Near-Infrared Fluorescent Probe with Remarkable Large Stokes Shift and Favorable Water Solubility for Real-Time Tracking Leucine Aminopeptidase in Living Cells and in Vivo

Article abstract of DOI:10.1021/acs.analchem.7b03332

Leucine aminopeptidase (LAP) is a kind of proteolytic enzymes and associated closely with pathogenesis of cancer and liver injury. Accurate detection of LAP activity with high sensitivity and selectivity is imperative to detect its distribution and dynamic changes for understanding LAP's function and early diagnosing the disease states. However, fluorescent detection of LAP in living systems is challenging. To date, rarely fluorescent probes have been reported for imaging LAP in vivo. In this study, a novel probe (TMN-Leu) was developed by conjugating a near-infrared dicyanoisophorone derivative fluorophore with LAP activatable l-leucine amide moiety for the first time. TMN-Leu featured large Stokes shift (198 nm), favorable water solubility, ultrasensitive sensitivity (detection limit of ～0.38 ng/mL), good specificity, excellent cell membrane permeability, low toxicity, and a prominent near-infrared emission (658 nm) in response to LAP. TMN-Leu has been successfully applied to track LAP of cancer cells and normal cells, monitor LAP changes in different disease models, and rapidly evaluate LAP inhibitor in cell-based assay. Notably, this probe firstly revealed that HCT116 cells with higher LAP activity were more invasive than LAP siRNA transfected HCT116 cells, suggesting that LAP might serve as an indicator reflecting the intrinsic invasion ability of cancer cells. Finally, TMN-Leu was also employed for in vivo real-time imaging LAP in living tumor-bearing nude mice with low background interference. All together, our probe possesses potential value as a promising tool for diagnostic application, cell-based screening inhibitors and in vivo real-time tracking enzymatic activity in preclinical applications.

Full text of DOI:10.1021/acs.analchem.7b03332

Subscriber access provided by University of Florida | Smathers Libraries
Article
Near-infrared Fluorescent Probe with Remarkable Large
Stokes Shift and Favorable Water Solubility for Real-time
Tracking Leucine Aminopeptidase In Living Cells and In Vivo
Wenda Zhang, Feiyan Liu, Chao Zhang, Jian-Guang Luo, Jun Luo, Wenying Yu, and Lingyi Kong
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03332 • Publication Date (Web): 19 Oct 2017
Downloaded from http://pubs.acs.org on October 19, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth
Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
Analytical Chemistry
1
2
3
4
5
6
7
8
9
Near-infrared Fluorescent Probe with Remarkable Large Stokes
Shift and Favorable Water Solubility for Real-time Tracking Leu-
cine Aminopeptidase In Living Cells and In Vivo
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Wenda Zhang, Feiyan Liu, Chao Zhang, JianꢀGuang Luo, Jun Luo, Wenying Yu*, and Lingyi Kong*
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China
Abstract: Leucine aminopeptidase (LAP) is a kind of proteolytic enzymes and associated closely with pathogenesis of cancer and
liver injury. Accurate detection of LAP activity with high sensitivity and selectivity is imperative to detect its distribution and dyꢀ
namic changes for understanding LAP’s function and early diagnosing the disease states. However, fluorescent detection of LAP in
living systems is challenging. To data, rarely fluorescent probes have been reported for imaging LAP in vivo. In this study, a novel
probe (TMN-Leu) was developed by conjugating a nearꢀinfrared dicyanoisophorone derivative fluorophore with LAP activatable
Lꢀleucine amide moiety for the first time. TMN-Leu featured large Stokes shift (198 nm), favorable water solubility, ultrasensitive
sensitivity (detection limit of ~0.38 ng/mL), good specificity, excellent cell membrane permeability, low toxicity and a prominent
nearꢀinfrared emission (658 nm) in response to LAP. TMN-Leu has been successfully applied to track LAP of cancer cells and
normal cells, monitor LAP changes in different disease models and rapidly evaluate LAP inhibitor in cellꢀbased assay. Notably, this
probe firstly revealed that HCT116 cells with higher LAP activity were more invasive than LAP siRNA transfected HCT116 cells,
suggesting that LAP might serve as an indicator reflecting the intrinsic invasion ability of cancer cells. Finally, TMN-Leu was also
employed for in vivo realꢀtime imaging LAP in living tumorꢀbearing nude mice with low background interference. All together, our
probe possesses potential value as a promising tool for diagnostic application, cellꢀbased screening inhibitors and in vivo realꢀtime
tracking enzymatic activity in preclinical applications.
19ꢀ22
INTRODUCTION
cal diagnosis.
For detection of LAP activity in living cells,
fluorescent probes have been attracted much more attention as
well. However, it is still challenging to monitor the trace of the
intracellular LAP with existing LAP probes because of the
insufficient sensitivity (detection limit > 1 ng/mL), and some
of the probes cannot be applied in living systems due to the
short emission wavelengths and short Stokes Shift (Table S1).
Leucine aminopeptidase (LAP) is one of the important proteoꢀ
lytic enzymes. It belongs to the M1 and M17 peptidase famiꢀ
1
lies, which can cleave Nꢀterminal leucine residues from subꢀ
2
strates. Importantly, LAP is involved in various physiological
and biological processes ranging from tumor cell invasion,
3ꢀ5
9, 17, 23ꢀ25
proliferation, drug resistance, angiogenesis to liver injury.
To data, only one LAP probe was used for in vivo
Therefore, in situ dynamic monitoring and indentifying intraꢀ
cellular LAP is imperative for LAPꢀrelated pathophysiology
elucidation and disease diagnosis.
imaging, but its relatively small Stokes Shift limited further
2
5
biological application. The NIR fluorphores have been paid
increasing attention for in vivo imaging application with
avoided interference from autoꢀfluorescence of indigenous
biomolecules and higher penetrability to biological systems.
