Curcumin-based polarity fluorescent probes: Design strategy and biological applications

Article abstract of DOI:10.1016/j.dyepig.2020.108320

Polarity is a crucial characteristic of indispensable organelles, which has been indicated to be closely related to many pathological and biological processes. Hence, a novel design strategy of curcumin-based polarity fluorescent probes was presented. Cur

Full text of DOI:10.1016/j.dyepig.2020.108320

Journal Pre-proof
Curcumin-based polarity fluorescent probes: Design strategy and biological
applications
Linghao Hu, Donglei Shi, Xinming Li, Jin Zhu, Fei Mao, Xiaokang Li, Conglong Xia,
Bei Jiang, Yuan Guo, Jian Li
PII:
S0143-7208(19)32510-0
DOI:
https://doi.org/10.1016/j.dyepig.2020.108320
DYPI 108320
Reference:
To appear in: Dyes and Pigments
Received Date: 25 October 2019
Revised Date: 26 February 2020
Accepted Date: 28 February 2020
Please cite this article as: Hu L, Shi D, Li X, Zhu J, Mao F, Li X, Xia C, Jiang B, Guo Y, Li J, Curcumin-
based polarity fluorescent probes: Design strategy and biological applications, Dyes and Pigments
(2020), doi: https://doi.org/10.1016/j.dyepig.2020.108320.
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Author Statement
Linghao Hu: Methodology, Investigation, Formal analysis, Writing-Original Draft. Donglei
Shi: Conceptualization, Investigation, Formal analysis, Writing-Original Draft. Xinming Li:
Investigation, Validation, Visualization. Jin Zhu: Investigation, Validation. Fei Mao: Visualization,
Software. Xiaokang Li: Visualization, Software. Conglong Xia: Visualization, Software. Bei
Jiang: Visualization, Software. Yuan Guo: Supervision, Writing–Review & Editing, Project
administration, Funding acquisition. Jian Li: Supervision, Writing–Review & Editing, Project
administration, Funding acquisition.

Curcumin-based polarity fluorescent probes: design strategy
and biological applications
Graphical abstract
A novel design strategy of polarity-specific fluorescent probes based on curcumin
was presented.

Curcumin-based polarity fluorescent probes: design strategy and
biological applications
Linghao Hu,a,‡ Donglei Shi,a,b,‡ Xinming Li,a,‡ Jin Zhu,a Fei Mao,a Xiaokang Li,a Conglong Xia,c Bei Jiang,c
Yuan Guo,b,* Jian Lia,c,
*
aState Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New Drug Design, School of
Pharmacy, East China University of Science and Technology, Shanghai 200237, China
bKey Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry of Education,
College of Chemistry and Materials Science, Northwest University, Xi’an 710127, China
cCollege of Pharmacy and Chemistry, Institute of Materia Medica, Dali University, 5 Xue Ren Road, Dali
(Yunnan) 671000, China
‡These authors contributed equally to this work.
E-mail address: jianli@ecust.edu.cn (J. Li); guoyuan@nwu.edu.cn (Y. Guo).
1

Abstract
Polarity is a crucial characteristic of indispensable organelles, which has been indicated to be closely related to
many pathological and biological processes. Hence, a novel design strategy of curcumin-based polarity
fluorescent probes was presented. Curcumin attracted much attention due to its environment-sensitivity and
optical properties. We reformed the structure of curcumin by attaching a galactose moiety that was used to
increase the water solubility and regulate the probe working mode. Furthermore, we also introduced a
diethylamino group, which played a substantial role in adjusting the polarity specificity and sensitivity of the
probes. We finally screened two polarity-specific fluorescent probes 0301 and 0302. Both probes exhibited
remarkable changes in fluorescence from red to green upon increasing polarity of environment. Using 0301 in
confocal imaging, we obtained a clear and exact view of lysosomal polarity in different cells, suggesting that
the lysosomal polarity in normal cells is higher than that in cancer cells. Also, we use 0301 to monitor variations
in lysosomal polarity in live cells induced by dimethylsulfoxide and sucrose, indicating that our probe could
specifically monitor the changes of polarity in live cells to elucidate polarity-related biological processes.
Keywords: Curcumin; Polarity-specificity; Polarity fluorescent probe; Lysosome; Live-cell imaging; Design
strategy
1. Introduction
Polarity, an extremely significant factor in the biological microenvironment, plays a pivotal role in many
biological processes. Differentiation, activation of the immune response, vectorial transport of molecules across
cell layers, localized membrane growth and cell migration may all lead to changes in the polarity of cells [1-14].
In view of the complexity of the intracellular microenvironment, local polarity differs greatly in different
regions. Moreover, cell polarity changes with the variations of cell status in some extent, which is the
feedback of cell operation and regulation mechanism [15]. Thus, accurate detection of polarity variations in
2

different subcellular organelles (e.g., lysosomes) has attracted much attention. In particular, changes in the
polarity of lysosomes have been regarded as a key indicator of the state of cells. Moreover, recent works have
reported that the level of lysosome polarity in cancer cells is significantly different from that in normal cells,
suggesting that the polarity of lysosomes is closely related to cancer and is a potential versatile biomarker for
cancer diagnosis [16-19]. Consequently, it is of great significance to develop methods and strategies to monitor
lysosome polarity.
Fig. 1. The previous design strategy of polarity fluorescent probes and our design strategy of curcumin-based polarity fluorescent probes.
Fluorescence imaging has become an emerging and powerful technique for monitoring different bioactive
molecules because of its high spatiotemporal resolution and nondestructive detection characteristics [20-25].
Currently, a number of excellent fluorescent probes have been developed for the detection and imaging of the
polarity in cells [26-38]. However, only a few of these probes can target live-cell lysosomes and monitor
lysosome polarity. More importantly, a limited number of fluorescent probes can monitor lysosome polarity
with a ratiometric response; however, these probes exhibit short wavelength emission, poor solubility, low
polarity-specificity leading to a limited penetration depth and excessive self-absorption of the fluorescence,
which limit their wide range of applications. Therefore, it remains an urgent need to develop excellently
3

