BODIPY derivatives bearing borneol moieties: Enhancing cell membrane permeability for living cell imaging

Article abstract of DOI:10.1016/j.dyepig.2019.01.012

Three novel boron-dipyrromethene (BODIPY) dyes bearing borneol moieties have been designed, synthesized and characterized. The single-crystal structure of the compound 3 was also elucidated for the first time. Their photophysical properties and confocal f

Full text of DOI:10.1016/j.dyepig.2019.01.012

Accepted Manuscript
BODIPY derivatives bearing borneol moieties: Enhancing cell membrane permeability
for living cell imaging
Jian Yang, Yunshi Fan, Fangjian Cai, Xu Xu, Bo Fu, Shifa Wang, Zhen Shen,
Jiangwei Tian, Haijun Xu
PII:
S0143-7208(18)32422-7
https://doi.org/10.1016/j.dyepig.2019.01.012
DYPI 7285
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 2 November 2018
Revised Date: 31 December 2018
Accepted Date: 9 January 2019
Please cite this article as: Yang J, Fan Y, Cai F, Xu X, Fu B, Wang S, Shen Z, Tian J, Xu H, BODIPY
derivatives bearing borneol moieties: Enhancing cell membrane permeability for living cell imaging, Dyes
and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.01.012.
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ACCEPTED MANUSCRIPT
BODIPY derivatives bearing borneol moieties: Enhancing cell membrane
permeability for living cell imaging
a,#
b,#
a
a
a
a
Jian Yang , Yunshi Fan , Fangjian Cai , Xu Xu , Bo Fu , Shifa Wang , Zhen
c,
b,
a,d,
Shen *, Jiangwei Tian *, Haijun Xu
*
a
College of Chemical Engineering, Jiangsu Key Lab of Biomass-based Green Fuels and
Chemicals, Nanjing Forestry University, Nanjing 210037, P. R. China.
b
State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of TCM
Evaluation and Translational Research, School of Traditional Chinese Pharmacy,
China Pharmaceutical University, Nanjing 211198, P. R. China.
c
Nanjing Natl Lab Microstruct, State Key Lab Coordinat Chem, Nanjing University
2
10046, Jiangsu, P. R. China.
d
Jiangsu Key Laboratory for Biomass Energy and Material, Jiangsu Province, Nanjing
2
10042, P. R. China.
#
These authors equally contribute to this work.
*
Corresponding author: Dr. and Prof. Haijun Xu
College of Chemical Engineering, Jiangsu Key Lab of Biomass-based Green Fuels and
Chemicals, Nanjing Forestry University, Nanjing 210037, P. R. China.
Jiangsu Key Laboratory for Biomass Energy and Material, Jiangsu Province, Nanjing
2
10042, P. R. China.
Email: xuhaijun@njfu.edu.cn, Phone: +86-25-85427432;
1

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*
*Corresponding author: Dr. and Prof. Jiangwei Tian
State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of TCM
Evaluation and Translational Research, School of Traditional Chinese Pharmacy, China
Pharmaceutical University, Nanjing 211198, P. R. China.
Email: jwtian@cpu.edu.cn，Phone: 86-18260088865
*
**Corresponding author: Dr. and Prof. Zhen Shen
Nanjing Natl Lab Microstruct, State Key Lab Coordinat Chem, Nanjing University
10046, Jiangsu, P. R. China.
Email: zshen@nju.edu.cn, Phone: 86-25-83621250;
2
2

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Abstract
Three novel boron-dipyrromethene (BODIPY) dyes bearing borneol moieties have
been designed, synthesized and characterized. The single-crystal structure of the
compound 3 was also elucidated for the first time. Their photophysical properties and
confocal fluorescence images were investigated by the optical spectroscopy and
Confocal fluorescence microscopy. The results indicate that the ompounds 2-3 take on
high fluorescence quantum yield and cell membrane permeability, which can be utilized
as fluorescent visualizers for cell and lysosome fluorescence imaging.
Keywords: BODIPY; cell imaging; borneol; photophysical properties; membrane
permeability
3

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1
. Introduction
Fluorescence imaging has emerged as a very facile and powerful tool to enable the
visualization, characterization, and quantification of biological processes that occur at
[1-5]
cellular levels without disturbing living subjects in complex biosystems
. This
imaging technique has been widely applied in tumor diagnosis, biomolecule detection,
tracking therapeutic effects, the distribution of drug in vivo due to its noninvasion, high
spatiotemporal resolution, and real-time visual tracking of biological structures and
[5-8]
processes in the living systems
.
Over the past few decades, various of fluorophores with different excitation and
emission wavelengths have been designed and developed including coumarin, 1,8-
naphthalimide, boron dipyrromethene difluoride (BODIPY), rhodamine, fluoroscein,
[
2,9-12]
cyanine, etc
. Among them, the BODIPY derivatives have attracted considerable
interest as one of the most promising candidates for fluorescent bio-imaging due to their
excellent photophysical properties, such as remarkable fluorescence quantum yields,
good biocompatibility, easy modification, sharp fluorescence emission, high
photostability, large molar extinction coefficients, and relative insensitivity to
[
13-15]
environmental conditions
. In particular, the parent structure of the BODIPY is
easily modified, and functional groups can be introduced to the BODIPY core, while
retaining their inherent properties. As a result, the BODIPY dyes are widely used for
[
16-18]
fluorescent bioimaging in medicine and biochemistry
. However, the big challenge
is the poor cell membrane permeability of BODIPY dyes, which limits their application
[
16,19]
in cell biological imaging
. So, it is still highly necessary and urgent to design
fluorescent BODIPY derivatives with good cell permeability.
4

