Exploiting novel rotors with auxochromic dynamic motors for monitoring lysosomal viscosity

Article abstract of DOI:10.1016/j.dyepig.2020.108974

Organelles like lysosomes usually exhibit dynamic changes of locations, morphologies, and components, which could be reflected by the local viscosity. For monitoring lysosomal viscosity, a novel rotor P7-B was introduced with an improved auxochromic dynam

Full text of DOI:10.1016/j.dyepig.2020.108974

Journal Pre-proof
Exploiting novel rotors with auxochromic dynamic motors for monitoring lysosomal
viscosity
Jian-Fei Xu, Jin Gu, Ya-Lin Qi, Meng Zhang, Jian Chen, Dong-Dong Li, Yu-Shun
Yang, Peng-Cheng Lv, Hai-Liang Zhu
PII:
S0143-7208(20)31671-5
DOI:
https://doi.org/10.1016/j.dyepig.2020.108974
DYPI 108974
Reference:
To appear in: Dyes and Pigments
Received Date: 25 July 2020
Revised Date: 20 October 2020
Accepted Date: 27 October 2020
Please cite this article as: Xu J-F, Gu J, Qi Y-L, Zhang M, Chen J, Li D-D, Yang Y-S, Lv P-C, Zhu H-L,
Exploiting novel rotors with auxochromic dynamic motors for monitoring lysosomal viscosity, Dyes and
Pigments, https://doi.org/10.1016/j.dyepig.2020.108974.
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Jian-Fei Xu: Methodolog, Formal analysis;
Jin Gu: Resources, Investigation;
Ya-Lin Qi: Validation, Visualization;
Meng Zhang: Writing - Original Draft, Validation;
Jian Chen: Writing - Review & Editing;
Dong-Dong Li: Funding acquisition;
Yu-Shun Yang: Conceptualization;
Peng-Cheng Lv: Supervision;
Hai-Liang Zhu: Project administration.

Exploiting novel rotors with auxochromic dynamic motors for
monitoring lysosomal viscosity
a,‡
a,‡
a
a
a
b
Jian-Fei Xu, Jin Gu, Ya-Lin Qi, Meng Zhang, Jian Chen, Dong-Dong Li,
a,*
c,*
a,*
Yu-Shun Yang, Peng-Cheng Lv, and Hai-Liang Zhu
a
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing,
10023, China
2
b
College of Chemical Engineering, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, China.
The Joint Research Center of Guangzhou University and Keele Univeristy for Gene Interference and
c
Application, School of Life Science, Guangzhou University, Guangzhou, 510006, China.
*
Corresponding authors. Tel. & Fax: +86-25-89682572; E-mail: ys_yang@nju.edu.cn; lpengc8283@163.com;
zhuhl@nju.edu.cn.
‡
Both authors contributed equally to the work
For monitoring lysosomal viscosity, a novel rotor P7-B was introduced with an
improved auxochromic dynamic motor of azepanyl. All through the evaluating
processes including optical tests, wave-band optimization and intracellular
applications, P7-B exhibited comparable targeting capability and better auxochroming
potential than morpholine-containing rotors. More than offering practical lysosomal
viscosity monitoring tools or referential auxochromic dynamic motors, this work
raised significant information for transcending the structural limits in future
investigations.

Exploiting novel rotors with auxochromic dynamic motors for
monitoring lysosomal viscosity
a,‡
a,‡
a
a
a
b
Jian-Fei Xu, Jin Gu, Ya-Lin Qi, Meng Zhang, Jian Chen, Dong-Dong Li,
a,*
c,*
a,*
Yu-Shun Yang, Peng-Cheng Lv, and Hai-Liang Zhu
a
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing,
10023, China
2
b
College of Chemical Engineering, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, China.
The Joint Research Center of Guangzhou University and Keele Univeristy for Gene Interference and
c
Application, School of Life Science, Guangzhou University, Guangzhou, 510006, China.
*
Corresponding authors. Tel. & Fax: +86-25-89682572; E-mail: ys_yang@nju.edu.cn; lpengc8283@163.com;
zhuhl@nju.edu.cn.
‡
Both authors contributed equally to the work
Abstract: Organelles like lysosomes usually exhibit dynamic changes of locations,
morphologies, and components, which could be reflected by the local viscosity. For
monitoring lysosomal viscosity, a novel rotor P7-B was introduced with an improved
auxochromic dynamic motor of azepanyl. All through the evaluating processes
including optical tests, wave-band optimization and intracellular applications, P7-B
exhibited comparable targeting capability and better auxochroming potential than
morpholine-containing rotors. More than offering practical lysosomal viscosity
monitoring tools or referential auxochromic dynamic motors, this work raised
significant information for transcending the structural limits in future investigations.
Keywords: Lysosomal viscosity; Dynamic motor; Auxochroming potential; Azepanyl
rotor; Optical tests; Intracellular applications.
1