polarity-sensitive fluorescent probes with long wavelength emission, high water solubility and a sensitive
response.
To solve these issues, we designed and synthesized four novel polarity-sensitive fluorescent probes
(Scheme 1), 0301-0304, to monitor lysosomal polarity based on a curcumin core, which showed long
wavelength emission, high water solubility and high sensitivity to polarity. Curcumin is a satisfactory
environmentally sensitive fluorophore that exhibits outstanding optical properties and biocompatibility.
Although curcumin showed high environmental sensitivity and many eminent properties, its application has
been restricted due to low polarity specificity and water insolubility [39-41]. Herein, we reformed the
structure of curcumin by attaching a galactose moiety that was used to increase the water solubility and
regulate the probe working mode (Fig. 1). Probes 0301 and 0302 exhibited a good polarity dependence in
their emission spectra, which showed green fluorescence emission in high polarity media, in contrast, red
fluorescence emission in low polarity media, revealing that they could monitor the variation of polarity with a
remarkable changes of fluorescence. To further systematically investigate the factors affecting their sensitivity
to polarity, we also fabricated additional types of candidate probe molecules (0303, 0304, M1, 0302D). The
results showed that the introduction of galactose and diethylamino moieties significantly increased the
polarity sensitivity and regulate the detection mode of the curcumin-based probes. The results of optical
experiments indicated that probes 0301 and 0302 were insensitive to pH and viscosity, which means our
probes are able to monitor polarity specifically. Using 0301 in live cell confocal imaging, we obtained a clear
and exact view of the difference in lysosome polarity between normal cells and cancer cells. The results
indicated that the level of lysosome polarity in normal cells is higher than that in cancer cells. We also studied
the changes in lysosome polarity in lysosomal storage disorders, and the results indicated that lysosome
polarity decreases in lysosomal storage disorders.
4

2. Materials and Methods
2.1 Materials and measurements
Reagents and solvents were purchased from Adamas-beta, J&K, Energy Chemical, TCI, Alfa Aesar, and
Bide Pharmatech and were used without further purification. Distilled water was used in the experiment and all
solvents were spectral grade unless otherwise noted. All inorganic salts used were of analytical grade. A Hitachi
F-4700 fluorescence spectrometer and a Hitachi U-2910 spectrometer with a 10 mm quartz cuvette were
employed to record the fluorescence spectra and absorption spectra of all probes, respectively. The cell viability
assays were dected by BioTek SynergyH1 multimode reader. A Bruker AMX-400 and AMX-600 spectrometer
were used to record the 1H and 13C NMR spectra, using TMS as an internal standard. Electric ionization (EI) and
electrospray ionization (ESI) with a Waters LCT Premier XE spectrometer were employed to record High
resolution mass spectrometry (HRMS) data. The fluorescence imaging studies of cells were observed with
Leica TCS SP8 Confocal Laser Scanning Microscope.
2.2 Synthesis
The synthetic route for all compounds is shown in Scheme 1. Compounds 0301 and 0302 were prepared
through synthetic route I. The reaction of acetobromogalactose with 4-hydroxybenzaldehyde in the presence
of Cs2CO3 gave M0. Using 2-hydroxy-1-naphthaldehyde and acetobromogalactose as reactants, 0301M1 was
prepared in a similar way. M1 was obtained through the condensation of 4-diethylaminobenzaldehyde with an
acetylacetone-B2O3 complex. Then, M1 reacted with B2O3, and the resulting complex subsequently reacted
with M0 in the presence of tributyl borate and n-butylamine to afford 0302AC. Similarly, 0301AC was
prepared through the condensation of M0 with 0301M1. Finally, 0301 and 0302 were obtained through the
deprotection of 0301AC and 0302AC employing sodium methylate as the base. As shown in synthetic route II,
0302D, unlike 0302, did not contain a galactose structure. Compound 0302D was prepared through the
5

Scheme 1. Synthetic routes of all compounds.
condensation of 4-diethylaminobenzaldehyde with M2, which was the product of the condensation reaction of
4-hydroxybenzaldehyde and acetylacetone. Derivatives 0303 and 0304 were prepared through synthetic route
III. M0 reacted with acetylacetone to afford M4. 4-(4-Methylpiperazin-1-yl)benzaldehyde and
9-julolidinecarboxaldehyde reacted with M4 to afford 0303AC and 0304AC, respectively. These two
intermediates spontaneously formed 0303 and 0304 in the presence of sodium methylate.
General method A (condensation of acetylacetone with corresponding aldehydes, reaction condition a in
the scheme 1): B2O3 (1 eq.) and acetylacetone (4 eq.) was mixed in ethyl acetate (2 mL per mmol) and
refluxed for 30 minutes to afford the acetylacetone-B2O3 complex. The mixture was cooled to 50 °C, then the
corresponding aldehyde (1 eq.) and tributyl borate (2 eq.) was added. After stirring for 30 minutes,
n-butylamine (1.1 eq. dissolved in a small amount of ethyl acetate) was added dropwise over 10 minutes. The
6

reaction was then cooled to room temperature, and it was stirred overnight. 1 mmol/mL HCl solution (3 eq.)
was added to quench the reaction (stirring for 5 minutes), and the resulting mixture was neutralized with
saturated NaHCO3 and extracted with ethyl acetate. The ethyl acetate solution was concentrated through
rotoevaporation to give the crude product. It was subsequently purified by column chromatography to give
M1/M2 or M4.
General method B (reaction condition b in the scheme 1 for the preparation of galactosyl aldehyde):
Acetobromogalactose (1 eq.) and corresponding aldehyde (1 eq.) were dissolved in acetonitrile (2.5 mL per
mmol), then Cs2CO3 (2.5 eq.) was added. After stirring overnight, the mixture was filtered, and the filtrate
was condensed to afford the crude product. It was subsequently purified by column chromatography.
General method C (reaction condition c in the scheme 1): The corresponding intermediate (M1/M2 or
M4) and B2O3 (0.5 eq.) were suspended in ethyl acetate (5 mL per mmol). The reaction was refluxed for 1 h,
and cooled to 50 °C. The aldehyde (1 eq.) and tributyl borate (1 eq.) was then added, the resulting mixture
was stirring for 1h. 1.1 equivalent n-butyl amine (1.1 eq. dissolved in a small amount of ethyl acetate) was
added dropwise over 10 minutes. The reaction was allowed to cooled to room temperature and stirring for 24
h before the addition of 1 mmol/mL HCl solution (3 eq. stirring for 30 minutes to quench the reaction). The
mixtrure was neutralized with saturated NaHCO3 and extracted with ethyl acetate and condensed to give the
crude product, and it was then purified by column chromatography to give 0301AC-0304AC or 0302D.
General method D (deprotection, reaction condition d in the scheme 1) ： The intermediate
(0301AC-0304AC) was dissolved in methanol (10-20 mL per mmol), and sodium methylate (4 eq.) was then
added. After stirring for 10 minutes, the mixture was neutralized with cation-exchange resin. After filtering
the resin, the mixture was condensed to give the crude product. The crude product was purified through
column (silica gel or C18 silica gel) chromatography to give 0301-0304.
7

