Dicyanoboron diketonate dyes: Synthesis, photophysical properties and bioimaging

Article abstract of DOI:10.1016/j.dyepig.2014.07.001

A new class of dicyanoboron diketonates (B(CN)₂) was synthesized and the photophysical properties were investigated. The B(CN)₂ complexes exhibited high molar absorption coefficients, large Stokes shifts, high photostability and low cytotoxicity. Especially, the emission of the B(CN)₂ extended into the deep red region. The extensive π conjugation and the presence of intramolecular charge transfer (ICT) transitions are responsible for their red-shifted emission. Their fluorescence are very sensitive to the polarity of the solvents. They are highly emissive in low polarity solvents, but weakly fluorescent in polar solvents. Cell imaging experiments demonstrated its potential application as a probe in bioorganisms due to its excellent imaging contrast. This strategy represents a facile approach to modulate the photophysical properties of dyes.

Full text of DOI:10.1016/j.dyepig.2014.07.001

Accepted Manuscript
Dicyanoboron Diketonate Dyes: Synthesis, Photophysical Properties and Bioimaging
Yan Zhou, Yu-Zhe Chen, Jian-Hua Cao, Qing-Zheng Yang, Li-Zhu Wu, Chen-Ho
Tung, Da-Yong Wu
PII:
S0143-7208(14)00278-2
10.1016/j.dyepig.2014.07.001
DYPI 4444
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 26 May 2014
Revised Date: 30 June 2014
Accepted Date: 2 July 2014
Please cite this article as: Zhou Y, Chen Y-Z, Cao J-H, Yang Q-Z, Wu L-Z, Tung C-H, Wu D-Y,
Dicyanoboron Diketonate Dyes: Synthesis, Photophysical Properties and Bioimaging, Dyes and
Pigments (2014), doi: 10.1016/j.dyepig.2014.07.001.
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ACCEPTED MANUSCRIPT
Graphical abstract
We report a new class of dicyanoboron diketonates complexes which exhibit high
molar absorption coefficients, large Stokes shifts, high photostability and low
cytotoxicity.

ACCEPTED MANUSCRIPT
Dicyanoboron Diketonate Dyes: Synthesis, Photophysical
Properties and Bioimaging
a,b
a
a
a
a
Yan Zhou , Yu-Zhe Chen* , Jian-Hua Cao , Qing-Zheng Yang , Li-Zhu Wu ,
a
a
Chen-Ho Tung , Da-Yong Wu*
a
Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, 29 Zhongguancun
East Road, Beijing, 100190, China.
b
University of The Chinese Academy of Sciences, Beijing, 100049, China
*
Corresponding author. Fax: +86 10 62554670, Tel: +86 10 82543772.
E-mail: chenyuzhe@mail.ipc.ac.cn (Y. Chen), dayongwu@mail.ipc.ac.cn (D. Wu).
Abstract
A new class of dicyanoboron diketonates (B(CN) ) was synthesized and the
2
photophysical properties were investigated. The B(CN) complexes exhibited high
2
molar absorption coefficients, large Stokes shifts, high photostability and low
cytotoxicity. Especially, the emission of the B(CN) extended into the deep red region.
2
The extensive π conjugation and the presence of intramolecular charge transfer (ICT)
transitions are responsible for their red-shifted emission. Their fluorescence are very
sensitive to the polarity of the solvents. They are highly emissive in low polarity
solvents, but weakly fluorescent in polar solvents. Cell imaging experiments
demonstrated its potential application as a probe in bioorganisms due to its excellent
imaging contrast. This strategy represents a facile approach to modulate the
photophysical properties of dyes.
1

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Keywords
Dicyanoboron diketonate, fluorescence dye, optical properties, solvent effect
1
. Introduction
Organoboron complexes are one of the most important types of fluorescent dyes
[
1]. Notably, because of their large molar extinction coefficients, high emission
quantum yields, and sensitivity to the surrounding medium, boron dipyrromethene
dyes (BODIPYs) and boron diketonates have attracted much interest in the fields of
molecular probes [2,3], optical imaging [4,5], sensing applications [6], and laser dyes
[
7,8]. In addition to the rich photophysical properties which they share in common
with BODIPYs, boron diketonates also show large two photon absorption cross
sections, air stability, pronounced room temperature phosphorescence and delayed
fluorescence when incorporated with polymer matrices in the solid state [9,10].
Within this context, the synthesis and studies of boron diketonates have gained much
attention recently [11]. Various efforts have been made to tune the photophysical
properties of the organoboron complexes, which is in essence, the alteration of the
transition energy levels of the dye molecules [12]. This change is often effected by
chemically modifying the π conjugation or the substituent groups. Extending the
absorption and emission properties of the dyes to the deep red/near-infrared region
would facilitate their application in biological systems by avoiding interferences from
background autofluorescence, minimizing photodamage to samples, and increasing
tissue penetration [13]. For organoboron complexes, replacing fluorine with other
functional groups such as aryl, ethynylaryl etc. represents a very simple and effective
2

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method for such a purpose. For example, Ziessel et al. have reported on the
substitution of fluorine of BODIPY by alkynyl, alkynylaryl, ethynylaryl and aryl
groups [14]. Such derivatives share not only improvement of their stabilities but also
large Stokes shifts in their emission spectra. In contrast, very few difluoroboron
diketonate derivatives, with fluorine replaced by other groups as analogous
derivatives, have been reported [15]. Information such as emission properties in
solution and the solid state have not yet been studied.
In line with our ongoing work on BODIPY and boron diketonates as fluorescent
sensors for the detection of biological thiols and various metal ions in solution and in
living cells [16], we decided to extend our investigation toward tuning the
photophysical properties of diketonate derivatives. In particular, we intended to
design boron 1, 3-diketonate dyes which are fluorescent in the deep red/near-infrared
region by simple methods and to apply them for in vivo studies. In this paper, we
present the synthesis, absorption and fluorescence emission properties, and theoretical
calculations of dicyanoboron diketonates (B(CN) , 1c-3c, Scheme 1). The substitution
2
of fluorine by cyano group red shifted the emission > 25 nm. Especially, the emission
of 3c extended into the deep red region. The B(CN) complexes exhibited high molar
2
absorption coefficients, large Stokes shifts, good imaging contrast, high photostability
and low cytotoxicity.
Scheme 1. Structures of the difluoroboron diketonates 1b-3b and dicyanoboron diketonates 1c-3c.
3

