Syntheses, kinetics and thermodynamics of BODIPY-based fluorescent probes with different kinds of hydrophilic groups for the detection of biothiols

Article abstract of DOI:10.1016/j.dyepig.2020.108434

A series of BODIPY-based fluorescent probes incorporating different kinds of hydrophilic groups were synthesized for the detection of biothiols (GSH as a representative target), namely BDP (control), BDP-OH (acidic), BDP-OEG (neutral and hydrophilic) and

Full text of DOI:10.1016/j.dyepig.2020.108434

Journal Pre-proof
Syntheses, kinetics and thermodynamics of BODIPY-based fluorescent probes with
different kinds of hydrophilic groups for the detection of biothiols
Lian-Xun Gao, Ming Tian, Lu Zhang, Yi Liu, Feng-Lei Jiang
PII:
S0143-7208(20)30744-0
DOI:
https://doi.org/10.1016/j.dyepig.2020.108434
DYPI 108434
Reference:
To appear in: Dyes and Pigments
Received Date: 23 March 2020
Revised Date: 2 April 2020
Accepted Date: 5 April 2020
Please cite this article as: Gao L-X, Tian M, Zhang L, Liu Y, Jiang F-L, Syntheses, kinetics and
thermodynamics of BODIPY-based fluorescent probes with different kinds of hydrophilic groups for the
detection of biothiols, Dyes and Pigments (2020), doi: https://doi.org/10.1016/j.dyepig.2020.108434.
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Author Statement
Lian-Xun Gao: Methodology, Investigation, Formal analysis, Writing - original draft.
Ming Tian: Methodology, Investigation. Lu Zhang: Investigation. Yi Liu: Resources,
Supervision, Funding acquisition. Feng-Lei Jiang: Supervision, Writing - review &
editing, Funding acquisition.

Table of contents
Hydrophilic group
Thiol addition site

Syntheses, kinetics and thermodynamics of BODIPY-based
fluorescent probes with different kinds of hydrophilic groups
for the detection of biothiols
Lian-Xun Gao,a Ming Tian, a Lu Zhang, a Yi Liu a,b,c and Feng-Lei Jiang a,*
a
Sauvage Center for Molecular Sciences, College of Chemistry and Molecular
Sciences, Wuhan University, Wuhan 430072, P. R. China.
b
Hubei Province Key Laboratory for Coal Conversion and New Carbon Materials,
School of Chemistry and Chemical Engineering, Wuhan University of Science and
Technology, Wuhan 430081, P. R. China
c
Guangxi Key Laboratory of Natural Polymer Chemistry, College of Chemistry and
Materials Science, Nanning Normal University, Nanning 530001, P. R. China
* Corresponding author. Email: fljiang@whu.edu.cn (F.-L. Jiang)

Abstract
A series of BODIPY-based fluorescent probes incorporating different kinds of
hydrophilic groups were synthesized for the detection of biothiols (GSH as a
representative target), namely BDP (control), BDP-OH (acidic), BDP-OEG (neutral
and hydrophilic) and BDP-QA (cationic). The incorporation of nitroolefin unit
(-CH=CH-NO2) to the BODIPY core enabled the OFF-ON fluorescent probes. The
results indicated that the absorption and fluorescence emission spectra of the probes
were essentially not affected by the hydrophilic groups attached on the para position
of the meso phenyl group. The reaction rate constants were affected by the
hydrophilicity of probes. It was highly worth noting that the cationic BDP-QA had
the fastest response toward biothiols owing to the best water solubility and the
possible formation of ion pairs with thiolate (R-S-). Thermodynamics illustrated that
the reactions of probes with GSH all had negative enthalpy changes and negative
entropy changes. Moreover, BDP-QA had the highest affinity toward GSH (K = 2.54
× 104 M-1) and the smallest LOD value (182 nΜ), which benefited from its best water
solubility. This work has primarily elucidated the effects of hydrophilic groups from
the kinetic and thermodynamic perspectives. It will promote better design of
fluorescent probes with fast response and high affinity.
Key words: fluorescent probe; water solubility; hydrophilic group; kinetics;
thermodynamics

1. Introduction
Biothiols, including cysteine (Cys, 30 ~ 200 μM), homocysteine (Hcy, 5 ~ 15 μM)
and glutathione (GSH, γ-glutamyl-cysteinyl-glycin, 1 ~ 10 mM), play crucial roles for
the maintenance of cellular redox status and healthy operations of life systems [1-4].
GSH, the most abundant biothiol and a non-protein thiol in living cells, is regarded as
a detoxifying antioxidant that protects cells against damage caused by harmful heavy
metals, peroxides and free radicals and so on [5]. So, it is highly necessary to monitor
and detect GSH level in living cells and tissues. In the past few decades, there were
many methods to detect biothiols, such as electrochemical detection [6], high
performance liquid chromatography (HPLC) [7], electrospray ionization mass
spectrometry [8] and so on. Although these methods could realize the detection of
biothiols, they have suffered from some drawbacks, such as complicated preparations
and operations, expensive testing facilities and restricted applications in living cells
[9]. It was worth noting that fluorescent probes attracted growing interest owing to
their excellent properties, such as simple operation, low detection limit, applicability
in living cells and tissues, etc [10-13]. Over the last decades, researchers have
exploited plentiful fluorescent probes based on different fluorophores, such as
rhodamine
[14-16],
coumarin
[17-20],
cyanine
[21,22],
BODIPY
(boron-dipyrromethene) derivatives [23-28], etc. Among them, BODIPY fluorophores
have advantages such as facile syntheses, structural versatility, tunable spectroscopic
properties and high quantum yields [29]. Thus, it is a good choice for BODIPY probes
to detect and monitor thiols in cells and tissues.
However, BODIPY fluorophores have some drawbacks: ( ) poor water solubility
[30], ( ) slow response [28,31], and ( ) intense fluorescent background [31]. These
problems seriously impeded the applications of fluorescent probes in living cells.
Therefore, many researchers have made an enormous number of achievements in the
past decades. To resolve the water solubility of probes, researchers have employed
many methods including introduction of ionic hydrophilic groups (anionic [32,33] and
cationic [34,35]), neutral hydrophilic groups (polyethylene ethanol or polyether chain)
1