While the large emission wavelength of NIR probes always
with conjugated system of polycyclic aromatic rings leads to
poor water solubility, and the high proportion of dimethyl
sulphoxide or other organic solvent in test system may cause
enzyme degeneration. The aboveꢀmentioned concerns encourꢀ
aged us to develop novel NIR fluorescent LAP probes with
large Stokes Shift, high sensitivity and good water solubility.
In this work, a novel NIR emission fluorescent probe TMN-
Leu with large Stokes Shift and favorable water solubility was
successfully developed. In our design, the Lꢀleucine amide
Molecular bioimaging of enzyme activity in vitro and in vi-
vo has been emerging as a powerful strategy for accurate disꢀ
6ꢀ15
ease diagnostics.
The development of “smart” noninvasive
imaging probes for the detection of specific enzyme activity in
vivo is urgently required for cancer diagnosis and related disꢀ
1
6ꢀ18
ease detection.
However, the complexity of dynamic living
system makes it difficult to trace and visualize enzyme activity
in vivo. In past years, fluorescent spectroscopy, a noninvasive
imaging technique, has increasingly attracted interest attributꢀ
ed to its excellent advantages of high sensitivity, good selecꢀ
tivity, operation simplicity and in situ response. It has been
applied in many fields such as biological monitoring and cliniꢀ
ACS Paragon Plus Environment

Analytical Chemistry
Page 2 of 8
moiety was chosen as the enzyme active trigger and the dicyꢀ
anoisophorone group was utilized as the NIR chromophore
due to its advantages of excellent membrane permeability,
applicable fluorescence and especially the NIR emission
wavelength. Which in turn allows for in vivo realꢀtime imagꢀ
ing of LAP activity in colorectal tumorꢀbearing nude mice.
The enzymeꢀtriggered amide moiety of TMN-Leu was
cleaved upon interaction with a suitable LAP, resulting in a
distinct turnꢀon broad emission fluorescence signal at the NIR
region. TMN-Leu is a high sensitivity and selectivity LAP
probe with excellent features such as low cytotoxicity, lightꢀup
NIR emission, ultrasensitive response, large Stokes shift, faꢀ
vorable water solubility, realꢀtime and high signalꢀtoꢀnoise
ratio in bioimaging of trace LAP in living cells and in vivo. To
the best of our knowledge, the NIR lightꢀup TMN-Leu is the
first mitochondriaꢀtargetable fluorescence probe for realꢀtime
in vivo tracking of LAP activity in tumorꢀbearing nude mice.
EXPERIMENTAL SECTION
with 10% fetal bovine serum at 37 °C in a 5% CO /95% air
incubator. The cytotoxicity of the probe to HCT116 cells and
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
HepG2 cells was evaluated through MTT assays. The cells
3
were initially seeded into 96ꢀwell plate at a density of 6×10
cells/well, and various concentrations (0, 2.5, 5.0, 10, 20 ꢁM)
of the probe in 200 ꢁL DMEM mediums were added into each
well. After incubation at 37°C in a 5% CO /95% atmosphere
2
for 24 h. 0.02 mL of the MTT (0.5 mg/mL) were diluted in the
plates and followed by culture for another 4 h. After treatment,
the formed formazan were dissolved after placing 150 ꢁL
DMSO in the wells. Then, the cell viability rate was calculated
by measuring the absorbance values at 570 nm with a referꢀ
ence wavelength at 630 nm by ELISA reader (Spectra Max
Plus 384, Molecular Devices, Sunnyvale, CA).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Fluorescence Microscopy Imaging of LAP in Cells. Iniꢀ
tially, cells were seeded into a 96ꢀwell plate and cultured in
DMEM medium supplemented with 10% FBS in an atmosꢀ
Materials and Methods. Unless special stated, all chemiꢀ
cals and solvents including 4ꢀnitrobenzaldehyde, petroleum
ether, isophorone, acetonitrile, dichloromethane, malonitrile,
ethyl acetate, was supplied by Sinopharm chemical reagent
Co. Ltd., Nitroreductase, GGT, Monoamine oxidase A (MAOꢀ
A) and Leucine aminopeptidase (LAP) were obtained from
SigmaꢀAldrich Co. Ltd.
phere of 5% CO at 37°C. Before imaging, 100 ꢁL of media
2
containing 10 ꢁM of TMN-Leu was added to the wells and
incubated. After treatment, the images of cells were taken
under an ImageXpress Micro Confocal analysis.
For Cisplatin stimulation, a concentration of Cisplatin (2
mg/L) solution was added to the 96ꢀwell plates containing
adherent cells in DMEM medium contained with 10% (v/v)
1
H NMR spectra were measured using a Bruker spectromeꢀ
FBS at 37°C in a humidified 5% CO
were incubated for 12 h.
2
incubator, and the cells
ter (500 MHz) and referenced to TMS. HRMS analysis was
performed on a Mariner ESIꢀTOF spectrometer. All pH measꢀ
urements were measured with a sartorius basic pHꢀmeter PBꢀ
For Ace (acetamidophenol) stimulation, an appropriate conꢀ
centration of Ace solution was added to the 96ꢀwell plates
containing adherent cells in DMEM supplemented with 10%
1
0. HPLC analysis was obtained on an Agilent 1100 series.
The UVꢀvisible absorption spectra were performed on a Shiꢀ
madzu UVꢀ1700 spectrometer and the fluorescence spectra
were performed on a Hitachi Fꢀ7000 luminescence spectromeꢀ
ter respectively or MultiꢀMode Detection Platform. Cell imagꢀ
ing was observed under an ImageXpress Micro Confocal analꢀ
ysis. In vivo imaging was obtained using a CRI Maestro small
animal in vivo imaging system.
(v/v) FBS at 37°C in a humidified 5% CO incubator, and the
2
cells were incubated for 48 h.
