Structurally-thrifty and visible-absorbing fluorophores

Article abstract of DOI:10.1016/j.saa.2020.118907

Fluorophores with a minimal push-pull backbone are actively pursued due to their potentials in biological labelling. Herein a series of structurally-thrifty and visible-absorbing fluorophores (SDXs) were successfully constructed following the D′D-π-A design strategy, in which a secondary donor (D′) was introduced in conjugation with the donor (D) to enhance its electron donating capability. For a very small scaffold, SDXs exhibit a surprisingly long-wavelength absorption band in the visible spectral range (λ_abs = 420 nm) and a strong green fluorescence emission (λ_em = 530 nm) with a fluorescence quantum yield up to 0.84. Notably, fluorescence of SDXs was quenched in hydrogen-bonding solvents, e.g. MeOH and H₂O. This phenomenon renders SDXs feasibility for imaging of cellular non-hydrogen-bonding microenvironment, as demonstrated with BEAS-2B cells. These results proved that the D′D-π-A is a powerful design strategy to construct novel structurally-thrifty fluorophores.

Full text of DOI:10.1016/j.saa.2020.118907

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118907
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Structurally-thrifty and visible-absorbing fluorophores
d,
a
a,
Xiao Luo a,b, Yan Chen a, Yanchun Li a, Zhenglong Sun c, , Weihong Zhu , Xuhong Qian , Youjun Yang
⁎
⁎
⁎
a
State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
b
School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China
c
Suzhou Institute of Biomedical Engineering and Technology (SIBET), Chinese Academy of Sciences, Suzhou, Jiangsu 215163, China
Key Laboratory for Advanced Materials, Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, Institute of
Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2020
Received in revised form 13 August 2020
Accepted 29 August 2020
Available online 4 September 2020
Fluorophores with a minimal push-pull backbone are actively pursued due to their potentials in biological label-
ling. Herein a series of structurally-thrifty and visible-absorbing fluorophores (SDXs) were successfully con-
structed following the D′D-π-A design strategy, in which a secondary donor (D′) was introduced in
conjugation with the donor (D) to enhance its electron donating capability. For a very small scaffold, SDXs exhibit
a surprisingly long-wavelength absorption band in the visible spectral range (λabs = 420 nm) and a strong green
fluorescence emission (λem = 530 nm) with a fluorescence quantum yield up to 0.84. Notably, fluorescence of
SDXs was quenched in hydrogen-bonding solvents, e.g. MeOH and H2O. This phenomenon renders SDXs feasibil-
ity for imaging of cellular non-hydrogen-bonding microenvironment, as demonstrated with BEAS-2B cells. These
results proved that the D′D-π-A is a powerful design strategy to construct novel structurally-thrifty fluorophores.
© 2020 Elsevier B.V. All rights reserved.
Keywords:
Structurally-thrifty
D′D-π-A
Visible-absorbing
Green emission
Cell imaging
1. Introduction
induce injuries to biological molecules [14]. Therefore, it is our goal to
develop small molecules absorbing and emitting beyond 400 nm
Fluorescent dyes are particularly suitable for biological imaging due
to its sensitivity for detection, versatility for manipulation, and feasibil-
ity for complex matrix [1–3]. Over the years, they have become indis-
pensable for a variety of basic, translational and clinical applications
[4–6]. For a fluorescent dye to find practical applications, it should ex-
hibit localization-specificity toward the structure-of-interest in the
complex biological matrix. Target-specificity in biological labelling is
typically achieved harnessing preferential solubility, dipole-dipole in-
teraction, antibody-antigen interaction, ligand-receptor interaction, ge-
netic labelling, or covalent bond modification [7–9]. Recently, metabolic
labelling of oligosaccharides with unnatural saccharides or proteins
with unnatural amino acids by the cell biosynthesis machinery has
attracted tremendous attention [10,11]. A high degree of structural sim-
ilarity to the native substrate is necessary to bypass the specificity of en-
zyme. Even the relatively small ones of the existing classic fluorophores,
e.g. coumarin, NBD, dansyl, etc., are still not small enough, not to men-
tion naphthalimides, BODIPY, xanthenes, cyanines, etc. [12,13]. While
the structure of a dye should be kept small, its absorbing wavelength
should be long enough, e.g. the visible spectral region, not to readily
(Fig. 1).