1
. Introduction
Intracellular viscosity has been regarded as a significant parameter to reflect the
micro-environment in various biological systems [1,2]. It affected the physiological
procedures by regulating the mobility of substances [3-5], and was reported to be
connected with diseases such as diabetes, infarction, and hypertension [6-9]. Rotors
for viscosity required more elaborate design because tools for macroscopic fluids
could hardly leap over the obstacle of the diversity between highly inhomogeneous
organelles including lysosome and mitochondria [10-14]. Monitoring viscosity within
certain organelles rather than the unknown intracellular area was beneficial due to the
differences between specific regions, which could provide consulting information for
other micro-environmental parameters such as pH and polarity [15-18].
Organelles like lysosomes usually exhibit dynamic changes of locations,
morphologies, and components, which leads to the corresponding causations of their
status and functions. These variations could be reflected by the local viscosity [19].
During lysosomal-related disorders, macromolecular substances accumulate in
lysosomes instead of decomposing, thus their increasing amounts or density affects
the lysosomal viscosity. Reversely, diagnosis of lysosome-related disorders could also
be hinted by real-time and in-situ monitoring of lysosomal viscosity [20,21]. Early
fluorescent viscosity-sensitive rotors failed mapping trials in organelles mainly
because of their uneven or nonspecific distributions [22-24]. From then on,
investigators have introduced targeting moiety to realize the imaging in lysosome
[
25-30] and mitochondria [31,32]. Especially for lysosome, the morpholine moiety
seemed an intrinsic component [25-29]. Although morpholine is an excellent targeting
group, its properties as an auxochromic group are mediocre, with a restricted rotating
angle [33]. Therefore, the evolution of lysosomal viscosity rotors should transcend the
structural limits to achieve more capabilities.
In this work, based on our previous work of aminoquinoline fluorophore [34,35],
we intended to improve the auxochromic dynamic motor from morpholine to a
better-integrated one with three functions - targeting, auxochroming and rotating. By
rational designing, optical evaluating, performance testing, wave-band optimization
2

and intracellular application, novel rotors have been exploited for monitoring
lysosomal viscosity (Figure 1), offering referable information for developing
micro-environmental-parameter trackers focusing on lysosome.
Figure 1. Schematic illustration of procedure for exploiting novel rotors with
auxochromic dynamic motors for monitoring lysosomal viscosity.
2
2
. Experimental
.1. The experimental materials and equipment
All chemicals purchased were used as received with no further purification.
3

Chromatographic purification was conducted through 300-400 mesh chromatography
silica gel. The NMR data were collected in DMSO-d or CDCl using Bruker
6
3
(
Rhenistetten-Forchheim, Germany) DRX-600 and Advance III HD spectrometer,
then dealt with MestReNova software. Mass spectra were acquired from an Agilent
540 UHD Accurate Mass Q-TOF LC/MS. UV-vis spectra were accomplished on
6
Shimadzu UV-2550 spectrometer. All the fluorescence measurements were recorded
on Hitachi Fluorescence Spectrophotometer F-7000. The imaging experiments were
performed using two-photon confocal fluorescent microscope (Leica TCS SP8 MP)
and analyzed with LASX software.
Scheme 1. The scheme for the general synthesis of PX series and PX-B series.
2
.2. Synthesis experiments
The general synthesis was shown in Scheme 1. First, 2-nitrobenzaldehyde (500
mg, 3.31 mmol) was dissolved in 5 mL 95% ethanol, then iron powder (1.48 mg,
2
6.4 mmol, 8 eq) and 1M HCl (200 μL) were added to the solution. The mixture was
4