2.2.1 Synthesis of compound M0
M0, 4-((2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy)-benzaldehyde, was prepared through general
method A using acetobromogalactose and 4-hydroxybenzaldehyde as reactants, yield = 73%. Column
1
chromatography condition for M0: silica gel (200 meshes), ethyl acetate: petrol ether, 1:5 to 1:3 (v/v). H
NMR (400 MHz, Chloroform-d) δ 9.93 (s, 1H), 7.93-7.81 (m, 2H), 7.18-7.05 (m, 2H), 5.61-5.45 (m, 2H),
5.23-5.10 (m, 2H), 4.35-4.06 (m, 3H), 2.20 (s, 3H), 2.08 (s, 6H), 2.03 (s, 3H). 13C NMR (151 MHz,
Chloroform-d) δ 190.73, 170.34, 170.18, 169.32, 161.30, 131.83, 116.75, 98.59, 71.34, 70.68, 68.41, 66.78,
61.38, 20.64. The NMR spectra of M0 is consistent with the published data.
2.2.2 Synthesis of compound M1
M1, 6-(4-(diethylamino)phenyl)hex-5-ene-2,4-dione, was prepared through general method B using
4-diethylaminobenzaldehyde and acetylacetone as reactants, yield = 31%. Column chromatography condition
1
for M1: silica gel (200 meshes), ethyl acetate: petrol ether, 1:50 to 1:15 (v/v). H NMR (400 MHz,
Chloroform-d) δ 15.73 (s, 1H), 7.55 (d, J = 15.6 Hz, 1H), 7.40 (d, J = 8.7 Hz, 2H), 6.63 (d, J = 8.5 Hz, 2H), 6.25
(d, J = 15.7 Hz, 1H), 5.59 (s, 1H), 3.40 (q, J = 7.1 Hz, 4H), 2.13 (s, 3H), 1.19 (t, J = 7.1 Hz, 6H). The NMR
spectra of M1 is consistent with the published data
2.2.3 Synthesis of compound M2
M2, 6-(4-hydroxyphenyl)hex-5-ene-2,4-dione, was prepared through general method B using
4-hydroxybenzaldehyde and acetylacetone as reactants, yield = 28%. Column chromatography condition for
M2: silica gel (200 meshes), ethyl acetate: petrol ether, 1:10 (v/v). 1H NMR (400 MHz, Chloroform-d) δ 7.55
(d, J = 15.8 Hz, 1H), 7.44 (d, J = 8.6 Hz, 2H), 6.94-6.79 (m, 2H), 6.34 (d, J = 15.8 Hz, 1H), 5.62 (s, 1H), 2.16
(s, 3H). The NMR spectrum of M2 is consistent with the published data.
2.2.4 Synthesis of compound M3
8

M3, 2-((2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy)-1-naphthaldehyde, was prepared through
general method A using 2-hydroxy-1-naphthaldehyde and acetobromogalactose as reactants, yield = 78%.
Column chromatography condition for M3: silica gel (200 meshes), ethyl acetate: petrol ether, 1:5 to 1:3 (v/v).
1H NMR (400 MHz, Chloroform-d) δ 10.78 (s, 1H), 9.24 (dq, J = 8.7, 0.9 Hz, 1H), 8.07 (d, J = 9.1 Hz, 1H),
7.89-.76 (m, 1H), 7.66 (ddd, J = 8.5, 6.9, 1.5 Hz, 1H), 7.50 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.35 (d, J = 9.1 Hz,
1H), 5.64 (dd, J = 10.5, 7.9 Hz, 1H), 5.50 (dd, J = 3.4, 1.1 Hz, 1H), 5.23 (d, J = 7.9 Hz, 1H), 5.16 (dd, J = 10.5,
3.4 Hz, 1H), 4.31-4.06 (m, 3H), 2.23 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H). 13C NMR (151 MHz,
Chloroform-d) δ 191.95, 170.23, 169.36, 160.61, 137.21, 131.03, 130.09, 130.01, 128.21, 125.87, 125.41,
119.15, 115.94, 100.15, 71.39, 70.69, 68.52, 66.74, 61.24, 20.69, 20.66, 20.58. HRMS (ES) m/z calcd for
C25H26NaO11+ [M+Na]+ 525.1373, found 525.1372.
2.2.5 Synthesis of compound M4
M4, 6-(4-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxyphenyl)hex-5-ene-2,4-dioe, was prepared
through general method A using M0 and acetylacetone as reactants, yield = 38%. Column chromatography
condition for M4: silica gel (200 meshes), ethyl acetate: petrol ether= 1:4 (v/v), dichloromethane, then ethyl
acetate: dichloromethane = 1:10 (v/v). 1H NMR (400 MHz, Chloroform-d) δ 7.62 (d, J = 15.8 Hz, 1H), 7.51 (d,
J = 8.4 Hz, 2H), 7.02 (d, J = 8.4 Hz, 3H), 6.53 (d, J = 15.8 Hz, 1H), 5.61-5.43 (m, 2H), 5.22-5.07 (m, 2H),
4.35-4.01 (m, 5H), 2.19 (s, 3H), 2.10-2.00 (m, 12H). 13C NMR (151 MHz, Chloroform-d) δ 183.23, 170.37,
170.23, 170.14, 169.38, 158.19, 139.67, 130.22, 129.66, 123.05, 117.11, 101.69, 99.13, 71.17, 70.77, 68.53,
66.8, 61.33, 20.74, 20.67, 20.59. HRMS (ES) m/z calcd for C26H30NaO12+ [M+Na]+ 557.1635, found 557.1636.
2.2.6 Synthesis of compound 0301AC
0301AC, 1-(4-(diethylamino)phenyl)-7-[2-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxynaphthalen
-1-yl]hepta-1,6-diene-3,5-dione, was prepared through general method C using M3 and M1 as reactants, yield =
9