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2
. Experimental Section
.1 General Experimental Methods
2
1
13
H and C NMR spectra were recorded on an Advance Bruker 400M
spectrometer (400 MHz) using tetramethylsilane (TMS) as an internal standard in
CDCl at room temperature. Mass spectra (ESI) were obtained on Bruker Apex IV
3
Fourier Transform Mass Spectrometer. Mass spectra (EI) were obtained in the
positive ion mode on a Waters GCT premier. Fourier-Transform Infrared (FT-IR)
Spectra were recorded on a Varian Excalibur 3100 infrared spectrometer.
Fluorescence spectra were determined on a Hitachi F-4600 spectrophotometer.
Absorption spectra were determined on a Hitachi U-3900 UV-Visible
spectrophotometer. Fluorescence lifetimes were recorded on Edinburgh F900
spectrometer. Tetrahydrofuran (THF) was refluxed with sodium in the presence of
benzophenone as indicator for 5 h and then distilled under nitrogen. Other reagents
were analytical grade and were used without further purification.
2
2
.2 Synthesis
.2.1 Synthesis of 1a
1
a was synthesized by a standard Claisen condensation under basic conditions
according to the literature [17] (Scheme S1). The crude product was recrystallized in
1
ethanol to give 1a as yellow needle crystals (2.06 g, 72.5%). H NMR (400 MHz,
CDCl ) δ 17.11 (s, 1H, enol-OH), 7.96 (d, 4H), 7.00 (d, 4H), 6.74 (s, 1H, COCHCO),
3
1
3
3
1
.90 (s, 6H, OCH ). C NMR (100 MHz, CDCl ) δ 192.90, 184.74, 164.09, 163.18,
3 3
31.43, 129.70, 129.21, 128.30, 114.09, 91.60, 55.62, 55.57. MS (EI) m/z calcd for
+
C H O [M] 284.10, found 284.11.
17
16
4
4

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2
.2.2 Synthesis of 1b
To a solution of 1a (1.42 g, 5 mmol) in CH Cl (40 mL), boron trifluoride diethyl
2
2
etherate (1.25 mL, 10 mmol) and triethylamine (1 mL, 7.5 mmol) was added. After
being stirred for 6 h at room temperature in the dark, the reaction solution was washed
with water (50 mL) and brine (50 mL). The organic layer was dried over anhydrous
MgSO and the solvent was removed in vacuo to give the crude product. Purification
4
by flash column chromatography (silica gel, petroleum/CH Cl 1:1) gave 1b as a
2
2
1
yellow green powder (1.56 g, 94%) [17]. H NMR (400 MHz, CDCl ) δ 8.14 (d, J =
3
13
9
.0 Hz, 4H), 7.11 – 6.93 (m, 5H), 3.94 (s, 6H, OCH ). C NMR (100 MHz, CDCl ) δ
3 3
1
80.84, 165.32, 131.25, 124.56, 114.57, 91.53, 55.75. MS (EI) m/z calcd for
+
C H O BF [M] 332.10, found 332.11.
17
15
4
2
2
.2.3 Synthesis of 1c
To a solution of 1b (100 mg, 0.3 mmol) in CH Cl (20 mL), trimethylsilyl cyanide
2
2
(120 ꢀL, 1 mmol) and boron trifluoride diethyl etherate (125 ꢀL, 1 mmol) were added.
After stirring at room temperature for 1 h, the reaction solution was washed with
water (30 mL) and brine (30 mL). The organic layer was dried over anhydrous
MgSO and the solvent was removed in vacuo to give the crude product. Purification
4
by flash column chromatography (silica gel, petroleum/CH Cl 1:3) gave 1c as a
2
2
1
yellow powder (55 mg, 53%). H NMR (400 MHz, CDCl ) δ 8.12 (d, J = 9.0 Hz, 4H),
3
1
3
7
1
.14 – 6.94 (m, 5H), 3.96 (s, 6H). C NMR (100 MHz, CDCl ) δ 179.78, 166.66,
3
32.08, 123.21, 115.06, 93.65, 55.99. HRMS (ESI) m/z calcd for C H BN NaO
1
9
15
2
4
+
[
M+Na] 369.10205, found 369.10210.
5

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2
.2.4 Synthesis of 2a
a was synthesized by a standard Claisen condensation as described for 1a [17]
2
(Scheme S1). Purification by flash column chromatography (silica gel,
1
petroleum/ethyl acetate 4:1) gave 2a as a dark yellow solid. Yield 70%. H NMR (400
MHz, CDCl ) δ 17.09 (s, 1H, enol-OH), 7.94 (d, J = 8.9 Hz, 4H), 6.98 (d, J = 8.9 Hz,
3
4
H), 6.72 (s, 1H, COCHCO), 6.06 (ddd, J = 22.5, 10.6, 5.3 Hz, 2H), 5.44 (d, J = 17.3
1
3
Hz, 2H), 5.33 (d, J = 10.5 Hz, 2H), 4.61 (d, J = 5.3 Hz, 4H, OCH ). C NMR (100
2
MHz, CDCl ) δ 192.55, 184.57, 162.97, 162.07, 132.68, 132.47, 131.27, 129.79,
3
1
29.04, 128.44, 118.11, 117.99, 114.71, 114.63, 91.54, 68.93. MS (EI) m/z calcd for
C H O 336.14, found 336.14.
21
20
4
2
.2.5 Synthesis of 2b
b was synthesized by the same method as 1b. Purification by flash column
chromatography (silica gel, petroleum/CH Cl 1:2) gave 2b as a bright yellow powder
2
2
2
1
[
17]. Yield 90%. H NMR (400 MHz, CDCl ) δ 8.10 (d, J = 8.9 Hz, 4H), 7.11 – 6.91
3
(m, 5H), 6.06 (ddd, J = 22.4, 10.5, 5.3 Hz, 2H), 5.45 (d, J = 17.3, 2H), 5.35 (d, J =
1
3
1
1
1
0.5, 2H), 4.65 (d, J = 5.2 Hz, 4H, OCH ). C NMR (100 MHz, CDCl ) δ 184.64,
2
3
80.94, 164.40, 162.14, 132.74, 132.18, 131.34, 131.23, 129.10, 128.50, 124.77,
18.50, 118.07, 115.31, 114.78, 91.65, 69.23, 69.00. MS (EI) m/z calcd for
+
C H O BF [M] 384.13, found 384.14.
21
19
4
2
2
.2.6 Synthesis of 2c
c was synthesized by the same method as 1c. Purification by flash column
chromatography (silica gel, petroleum/CH Cl 1:2) gave 2c as a yellow solid. Yield
2
2
2
6