stirred at 90 ℃ for 1 h. After removal of solvent under vacuum, the yellow solid
2
2
-aminobenzaldehyde (489 mg, yield 97.8%) was obtained. Second,
-aminobenzaldehyde (400 mg, 3.7 mmol) and 4'-bromoacetophenone (736 mg, 3.7
mmol) were dissolved in 5 mL ethanol, dropped with KOH aqueous solution (0.1 eq).
The mixture were refluxed at 80 ℃ for 2 h. The reaction was monitored by TLC.
After removal of solvent under vacuum, the residue was purified by column
chromatography to give the white solid Intermediate C (378 mg, yield 95.5%). From
2
-aminobenzaldehyde or Intermediate C, PX series were synthesized using various
nitrogen-containing compounds.
PN: 2-aminobenzaldehyde
'-dimethylaminoacetophenone (921 mg, 3.7 mmol) were dissolved in 5 mL ethanol,
dropped with KOH aqueous solution (0.1 eq). The mixture were refluxed at 80 ℃ for
h. The reaction was monitored by TLC. After removal of solvent under vacuum, the
(400
mg,
3.7
mmol)
and
4
2
residue was purified by column chromatography to give the white solid PN (380 mg,
1
yield 95%). H NMR (600 MHz, DMSO-d ) δ 8.32 (d, J = 8.7 Hz, 1H), 8.16 (d, J =
6
9.0 Hz, 2H), 8.05 (d, J = 8.7 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 7.2 Hz,
1H), 7.72 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.51 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H),
6.91-6.80 (m, 2H), 3.01 (s, 6H).
P4: Intermediate C (200mg, 0.699 mmol) was dissolved in 10 mL toluene, then
palladium acetate (9.4 mg, 0.042 mmol , 0.06 eq), cesium carbonate (273 mg, 0.84
mmol , 1.2 eq), BINAP (26.12 mg, 0.042 mmol, 0.06 eq) were added to the solution.
After adding azetidine (461 μL, 20 eq), the mixture were stirred at 100 ℃ for 8 h
under Argon atmosphere. The reaction was monitored by TLC. The solution was
filtered with diatomite, and concentrated by rotary evaporator. The residue was
purified by column chromatography to give the yellow solid P4 (30 mg, yield 15%).
1
1
H NMR (600 MHz, DMSO-d ) δ H NMR (400 MHz, DMSO-d ) δ 8.33 (d, J = 8.7
6
6
Hz, 1H), 8.15 (d, J = 8.6 Hz, 2H), 8.04 (d, J = 8.7 Hz, 1H), 7.98 (d, J = 8.5 Hz, 1H),
7
.92 (d, J = 7.9 Hz, 1H), 7.72 (t, J = 7.2 Hz, 1H), 7.51 (t, J = 7.3 Hz, 1H), 6.54 (d, J =
.6 Hz, 2H), 3.92 (t, J = 7.3 Hz, 4H), 2.41-2.32 (m, 2H).
8
P5: With the same method of synthesizing P4, azetidine was changed into
5