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It is well known that borneol is a kind of natural chiral compounds. It has many good
pharmacological activities and functions, such as analgesic, anti-inflammatory,
[
20]
antibacterial
. It can be used as chiral source to synthesize new functional organic
compounds. Recently, borneol compounds have been used to improve the cell
[
21]
permeability of drugs which have attracted widespread attention . Borneol can also
carry a variety of drugs through the blood-brain barrier and make drugs work better for
[
22]
target organs . To the best of our knowledge, the research has not been published on
the synthesis of fluorescent BODIPY derivatives containing borneol moiety and their
application in fluorescent imaging yet. In this paper, we have successfully synthesized
fluorescent BODIPY derivatives containing borneol group, and report the synthesis,
basic photophysical properties and application of these molecules.
2
. Results and discussion
2
.1. Synthesis
The compounds 1-4 were synthesized according to the routes outlined in Scheme 1.
4
-formylbenzoic acid was reacted with excess SOCl in the presence of catalytic amount
2
of DMF under reflux to yield 4-formylbenzoyl chloride. Bornyl 4-formylbenzoate was
synthesized by treating 4-formylbenzoyl chloride with (-)-Bomeol in the presence of
pyridine in THF at room temperature. 1 and 2 were obtained by the reaction of two
equivalents of 2, 4-dimethy-pyrrole and 1 equivalent of 4-methoxybenzaldehyde or
bornyl 4-formylbenzoate in the presence of a catalytic amount of TFA and subsequently
addition of an excess of TEA and BF ⋅OEt . 3 and 4 were acquired by the condensation
3
2
[23,24]
of 2 with bornyl 4-formylbenzoate using the Knoevenagel condensation method
.
1
All compounds were characterized by elemental analyses, H-NMR and HRMS with
5

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satisfactory results. The detailed experimental procedure and characterization data are
provided in the ESI.
2
.2. X-ray structure of the compound 3
The structure of 3 has been determined by X-ray analysis. Fig. 1 shows the
perspective view of the molecular conformation of 3, and there are two
crystallographically independent BODIPY molecules in the asymmetric unit. The
selected bond lengths and angles are listed in Table S2. The bond lengths for B-N and
B-F and the bond angles of N-B-N and F-B-F indicate a tetrahedral BF2N2 geometry
[
25,26]
and are in good agreement with previously reported data
. The C20-C21 and C58-
C59 bond lengths are both 1.319(5) Å, indicating a double bond character in a trans
conformation formed by the condensation reaction between 4,4-difluoro-1, 3, 5, 7-
tetramethyl-8-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene
and
bornyl
4-
formylbenzoate. The bond lengths of C28-O2 and C66-O5 are 1.330(4) Å and 1.317(5)
Å, respectively, which is much shorter with respect to the bond distances of C29-
O2(1.439(5) Å) and C67-O5 (1.455(5) Å), showing that there are π-electron
delocalization within the ester moiety. The meso-C2-C7 phenyl ring and the meso-C40-
C45 phenyl ring on the BODIPY core are virtually perpendicular to the corresponding
indacene plane with the dihedral angle of 86.38 (0.13) ºand 88.28 (0.13) º, respectively,
resulting from the steric hindrance between the 1,7-methyl groups at the 1,7-positions
on the indacene moiety and the hydrogen atoms at the 8-position of the meso-phenyl
moiety. In addition, the average lengths of the C5-C14 and C43-C52 bond are almost
the same length as a single C-C bond, further suggesting almost no π-electron
delocalization between the meso-aryl group and the BODIPY core (indacene plane).
two indacene planes are highly planar with average root-mean-square (rms) deviation of
6