The UV–Vis absorption of
a
fluorochrome is the result of
HOMO-LUMO electronic transition, i.e. π-π* and n-π* (Fig. 1). Since
n-π* is typically of low absorptivity due to its spin-forbidden nature, it
is not considered further [15]. The Lewis structure of a given fluoro-
chrome with a π-π* transition typically exhibits a theme of D-π-A. To
render long-wavelength absorption for a D-π-A scaffold, the following
three approaches may be adopted, enhancing the electron-donating/
withdrawing capability of the donor (D)/acceptor (A), respectively,
elongating the π backbone in between, and substituting the π backbone
to destabilize the ground state (S0) or stabilize the first singlet excited
state (S1) following the perturbation theory [16,17]. Since it is the goal
of this manuscript to develop structurally thrifty dyes, we wish to
keep the π backbone as small as possible, i.e. an ethylene unit. In this
case, a viable approach to red-shift the absorption maxima is to make
the donor more donating and the acceptor more withdrawing [18]. A
secondary donor (D′) in conjugation destabilizes the lone-pair of D
and renders D more electron-donating. Analogously, a secondary accep-
tor (A′) in conjugation destabilizes the empty orbital of A and renders A
more electron-withdrawing [19]. Not surprisingly, the generic structure
of D′-π-D-π-A-π-A′ has been proposed as a guideline to develop dyes
exhibiting an even longer-wavelength absorption than what D-π-A
could possibly render [20,21]. However, a D′-π-D-π-A-π-A′ type dye
will not be structurally thrifty, without a doubt. We were further
⁎
Corresponding authors.
E-mail addresses: sunzhl@sibet.ac.cn (Z. Sun), weihongzhu@ecust.edu.cn (W. Zhu),
youjunyang@ecust.edu.cn (Y. Yang).
https://doi.org/10.1016/j.saa.2020.118907
1386-1425/© 2020 Elsevier B.V. All rights reserved.

2
X. Luo et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118907
Fig. 1. Folded D′D-π-A: the optimal design strategies of developing structurally-thrifty fluorophores with long absorption and emission wavelength.
inspired by the structural features of a special class of fluorophores,
commonly referred to as H-chromophores [22]. In such dyes, the π scaf-
fold of the D-π-A is also the π scaffold of the D′-π-D and/or A′-π-A. We
call such a design principle, folded D′D-π-AA′. The most notable dye of
this class is indigo [23], a red-absorbing dye with a rather small scaffold,
quinone dyes [24], and many single-benzene fluorophores with two do-
nors at C-1,4 and two acceptors at C-2,5, i.e. D′D-π-AA′ [25,26], are also
dyes of this type. We wish to further shrink the scaffold of folded D′D-π-
AA′ to folded D-π-AA′ [27,28] or folded D′D-π-A [29], by eliminating a D′
or a A′ group. While minimizing the structure, we also expect that the
maximal absorption wavelength should be kept longer than 400 nm,
the light of which spectral region is comparatively less absorbed and
less toxic to the biological macromolecules than the light of shorter
spectral region.
HRMS spectra were acquired on a Micromass GCT spectrometer. Ab-
sorption spectra were collected by a SHIMADZU UV-2600 UV–vis spec-
trophotometer. Fluorescence excitation and emission spectra were
collected in a PTI-QM4 steady-stead fluorimeter with a 75 W Xenon
arc-lamp and a model 810 PMT. The voltage of PMT was 950 V. The ex-
citation and emission slits were set to 2 nm.