[36-39], water-soluble polypeptide [40-42], saccharides [43], amine [44] and
amphiphilic groups [45], etc. These methods improved water solubility of probes and
raised dispersion coefficients of probes by introducing hydrophilic groups. So the
collision probability of reactant molecules would increase and the response could be
faster according to collision theory. Though these achievements have been made, it is
still unclear how the different hydrophilic groups affect the efficacies of
BODIPY-based fluorescent probes. To address this challenge, we focused on the
effects of hydrophilic groups on the reaction kinetics and thermodynamics. Obviously,
we did not mean to develop an excellent fluorescent probe with high sensitivity and
selectivity in this work. However, this mechanism study could promote better design
of fluorescent probes with fast response and high affinity.
Because sulfydryl group (-SH) has strong nucleophilicity and coordination
capability, many sensing mechanisms have been exploited to detect the biothiols,
including cleavage of sulphonamide [46,47], sulfonate ester [48], Se-N bond cleavage
[49], aryl substitution reactions [3], disulfide bond cleavage and cyclization [50,51],
and Michael additions [52,53]. Many strategies have employed the Michael
addition-based thiol sensing protocols [54-58]. In this work, the incorporation of
nitroolefin unit (-CH=CH-NO2) to the parent BODIPY dye would introduce a strong
Micheal acceptor (Scheme 1), which would be highly susceptible to sulfhydryl
nucleophiles [59,60]. Besides, the nitroolefin unit can improve the response rates of
probes toward biothiols. When biothiols were reacted with nitroolefin by Michael
addition, the photo-induced electron transfer (PET) and intramolecular charge transfer
(ICT) were blocked and thereby the fluorescence of probes was switched on [31]. The
effects of different hydrophilic groups (the R group in Scheme 1) were investigated on
the sensing efficacies for in vitro biothiols from the kinetic and thermodynamic
perspectives. Since we had already demonstrated the fluorescent imaging of
intracellular biothiols with probes of similar structures in our previous work [28,31],
we were convinced these probes can be applied in cell imaging as well.
2

Hydrophilic group
Thiol addition site
Scheme 1. The structures of the BODIPY-based fluorescent probes. The R group is
modified for water solubility. The nitroolefin unit (-CH=CH-NO2) is designed for
sensing biothiols.
2. Experimental
2.1 Apparatus and reagents
The NMR spectra were measured in appropriate deuterated solvents on a Bruker
AVANCE III HD 400 MHz spectrometer, using tetramethylsilane (TMS) as the
internal standard. The electrospray ionization mass spectra (ESI-MS) were measured
on an Agilent Q-TOF high resolution liquid chromatography mass spectrometer. The
fluorescence spectra were measured by a LS-55 fluorescence spectrometer
(PerkinElmer). Absolute quantum yields were determined by a FLS1000 fluorescence
spectrometer with an integrating sphere (Edinburgh Instruments). The absorption
spectra were measured by an Agilent Cary 100 UV-Vis double-beam
spectrophotometer. All reagents and chemicals, unless stated otherwise, were
purchased from commercial suppliers and used without further purification. Solvents
were dried and distilled by drying agents under an inert atmosphere prior to use.
Twice-distilled water was used throughout all experiments. The stock solution of
probes was prepared at 1 mM in dimethylsulfoxide (DMSO). Testing solutions were
prepared in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer
solution (20 mM, pH = 7.4).
2.2 Syntheses of probes
3

