A Fluorescent Visual Proton Donor and Photoacid Sterilant Based on Sulfonate‐conjugated BODIPY

Article abstract of DOI:10.1007/s10895-021-02682-8

Increasing acidity is an effective method for bacterial inactivation by inhibiting the synthesis of intracellular proteins at low pH. Photo-driven proton release probe can be used for the measurement of proton in hydrophobic condition. To develop fluorescent proton donor, two boron dipyrromethene derivatives (BDP-S and BDP-S2) were characterized by spectroscopic methods. Irradiation of BDP-S by white LED light resulted in efficient generation of acidic species with changes of fluorescence emission. The linear relationship between the pH value and the fluorescence intensity of BDP-S was obtained, indicating that BDP-S is a fluorescent visual proton donor. Light-induced antibacterial results indicate that BDP-S can significantly inhibit the growth of E. coli. The results prove that BDP-S is a very promising photoacid sterilant.

Full text of DOI:10.1007/s10895-021-02682-8

Journal of Fluorescence (2021) 31:501–507
https://doi.org/10.1007/s10895-021-02682-8
ORIGINAL ARTICLE
A Fluorescent Visual Proton Donor and Photoacid Sterilant Based
on Sulfonate‐conjugated BODIPY
Abbas Mohammed Ali1 & Jian Shao1 & Jia-Xin Wang1 & Qiu-Yun Chen1 & Yang Li1 & Ling-Ling Qu1
Received: 18 October 2020 /Accepted: 5 January 2021
/ Published online: 15 January 2021
#
The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
Increasing acidity is an effective method for bacterial inactivation by inhibiting the synthesis of intracellular proteins at low pH.
Photo-driven proton release probe can be used for the measurement of proton in hydrophobic condition. To develop fluorescent
proton donor, two boron dipyrromethene derivatives (BDP-S and BDP-S2) were characterized by spectroscopic methods.
Irradiation of BDP-S by white LED light resulted in efficient generation of acidic species with changes of fluorescence emission.
The linear relationship between the pH value and the fluorescence intensity of BDP-S was obtained, indicating that BDP-S is a
fluorescent visual proton donor. Light-induced antibacterial results indicate that BDP-S can significantly inhibit the growth of
E. coli. The results prove that BDP-S is a very promising photoacid sterilant.
.
.
.
Keywords BODIPY Antibacterial Proton donor Photoacid generator
Introduction
to controlling various proton transfer processes, such as acid-
catalyzed reactions [14–16], enzyme activity [17], biological
probing [18], bacteria killing [19], etc.. Although the acid
release properties of PAGs have been studied, there have been
few PAGs with fluorescence response. Therefore, the devel-
opment of new PAGs with fluorescence response is still a
work of great significance.
Boron dipyrromethene (BODIPY) dyes have characteris-
tics such as large molar absorption coefficient, high fluores-
cence quantum yield and easy modification [20–24]. If boron
dipyrromethene (BODIPY) dyes could be potential photo-
acids, a sensitive method for the measurement of in-site pro-
duced proton will be developed. In this work, a BODIPY
conjugated sulfonate (BDP-S) was reported as a fluorescence
responsive photoacid. The light-induced emission changes,
photoacid generation and antibacterial activity were studied.
The effectiveness of traditional antibiotics is decreasing due to
variations of pathogens and enhancement of drug resistance in
recent years. This has given rise to the search for a more
effective way of bacterial inactivation. Light-driven bacterial
inactivation has become the main treatment method in the
fields of biomedicine, environmental science and materials
chemistry because of its non-invasiveness, less toxicity and
low drug resistance [1–4]. Photo-driven antibacterial by
photoacid is a promising approach in photo-driven bacteria
killing [5]. Photoacid generators (PAGs) are a class of com-
pounds that undergo dissociation or rearrangement reactions
upon illumination, resulting in acidic species eventually
[6–10]. Proton-sensitive reactions can be controlled in space
and time by PAGs which also provide a new method for
converting light energy into chemical energy [11, 12]. PAGs
can generally be activated by LED or visible light either in
solution or the solid state [13], which makes PAGs applicable
Experimental
Chemicals and Measurements
* Qiu-Yun Chen
chenqy@ujs.edu.cn
All chemicals were purchased from commercial suppliers and
were used without further purification. 2,4-dimethylpyrrole,
p-hydroxybenzaldehyde, trifluoroacetic acid (TFA), 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), p-
nitrobenzenesulfonyl chloride, 4-nitro-α-toluenesulfonyl
* Ling-Ling Qu
llqu@ujs.edu.cn
1
School of Chemistry and Chemical Engineering, Jiangsu University,
Zhenjiang, China