25%. Column chromatography condition for 0301AC: silica gel (200 meshes), ethyl acetate: petrol ether= 1:5
(v/v), dichloromethane, then ethyl acetate: dichloromethane= 1:10 (v/v). 1H NMR (600 MHz, Methanol-d4) δ
10.72 (d, J = 2.1 Hz, 1H), 8.20 (dd, J = 9.1, 2.2 Hz, 2H), 8.14 (d, J = 16.1 Hz, 1H), 7.94-7.88 (m, 1H), 7.90-7.83
(m, 1H), 7.64 (d, J = 15.8 Hz, 1H), 7.58 (ddd, J = 8.5, 6.7, 1.4 Hz, 1H), 7.55-7.49 (m, 2H), 7.51-7.46 (m, 2H),
7.49-7.40 (m, 1H), 6.86 (d, J = 16.1 Hz, 1H), 6.74-6.67 (m, 2H), 6.56 (d, J = 15.6 Hz, 1H), 5.58 (d, J = 7.9 Hz,
1H), 5.53-5.50 (m, 1H), 5.51-5.44 (m, 1H), 5.30 (dd, J = 10.3, 3.5 Hz, 1H), 4.39 (dd, J = 6.5, 1.2 Hz, 2H), 4.23
(d, J = 6.6 Hz, 1H), 4.22-4.21 (m, 1H), 3.45 (q, J = 7.1 Hz, 4H), 2.08 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H), 1.99 (s,
3H), 1.19 (t, J = 7.1 Hz, 6H). 13C NMR (151 MHz, Methanol-d4) δ 191.89, 186.52, 179.84, 170.63, 170.56,
170.14, 170.05, 161.04, 149.76, 142.49, 137.42, 132.46, 131.35, 130.94, 130.32, 130.27, 130.18, 130.02,
129.44, 128.35, 128.17, 127.91, 127.36, 127.22, 125.35, 124.60, 124.53, 123.26, 121.80, 117.94, 115.73,
111.20, 99.03, 71.14, 71.06, 71.03, 70.93, 70.64, 68.72, 68.68, 67.40, 67.23, 61.29, 61.15, 44.03, 19.42, 19.15,
19.09, 11.51. HRMS (ES) m/z calcd for C41H46O12+ [M+H]+ 744.2932, found 744.3201.
2.2.7 Synthesis of compound 0301
0301,
1-(4-(diethylamino)phenyl)-7-(2-β-D-galactopyranosyloxynaphthalen-1-yl)hepta-1,6-diene-3,5
-dione, was prepared through general method D using 0301AC as reactant, yield = 40%. Column
chromatography condition for 0301: silica gel (200 meshes), methanol: dichloromethane = 1:20 to 1:15 (v/v).
1H NMR (600 MHz, Methanol-d4) δ 8.25 (d, J = 16.1 Hz, 1H), 8.21 (d, J = 8.6 Hz, 1H), 7.88 (d, J = 9.1 Hz, 1H),
7.85 (dd, J = 8.2, 1.2 Hz, 1H), 7.62 (d, J = 15.6 Hz, 1H), 7.58 (d, J = 9.1 Hz, 1H), 7.55 (ddd, J = 8.4, 6.8, 1.3 Hz,
1H), 7.48 (d, J = 8.9 Hz, 2H), 7.44-7.39 (m, 1H), 7.17 (d, J = 16.1 Hz, 1H), 6.71 (d, J = 9.0 Hz, 2H), 6.54 (d, J
= 15.7 Hz, 1H), 5.17 (d, J = 7.7 Hz, 1H), 3.98 (dd, J = 9.7, 7.7 Hz, 1H), 3.95 (d, J = 3.4 Hz, 1H), 3.83-3.74 (m,
3H), 3.65 (dd, J = 9.6, 3.6 Hz, 1H), 3.45 (q, J = 7.1 Hz, 4H), 1.19 (t, J = 7.1 Hz, 6H). 13C NMR (151 MHz,
Methanol-d4) δ 185.58, 181.51, 154.25, 149.72, 142.23, 132.44, 132.05, 130.80, 130.21, 130.03, 128.30,
10

126.94, 124.09, 123.14, 121.83, 118.64, 117.62, 116.11, 111.20, 101.52, 75.80, 73.86, 71.00, 68.91, 61.02,
44.04, 11.51. HRMS (ES) m/z calcd for C33H38NO8 [M+H]+ 576.2597 found 576.2598.
2.2.8 Synthesis of probe 0302D
0302D, 1-(4-(diethylamino)phenyl)-7-(4-hydroxyphenyl)hepta-1,6-diene-3,5-dione, was prepared
through general method C using M2 and 4-diethylaminobenzaldehyde as reactants, yield = 41%. Column
chromatography condition for 0302D: silica gel (200 meshes), ethyl acetate: petrol ether, 1:25 to 1:8 (v/v). 1H
NMR (400 MHz, Chloroform-d) δ 7.59 (dd, J = 18.0, 15.7 Hz, 2H), 7.45 (d, J = 3.4 Hz, 2H), 7.43 (d, J = 3.8 Hz,
2H), 6.98 (d, J = 8.6 Hz, 1H), 6.64 (d, J = 8.5 Hz, 2H), 6.47 (d, J = 15.8 Hz, 1H), 6.41 (d, J = 15.6 Hz, 1H), 5.76
(s, 1H), 3.41 (q, J = 7.2 Hz, 5H), 1.20 (t, J = 7.1 Hz, 9H). The NMR spectrum of 0302D is consistent with the
published data.
2.2.9 Synthesis of probe 0302AC
0302AC,
1-(4-(diethylamino)phenyl)-7-(4-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxyphenyl)
hepta-1,6-diene-3,5-dione, was prepared through general method C using M0 and M1 as reactants, yield = 28%.
Column chromatography condition for 0302AC: silica gel (200 meshes), ethyl acetate: petrol ether= 1:5 (v/v),
dichloromethane, then ethyl acetate: dichloromethane= 1:10 (v/v). 1H NMR (600 MHz, Chloroform-d) δ 9.93
(s, 1H), 7.95-7.79 (m, 2H), 7.62 (d, J = 15.7 Hz, 1H), 7.58 (d, J = 15.7 Hz, 1H), 7.54-7.42 (m, 3H), 7.15-7.09 (m,
2H), 7.04-6.98 (m, 2H), 6.52 (d, J = 15.8 Hz, 1H), 5.78 (s, 1H), 5.56–5.49 (m, 1H), 5.48 (ddt, J = 6.8, 3.5, 1.6
Hz, 2H), 5.17 (d, J = 7.9 Hz, 1H), 5.13 (ddd, J = 10.4, 8.1, 3.5 Hz, 1H), 5.10 (d, J = 7.9 Hz, 1H), 4.26-4.15 (m,
2H), 3.50-3.30 (brs, 4H), 2.19 (s, 3H), 2.07 (s, 6H), 2.02 (s, 3H), 1.21 (t, J = 7.1 Hz, 6H). 13C NMR (151 MHz,
DMSO-d6) δ 170.41, 170.29, 169.99, 169.66, 158.19, 130.35, 130.10, 123.53, 117.10, 97.70, 70.93, 70.59,
68.74, 67.68, 61.79, 21.00, 20.93, 20.88, 20.83. HRMS (ES) m/z calcd for C37H44NO12 [M+H]+ 694.2864 found
694.286.
11