Scheme 2. Synthetic routes for BDP, BDP-OEG, BDP-OH and 3d. (i) 2,
4-dimethyl-1H-pyrrole, TFA, DDQ, Et3N, BF3.OEt2, DCM; (ii) phosphorus
oxychloride, DMF, DCE, 62 ; (iii) piperidine, acetic acid glacial, CH3NO2, toluene.
2.2.1 Synthesis of 1a
Trifluoroacetic acid (TFA) (150 μL) was added to a solution of benzaldehyde (425
mg, 4 mmol) and 2,4-dimethylpyrrole (827 μL, 8 mmol) in 100 mL dichloromethane
(DCM) under argon atmosphere. The reaction mixture was stirred at room
temperature for 5 h. Then 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (0.908
g, 4 mmol) was added to the mixture and stirred for 15 min. Triethylamine (Et3N) (6.4
mL) was added dropwise to the reaction mixture and stirred for 15 min. Finally,
BF3.OEt2 (7.0 mL) was added dropwise to the reaction mixture. The mixture was
stirred at room temperature for 12 h. The resulting solution was extracted with water
(2 × 200mL). The organic phase was dried over anhydrous Na2SO4. After removing
the solvent in vacuum, the residue was purified by flash column chromatography on
silica gel using DCM as the eluent. 1a was obtained as a red solid (400 mg, yield:
31 %). 1H NMR (400 MHz, CDCl3): δ = 7.48 (t, 3H, J = 4.0 Hz), 7.29 - 7.26 (m, 2H),
5.98 (s, 2H), 2.56 (s, 6H), 1.37 (s, 6H). 13C NMR (100 MHz, CDCl3) δ = 155.44,
143.19, 141.75, 135.01, 131.45, 129.17, 128.96, 127.95, 121.22, 14.67, 14.65, 14.62,
14.40. ESI-MS: m/z calcd for C19H19BF2N2 [M + H] +: 325.1682; found 325.1685.
2.2.2 Synthesis of 2a
The N,N-dimethylformamide (DMF) (3.0 mL) was stirred with ice bath under
argon. The POCl3 (3.0 mL) was dropwise added into the DMF and stirred for 10 min.
4

Then the mixture was stirred at room temperature for 30 min. Then 1a (200 mg, 0.62
mmol) dissolved in 1,2-dichloroethane (DCE) (30 mL) was added dropwise in the
mixture. The resulting mixture was stirred at 62 for 3 h. The reaction solution was
cooled to room temperature and slowly dropped into saturated NaHCO3 solution (150
mL) with an iced bath. Then the mixture was stirred for 30 min and washed with
water (100 mL). The combined organic phase was dried over anhydrous Na2SO4 and
evaporated in vacuum. The residue was further purified with column chromatography
(CH2Cl2/petroleum = 25 :1) to gain 2a as a red solid (160 mg, yield: 73 %). 1H NMR
(400 MHz, CDCl3): δ = 10.00 (s, 1H), 7.54 - 7.52 (t, 3H, J = 4.0 Hz), 7.29 - 7.26 (m,
2H), 6.15 (s, 1H), 2.82 (s, 3H), 2.62 (s, 3H), 1.65 (s, 3H), 1.42 (s, 3H). 13C NMR (100
MHz, CDCl3): δ = 186.00, 161.69, 156.54, 147.35, 143.59, 142.98, 142.96, 134.17,
129.59, 129.53, 127.71, 126.32, 124.06, 15.16, 14.90, 13.08, 11.61. ESI-MS: m/z
calcd for C20H19BF2N2O [M + H] +: 353.1631; found 353.1626.
2.2.3 Synthesis of 3a (BDP)
2a (170 mg, 0.49 mmol) was dissolved in toluene under argon. Then nitromethane
(262 μL, 4.9 mmol), piperidine (300 μL) and glacial acid (300 μL) were added
successively into the mixture. The mixture was stirred under reflux for 2 h. Then the
reaction mixture was cooled to room temperature. Then the solvent was removed in
vacuum. The crude product was purified by column chromatography
1
(CH2Cl2/petroleum = 1:1) to gain 3a (BDP) as a red solid (67 mg, yield: 35 %). H
NMR (400 MHz, CDCl3): δ = 8.03 (d, 1H, J = 12.0 Hz), 7.54 (t, 3H, J = 4.0 Hz), 7.34
(d, 1H, J = 12.0 Hz), 7.30 - 7.26 (m, 2H), 6.15 (s, 1H), 2.71 (s, 3H), 2.61 (s, 3H), 1.47
(s, 3H), 1.42 (s, 3H). 13C NMR (100 MHz, CDCl3): δ = 161.55, 154.19, 147.37,
142.72, 140.41, 134.58, 134.16, 130.92, 129.67, 129.56, 127.82, 124.05, 120.08,
15.15, 14.95, 13.98, 12.84. ESI-MS: m/z calcd for C21H20BF2N3O2 [M + H]+:
396.1689; found 396.1702.
Compounds 1b, 2b and 3b (BDP-OEG) were synthesized in the same ways as 1a,
2a and 3a (BDP).
2.2.4 Synthesis of 1b
5