For Bestatin inhibition, Bestatin (100 ꢁM) solution was preꢀ
treated to the 96ꢀwell plates containing adherent cells in
DMEM supplemented with 10% (v/v) FBS at 37°C in a huꢀ
midified 5% CO incubator before the probe was added, and
2
the cells were incubated for 1 h.
Synthesis. LAP activatable NIR fluorescence imaging
probe TMN-Leu was synthesized according to the procedure
in Scheme S1, and its structure characterization were deꢀ
scribed in the Supporting Information.
Evaluation of Inhibition Efficacy of Bestatin in Living
Cells. To evaluate the inhibitory efficacy of Bestatin towards
LAP in cellꢀbased assay, HCT116 cells were firstly treated
with Bestatin with concentration ranges from 0ꢀ100 ꢁΜ for 1
h. Then, the medium was removed and the cells were washed
with PBS for three times. After that, cells were incubated with
TMN-Leu (10 ꢁM, 100 ꢁL) for another 65 min. The fluoresꢀ
cence images were taken on an an ImageXpress Micro Confoꢀ
cal analysis without washing operation. Each cell was taken
as a region of interest, and fluorescence intensities were evaluꢀ
ated and averaged (Fluorescence intensities were measured in
General Procedure to Monitor LAP Level in Vitro. All
spectrum measurements were performed in PBS buffer (10
mM, pH 7.4) at 37 °C. The stock solution of probe TMN-Leu
(10 mM) was prepared in DMSO. Various physiologically
2
+
3+
2+
2+
important species (Ca , Fe , Mg , Mn , H O , GSH, Cys,
2
2
Hcy, MAOꢀA, GGT, Nitroreductase) were prepared in doubleꢀ
distilled water. The test solution was prepared by mixing the
calculated amount of probe into PBS to obtain a final concenꢀ
tration of 10 ꢁM. LAP and other analytes were also dissolved
in test solution with an appropriate concentration. The UVꢀvis
absorption spectra data was recorded from 300 to 550 nm, and
the fluoresecence emission spectra was collected in the range
from 550 to 750 nm (λ = 460 nm, λ = 658 nm, slit widths: 5
3
regions in each well).
4
Trans-well Invasive Assays. Cells were seeded at 2 x 10
cells per well (0.2 mL) and allowed to grow for 24 h. After
being fixed in 4% of paraformaldehyde for 10 min. Crystal
violet solution (Sigma, St. Louis, MO, USA) was used to stain
the colonies for 4 h. The migration of cells was visualized at
x200 magnification using a Leica Microscope.
ex
em
nm/5 nm) after incubation for 70 min.
Cell Culture and Cytotoxicity Assay. HCT116 cells, L02
cells, MCFꢀ10A cells, HepG2 cells and MDAꢀMBꢀ231 cells
were provided by Cell Bank of Shanghai Institute of Biochemꢀ
istry and Cell Biology, Chinese Academy of Sciences (Shangꢀ
hai, China), and were grown in DMEM medium supplemented
Fluorescence Imaging and Colocalization Studies. Initialꢀ
ly, cells were seeded into a 96ꢀwell plate and cultured in
DMEM medium supplemented with 10% FBS in an atmosꢀ
ACS Paragon Plus Environment

Page 3 of 8
Analytical Chemistry
phere of 5% CO at 37 °C. After 24 h, 100 ꢁL of fresh media
containing 10 ꢁM of TMN-Leu was added to the wells and
cence intensity increase (Figure 1C), indicating that the enꢀ
zymeꢀtriggered cleavage reaction led to the release of free
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
incubated for 65 min. After treatment, the medium was reꢀ
moved, and the cells were washed with PBS for three times.
Cells were then incubated with 1 ꢁM MitoꢀTracker Green or 1
ꢁM LysoꢀTracker Green for another 30 min. After being fixed
in 4% of paraformaldehyde for 10 min. The images of cells
were taken under an ImageXpress Micro Confocal analysis.
TMN-NH . Together, this probe could be applied as a “turnꢀ
2
on” fluorescent sensor which possessed a sensitive response to
LAP. More importantly, the longꢀwavelength emission of the
probe toward LAP with an obvious large Stokes shift (198 nm)
was highly favorable for bioimaging analysis and this effiꢀ
ciently decreased the measuring error induced by the excitaꢀ
tion light and scattered light.
In vivo Imaging of LAP Activity in Tumor-bearing Nude
Mice. All Animal procedures were performed in accordance
with the requirements of the Animal Ethics Committee of
China Pharmaceutical University, and the guidelines of the
National Institutes of Health. All animal experiments were
approved by the Animal Ethics Committee of China Pharmaꢀ
ceutical University.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
For in vivo imaging, 5 weeks old BALB/c nude mice were
selected and inoculated with HCT116 cells on the auxiliary
6
region by injecting 3×10 cells subcutaneously to establish
tumor model. Tumors were allowed to grow to 8ꢀ12 mm in
diameter and then used for experiment. To restrain the endogꢀ
enous LAP activity, the tumor region was given a subcutaneꢀ
ous injection with or without Bestatin (100 ꢁM, 50 ꢁL) and
incubate for 1 h, subsequently, the probe TMN-Leu (50 ꢁM,
5
0 ꢁL) was injected subcutaneously into tumor region of the
tumorꢀbearing nude mice. The in vivo imaging was obtained
by using a CRI Maestro small animal in vivo imaging system,
with excitation of red filter (460 nm~560 nm).