2.2.10 Synthesis of probe 0302
0302, 1-(4-(diethylamino)phenyl)-7-(4-β-D-galactopyranosyloxyphenyl)hepta-1,6-diene-3,5-dione, was
prepared through general method D using 0302AC as reactant, yield = 25%. Column chromatography condition
for 0302: C18 silica gel (40-70 µm), water: methanol= 1:1 to 1:4 (v/v). 1H NMR (600 MHz, DMSO-d6) δ 7.66
(d, J = 8.3 Hz, 2H), 7.60-7.40 (m, 4H), 7.07 (d, J = 8.3 Hz, 2H), 6.77 (d, J = 15.9 Hz, 1H), 6.73-6.65 (m, 3H),
6.59 (d, J = 15.6 Hz, 1H), 6.02 (s, 1H), 5.19 (d, J = 5.2 Hz, 1H), 4.95-4.85 (m, 2H), 4.66 (t, J = 5.5 Hz, 1H), 4.52
(d, J = 4.6 Hz, 1H), 3.71 (t, J = 4.0 Hz, 1H), 3.63-3.53 (m, 3H), 3.52-3.47 (m, 1H), 3.41 (q, J = 7.3, 6.7 Hz, 4H),
1.12 (t, J = 6.9 Hz, 6H). 13C NMR (151 MHz, DMSO-d6) δ 181.45, 159.44, 149.76, 131.10, 130.77, 130.19,
129.02, 121.67, 117.03, 111.75, 101.08, 76.08, 73.74, 70.68, 68.60, 60.84, 44.30, 12.95. HRMS (ES) m/z calcd
+
for C29H36NO8 [M+H]+ 526.2441, found 526.2442.
2.2.11 Synthesis of probe 0303AC
0303AC, 1-(4-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxyphenyl)-7-(4-(4-methylpiperazin-1-yl)
phenyl)hepta-1,6-diene-3,5-dione, was prepared through general method
C
using M4 and
4-(4-methylpiperazin-1-yl)benzaldehyde as reactants, yield = 35%. Column chromatography condition for
0303AC: silica gel (200 meshes), ethyl acetate: methanol: dichloromethane= 1:50 to 1:15 (v/v).1H NMR (400
MHz, Chloroform-d) δ 7.62 (d, J = 9.0 Hz, 1H), 7.58 (d, J = 9.1 Hz, 1H), 7.55-7.39 (m, 5H), 7.05-6.98 (m, 2H),
6.93-6.85 (m, 3H), 6.50 (t, J = 15.8 Hz, 2H), 5.79 (s, 1H), 5.63-5.43 (m, 2H), 5.18-5.01 (m, 2H), 4.32-4.03 (m,
3H), 3.36 (t, J = 5.1 Hz, 5H), 2.63 (t, J = 5.2 Hz, 6H), 2.40 (s, 4H), 2.19 (s, 3H), 2.08 (s, 5H), 2.02 (s, 3H). 13
C
NMR (151 MHz, Chloroform-d) δ 179.86, 177.12, 165.63, 165.50, 165.39, 164.65, 153.29, 147.53, 136.25,
135.29, 134.16, 125.67, 125.04, 124.78, 124.73, 124.24, 120.84, 118.44, 115.77, 114.41, 112.35, 110.33,
110.25, 96.73, 95.73, 94.41, 66.41, 66.03, 63.80, 62.07, 56.60, 49.94, 42.94, 42.87, 41.19, 27.29, 24.96, 21.97.
HRMS (ES) m/z calcd for C38H45N2O12+ [M+H]+ 721.2967, found, 721.2971.
12

2.2.12 Synthesis of probe 0303
0303, 1-(4-β-D-galactopyranosyloxyphenyl)-7-(4-(4-methylpiperazin-1-yl)phenyl)hepta-1,6-diene-3,5-
dione, was prepared through general method D using 0303AC as reactant, yield = 20%. Column
chromatography condition for 0303: C18 silica gel (40-70 µm), water: methanol= 1:1 to 1:4 (v/v). 1H NMR (600
MHz, DMSO-d6) δ 8.50 (s, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.58-7.45 (m, 4H), 7.06 (d, J = 8.3 Hz, 2H), 6.96 (d, J
= 8.5 Hz, 2H), 6.78 (t, J = 15.3 Hz, 1H), 6.69 (d, J = 16.6 Hz, 1H), 5.95 (s, 1H), 4.90 (d, J = 7.7 Hz, 1H),
4.78-4.65 (brs, 1H), 3.72 (d, J = 3.5 Hz, 1H), 3.64-3.45 (m, 5H), 3.27 (t, J = 5.1 Hz, 4H), 2.43 (t, J = 5.0 Hz, 4H),
2.22 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 166.33, 130.26, 117.01, 114.94, 101.10, 76.09, 73.78, 70.72,
68.51, 60.78, 54.85, 47.40, 46.22. HRMS (ES) m/z calcd for C30H37N3O8 [M+H]+ 553.2550, found 553.2551.
2.2.13 Synthesis of probe 0304AC
0304AC,
1-(4-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxyphenyl)-7-(2,3,6,7-tetrahydro-1H,5H
-pyrido[3,2,1-ij]quinolin-9-yl)hepta-1,6-diene-3,5-dione, was prepared through general method C using M4
and 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde as reactants, yield = 28%. Column
chromatography condition for 0304AC: silica gel (200 meshes), ethyl acetate: petrol ether= 1:5 (v/v),
dichloromethane, then ethyl acetate: dichloromethane= 1:10 (v/v). 1H NMR (600 MHz, Methanol-d4) δ 7.90 (d,
J = 8.7 Hz, 1H), 7.57 (d, J = 9.1 Hz, 2H), 7.56-7.42 (m, 3H), 7.32 (d, J = 15.9 Hz, 1H), 7.19 (d, J = 8.7 Hz, 1H),
7.10-6.98 (m, 3H), 6.60 (dd, J = 52.6, 15.9 Hz, 1H), 5.50-5.43 (m, 2H), 5.41-5.32 (m, 3H), 5.31-5.22 (m, 2H),
4.58 (s, 1H), 4.36-4.27 (m, 1H), 4.21-4.16 (m, 4H), 3.39 (t, J = 6.9 Hz, 1H), 3.26 (t, J = 5.7 Hz, 4H), 2.73 (t, J =
6.4 Hz, 4H), 2.18 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H), 1.94 (m, 4H). 13C NMR (151 MHz,
Methanol-d4) δ 170.64, 170.58, 170.05, 169.91, 142.87, 138.73, 138.04, 135.94, 131.49, 129.23, 128.77,
127.68, 121.03, 116.60, 116.53, 116.36, 98.27, 97.72, 70.84, 68.73, 67.31, 61.19, 49.59, 29.37, 27.33, 21.39,
19.18. HRMS (ES) m/z calcd for C39H44NO12+ [M+H]+ 718.2858, found 718.2866.
13