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0
.0101 and 0.0248 Å, respectively. The two dihedral angles of 27.54 (0.17) and 26.92
(0.18) º between the indacene planes and the styryl groups in the two independent
BODIPY molecules show partial conjugation within the entire chromophore. There are
no π-π stacking interactions between the asymmetrical molecules in compound 3, which
clearly indicates that the introduction of borneol groups can effectively restrain
intermolecular π-π stacking.
2
.3. Optical properties in solution
The photophysical properties of the compounds 1-4 were investigated by absorption
as well as steady-state and time-resolved fluorescence spectroscopy in toluene. The
photophysical data are summarized in Table 1. The normalized absorption and
fluorescence spectra of the compounds 1-4 are depicted in Fig. 2. The compounds 1 and
2
share the common BODIPY framework, and the only difference is the substituents of
the meso position. Therefore, they exhibit similar absorption and emission spectra. As
shown in Fig. 2, the absorption maxima of the compounds 1 and 2 are centred at
approximately 518 nm and 514 nm with molar absorption coefficients in the range
−
1
−1
2
3000-72000 M cm , respectively, which can be ascribed to the strong S →S
0 1
transitions. For the compound 3 and 4, with one or two phenyl styryl groups extending
the π-conjugation, their absorption maxima were found to redshift to 568 nm and 640
nm, respectively. At higher energies, the compounds 3 and 4 also show strong
absorption at 333 nm and 362 nm, which are are attributed to S -Sn (n ≥ 2) transitions
0
[
27]
.
In addition, the weaker and broader shoulders appearing at around 528 nm and 589
nm for 3 and 4 were observed at low wavelength, which are attributed to the 0–1
[
28,29]
vibrational transition of the BODIPY moiety
7
. As shown in Fig. 2, the emission

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bands of the compounds 1-4 are good mirror images of their S →S transition bands
0
1
-1
with a small Stokes shift between 240 and 458 cm , similar to classical meso-aryl
BODIPYs and centred at 518 nm, 514 nm, 577 nm and 650 nm, respectively. The
fluorescence quantum yields of compounds 1-3 determined in non-polar toluene were
were higher than that of compound 4. Thus compounds 1-3 were beneficial to biological
fluorescence imaging research. The fluorescence quantum yields of 1 and 2 are 0.39 and
0
.37, respectively, and they are almost equal. It was noteworthy that the fluorescence
quantum yield of 3 is 0.41, which are higher than that of 2. Such an improvement is
different from the ordinary mono- or di-styryl-substituted BODIPY analogues and
mainly attributed to the rigid steric effects of borneol moieties which limited the
nonradioactive relaxation, thus the fluorescence quantum yield is increased. However, 4
exhibits much weaker fluorescence compared with 2 and 3. 4-methoxylphenyl group is
typical electron-donating group, whereas ester group was a strong electron-withdrawing
group. In the excited state, strong intramolecular charge transfer would happen between
meso-4-methoxylphenyl group and two ester groups, which led to low quantum yields
[
30]
.
In addition, fluorescence lifetimes of 1-3 were also measured with τ values in the
range of 2.6 to 4.86 ns. These photophysical properties account for a very rigid system
[
31,32]
exhibiting a very little loss of energy via non radiative pathway
confirmed by low non radiative kinetic constants compared with the radiative ones
Table 1). The rate constant of radiative (k ) and nonradiative (k ) deactivation can be
. This last point is
(
r
nr
calculated from the measured Φ f and the single-exponential fluorescence lifetime τ
[
33]
according to equations in the literature
. Non-radiative (k ) rate constants are far
nr
[
34]
more dependent on the Bodipy structure . According to Table 1, the very rigid system
in compound 2 makes the compound reducing loss of energy via non radiative pathway
8

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compared to compound 1, the same with conpounds 3 and 4.
2
.4. Cell-imaging of the compounds 1-4
HeLa cells are human cervical carcinoma cells with strong proliferative capacity and
[
35,36]
easy to culture for cell imaging research
. Therefore, HeLa cell line is chosen as the
model to investigate the intracellular localization of BODIPY derivatives by confocal
fluorescence imaging. HeLa cells were incubated with BODIPY compounds bearing
borneol and performed with confocal fluorescence imaging (Fig.3-4). Compound 1 as a
control incubated with HeLa cells showed that the fluorescence of BODIPY was in
cytoplasm (Fig. 3). After incubated with 2.0 µM compound 3 for 1 h, the fluorescence
almost filled the entire cell and distributed throughout the cytoplasm and nucleus,
indicating excellent membrane penetrability of compound 3. In contrast, the
intracellular fluorescence appeared very weak after incubated with 2.0 µM compound 4
under the same condition, which could be attributed to the low fluorescence quantum
yield of compound 4. Notably, cells stained with compound 2 showed remarkable
fluorescence in discrete subcellular locations. To further determine the distribution site
of compound 2, co-staining experiments with LysoTracker Green and Hoechst 33342
has been carried out. As seen from the overlay images, the fluorescence ascribable to
compound 2 co-localized well with LysoTracker Green (Fig. 4). The result was further
confirmed by stack image for Z-depth scanning of Compound 2-loaded cells, suggesting
that Compound 2 has high lysosome-targeting capability. The different cellular
localization of compounds 2 and 3 in HeLa cells may be due to the different
substitutional positions of borneol group.
9

