Construction of red-emitting iminocoumarin-based fluorescent borate complexes with a large Stokes shift

Article abstract of DOI:10.1016/j.dyepig.2019.108007

Tetra-hydro-quinoxaline (THQ) moiety was used as the electron donor in the construction of a series of red-emitting iminocoumarin-based borate dyes with good fluorescent quantum yields, large Stokes shifts and long-wavelength emissions. These dyes could s

Full text of DOI:10.1016/j.dyepig.2019.108007

Journal Pre-proof
Construction of red-emitting iminocoumarin-based fluorescent borate complexes with
a large Stokes shift
Xiaojie Ren, Man Guo, Junyi Gong, Yun Zhang, Lei Yang, Xingjiang Liu, Xiangzhi
Song
PII:
S0143-7208(19)31867-4
DOI:
https://doi.org/10.1016/j.dyepig.2019.108007
DYPI 108007
Reference:
To appear in: Dyes and Pigments
Received Date: 6 August 2019
Revised Date: 27 October 2019
Accepted Date: 28 October 2019
Please cite this article as: Ren X, Guo M, Gong J, Zhang Y, Yang L, Liu X, Song X, Construction of
red-emitting iminocoumarin-based fluorescent borate complexes with a large Stokes shift, Dyes and
Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.108007.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Ltd.

Graphical Abstract
Aseries of red-emitting iminocoumarin-based borate dyes with tetra-hydro-quinoxaline
(THQ) moiety as the electron donor, THQ-BF2-R, were developed. These borate dyes
exhibited fairly high fluorescent quantum yields, large Stokes shifts and long-
wavelength emissions. These dyes could be potentially used as staining agents for
biological applications.

Construction of Red-emitting Iminocoumarin-based Fluorescent
Borate Complexes with A Large Stokes Shift
Xiaojie Ren,a Man Guo,a Junyi Gong,a Yun Zhang,a Lei Yang,a Xingjiang Liub,* and Xiangzhi
Song a, c,
*
a College of Chemistry & Chemical Engineering, Central South University, Changsha, Hunan
Province, China, 410083.
* Corresponding author. Email: song@rowland.harvard.edu. (Xiangzhi Song)
b
College of Chemistry & Chemical Engineering, Zhengzhou University, Zhengzhou, Henan,
Province, China, 450001.
* Corresponding author. Email: xingjiangliu@zzu.edu.cn (Xingjiang Liu).
c Key Laboratory of Hunan Province for Water Environment and Agriculture Product Safety,
Changsha, Hunan Province, China, 410083
Abstract: Tetra-hydro-quinoxaline (THQ) moiety was used as the electron donor in the
construction of a series of red-emitting iminocoumarin-based borate dyes with good
fluorescent quantum yields, large Stokes shifts and long-wavelength emissions. These dyes
could stain cells and image zebrafish with good performance.
Key words: borate, tetra-hydro-quinoxaline, Stokes shift, red-emitting, fluorescent dyes
1. Introduction
4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) have been widely utilized
as biological sensing agents[1-5], photodynamic therapeutic agents[6], laser dyes[7],
and optoelectronic materials[8] because of their advantageous photo-physical
properties such as good photo-stability, high molar absorption coefficient and high
fluorescence quantum yield.[9, 10] While being valuable, most of BODIPY dyes
suffer from very small Stokes shifts (usually < 30 nm), which usually causes serious
self-absorption and thereby leads to measurement errors in fluorescence sensing.
Moreover, most of BODIPY dyes exhibited fluorescence quenching in aggregation
form and/or solid state due to the strong intermolecular π-π stacking.[11, 12]

In principle, asymmetrical donor–acceptor (D-A) type fluorescent dyes display
larger Stokes shift and longer emission than their symmetrical analogues.[13, 14] In
2013, Ziessel’s group[15] pioneered a new kind of iminocoumarin-based borate
complexes, Borico dyes (DEA-BF2-R), having asymmetrical structure with
diethylamino group as the electron donor and “N-BF2-N” moiety as the electron
acceptor (Scheme 1). These dyes possess high fluorescent quantum yields in solution
(up to 80%). However, their emissions are in relatively short wavelength spectral
regions (blue and green) and their Stokes shifts are still small (< 50 nm), which affect
their applications for bioimaging and sensing due to the weak tissue penetration ability
and strong background interference.[16-21]
In the design of fluorescent dyes and fluorescent probes, introducing strong
electron donors such tetra-hydro-quinoxaline group (THQ) and julolidine moiety (Julo)
has been proved to be an efficient stragety to lengthen emission wavelengths as well as
Stokes shifts due to the enhanced ICT process.[22-26] Besides, fluorescent dyes with
such electron donors can retain a relatively high fluorescent quantum yield because the
rigid structure of THQ and Julo moieties can effectively prohibit the formation of
TICT (twisted intramolecular charge transfer) state.[27-29]
In this work, we used tetra-hydro-quinoxaline group (THQ) to replace
diethylamino group (DEA) in DEA-BF2-R dyes to obtain a series of new
iminocoumarin-based borate complexes, THQ-BF2-R, which are expected to exhibit
long-wavelength emission and large Stokes shift, as shown in Scheme 1. In addition,
we also used julolidine moiety (Julo) to replace diethylamino group (DEA) in
DEA-BF2-R to prepare another rigid borate complexes, Julo-BF2-R.
Scheme 1. Tuning the optical properties of iminocoumarin-based borate dyes by enhancing the
electron-donating ability of donors.