2.2.14 Synthesis of probe 0304
0304, 1-(4-β-D-galactopyranosyloxyphenyl)-7-(2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-9-yl)
hepta-1,6-diene-3,5-dione, was prepared through general method D using 0304AC as reactant, yield = 30%.
Column chromatography condition for 0304: silica gel (200 meshes), methanol: dichloromethane = 1:20 to 1:10
(v/v). 1H NMR (600 MHz, DMSO-d6 : Methanol-d4 = 4 : 1 (v/v)) δ 7.65 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 15.8 Hz,
1H), 7.44 (d, J = 15.5 Hz, 1H), 7.10 (s, 2H), 7.07 (d, J = 8.5 Hz, 2H), 6.75 (d, J = 15.8 Hz, 1H), 6.52 (d, J = 15.5
Hz, 1H), 5.96 (s, 1H), 5.19 (d, J = 5.3 Hz, 1H), 4.89 (t, J = 7.2 Hz, 2H), 4.66 (t, J = 5.4 Hz, 1H), 4.52 (d, J = 4.6
Hz, 1H), 3.70 (t, J = 4.2 Hz, 1H), 3.57 (ddt, J = 26.0, 11.2, 5.9 Hz, 3H), 3.49 (dt, J = 11.2, 6.0 Hz, 1H), 3.42 (dt,
J = 9.0, 4.3 Hz, 1H), 3.23 (t, J = 5.7 Hz, 4H), 2.68 (t, J = 6.3 Hz, 4H), 1.87 (p, J = 6.3 Hz, 4H). 13C NMR (151
MHz, Methanol-d4) δ 185.22, 166.65, 158.67, 136.33, 130.00, 129.45, 128.65, 127.64, 126.67, 124.52, 123.83,
118.65, 116.57, 101.22, 75.65, 73.44, 70.82, 68.81, 61.01, 49.60, 42.54, 30.41, 29.37. HRMS (ES) m/z calcd for
C31H36NO8 [M+H]+ 550.2441, found 550.2440.
2.3 Spectroscopic measurements
A stock solution of the probe 0301-0304 (1 mM) were dissolved in DMSO and stored at 4 ºC. Stock
2-
-
solutions of varieties of testing species (including Mn2+, Al3+, Mg2+, Na+, CO3 , HCO3 , GSH, Hcy, Cys and
DTT) were prepared in doubly distilled water. 3 mL of the test solutions were taken out a quartz cuvette (path
length = 1 cm) to measure UV-Vis absorption and fluorescence spectra at room temperature.
2.4 Cell culture and fluorescence imaging
A549, HepG2, HL-7702 and 293T cells were obtained from Procell Life Science&Technology Co., Ltd
(Wuhan, China). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal
bovine serum (FBS), 1% penicillin and streptomycin in an atmosphere of 5% CO2 and 95% air at 37 ºC in the
incubator. For confocal microscopy imaging, cells were adjusted to the density of 1×106 cells per mL in a 35
14

mm confocal dish (15 mm glass bottom well) and incubated for 24 h. Subsequently, different kinds of cells
were incubated with 0301 (4 µM) for 10 min. After being washed three times with PBS buffer, cells were
imaged under a confocal laser scanning microscope (Leica TCS SP8).
2.5 Cytotoxicity assay
The cytotoxicity of 0301 and 0302 were evaluated in 293T and HepG2 cells by the CCK-8 assays. 90%
HepG2 cells (293T cells) were seeded into a 96-well cell-culture plate with density of 8000 cells per well. After
incubating for 24 h, different concentrations of 0301 or 0302 (0, 4, 10, 15, 20, 25 µM) were added to the each
well, respectively. The cells were treated and incubated in the same conditions for 24 h. CCK-8 solution (100
µL) was then added to each well and kept for another 1 h. Absorbance at 450 nm was measured using the
microplate spectrophotometer system (Varioskan Flash). The viability of the cells was calculated by the ratio
of mean of the absorbance value of the treatment group and the control group.
3. Results and Discussion
3.1 Design and synthesis of probe 0301-0304
Curcumin has been widely developed as environment-sensitive fluorescent probes due to its eminent
properties (high biocompatibility, high fluorescence quantum yields and sufficient photostability) [39-41]. We
speculated that replacing the methoxyl group at the terminal of curcumin with potentially polarity-sensitive
groups could increase its polarity-specificity and polarity-sensitivity. Hence, probes 0301 and 0302 were
engineered by introducing a no-rigid group (diethylamino) and a galactose group into the molecular skeleton of
curcumin, respectively. To further verify our hypothesis, the no-rigid group was replaced with a middle-rigid
group (1,4-dimethylpiperazino) and a rigid group (julolidino) to obtain probes 0303 and 0304, respectively. All
15

probes were characterized by 1H NMR, 13C NMR, and HRMS and the spectra were displayed in Supporting
Information.
3.2 Spectral properties
First, we screened the responses of probes 0301-0304 to polarity changes by exploring their optical
properties in detail. The emission and absorption spectra of each derivative were investigated in various
solvents (Figs S1 and S2). It is obvious that both absorption and emission maxima of all probes exhibited
significant changes with the increase in polarity of different solvents, indicating that these probes were
polarity-sensitive. To accurately quantify the relationship between the optical properties and polarity, the
changes in the optical properties of 0301-0304 (10 µM) as a function of polarity changes were studied in a
H2O/dioxane system, in which the polarity of solution was adjusted by varying the volume fraction of dioxane
(Table S1). The polarity of the H2O/dioxane system was estimated by the Lippert Mataga polarity parameter ∆f
[15]. The absorption of probes 0301-0304 were shown in Fig. S3 and emission spectra were shown in Fig. 2. For
probe 0301, a continued increase in the polarity of the solution (∆f = 0.320-0.397) caused a blueshift in the
fluorescence from 602 nm to 554 nm and a 12.9-fold decrease in fluorescence intensity of maximum emission
peak (Fig. 2a and Table S2). Consistently, a clear blueshift from 606 nm to 545 nm can be observed in the
fluorescence spectrum of 0302 upon increasing the solvent polarity from ∆f = 0.278 to ∆f = 0.397 (Fig. 2b). The
fluorescence intensity of maximum emission peak of 0302 gradually decreased (4.7-fold) with the increase of
polarity from ∆f = 278 to ∆f = 0.381. On continued increase of polarity (from ∆f = 381 to ∆f = 0.397), 0302
showed a 3.5-fold enhancement in fluorescence intensity of maximum emission peak. However, 0303 and
0304 displayed slight fluorescence changes after varying the polarity of the systems (Fig. 2d-e), indicating that
the no-rigid diethylamino moiety is essential for polarity detection. The fluorescence changes could also be
obviously observed under ultraviolet light (Fig. 2f). Moreover, 0301 and 0302 exhibited a good
16