1
1b was gained as an orange yellow solid (225 mg, yield: 12 %). H NMR (400
MHz, CDCl3): δ = 7.15 (d, 2H, J = 12.0 Hz), 7.01 (d, 2H, J = 8.0 Hz), 5.96 (s, 2H),
4.18 (t, 2H, J = 6.0 Hz), 3.90 (t, 2H, J = 4.0 Hz), 3.78 - 3.76 (m, 2H), 3.72 - 3.70 (m,
2H), 3.68 - 3.66 (m, 2H), 3.57 - 3.55 (m, 2H), 3.38 (s, 3H), 2.54 (s, 6H), 1.41 (s, 6H).
13C NMR (100 MHz, CDCl3): δ = 159.35, 155.25, 143.20, 141.83, 131.84, 129.15,
127.20, 121.11, 115.19, 71.95, 70.90, 70.69, 70.62, 69.76, 67.48, 59.12, 14.65;
ESI-MS: m/z calcd for C26H33BF2N2O4 [M + H] +: 487.2574; found 487.2589.
2.2.5 Synthesis of 2b
2b was gained as a yellow solid (95 mg, yield: 58 %). 1H NMR (400 MHz, CDCl3):
δ = 9.99 (s, 1H), 7.14 (d, 2H, J = 8.0 Hz), 7.04 (d, 2H, J = 8.0 Hz), 6.13 (s, 1H), 4.19
(t, 2H, J = 6.0 Hz), 3.90 (t, 2H, J = 6.0 Hz), 3.78 - 3.75 (m, 2H), 3.71 - 3.67 (m, 2H),
3.66 - 3.65 (m, 2H), 3.57 - 3.55 (m, 2H), 3.37 (s, 3H), 2.80 (s, 3H), 2.59 (s, 3H), 1.69
(s, 3H), 1.46 (s, 3H). 13C NMR (100 MHz, CDCl3): δ = 184.97, 165.65, 160.41,
158.73, 155.31, 146.35, 142.69, 141.85, 133.45, 129.14, 127.92, 125.20, 122.90,
114.48, 70.89, 69.86, 69.63, 69.56, 68.65, 66.51, 58.06, 14.13, 13.05, 12.01, 10.80;
ESI-MS: m/z calcd for C27H33BF2N2O5 [M + H] +: 515.2523; found 515.2537.
2.2.6 Synthesis of 3b (BDP-OEG)
1
3b (BDP-OEG) was gained a yellow solid (25 mg, yield: 25 %). H NMR (400
MHz, CDCl3): δ = 8.01 (d, 1H, J = 16.0 Hz), 7.33 (d, 1H, J = 12 Hz), 7.14 (d, 2H, J =
8.0 Hz), 7.05 (d, 2H, J = 8.0 Hz), 6.13 (s, 1H), 4.19(t, 2H, J = 4.0 Hz), 3.91 (t, 2H, J =
4.0 Hz), 3.77 (t, 2H, J = 4 Hz), 3.71 - 3.69 (m, 2H), 3.67 - 3.65 (m, 2H), 3.57 - 3.55
(m, 2H), 3.38 (s, 3H), 2.68 (s, 3H), 2.58 (s, 3H), 1.51 (s, 3H), 1.45 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ = 161.35, 159.83, 154.03, 147.44, 142.86, 140.38, 134.46,
134.24, 131.11, 130.99, 129.20, 129.09, 126.20, 123.96, 119.99, 115.70, 115.56, 71.93,
70.90, 70.66, 70.59, 69.89, 67.56, 59.10, 15.23, 15.11, 13.94, 13.06; ESI-MS: m/z
calcd for C28H34BF2N3O6 [M + H] +: 558.2581; found 558.2594.
Compound 3c (BDP-OH) was synthesized according to the previous literature [31].
2.2.7 Synthesis of 3c (BDP-OH)
6

3c (BDP-OH) was gained as a dark red solid (45 mg, yield: 34 % with step iii in
Scheme 2). 1H NMR (400 MHz, CDCl3): δ = 8.05 (d, 1H, J = 16.0 Hz), 7.36 (d, 1H, J
= 16.0 Hz), 7.13 (d, 2H, J = 8.0 Hz), 7.02 (d, 2H, J = 8.0 Hz), 6.16 (s, 1H), 2.71 (s,
3H), 2.62 (s, 3H), 1.51 (s, 3H), 1.26 (s, 3H). 13C NMR (100 MHz, CDCl3): δ = 160.35,
156.04, 153.04, 146.39, 141.84, 139.33, 133.39, 133.23, 133.23, 130.07, 128.26,
124.98, 122.96, 122.94, 118.95, 118.93, 115.52, 14.17, 14.09, 12.91, 12.01; ESI-MS:
m/z calcd for C21H20BF2N3O3 [M - H] -: 410.1493; found 410.1498.
Compounds 1d, 2d and 3d were synthesized in same ways as 1a, 2a and 3a (BDP).
2.2.8 Synthesis of 1d
1d was gained as a red solid (300 mg, yield: 21 %). 1H NMR (400 MHz, CDCl3): δ
= 7.06 (d, 2H, J = 12.0 Hz), 6.77 (d, 2H, J = 12.0 Hz), 5.97 (s, 2H), 3.02 (s, 6H), 2.55
(s, 6H), 1.48 (s, 6H). 13C NMR (100 MHz, CDCl3): δ =154.73, 150.66, 143.29,
143.24, 132.21, 128.74, 122.16, 120.86, 112.34, 40.37, 14.75; ESI-MS: m/z calcd for
C21H24BF2N3 [M + H] +: 368.2104; found 368.2114.
2.2.9 Synthesis of 2d
2d was gained as a black solid (135 mg, yield: 61 %). 1H NMR (400 MHz, CDCl3):
δ = 10.01 (s, 1H), 7.05 (d, 2H, J = 12.0 Hz), 6.79 (d, 2H, J = 8.0 Hz), 6.13 (s, 1H),
3.04 (s, 6H), 2.82 (s, 3H), 2.60 (s, 3H),1.77 (s, 3H),1.54 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ = 186.11, 160.71, 156.03, 156.01, 150.97, 147.46, 145.23, 142.95,128.63,
123.60, 123.58, 123.55, 120.99, 112.38, 112.31, 40.30, 15.32, 15.09, 13.03, 13.05,
12.00; ESI-MS: m/z calcd for C22H24BF2N3O [M + H] +: 396.2053; found 396.2065.
2.2.10 Synthesis of 3d
1
3d was gained as a dark red solid (74 mg, yield: 52 %). H NMR (400 MHz,
CDCl3): δ = 8.05 (d, 1H, J = 12.0 Hz), 7.36 (d, 1H, J = 12.0 Hz), 7.05 (d, 2H, J = 8.0
Hz), 6.80 (d, 2H, J = 8.0 Hz), 6.14 (s, 1H), 3.05 (s, 6H), 2.70 (s, 3H), 2.60 (s, 3H),
13
1.69 (s, 3H), 1.54 (s, 3H). C NMR (100 MHz, CDCl3): δ = 160.68, 153.79, 150.95,
147.52, 144.34, 140.44, 134.24, 131.61, 131.28, 128.80, 123.66, 121.02, 119.89,
119.87, 119.83, 112.44, 40.35, 15.39, 15.09, 13.87, 13.28; ESI-MS: m/z calcd for
C23H25BF2N4O2 [M + H] +: 439.2111; found 439.2121.
7