Figure 1. (A) Proposed sensing mechanism of TMN-Leu for
LAP enzymatic activation. (B) Absorption spectra of TMN-Leu
RESULTS AND DISCUSSION
(10 ꢁΜ) before and after incubation with LAP (165 ng/mL) in
Design and Synthesis. The NIR fluorphores have been paid
increasing attention for in vivo imaging application with
avoided interference from autoꢀfluorescence of indigenous
biomolecules. Moreover, due to their higher penetrability to
biological samples compared with the shorter wavelength, the
dicyanoisophorone derivatives were used as an NIRꢀactive
fluorophores to detect biomarker for disease diagnosis and
pathophysiology elucidation. Here, we envisioned that the
merge of the Lꢀleucine moiety and a fluorophore TMN-NH2
could quench the emission intensity of the probe by the strong
electronꢀwithdrawing amide bond. After treated with LAP, the
PBS buffer (10 mM, pH 7.4, 0.1% DMSO). (C) Fluorescence
response of 10 ꢁΜ TMN-Leu (λex = 460 nm, λem = 658 nm) after
incubation with various concentrations of LAP (0ꢀ330 ng/mL) for
o
70 min in PBS buffer (10 mM, pH 7.4, 0.1% DMSO) at 37 C. (D)
Line relationship between the fluorescence intensity of TMN-Leu
(658 nm) and the concentration of LAP (0.4ꢀ14.0 ng/mL). Error
bars represent standard deviation (n = 3).
The timeꢀdependent fluorescence response of the probe toꢀ
ward LAP was tested. As shown in Figure S1, the fluorescent
intensity at 658 nm increased rapidly in a timeꢀdependent
manner, and then obtained a maximum intensity in approxiꢀ
mately 70 min. Subsequently, the fluorescence intensity of the
probe was varied upon addition of different concentrations of
LAP (0ꢀ330 ng/mL) which was examined and recorded in
Figure 1C, the continuous increase of LAP resulted in an obꢀ
vious fluorescence intensity enhancement (658 nm), and
reached a plateau at 165 ng/mL of LAP. Moreover, the fluoꢀ
rescent signal intensities were linearly proportional to LAP at
free TMN-NH was specially activated and the electronꢀrich
2
amine moiety was released, then yielded a remarkable fluoresꢀ
cence signal (Figure 1A). To test our hypothesis, the probe
TMN-Leu was designed, synthesized and confirmed by
1
13
HNMR, C NMR and HRMS. All the intermediates were
1
13
also characterized by HNMR, C NMR and HRꢀESIꢀMS
(Scheme S1 and Figure S13ꢀS23).
2
Spectroscopic Properties and Optical Response to LAP.
the concentration range of 0.4ꢀ14.0 ng/mL (R = 0.9972, Figꢀ
To investigate the ability to detect LAP, the spectral character
of this probe responding to LAP was initially measured in the
buffer solution (PBS/DMSO = 999:1, v/v, 10 mM, pH = 7.4),
implying its favorable water solubility. As Figure 1B illustratꢀ
ed, the probe exhibited a faint yellow color and a maximum
absorption wavelength at 395 nm, but upon the addition of
LAP (165 ng/mL), the absorption maximum decreased, a disꢀ
tinctive increase of the absorption band centered at 460 nm
could be clearly visualized with the solution color changed to
pink. Simultaneously, the fluorescent spectra of probe TMN-
Leu treated with LAP was also systematically investigated,
the maximum emission at 658 nm had a significant fluoresꢀ
ure 1D), the detection limit of TMN-Leu (3σ/slope method)
was calculated to be as low as 0.38 ng/mL. This may enable
the probe to trace amounts of intracellular LAP. In addition,
the kinetic parameters of probe toward LAP was also deterꢀ
mined by the LineweaverꢀBurk plot, 1/V (reaction rate) versus
1/concentration (Figure S2). The Michaelis constant (K ) of
m
the probe was estimated to be 24.1 ꢁM with the Michaelisꢀ
Menten equation, the calculated small K indicating the probe
m
possessing a strong binding affinity with LAP.
Moreover, we also evaluated the fluorescence profiles of
TMN-Leu verses different pH values to investigate its practiꢀ
ACS Paragon Plus Environment

Analytical Chemistry
cability for biological imaging (Figure S3). No obvious emisꢀ
sion was changed at 658 nm above the pH range of 5ꢀ9, indiꢀ
cating the stable structure of the probe during acidic to alkaꢀ
line conditions. As expected, upon addition of LAP (165
ng/mL), the emission intensity increase was sensitive to the
pH range from 5ꢀ9, especially remained the maximum intensiꢀ
ty at the physiological pH window. The results indicated that
the probe had obvious advantage and highly practical value for
imaging application in biological systems.
Page 4 of 8
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 3. (A) Fluorescence response of TMN-Leu (10 ꢁΜ) toꢀ
ward LAP (165 ng/mL) in the absence and presence of Bestaint at
concentration of (a) 0, (b) 5, (c) 15, (d) 30 ꢁΜ for 70 min in PBS
buffer (10 mM, pH 7.4, 0.1% DMSO) at 37 C. Error bars repreꢀ
sent standard deviation (n = 3). (B) Fluorescence response of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
TMN-Leu (10 ꢁΜ) and TMN-NH in the absence of Bestatin (5ꢀ
2
3
0 ꢁΜ) for 70 min in PBS buffer.
Enzyme-Catalytic Activation Mechanism. As aforemenꢀ
tioned, the cleavage of Lꢀleucine amide moiety by the enzymeꢀ
reactive reaction could liberate the electroꢀdonating amino
group accompanying by the generation of TMN-NH . To veriꢀ
2
fy the activation mechanism, HPLC analysis were used. As
shown in Figure 4A, the chromatographic peak of TMN-NH2
and TMN-Leu were found at 11.08 min and 6.58 min respecꢀ
tively. Additionally, upon addition of LAP (165 ng/mL) and
incubation with TMN-Leu for 30 min, the reaction product
exhibited a chromatographic peak at 11.08 min which matched
perfectly with compound TMN-NH , a weak peak was also
observed at 6.58 min, corresponding to probe TMN-Leu.