1
pyrrolidine (936 μL, 20 eq) to obtain the yellow solid P5 (18 mg, yield 9%). H NMR
(
600 MHz, DMSO-d ) δ 8.30 (d, J = 8.7 Hz, 1H), 8.16 (d, J = 8.8 Hz, 2H), 8.03 (d, J
6
=
8.7 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 7.4 Hz, 1H), 7.71 (ddd, J = 8.3,
6
.9, 1.4 Hz, 1H), 7.50 (t, J = 7.4 Hz, 1H), 6.68 (d, J = 8.8 Hz, 2H), 2.00 (t, J = 6.6 Hz,
4
H).
P6: With the same method of synthesizing P4, azetidine was changed into
1
piperidine (1188 μL, 20 eq) to obtain the yellow solid P6 (28 mg, yield 14%). H
NMR (600 MHz, DMSO-d ) δ 8.34 (d, J = 8.6 Hz, 1H), 8.16 (d, J = 8.9 Hz, 2H), 8.06
6
(d, J = 8.7 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.93 (d, J = 7.3 Hz, 1H), 7.73 (t, J = 7.6
Hz, 1H), 7.53 (t, J = 7.4 Hz, 1H), 7.06 (d, J = 9.0 Hz, 2H), 3.33-3.27 (m, 4H),
1
.68-1.55 (m, 6H).
P7: With the same method of synthesizing P4, azetidine was changed into
hexamethyleneimine (1384 μL, 20 eq) to obtain the yellow solid P7 (90 mg, yield
1
4
2
5%). H NMR (600 MHz, DMSO-d ) δ 8.30 (d, J = 8.7 Hz, 1H), 8.12 (d, J = 9.0 Hz,
6
H), 8.01 (d, J = 8.7 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 7.5 Hz, 1H), 7.71
(ddd, J = 8.3, 6.9, 1.4 Hz, 1H), 7.53-7.47 (m, 1H), 6.82 (d, J = 9.0 Hz, 2H), 3.54 (t, J
=
6.0 Hz, 4H), 1.76 (s, 4H), 1.56-1.44 (m, 4H).
PM: With the same method of synthesizing P4, azetidine was changed into
1
morpholine (1131 μL, 20 eq) to obtain the yellow solid PM (59 mg, yield 29.5%). H
NMR (400 MHz, DMSO-d ) δ 8.37 (d, J = 8.7 Hz, 1H), 8.20 (d, J = 8.8 Hz, 2H), 8.09
6
(d, J = 8.7 Hz, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.95 (d, J = 7.9 Hz, 1H), 7.74 (t, J = 7.3
Hz, 1H), 7.54 (t, J = 7.3 Hz, 1H), 7.10 (d, J = 8.8 Hz, 2H), 3.81-3.75 (m, 4H),
3
.28-3.22 (m, 4H).
PX-B series were synthesized using PX series as upstream material.
PN-B: Compound PN (300 mg, 1.21 mmol) was dissolved in 10 mL DCM.
DIPEA (150 mg, 1.21 mmol, 1 eq) was added to the solution at 0 ℃ under Argon
atmosphere, then BBr was added quickly. The reaction was monitored by TLC. After
3
removal of solvent under vacuum, the residue was purified by column
1
chromatography to give the brick red solid PN-B (11 mg, yield 3.6%). H NMR (600
MHz, DMSO-d ) δ 8.78 (t, J = 8.4 Hz, 2H), 8.21 (d, J = 8.8 Hz, 1H), 8.15 (dd, J = 8.0,
6
6

1
.1 Hz, 1H), 8.02 (ddd, J = 8.8, 7.1, 1.4 Hz, 1H), 7.97 (d, J = 8.6 Hz, 1H), 7.69-7.65
(m, 1H), 6.90 (d, J = 2.0 Hz, 1H), 6.68 (dd, J = 8.6, 2.2 Hz, 1H), 3.67 (t, J = 6.6 Hz,
1
3
2
H), 3.46 (s, 2H), 2.16-2.10 (m, 2H). C NMR (101 MHz, DMSO) δ 156.73, 151.76,
1
48.15, 137.02, 130.06, 129.09, 128.59, 128.15, 126.84, 126.33, 125.85, 118.34,
+
1
12.40. HRMS (ESI-TOF) m/z: [M+H] Calcd. for C H BBr N 416.9773, Found
1
7
16
2
2
4
16.9768.
P7-B: Compound P7 (300 mg, 0.99 mmol) was dissolved in 10 mL DCM.
DIPEA (123 mg, 0.99 mmol, 1 eq) was added to the solution at 0 ℃ under Argon
atmosphere, then BBr was added quickly. The reaction was monitored by TLC. After
3
removal of solvent under vacuum, the residue was purified by column
1
chromatography to give the brick red solid P7-B (16 mg, yield 5.3%). H NMR (600
MHz, CDCl ) δ 9.00 (d, J = 8.8 Hz, 1H), 8.22 (d, J = 8.7 Hz, 1H), 7.83 (t, J = 7.9 Hz,
3
1
H), 7.77 (d, J = 7.9 Hz, 1H), 7.62 (d, J = 8.7 Hz, 1H), 7.58 (d, J = 8.7 Hz, 1H), 7.47
(t, J = 7.5 Hz, 1H), 6.74 (s, 1H), 3.60-3.52 (m, 4H), 1.83 (s, 4H), 1.62-1.47 (m, 5H).
1
3
C NMR (101 MHz, DMSO) δ 156.79, 150.03, 148.17, 136.94, 130.02, 128.94,
1
28.13, 126.76, 125.71, 125.41, 118.24, 111.46, 49.28, 27.30, 26.79. HRMS
+
(
ESI-TOF) m/z: [M+H] Calcd. for C H BBr N 471.0243, Found 471.0215.
2
1
22
2
2
2
.3. Determination of the fluorescence quantum yield
The participation ratio method was used in estimating the fluorescence quantum
yield Φ with the ethanol solution of rhodamine B as control. The fluorescence
u
quantum yields were calculated as 0.25 for PN-B and 0.29 for P7-B.
2
.4. Ethic statement
All cell lines were cultured and passaged in accordance with the guidelines
established by the National Science Council of Republic China. The primary cell lines
were purchased from ATCC. All experimental protocols were approved by Academic
Committee of Nanjing University.
2
.5. Cell viability assay
HeLa (human cervical cancer cell line) cells were cultured in 96-well plates at a
5
density of 5 × 10 cells and different concentrations of tested compound were added.
Cytotoxicity evaluation was performed after culturing for 48 h at 37 ℃ in a 5% CO2
7