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1
4
6
2
1
8%. H NMR (400 MHz, CDCl ) δ 8.09 (d, J = 8.8 Hz, 4H), 7.18 – 6.90 (m, 5H),
3
.06 (ddd, J = 22.2, 10.4, 5.2 Hz, 2H), 5.46 (d, J = 17.3 Hz, 2H), 5.37 (d, J = 10.5 Hz,
13
H), 4.69 (d, J = 4.9 Hz, 4H, OCH ). C NMR (100 MHz, CDCl ) δ 179.71, 165.66,
2
3
32.07, 131.72, 129.07, 123.23, 118.91, 118.17, 115.70, 114.69, 93.70, 69.43, 68.94.
+
HRMS (ESI) m/z calcd for C H BN NaO [M+Na] 421.13342, found 421.13403.
2
3
19
2
4
2
.2.7 Synthesis of 3a
3
a was obtained as a white powder by reaction of benzoylacetone and
1
trifluoroboron diethyl etherate. Yield 92%. H NMR (400 MHz, CDCl ) δ 8.07 (d, J =
3
7
3
1
2
.3 Hz, 2H), 7.70 (t, J = 7.5 Hz, 1H), 7.54 (t, J = 7.9 Hz, 2H), 6.60 (s, 1H), 2.43 (s,
1
3
H, CH ). C NMR (100 MHz, CDCl ) δ 192.69, 182.91, 135.47, 131.21, 129.21,
3
3
+
29.01, 97.51, 24.75. MS (EI) m/z calcd for C H O BF [M] 210.07, found 210.07.
1
0
9
2
2
.2.8 Synthesis of 3b
To a solution of 3a (1.05 g, 5 mmol) in EtOH (60 mL), was added
dimethylaminobenzaldehyde (0.89 g, 6 mmol). After it had dissolved completely,
hexahydropyridine (60 ꢀL, 6 mmol) was added into the solution. After refluxing at 80ꢀ
for 16 h, EtOH was removed in vacuo. The residue was washed with water and
extracted with CH Cl . The organic layer was dried over anhydrous MgSO and the
2
2
4
solvent was removed in vacuo to give the crude product. Purification by flash column
chromatography (silica gel, petroleum ether/CH Cl 1:4) gave 3b as a blue powder
2
2
1
(
600 mg, 35%). H NMR (400 MHz, CDCl ) δ 8.12 (d, J = 15.2 Hz, 1H, Ar'-CH=C),
3
8
.05 (d, J = 7.5 Hz, 2H, Ar-H), 7.62 (t, J = 7.4 Hz, 1H, Ar-H), 7.56 (d, J = 8.9 Hz, 2H,
Ar'-H), 7.51 (t, J = 7.7 Hz, 2H, Ar-H), 6.71 (d, J = 8.9 Hz, 2H, Ar'-H), 6.57 (d, J =
7

ACCEPTED MANUSCRIPT
13
1
5.2 Hz, 1H, CH=CH(CO)), 6.53 (s, 1H, (CO)CH(CO)), 3.11 (s, 6H, CH ). C NMR
3
(
100 MHz, CDCl ) δ 180.99, 178.86, 153.31, 149.67, 133.92, 132.72, 132.18, 130.91,
3
1
29.93, 128.90, 128.55, 128.28, 127.14, 121.91, 114.16, 111.98, 97.06, 40.14. HRMS
+
(
ESI) m/z calcd for C H BF NNaO [M+Na] 364.12943, found 364.12937.
1
9
18
2
2
2
.2.9 Synthesis of 3c
c was synthesized from 3b by the same method as 1c. Purification by flash
column chromatography (silica gel, petroleum ether/CH Cl 1:2) gave 3c as a dark
3
2
2
1
blue powder. Yield 42%. H NMR (400 MHz, CDCl ) δ 8.12 (d, J = 14.8 Hz, 1H,
3
Ar'-CH=C), 7.97 (d, J = 7.4 Hz, 2H, Ar-H), 7.68 – 7.56 (m, 3H), 7.51 (t, J = 7.8 Hz,
2
H, Ar-H), 6.73 (d, J = 9.0 Hz, 2H, Ar'-H), 6.58 – 6.49 (m, 2H), 3.17 (s, 6H, CH3).
1
3
C NMR (100 MHz, CDCl ) δ 178.91, 176.01, 154.50, 153.07, 134.59, 133.81,
3
1
31.72, 129.11, 128.36, 121.98, 112.44, 112.34, 99.14, 40.27. HRMS (ESI) m/z calcd
+
for C H BN NaO [M+Na] 378.13788, found 378.13880.
2
1
18
3
2
2
.3 Fluorescence quantum yields measurement
The fluorescence quantum yields Φ were calculated from the relation shown in the
following equation:
2
2
s
Φ = Φ × (F /A ) × (A /F ) × (n /n )
u
s
u
u
s
s
u
in which u and s denote a dye and a standard, respectively, A is the absorbance at the
excitation wavelength, F is the integrated emission area, and n is the refractive index
for the solvent [18]
.
Fluorescence quantum yields, Φ , for β-diketonate derivatives (1b, 1c, 2b, 2c) in
f
8

ACCEPTED MANUSCRIPT
CH Cl were calculated versus anthracene in EtOH as a standard method using the
2
2
2
D
0
20
following values: Φ (anthracene) =0.27, n (EtOH) = 1.360, and nD (CH Cl ) =
f
2
2
1
.424 [19,20]. Optically dilute CH Cl solutions of β-diketonate derivatives and EtOH
2 2
solutions of the anthracene standard were prepared in 1 cm path length quartz cuvettes
with absorbances < 0.1. Quantum yield measurements were performed with excitation
at λ = 356 nm and emission integration range = 370-700 nm. Fluorescence quantum
ex
yields for β-diketonate derivatives (3b, 3c) in CH Cl were calculated versus
2
2
rhodamine B in EtOH as a standard method using the following values: Φf
2
0
20
(
rhodamine B) =0.5, nD (EtOH) = 1.360, and nD (CH Cl ) = 1.424 [19,20]. Optically
2 2
dilute CH Cl solutions of β-diketonate derivatives and EtOH solutions of the
2
2
rhodamine B standard were prepared in 1 cm path length quartz cuvettes with
absorbances < 0.1. Quantum yield measurements were performed with excitation at
λex = 500 nm and an emission integration range = 520-750 nm.
2
.4 Cell culture and cell imaging
HeLa cells were cultured in Dulbecco's Modified Eagle’s Medium (DMEM),
supplemented with 10% Fetal Bovine Serum (FBS), penicillin (100 units/mL), and
streptomycin (100 ꢀg/mL) at 37 °C in a humidified atmosphere of 5% CO for 24 h.
2
The cells were seeded in a glass-bottom cell culture dish (NEST Biotechnology Co.
LTD.) and incubated with 1 ꢀM compound 3c at 37 °C in a humidified atmosphere of
5
% CO for 6 h, then were replaced three times with fresh growth medium. Confocal
2
cell imaging was performed on a Nikon Eclipse Ti confocal laser scanning
microscopy (CLSM) with a TDKAI HIT live cell imaging system. Fluorescence was
9

