE2
materials 35 was synthesized by the Suzuki coupling reaction contributes to the formation of a coplanar conjugated
between 4-bromo-5-nitroacenaphthene 33 and 4- structure composed of five six-membered rings, namely, three
methoxyphenylboronic acid 34 RE2 and RE3 contain an fused benzene rings separated by an oxazine ring and a pyran
-RE6 were prepared. For RE7, the critical starting intramolecular annulation. The formation of the N1=C8 bond
.
electron-donating methoxyl group and an electron- ring; the structure is analogous to that of pentacene (Fig. 1).
withdrawing nitro group, respectively. RE4 and RE5 are The mean plane of the five fused rings was almost orthogonal
pyridine- and quinoline-fused rhodamines, respectively. In (dihedral angle 89.10°) to the plane of the benzene ring of the
RE6, the oxygen atom is replaced with a sulfur atom, and the benzoic acid methyl ester group.
benzene ring is replaced with a pyridine ring. Different from
RE1–RE6, the acenaphthene moiety in RE7 is linked to
xanthene directly without a linker atom, such as oxygen or
H
sulfur, and the intramolecular S
smoothly under very mild conditions (Scheme S7). Our
successful syntheses of RE1 RE7 confirmed the feasibility of
N
Ar reaction still proceeds
–
H
the SNAr C–N annulation strategy for the efficient
construction of rectilinearly π-extended rhodamine dyes.
Scheme 3 The control experiment of synthesis of 43. Reagents and conditions: (i) DMF,
o
Na
2
CO
3
, 100 C for 14 h; (ii) 2-(4-(N,N-diethylamino)-2-hydroxybenzoyl)benzoic acid,
o
CH
3 3
SO
H, 85 C for 3 d; (iii) EtOH, H
2
SO
4
, refluxed for 24 h; (iv) SnCl
2 2
·2H O, EtOH,
o
o
refluxed for 10 h; (v) Na
2 3
CO
, EtOAc, 50 C for 4 h; (vi) Na CO , toluene, 110 C for 4 h.
2
3
Fig. 1 Single crystal X-ray structure of RE1. a) Top view along the xanthylium plane and
Control experiment
b) side view along the xanthylium plane.
H
N
Ar
To verify our speculation that the intramolecular S
Optical properties of dyes RE1–RE7
reaction proceeds only at the highly electron-deficient
transient carbocation of the xanthylium skeleton, we The photophysical properties of RE1–RE7 in ethanol were
conducted a control experiment (Schemes 3 and S8). investigated (Fig. 2 and S1, Table 1). RE1 showed intense
Specifically, we prepared compound 43, in which the 2'- absorption and emission bands at 628 and 680 nm,
aminophenoxyl moiety is attached to C-7 of the xanthylium ion, respectively, the latter representing a large (108 nm) red shift
and then we attempted to prepare annulation product 44a or relative to the emission band of rhodamine B (Rho B). RE2
H
,
4
4b by means of the intramolecular S
H
N
Ar reaction. As which has an electron-donating methoxyl group, showed even
N
Ar reaction of 43 did not proceed even at larger red shifts in the absorption and emission wavelengths
expected, the S
increased temperature (110 °C), because a highly electron- (to 649 and 716 nm, respectively). Conversely, introduction of
deficient transient carbocation cannot form at either the 6- an electron-withdrawing nitro group (RE3) induced a clear blue
position or the 8-position.
shift relative to RE1, as well as a remarkable increase of the
fluorescence quantum yield (Φ = 0.33, λabs/λem = 604/647 nm).
X-Ray crystal structure analysis of RE1
f
Like RE3, the electron-deficient pyridine-fused dye RE4 also
showed blue- shifted spectra relative to those of RE1 and an
To confirm the formation of the C–N bond, we obtained a
single crystal of RE1 by slow evaporation of 2:1 (v/v)
f f
increased Φ (λabs/λem = 602/665 nm, Φ = 0.108). Compared
2 2
MeOH/CH Cl and subjected it to X-ray crystallographic
with RE4, the pyridine-fused RE6, in which the oxygen atom
was replaced with a sulfur atom, showed prominent red shifts
of both the absorption and the emission wavelengths (λabs/λem
analysis (Table S1 and Fig. 1). We were encouraged to observe
that the amino nitrogen was bonded to C-8 of the xanthene
H
Ar annulation. The
moiety, which is solid evidence for the S
N
=
654/745 nm). Quinoline-fused RE5 (λabs/λem = 618/721 nm,
N1–C8 bond length was 1.3414 Å, which is typical for a C=N
bond and is much shorter than the N1–C1 bond length (1.3936
Å); this means that the N1=C8 bond formed after the
Φ
Φ
f
< 3%) and acenaphthene-fused RE7 (λabs/λem = 677/830 nm,
f
< 3%), which have π systems that are even more extended
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than that of RE1, exhibited remarkably red-shifted absorption
and emission wavelengths, but their Φ values were
Table 1 Photophysical properties of rhodamine B (Rho B) and RE1–RE7 in ethanol.