atmosphere. 4 h before the end of the incubation, 20 µL MTT reagent (5 mg/mL) was
added to each well. Then the plate was centrifuged at 1200 rpm for 5 min. The
supernatant were removed and 150 µL DMSO was added. The absorbance was
measured at a wavelength of 490 nm on an ELISA microplate reader. The experiment
was conducted in triplicate.
2
.6. Confocal microscopy experiments
HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM)
supplemented with 10% FBS (Fetal Bovine Serum), 100 U/mL penicillin and 100
µg/mL streptomycin in a 5% CO , water saturated incubator at 37 ℃. In the
2
co-localization experiment, the prepared cells were incubated for 30 min with
Lyso-Tracker Red, washed by PBS carefully for three times, and then incubated for
another 30 min with PN-B or P7-B. The signals of Lyso-Tracker Red, Mitotracker
and ER-tracker were presented in the red channel (620-700 nm) while that of PN-B
and P7-B were both exhibited in the green channel (500-570 nm). In the oxidative
stress experiment, the prepared cells were incubated for 30 min with buffer or 500 μM
H O , washed by PBS carefully for three times, and then incubated for another 30 min
2
2
with PN-B or P7-B. The signals were collected in the green channel (500-570 nm).
3
3
. Results and discussion
.1. Synthesis
With familiar aminoquinoline fluorophore, we focused on the auxochromic
dynamic motor here to synthesize PX series and PX-B series. Besides a novel sensor
PM who still employed morpholine, other sensors were designed with alternative
motors as shown in Figure 1. Subsequent red-shift optimized sensors PN-B and P7-B
were selected due to the general optical performances of PX series. All the mentioned
compounds were synthesized as illustrated in Scheme 1 and confirmed by satisfactory
spectroscopic data (Figure S2-11, SI).
3
.2. General fluorescent evaluation for PX series
Since various auxochromic dynamic motors had been installed in PX series, the
initial screening was conducted by evaluating the Stoke's Shift in different solutions
(Table 1). Larger Stoke's Shift is beneficial for reducing background interferences and
8