2. Experimental
2.1 General methods
All reagents were purchased from commercial suppliers without further purification. The
absorption spectra were detected using an Agilent UV-2450 spectrophotometer and the
corrected fluorescence spectra were recorded on a Hitachi F-7000 spectrophotometer. The
fluorescent lifetimes were determined on a Pico Quant GmbH Fluo Time 100 lifetime
spectrometer. The absolute fluorescent quantum yields for the solid samples were detected on
a Hamamatsu Quantarus-QY C11347. The fluorescent quantum yields in solution were
measured on a Hitachi F-7000 spectrophotometer using a standard reference and calculated
from the following equation:
Fꢀ Aꢃ nꢄꢀ
Φꢀ ꢁ Φꢂ
Fꢃ Aꢀ nꢄꢃ
Φ denotes the fluorescent quantum yield; F means the integral intensity of fluorescence,
A refers to the absorbance at the excitation wavelength and n is the refraction index of
solvents. u and s represent the testing sample and the standard sample, respectively.
13
1H NMR and C NMR were obtained on Bruker 400/500 spectrometers (using TMS as
an internal standard). Mass spectra were measured using a Bruker ESI-TOF-Q spectrometer.
Fluorescence photos of solid samples were obtained on an Olympus IX83 inverted
microscope. Fluorescence imaging experiments in living cells and zebrafish were performed
on an Opera Phenix/Operetta CLS from PerkinElmer, Inc. All the tests were conducted at
room temperature.
2.2 Synthesis
These dyes were prepared according to the procedure shown in Scheme 2. Firstly,
aminosalicylaldehydes reacted with 2-cyanomethylbenzimidazole in ethanol to afford
iminocoumarins,[30] which condensed with substituted anilines to give compounds 4a-d,
5a-d and 6a-d. Without further purification, compounds 4a-d, 5a-d and 6a-d were treated
with BF3•OEt2 to yield the borate complexes DEA-BF2-R, Julo-BF2-R and THQ-BF2-R. All
these new compounds were fully characterized by NMR and HRMS analysis.
Scheme 2. General synthetic procedure of borate dyes.

2.2.1 General procedure for the synthesis of DEA-BF2-R
Dyes DEA-BF2-CH3, DEA-BF2-OCH3, DEA-BF2-H were prepared according to the
literature methods.[15]
Synthesis of DEA-BF2-I: Under an argon atmosphere,
a
solution of
4-diethylaminosalicylaldehyde (1 mmol) (compound 1), 2-cyanomethylbenzimidazole (1.1
mmol) and piperidine (15 µL) in ethanol was stirred for 2 hours. Then, the reaction solution
was diluted with 10 mL anhydrous ethanol and p-toluenesulfonic acid (p-TsOH, 5 mg) and
4-iodoaniline (2 mmol) were added. After stirring for 15 hours, the mixture was cooled down
and the solvent was removed and the residue was purified by column chromatograph (CH2Cl2:
C2H5OH 100:1) to afford compound 4d.
Without further purification, compound 4d was dissolved in 1,2-dichloroethane (10 mL),
then N, N-di-isopropyl-amine (100 µL) and BF3•OEt2 (100 µL) were added. The mixture was
refluxed for 2 hours under an argon atmosphere. After cooling to room temperature, the
reaction was quenched with saturated aqueous NaHCO3 solution and extracted with CH2Cl2
three times (3 x 30 mL). The organic layers were collected, combined and dried over
anhydrous Na2SO4 for 2 hours. After the removal of solvent, the obtained residue was purified
by silica gel column chromatograph twice (CH2Cl2: C2H5OH = 100:1) to give a solid, which
was then recrystallized in the mixture of DCM: PE (5:1) to give pure DEA-BF2-I as a red
powder. Yield 6.8%.
1H NMR (400 MHz, CDCl3) δ 9.09 (s, 1H), 7.97-7.86 (m, 2H), 7.71 (t, J = 8.5 Hz, 1H), 7.66
(s, 1H), 7.56-7.47 (m, 1H), 7.31 (t, J = 10.5 Hz, 2H), 7.25-7.11 (m, 2H), 6.71 (dd, J = 9.1, 2.4 Hz,
1H), 6.19 (d, J = 2.1 Hz, 1H), 3.43 (q, J = 7.1 Hz, 4H), 1.23 (t, J = 7.1 Hz, 6H). 13C NMR (100
MHz, CDCl3) δ 159, 154.5, 152.9, 145.3, 143.7, 140.6, 138.6, 136.56, 135.9, 131.4, 129.0, 122.8,
117.5, 113.9, 112.2, 109.3, 104.3, 96.42, 93.8, 45.16, 12.5. HRMS (ESI) m/z
[C26H22BF2IN4O+H]+ calcd.: 582.0899, found: 583.0983.
2.2.2 General procedure for the synthesis of Julo-BF2-R
Dissolved
compound
2
(julolidine-salicylaldehyde,
1
mmol)
and
2-cyanomethyl-benzimidazole (1.1 mmol) in 10 mL anhydrous acetonitrile, then 15 µL
piperidine was added into above solution. The obtained solution was refluxed under argon
atmosphere for 2 hours to precipitate a yellowish-brown solid. To the above reaction solution
were added 10 mL anhydrous acetonitrile, 5 mg p-TsOH and 2 mmol p-R-aniline (R = -CH3,
-OCH3, -H, -I). Then the reaction mixture was refluxed under an argon atmosphere until the
solid disappeared (about 15 hours). Cooling the reaction mixture to room temperature, and the
solution was evaporated in vacuum. The residue was purified by silica gel column
chromatograph (CH2Cl2: C2H5OH from 100:1 to 50:1) to give crude products 5a/5b/5c/5d as