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excited at 561 nm and emission was collected by a 570-620 nm band pass filter.
3
. Results and Discussion
3
.1 Synthesis and Characterization
The Claisen condensation reaction of acetophenone and benzoate gave the ligands
1
a-2a (Scheme S1 in supplementary data) [17], which exist as their enol forms in
1
solution as indicated by the H NMR. 1a-2a were then allowed to react with the boron
trifluoride-diethyl ether complex in the presence of triethylamine in dichloromethane
to afford the corresponding BF complexes 1b-2b. The B(CN) (1c, 2c, 3c) were
2
2
synthesized by treatment of the corresponding difluoroboron diketonates (1b, 2b, 3b)
with trimethylsilyl cyanide (TMSCN) in the presence of boron trifluoride diethyl
etherate in CH Cl at room temperature (Scheme 2). The successful substitutions were
2
2
1
13
confirmed by H and C NMR, IR and HRMS (see supplementary data).
Scheme 2. Synthesis of dicyanoboron diketonate complexes.
3
.2 Optical Properties
Figure 1. (a) UV-vis absorption and (b) normalized fluorescence spectra of 1b-3b, 1c-3c in
-5
dichloromethane (1.0 × 10 M).
1
0

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Table 1. Photophysical Properties of Difluoroboron Diketonates and Dicyanoboron Diketonates.
a
in dichloromethane
solid state
Energy
gapcalc
[eV]
g
compd
b
d
e
f
Fmax
λmax
Fmax
Stokes
τs
kf
9
knr
9
b,c
Φf
-1
-1
[nm]
501
[
(
(
(
(
(
(
nm] (ε)
[nm]
shift [nm]
[ns]
[10 s ]
[10 s ]
4
12
78200)
39
78500)
11
63200)
40
69700)
35
87800)
85
97600)
1
b
438
26
0.85
0.66
0.72
0.24
2.15
0.40
0.07
3.7144
3.5076
3.6409
3.4123
3.0287
2.8491
4
1
c
464
438
466
621
664
25
27
26
86
79
2.23
1.96
2.05
0.51
0.30
0.37
0.12
0.15
0.14
0.37
1.91
0.70
547
501
550
4
2
b
4
2
c
5
h
3
b
0.026
<0.00
0.05
——
5
h
h
3
c
1.43 <0.001
——
h
1
a
-5
-3 b
Measured at a concentration of 1.0 × 10 mol dm . The excitation wavelengths (λ ) were as follows: 1b (c)
ex
c
(
356 nm), 2b (c) (356 nm), 3b (450 nm) and 3c (530 nm). Φ is the relative fluorescence quantum yield estimated
f
using anthracene (Φ = 0.27 in ethanol) for 1b, 1c, 2b, 2c or rhodamine B (Φ = 0.5 in ethanol) for 3b-3c as
f
f
d
e
f
standard. Measured using time-resolved fluorescence measurements. Radiative rate constant (k = Φ /τ ).
f
f f
g
Nonradiative rate constant (k = (1 − Φ )/τ ). The λ were as follow: 1b (410 nm), 1c (440 nm), 2b (410 nm), 2c
nr
f
f
ex
h
(
440 nm). Too weak to be measured.
To examine the optical properties of the obtained boron dicyanodiketonates,
UV-vis absorption and emission spectra were carried out in CH Cl (Figure 1). The
2
2
data are compared to those of the model BF complexes 1b-3b (Table 1). Substitution
2
of fluorine by cyano group strongly influences the absorption and emission spectra.
-1
-1
1
c-3c exhibited strong absorption (ε > 60000 M cm ) in the 440-580 nm region,
which are red-shifted > 25 nm compared to those of BF complexes. Especially in 3c,
2
1
1

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the absorption maxima shifted as much as 43 nm compared to that of 3b. The molar
absorption coefficients of B(CN) complexes are generally higher than those of the
2
BF compounds. The emission maxima of B(CN) complexes are red-shifted
2
2
proportionally with the absorption changes. In other words, the Stokes shift didn’t
change much after cyano substitution.
As shown in Table 1, the organoboron BF dyes 1b and 2b are highly emissive,
2
with fluorescence quantum yields in CH Cl at room temperature are 0.85 and 0.72,
2
2
respectively. After substitution with cyano group, the quantum yields of 1c and 2c
decrease to 0.66 and 0.24. In light of the fact that the nonradiative rate constants (knr)
9
-1
9
-1
of 1c (0.15×10 s ) and 2c (0.37×10 s ) are higher compared with those of 1b
9
-1
9
-1
(
0.07×10 s ) and 2b (0.14×10 s ), there may be other processes in the nonradiative
decay of B(CN) compounds responsible for the lower quantum yields [21,22].
2
The absorption and emission of 3b and 3c in CH Cl are in the deep red region
2
2
due to their more extensive π conjugation and the presence of intramolecular charge
transfer (ICT) process. The molar absorption coefficient (ε) of 3c is significantly
higher than those of 1b (c) and 2b (c), which might be due to the extension of π
conjugation and the two substituted cyano groups. Very weak fluorescence was
observed for 3b and 3c in CH Cl for reasons to be discussed below. The Stokes shifts
2
2
for 3b and 3c are as large as 86 nm, which implies that there is a large difference in
the molecular structures between the ground and excited states. The presence of ICT
process is indicated [23]. The excitation spectra of 3b and 3c almost match well with
their absorption spectra. (Figure S1)The emission of B(CN) complexes (1c and 2c)
2
1
2