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3
. Conclusion
In summary, we have designed and synthesized three new BODIPY derivatives
Compounds 2-4) containing borneol moieties which show excellent fluorescence
(
properties. Confocal fluorescence microscopy experiments have indicated the
compounds 2 and 3 possess good cell membrane permeability. In particular, 2 has high
lysosome-targeting capability, which is highly promising for practical applications such
as tumor diagnosis.
4
. Experimental section
4
.1. General
All chemicals and solvents were of commercial quality and used without further
purification unless otherwise indicated. Dry CH Cl was distilled from CaH under
2
2
2
nitrogen. The reactions to synthesize the compounds 1 and 2 were carried out under an
N atmosphere. Analytical thin-layer chromatography was performed on Kieselgel 60
2
F254 (Merck). The photo-physical properties was mesured by LAMBDA 950 UV/Vis
Spectrophotometer, PerkinElmer LS 55 Fluorescence spectrometer and a FluoroLog-
1
UltraFast (HORIBA Instrument Inc, Edison) spectrometer. H NMR spectra were
1
recorded on a Bruker DRX-600 AVANCE III spectrometer. Chemical shifts for H
NMR spectra were expressed in parts per million (ppm) relative to CDCl (δ = 7.26
3
ppm) as the internal standard. The High Resolution Mass Spectra (HRMS) data were
obtained on a LTQ Orbitrap XL spectrometer in ESI mode. All the solvents employed
for the spectroscopic measurements were of spectroscopic grade (Aldrich).
Cell culture. Human cervical carcinoma HeLa cell lines were obtained from KeyGEN
Biotech (Nanjing, China). Both cell lines were cultured in Dulbecco's modified Eagle's
–
1
medium (DMEM) containing 10% fetal bovine serum (FBS), 100 µg·mL streptomycin
1
0

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–
1
and 100 U·mL penicillin at 37 °C in a humidified incubator containing 5% CO and
2
9
5% air. The medium was replenished every other day and the cells were subcultured
after reaching confluence. Cell numbers were determined with a Petroff-Hausser cell
counter (USA).
HeLa cells were seeded into 35-mm confocal dishes (Glass Bottom Dish) at a density
4
of 1 × 10 per dish and incubated for 24 h at 37 °C. The medium was then replaced with
fresh serum-free culture medium containing 2.0 µM compound and incubated at 37 °C
for 1 h. Before imaging, the cells were washed with PBS and further incubated with 1.0
µM LysoTracker® Green and 1.0 µM Hoechst 33342 for 15 min. The fluorescence of
cells was visualized with a confocal laser scan microscopy at stationary parameters
including the laser intensity, exposure time and objective lens. Compound 1 and 2 was
excited with a 488 nm laser and emission was collected from 550 to 600 nm. Compound
3
was excited with a 532 nm laser and emission was collected from 550 to 600 nm.
Compound 4 was excited with a 532 nm laser and emission was collected from 600 to
6
50 nm.
X-ray structure determination. X-ray single-crystal diffraction data were collected on
a Bruker smart II diffractometer with Mo–Kα radiation (λ = 0.71073 Å). Data collection
and procees was completed by using the Bruker Smart and Saint (Bruker). Empirical
absorption corrections were performed with SADADS. The structures were solved by
2
direct methods and refined by the full-matrix method based on F by means of the
SHELXLTL software package. Non-H atoms were refined anisotropically using all
reflections with I > 2σ (I). All H atoms were generated geometrically and refined using
a "riding" model with Uiso = 1.2Ueq (C and N). The CCDC deposition No. 1859519 for
3
containing the supplementary crystallographic data for this paper. These data can be
1
1

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obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK;
fax: (+44)1223–336–033; E-mail: deposit@ccdc.cam.ac.uk).
Spectroscopic measurements. UV-visible absorption spectra were carried out on a
Shimadzu UV-3100 spectrophotometer. Fluorescence spectra were measured
PerkinElmer LS 55 Fluorescence spectrometer. The fluorescence life time of the
samples were determined with a FluoroLog-UltraFast (HORIBA Instrument Inc,
Edison) spectrometer equipped with a 450 W CW xenon lamp and an Open-Electrode
TECooled CCD Detector (Syncerity). Nanosecond lifetime and TRES studies were
conducted using a TCSPC MCA model equipped with a picosecond photodetector
(<200 ps) (PPD850) and picosecond laser (duration is 180 ps, Deltadiode, 100 MHz
laser). TRES data were measured by incrementing the monochromator on the emission
channel of the time-resolved fluorometer in fixed wavelength intervals at each
wavelength. Slices of data were taken in the intensity-wavelength plane to obtain
spectra at different times during the decay. Microsecond lifetime decays were collected
by a MCS mode on TCSPC HUB（DeltaHUB）with a LED source (SpectraLED) as a
sample excitation source. DeltaDiode-405 and -635 were used for lifetime
measurements. Absorption and emission measurements were carried out in 1×1 cm
quartz cuvettes. For all measurements, the temperature was kept constant at 298 K.
Dilute solutions with absorbance of less than 0.05 at the excitation wavelength were
used for the measurement of fluorescence quantum yields. Rhodamine B was used as
[
37]
the standard (Φ = 0.5 in ethanol)
. The quantum yield, Φ, was calculated using
F
equation (1):
Фsample=Ф ×(I
/I )× (A /Asample) × (nsample/n )
(1)
std
sample std
std
std
1
2