2.2. Synthesis of 8-nitrochroman-4-one (2) and 6-nitrochroman-4-one (3)
To a 25 mL round bottomed flask was added 4-chromanone (1, 5.0 g,
1 equiv., 33.97 mmol) and concentrated sulfuric acid (10 mL). The mix-
ture was cooled to 0 °C, and then a mixture of concentrated sulfuric acid
(2 mL) and concentrated nitric acid (2.25 mL, 1.00 equiv., 33.97 mmol)
was added dropwise over 15 min. The resultant solution was stirred at
25 °C for 30 min before poured into ice water. Light yellow precipitates
were collected via a suction filtration and then purified by a flash col-
umn using a mixture of petroleum ether and ethyl acetate [5:1, v/v] as
an eluent to afford 2 [30] (1.83 g, 28%) and 3 (3.98 g, 61%) as white
solid. Compound 2: 1H NMR (400 MHz, CDCl3): δ 8.19 (dd, J = 7.8,
1.8 Hz, 1H), 8.11 (dd, J = 8.0, 1.8 Hz, 1H), 7.14 (t, J = 7.9 Hz, 1H), 4.73
(t, J = 6.5 Hz, 2H), 2.96 (t, J = 6.5 Hz, 2H); EI-MS (m/z): [M] calcd. for
C9H7NO4, 193.0375; found 193.0376. Compound 3: 1H NMR
(400 MHz, CDCl3): δ 8.74 (d, J = 2.8 Hz, 1H), 8.30 (dd, J = 9.1, 2.9 Hz,
1H), 7.10 (d, J = 9.1 Hz, 1H), 4.66 (t, J = 6.5 Hz, 2H), 2.89 (t, J =
6.5 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 189.7, 165.8, 142.1, 130.3,
123.8, 120.8, 119.4, 67.7, 37.2; EI-MS (m/z): [M] calcd. for C9H7NO4,
193.0375; found 193.0374.
Herein, we developed a series of structurally-thrifty fluorophores
(SDX) utilizing
a rigidified folded D′D-π-A design strategy.
Rigidification mandates coplanarity of the D and A, enhances the
electron-delocalization, and is the highlight of this approach compared
to the literature analogous. SDXs exhibit visible absorption and fluores-
cence emission maxima. SDXs showed high fluorescent quantum yields,
among which SDX4 possessed a reddest absorption wavelength of ca.
420 nm and emitted strong green fluorescence with a large Stokes
shift over 100 nm. The fluorescence emission of SDXs was highly sensi-
tive to hydrogen bonding. Finally, SDXs were feasible live cell imaging.
2. Experimental
2.1. Material and instruments
2.3. Synthesis of 8-aminochroman-4-one (SDX1)
All chemical regents and solvents were purchased from commercial
suppliers and were used without further purification. The 1H NMR and
13C NMR spectra were collected on a Bruker AV-400 spectrometer.
Compound 2 (1 g, 1 equiv., 5.18 mmol) was dissolved in 20 mL of
methanol, and Pd/C (100 mg) was added. The suspension was

X. Luo et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118907
3
deoxygenated and stirred under a hydrogen atmosphere (balloon) for
6 h at 25 °C. Powder of Pd/C was removed by filtration. The organic sol-
vent in the filtrate was removed under reduced pressure to yield a yel-
low solid SDX1 (778 mg) in a 92% yield and used without further
purification. 1H NMR (400 MHz, CDCl3): δ 7.30 (ddd, J = 7.7, 1.6,
0.9 Hz, 1H), 6.90–6.80 (m, 2H), 4.57 (td, J = 6.4, 0.64 Hz, 2H), 2.81
(td, J = 6.4, 0.64 Hz, 2H); 13C NMR (101 MHz, CDCl3): δ 192.2, 150.0,
136.2, 121.4, 120.2, 116.1, 67.4, 38.0; EI-MS (m/z): [M] calcd. for
C9H9NO2, 163.0633; found 163.0634.
to calculate the fluorescence quantum yields. The absorbance values
were plotted against the dye concentration. The slope was calculated,
which is the extinction coefficient of SDX. The relative fluorescence
quantum yield of SDX in different solvents is calculated following a pro-
tocol [31]. A fluorescence quantum yield of 0.58 for the cation Coumarin
102 in EtOH was used as a reference to calculate the fluorescence quan-
tum yields of SDX [32].