red solid, which were used for the next reactions without further purification.
The respective 5a/5b/5c/5d was dissolved in 20 mL 1,2-dichloroethane and 100 µL N,
N-di-isopropyl-amine and 100 µL BF3•OEt2 were added. The mixture was stirred at 80
under an argon atmosphere until the dark-red solution turned into yellow-green (about 2
hours). After cooling to room temperature, the reaction was quenched with saturated aqueous
NaHCO3 solution and the reaction mixture was extracted with CH2Cl2 three times (3 x 30 ml).
The organic layers were collected and dried over anhydrous Na2SO4 for 2 hours. The solvent
was removed by distillation and the residue was purified with column chromatograph to yield
a red solid, which was further purified by recrystallization from the mixture DCM: PE (5: 1)
to yield the pure products.
Julo-BF2-CH3: Silica gel, CH2Cl2: C2H5OH (from 100: 0 to 100: 1). Yield: 10.3%. 1H NMR
(400 MHz, CDCl3) δ 8.92 (d, J = 55.3 Hz, 1H), 7.72 (dd, J = 15.2, 7.6 Hz, 2H), 7.46 (d, J = 8.2 Hz,
2H), 7.38 (d, J = 8.2 Hz, 2H), 7.27-7.17 (m, 2H), 7.09 (s, 1H), 3.32-3.19 (m, 4H), 2.75 (t, J = 6.1
Hz, 2H), 2.49 (s, 3H), 2.22 (dd, J = 13.1, 6.7 Hz, 2H), 1.98-1.87 (m, 2H), 1.87-1.75 (m, 2H). 13
C
NMR (100 MHz, CDCl3) δ 149.5, 148.9, 145.6, 140.7, 137.9, 135.7, 134.0, 129.91, 127.4, 126.6,
123.2, 122.9, 122.0, 116.9, 114.1, 109.2, 105.9, 50.4, 49.8, 27.3, 21.3, 20.7, 19.7, 19.0. HRMS
(ESI) m/z [C29H25BF2N4O+H] + calcd.: 495.2168, found: 495.2100.
1
Julo-BF2-OCH3: Silica gel, CH2Cl2: C2H5OH (from 100: 0 to 100: 1). Yield: 10.8%. H
NMR (500 MHz, CDCl3) δ 7.74 (t, J = 8.6 Hz, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.24 (dd, J = 16.9,
8.9 Hz, 1H), 7.09 (d, J = 8.6 Hz, 1H), 3.92 (s, 1H), 3.36-3.30 (m, 1H), 3.30-3.22 (m, 1H), 2.80 (t,
J = 6.1 Hz, 1H), 2.27 (t, J = 6.4 Hz, 1H), 2.00-1.93 (m, 1H), 1.88-1.81 (m, 1H). 13C NMR (100
MHz, CDCl3) δ 159.15, 149.40, 148.64, 146.27, 139.70, 136.06, 129.53, 128.00, 127.16, 122.54,
122.34, 121.95, 117.37, 114.49, 113.80, 109.21, 105.75, 77.54, 77.08, 76.76, 55.57, 50.26, 49.90,
+
49.38, 49.16, 48.95, 27.31, 20.67, 19.68, 19.06. HRMS (ESI) m/z [C29H25BF2N4O2+H] calcd.:
511.2117, found: 511.2025.
1
Julo-BF2-H: Silica gel, CH2Cl2: C2H5OH (from 100: 0 to 50: 1). Yield: 10.5%. H NMR
(500 MHz, CDCl3) δ 8.84 (s, 1H), 7.72 (dd, J = 12.1, 7.8 Hz, 2H), 7.59 (d, J = 4.4 Hz, 4H), 7.49
(dt, J = 8.7, 4.2 Hz, 1H), 7.22 (dt, J = 20.5, 6.9 Hz, 2H), 7.07 (s, 1H), 3.33-3.27 (m, 2H), 3.27-3.20
(m, 2H), 2.78 (t, J = 6.2 Hz, 2H), 2.18 (t, J = 6.4 Hz, 2H), 1.99-1.92 (m, 2H), 1.80 (dd, J = 11.7,
6.1 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 149.3, 148.4, 146.2, 144.7, 139.5, 137.0, 136.3, 129.2,
128.0, 127.0, 122.6, 117.6, 113.7, 109.1, 105.7, 103.5, 50.2, 49.6, 27.3, 20.7, 19.6, 18.9. HRMS
(ESI) m/z [C29H25BF2N4O2+H]+ calcd.: 511.2117, found: 511.2025.
1
Julo-BF2-I: Silica gel, CH2Cl2: C2H5OH (from 100: 0 to 25: 1). Yield: 10%. H NMR (500
MHz, CDCl3) δ 9.06 (s, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 7.8 Hz,
1H), 7.35 (d, J = 8.3 Hz, 2H), 7.25 (dd, J = 13.4, 5.9 Hz, 2H), 7.16 (s, 1H), 3.41-3.35 (m, 2H),
3.35 -3.28 (m, 2H), 2.80 (t, J = 6.1 Hz, 2H), 2.26 (t, J = 6.3 Hz, 2H), 1.99 (dd, J = 11.3, 5.8 Hz,
2H), 1.88 (dd, J = 11.9, 6.3 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 153.48, 144.17, 142.38,
133.02, 132.71, 131.31, 126.79, 126.54, 121.04, 117.68, 113.44, 109.64, 96.87, 81.48, 81.16,