f
a
abs
b
c
f
λ
ε
λ
em
Φ
Stokes shift
diminished. It is noteworthy that all seven of the dyes we
synthesized exhibited larger Stokes shifts (43 nm) than that of
Rho B (19 nm), demonstrating their potential utility for
bioimaging applications requiring a high signal-to-noise ratio
with enhanced contrast discrimination. The emission maxima
Dye
(
nm)
(M-1 cm-1)
117000
93600
(nm)
572
680
716
647
665
728
745
830
(%)
53
(nm)
19
Rho B
RE1
RE2
RE3
RE4
RE5
RE6
RE7
553
628
649
604
602
618
654
677
5.3
< 3
33
52
44000
67
94000
43
of the seven dyes and Rho B decreased in the order RE7
>
146400
43400
10.8
< 3
< 3
< 3
63
RE6 > RE5 > RE2 > RE1 > RE4 > RE3 Rho B, suggesting that
>
110
91
extending the π-conjugation system, introducing an electron-
donating group, or replacing the bridging oxygen atom with
sulfur atom could increase the absorption and emission
wavelengths.
31000
22000
153
a
b
c
Maximum absorption wavelength. Maximum emission wavelength. Relative
f
fluorescence quantum yield estimated by using Nile Blue (Φ = 0.27 in ethanol)
1
3
as a fluorescence standard.
available subcellular organelle markers. In images obtained
with RE1 RE3, and RE4, the fluorescence of all three dyes
,
Fig. 3 Colocalization of RE1, RE3, and RE4 (50 nM) with LysoTracker Green DND-26
Lyso-TG) (200 nM) in L929 cells: (a1-c1) fluorescence images from Lyso-TG; (a2-c2)
fluorescence images from RE1, RE3, and RE4, respectively; (a3-c3) merged images; and
a4-c4) bright-field images.
(
(
colocalized perfectly with that of LysoTracker Green DND-26
the Pearson's coefficients were 0.94, 0.94 and 0.93,
Fig. 2 Normalized UV–vis absorption a) and fluorescence b) spectra of RE1–RE7 in
(
ethanol.
respectively; Fig. 3). In contrast, the fluorescence of the dyes
did not colocalize with that of ER-Tracker Green, MitoTracker
Green, or Golgi-Tracker Green (Fig. S3–S5). These findings
Biological imaging and cell location studies
NIR dyes are reportedly favorable for fluorescence imaging of demonstrate that RE1
living cells because NIR photons cause minimal photodamage in the lysosomes. In an MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-
to living cells and minimum interference from sample diphenyl-2H-tetrazolium) assay, RE1 RE3, and RE4 exhibited
values, we low cytotoxicity (cell viability >95%) at concentrations up to
RE3, and RE4 for 250 nM after incubation with L929 cells, a murine aneuploid
bioimaging applications. First, costaining of HeLa cells with fibrosarcoma cell line, for 24 h (Fig. S6). Because the pH within
',6–diamidino–2–phenylindole (DAPI), RE1
RE3, and RE4 led lysosomes is in the 4.5–5.5 range, we evaluated the
to obvious intracellular fluorescence (Fig. S2), indicating that photostabilities of RE1
, RE3, and RE4 accumulated specifically
,
1
4
autofluorescence. On the basis of the Φ
evaluated the potential utility of RE1
f
,
4
,
1
6
,
RE3, RE4, Rho B, and Lyso-TG in
the
molecules
were
membrane-permeable.
6b
Some phosphate-buffered saline (pH 5.5). After continuous
reportedly localize in irradiation for 2000 s, the fluorescence intensity of Lyso-TG
1
rhodamines and Si-rhodamines
5
mitochondria owing to their lipophilic and cationic character.