2.2.11 Synthesis of BDP-QA
Scheme 3. Synthetic route for BDP-QA. (i) iodomethane, acetonitrile, argon, room
temperature.
3d (57 mg, 0.13 mmol) was dissolved in acetonitrile under argon atmosphere.
Iodomethane (828 μL, 13 mmol) was dropwise added into the solution. The reaction
mixture was stirred at room temperature for 24 h. Then the solvent was removed in
vacuum. The crude product was purified by column chromatography (methyl alcohol
1
as eluent) to gain BDP-QA as a yellow solid (25 mg, yield: 42 %). H NMR (400
MHz, DMSO): δ = 8.24 (d, 2H, J = 12.0 Hz), 7.97 (d, 1H, J = 12.0 Hz), 7.76 (d, 2H, J
= 8.0 Hz), 7.60 (d, 1H, J = 12.0 Hz), 6.45 (s, 1H), 3.72 (s, 9H), 2.67 (s, 3H), 2.55 (s,
3H), 1.46 (s, 3H), 1.37 (s, 3H); ESI-MS: m/z calcd for C24H28BF2N4O2 [M]+:
453.2273; found 453.2279.
2.3 Kinetic and thermodynamic experiments
For kinetic parameters, the concentrations of Cys, Hcy and GSH were 100-fold of
those of probes. So, the reactions of probes with biothiols could be regarded as
pseudo-first-order kinetics. We added GSH into the HEPES buffer solution of probes
and detected the fluorescence intensity as time by a fluorescence spectrometer at
varied temperatures. For thermodynamic parameters, we conducted the titration
experiments in HEPES buffer at varied temperatures.
3. Results and discussion
8

To investigate the effects of hydrophilic groups on the efficacies of probes, we
chose BODIPY as the fluorophore and nitroolefin (-CH=CH-NO2) as the sensing unit
for biothiols (Scheme 1). We synthesized four probes conjugating different
hydrophilic groups, namely BDP (control), BDP-OH (acidic), BDP-OEG (neutral
and hydrophilic) and BDP-QA (cationic). The neutral BDP was synthesized as the
control to study the properties of different substituents at the para position of phenyl
group attached to the BODIPY core. The neutral (oligo ethylene glycol, -OEG),
acidic (-OH) and quaternary ammonium (-QA) substituents at the para position of
phenyl group endowed the BODIPY-based fluorescent probes water-solubility. All the
1
13
compounds reported herein were carefully characterized by H NMR, C NMR and
high resolution ESI mass spectrometry (Figs. S1 - 32 in the Supporting Information).
The 13C NMR data of BDP-QA was not available yet because its 13C NMR signal was
1
very weak. However, the H NMR (Fig. S31) and HRMS (Fig. S32) characterization
of BDP-QA can well confirm its chemical structure. The intracellular concentrations
of Cys, Hcy and GSH were in the range of 30 ~ 200 μM, 5 ~ 15 μM, 1 ~ 10 mM,
respectively. Since GSH was the predominant biothiol in cells, it was selected as the
sensing target in this work.
First, we investigated whether the hydrophilic groups affected the optical properties
of probes. The fluorescence and UV-Vis absorption spectra of probes were measured
in HEPES buffer solutions containing ~ 1% DMSO brought by dilution from the stock
solution (prepared in DMSO), i.e. almost 100% aqueous solutions. Most of the
previous works conducted the sensing of biothiols in a mixture solution of water and
organic solvents, acetonitrile (ACN) from example [5,11,28,31,61]. As shown in Fig.
1a, when BDP-QA was reacted with excess GSH in the HEPES buffer, the absorption
maximum was slightly blue-shifted for 1 nm. Besides, the molar absorption
coefficient slightly increased, presumably owing to the enhancement of dispersion
coefficient of the adduct BDP-QA-GSH. The emission maximum of BDP-QA was
red-shifted for 12 nm when reacted with GSH in the HEPES buffer, possibly due to
the aggregation of BODIPY fluorophores in water. In contrast, the absorption and
emission maxima were blue-shifted in DMSO for 19 and 25 nm (Table S1),
9