80.84, 54.31, 53.73, 53.35, 53.14, 52.93, 52.71, 52.50, 52.28, 52.07, 31.26, 24.52, 23.51, 22.99.
HRMS (ESI) m/z [C28H22BF2IN4O+H]+ calcd.: 607.0978, found: 607.0871.
2.2.3 General procedure for the synthesis of THQ-BF2-R
To a solution of compound 3 (2,4-diethylamino-1,2,3,4-tetralhydro-quinoxaline, 1 mmol)
and 2-cyanomethyl-benzimidazole (1.1 mmol) in 5 ml anhydrous ethanol was added 15 µL
piperidine. The mixture was stirred for 2 hours at 80 under an argon atmosphere. 10 mL
anhydrous ethanol was added to dilute the reaction mixtute, then 5 mg p-TsOH and 2 mmol
p-R-aniline (R = -CH3, -OCH3, -H, -I) were added. The resulting reaction solution was
refluxed for 15 hours. After cooling to room temperature, the solvent was distilled in vacuum.
The obtained residue was purified by column chromatography using CH2Cl2: C2H5OH (100: 1)
as the eluent to yield a red solid, which was used for the next step without further purification.
A solution of 6a/6b/6c/6d, N, N-di-isopropyl-amine (100 µL) and BF3•OEt2 (100 µL) in
1,2-dichloroethane (20 mL) was refluxed for 2 hours under an argon atmosphere. After
cooling to room temperature, dichloromethane (20 mL) was added into the reaction mixture,
which was then quenched with saturated aqueous NaHCO3 solution and extracted with
CH2Cl2 three times (3 x 30 ml). The organic layers were combined and dried over anhydrous
Na2SO4 for 2 hours. After the removal of solvent, the residue was purified by column
chromatograph twice using CH2Cl2: C2H5OH (from 100: 1 to 50: 1) as the eluent to give the
pure products.
1
THQ-BF2-CH3: Dark red solid. Yield: 6.16%. H NMR (400 MHz, CDCl3) δ 9.12 (s, 1H),
7.81 (dd, J = 10.7, 4.3 Hz, 2H), 7.41 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 8.5 Hz, 2H), 7.35-7.29 (m,
2H), 6.65 (s, 1H), 6.25 (s, 1H), 3.63-3.53 (m, 2H), 3.42 (q, J = 7.1 Hz, 4H), 3.34-3.25 (m, 2H),
2.50 (s, 3H), 1.32-1.11 (m, 5H). 13C NMR (100 MHz, CDCl3) δ 142.95, 141.76, 138.23, 133.88,
130.70, 126.64, 126.42, 121.25, 117.78, 114.66, 110.29, 98.93, 81.42, 81.10, 80.78, 53.49, 53.28,
53.07, 52.85, 52.64, 52.43, 52.21, 51.25, 50.16, 49.37, 47.99, 25.04, 14.55, 13.50. HRMS (ESI)
m/z [C29H29BF2N5O+H] + calcd.: 512.2433, found: 512.2549.
THQ-BF2-OCH3: Dark red solid. Yield: 6.35%. 1H NMR (400 MHz, CDCl3) δ 9.69 (s, 1H),
7.93 (d, J = 7.5 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.55 (d, J = 9.0 Hz, 1H), 7.41 (p, J = 7.1 Hz,
3H), 7.18-7.03 (m, 2H), 6.78 (s, 2H), 6.26 (s, 1H), 3.94 (s, 3H), 3.71-3.56 (m, 2H), 3.45 (dq, J =
14.4, 7.2 Hz, 4H), 3.36-3.25 (m, 2H), 1.26 (dd, J = 12.3, 5.1 Hz, 6H). 13C NMR (100 MHz, CDCl3)
δ 163.19, 154.22, 148.98, 145.07, 144.91, 144.68, 138.34, 132.97, 132.00, 128.77, 128.17, 123.30,
121.08, 118.97, 118.62, 115.02, 110.70, 107.51, 98.87, 59.46, 51.57, 50.54, 49.38, 47.60, 14.76,
13.52. HRMS (ESI) m/z [C29H29BF2N5O2+H] + calcd.: 528.2382, found: 528.2352.
THQ-BF2-H: Dark red solid. Yield: 6.11%. 1H NMR (400 MHz, CDCl3) δ 9.69 (s, 1H), 7.93
(d, J = 7.5 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.55 (d, J = 9.0 Hz, 1H), 7.41 (p, J = 7.1 Hz, 3H),
7.18-7.03 (m, 2H), 6.78 (s, 2H), 6.26 (s, 1H), 3.94 (s, 3H), 3.71-3.56 (m, 2H), 3.45 (dq, J = 14.4,
7.2 Hz, 4H), 3.36 -3.25 (m, 2H), 1.26 (dd, J = 12.3, 5.1 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ

159.31, 150.33, 145.07, 143.24, 141.53, 134.59, 133.33, 128.95, 128.09, 125.00, 124.41, 115.01,
114.79, 114.73, 111.10, 106.99, 94.96, 77.38, 77.26, 77.06, 76.74, 55.58, 49.92, 49.70, 49.49,
+
49.28, 49.06, 47.66, 46.62, 45.48, 43.67, 10.89, 9.72. HRMS (ESI) m/z [C28H26BF2N5O2+H]
calcd.: 498.2277, found: 498.2331.
THQ-BF2-I: Dark red solid. Yield: 6.48%. 1H NMR (500 MHz, DMSO-d6) δ 7.93 (d, J = 8.5
Hz, 2H), 7.64 (d, J = 7.7 Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.18 (dt, J =
15.9, 6.8 Hz, 2H), 7.07 (s, 1H), 6.38 (s, 1H), 5.77 (s, 1H), 3.63-3.54 (m, 2H), 3.48 (q, J = 7.0 Hz,
2H), 3.41 (q, J = 7.0 Hz, 2H), 3.26 (t, J = 4.9 Hz, 2H), 1.25-1.12 (m, 3H), 1.09 (t, J = 7.1 Hz, 3H).
13C NMR (100 MHz, CDCl3) δ 153.9, 147.9, 143.7, 142.5, 138.6, 133.1, 129.8, 127.2, 118.16,
115.6, 110.3, 103.8, 101.6, 99.9, 98.8, 94.5, 88.36, 51.4, 50.4, 49.4, 47.9, 14.6, 13.7. HRMS (ESI)
m/z [C28H25BF2IN5O+H] + calcd.: 624.1243, found: 624.1165.
2.3 Cell and zebrafish culture and fluorescence imaging
Hela cells were used for imaging and toxicity experiments. HeLa cells were cultured
using DMEM (GIBCO, Invitrogen) medium with 10% fetal bovine and 1% dual antibody
(streptomycin and penicillin) at 37 in a humidified 5% CO2 incubator. When the number of
adherent cells grow steadily in the culture bottle reaches 85-95%, the digestion is passed on.
Discard the original culture medium, wash the adhered cells three times with phosphate buffer
solution (PBS), add trypsin to digest the cells for three minutes, and then add culture medium
containing 10% serum to stop digestion. The digested cell suspension was transferred to the
centrifugal tube for centrifugation (800 r/min, 3min), the supernatant was discarded, and cells
were suspended and precipitated by a mixture of serum and dual antibody media. The cell
concentration (about 2*105 cells/mL) was adjusted and transferred to 96 orifice plate and
co-focusing plate for MTT and bioimaging experiment, respectively.
Zebrafish was cultured at 28 in E3 embryo media, consisting of 15 mM NaCl, 0.5 mM
MgSO4, 1 mM CaCl2, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, 0.7 mM NaHCO3 and 1%
methylene blue, whose pH = 7.5. Three-day old zebrafish was selected for the imaging
experiments.
3. Results and discussion
3.1 Photophysical properties in various solvents
With these dyes in hands, we firstly investigated their photophysical property in solutions
(DCM, EtOH, CH3CN and DMSO). DEA-BF2-R dyes absorbed in a range from 482 nm to
497 nm with large molar extinction coefficients (about 104 L*mol-1cm-1) and displayed green
fluorescence with maxima ranging from 520 nm to 550 nm (Table 1 and Figures S1-S4).
Compared with DEA-BF2-R, Julo-BF2-R displayed about 20 nm red-shift in their absorption
spectra and 10 nm red-shift in emission spectra (shown in Table 1 and Figures S5-S8). We
were very pleased that the replacement of diethylamino group with THQ group in borate dyes

significantly extended the emission to red spectral region (around 90 nm red shift) and
resulted in about 40 nm red shift in their absorption spectra (Table 1 and Figures S9-S12). For
example, the absorption maxima of DEA-BF2-CH3 and THQ-BF2-CH3 in DMSO were 483
nm and 526 nm and their fluorescence spectra peaked at 548 nm and 646 nm, respectively
(shown in Figure 1). The Stokes shift for THQ-BF2-CH3 was remarkably increased to 120
nm. As a result, we successfully tuned the optical properties of iminocoumarin-based borate
complexes to obtain long-wavelength emission (> 600 nm) as well as large Stokes shift (>100
nm) through introducing THQ electron donor to enhance intracellular charge ICT process.
Table 1 Photophysical properties of DEA-BF2-R, Julo-BF2-R and THQ-BF2-R in various solvents.
DEA-BF2-R
Julo-BF2-R
THQ-BF2-R
λabs
/
∆ss/
nm
35
47
54
65
38
50
53
65
35
46
54
65
35
50
56
68
λabs
/
∆ss/
nm
28
38
50
54
26
41
50
54
26
39
39
53
27
42
52
56
λabs
/
∆ss/
nm
73
a
a
b
-R
Solvents
ε/M-1cm-1
Φf
τ/ ns
X c
ε/M-1cm-1
Φf
τ/ ns
X c
ε/M-1cm-1
Φf
τ/ ns
X c
λem/ nm
490/525
487/534
483/537
483/548
493/531
485/535
484/537
483/548
493/528
488/534
482/536
482/547
497/532
490/540
486/542
486/554
λem/ nm
512/540
507/545
505/555
505/559
513/539
505/546
505/555
505/559
513/538
506/545
505/554
505/558
514/541
508/550
507/559
508/564
λem/ nm
534/607
527/620
522/626
526/646
538/610
527/619
521/626
526/643
538/608
528/621
525/627
528/650
537/611
532/626
532/632
530/648
DCM
EtOH
41300
31300
34400
33000
42700
43900
41600
37400
39300
34300
36300
31700
34300
33000
34800
29500
0.84
0.77
0.70
0.72
0.18
0.11
0.09
0.24
0.84
0.81
0.73
0.71
0.87
0.83
0.73
0.71
4.09
3.82
4.03
3.53
0.92
0.63
0.98
1.35
4.20
4.01
4.09
3.51
4.34
4.12
4.29
3.61
1.32
1.20
1.13
1.34
1.19
1.38
1.20
1.19
1.02
1.17
1.11
1.09
1.32
1.33
1.29
1.26
86500
31300
53300
53100
69800
39900
48500
38500
63000
35600
37900
37700
69200
18400
37700
38900
0.79
0.71
0.69
0.70
0.65
0.50
0.60
0.57
0.80
0.73
0.67
0.65
0.84
0.83
0.69
0.64
4.19
4.28
4.43
4.17
3.54
3.20
3.92
3.84
4.20
4.25
4.48
4.15
4.45
4.47
4.68
4.33
1.25
1.03
1.24
1.31
1.08
1.15
1.03
1.27
1.15
1.04
1.31
1.47
1.22
1.59
1.24
1.23
31600
34000
30900
34400
24400
22300
21300
21700
28800
24700
25900
26600
40900
37600
41600
35200
0.76
0.44
0.40
0.29
0.72
0.46
0.42
0.32
0.75
0.40
0.39
0.28
0.66
0.37
0.36
0.16
5.38
4.05
3.98
3.11
5.35
4.15
4.11
3.29
5.35
3.94
3.85
2.92
5.37
3.77
3.69
2.80
1.37
1.22
1.05
1.11
1.15
1.07
1.09
1.02
1.29
1.08
1.14
1.12
1.21
1.03
0.95
1.46
93
-CH3
CH3CN
DMSO
DCM
104
120
72
EtOH
92
-OCH3
CH3CN
DMSO
DCM
105
117
70
EtOH
93
-H
CH3CN
DMSO
DCM
102
122
74
EtOH
94
-I
CH3CN
DMSO
100
118
a Φf is the relative fluorescent quantum yield determined by using fluorescein as the reference (Φ = 0.92 in 0.1 M NaOH at 25 ).[31]
b Φf is the relative fluorescent quantum yield determined using Julo-BF2-CH3 as the reference (Φ = 0.79 in DCM at 25 ).
c X is the linear fitting coefficient of the fluorescent lifetime.
Figure 1. Normalized absorbance and fluorescence spectra of dyes (a) DEA-BF2-CH3 and (b)
THQ-BF2-CH3 in DMSO.