Moreover, the product of the reaction of TMN-Leu and LAP
Figure 2. Fluorescence spectra of TMN-Leu (10 ꢁΜ) upon interꢀ
action with LAP or other analytes in PBS buffer (10 mM, pH 7.4,
2
+
3+
2+
2+
2
0.1% DMSO). Columns 1ꢀ9: PBS, Ca , Fe , Mg , Mn , H O ,
2
2
GSH, Cys, Hcy; 10ꢀ12: MAOꢀA, GGT, Nitroreductase. 13: LAP.
o
All measurements were taken at 37 C. Error bars represent standꢀ
was further examined by HRMS. The peak at m/z 290.1651
ard deviation (n = 3).
+
[
[
(
M+H] , corresponding to TMN-NH and at m/z 403.2494
2
+
M+H] corresponding to TMN-Leu were clearly observed
To further confirm the fluorescence monitoring specificity
Figure 4B), indicating the evolution of TMN-NH upon the
2
of TMN-Leu toward LAP, the fluorescence signal effect of
2+
enzymeꢀtriggered cleavage reaction. These data confirmed the
fact that TMN-Leu could reacted with LAP to form TMN-
the probe to other species (including 10 equivalent of Ca ,
3+
2+
2+
Fe , Mg , Mn , H O , GSH, Cys, Hcy, 10 U/L of MAOꢀA,
2
2
NH which emitted at NIR region with a distinct fluorescence
2
GGT, Nitroreductase) were detected in the PBS buffer soluꢀ
tion (0.1% DMSO, 10 mM, pH = 7.4). As Figure 2 illustrated,
the addition of other species exhibited a negligible fluoresꢀ
cence response, only LAP could induce a remarkable fluoresꢀ
cence enhancement. Furthermore, the addition of these chemiꢀ
cal species had little effect on the proteolytic activity of LAP
(Figure S4). All data revealed that TMN-Leu exerted excelꢀ
lent specificity under physiological conditions.
signal.
To gain more insights into the specificity of the probe toꢀ
ward LAP, a wellꢀknown LAP inhibitor (Bestatin) was preꢀ
treated with LAP before treated the enzyme with TMN-Leu
Figure 4. (A) HPLC analysis of TMN-Leu (a) the reaction prodꢀ
uct of TMN-Leu by LAP (b) and TMN-NH (c). (B) HRꢀESIꢀMS
of TMN-Leu and (C) the reaction product of TMN-Leu by LAP.
(10 ꢁM). As shown in Figure 3A, Bestatin could efficiently
2
suppress the fluorescence response of LAP in a doseꢀ
dependent manner, while it hardly affected the fluorescence of
TMN-NH and TMN-Leu in the same concentration (Figure
2
In Situ Tracking of Endogenous LAP Activity in Living
Cells. Considering the excellent sensing properties of the
probe in vitro system, we further studied its capability of
tracking LAP in living cells. Before cellular application, the
biocompatibility of the probe was evaluated with two cell lines
by MTT assays (Figure S5), the viability of HCT116 and
HepG2 cells was almost 95% after incubation with a high conꢀ
centration of 20 ꢁM for 24 h, confirming the ultra low cytoꢀ
toxicity of the probe to cells.
3
B). These data indicated that TMN-Leu could be applied as
an excellent candidate for specific LAP detection in bioꢀ
samples.
ACS Paragon Plus Environment

Page 5 of 8
Analytical Chemistry
We further applied the probe to assess the applicability of
therapy. To investigate the inhibitory effect of LAP inhibitor
on the LAP activity in cells, the currently common detection
methods such as western blot and RTꢀPCR employed in vitro
need complex manipulation process and have relative low
sensitivity. The fluorescence probe with high sensitivity, simꢀ
ple operation and excellent specificity may provide a concise
method to detect LAP in situ. Subsequently, we investigated
whether TMN-Leu could test the inhibition efficacy towards
LAP activity using a fluorescence imaging experiment.
HCT116 cells were pretreated with various concentrations of
Bestatin for 1 h, following by incubation with TMN-Leu for
another 65 min. The resulting fluorescence images were diꢀ
rectly recorded without washing and fixing. As shown in Figꢀ
ure 6A, the fluorescence intensity decreased stepwise with the
increasing concentration of Bestatin. To transfer the change of
fluorescence intensity into I/I (I and I respectively represent
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
probe toward intracellular LAP in HCT116 cells under cultural
condition using an ImageXpress Micro Confocal analysis. The
experimental results were illustrated as Video S1 (cell images
in Living Cell Station) and Figure S6, after addition with the
probe 10 ꢁM, a gradually increasing NIR fluorescence signal
of living cells was investigated in a timeꢀdependent manner.
Through the Video S1, we found that the fluorescence signal
reached a plateau after incubated with TMN-Leu for 65 min,
which was almost consistent with in cell free LAP activity
test, indicating good cell permeability of the probe and its
reaction with the intracellular LAP in cells. This is the first
time for the LAP probe to in situ image LAP activity in living
cells under cultural condition. Meanwhile, the same concluꢀ
sion was obtained on MDAꢀMBꢀ231 cells (Figure S7). Next,
we investigated the response of the probe at the different conꢀ
centrations of TMN-Leu in HCT116 cells for 65 min. As Figꢀ
ure 5 illustrated, the fluorescence signal was increased in a
good doseꢀdependent manner, which was further confirmed
using flow cytometry (Figure S8). LAP was always overꢀ
expressed in cancer cells compared with normal cells. To furꢀ
ther investigate the difference LAP content between cancer
cells and normal cells, cancer cells HCT116 (Hoechst 33342
preꢀstained) and normal cells MCFꢀ10A were coꢀcultured and
their LAP activity was detected by TMN-Leu, the fluoresꢀ
cence signal intensity in HCT116 was much stronger than that
in MCFꢀ10A cells (Figure S9), indicating the LAP in cancer
cells HCT116 was more active than that in normal cells MCFꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
0
the fluorescence data of inhibitor treated and untreated cells),
we plotted the inhibitory histogram to give the IC50 value
(concentration for half maximal inhibitory fluorescence intenꢀ
sity) to be ~5.7 ꢁM (Figure 6B). Thus, TMN-Leu may have a
great potential to be applied for the highꢀthroughput cellꢀbased
assay to rapidly screen and evaluate new LAP inhibitors.