2.8. Cell culture and cell cytotoxicity
2.4. Synthesis of 6-aminochroman-4-one (SDX2)
Human lung epithelial BEAS-2B cells were purchased from Cell Bank
of Type Culture Collection of Chinese Academy of Sciences. BEAS-2B
cells were maintained in Dulbecco's modified Eagle's medium (DMEM,
Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone) and
1% penicillin-streptomycin. The cells were cultured in a humidified at-
mosphere of 5% CO2/95% air at 37 °C and grown on 25 mm cover slips
(Fisherbrand, 12-545-102) for 1–2 days to reach 70–90% confluency be-
fore use. Cytotoxicity study of SDX2 was performed using Cell Counting
Kit-8 (CCK-8) assay. BEAS-2B cells were grown to 70%–80% confluency
before they were passaged. BEAS-2B cells were seeded into 96-well
cell culture plate at 104/well, with 100 μL complete media for 24 h. A
10 mM stock solution of SDX2 was diluted with complete medium to
obtain different concentrations (0, 5, 10, 20, 30, 40 μM). The culture me-
dium was carefully removed, and different concentrations of SDX2 were
added into each well. After incubation at 37 °C for 24 h, 10 μL Cell
Counting Kit-8 (CCK-8) solution was added per well and the cells
were incubated for another 2 h, then the absorbance at 450 nm was
read by Microplate reader (Multiskan, Thermo Scientific, Waltham,
MA, USA).
Compound 3 (1 g, 1 equiv., 5.18 mmol) was dissolved in 20 mL of
methanol, and Pd/C (100 mg) was added. The suspension was deoxy-
genated and stirred under a hydrogen atmosphere (balloon) for 6 h at
25 °C. Powder of Pd/C was removed by filtration. The organic solvent
in the filtrate was removed under reduced pressure to yield a yellow
solid SDX2 (811 mg) in a 96% yield and used without further purifica-
tion. 1H NMR (400 MHz, CDCl3): δ 7.16 (d, J = 2.9 Hz, 1H), 6.88 (dd,
J = 8.7, 2.9 Hz, 1H), 6.81 (d, J = 8.7 Hz, 1H), 4.45 (t, J = 6.4 Hz, 2H),
3.55 (s, 2H), 2.76 (t, J = 6.4 Hz, 2H); 13C NMR (101 MHz, CDCl3): δ
192.4, 155.5, 140.6, 124.6, 121.7, 118.8, 111.2, 67.1, 38.1, 29.8; EI-MS
(m/z): [M] calcd. for C9H9NO2, 163.0633; found 163.0631.
2.5. Synthesis of 8-(diethylamino)chroman-4-one (SDX3)
SDX1 (500 mg, 1 equiv., 3.06 mmol), EtI (1.00 g, 2.1 equiv.,
6.43 mmol), Na2CO3 (324 mg, 1 equiv., 3.06 mmol) and anhydrous ace-
tonitrile (30 mL) were added into a 100 mL flask. The resulting mixture
was heated to 80 °C with rigorous stirring for 10 h before being cooled to
room temperature. Solid materials filtered off using a Celite cake under
reduced pressure to give the crude product as a dark brown liquid,
which was purified by a flash column using a mixture of petroleum
ether and EtOAc [20:1, v/v] as an eluent to afford SDX3 (624 mg) as a
light yellow liquid in a 93% yield. 1H NMR (400 MHz, CDCl3): δ 7.14
(d, J = 3.2 Hz, 1H), 6.95 (dd, J = 9.1, 3.2 Hz, 1H), 6.86 (d, J = 9.0 Hz,
1H), 4.46 (t, J = 6.5 Hz, 2H), 3.30 (q, J = 7.1 Hz, 4H), 2.77 (t, J =
6.5 Hz, 2H), 1.11 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ
192.8, 153.8, 143.1, 122.7, 121.6, 118.6, 108.7, 67.1, 44.9, 38.3, 12.5. EI-
MS (m/z): [M] calcd. for C13H17NO2, 219.1259; found 219.1258.