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Where the sample and std subscripts denote the sample and standard, respectiveluy. l
is the integrated emission intensity. A stands for the absorbance, and n is the refractive
index.
When the fluorescence decays were monoexponential, the rate constants of radiative
(
k ) and nonradiative (k )deactivation were calculated from the measured fluorescence
r nr
quantum yield (Φ ) and fluorescence lifetime (τ) according to equations (2) and (3):
F
k = Φ /τ
(2)
(3)
r
F
k = (1-Φ )/τ
nr
F
4
.2. Synthesis of Compounds 1-4
4
.2.1. Compound 1. Compound 1 was synthesized according to procedures described
[
38]
previously
.
4
.2.2 The synthesis of Bornyl 4-formylbenzoate. Terephthalaldehydic acid 1.58 g (10
mmol), thionyl chloride 10 mL and N,N-dimethylformamide in a catalytic amount were
added to a 100 ml eggplant-shaped flask equipped with a condenser and calcium
chloride tube. The reaction mixture was refluxed for 10 hours. After cooling to room
temperature, and the solvents were removed in vacuo, and the crude product was used
immediately without further purification.
A solution of (-)-Bomeol 1.62 g (10 mmol) and pyridine in a catalytic amount in 15
mL dry CH Cl was cooled to 0 °C in an ice bath while stirring under a nitrogen
2
2
atmosphere. To this solution 4-formylbenzoyl chloride in CH Cl (10 mL) was added
2
2
dropwise under ice-bath during 1 hour, and the reaction was left to stir for further 12
hours at room temperature. After the reaction was completed, water was added and the
mixture was extracted three times with CH Cl . The combined organic phases were
2
2
washed with saturated NaHCO solution, and dried over Na SO . After filtration and the
3
2
4
solvent was removed in vaccuo to obtain a pale yellow oil. The product was purified by
column chromatography[SiO , CH Cl -hexane (1:1)] to yield the white solid product
2
2
2
1
3
1
.71g ( Yield: 60%) H NMR (600 MHz, CDCl ) δ 10.11(s, 1 H), 8.21(d, J = 8.4 Hz,
3 HH
1
3

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3
2
1
1
H), 7.96(d, J = 7.8 Hz, 2 H), 5.16- 5.13(m, 1 H), 2.53- 2.47(m, 1 H), 2.14- 2.09(m,
HH
3
H), 1.86- 1.80(m, 1 H), 1.76(t, J = 4.8 Hz, 1 H), 1.47- 1.41(m, 1 H), 1.35- 1.30(m,
HH
3
H), 1.13(dd, J = 3.0, 13.8 Hz, 1 H), 0.98(s, 3 H), 0.93(s, 6 H).
HH
4
.2.3. Compound 2. A 250 mL three necked flask was used for the reaction. Bornyl 4-
formylbenzoate (2 mmol, 0.5727 mg) was dissolved in dry dichloromethane. After the
solvent was bubbled by N for 20 min, 2,4-dimethyl-1H-pyrrole (4 mmol, 412 µL) was
2
added. Then trifluoroacetic acid (50 µL) was added drop by drop with a syringe while
covered from light. After the reaction continued for 5 h, DDQ (2 mmol, 0.454 g) was
added and the solvent was stirred for 1 h. After that, Et N and BF •OEt were followed
3
3
2
and stirred for 2 h. The solvent was washed by water and Organic phase dried over
Na SO , evaporated and residue was purified by silica gel column chromatography
2
4
using DCM: PE (3 : 7) as the eluent, yielded the desired compound 2 as orange powder
1
3
(
139.4 mg, 13.8%). H NMR (600 MHz, CDCl ): δ 8.18 (d, J = 8.4 Hz, 2H), 7.41 (d,
3 HH
3
JHH = 8.4 Hz, 2 H), 6.00 (s, 2 H), 5.17-5.14 (m, 1 H), 2.56 (s, 6 H), 2.54-2.49 (m, 1 H),
3
2
.18-2.13 (m, 1 H), 1.87-1.81 (m, 1 H), 1.78 (t, JHH = 16.2 Hz, 1 H), 1.56-1.55 (m, 1
4
H), 1.38 (s, 6 H), 1.36-1.33 (m, 1 H), 1.19-1.16 (m, 1 H), 0.99 (s, 3 H), 0.95 (d, JHH
=
-1
-1
9
.6 Hz, 6 H). λmax (ε)(in Toluene) = 514 nm (72420 L·mol ·cm ). m/z (HRMS): calcd
+
[
M ] for C H BF N O : 504.2760, found: 504.2820.
30
35
2
2
2
4
.2.4. Compounds 3 and 4. Compound 1 (0.2 mmol, 70.8 mg) and borneol aldehyde
(0.4 mmol, 114.4 mg) were added to a 50 mL round bottomed flask containing 25 mL
toluene and to this solution was added piperidine (2 mL) and PTSA (35 mg). The
mixture was heated under reflux by using a Dean-Stark trap and reaction was monitored
by TLC DCM : PE (1 : 1). When the starting material compound 1 had been consumed,
the mixture was cooled to room temperature and solvent was evaporated. Water (100
mL) added to the residue and the product was extracted into the CH Cl (3×100 mL).
2
2
Organic phase dried over Na SO , evaporated and residue was purified by silica gel
2
4
column chromatography using DCM: PE (1 : 1) as the eluent, yielded the desired
compound 3 as black powder (10 mg, 8%) and compound 4 (23 mg, 12.9 %).
1
3
3
Compound 3 H NMR (600 MHz, CDCl ): δ 8.04 (d, J = 8.4 Hz, 2 H), 7.77 (d, J
HH
3
HH
3
3
=
16.8 Hz, 1 H), 7.64 (d, J = 7.8 Hz, 2 H), 7.23-7.20 (m, 3 H), 7.03 (d, J = 9.0
HH HH
Hz, 2 H), 6.62 (s, 1 H), 6.04 (s, 1 H), 5.14-5.11 (m, 1 H), 3.89 (s, 3 H), 2.61 (s, 3 H),
3
2
.51-2.46 (m, 1 H), 2.17-2.12 (m, 1 H), 1.85-1.79 (m, 1 H), 1.75 (t, J = 9.0 Hz, 1 H),
HH
1
4