respectively, which was clearly resulted from the decrease of π-conjugation after the
addition of GSH to BDP-QA (Fig. S33a). The solvent had obvious influence on the
optical response of BODIPY probes. Compromises between the aggregation (red shift)
and the decreased π-conjugation (blue shift) were present in the absorption and
emission spectra when the probes were applied in the HEPES buffer. This can well
explain the blue shift of only 1~4 nm in the absorption spectra of probes in the
HEPES buffer after the conjugate addition with GSH. The optical properties of other
probes were similar as that of BDP-QA (Table 1 and Figs. S33 - 36). The absorption
and emission maxima of these four probes were all at 507 ± 1 nm and 512 ± 1 nm,
respectively, obviously demonstrating that the substitution with hydrophilic groups on
the para position of meso-phenyl group of BODIPY essentially did not affect the
π-conjugation and the HOMO-LUMO gap. The low quantum yields (QYs) of these
four fluorescent probes in the HEPES buffer were presumably caused by PET and
ICT as well as aggregation-caused quenching (ACQ). After they were reacted with
GSH, the quantum yields of adducts were obviously different, maybe due to their
different water solubility. As shown in Fig. 1b, when BDP-QA was reacted with
different small biological molecules with corresponding biological concentrations,
fluorescent enhancement up to 28-fold was observed in the presence of 10 mM GSH,
the concentration likewise in cells. In the cellular conditions, the fluorescent probes
would mostly report the signal caused by GSH. BDP, BDP-OH and BDP-OEG had
similar results (Figs. S34 - 36). These results indicated that all the probes could
recognize GSH in the cellular conditions.
10

Fig. 1. The optical properties of BDP-QA. (a) The fluorescence and absorption
spectra of BDP-QA in the HEPES buffer solution and (b) the selectivity of BDP-QA
(10 μM). Except for Cys (200 μΜ), Hcy (15 μΜ) and GSH (10 mM), the
concentration of other analytes was 1 mM respectively. The concentrations of Cys,
Hcy and GSH are close to those in cellular conditions.
Table 1. Photophysical properties of probes in the absence and presence of GSH in
the HEPES buffer solution. QY represents the absolute quantum yield.
λabs (nm)
506
λem (nm)
510
QY (%)
< 0.01
3.97
BDP
BDP + GSH
BDP-OEG
505
516
507
513
< 0.01
4.57
BDP-OEG + GSH
BDP-OH
503
518
505
512
< 0.01
1.64
BDP-OH + GSH
BDP-QA
503
517
508
512
0.58
BDP-QA + GSH
507
524
15.21
Then, we explored whether hydrophilic groups affected the reaction rates of probes
toward GSH. When the concentration of GSH was 100 times larger than that of a
probe, the reaction can be regarded as pseudo-first-order kinetics. So, we can describe
the dependence of the probe concentration (c) on the reaction time (t) by eq. (1):
ꢂ
ꢅ
ꢀ = ꢀꢁ × exp − ꢃꢄ
where c0 represents the original concentration of probe (reactant), k is the reaction rate
constant. When the probes were reacted with GSH, the PET and ICT processes would
be blocked and thereafter the fluorescence of probes was switched on. So the
enhancement of fluorescence intensity can be used to reflect the product concentration.
The kinetics can be fitted by eq. (2):
ꢂ ꢅ
ꢆ = a × exp ꢃꢄ + b
11

where a and b are constants without physical significance, I represents fluorescence
intensity of the resulting product, k represents the rate constant, and t is the reaction
time. As shown in Fig. 2a, when BDP-QA was reacted with Cys, Hcy and GSH at 298
K, their apparent rate constants were 0.102 s-1, 0.0259 s-1 and 0.0162 s-1, respectively.
The results indicated that a biothiol of larger molecular weight had smaller rate
constant. The reaction rate constants were affected by the steric hindrance of biothiols.
Among the three biothiols, GSH (a tripeptide) had the largest molecular weight and
therefore the largest steric hindrance as well as the smallest diffusion rate. So, the rate
constant for GSH was the smallest. Besides, the half-time (t1/2) of the pseudo-first-
order reaction obeys the following eq. (3):
ꢉꢊ2
ꢄꢇ/ꢈ
=
ꢃ
So the half-time t1/2 of the reaction of BDP-QA with GSH was 43 s at 298 K. From
Fig. 2b, as the temperature increased, the rate constants gradually increased owing to
more vigorous molecular movement in the reaction system. However, the more
vigorous collision would lead to strengthened non-radiative relaxation and therefore
weakened fluorescence. The dependence of k on temperature T is simulated by the
Arrhenius equation, eq. (4):
ꢌꢍ
ꢃ = A × exp ꢋ−
ꢐ
ꢎꢏ
where A is the pre-exponential factor that represents the frequency of collisions
between reactive molecules, Ea represents the activation energy, and R is the gas
constant (8.314 J mol−1 K−1). As shown in Fig. 2c, the apparent activation energy (Ea)
was 75.24 kJ mol-1 and pre-exponential factor (A) was 2.56 × 1011 s-1 for the reaction
of BDP-QA with GSH. We also completed kinetic investigations for other probes
(BDP, BDP-OEG and BDP-OH) (Table 2 and Figs. S37 - 39). The kinetic
parameters were the mean of three independent experiments. BDP-QA had the largest
rate constant. The activation energy had no obvious changes, so these probes should
have the same reaction mechanism and hydrophilic groups had no obvious effect on it.
The reactions of these probes with Hcy showed the similar trend (Table S2).
12

Fig. 2. Kinetics of BDP-QA (10 μΜ) and thiols (1 mM) in HEPES buffer. (a) The
reactions of BDP-QA with Cys, Hcy and GSH at 298K; (b) The reactions of
BDP-QA with GSH at different temperatures; (c) The relationship of ln k with 1/T for
the reaction of BDP-QA with GSH.
Table 2. The kinetic parameters of probes reacted with GSH in the HEPES buffer.
The data are the mean of three independent experiments.
Probes
BDP
k a (10-3 s-1)
3.06 ± 0.23
3.94 ± 0.34
5.24 ± 0.73
18.53 ± 2.77
t1/2 a, b (s)
228 ± 18
177 ± 16
133 ± 21
38 ± 6
Ea (kJ mol-1)
73.49 ± 8.27
59.84 ± 8.60
73.33 ± 8.27
69.92 ± 4.61
lnA
23.85 ± 3.26
18.58 ± 3.29
23.27 ± 3.54
24.26 ± 1.74
BDO-OEG
BDP-OH
BDP-QA
a The reactions proceed at 298 K. b The half-time of pseudo-first-order reaction.
13

Next, we investigated the reaction mechanism of probes with biothiols, GSH for
example. As shown in Scheme 4, speculative reaction mechanism of probes with
biothiols was proposed according to the nucleophilic addition [62-64]. The nitro unit
(-NO2) was a very strong electron-withdrawing group (EWG), which could make the
nucleophilic addition happen. First, GSH dissociated as GS- and H+ (Step 1). Then
GS- attacked the olefinic bond and formed the carbanion (Step 2), which should be the
rate-determining step (RDS) for this reaction [62-64]. The conjugate addition would
result in the turn-on fluorescence. Finally, protonation of the carbanion resulted in the
addition product (Step 3). In the HEPES buffer with a pH of 7.4, there might be only a
minor concentration of GS-. Besides the above speculative mechanism, GSH can
possibly attack the nitroolefin and then proton transfer occurred from the intermediate
since the -SH group was highly nucleophilic.
Scheme 4. The speculative reaction mechanism of probes with GSH. Step 1: Thiol
deprotonation; Step 2: Conjugate addition; Step 3: Protonation.
We used BDP-QA to certify this speculation. First, we detected the final product of
1
BDP-QA reacted with GSH in the HEPES buffer (Fig. S40). H NMR spectra of
BDP-QA exhibited protons of olefin double bond at 7.99, 7.96, 7.62 and 7.58 ppm,
respectively (Fig. S40a). When reacted with GSH, the protons of olefin double bond
disappeared (Fig. S40b). So we speculated that the reaction site should be the olefin
double bond. As shown in Fig. S41, we performed the ESI-MS experiment of the
reaction solution. After reacting with GSH, the peaks of [GSH + H] +, [BDP-QA] +,
[2GSH + H] +, [2GSH + Na] +, [2GSH + K] + and [BDP-QA-GSH] + appeared in the
mass spectrum. Due to the far excess of GSH, the signal corresponding to the adduct
was relatively weak. These results proved our speculation of the conjugate addition.
14

Then, we carried out the kinetic experiments under basic conditions. As shown in Fig.
3, the kinetic experiments were performed at pH 7.4 and 13.0, respectively. It was
found that the apparent reaction rate constants were 0.0211 s-1 at pH 7.4 and 0.000107
s-1 at pH 13.0, respectively. When the pH increased, the rate constant sharply
decreased. This might be due to the difficult protonation of the carbanion (Step 3 in
Scheme 4) under such a basic condition (pH = 13.0).
Fig. 3. Kinetics for the reactions of BDP-QA with GSH at pH 7.4 (a) and pH 13.0 (b).
With the above results in hand, we considered the effects of hydrophilicity on the
reaction rates. Among the four probes, we knew that BDP-QA had a positive charge,
so we can make a judgment that its hydrophilicity was the largest. Then, we
determined the hydrophilicity of other three probes by liquid chromatography mass
spectrometry (LC-MS). As shown in Fig. 4, the retention time of BDP, BDP-OEG
and BDP-OH were 4.382 min, 2.533 min and 1.550 min, respectively, by extracting
extracted ion chromatography (EIC) from total ion chromatography (TIC). Because
the chromatographic column of LC-MS apparatus was C-18 reversed-phase column,
the sequence of polarity was BDP-OH > BDP-OEG > BDP. The sequence of
hydrophilicity would follow this order. So, the hydrophilicity of four probes was in
the order of BDP-QA > BDP-OH > BDP-OEG > BDP. In combination with the
reaction rate constants in Table 2, we could infer that the rate constants were
positively correlated with the hydrophilicity of probes.
15