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J Fluoresc (2021) 31:501–507
chloride, rhodamine B base and other chemicals were pur-
chased from Energy Chemical Reagent Co., Ltd. 4-(5,5-
Difluoro-1,3,7,9-tetramethyl-5H-4λ4,5λ4- dipyrrolo[1,2-
c:2’,1’-f][1,3,2]diazaborinin-10-yl)phenol (BDP-OH) was
synthesized according to the reported procedure [25].
The electronic absorption spectra were measured at room
temperature by a UV-2450 UV–vis spectrophotometer.
Fluorescence measurements were carried out on a fluores-
cence spectrofluorometer Model CARY Eclipse (VARIAN,
USA), a 1.0 cm quartz cell (slit width = 5 nm). The 1H NMR
(400 MHz) and 13C NMR (100 MHz) data was recorded on a
Bruker AVANCE II 400 MHz spectrometer using CDCl3 as a
solvent. The chemical shifts (δ) were reported in ppm and
coupling constants (J) in Hz. The pH of solution was mea-
sured using a Fisher Scientific Accumet AR15 benchtop meter
and a pH combination electrode.
6.01 (s, 2H), 2.55 (s, 6H), 1.33 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 156.22, 151.14, 149.67, 142.44, 140.42, 139.24,
134.75, 131.12, 130.03, 129.97, 124.37, 123.05, 121.66,
14.60, 14.49.
Synthesis of 4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-
4λ4,5λ4-dipyrrolo[1,2-c:2’,1’-f] [1,3,2]diazaborinin-10-
yl)phenyl (4-nitrophenyl)methanesulfonate (BDP-S2)
To a solution of BDP-OH (170 mg, 0.5 mmol) and
triethylamine (140 µL, 1 mmol) in dry DCM (10 mL) was
added 4-nitro-α-toluenesulfonyl chloride (141 mg, 0.6 mmol)
dropwise at 0 °C under nitrogen. The reaction mixture was
stirred for 10 h at room temperature. The solvent was washed
with saturated sodium chloride (3 × 10 mL), dried with sodi-
um sulfate and concentrated in vacuo. The residue was loaded
onto a column of silica gel to give BDP-S2 (235 mg, 87.1%
yield) as an orange solid. 1H NMR (400 MHz, CDCl3) δ 8.36
(d, J = 8.7 Hz, 2H), 7.75 (d, J = 8.7 Hz, 2H), 7.37 (q, J =
8.8 Hz, 4H), 6.04 (s, 2H), 4.72 (s, 2H), 2.60 (s, 6H), 1.42 (s,
6H). 13C NMR (100 MHz, CDCl3) δ 156.13, 149.06, 148.63,
142.77, 139.48, 134.44, 134.12, 131.88, 131.22, 130.04,
124.20, 122.76, 121.60, 56.43, 14.60, 14.52.
Synthesis of 4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-
4λ4,5λ4- Dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-10-
yl)phenyl 4-nitrobenzenesulfonate (BDP-S)
To a solution of BDP-OH (170 mg, 0.5 mmol) and
triethylamine (140 µL, 1 mmol) in dry DCM (10 mL) was
added p-nitrobenzenesulfonyl chloride (133 mg, 0.6 mmol)
dropwise at 0 °C under nitrogen. The reaction mixture was
stirred for 4 h at room temperature. The solvent was then
washed with saturated sodium chloride (3 × 10 mL), dried
with sodium sulfate and concentrated in vacuo. The residue
was loaded onto a column of silica gel to give BDP-S
(219 mg, 83.5% yield) as an orange solid. 1H NMR
(400 MHz, CDCl3) δ 8.38 (d, J = 8.8 Hz, 2H), 8.05 (d, J =
8.8 Hz, 2H), 7.28 (d, J = 8.5 Hz, 2H), 7.19 (d, J = 8.5 Hz, 2H),
Detection of H+ Formed By Photolysis
The photogenerated acid (H+) was captured by the
added rhodamine B base whose lactone ring will open
after accepting a proton, with a new absorption peak rising at
555 nm [26]. A solution of BDP-S or BDP-S2 (1 mM, MeCN)
and rhodamine B base (20 µM, MeCN) was degassed with
nitrogen for 15 min and irradiated with an LED lamp (365 nm,
Scheme 1 Schematic illustration
of antibacterial mechanism for
photoacid