2.9. Fluorescent imaging
Confocal fluorescent images were recorded on Leica SP5 confocal
microscope and 60× oil-immersion objective lens was used. BEAS-2B
cells (approximately 5 ∗ 104) were seeded and cultured 24 h for adhe-
sion in 15 mm glass-bottomed dishes. BEAS-2B cells were incubated
with SDXs (20 μM) for 60 min and washed with PBS for three times to
remove excess dyes. The excitation light source of is a 405 nm laser,
and the emission wavelength collection range were 410–650 nm. The
data obtained were analyzed and processed using Leica's own software
and Image J.
2.6. Synthesis of 6-(diethylamino)chroman-4-one (SDX4)
3. Results and discussion
SDX2 (500 mg, 1 equiv., 3.06 mmol), EtI (1.00 g, 2.1 equiv.,
6.43 mmol), Na2CO3 (324 mg, 1 equiv., 3.06 mmol) and anhydrous ace-
tonitrile (30 mL) were added into a 100 mL flask. The resulting mixture
was heated to 80 °C with rigorous stirring for 10 h before being cooled to
room temperature. Solid materials filtered off using a Celite cake under
reduced pressure to give the crude product as a dark brown liquid,
which was purified by a flash column using a mixture of petroleum
ether and EtOAc [20:1, v/v] as an eluent to afford SDX4 (618 mg) as a
light yellow liquid in a 93% yield. 1H NMR (400 MHz, CDCl3): δ 7.57
(dd, J = 7.9, 1.6 Hz, 1H), 7.11 (dd, J = 7.8, 1.6 Hz, 1H), 6.94 (t, J =
7.8 Hz, 1H), 4.59 (t, J = 6.4 Hz, 2H), 3.17 (d, J = 7.1 Hz, 4H), 2.81 (t,
J = 6.4 Hz, 2H), 1.04 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ
192.5, 156.5, 139.6, 127.4, 122.3, 120.8, 120.4, 67.2, 46.3, 37.8, 12.0. EI-
MS (m/z): [M] calcd. for C13H17NO2, 219.1259; found 219.1260.
3.1. Molecular design and synthesis
Our approach to develop a structurally-thrifty dye with visible ab-
sorption and fluorescent emission wavelength was based on the design
concept of D′D-π-A, i.e. further substituting a D-π-A scaffold with an
electron donating group (D′) in conjugation with the electron donor
(D). Such a secondary electron donor (D′) destabilizes the lone-pair of
D and make D more electron-donating. The D-π-A scaffold to start
with was 4-chromanone (1), in which the oxygen atom is the electron
donor (D), the carbonyl group is the electron acceptor (A), and the dou-
ble bond in between is the π conjugation system. Such a D-π-A scaffold
is further rigidified by tethering the D and the A groups with an ethylene
bridge, to increase the fluorescence quantum yield. A strongly electron-
donating amino or diethylamino group was then introduced ortho or
para to the electron-donating oxygen atom as the secondary electron
donor.
2.7. Molar absorptivity and fluorescence quantum yields determinations
The DMSO solution of dyes at 10 mM were prepared and used as a
stock for further preparation of dilute dye solutions. The absorption
spectrum and the emission spectrum of the resulting solution with dif-
ferent dye concentration (in a 4 mL micro-fluorescence cuvette) were
acquired until five sets of abs/em spectra with an absorbance below
0.1 were available. The abs/em spectra from these five sets were used
SDXs were conveniently synthesized with high yields according to
Scheme 1. Commercially available 4-chromanone (1) was nitrated
with mixed acids of HNO3 and H2SO4, resulting in a mixture of ortho-
and para-nitration isomers, i.e. compound 2 and 3, in a ca. 1:2 ratio.
The mononitrated products (2 and 3) were reduced with catalytic hy-
drogenation to yield their corresponding amino derivatives (SDX1 and

4
X. Luo et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118907
Scheme 1. Synthetic pathway of structurally-thrifty SDXs.