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1
.49 (s, 3 H), 1.47 (s, 3 H), 1.35-1.30 (m, 2 H), 1.16-1.13 (m, 1 H), 0.98 (s, 3 H), 0.93
-1
-1
+
(
s, 6 H). λmax (ε)(in Toluene) = 568 nm (72330 L·mol ·cm ). m/z (HRMS): calcd [M ]
1
for C H BF N O : 622.3178, found: 622.3176. Compound 4 H NMR (600 MHz,
3
8
41
2
2
3
3
3
3
CDCl ): δ 8.09 (d, J = 8.4 Hz, 4 H), 7.84 (d, J = 16.2 Hz, 2 H), 7.70 (d, J = 7.8
3
HH
HH
HH
3
3
Hz, 4 H), 7.29 (s, 2 H), 7.23 (d, JHH = 8.4 Hz, 2 H), 7.05 (d, J = 8.4 Hz, 2 H), 6.68
HH
(s, 2 H), 5.15-5.13 (m, 2 H), 3.90 (s, 3 H), 2.52-2.47 (m, 2 H), 2.19-2.14 (m, 2 H), 1.86-
3
1
.80 (m, 2 H), 1.76 (t, J = 9.0 Hz, 2 H), 1.52 (s, 6 H), 1.37-1.26 (m, 4 H), 1.17-1.14
HH
-
(m, 2 H), 0.99 (s, 6 H), 0.94 (s, 12 H). λmax (ε)(in Toluene) = 640 nm (79930 L·mol
1
-1
+
·
cm ). m/z (HRMS): calcd [M ] for C H BF N O : 890.4642, found: 890.4690.
56 61 2 2 5
Acknowledgements
Financially supported by National Key R&D Program of China (2016YFD0600804),
National Natural Scientific Foundation of China (No. 21301092, 21606133), Natural
Scientific Foundation of Jiangsu Province, P. R. China (No. BK20151513), the Fok
Ying Tung Education Foundation (Grant No. 141030), and the open Fund of Jiangsu
Key Laboratory for Biomass Energy and Material(JSBEM201702).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http:// xxxxxxxxxxxxx.
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References
[
1] Zheng Q, Xu G, Prasad P N. Conformationally restricted dipyrromethene boron
difluoride (BODIPY) dyes: highly fluorescent, multicolored probes for cellular
imaging. Chem-Eur J 2010; 14: 5812-5819.
[
2] Zhang M, Zheng S T, Liu X J, et al. A series of novel NIR fluorescent dyes:
Synthesis, theoretical calculations and fluorescence imaging applications in living
cells. Dyes Pigments 2016; 125: 220-228.
[
[
3] Cho D G, Sessler J L. Modern Reaction‐Based Indicator Systems. Chem Soc Rev
2
009; 38: 1647-1662.
4] Qian X, Xiao Y, Xu Y, et al. “Alive” Dyes as Fluorescent Sensors: Fluorophore,
Mechanism, Receptor and Images in Living Cells. Chem Commun 2010; 46: 6418-
6
436.
[
5] Liu B, Luo Z, Si S, et al. A photostable triphenylamine-based flavonoid dye:
Solvatochromism, aggregation-induced emission enhancement, fabrication of
organic nanodots, and cell imaging applications. Dyes Pigments 2017; 142: 32-38.
[
[
[
[
[
[
[
6] Chan J, Dodani S C, Chang C J. Reaction-based small-molecule fluorescent probes
for chemoselective bioimaging. Nat Chem 2012; 4: 973-984.
7] Hong G, Lee J C, Robinson J T, et al. Multifunctional in vivo vascular imaging
using near-infrared II fluorescence. Nat Med 2012; 18: 1841-1846.
8] Alford R, Ogawa M, Hassan M, et al. Fluorescence lifetime imaging of activatable
target specific molecular probes. Contrast Media Mol Imaging 2010; 5: 1-8.
9] Ashton T D, Jolliffe K A, Pfeffer F M. Luminescent probes for the bioimaging of
small anionic species in vitro and in vivo. Chem Soc Rev 2015; 44: 4547-4595.
10] Lee M H, Kim J S, Sessler J L. Small molecule-based ratiometric fluorescence
probes for cations, anions, and biomolecules. Chem Soc Rev 2015; 44: 4185-4191.
11] Zhao Y, Ren Y, Li H, et al. A new far-red naphthorhodamine dye: Synthesis,
fluorescent probe and bioimaging applications. Dyes Pigments 2016; 132: 255-261.
12] Gao Z, Zhang X, Chen Y. One-pot synthesis of a near-infrared fluorophore for
living cells imaging. Dyes Pigments 2017; 140: 56-61.
1
6