Therefore, we used RE1
RE3, and RE4 for subcellular intensities of RE1
localization experiments involving colocalization of these dyes this result indicates that RE1
at a low concentration (50 nM) with various commercially superior photostability of the parent xanthene dye. Under the
had decreased to 93%, but little decrease in the emission
RE3 RE4, and Rho B were observed (Fig. S7);
RE3, and RE4 retained the
,
,
,
,
4
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2
, RE5, and RE6 all showed good the real-time detection of Cu . In addtion, probe RE1-Cu
+
same conditions, RE2
2
+
photostabilities as well; no obvious decreases in fluorescence exhibited good selectivity towards Cu over other metal ions
intensity were observed after 2000 s of irradiation (Fig. S7). (Fig. S13a), and the competition experiment (Fig. S13b)
Meanwhile, the photostabilities of RE1, RE3, RE4, and Lyso-TG revealed that all of the competitive metal ions had minor or no
were also investigated in cells. As shown in Fig. S8 and Videos interference with the fluorescence response of RE1-Cu
2
S1-S4, dyes RE1 and RE4 possess superior photostability. towards Cu . Then, the fluorescence response of RE1-Cu to
+
2
However, the fluorescence signal intensity of RE3 decayed Cu at different pH levels was explored and the results implied
+
2
gradually to 13% afer 30 min continuous irradiation and Lyso- that the probe can be used for detecting Cu in the pH range
+
TG became non-fluorescent only after 2 min.
of 4 to 9, which meets the requirements for fluorescent tracers
for use in biological applications (Fig. S14).
Scheme 4 Synthesis of the probe RE1-Cu.
Application of dye RE1 for designing fluorescent probe
Dyes RE1-RE7 possess rhodamine-like spirolactam structure,
which provides a universal platform for the design of efficient
NIR turn-on bioimaging probes for various targets. To
demonstrate the feasibility of this probe design, a NIR
2
+
fluorescent probe, RE1-Cu, was synthesized for sensing Cu by
utilizing dye RE1 as fluorophore and 2-hydroxyethylhydrazine
unit as recognition unit (Scheme 4). Copper is the third-most
abundant transition metal in the human body and plays a key
role in a variety of fundamental physiological processes in
organisms including enzyme functions and transcriptional
1
7
events. However, the disruption of copper homeostasis is
1
closely connected with human genetic disorders like Wilson’s
8
1
9
and Menkes disease; neurodegenerative diseases such as
2
0
21
Alzheimer’s,
Therefore, the detection of copper in living systems is very
Parkinson’s, and Huntington’s
diseases.
2
2
important.
Fig. 4 showed the spectroscopic properties of RE1-Cu at
2
+
various Cu concentrations in PBS buffer (0.01 M, pH 7.4,
Fig. 4 a) Absorption and b) fluorescence spectra of RE1-Cu (5 μM) with different
2
+
containing 25% CH CN as a cosolvent). No obvious absorption concentrations of Cu in PBS buffer (0.01 M, pH 7.4, containing 25% CH
3
CN as a
3
cosolvent).
and fluorescence of RE1-Cu were observed because of its
+
2
spirolactam form. The addition of Cu ions elicited marked
increases of absorption and fluorescence emission at 620 and
680 nm, respectively, along with an obvious change from
colorless to blue. HRMS confirmed that the spectral changes of
2
+
RE1-Cu were due to the Cu -mediated ring-opening and
spirolactam hydrolysis to form the fluorophore RE1 (Fig. S9).
2
The fluorescence turn-on constants (Kturn-on) were estimated
3
2
to be 10.67 ± 0.11 μM (R = 0.997) by fluorescence titration
2
+
(
Fig. S10). The detection limit of the probe RE1-Cu for Cu was
evaluated to be 9.1 nM based on the signal-to-noise ratio of
2
4
three method (Fig. S11), which is much lower than the upper
limit of cooper in drinking water defined by Administration of
the People’s Republic of China (ca. 15.7 μM) and U.S.
2
5
2+
Fig. 5 Confocal fluorescence images of Cu using RE1-Cu in L929 cells. (a1-a3) L929
Environmental Protection Agency (EPA) (ca. 20 μM).
The time-dependent fluorescence response of the probe
cells were incubated with RE1-Cu (3 μM, 30 min) only; (b1-b3) L929 cells were
2+
pretreated with Cu (10 μM, 30 min) and then were further incubated with RE1-Cu (3
2
RE1-Cu towards Cu ions was also measured. As shown in Fig.