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were red-shifted 84 nm in their solid state with respect to those in solution, while the
emission of BF complexes (1b and 2b) in the solid state were red shifted 63 nm
2
compared with those in solution. These changes might be due to different packing
structures in the solids with different substituents for the BF and B(CN) complexes.
2
2
Fluorescence of 3b and 3c in the solid state was too weak to be observed.
3
.3 Solvent Effects
Dye emission color tuning can also take advantage of solvatochromism. Polar
dyes in particular can be sensitive to the properties of their surroundings. The polarity
or dielectric constant of the local medium can affect the ground state or excited state
energies of fluorophores or both. To demonstrate the solvent effect, the absorption
and emission of 3b and 3c in different solvents were measured (Figure 2, Table 2 and
3
). The absorption spectra of 3b and 3c show minor changes in different solvents,
which suggests that the difference among their ground-state dipole moments in
different solvents is rather small. However, the color changes of 3b and 3c in different
solvents could easily be visualized by naked-eye (Figure S2 and S3). For example, the
color of 3c changed from pink to blue when the solvent was changed from toluene to
acetonitrile. The fluorescence emission spectra of 3b and 3c exhibit positive
solvatochromism in general. The fluorescence quantum yields of 3b and 3c, which are
high in toluene (0.5 and 0.28 respectively), are too low to be determined in the more
polar solvent such as methanol.
Typically, the fluorescence emission spectra from an ICT state are very dependent
on the polarity of solvents [24]. The fluorescence quantum yields are low in polar
1
3

ACCEPTED MANUSCRIPT
solvents but high in low polarity ones. Consequently, the presence of an ICT process
in 3b and 3c was indicated. Kinetic constants were also determined for radiative and
nonradiative deactivation pathways in various solvents and are collected in Table 2
and Table 3. The radiative constants k of 3b and 3c are found to decrease with the
f
polarity of solvents, accompanied by an increase of the nonradiative constant knr.
According to the energy gap rule [25], this decrease in fluorescence quantum yield
(
Φ ) and lifetime (τ ) is attributed to the acceleration of internal conversion (ic) as the
f s
energy gap between the excited and the ground state decreases. Thus, the observed
positive solvato-kinetic effect does not arise from the population of different emitting
species during the lifetime of the excited state, but the quenching of the highly polar
ICT state which is caused by an increase of kic.
-5
Figure 2. Fluorescence spectra of 3b (a) and 3c (b) in various solvents (1.0 × 10 M).
Table 2. Absorption and Emission Properties of 3b in Various Solvents.
c
e
f
g
Fmax
nm]
Stokes shift
τs
kf
9
knr
9
a
b
d
solvent
ꢁf
λmax [nm] (ε)
Φf
-
1
-1
-1
[
[nm/cm ]
54/1820
70/2200
89/2790
86/2590
52/1690
84/2600
58/1830
[ns]
2.42
2.55
0.52
0.51
1.97
0.35
1.26
[10 s ]
[10 s ]
Toluene
CHCl3
THF
0.02
0.15
0.21
0.22
0.28
0.30
0.31
519 (62400)
530 (72600)
522 (71200)
535 (87800)
530 (69500)
528 (63400)
535 (66600)
573
600
611
621
582
612
593
0.50
0.17
0.21
0.21
0.07
0.33
1.88
0.024
0.026
<0.001
<0.001
<0.001
0.05
CH Cl2
0.05
1.91
0.51
2
h
h
h
h
Acetone
<0.001
h
CH OH
<0.001
2.86
3
h
CH CN
<0.001
0.79
3
a
-5
-3
b
2
Measured at a concentration of 1.0 × 10 mol dm . Solvent polarity parameter ([(ε − 1)/(2ε + 1)] − [(n −
1
4

ACCEPTED MANUSCRIPT
2
c
d
1
)/(2n + 1)]) (ref 23). The excitation wavelengths (λ ) is 450 nm in all solvents. Φ is the relative fluorescence
ex f
e
quantum yield estimated by using rhodamine B (Φ = 0.5 in ethanol) as standard. Measured using time-resolved
f
f
g
h
fluorescence measurements. Radiative rate constant (k = Φ /τ ). Nonradiative rate constant (knr = (1 − Φ )/τ ).
f
f
f
f
f
Too weak to be measured.
Table 3. Absorption and Emission Properties of 3c in Various Solvents.
c
e
f
g
Fmax
Stokes shift
τs
kf
9
knr
9
a
b
d
solvent
ꢁf
λmax [nm] (ε)
Φf
-
1
-1
-1
[
nm]
[nm/cm ]
52/1457
59/1592
79/2067
79/2034
82/2102
79/2027
89/2250
[ns]
2.53
0.63
1.42
1.43
1.99
0.61
1.30
[10 s ]
[10 s ]
Toluene
CHCl3
THF
0.02
0.15
0.21
0.22
0.28
0.30
0.31
572 (104400)
580 (107700)
580 (92700)
585 (97600)
585 (96100)
586 (93100)
586 (92500)
624
639
659
664
667
665
675
0.28
0.021
0.11
0.28
0.03
1.55
0.70
0.002
0.001
h
h
h
h
h
CH Cl2
<0.001
<0.001
<0.001
<0.001
<0.001
0.70
2
h
Acetone
<0.001
0.50
h
CH OH
<0.001
1.64
3
h
CH CN
<0.001
0.77
3
a
-5
-3
b
2
Measured at a concentration of 1.0 × 10 mol dm . Solvent polarity parameter ([(ε − 1)/(2ε + 1)] − [(n −
2
c
d
1
)/(2n + 1)]). The excitation wavelengths (λ ) is 530 nm in all solvents. Φ is the relative fluorescence quantum
ex f
e
yield estimated by using rhodamine B (Φ = 0.5 in ethanol) as standard. Measured using time-resolved
f
f
g
h
fluorescence measurements. Radiative rate constant (k = Φ /τ ). Nonradiative rate constant (knr = (1 − Φ )/τ ).
f
f
f
f
f
Too weak to be measured.
3
.4 Theoretical Calculations
The Gaussian 03 software package [26] was used for all computational modeling.
The geometries for 1b-3b and 1c-3c were optimized with a density functional theory
DFT) method, using the B3LYP//6-311+G* basis set. Figure 3 shows the lowest
unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital
(
1
5