1
0A. The above data showed that probe TMN-Leu possessed
striking capability of tracking LAP in living cells and revealꢀ
ing the different LAP activity between cancer cells and normal
cells.
Figure 6. Fluorescence imagingꢀbased LAP inhibitor assay with
TMN-Leu. (A) Fluorescence images of HCT116 cells treated
with LAP inhibitor Bestatin at concentration of (a) 100, (b) 50, (c)
2
5, (d)12.5, (e) 6.25, (f) 3.125 and (g) 0 ꢁΜ for 1 h, and then
incubated with TMN-Leu (10 ꢁM) for another 65 min. (h)ꢀ(n)
Differential interference contrast (DIC) images of the above corꢀ
responding cells. The images were captured without washing.
Scale bar: 20 ꢁm. (B) Plot of the change in intracellular fluoresꢀ
cence intensity (I/I ) versus Bestatin concentration based on the
0
fluorescence images. Fluorescence intensities were evaluated at
three regions in each dish. Error bars represent standard deviation
(n = 3). The IC50 values of Bestatin for LAP in culturing HCT116
cells was found to be ~5.7 ꢁM.
Figure 5. Fluorescence imaging of endogenous LAP in HCT116
cells using different concentrations of TMN-Leu. (A) Fluoresꢀ
cence images of HCT116 cells treated with TMN-Leu at concenꢀ
tration of (a) 0, (b) 1.25, (c) 2.5, (d) 5, (e) 10 and (f) 20 ꢁΜ for 65
min. (g)ꢀ(l) Differential interference contrast (DIC) images of the
above corresponding cells. The images were captured without
washing and imaged using an ImageXpress Micro Confocal analꢀ
ysis. Scale bar: 20 ꢁm. (B) Fluorescence intensities were evaluatꢀ
ed at three regions in each dish. Error bars represent standard
deviation (n = 3).
In Situ Tracking of Endogenous LAP Activity in Cispla-
tin-induced Cells. Treatment cancer cells with Cisplatin causꢀ
es an increase of LAP, and the increased LAP plays a role in
1
intrinsic resistance of cancer cells towards Cisplatin. Next,
the effect of Cisplatin on the intracellular LAP change in
HepG2 cells was investigated by TMN-Leu. As shown in
Figure S10, the fluorescence intensity in Cisplatinꢀtreated cells
was almost 2ꢀfold increased compared with the untreated
HepG2 cells. To confirm that the increased fluorescence enꢀ
hancement response was derived from the elevated LAP enꢀ
zymeꢀcatalyzed cleavage reaction, a control enzyme inhibition
experiment was further performed by treating the fluoresꢀ
cenceꢀincreased HepG2 cells with Bestatin. The cells treated
Evaluation of Inhibition Efficacy of Bestatin in Living
Cells. LAP is also confirmed as an efficient target and increasꢀ
ing number of LAP inhibitors have been developed for cancer
ACS Paragon Plus Environment

Analytical Chemistry
Page 6 of 8
with Bestatin exhibited a marked fluorescence signal decrease, transwell assays. The fluorescence in LAP knockdown
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
clearly indicating that Bestatin can effectively inhibit the LAP
activity induced by Cisplatin in living cells. In other words,
the fluorescence of the HepG2 cells indeed arose from the
catalytic reaction of LAP. Thus, this probe could detect the
intrinsic resistance of cancer cells by monitoring their LAP
changes.
HCT116 cells was lower than that in parent cells which exertꢀ
ed more invasive (Figure 7A and 7B). These data suggested a
positive correlation between the intrinsic invasion ability of
cancer cells and LAP activity. Furthermore, their LAP level
was also confirmed by RTꢀPCR (Figure 7C). Together, LAP
may act as a simple indicator to monitor the intrinsic invasive
ability of cancer cells.
In Situ Tracking of Endogenous LAP Activity in Drug-
induced Hepatotoxicity Model. Human body will be exposed
to a wide of toxic substance from environmental and pharmaꢀ
ceuticals (such as Ace ) in one’s lifetime. While the main deꢀ
toxification organ liver is related to a complex enzymatic sysꢀ
tem. Among them, LAP was confirmed as an indicator of liver
dysfunction. Next, we examined the capability of the probe to
image the endogenous LAP in an Aceꢀinduced human normal
hepatocyte cell line (L02). As shown in Figure S11, when L02
cells were incubated with TMN-Leu, the fluorescence in the
cells gradually increased. Moreover, under the same imaging
condition the Aceꢀpretreated L02 cells exhibited a largely inꢀ
creased fluorescence as compared to the control, which was
further suppressed by the LAP inhibitor Bestatin. In short, this
fluorescence probe can be used for detecting the alteration of
the intracellular LAP activity in liver injury diseases.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. Intracellular localization of HCT116 cells incubated
with TMN-Leu. Images of cells pretreated respectively with 10
ꢁM TMN-Leu for 65 min and subsequently 1 ꢁM MitoꢀTracker
Green (or 1 ꢁM LysoꢀTracker Green) for 30 min. Red channel,
probe fluorescence (a) and (d); MitoꢀTracker, Green channel (b);
LysoꢀTracker, Green channel (e); the overlap (c) and (f); (g)ꢀ(h)
Differential interference contrast (DIC) images of the correspondꢀ
ing cells. Scale bar: 20 ꢁm.