As seen in Table 1, these iminocoumarin-based borate complexes exhibited relatively
high fluorescent quantum yields in solutions. For DEA-BF2-R, the fluorescent quantum
yields were up to 0.7 and their fluorescent lifetimes are around 4.0 ns. However, there was an
exception for DEA-BF2-OCH3, which was weakly fluorescent (Φf = 0.11, τ = 0.63 ns in
EtOH). Similar to DEA-BF2-R, Julo-BF2-R exhibited high fluorescent quantum yields and
long lifetimes. It’s interesting that Julo-BF2-OCH3 was much more fluorescent than
DEA-BF2-OCH3. In particular, we were very pleased that red-emitting THQ-BF2-R dyes
were strongly fluorescent, especially in DCM (Φf = 0.66-0.76), and their fluorescent quantum
yields were up to 0.4 even in polar solution, which should be ascribed to the rigidity of THQ
moiety. It’s noted that THQ-BF2-OCH3 displayed a similar fluorescent quantum yield to
other THQ-BF2-R dyes.
In addition, a bathochromic shift in the emission spectra and a decrease in the fluorescent
quantum yields were observed for THQ-BF2-R dyes with increasing polarity of the solvents.
For instance, when the solvents went from DCM, EtOH, CH3CN to DMSO, THQ-BF2-CH3
exhibited red-shifts in their emissions from 607 nm, 620 nm, 626 nm to 646 nm whereas the
fluorescent quantum yields dropped from 0.76, 0.44, 0.40 to 0.29. The same phenomena were
observed in the optical properties of DEA-BF2-R and Julo-BF2-R dyes. Moreover, when
DEA-BF2-CH3, Julo-BF2-CH3 and THQ-BF2-CH3 were irradiated in acetonitrile under a
500 W Xe-lamp light, all the dyes can keep 80% absorption strength after 80 min of radiation,
indicating the good photostability, as shown in Figure S13.
3.2 Theotetical calculations
Figure 2. Molecular orbital plots (LUMO and HOMO) and HOMO/LUMO energy gaps of
DEA-BF2-CH3, Julo-BF2-CH3 and THQ-BF2-CH3 in DCM solvent.

To obtain more evidence to support our design strategy, DFT calculations were
performed on DEA-BF2-CH3, Julo-BF2-CH3 and THQ-BF2-CH3 using Gaussian 09 program,
whose geometries were optimized under B3LYP/6-31G level, and the frontier energy levels
(HOMO and LUMO) were calculated in DCM solvent. As depicted in Figure 2, the π
electrons of the HOMO of DEA-BF2-CH3 were mainly located on the coumarin core, N,
N-diethyl donor and benzimidazole moiety. At the excited state, the electron clouds were
mostly located on coumarin core and BF2 bridge linking iminocoumarin core. For
DEA-BF2-CH3, Julo-BF2-CH3 and THQ-BF2-CH3, the energy gaps between HOMOs and
LUMOs decreased (from 3.06 eV, 2.99 eV to 2.86 eV) owing to the enhanced conjugation of
donors (Julo and THQ) to iminocoumarin-BF2 framework, which was well in agreement with
the red-shift in their absorption wavelength.
3.3 Solid fluorescence
Figure 3. Photographs of (A) DEA-BF2-CH3, (B) Julo-BF2-CH3 and (C) THQ-BF2-CH3 (1) in a
solution of DCM under UV-bench lamp (λex = 365 nm), (2) in solid state under 10× fluorescence
inverse microscope.
Table 2 Photo-physical property of DEA-BF2-R, Julo-BF2-R and THQ-BF2-R in solid state.
Dyes
DEA-BF2
DEA-BF2
λem/ nm
634
608
604
614
624
622
635
636
713
665
666
719
Φf
τ/ ns
4.95
4.27
3.43
5.75
1.08
1.36
0.95
1.16
0.81
0.39
0.82
0.97
X
0.08
0.08
0.07
0.10
0.02
0.01
0.01
0.02
0.01
0.01
0.01
0.01
1.47
1.47
1.42
1.19
1.32
1.22
1.08
1.41
1.54
1.38
1.23
0.86
-
CH3
-
OCH3
DEA-BF2
DEA-BF2
Julo-BF2 CH3
Julo-BF2 OCH3
Julo-BF2
Julo-BF2
THQ-BF2
THQ-BF2
THQ-BF2
THQ-BF2
-H
-
I
-
-
-
H
-
I
-
CH3
-
OCH3
-
H
-
I
Next, the solid fluorescence of these borate dyes was investigated. DEA-BF2-R,
Julo-BF2-R and THQ-BF2-R all exhibited strong red solid fluorescence. As shown in Figure
3, DEA-BF2-CH3, Julo-BF2-CH3 and THQ-BF2-CH3 at solid state showed different