ACCEPTED MANUSCRIPT
(HOMO) for 1b-3b, 1c-3c. The corresponding energy gaps between LUMO and
HOMO are included in Table 1.
The substitution of fluorine by cyano group in the boron diketonates did not
change the location of the π orbital of the 1,3-diketone group. As a result, the
transition between the excited state and ground state is allowed because the π,π*
excited states of these six compounds occupy the lowest excited state. In contrast, the
bathochromic shift experimentally observed for B(CN) complexes after cyano
2
substitution can be attributed to more pronounced lowering of the LUMO rather than
elevation of the HOMO. On the other hand, as indicated by the DFT calculations,
extended conjugation in 3b and 3c leads to elevation of the HOMO levels and thus the
observed bathochromic shift of the emission compared with 1b (c) and 2b (c) .
Figure 3. Calculated molecular orbital energy diagram and isodensity surface plots of the frontier
orbitals (HOMO and LUMO) of 1b-3b, 1c-3c.
3
.5 Confocal Fluorescent Images of Cells
Fluorescence probes are useful for imaging various biological functions and
diagnostic application. Considering the promising biological applications, some
cell-imaging experiments of 3c were conducted. In our experiment, HeLa cells were
incubated with 3c at the concentration of 1 ꢀM for 6 h. We chose this dye because its
deep red fluorescence had minimal interference from background autofluorescence of
the cells. It is apparent from Figure 4 that 3c could readily penetrate cell membranes,
1
6

ACCEPTED MANUSCRIPT
and the strong intracellular fluorescence may be attributed to the distribution of the
dyes in the lipophilic environment of the cytoplasm. Although 3c shows almost no
fluorescence (Φ < 0.001) in polar solvents, the fluorescence intensity in cytoplasm
was sufficient to provide strong contrast for imaging due to its possible enrichment
inside the low polarity microenvironment of cytoplasm. Almost no background
fluorescence could be observed.
Figure 4. Confocal fluorescence images of living HeLa cells: (a) bright field image of living HeLa
cells; (b) fluorescence image of living HeLa cells incubated with 1 ꢀM 3c for 6 h; (c) the overlap
of bright field and fluorescence images.
For a fluorescence probe to be of practical value, photostability is very important
because of the possible temporal monitoring of dynamic events inside cells. To
evaluate the photostability of the probe, the deep red dyes 3b and 3c were irradiated in
toluene continuously at λ > 420 nm using a 500 W Xe lamp as the collimated light
source. As monitored by UV spectra, the absorption intensity of 3c could keep 93% at
5
67 nm after 15 min continuous irradiation, while the intensity of 3b dropped to 85%
(Figure 5). This indicates that the photostability of the cyano group substituted boron
diketonates derivatives is much higher than that of BF compounds. The cytotoxicity
2
of 3c in HeLa cells was examined using the Cell Counting Kit-8 (CCK-8) assay.
The results indicate that 3c has low cytotoxicity (cell viability > 95%) after incubation
for 4 hours, even at 10 ꢀM (Figure S4 in supplementary data).
1
7

ACCEPTED MANUSCRIPT
-5
Figure 5. Photostability of 3b and 3c (1.0 × 10 M) in toluene.
4
. Conclusions
In summary, we have synthesized a new class of dicyanoboron diketonates from
difluoroboron diketonate derivatives. Dicyanoboron diketonates show relatively high
-1
-1
molar absorption coefficients (> 60000 M cm ) and larger red-shifts (> 25 nm) in
their absorption and emission spectra than do the corresponding difluoroboron
diketonates. Especially, the fluorescence emission wavelength of 3c extended to the
deep red region (664 nm in CH Cl ). The emission of 3b and 3c are very sensitive to
2
2
the polarity of solvents. The fluorescence quantum yields of 3b and 3c are reasonably
high in low polarity solvents, but too low to be determined in high polarity solvents.
Cell imaging experiments demonstrated the potential application of 3c as a probe in
bioorganisms due to its good imaging contrast, large Stokes shift, low cytotoxicity
and high photostability. This strategy represents a facile approach to modulate the
photophysical properties of dyes.
Acknowledgments
This work was financially supported by the National Natural Science Foundation
of China (21272243, 21102155, 91027041) and the 973 Program (2013CB933800).
We thank Dr. Ye Tian (TIPC, CAS) for her help in cell imaging and Dr. Man Wu
(TIPC, CAS) for her help in the cytotoxicity test.
Appendix A. Supplementary data
1
8

ACCEPTED MANUSCRIPT
Absorption spectra of 3b and 3c in various solvents; Cytotoxicity; Synthetic route
1
13
for 1a and 2a; H NMR, C NMR, MS-EI , HRMS-ESI and FT-IR spectra of 1a-3c.
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/xxx.
References
[
1] (a) Loudet A, Burgess K. BODIPY Dyes and Their Derivatives: Syntheses and Spectroscopic
Properties. Chem Rev 2007;107(11):4891-932.
(
b) Kamkaew A, Lim SH, Lee HB, Kiew LV, Chung LY, Burgess K. BODIPY dyes in
photodynamic therapy. Chem Soc Rev 2013;42(1):77-88.
(
c) Boens N, Leen V, Dehaen W. Fluorescent indicators based on BODIPY. Chem Soc Rev
2
012;41(3):1130-72.
(
d) Wade CR, Broomsgrove AEJ, Aldridge S, Gabbaı FoP. Fluoride Ion Complexation and
Sensing Using Organoboron Compounds. Chem Rev 2010;110:3958-84.
(
e) Benniston AC, Copley G. Lighting the way ahead with boron dipyrromethene (Bodipy) dyes.
Phys Chem Chem Phys 2009;11(21):4124-31.
[
[
[
[
2] Zhai D, Lee SC, Yun SW, Chang YT. A ratiometric fluorescent dye for the detection of glutathione
in live cells and liver cancer tissue. Chem Commun 2013;49(65):7207-9.
3] Zhang HX, Chen JB, Guo XF, Wang H, Zhang HS. Highly sensitive low-background fluorescent
probes for imaging of nitric oxide in cells and tissues. Anal Chem 2014;86(6):3115-23.
4] Zhang G, Palmer GM, Dewhirst MW, Fraser CL. A dual-emissive-materials design concept
enables tumour hypoxia imaging. Nat Mater 2009;8(9):747-51.
5] Liu W, Li F, Chen X, Hou J, Yi L, Wu YW. A Rapid and Fluorogenic TMP-AcBOPDIPY Probe for
1
9