Fluorescence Imaging and Colocalization Studies. Havꢀ
ing performed the LAP imaging in cells, we proceeded to inꢀ
vestigate the detected distribution of LAP at subcellular levels
by TMN-Leu using colocalization experiments in HCT116
cells. Cells were pretreated with 10 ꢁM TMN-Leu for 65 min
and subsequently 1 ꢁM MitoꢀTracker Green or 1 ꢁM Lysoꢀ
Tracker Green for another 30 min. Interestingly, the fluoresꢀ
cence of released TMN-NH in red channel overlapped exactꢀ
2
ly with that of MitoꢀTracker Green in green channel but that of
lysosome indicating that the enzyme cleavage was mainly
occurred in mitochondria (Figure 8). Meanwhile, the same
subcellular distribution of released TMN-NH can also be
2
confirmed in MDAꢀMBꢀ231 cells (Figure S12). Thus, this
probe may provide an excellent tool for tracking mitochondrial
LAP.
Figure 7. Fluorescence imaging of endogenous LAP in HCT116
cells and siRNAꢀtransfected HCT116 cells and their relative invaꢀ
sion activity. (A) Fluorescence images of HCT116 cells (b),
HCT116 cells transfected with LAP siRNA (d), (a) and (c) Differꢀ
ential interference contrast (DIC) images of the corresponding
cells. The images were captured without washing and recorded
using an ImageXpress Micro Confocal analysis. Scale bar: 20 ꢁm.
Fluorescence intensities were evaluated at three regions in each
dish. Error bars represent standard deviation (n = 3). (B) HCT116
cells (a and c) and siRNAꢀtransfected HCT116 cells (b and d)
were seeded in transwell chamber and incubation for 24 h, then
fixed by 4% paraformaldehyde and dyed by crystal violet and
imaged under microscope (magnification, x200). (C) The relative
mRNA level of LAP in HCT116 cells and siRNAꢀtransfected
HCT116 cells, ***p < 0.001.
In Vivo Real-time Tracking of Endogenous LAP Activity
in the HCT116 Tumor-bearing Mice. In vivo realꢀtime imꢀ
aging offers a powerful tool for accurately diagnosing disease
and suspicious lesions with valuable spatiotemporal precision.
Almost, the current fluorescent probes for imaging LAP acꢀ
tivity are not suitable for in vivo experiments because their
emissions are not located in NIR region and short Stokes Shift,
thus fail to penetrate deeper tissue and are disturbed by intrinꢀ
sic autoꢀfluorescence background signals. Therefore, with the
prominent performance of the LAP probe in cellular fluoresꢀ
cence imaging, we further attempted to examine the applicaꢀ
bility of our probe in vivo realꢀtime visualizing the endogeꢀ
nous LAP activity in the HCT116 tumorꢀbearing mice. Before
in vivo imaging application, a drug tolerance test was perꢀ
formed. Ten ICR mice were respectively given TMN-Leu at 5
mg/kg (0.2 mL) by intraperitoneal injection in a single dose,
and no obvious cytotoxicity was observed. Next, the probe
was applied to inject into the tumor region in HCT116 tumorꢀ
bearing mice, followed by fluorescence imaging using a small
The Relationship between LAP and Intrinsic Invasion
Ability. It has been found that LAP is related to the invasive
ability of cancer cells. However, it is indistinct whether the
endogenous LAP plays a great role in the intrinsic invasive
ability of colorectal cancer cells. Subsequently, we knocked
down the LAP in HCT116 cells, the LAP activity and invasive
ability of them were evaluated by probe TMN-Leu and
ACS Paragon Plus Environment

Page 7 of 8
Analytical Chemistry
ASSOCIATED CONTENT
animal in vivo imaging system. As shown in Figure 9, a gradꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ually increased fluorescence response was observed in tumor
region in a timeꢀdependent manner. The fluorescence signal
rapid responded to LAP activity and reached the plateau at 60
min. In the other group, after subcutaneously injection of
Bestatin in tumor region of nude mice for 1 h, the probe was
injected into the tumor region, the region treated with Bestatin
exhibited little fluorescence signal under the same condition,
indicating the probe was catalyzed by LAP to release TMN-
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
A comparison of the fluorescent probes for LAP detection, synꢀ
thesis of probe TMN-Leu, timeꢀdependent fluorescence intensity
of TMN-Leu with LAP, LineweaverꢀBurk reciprocal plot, fluoꢀ
rescence intensity at different pH conditions, fluorescence spectra
of TMN-Leu with LAP affected by other analytes, the MTT asꢀ
says, timeꢀdependently fluorescence imaging of endogenous LAP
in HCT116 cells, fluorescence imaging of LAP in MDAꢀMBꢀ231
cells, fluorescence monitor using an BD flow cytometry, fluoresꢀ
NH in the tumor. The result demonstrated that our probe
2
could be utilized as a prominent bioimaging tool for in vivo
realꢀtime detecting LAP activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
cence imaging of LAP in coꢀcultural cells,
fluoꢀ
rescence imaging of endogenous LAP in Cisplatinꢀinduced
HepG2 cells, fluorescence imaging of endogenous LAP in Aceꢀ
induced L02 cells, intracellular localization of MDAꢀMBꢀ231
1
13
cells incubated with TMN-Leu, the spectrums of H NMR,
C
NMR and HRꢀESIꢀMS.
Supporting Information (PDF)
Video S1 (avi)
AUTHOR INFORMATION
Corresponding Author
* Lingyi Kong: Eꢀmail: lykong@cpu.edu.cn.
Figure 9. In vivo realꢀtime fluorescence imaging of endogenous
LAP in HCT116 tumorꢀbearing mice. (Group A) In vivo realꢀtime
fluorescence imaging of endogenous LAP in HCT116 tumorꢀ
bearing mice after tumor injection TMN-Leu (50 ꢁM, 50 ꢁL).
*
Wenying Yu: Eꢀmail: ywy@cpu.edu.cn.
Author Contributions
(Group B) Tumor region were pretreated with Bestatin (100 ꢁM,
All authors have given approval to the final version of the manuꢀ
script.
Notes
5
0 ꢁL) for 1h, then tumor injection subcutaneously with TMN-
Leu (50 ꢁM, 50 ꢁL). And the images were acquired 5 min, 15
min 30 min, 45 min, 60 min, 75 min and 90 min after injection.