improving the sensitivity. In the orthogonal comparison, weighting algorithm was
conducted to gain the top 3 choices in each solution except water (P7, P5 and P6 for
n-Hexane; P7, P5 and PN for DMSO; P7, P5 and PN for DCM; P5, P7 and PN for
MeCN; P7, PN and P5 for EtOH). Therefore, PN, P5 and P7 were potential
candidates for this parameter. A further glance at the fluorescence intensity of PX
series (Figure 2) offered a more rigid restriction. Only PN and P7 would be qualified
for the subsequent modification of generating PN-B and P7-B because P5 did not
show enough intensity in DMSO or MeCN, two major solutions for biological
imaging with a certain viscosity.
Table 1. The Stoke's Shift values of PX series in various solutions.
Rotors
Ex(nm)
n-Hexane
375
392
17
DMSO
400
501
101
416
500
84
DCM
399
446
47
MeCN
394
494
100
410
492
82
EtOH
398
491
93
H O
2
367
419
52
PN
P4
Em(nm)
Δλ(nm)
Ex(nm)
Em(nm)
Δλ(nm)
Ex(nm)
Em(nm)
Δλ(nm)
Ex(nm)
Em(nm)
Δλ(nm)
Ex(nm)
Em(nm)
Δλ(nm)
Ex(nm)
Em(nm)
Δλ(nm)
381
391
10
415
442
27
414
488
74
330
418
88
366
393
27
385
492
107
419
496
77
376
452
76
374
485
111
414
489
75
367
459
92
357
418
61
P5
366
392
26
415
454
39
415
486
71
306
418
112
352
419
94
P6
357
392
51
390
498
108
408
498
90
376
452
76
389
492
103
400
489
98
389
491
102
405
486
81
P7
375
387
12
405
451
46
322
419
97
PM
9

Figure 2. The fluorescence intensity of PX series in various solutions (n-Hexane,
DMSO, DCM, MeCN, EtOH and H O).
2
PN-B and P7-B were modified by fixing of quinoline and benzene with boride. This
optimization caused the enhancement of conjugation and corresponding bathochromic
shift, limiting the auxochromic dynamic motors as the only rotating units for sensing
viscosity. The fluorescent quantum yields of PN-B and P7-B were determined as 0.25
and 0.29 by participation ratio method, respectively. Although in vitro testing was not
only affected by the viscosity but also influenced by the types of solutions, the
dynamic viscosity changes could still be characterized in a two-solution-mixed system.
In Figure 3, both PN-B and P7-B were tested in Isopropanol/Water and
Glycerol/Water systems. In both systems, along with the increasing of the viscosity,
the fluorescence intensity enhanced gradually. Compared with the 10% organic
solvent status, the 100% status indicated a 500-fold enhancement of fluorescence
intensity in Isopropanol/Water system and a 50-fold enhancement in Glycerol/Water
system, with a peak at 570 nm and 580 nm for PN-B and P7-B, respectively. In
addition, to monitor the variations of viscosity in complex microenvironments, the
fluorescence response of probe P7-B to the micro-environmental parameters such as
pH and polarity were investigated. Accordingly, a relatively wide range of pH
applications was observed (Figure S12). Therefore, the increase of viscosity could
impede the rotating of our rotors and exhibit the real-time situation accordingly.
1
0

Figure 3. The fluorescence intensity changes of PN-B and P7-B in different solvent
systems. (a) PN-B in Isopropanol/Water system (λex= 367 nm); (b) PN-B in
Glycerol/Water system (λex= 367 nm); (c) P7-B in Isopropanol/Water system (λex=
3
52 nm); (d) P7-B in Glycerol/Water system (λex= 352 nm).
3
.3. Biological imaging
Before imaging, the cytotoxicity of PN-B and P7-B were examined in HeLa
cells by MTT method (Figure S1, SI). Even at the concentration higher (20 μM) than
the working one (10 μM), almost 80% of cells remained alive, which guaranteed the
basic safety of both rotors. Then we used 10 μM rotors in PBS buffer (10 mM, pH 7.4,
5
% DMSO) to conduct the co-localization experiment. In this stage, we came to the
topic of lysosomal targeting. Since neither the auxochromic dynamic motors of PN-B
nor P7-B was morpholine, if they succeeded here, the major purpose of this work
would be achieved. The commercial lysosomal targeting tag Lyso-Tracker Red was
employed. The HeLa cells were incubated with Lyso-Tracker Red for 30 min, washed
three times by PBS buffer and further incubated with PN-B (Figure 4a) or P7-B
11