crystalline morphologies. The emission spectra, fluorescent quantum yields (Φf) and lifetimes
(τ) of all three kinds of dyes were summarized in Table 2 and Figure S14. The emission
wavelength of these dyes increased in an order of DEA-BF2-R, Julo-BF2-R and THQ-BF2-R
and their fluorescent quantum yields and fluorescent lifetimes dropped in the same order. it’s
noteworthy that THQ-BF2-CH3 and THQ-BF2-I had emissions in near-infrared spectra
region (λmaxEm= 713 and 717 nm, respectively). The solid-state fluorescence might be ascribed
to the existence of twisted 3D structure in these dyes, which could prevent π-π stacking
interactions.[12, 32]
3.4 Cell and zebrafish imaging
Figure 4. Cytotoxicity assay of THQ-BF2-CH3 at different concentrations in HeLa cells.
Figure 5. High content screening images of HeLa cells after incubation with dye THQ-BF2-CH3 (2.0
µM) and Hoechst 33342 (1.0 µM) for 15 min, respectively, at 37 °C. Red channel: THQ-BF2-CH3: λex
= 490-515 nm, λem = 570-650 nm; Blue channel: Hoechst 33342: λex = 350 nm, λem = 430-500 nm. (a)
Bright field, (b) merged of red channel and blue channel, (c) overlay of bright field and dark field.
Scale bar: 50 µm.

Figure 6. High content screening images of zebrafish after incubation with dye THQ-BF2-CH3 (2.0
µM) for 20 min at 28 °C. (a) Bright field; (b) Red channel: THQ-BF2-CH3: λex = 490-515 nm, λem
=
570-650 nm.
The excellent optical properties of these dyes encouraged us to explore their applications
for bioimaging. First, we evaluated the cytotoxicity of THQ-BF2-CH3 by MTT assays. As
shown in Figure 4, the cell viabilities were over 94% when cells were incubated with
THQ-BF2-CH3 at a concentration below 5.0 µM, indicating the low cytotoxicity of dye
THQ-BF2-CH3. Then, we used THQ-BF2-CH3 to image living HeLa cells co-stained with
Hoechst 33342 (nucleus indicator). When cells were incubated with THQ-BF2-CH3 for 15
min, strong red fluorescence was viewed from cytoplasm (shown in Figure 5). Furthermore,
we fed a 3-day-old zebrafish with THQ-BF2-CH3 for 20 min at 28 and remarkable red
fluorescence was observed (shown in Figure 6). The imaging experiments in cells and
zebrafish suggested THQ-BF2-R dyes were cell-membrane penetrable and could be used as
staining agents for biological applications.
4. Conclusions
In conclusion, we developed a series of borate fluorescent dyes, THQ-BF2-R, using
tetra-hydro-quinoxaline as the electron donor, which displayed large Stokes shifts, red
emissions and fairly high fluorescent quantum yields in solution, as well as exhibited red/NIR
solid fluorescence and could stain living cells and zebrafish with good performance and low
cytotoxicity.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.
U1608222), the State Key Laboratory of Fine Chemicals (KF1606), the State Key Laboratory

of Chemo/Biosensing and Chemometrics (2016005), Special Fund for Agro-scientific
Research in the Public Interest of China (No. 201503108) and the Fundamental Research
Funds for the Central Universities of Central South University (No. 2019zzts002 and No.
2019zzts438).
References
[1] Kowada T, Maeda H, Kikuchi K. BODIPY-based probes for the fluorescence imaging of
biomolecules in living cells. Chemical Society Reviews. 2015;44(14):4953-72.
[2] Boens N, Leen V, Dehaen W. Fluorescent indicators based on BODIPY. Chemical Society
Reviews. 2012;41(3):1130-72.
[3] Yue Y, Huo F, Cheng F, Zhu X, Mafireyi T, Strongin RM, et al. Functional synthetic probes
for selective targeting and multi-analyte detection and imaging. Chemical Society Reviews.
2019;48(15):4155-77.
[4] Xu M, Kelley SP, Glass TE. A Multi-Component Sensor System for Detection of Amphiphilic
Compounds. Angewandte Chemie-International Edition. 2018;57(39):12741-4.
[5] Zhang L, Liu XA, Gillis KD, Glass TE. A High-Affinity Fluorescent Sensor for Catecholamine:
Application to Monitoring Norepinephrine Exocytosis. Angewandte Chemie-International
Edition. 2019;58(23):7611-4.
[6] Kamkaew A, Lim SH, Lee HB, Kiew LV, Chung LY, Burgess K. BODIPY dyes in
photodynamic therapy. Chemical Society Reviews. 2013;42(1):77-88.
[7] Benniston AC, Copley G. Lighting the way ahead with boron dipyrromethene (Bodipy) dyes.
Physical Chemistry Chemical Physics. 2009;11(21):4124-31.
[8] Bessette A, Hanan GS. Design, synthesis and photophysical studies of
dipyrromethene-based materials: insights into their applications in organic photovoltaic