Fig. 2. (a) The fluorescence spectra of 0301 (10 µM) in dioxane/water systems (dioxane from 10-90%); (b) The fluorescence spectra of
0302 (10 µM) in dioxane/water systems (dioxane from 10-95%); (c) The strcutures of probes 0301-0304; (d) The fluorescence spectra of
0303 (10 µM) in dioxane/water systems (dioxane from 10-80%); (e) The fluorescence spectra of 0304 (10 µM) in dioxane/water systems
(dioxane from 10-80%); (f) Photographs of 0301-0304 in dioxane/water systems (dioxane from 10-90%) under a UV lamp.
linear relationship between fluorescence ratio (I602/I554, I606/I545) and solvent parameter ∆f and the linear
correlation coefficients (R2) of were calculated to be 0.95 and 0.99 (Fig. S4), respectively. These results
indicated that due to the introduction of galactose and diethylamino moieties, the curcumin-based fluorescent
probes 0301 and 0302 exhibited excellent polarity sensitivity and remarkable fluorescence responses. In other
words, as designed, 0301 and 0302 could be applied to monitor the variation of polarity with high sensitivity.
To further estimate the ability of the probes to monitor polarity, we then investigated the fluorescence
17

Fig. 3. (a) The fluorescence spectra of 0301 (10 µM) in water/dioxane systems at different pH values; (b) The fluorescence spectra of 0302
(10 µM) in water/dioxane systems at different pH values; (c) The fluorescence spectra of 0301 (10 µM) in methanol-glycerol (glycerol
from 0-30%) systems under different viscosities; (d) The fluorescence spectra 0302 (10 µM) in methanol-glycerol (glycerol from 0-30%)
systems under different viscosities.
response of the probes to pH changes and viscosity. In the pH titration experiment (Fig. 3a-b), the fluorescence
intensities of 0301 and 0302 were essentially unchanged at pH values ranging from 4.0 to 9.0 in water/dioxane
mixtures (30% and 50% dioxane), indicating that the two probes were pH-insensitive over that pH range and
can be employed to monitor the polarity in lysosomes. In the viscosity titration experiment (Fig. 3c-d), the
emission intensity of probes 0301 and 0302 slightly enhanced with increased viscosity, suggesting that the two
probes were insensitive to viscosity. Also, the photostability of the probes in water/dioxane mixtures (10% and
30% dioxane) was measured by continuous irradiation with a Xe lamp (150W) at 5 nm slit width. Both probes
exhibited good photostability (Fig. S5a-b), indicating that they could be used in long-time cell tracing.
Subsequently, to investigate the fluorescence changes of 0301 and 0302 at around 550 nm after the addition of
other interferences, we recorded the variations in fluorescence of probes in 30% dioxane system. The
18

fluorescence of the probes exhibited negligible changes in the water/dioxane system (30% dioxane) after the
addition of different interfering substances (Fig. S5c-d). All of the above experiments proved that probes 0301
and 0302 possessed high polarity selectivity and desired optical properties, which can be employed to monitor
polarity in complex systems.
Fig. 4. (a) Proposed detection mechanism of 0302 in systems of polarities; (b) HMQC spectra of 0302 in methanol-d4 (top) and acetone-d6
(bottom). Acetone-d6: the box indicates features from a keto-enol tautomer. Methanol-d4: the middle box indicates the features from a
β-diketone tautomer and the lower left box indicates that there is no keto-enol tautomer in methanol-d4; (c) The fluorescence spectra of 0302
in MeOH and MeOD.
3.3 The sensing mechanism
Previous research has reported that most polarity-sensitive probes are composed of an intramolecular
electron donor, a conjugating spacer, and an electron receptor, forming an intramolecular charge transfer (ICT)
system. These probes usually display a significant redshift and a decrease in fluorescence intensity upon the
increase in surrounding polarity. Interestingly, our probes (0301, 0302 and 0303) all showed a blueshift with
increased polarity, which is not in accordance with most polarity responsive principles. Curcumin-based probes,
19

possess an excited-state intramolecular hydrogen atom transfer process (ESIPT) [40, 42], bearing
α,β-unsaturated β-diketone (heptadiene-dione) moiety, resulting in their predominant existence as β-diketone
or keto-enol tautomers in various polar solvents. Thus, a possible detection mechanism for polarity was
proposed (Fig. 4a). NMR spectroscopy was employed to distinguish the changes in the structures of the probes
in different polar environments. As shown in the Fig. 4b (lower right), the cross peak (δH 5.97, δC 100.85)
assigned to the methine (=C-H) in keto-enol tautomer of 0302 in the central box indicates that 0302 exists
mainly in form of keto-enol tautomer in acetone-d6. Whereas, in the HMQC spectrum of 0302 in methanol-d4
(Fig. 4b upper right), there is a cross peak (δH 3.88-3.72, δC 61.02) assigned to the methylene (CH2) in
β-diketone tautomer of 0302 in the central box, indicating that 0302 exists completely in form of β-diketone
tautomer in methanol-d4. The interpretation of HMQC spectra confirmed the proposed detecting mechanism.
Furthermore, 0302 displayed a clear blueshift (12 nm) in the fluorescence spectrum in MeOD compared to
MeOH (Fig. 4c), indicating that strong intramolecular hydrogen bonding is present in the keto-enol tautomer of
curcumin-based probes. Also, two candidate probes (0302D and M1) were synthesized by removing the
galactose group to further study the effects of substitutions to the polarity-sensitivity of 0301-0304 (Scheme 1).
Both 0302D and M1 exhibited a fluorescence change trend that was similar to most polarity probes (Fig. S6),
which was different from that of 0302, indicating that introduction of the galactose moiety not only enhanced
the water solubility but also regulated the detection mode to polarity. Moreover, the diethylamino group played
a substantial role in adjusting the polarity specificity and sensitivity of the probes, as replacing this group with
others, such as 1,4-dimethylpiperazine or julolidine, completely decreasing the polarity sensitivity.
3.4 Fluorescence imaging in live cells
We then investigated whether probes 0301 and 0302 could be employed to monitor changes in lysosome
polarity in live cells. The cytotoxicity of probe was studied by a CCK-8 assay. Compared to probe 0302, 0301
20