ACCEPTED MANUSCRIPT
Covalent Labeling of Proteins in Live Cells. J Am Chem Soc 2014;136(12):4468-71.
6] (a) Karakus E, Ucuncu M, Emrullahoglu M. A rhodamine/BODIPY-based fluorescent probe for
the differential detection of Hg(II) and Au(III). Chem Commun 2014;50(9):1119-21.
[
(
b) Madhu S, Ravikanth M. Boron-dipyrromethene based reversible and reusable selective
chemosensor for fluoride detection. Inorg Chem 2014;53(3):1646-53.
(
c) P CA, Thilagar P. Triarylborane-appended new triad and tetrad: chromogenic and fluorogenic
anion recognition. Inorg Chem 2014;53(6):2776-86.
[
[
[
[
[
7] Garcı´a-Moreno I, Costela A, Campo L, Sastre R, Amat-Guerri F, Liras M, et al.
8
-Phenyl-Substituted Dipyrromethene·BF Complexes as Highly Efficient and Photostable Laser
2
Dyes. J Phys Chem A 2004;108:3315-23.
8] García-Moreno I, Amat-Guerri F, Liras M, Costela A, Infantes L, Sastre R, et al. Structural
Changes in the BODIPY Dye PM567 Enhancing the Laser Action in Liquid and Solid Media. Adv
Funct Mater 2007;17(16):3088-98.
9] Xu S, Evans RE, Liu T, Zhang G, Demas JN, Trindle CO, et al. Aromatic difluoroboron
β-diketonate complexes: effects of π-conjugation and media on optical properties. Inorg Chem
2
013;52(7):3597-610.
10] Zhang G, Chen J, Payne SJ, Kooi SE, Demas JN, Fraser CL. Multi-Emissive Difluoroboron
Dibenzoylmethane Polylactide Exhibiting Intense Fluorescence and Oxygen-Sensitive
Room-Temperature Phosphorescence. J Am Chem Soc 2007;129:8942-3.
11] (a) Mayoral MJ, Ovejero P, Cano M, Orellana G. Alkoxy-substituted difluoroboron
benzoylmethanes for photonics applications: a photophysical and spectroscopic study. Dalton
Trans 2011;40(2):377-83.
2
0

ACCEPTED MANUSCRIPT
(
b) Zhang G, Kooi SE, Demas JN, Fraser CL. Emission Color Tuning with Polymer Molecular
Weight for Difluoroboron Dibenzoylmethane-Polylactide. Adv Mater 2008;20(11):2099-104.
(
c) Yoshii R, Nagai A, Tanaka K, Chujo Y. Highly emissive boron ketoiminate derivatives as a
new class of aggregation-induced emission fluorophores. Chem Eur J 2013;19(14):4506-12.
(
d) Wang DJ, Kang YF, Xu BP, Zheng J, Wei XH. Synthesis, characterization and fluorescence
properties of boron difluoride pyridyl-β-diketonate derivatives. Spectrochim Acta A Mol Biomol
Spectrosc 2013;104:419-22.
(
e) Kumar GR, Thilagar P. Triarylborane conjugated acacH ligands and their BF complexes:
2
facile synthesis and intriguing optical properties. Dalton Trans 2014;43(10):3871-9.
[
12] (a) Sanchez-Carnerero EM, Moreno F, Maroto BL, Agarrabeitia AR, Ortiz MJ, Vo BG, et al.
Circularly Polarized Luminescence by Visible-Light Absorption in a Chiral O-BODIPY Dye:
Unprecedented Design of CPL Organic Molecules from Achiral Chromophores. J Am Chem Soc
2
014;136(9):3346-9.
(
b) Rohand T, Baruah M, Qin W, Boens N, Dehaen W. Functionalisation of fluorescent BODIPY
dyes by nucleophilic substitution. Chem Commun 2006(3):266-8.
(
c) Kubota Y, Ozaki Y, Funabiki K, Matsui M. Synthesis and fluorescence properties of
pyrimidine mono- and bisboron complexes. J Org Chem 2013;78(14):7058-67.
(
d) Zhao C, Feng P, Cao J, Zhang Y, Wang X, Yang Y, et al. 6-Hydroxyindole-based
borondipyrromethene: synthesis and spectroscopic studies. Org Biomol Chem 2012;10(2):267-72.
(
e) Boens N, Wang L, Leen V, Yuan P, Verbelen B, Dehaen W, et al. 8-HaloBODIPYs and Their
8
-(C, N, O, S) Substituted Analogues: Solvent Dependent UV-Vis Spectroscopy, Variable
Temperature NMR, Crystal Structure Determination, and Quantum Chemical Calculations. J Phys
2
1

ACCEPTED MANUSCRIPT
Chem A 2014;118(9):1576-94.
(
f) Hao Q., Yu S., Li S., Chen J., Zeng Y., Yu T., Yang G., Li Y. Locked Planarity: A Strategy for
Tailoring Ladder-Type π-Conjugated Anilido–Pyridine Boron Difluorides. J. Org. Chem., 2014,
7
9 (1): 459–464.
[
[
13] Chen J, Liu W, Zhou B, Niu G, Zhang H, Wu J, et al. Coumarin- and rhodamine-fused deep red
fluorescent dyes: synthesis, photophysical properties, and bioimaging in vitro. J Org Chem
2
013;78(12):6121-30.
14] (a)
Goeb
Sb,
Ziessel
R.
Convenient
Synthesis
of
Green
the
Diisoindolodithienylpyrromethene-Dialkynyl Borane Dyes. Org Lett 2007;9(5):737-40.
(
b)
Li
L,
Nguyen
B,
Burgess
K.
Functionalization
of
4
2
,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) core. Bioorg Med Chem Lett
008;18(10):3112-6.
(
c) Nagai A, Miyake J, Kokado K, Nagata Y, Chujo Y. Highly Luminescent BODIPY-Based
Organoboron Polymer Exhibiting Supramolecular Self-Assemble Structure. J Am Chem Soc
2
008;130(46):15276-8.
(
d) Ulrich G, Goze C, Goeb Sb, Retailleau P, Ziessel R. New fluorescent aryl- or
ethynylaryl-boron-substituted indacenes as promising dyes. New J Chem 2006;30(7):982.
(
e) Goze C, Ulrich G, Mallon LJ, Allen BD, Harriman A, Ziessel R. Synthesis and Photophysical
Properties of Borondipyrromethene Dyes Bearing Aryl Substituents at the Boron Center. J Am
Chem Soc 2006;128(31):10231-9.
(
f) Bonardi L, Ulrich G, Ziessel R. Tailoring the Properties of Boron-Dipyrromethene Dyes with
Acetylenic Functions at the 2,6,8 and 4-B Substitution Positions. Org Lett 2008;10(11):2183-6.
2
2