(Figure 4b) for 30 min, respectively. The signals of Lyso-Tracker Red were presented
in the red channel (Figure 5a and 5b) while that of PN-B or P7-B were exhibited in
the green channel. Analyzed by LASX software, Pearson Correlation Coefficient
between Lyso-Tracker Red and PN-B was determined as 0.82 (Figure 4c) while that
between Lyso-Tracker Red and P7-B was an even higher 0.90 (Figure 4d). However,
the average Pearson's co-localization coefficients of probe P7-B with endoplasmic
reticulum (ER) and mitochondria are 0.54552, 0.38593, respectively, the fluorescence
colocalization experiment effects are poor (Figure S13). This result verified the
capability of both rotors for monitoring lysosomal viscosity. Moreover, P7-B was
slightly more potential due to its targeting, auxochroming and rotating advantages.
Figure 4. The co-localization experiment of PN-B (a & c) or P7-B (b & d) with
commercial lysosomal targeting tag Lyso-Tracker Red. The signals of Lyso-Tracker
Red were presented in the red channel (620-700 nm) while that of PN-B or P7-B were
exhibited in the green channel (500-570 nm). Images for PN-B/P7-B and
1
2

Lyso-Tracker Red were then recorded using excitation wavelengths of 405 and 594
nm, respectively.
Subsequently, the rotors PN-B and P7-B were applied in sensing the dynamic
variation of intracellular viscosity in an oxidative stress event. The oxidative situation
was induced by H O . After the incubation with buffer or 500 μM H O for 30 min,
2
2
2
2
further treatment with the rotors led to different fluorescence intensity in the green
channel. When experiencing the cellular disorder caused by oxidative stress, the
intracellular viscosity increased, which was reflected by the enhancement of
fluorescent intensity by PN-B (Figure 5b) and P7-B (Figure 5d) in comparison of
their normal status (Figure 5a & 5c). Still, P7-B was slightly more potential due to its
stronger intensity in this experiment. Thus, we might infer that azepanyl was a
practical auxochromic dynamic motor other than morpholine, suggesting comparable
targeting capability but better auxochroming potential.
Figure 5. The confocal images of HeLa cells in oxidative stress event with PN-B (a &
b) or P7-B (c & d). (a) Buffer, 30 min; PN-B, 30 min; (b) 500 μM H O , 30 min;
2
2
PN-B, 30 min; (c) Buffer, 30 min; P7-B, 30 min; (d) 500 μM H O , 30 min; P7-B, 30
2
2
1
3

min. Green channel: 500-570 nm. Images for PN-B/P7-B were recorded using
excitation wavelengths of 405 nm.
4
. Conclusion
In conclusion, we selected the quinoline fluorophore then improve the
auxochromic dynamic motor from morpholine to a better integrated one with three
functions, including targeting, auxochroming and rotating. After evaluating the optical
properties of PX series, two wave-band optimized rotors, PN-B and P7-B, were
obtained by introducing a red shift of emission. Both rotors indicated applicable
capability for sensing the viscosity in vitro. The co-localization with Lyso-Tracker
Red convinced the capability of both rotors for monitoring viscosity in lysosome.
They could also be applied in sensing the dynamic variation of intracellular viscosity
in an oxidative stress event. Moreover, since P7-B exhibited attractive performance in
each stage, we could infer that azepanyl was a practical auxochromic dynamic motor
other than morpholine, suggesting comparable targeting capability but better
auxochroming potential. More than offering practical lysosomal viscosity monitoring
tools or referential auxochromic dynamic motors, this work raised significant
information for transcending the structural limits, which might encourage the
investigators when facing scientific joseki or dilemma.
Acknowledgments
This work was supported by the Natural Science Foundation of Jiangsu Higher
Education Institutions, China (No. 18KJB350004), and the Project for Young
Teachers of Nanjing Forestry University, China (No. CX2017005).
Conflicts of interest
There are no conflicts to declare.
Supplementary data
Supplementary data associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.dyepig.2020.XX.XXX.
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>
Introducing a novel rotor P7-B for monitoring lysosomal viscosity with better
auxochroming potential
Establishing the exploiting processes including optical tests, wave-band
optimization and intracellular applications
Originally indicating that azepanyl could be an practical choice other than
morpholine for developing lysosomal targeting tools
>
>

Here we claim the major novelty of this submission.
To our knowledge, monitoring viscosity within lysosome was essential for reflect
its dynamic status and functions. The morpholine moiety seemed an intrinsic
component for lysosome-targeting. This work exploited novel rotors with
auxochromic dynamic motors, and reported azepanyl as a practical choice with
better potential for the first time. It could offer referable information for developing
micro-environmental-parameter trackers focusing on lysosome.