devices. Chemical Society Reviews. 2014;43(10):3342-405.
[9] Loudet A, Burgess K. BODIPY dyes and their derivatives: Syntheses and spectroscopic
properties. Chemical Reviews. 2007;107(11):4891-932.
[10] Ulrich G, Ziessel R, Harriman A. The chemistry of fluorescent bodipy dyes: Versatility
unsurpassed. Angewandte Chemie-International Edition. 2008;47(7):1184-201.
[11] Hong Y, Lam JWY, Tang BZ. Aggregation-induced emission. Chemical Society Reviews.
2011;40(11):5361-88.
[12] Mei J, Leung NL, Kwok RT, Lam JW, Tang BZ. Aggregation-Induced Emission: Together
We Shine, United We Soar! Chemical Reviews. 2015;115(21):11718-940.
[13] Araneda JF, Piers WE, Heyne B, Parvez M, McDonald R. High Stokes shift
anilido-pyridine boron difluoride dyes. Angewandte Chemie-International Edition.
2011;50(51):12214-7.
[14] Frath D, Massue J, Ulrich G, Ziessel R. Luminescent Materials: Locking pi-Conjugated
and Heterocyclic Ligands with Boron(III). Angewandte Chemie-International Edition.
2014;53(9):2290-310.
[15] Frath D, Poirel A, Ulrich G, De Nicola A, Ziessel R. Fluorescent boron(III) iminocoumarins
(Boricos). Chemical Communications. 2013;49(43):4908-10.
[16] Peng XJ, Song FL, Lu E, Wang YN, Zhou W, Fan JL, et al. Heptamethine cyanine dyes
with a large stokes shift and strong fluorescence: A paradigm for excited-state intramolecular
charge transfer. Journal of the American Chemical Society. 2005;127(12):4170-1.
[17] Liu J, Sun YQ, Zhang H, Shi H, Shi Y, Guo W. Sulfone-Rhodamines: A New Class of
Near-Infrared Fluorescent Dyes for Bioimaging. ACS applied materials & interfaces.

2016;8(35):22953-62.
[18] Yuan L, Lin W, Chen H. Analogs of Changsha near-infrared dyes with large Stokes Shifts
for bioimaging. Biomaterials. 2013;34(37):9566-71.
[19] Xiong K, Huo F, Chao J, Zhang Y, Yin C. Colorimetric and NIR Fluorescence Probe with
Multiple Binding Sites for Distinguishing Detection of Cys/Hcy and GSH in Vivo. Analytical
Chemistry. 2019;91(2):1472-8.
[20] Zhang W, Huo F, Zhang Y, Yin C. Dual-site functionalized NIR fluorescent material for a
discriminative concentration-dependent response to SO2 in cells and mice. Journal of
Materials Chemistry B. 2019;7(11):1945-50.
[21] Zhang W, Huo F, Yin C. Photocontrolled Single-/Dual-Site Alternative Fluorescence
Probes Distinguishing Detection of H2S/SO2 in Vivo. Organic Letters. 2019;21(13):5277-80.
[22] Hou P, Chen S, Wang H, Wang J, Voitchovsky K, Song X. An aqueous red emitting
fluorescent fluoride sensing probe exhibiting a large Stokes shift and its application in cell
imaging. Chemical Communications. 2014;50(3):320-2.
[23] Ren TB, Xu W, Zhang W, Zhang XX, Wang ZY, Xiang Z, et al. A general method To
increase Stokes shift by introducing alternating vibronic structures. Journal of the American
Chemical Society. 2018;140(24):7716-22.
[24] Liu X, Li Y, Ren X, Yang Q, Su Y, He L, et al. Methylated chromenoquinoline dyes:
synthesis, optical properties, and application for mitochondrial labeling. Chemical
Communications. 2018;54(12):1509-12.
[25] Geng Y, Tian H, Yang L, Liu X, Song X. An aqueous methylated
chromenoquinoline-based fluorescent probe for instantaneous sensing of thiophenol with a red

emission and a large Stokes shift. Sensors and Actuators B-chemical. 2018;273:1670-5.
[26] Liu X, Cole JM, Xu Z. Substantial Intramolecular Charge Transfer Induces Long Emission
Wavelengths and Mega Stokes Shifts in 6-Aminocoumarins. Journal of Physical Chemistry C.
2017;121(24):13274-9.
[27] Chen W, Xu S, Day JJ, Wang DF, Xian M. A General Strategy for Development of
Near-Infrared Fluorescent Probes for Bioimaging. Angewandte Chemie-International Edition.
2017;56(52):16611-5.
[28] Grabowski ZR, Rotkiewicz K, Rettig W. Structural changes accompanying intramolecular
electron transfer: focus on twisted intramolecular charge-transfer states and structures.
Chemical reviews. 2003;103(10):3899-4032.
[29] Liu X, Qiao Q, Tian W, Liu W, Chen J, Lang MJ, et al. Aziridinyl Fluorophores
Demonstrate Bright Fluorescence and Superior Photostability by Effectively Inhibiting Twisted
Intramolecular Charge Transfer. Journal of the American Chemical Society.
2016;138(22):6960-3.
[30] Rajagopal R, Shenoy VU, Padmanabhan S, Sequeira S, Seshadri S. Synthesis of
fluorescent 2, 3-fused coumarin derivatives. Dyes and Pigments. 1990;13(3):167-75.
[31] Weber G, Teale FWJ. Determination of the absolute quantum yield of fluorescent
solutions. Transactions of the Faraday Society. 1957;53:646.
[32] Luo J, Xie Z, Lam JWY, Cheng L, Tang BZ, Chen H, et al. Aggregation-induced emission
of 1-methyl-1,2,3,4,5-pentaphenylsilole. Chemical Communications. 2001;18(18):1740-1.

Highlights
1. A series of new iminocoumarin-based fluorescent borate complexes was developed
by introducing a very strong electron donor, tetrahydroquinoline group.
2. These dyes displayed red/NIR emissions (up to 650 nm in DMSO) and large Stokes
shift (up to 122 nm in DMSO).
3. These dyes had good fluorescent quantum yields (0.65-0.84 in DCM) and large
absorption molecular extinction coefficients (104 M-1cm-1).
4. These dyes showed red/NIR solid fluorescence.
5. These dyes had potential application for bioimaging in cells and zebrafish.

Declaration of interests
☑ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☒The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: