L. Dai, M. Ren and W. Lin
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 254 (2021) 119627
1
. Introduction
cosity [30,31]. We developed a novel near-infrared probe, NI-VD,
that belongs to the classic molecular rotor probe. The classic fluo-
rophore phenolic dihydroxanthene is used as the main body of the
probe. It is easy to modify and synthesize [32]. We introduced a
methoxy electron-donating group to improve its structure-
activity relationship. Quinoline is the positioning group of mito-
chondria—it locates the negative potential on the inner mitochon-
drial membrane with a positive charge on N and also extends the
conjugate structure to obtain a larger emission wavelength [33–
35]. NI-VD has weak fluorescence in low-viscosity solutions and
strong fluorescence in high-viscosity solutions. The maximum
emission of NI-VD is 740 nm with a maximum response multiple
of 13-fold. It can sensitively monitor viscosity changes in living
cells. The background fluorescence interference during the imaging
process is minimal and benefited from a large Stokes’ shift. We also
used fluorescence lifetime imaging microscopy (FLIM) to confirm
our experimental results, fluorescence lifetime is a stable physical
property of fluorophores [36]. NI-VD can measure the kidneys of
diabetic mice and normal mice for the first time.
Cells are as precise as machines, where each organelle plays a
unique function. There are many essential factors that ensure that
normal physiological activity can be maintained in cells such as
ROS, RNS, small molecular thiols, and viscosity [1–7]. At the micro-
scopic level, viscosity is key to diffusion control and plays a signif-
icant role in the chemical behavior and biological behavior. Some
normal chemical and biological activities including signal trans-
mission, protein folding, and enzyme catalysis work on the basis
of viscosity balance. Studies have shown that abnormal activities
are usually caused by abnormal changes in viscosity in cells. Con-
sequently, viscosity is an important indicator of healthy cells.
The viscosity of cells is 1–2 cP, but it can significantly increase
in diseased cells reaching 140 cP and even more [8–11]. At the
macroscopic level, the normal activities in different organelles like
mitochondria are affected by viscosity. There can be poor organelle
performance when the viscosity is out of range. Many common dis-
eases such as diabetes, Alzheimer’s disease, and neurodegenerative
diseases all feature abnormal cell viscosity [12,13].
Mitochondria dominate many cellular processes include ATP
production, central metabolism, and apoptosis [14–16]. Some com-
mon functions of mitochondria will be inhibited by the alteration
in mitochondrial membrane fluidity including a reduction in the
electron transport chain activation, and release of cytochrome c
2
. Experimental section
2.1. Synthesis
Twisted internal charge transfer (TICT) is a classic mechanism
[
17]. Therefore, for better medical care, it is important to monitor
for designing small molecule fluorescent probes that respond to
viscosity. Molecules devised by TICT usually have ‘‘D- -A” molec-
the viscosity in the mitochondria.
p
Drop ball, capillary, and rotational viscometers are traditional
viscosity measurement methods, but these methods cannot make
measurements at the cell level or in vivo [18–20]. Fluorescent
probes offer low phototoxicity, deep penetration, and high-
resolution biological imaging. They can measure changes in the
microenvironment of subcellular regions. After a long period of
development, the molecular rotor is still an important method
for studying the viscosity of cells [21–23]. The rotation of the
molecular rotor is not inhibited in a low-viscosity environment.
Faster rotation relaxes the excitation energy with little fluores-
cence. Rotation is hindered in a high-viscosity environment such
as glycerol, thus reducing the possibility of non-radiating path-
ways: This increases the fluorescence. In recent years, many rotor
probes have emerged, and some of them have been successfully
applied to detect changes in the viscosity of cells in organisms.
However, most of the reported probes have two major shortcom-
ings: One problem is the emission wavelength of probe is too small
and not in the near-infrared region. The second is that the Stokes-
shift is not long enough to prevent an overlap in excitation light
and emission light—thus, there is autofluorescence and a high
background.
ular configuration, and a strong electron withdrawing group is
used for the acceptor. The donor was a strong electron donor.
Scheme 1 shows that the two parts of NI-VD were linked though
a flexible-conjugated linker. Phenolic dihydroxanthene with a
methoxy was the donor, and quinolone was the acceptor, mean-
while, the quaternary ammonium salt group makes NI-VD water
soluble and helps NI-VD target mitochondria. The Structural char-
acterization data, including H NMR, C NMR and HR-MS (High
Resolution Mass Spectrometry) were provided in Supporting
Information.
NI-VD exhibits weak fluorescence in low-viscosity solutions
because the solvent cannot prevent the rotor from rotating freely
through the single bond. In high-viscosity solutions, the rotation
of the rotor was restrained, and the energy tend to return to the
ground state in the form of a radiative transition; thus, it has strong
fluorescence. Furthermore, the structure of a large conjugate chain
gives NI-VD a longer emission wavelength and lower background
fluorescence.
1
13
2.2. Synthesis of the compound NI-VD
Diabetes is a keystone disease with many related diseases. Dia-
betic nephropathy (DN) is a common complication. Little is known
about the mechanism and specific function and injury in vascular
endothelial cells after diabetes induction. A close relation has been
seen between the complications of diabetes (such as vascular com-
plications) and dysfunction of mitochondria in vascular endothelial
cells [24–27]. In the kidney, the glomerular vascular endothelial
cells are in a state of diabetic hyperglycemia for a long time, and
glucose metabolic disorders will appear in cells with this situation.
More reactive oxygen species (ROS) are produced in the mitochon-
dria in this situation. These undesirable changes will lead to an
increase in the permeability of the mitochondrial outer membrane
and can even lead to glomerular endothelial cell apoptosis and tis-
sue damage [28,29]. However, the present research by fluorescence
probe that can distinguish between abnormal diabetic kidneys and
normal kidneys is still vacant.
Compounds 1 and 2 were prepared as described [32,37]. Com-
pound 1 (0.5 g) and Compound 2 (1 g) were mixed in ethanol and
added to a dry clean round bottom flask. A drop of piperidine was
carefully added. The reaction was allowed to proceed. We used TLC
to monitor the progress; during the reaction, the temperature was
held at 50 °C. After the reaction was completed, the solvent was
removed by evaporation under reduced pressure. We purified the
remaining residue using column chromatography on a silica gel.
The polarity of the solvent is ‘‘CH Cl /methanol” = 50: 1, v/v. The
2
2
1
product was a purple powder (600 mg, 38% yield). H NMR
(400 MHz, CDCl and CD OD) d 9.70 (d, 1H), 8.63 (d, 1H), 8.49 (d,
3
3
1H), 8.36 (d, 1H), 8.06 – 7.98 (m, 2H), 7.85 – 7.80 (m, 1H), 7.18 –
7.06 (m, 3H), 6.78 – 6.72 (m, 2H), 4.56 (s, 3H), 4.02 (s, 3H), 2.69
1
3
(dt, 4H), 0.89 (d, 2H). C NMR (151 MHz, CDCl3 and CD OD) d
3
161.90, 155.61, 154.14, 153.82, 145.46, 139.23, 138.44, 134.52,
Our previous studies showed that the production of reactive
oxygen species (ROS) increases with abnormal mitochondrial vis-
128.37, 127.46, 127.21, 127.12, 126.10, 125.59, 118.09, 115.36,
113.39, 112.90, 112.51, 111.03, 100.31, 55.13, 43.19, 29.18, 24.47,
2