ACCEPTED MANUSCRIPT
[
[
13] Zhu S, Dorh N, Zhang J, et al. Highly water-soluble neutral near-infrared emissive
BODIPY polymeric dyes. J Mater Chem 2012; 22: 2781-2790.
14] Wang B, Li P, Yu F, et al. A near-infrared reversible and ratiometric fluorescent
probe based on Se-BODIPY for the redox cycle mediated by hypobromous acid and
hydrogen sulfide in living cells. Chem Commun 2013; 49: 5790-5792.
[
[
15] Lu H, Mack J, Yang Y, et al. Structural modification strategies for the rational
design of red/NIR region BODIPYs. Chem Soc Rev 2014; 43: 4778-4823.
16] Kowada T, Maeda H, Kikuchi K. ChemInform Abstract: BODIPY-Based Probes
for the Fluorescence Imaging of Biomolecules in Living Cells. Chem. Soc. Rev.
2
015; 44: 4953-4972.
[
[
17] Wang S S, Liu H, Mack J, et al. A BODIPY-based ‘turn-on’ fluorescent probe for
hypoxic cell imaging. Chem Commun 2015; 51: 13389-13392.
18] Lu H, Xiong L, Liu H, et al. A highly selective and sensitive fluorescent turn-on
2
+
sensor for Hg and its application in live cell imaging. Org Biomol Chem 2009; 7:
554-2558.
2
[
[
[
[
[
19] Wang K P, Xiao Y J, Wang Y Y, et al. Self-assembled hydrophobin for producing
water-soluble and membrane permeable fluorescent dye. Sci Rep 2016; 6: 23061-
2
3071.
20] Dai J, Chen J, Bei Y, et al. Influence of borneol on primary mice oral fibroblasts: a
penetration enhancer may be used in oral submucous fibrosis. J Oral Pathol Med
2
010; 38: 276-281.
21] Zhang Q L, Fu B M, Zhang Z J. Borneol, a novel agent that improves central
nervous system drug delivery by enhancing blood-brain barrier permeability. Drug
Delivery 2017; 24: 1037-1044.
22] Yu B, Ruan M, Dong X, et al. The mechanism of the opening of the blood-brain
barrier by borneol: A pharmacodynamics and pharmacokinetics combination study.
J Ethnopharmacol 2013; 150: 1096-1108.
23] Mani V, Krishnakumar V G, Gupta S, et al. Synthesis and characterization of
styryl-BODIPY derivatives for monitoring in vitro Tau aggregation. Sens Actuators
B 2017; 244: 673-683.
1
7