+
μM) for 30 min. a1) and b1): fluorescence images; a2) and b2): bright-field images; a3)
2
+
S12), the reaction between the probe and Cu could complete and b3) merged images.
in less than 2 min, which means that RE1-Cu can be used for
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The application of RE1-Cu for fluorescence imaging of Cu
Journal Name
2
+
1
2
(a) Z. Guo, S. Park, J. Yoon and I. Shin, Chem. Soc. Rev., 2014,
43, 16; (b) E. L. Que, D. W. Domaille and C. J. Chang, Chem.
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Leung, Acc. Chem. Res., 2014, 47, 3614; (c) M. Beija, C. A. M.
in cultured living cells was demonstrated. L929 cells incubated
with RE1-Cu (3 μM) for 30 min displayed almost no
fluorescence (Fig. 5, a1-a3), which is consistent with the low
brightness of RE1-Cu in aqueous solutions. In the presence of
2
+
Cu , probe-loaded L929 cells showed bright red fluorescence
Fig. 5, b1-b3), which means that RE1-Cu is cell membrane
Afonsoa and J. M. G. Martinhoa, Chem. Soc. Rev., 2009, 38
2
,
410-2433; (d) C. Liu, C. Yang, L. Lu, W. Wang, W. Tan, C.-H.
(
2
permeable and can be used for detecting Cu in living cells.
+
Leung and D.-L. Ma, Chem. Commun., 2017, 53, 2822; (e) Z.
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,
In summary, we synthesized a series of heterocyclic fused π-
4
extended rhodamine dyes
(RE1–RE7) with rectilinearly
arranged fused aromatic rings. The dyes were synthesized via
H
3
4
(a) N. Karton-Lifshin, E. Segal, L. Omer, M. Portnoy, R. Satchi-
Fainaro and D. Shabat, J. Am. Chem. Soc., 2011, 133, 10960;
N
an intramolecular S Ar reaction to cyclize heterocyclic rings to
(
b) J. M. Baumes, J. J. Gassensmith, J. Giblin, J.-J. Lee, A. G.
White, W. J. Culligan, W. M. Leevy, M. Kuno and B. D. Smith,
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the benzene ring of preorganized 2-aminoaryl linked
xanthylium under mild conditions without a transition metal
catalyst under mild conditions. The emission maxima of RE1–
RE7 were significantly red-shifted (to the NIR region) relative
to the corresponding maxima for traditional rhodamine dyes
2
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Shangguan and W. Tan, J. Am. Chem. Soc., 2016, 138, 12368.
(
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and RE1, RE3, and RE4 could be utilized for lysosome labeling.
2
+
Furthermore, a NIR fluorescent probe, RE1-Cu for Cu , was
synthesized using RE1 as a platform, and successfully applied
5
6
2
+
for the imaging of Cu in living cells, demonstrating the
potential bioimaging applications of the new probe. We expect
the synthesis strategy that used in our text can be extended to
the preparation of a wide variety of heterocyclic fused π-
extended NIR rhodamine dyes.
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1
,
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7
,
Experimental
1
Additional schemes, UV/Vis and fluorescence spectra figures;
subcellular localization and cytotoxicity experiment
procedures, and confocal microscopy fluorescence imaging;
,
1
J. L. Klocke, D. Kamin, D. N. H. Meineke, E. D’Este, P.-T.
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experimental procedures for synthesis of dyes RE1
-
RE7 and
H, C NMR and HRMS spectra for new
compounds are included in the Supporting Information. The
photostabilites of dyes RE1 RE3 RE4 and Lysotracker green in
L929 were recorded in videos S1-S4 (AVI).
1
13
probe RE1-Cu
,
,
,
CCDC 1582793 (dye RE1
)
contains the supplementary
8
crystallographic data for this paper. These data are provided
free of charge by The Cambridge Crystallographic Data Centre.
Fronczek and R. M. Strongin, J. Am. Chem. Soc., 2012, 134
0502; (b) Y. Yang, M. Lowry, X. Xu, J. O. Escobedo, M.
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,
1
,
8
Conflicts of interest
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Q. A. Best, A. E. Johnson, B. Prasai, A. Rouillere and R. L.
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Acknowledgements
2
0
We gratefully acknowledge the Natural Science Foundation of
China (NNSFC 21272172), and the Scientific Developing
Foundation of Tianjin Education Commission (2017ZD14).
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