Fig. 4. LC-MS experiments for BDP, BDP-OEG and BDP-OH. The
chromatographic column is C-18 reversed-phase column and the eluent is a mixture of
acetonitrile and water (v/v = 8:2).
We further considered whether the ionic strength affected the reaction rate
constants in the HEPES buffer. The kinetic experiments were performed under
different concentrations of NaCl (Fig. 5a). The ionic strength might influence the
quantum yields of fluorescent probes. Though the curves had different final intensities,
they would not affect the calculation of rate constants. The curves were fitted with
first-order reaction kinetics to obtain the rate constants. As shown in Fig. 5b, as the
concentration of NaCl increased (0 ~ 1 M), the rate constant first increased and then
slightly decreased. At first, the increased ionic strength would benefit the thiol
deprotonation (Step 1 in Scheme 4) by stabilization of thiolate anions, presumably
owing to the formation of ion pairs. To be noted, the cationic BDP-QA can form ion
pairs with the GS- anion. Exposed to further increased ionic strength, however, it
would be harmful to the protonation of the carbanion (Step 3 in Scheme 4) according
to the primary salt effect, so the rate constant would decrease. In contrast, the k values
of other probes increased with the increase of ionic strength (Figs. S42 - 44). This
may be due to the favorable stabilization of the thiolate anions (Scheme 4). These
16

results indicated that the cationic probe had the largest rate constant, possibly owing
to the cationic nature of BDP-QA and consequently the improved hydrophilicity.
Fig. 5. (a) The kinetic experiments of BDP-QA reacted with GSH in the presence of
different concentrations of NaCl. (b) Dependence of k on the concentration of NaCl
for the reaction system of BDP-QA and GSH.
To obtain the affinities of the BODIPY-based probes toward GSH, the reaction
thermodynamics was further investigated. Owing to the weak fluorescence of probes
(the reactants), the enhanced fluorescent intensities can represent the adducts formed
by the probes and GSH. The fluorescent intensity is proportional to the concentration
of adduct [65]. So, the dependence of fluorescence intensity change (ΔF) on the
concentration of reactant (c) can be fitted by eq. (5) [28]:
Bꢒꢀ
∆ꢑ =
ꢂ1 + ꢒꢀꢅ
where K is the equilibrium constants and B is a constant which has no physical
meaning. As shown in Fig 6a, the results indicated that the equilibrium constants
gradually decreased with increased temperature. The dependence of equilibrium
constant K on the temperature T can be fitted by the van’t Hoff equation, eq. (6):
∆ꢓ
ꢎꢏ
∆ꢔ
ꢎ
ꢉꢊꢒ = −
+
where ΔH is the enthalpy change and ΔS is the entropy change. From the linear fitting
in Fig. 6b, the enthalpy change and entropy change were - 52.13 kJ mol-1 and - 89.79
J mol-1 K-1, respectively. The negative ΔS was reasonable since an adduct was formed
17

by the reaction of BDP-QA with GSH. The negative ΔH illustrated that the reaction
was exothermic. The thermodynamic parameters of other probes with GSH were
similar with BDP-QA (Figs. S45 - 47). As shown in Table 3, all reactions were
exothermic. The more hydrophilicity of probes led to the larger equilibrium constant
K, i.e. the greater affinity. In addition, we calculated the limit of detection (LOD) by
eq. (7):
3ꢕ
LOD =
ꢔ
where σ is the standard deviation of blank sample values in 10 times, and S is the
slope of the linear regression equation. As shown in Table S3, the LOD values for
BDP, BDP-OEG, BDP-OH and BDP-QA were 14.5 μM, 3.81 μM, 1.55 μM and 182
nM, respectively. We found that the LOD values highly depended on their
hydrophilicity. With enhanced water solubility of probes, the LOD values would
decrease owing to their better dispersion in water. However, the linearity ranges were
gradually narrowed (Table S3).
Fig. 6. (a) The reaction of BDP-QA with GSH at different temperatures (299 ~ 310
K). (b) Relationship of lnK with 1/T.
18

Table 3. The reaction thermodynamic parameters of probes with GSH at ~ 298 K.
K (M-1)
ΔG (kJ mol-1)
ΔH (kJ mol-1)
ΔS (J mol-1 K-1)
- 152.80
-34.89
1467
- 18.06
- 63.57
BDP
5268
- 21.23
- 31.69
BDP-OEG
BDP-OH
BDP-QA
6681
- 21.82
- 49.15
- 90.54
25393
- 25.13
- 52.13
- 89.79
4. Conclusions
A series of BODIPY-based probes with different hydrophilic groups, namely BDP,
BDP-OEG, BDP-OH and BDP-QA, were designed and synthesized. The absorption
and fluorescence emission spectra were not affected by these hydrophilic groups.
These probes had 27~38 fold fluorescence enhancement in the presence of 10 mM
GSH. The cationic BDP-QA had the fastest response toward GSH owing to the best
water solubility and the possible formation of ion pairs with the thiolate anion. The
reaction rate constants of probes were positively correlated with their hydrophilicity.
Thermodynamics indicated that the reactions of probes with GSH all had negative ΔH
and negative ΔS. The LOD values would be lowered with the increased hydrophilicity.
Our work emphasized the importance of hydrophilicity of the fluorescent probes. This
work had built the fundamental basis for the design of fluorescent probes with fast
response and high affinity.
Acknowledgments
The authors gratefully acknowledge the financial support from National Natural
Science Foundation of China (21773178, 21973071, 21573168), the Natural Science
Foundation of Hubei Province (2018CFA090), Guangxi Science and Technology
Project (GuiKeAD17195081), and Bagui Scholar Program of Guangxi Province
(2016).
19

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