SDX2), respectively. The amino group of SDX1 and SDX2 was further
alkylated with ethyl iodide to afford the SDX3 and SDX4, respectively.
was not fluorescent in these solvents upon excitation at 320 nm. As ex-
pected, the absorption of SDX1 or SDX2 exhibited notable
a
bathochromic shift compared to 1. For example, the absorption maxi-
mum of SDX1 or SDX2 in THF localizes at 360 and 380 nm, respectively,
compared to the 320 nm of 1. SDX1 and SDX2 did not exhibit a strong
solvatochromism, exemplified by a ca. 10 nm bathochromic shift as
the solvent polarities gradually increased from THF to CH2Cl2, acetone,
EtOH, CH3CN, and DMSO. Fortunately, SDX1 and SDX2 were fluorescent
with a maximal emitting wavelength at 455 and 480 nm in THF upon
excitation at 360 and 380 nm. And the fluorescence quantum yields
3.2. Spectroscopic properties of SDXs
The spectral properties of SDXs were studied in various organic sol-
vents (Fig. 2 and Table 1). The maximal absorption wavelength of 4-
chromanone (1) localizes at ca. 320 nm with an extinction coefficient
of ca. 4.50 × 103 M−1 cm−1 (Fig. S1 and Table S1), in all six tested sol-
vents of THF, CH2Cl2, acetone, EtOH, CH3CN and DMSO. Compound 1
Fig. 2. The absorption and fluorescent emission spectra of SDXs in three solvents. In DMSO: (A) and (B). In CH3CN: (C) and (D). In CH2Cl2: (E) and (F).

X. Luo et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118907
5
Table 1
notable bathochromic shift compared to the 380 nm of SDX2. Similar
to SDX1/2, SDX4 did not display a strong solvatochromism, and a
slight bathochromic shift (ca. 10 nm) arose accompanied by an in-
crease in solvent polarities from THF to DMSO. SDX4 were strongly
fluorescent with a maximal emitting wavelength at 495 nm in THF
upon excitation at 410 nm and the fluorescent quantum yield was
0.53. With the solvent polarity increased, the maximal emitting
wavelength of SDX4 redshifted to 520 nm in DMSO with a high quan-
tum yield of 0.84. However, the lone pair of electrons on nitrogen
atom of the ortho-diethylamino group in SDX3 couldn't effectively
conjugate with the phenyl ring system due to the steric interaction.
This decreased coplanarity weakened the electron donating capabil-
ity of the second donor and the absorption of SDX3 might exhibit a
hypsochromic shift of ca. 50–60 nm, compared to SDX4. The maxi-
mal absorption wavelength of SDX3 localized at ca. 360 nm in vari-
ous solvent. The maximal emitting wavelength of SDX3 was only
ca. 20–30 nm shorter than SDX4. Therefore, a larger Stokes shift
was observed for SDX3 compared to SDX4.
The spectra properties of structurally-thrifty dyes (SDX) in different solvents.
max
max
Solvent
DMSO
CH3CN
EtOH
Compound
λ
abs.
ε/103
λ
Stokes shift
[nm]
Φa
em.
[nm]
[M−1 cm−1
]
[nm]
SDX1
SDX2
SDX3
SDX4
SDX1
SDX2
SDX3
SDX4
SDX1
SDX2
SDX3
SDX4
SDX1
SDX2
SDX3
SDX4
SDX1
SDX2
SDX3
SDX4
SDX1
SDX2
SDX3
SDX4
370
390
362
420
357
376
359
410
360
375
352
420
360
380
359
410
353
375
361
415
360
380
354
410
3.67
3.87
2.93
3.15
4.15
3.79
2.69
2.76
3.21
3.77
2.37
2.35
3.65
3.96
3.18
2.83
3.63
4.23
2.77
2.27
3.51
4.10
2.87
3.07
480
505
505
520
475
490
510
530
N.D.
N.D.
N.D.
N.D.
460
485
490
510
460
475
500
520
455
480
470
495
110
115
143
100
118
114
151
120
N.A.
N.A.