J Fluoresc (2021) 31:501–507
503
Scheme 2 The mechanism for the photolysis of BDP-S
10 mW/cm2) for 2 h. The UV-Vis spectrum of the solution
was recorded every 20 min. A rhodamine B base solution
without BODIPY was used as a negative control.
of emission and pH, three independent experiments were car-
ried out.
Antibacterial Assays
Fluorescence Emission and PH Changes Under
Irradiation
Gram-negative Escherichia coli (E. coli, ATCC 25,922) strain
was chosen as a standard bacterial model. The antibacterial
activity of BDP-S was evaluated in the dark condition or LED
light irradiation. All assays were conducted in 96-well, flat
bottom, sterile plates. Bacterial suspension was adjusted to
contain 1 × 106CFU/mL in fresh sterile PBS solution. The
as-prepared BDP-S solution (50, 40, 30, 20, 10 and 0 µM)
was mixed with bacterial suspension (1 × 106 CFU/mL) and
irradiated using a white LED lamp (35–500 nm, 4 mW/cm2)
for 2 h. For each test, the sample in a dark was set as a control
group. All tests were performed in triplicate. Survivors were
quantified using the viable count technique. Plates were incu-
bated at 37 °C for 24 h.
A BDP-S or BDP-S2 stock solution (70 µM, MeCN) was
nitrogen saturated for about 15 min firstly and then irradiated
with white LED lamps (350–500 nm, 4 mW/cm2) for 2 h.
Photolysis process of BDP-S and BDP-S2 was monitored by
fluorescence spectra (λex: 480 nm, time interval: 20 min). The
pH of the solution after different exposure times were mea-
sured by a Fisher Scientific Accumet AR15 benchtop meter
and a pH combination electrode. For determining the changes
1.0
0.8
Results and Discussion
0.6
a to g
Syntheses and Characterization of Compounds
0.4
0.2
0.0
Two new BODIPY derivatives (BDP-S and BDP-S2) were
synthesized by simply coupling hydroxyl-conjugated
BODIPY (BDP-OH) with p-nitrobenzenesulfonyl chloride
1
or 4-nitro-α-toluenesulfonyl chloride (Scheme 1). The H
NMR and 13C NMR spectra of the compounds confirm the
compounds with exact structures and high purity (Figure S1-
S6). BDP-S was found to decompose upon irradiation be-
tween the two compounds, whilst the pH of the solution was
changed. A mechanism for the photolysis of BDP-S is
520
540
560
580
600
Wavelength (nm)
Fig. 1 UV-Vis spectra changes of BDP-S (500 µM, MeCN) and RB Base
(10 µM, MeCN) mixture irradiated under 365 nm (a to g: 0 to 120 min,
time interval: 20 min)