showed almost no cytotoxicity toward live cells (Fig. S7), indicating that probe 0301 is suitable for bioimaging
experiments. We next further explored the subcellular localization ability of 0301 by performing colocalization
experiments with commercialized dyes for different subcellular organelles (LysoTracker® Green DND-99,
MitoTracker® Green FM and GolgiGreen). As shown in Fig. 5 and Fig. S8, the imaging and fluorescence
intensity correlation mapping overlaid very well with that of LysoTracker Green in HepG2 and 293T cells. The
Pearson’s co-localization coefficient (PCC) values were calculated as 0.90 and 0.88 in HepG2 and 293T cells,
respectively. In contrast, the fluorescence in red channel of probe 0301 partly overlapped with the
fluorescence in green channel of mitochondria and golgi apparatus, with their PCC values below 0.69. These
results demonstrated that probe 0301 possesses the ability to localize in lysosomes and monitor the variations of
polarity in lysosomes.
Fig. 5. Intracellular localization of 0301 was performed in HepG2 cells. Cells were incubated with 0301 and Lyso Tracker Green (λex
=
488 nm, collected 490-530 nm), Mito Tracker Green (λex = 488 nm, collected 500-530 nm) and Golgi Tracker (λex = 488 nm, collected
490-530 nm) for 30 min, respectively. Plots in column 4 represent the intensity correlation plot of dyes and probes. Column 5 represents
cross-sectional analysis along the white line in the insets. Red channel: λex = 488 nm, collected 570-650 nm Scale bars: 25 µm.
21

An increasing number of studies have found that normal cells and cancer cells exhibit a clear difference in
lysosome polarity or mitochondria polarity [16-18, 31-32]. Therefore, we used fluorescence imaging to assess
the feasibility of using 0301 to monitor the polarity in normal and cancer cells to further distinguish between
them. HepG2, HL-7702 and 293T cells were selected for the following imaging experiments. Green and red
channels were collected (Fig. 6a) and the relative fluorescence intensities of the green and red channels were
measured (Fig. 6b). According to the polarity-fluorescence relationship of 0301, the level of lysosome polarity
in normal cells was higher than that in cancer cells, which is consistent with the reported results. The results
demonstrated that 0301 can be used to monitor lysosome polarity in different live cells and discriminate
between cancer cells and normal cells.
Fig. 6. Confocal fluorescence images of cancer cells (HepG2 cells) and normal cells (HL-7702 and 293T cells) treated with 0301 (4 µM, 10
min); (b) Quantified relative fluorescent intensity ratio (Fred channel/Fgreen channel) of cells. Green channel: λex = 488 nm, collected 530-570 nm;
Red channel: λex = 488 nm, collected 570-650 nm. Scale bar: 25 µm. The values are the mean ± s.d. for n = 3, **p < 0.01, ***p<0.001.
To confirm the feasibility of 0301 for monitoring the variations of polarity in live cells, we then used
DMSO to stimulate cells, which could be taken up by cells and further decrease the lysosome polarity in live
cells. As shown in Fig. 7, DMSO-treated HepG2 cells showed an increased fluorescence intensity in the red
channel, indicating that the lysosome polarity in HepG2 cells decreased after stimulation with DMSO. Similar
22

changes in fluorescence in 293T cells were observed. Moreover, the lysosomal polarity will change as the
consequence of lysosomal storage disorders (LSDs) resulted from defective lysosomal acid hydrolysis of
macromolecules and their consequent accumulation [43, 44]. The Peng group verified in 2018 that lysosome
storage disorders caused by high concentrations of sucrose solution can increase lysosome polarity [15]. Thus,
HepG2 cells were stimulated with sucrose (80 mM) for 10 minutes. Then, sucrose-stimulated cells were stained
with 0301 (10 µM, 10 minutes). As shown in Fig. 7, a clear fluorescence increase in the red channel can be
observed, indicating that lysosome polarity increased in cells after stimulation by sucrose. Subsequently, we
also investigated whether sucrose could cause changes in the fluorescence of probe 0301. The fluorescence
intensity of 0301 was nearly identical before and after the addition of sucrose (Fig. S9). Together with the
results of the above experiments, these data indicate that 0301 is a highly sensitive polarity-specific fluorescent
probe that can be employed to quantitatively indicate variations in lysosome polarity in live cells.
23

Fig. 7. Lysosome polarity changes stimulated by sucrose and DMSO. (a) Confocal fluorescence images of 0301-stained 293T cells which
were stimulated by sucrose (80 mM) and DMSO (10 µL) for 10 minutes; (b) Quantified relative fluorescent intensity ratio (Fred channel/Fgreen
channel) of 293T cell; (c) Confocal fluorescence image of 0301-stained HepG2 cells stimulated by sucrose (80 mM) and DMSO (10 µL) for 10
minutes; (d) Quantified relative fluorescent intensity ratio (Fred channel/Fgreen channel) of HepG2 cells. Green channel: λex = 488 nm, collected
530-570 nm; Red channel: λex = 488 nm, collected 570-650 nm. Scale bar: 25 µm. The values are the mean ± s.d. for n = 3, *p < 0.05,
**p<0.01.
4. Conclusion
In conclusion, we presented a novel design strategy for curcumin-based polarity fluorescent and
eventually designed and synthesized four polarity candidate probes by modifying curcumin derivatives. After
screening their responses to variations in polarity, 0301 and 0302 exhibited preferable fluorescence responses
to polarity, indicating that diethylamino moiety is essential to monitoring polarity. Moreover, we found
introduction of the galactose group effectively improved the water solubility and regulated the detection mode
to polarity by comparing the optical properties between 0302 and 0302D (or M1). Probes 0301 and 0302 both
showed high sensitivity to polarity without significant interferences from pH, viscosity or some other
common interfering species (metal ions, anion species and biothiols). Additionally, both probes displayed
good photostability. Due to the low cytotoxicity and lysosome-targeting ability of 0301, we successfully
monitored and imaged the lysosome polarity in different cells (cancer cells and normal cells), suggesting that
probe 0301 possessed the potential ability to discriminate between cancer cells and normal cells. Using 0301,
we obtained an exact and clear image of the variations in lysosome polarity caused by DMSO and sucrose.
Hopefully, in the future, prominent curcumin-based fluorescent probes could be further improved to detect
lysosome polarity to elucidate polarity-related biological processes.
24

Acknowledgments
This work was supported by the National Natural Science Foundation of China (81872747, 21672064,
21977082), the National Key R&D Program of China (2017YFB0202600), the Innovative Research Team of
High-level Local Universities in Shanghai, the National Special Fund for State Key Laboratory of Bioreactor
Engineering (2060204), Open Funding Project of the State Key Laboratory of Bioreactor Engineering
(2018OPEN12), and the Shanghai Sailing Program (17YF1403600).
25

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