ACCEPTED MANUSCRIPT
(
g) Goze C, Ulrich G, Ziessel R. Tetrahedral Boron Chemistry for the Preparation of Highly
Efficient “Cascatelle” Devices. J Org Chem 2007;72(2):313-22.
(
h) Goze C, Ulrich G, Ziessel R. Unusual Fluorescent Monomeric and Dimeric Dialkynyl
Dipyrromethene-Borane Complexes. Org Lett 2006;8(20):4445-8.
15] (a) Nagai A, Kokado K, Nagata Y, Arita M, Chujo Y. Highly Intense Fluorescent Diarylboron
Diketonate. J Org Chem 2008;73:8605-7.
[
(
b) Kubota Y, Hara H, Tanaka S, Funabiki K, Matsui M. Synthesis and Fluorescence Properties of
Novel Pyrazine-Boron Complexes Bearing
a
β-Iminoketone Ligand. Org Lett
2
011;13(24):6544-7.
(
c) Poon C-T, Lam WH, Yam VW-W. Synthesis, Photochromic, and Computational Studies of
Dithienylethene-Containing β-Diketonate Derivatives and Their Near-Infrared Photochromic
Behavior Upon Coordination of a Boron(III) Center. Chem Eur J 2013;19(10):3467-76.
(
d) Nagai A, Kokado K, Nagata Y, Chujo Y. 1,3-Diketone-Based Organoboron Polymers:
Emission by Extending π-Conjugation along
a
Polymeric Ligand. Macromolecules
2
008;41:8295-8.
[
16] (a) Xin L, Chen YZ, Niu LY, Wu LZ, Tung CH, Tong QX, et al. A selective turn-on fluorescent
2+
probe for Cd based on a boron difluoride beta-dibenzoyl dye and its application in living cells.
Org Biomol Chem 2013;11(18):3014-9.
(
b) Niu LY, Guan YS, Chen YZ, Wu LZ, Tung CH, Yang QZ. A turn-on fluorescent sensor for the
discrimination of cystein from homocystein and glutathione. Chem Commun 2013;49(13):1294-6.
(
c) Niu LY, Guan YS, Chen YZ, Wu LZ, Tung CH, Yang QZ. BODIPY-based ratiometric
fluorescent sensor for highly selective detection of glutathione over cysteine and homocysteine. J
2
3

ACCEPTED MANUSCRIPT
Am Chem Soc 2012;134(46):18928-31.
[
[
[
[
[
17] Sun X, Zhang X, Li X, Liu S, Zhang G. A mechanistic investigation of mechanochromic
luminescent organoboron materials. J Mater Chem 2012;22(33):17332.
18] Resch-Genger U, Rurack K. Determination of the photoluminescence quantum yield of dilute dye
solutions (IUPAC Technical Report). Pure Appl Chem 2013;85(10).
19] Brouwer AM. Standards for photoluminescence quantum yield measurements in solution (IUPAC
Technical Report). Pure Appl Chem 2011;83(12).
20] Weast RC and Astle MJ. CRC Handbook of Data on Organic Compounds, Vol. I and II. CRC Press
1
985.
21] Caldwell RA, Jacobs LD, Furlani TR, Nalley EA, Laboy J. Conformationally Dependent Heavy
Atom Effect of Chlorine on Alkene Triplet Lifetimes. Experimental and ab Initio Calculations. J
Am Chem Soc 1992;114:1623-5.
[
[
[
22] Gorman A, Killoran J, Shea CO, Kenna T, Gallagher WM, Shea DFO. In Vitro Demonstration of
the Heavy-Atom Effect for Photodynamic Therapy. J Am Chem Soc 2004;126:10619-31.
23] Rurack K, Kollmannsberger M, Daub J. Molecular Switching in the Near Infrared (NIR) with a
Functionalized Boron-Dipyrromethene Dye. Angew Chem Int Ed 2001;40:385-7.
24] (a) Moyon NS, Mitra S. Fluorescence solvatochromism in lumichrome and excited-state
tautomerization: a combined experimental and DFT study. J Phys Chem A 2011;115(12):2456-64.
(
b) Hu R, Lager E, Aguilar-Aguilar Al, Liu J, Lam JWY, Sung HHY, et al. Twisted Intramolecular
Charge Transfer and Aggregation-Induced Emission of BODIPY Derivatives. J phys Chem C
2
009;113:15845-53.
[
25] Wardle B. Principles and Applications of Photochemistry. Wiley 2009.
2
4

ACCEPTED MANUSCRIPT
[
26] Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Montgomery JAJ,
Vreven T, Kudin KN, Burant JC, Millam JM, Iyengar SS, Tomasi J, Barone V, Mennucci B, Cossi
M, Scalmani G, Rega N, Petersson GA, Nakatsuji H, Hada M, Ehara M, Toyota K, Fukuda R,
Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Klene M, Li X, Knox JE,
Hratchian HP, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O,
Austin AJ, Cammi R, Pomelli C, Ochterski JW, Ayala PY, Morokuma K, Voth GA, Salvador P,
Dannenberg JJ, Zakrzewski VG, Dapprich S, Daniels AD, Strain MC, Farkas O, Malick DK,
Rabuck AD, Raghavachari K, Foresman JB, Ortiz JV, Cui Q, Baboul AG, Clifford S, Cioslowski J,
Stefanov BB, Liu G, Liashenko A, Piskorz P, Komaromi I, Martin RL, Fox DJ, Keith T, Al-Laham
MA, Peng CY, Nanayakkara A, Challacombe M, Gill PMW, Johnson B, Chen W, Wong MW,
Gonzalez C, Pople JA. Gaussian 03, Gaussian Inc., Wallingford CT, 2004.
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Captions
Scheme 1. Structures of the difluoroboron diketonates 1b-3b and dicyanoboron diketonates 1c-3c.
Scheme 2. Synthesis of dicyanoboron diketonate complexes.
Figure 1. (a) UV-vis absorption and (b) normalized fluorescence spectra of 1b-3b, 1c-3c in
-5
dichloromethane (1.0 × 10 M).
-5
Figure 2. Fluorescence spectra of 3b (a) and 3c (b) in various solvents (1.0 × 10 M).
Figure 3. The calculated molecular orbital energy diagram and isodensity surface plots of the
frontier orbitals (HOMO and LUMO) of 1b-3b, 1c-3c.
Figure 4. Confocal fluorescence images of living HeLa cells: (a) bright field image of living HeLa
cells; (b) fluorescence image of living HeLa cells incubated with 1 µM 3c for 6 h; (c) the overlap
of bright field and fluorescence images.
-5
Figure 5. Photostability of 3b and 3c (1.0 × 10 M) in toluene.
1

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Scheme 1
2

ACCEPTED MANUSCRIPT
Scheme 2
3