N.A.
N.A.
100
105
131
100
107
100
139
105
95
0.62
0.74
0.31
0.84
0.27
0.40
0.17
0.32
N.A.
N.A.
N.A.
N.A.
0.34
0.40
0.20
0.39
0.42
0.59
0.31
0.56
0.43
0.48
0.34
0.53
Acetone
CH2Cl2
Those four structurally-thrifty dyes (SDX1/2/3/4) exhibited dimin-
ished fluorescence emission in EtOH or H2O (Tables 1 and S2), presum-
ably due to hydrogen-bonding capability of the solvents to the carbonyl
group of SDXs. For the same reason, the presence of trace water in these
organic solvents will significantly lower the fluorescent quantum yields
of those four dyes.
100
116
85
THF
a
Based on this phenomenon, SDXs have potentials to be used as a mi-
croenvironment probe, to be fluorescent in non-hydrogen-bonding en-
vironment and non-fluorescent in hydrogen-bonding environment. To
test our hypothesis, a surfactant was added into H2O at a concentration
higher than its critical micellar concentration (CMC) [33]. Specially,
Tween-80 [34], a non-ionic surfactant, was used to prepare the micellar
aqueous media with different weight fractions of Tween-80 from 0.5%
to 10%. The fluorescence properties of SDX4 in micellar media were in-
vestigated. SDX4 was very weakly fluorescent in pure water (vide
supra). In micellar aqueous media containing 10%, 5%, 2.5% and 1%
Tween-80, a fluorescence enhancement of ca. 31-fold, 18-fold, 10-fold
and 5-fold was observed, relative to the fluorescence intensity of
SDX4 in aqueous solution (Fig. 3). We postulated that SDX4, which
are non-polar, prefers the non-polar and non-hydrogen-bonding
environment of the micelles of Tween-80. This prevented the
hydrogen-bonding between SDX4 with water molecules and resulted
in the fluorescent recovery of SDX2. This phenomenon demonstrated
the potentials of SDX2 in sensing the microenvironment associated
with hydrogen-bond interaction and might find applications in moni-
toring biological functions involved with spatial conformations of pro-
tein and DNA helix [35].
The quantum yields were determined with Coumarin 102 as the reference (Φ = 0.58
in EtOH). N.D.: Not determined. N.A.: Not applicable. (EtOH).
were 0.43 and 0.48, respectively. A bathochromic shift of the maximal
emitting wavelength of ca. 25 nm was observed as the solvent varied
from THF to DMSO. The fluorescence quantum yield was also mildly af-
fectedly, exemplified by a increased fluorescence quantum yield of 0.62
and 0.74 in DMSO for SDX1 and SDX2, respectively. This strongly sup-
ported the feasibility of our design principle of structurally-thrifty and
visible absorbing/emitting fluorophores, i.e. D′D-π-A. The spectral
wavelength of the para-substituted analog (SDX2) is consistently longer
than the ortho-substituted (SDX1). Presumably, the amino group in
SDX1 is closer to the oxygen atom and therefore exhibits a slightly
stronger inductive effect, which accounts for the slightly shorter absorp-
tion and emission wavelengths of SDX1, compared to SDX2.
Then the spectral properties of SDX3 and SDX4 were studied.
With the electron-donating amino group alkylated into a stronger
diethylamino group, a bathochromic absorption and fluorescent
emission spectra were anticipated. Unsurprisingly, SDX4 showed a
maximal absorbing wavelength at 410 nm in THF, exhibiting a
Fig. 3. (A) The fluorescent emission spectra of 10 μM SDX4 in H2O-Tween-80 mixtures measured with different weight fractions of Tween 80. (B) The plot of fluorescent intensity at
460 nm (λex = 390 nm) versus the weight faction of Tween-80.

6
X. Luo et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118907
Fig. 4. Confocal images of BEAS-2B cells co-stained with 20 μM SDX1/2/3/4. (λex = 405 nm, λem = 410–650 nm). Scale bar: 30 μm.
3.3. Imaging of live cells incubated with SDXs
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