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J Fluoresc (2021) 31:501–507
b
a
400
300
200
100
0
BDP-S
400
300
200
100
0
BDP-OH
BDP-S2
a to g
500
520
540
560
580
600
500 520 540 560 580 600
Wavelength (nm)
Wavelength (nm)
Fig. 2 a Fluorescence emission spectra of BDP-OH (70 µM, MeCN),
BDP-S (70 µM, MeCN) and BDP-S2 (70 µM, MeCN); b Fluorescence
emission spectra changes of BDP-S (70 µM, MeCN) irradiated with
white LED light (a to g: 0 to 120 min. Time interval: 20 min. Insert:
changes of intensity at 507 nm). Excitation wavelength: 480 nm,
Voltage: 500V
outlined in Scheme 2. After initial excitation, BDP-S un-
dergoes homolytic O-S bond cleavage to generate phenoxyl
and sulfonyl radical pairs [11]. The generated radical pairs,
escape from the cage and subsequently abstract trace water
or releases SO2 to produce the corresponding BDP-OH and
H2SO3.
gradually changed from light yellow to a final light orange
colour (Fig. S7). When the rhodamine B base was dissolved
in acetonitrile, it is colorless, whilst its protonated form is red.
The result also showed that rhodamine B base captured pro-
tons during LED irradiation and indirectly proved the release
of acid species by BDP-S.
Detection of H+ Formed By Photolysis
Fluorescence Emission and pH Changes of BDP-S After
Illumination
The generated H+ was captured by rhodamine B base [25].
Protonation of rhodamine B base leads to a new peak arising
at 555 nm. As shown in Fig. 1, a new peak rose gradually at
555 nm in the mixed solution of BDP-S and rhodamine B base
while BDP-S2 and the control group showed no significant
changes, indicating that an acid species was produced after
BDP-S was illuminated.
BODIPY dyes have the characteristics of large molar absorp-
tion coefficient and high photostability of the core while the
nitrosulfonate-conjugated one is generally not stable. As
shown in Fig. 2a, the integrated area of BDP-OH in the range
of 485–600 nm is 7.12 0.02 times higher than BDP-S and
0.98 0.01 times higher than BDP-S2 under the same condi-
tion. BDP-S2 shows no significant changes in emission com-
pared with BDP-OH while the emission of BDP-S is greatly
In addition, with the extension of irradiation time, the color
of the mixed solution of BDP-S and rhodamine B base
a 7.0
b
7.0
6.5
6.0
5.5
5.0
4.5
6.5
6.0
5.5
5.0
4.5
BDP-S
BDP-S2
50 100 150 200 250 300 350 400
Fluorescence Intensity
0
20 40 60 80 100 120
Time (min)
Fig. 3 a pH changes of BDP-S and BDP-S2 under illumination. b A fitted curve according to the measured pH and fluorescence intensity of BDP-S (y =
-0.00607 * a + 7.03523, R2 = 0.997). Data are expressed as the means of three independent experiments SD

J Fluoresc (2021) 31:501–507
505
Scheme 3 Schematic illustration
of antibacterial mechanism for
photoacid
quenched, indicating that the methylene group in BDP-S2
breaks the conjugated structure and blocks the electron-
withdrawing effect of the nitro group on BODIPY core. The
emission of BDP-S2 shows no obvious change under long-
term illumination while emission intensity of BDP-S at
507 nm increases with the extension of the illumination time
and the amplitude decreases (Fig. 2b). Furthermore, BDP-S
can be a fluorescence-response photo-acid. Besides, there is a
slight blue shift (508 nm → 504 nm) observed that further
proves that the structure of BDP-S changes under
illumination.
Also, the pH of the solution under different exposure times
was measured using a Fisher Scientific Accumet AR15 bench-
top meter with a pH combination electrode (Fig. 3a). The
results showed that the pH value of the BDP-S2 solution
remained basically unchanged under illumination, while the
photolysis of BDP-S occurred and the pH of the solution de-
creased significantly with the exposure time. A curve based on
the pH value and fluorescence intensity of BDP-S was fitted
(linear regression equation: y = -0.00607 * a + 7.03523, R2 =
0.997) (Fig. 3b), which allows us to roughly estimate of the
pH value of the irradiated solution according to fluorescence
emission intensity. Since the pH is the negative logarithm of
the hydrogen ion concentration, the number of protons in so-
lution can be easily calculated, which provides a new method
for estimating the number of protons in a hydrophobic envi-
ronment. This makes BDP-S a fluorescent visual proton
donor.
Increasing acidity is an effective method for bacterial
inactivation by inhibiting the synthesis of intracellular
proteins at low pH. Since BDP-S can lower the pH of the
solution with light, it can be applied for bacterial inactivation.
Fig. 4 a Growth of E. coli under
different conditions
(concentration of BDP-S:
50 µM); b Effects of increasing
concentrations of BDP-S on the
growth inhibition of E. coli. Data
are expressed as the means of
three independent experiments
SD. The white light source
(350–500 nm, 4 mW/cm2)
Dark
7
b
LED light
6
5
4
3
2
1
0
10
20
30
40
50
Concentration ( M)

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J Fluoresc (2021) 31:501–507
The proposed antibacterial mechanism for photoacid is
outlined in Scheme 3.
Compliance with Ethical Standards
Conflict of Interest The authors declare that there is no conflict of
interest.
Antibacterial Assays
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Fig. 4a, the bacteria can grow normally in the dark condition (-
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Funding This work was financially supported by the National Natural
Science Foundation of China (21,701,056).
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