ACCEPTED MANUSCRIPT
[
[
[
[
24] Wang C, Xia X, Luo J, et al. A novel near-infrared styryl-BODIPY fluorescent
probe for discrimination of GSH and its application in living cells. Dyes Pigments
2
018; 152: 85-92.
25] Yin Z, Tam A Y, Wong K M, et al. Functionalized BODIPY with various sensory
units--a versatile colorimetric and luminescent probe for pH and ions. Dalton Trans
2
012; 41: 11340-11350.
26] Shen Z, Röhr H, Rurack K, et al. Boron-diindomethene (BDI) dyes and their
tetrahydrobicyclo precursors--en route to a new class of highly emissive
fluorophores for the red spectral range. Chem-Eur J 2010; 10: 4853-4871.
27] Bura T., Retailleau P., Ulrich G., et al. Highly Substituted Bodipy Dyes with
Spectroscopic Features Sensitive to the Environment J Org Chem 2011; 76, 1109–
1
117.
[
[
28] Uppal T, Hu X, Fronczek F R, et al. Synthesis, Computational Modeling, and
Properties of Benzo-Appended BODIPYs. Chem-Eur J 2012; 18: 3893-3905.
29] Galangau O, Dumasverdes C, Méalletrenault R, et al. Rational design of visible
and NIR distyryl-BODIPY dyes from a novel fluorinated platform. Org Biomol
Chem 2010; 8: 4546-4553.
[
30] Xu J, Zhu L, Wang Q, et al. meso-C6F5, substituted BODIPYs with distinctive
spectroscopic properties and their application for bioimaging in living cells.
Tetrahedron 2014; 70: 5800-5805.
[
[
[
31] Guerin C, Gerard L J, Octobre G, et al. Bis-triazolyl BODIPYs: a simple dye with
strong red-light emission. RSC Adv. 2015; 5: 76342-76345.
32] Umezawa K, Nakamura Y, Makino H, et al. Bright, Color-Tunable Fluorescent
Dyes in the Visible-Near-Infrared Region. J Am Chem Soc 2008; 130: 1550-1551.
33] Qin W, Baruah M, Stefan A, et al. Photophysical Properties of BODIPY-Derived
Hydroxyaryl Fluorescent pH Probes in Solution. ChemPhysChem 2005; 6: 2343-
2
351.
34] Benniston A. C. and Copley G. Lighting the way ahead with boron dipyrromethene
Bodipy) dyes. Phys Chem Chem Phys 2009; 11: 4124-4131.
[
(
1
8

ACCEPTED MANUSCRIPT
[
[
35] Li D., Xu Y., Zhou N., Liu J., Wang R., Cheng T., Tang Y., Zhu W., Xu Y., Qian
X. A novel ‘‘donor-two-acceptor’’ type fluorophore-based probe for fast detection
and intracellular imaging of nitroreductase Dyes Pigment. 2017; 136: 627-632.
36] Li Z., Li X., Gao X., Zhang Y., Shi W., Ma H. Nitroreductase Detection and
Hypoxic Tumor Cell Imaging by a Designed Sensitive and Selective Fluorescent
Probe, 7-[(5- Nitrofuran-2-yl)methoxy]-3H-phenoxazin-3-one Anal. Chem., 2013,
8
5, 3926-3932.
[
[
37] Albert M. Brouwer. Standards for photoluminescence quantum yield measurements
in solution. Pure. Appl. Chem. 2011; 83: 2213–2228.
38] Qiao L, Nie H, Wu Y, et al. An ultrafast responsive BODIPY-based fluorescent
probe for the detection of endogenous hypochlorite in live cells. J Mater Chem B
2
016; 5: 525-530.
Figure Captions
Scheme 1. Structures and synthesis of Compounds 1-4.
Fig. 1. Perspective views of 3, showing 30% probability displacement ellipsoids
(
hydrogen atoms and ethyl ether molecule are deleted for clarity).
Fig. 2. Normalized UV/Vis (a) and fluorescence (b) spectra of BODIPYs 1 (blue), 2
green), 3 (black) and 4 (red) in toluene at 25 ℃.
(
Fig. 3. Confocal fluorescence images and bright-field images of HeLa cells incubated
with 2.0 µM compounds at 37 °C for 1 h.
Fig. 4. Confocal fluorescence images, bright-field images and three-dimensional
fluorescence images of compound 2-loaded HeLa cells stained with LysoTracker green
and Hoechst 33342 for 1 h at 37 °C.
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Table 1. Photophysical properties of the compounds 1-4 in toluene. [a] the compounds
1
and 2 were excited at 488 nm, the compounds 3 and 4 were excited at 520 nm, 570 nm
respectively.
Scheme 1.
Fig. 1.
Fig. 2.
2
0

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2
1

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Fig. 3.
Fig. 4.
2
2

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Table 1
[
a]
λ
abs
λ
em
∆υ
τ
F
κ
r
κ
nr
Φ
F
-
1
8
-1
8
-1
[nm]
[nm]
[
cm ]
[ns]
[10 s ]
[10 s ]
1
2
3
4
504
506
568
640
514
518
627
710
386
458
275
240
0.39
0.37
4.86
2.30
3.82
2.60
0.84
1.61
1.15
0.16
1.21
2.74
1.47
3.72
0.41
0.061
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4

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Highlights:
1
. Three novel boron-dipyrromethene (BODIPY) dyes bearing borneol moieties have
been synthesized and take on excellent cell membrane permeability.
. The compounds 2-3 show high fluorescence quantum yield, and can be utilized as
fluorescent visualizers for cell and lysosome fluorescence imaging.
2