The images were excited with a 460ꢀ560 nm filter, and acquired
with a 600ꢀ700 nm longꢀpass filter.
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This research work was supported by National Natural Science
Foundation of China (Grant No. 81673298 and No. 81402791),
Natural Science Foundation of the Jiangsu Higher Education Inꢀ
stitutions of China (Grant No. BK20140670), Jiangsu
Shuangchuang Talents for Oversea Ph.D.s, Postgraduate Research
CONCLUSIONS
In conclusion, we firstly designed a novel ultrasensitive
NIR fluorescent probe based on dicyanoisophorone for moniꢀ
toring endogenous LAP in mitochondria with a low detection
limit of 0.38 ng/mL. The probe featured striking characterisꢀ
tics in terms of large Stokes shift, favorable water solubility,
high specificity and sensitivity, well pH independency, good
cell membrane permeability, and low cytotoxicity enabling the
efficiently tracking of trace LAP activity in cells. We successꢀ
fully applied the probe to track LAP of cancer cells and norꢀ
mal cells, monitor the LAP changes on Aceꢀinduced liver injuꢀ
ry model and Cisplatinꢀinduced drugꢀresistant model, and in
situ to rapidly evaluate the LAP inhibitors on cellꢀbased assay.
Moreover, we found a positive relationship of the intrinsic
invasive ability of colorectal cancer cells and their LAP activiꢀ
ty by probe TMN-Leu, implying that LAP may act as a simple
indicator to reflect the intrinsic invasion ability of cancer cells.
More importantly, the NIR emission fluorescence of the probe
with fast response make it possible for in vivo realꢀtime visualꢀ
ization of endogenous LAP in living tumorꢀbearing mouse
model with negligible background interference. We believe
that the probe could serve as a useful indicator for distinguishꢀ
ing LAP in complex living systems and the diagnosis of LAPꢀ
associated diseases.
&
Practice Innovation Program of Jiangsu Province
(KYCX17_0731). Program for Changjiang Scholars and Innovaꢀ
tive Research Team in University (IRT_15R63), Project Funded
by the Priority Academic Program Development of Jiangsu Highꢀ
er Education Institutions (PAPD).
REFERENCES
(1) Gong, Q. Y.; Shi, W.; Li, L. H.; Ma, H. M. Chem. Sci. 2016, 7,
788ꢀ792.
(2) Shibata, K.; Kajiyama, H.; Mizokami, Y.; Ino, K.; Nomura, S.;
Mizutani, S.; Terauchi, M.; Kikkawa, F. Gynecol. Oncol. 2005, 98,
1
(
1ꢀ18.
3) Mizutani, S., Shibata, K.; Kikkawa, F.; Hattori, A.; Tsujimoto, M.;
Ishii, M.; Kobayashi, H. Expert Opin. Ther. Targets 2007, 4, 453ꢀ461.
4) Stephen, K.; Burley, P. R. D.; William, N. Lpscomb., Proc. Natl.
(
Acad. Sci. USA 1991, 88, 6916ꢀ6920.
(5) Taylor, A. The FASEB J. 1993, 7, 290ꢀ298.
(6) Gu, K.; Xu, Y.; Li, H.; Guo, Z.; Zhu, S.; Zhu, S.; Shi, P.; James, T.
D.; Tian, H.; Zhu, W. H. J. Am. Chem. Soc. 2016, 138, 5334ꢀ5340.
(
7) Guo, Z. Q.; Kim, G. H.; Yoon, J.; Shin, I. Nat. Protoc. 2014, 9,
245ꢀ1254.
8) Huang, C. S.; Jia, T.; Tang, M. F.; Yin, Q.; Zhu, W. P.; Zhang, C.;
Yang, Y.; Jia, N. Q.; Xu, Y. F.; Qian, X. H. J. Am. Chem. Soc. 2014,
36, 14237ꢀ14244.
1
(
1
ACS Paragon Plus Environment

Analytical Chemistry
Page 8 of 8
(
9) Huang, H.; Tanaka, H.; Hammock, B. D.; Morisseau, C. Anal.
414.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Biochem. 2009, 391, 11ꢀ16.
(18) Wang, X.; Li, S.; Zhang, P.; Lv, F.; Liu, L.; Li, L.; Wang, S. Adv.
Mater. 2015, 27, 6040ꢀ6045.
(19) Chen, H.; Dong, B. L.; Tang, Y.H.; Lin, W. Y. Chemistry 2015,
21, 11696ꢀ11700.
(20) Chen, X. X.; Wu, X.; Zhang, P.; Zhang, M.; Song, B. N.; Huang,
Y. J.; Li, Z.; Jiang, Y. B. Chem. Commun. 2015, 51, 13630ꢀ13633.
(21) Fan, J. L.; Hu, M. M.; Zhan, P.; Peng, X. J. Chem. Soc. Rev. 2013,
42, 29ꢀ43.
(22) Guo, Z. Q.; Park, S.; Yoon, J. Y.; Shin, I. J. Chem. Soc. Rev. 2014,
43, 16ꢀ29.
(23) Yoon, H. Y.; Shim, S. H.; Baek, L. J.; Hong, J. I. Bioor. Med.
Chem. lett. 2011, 21, 2403ꢀ2405.
(24) Gu, K. Z.; Liu, Y. J.; Guo, Z. Q.; Lian, C.; Yan, C. X.; Shi, P.;
Tian, H.; Zhu, W. H. ACS Appl. Mater. Interfaces 2016, 8, 26622−
26629.
(
10) Jung, H. S.; Chen, X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2013,
2, 6019ꢀ6031.
11) Koide, Y.; Urano, Y.; Hanaoka, K.; Piao, W.; Kusakabe, M.; Saito,
N.; Terai, T.; Okabe, T.; Nagano, T. J. Am. Chem. Soc. 2012, 134,
029ꢀ5031.
4
(
5
(12) Kong, F. P.; Ge, L. H.; Pan, X. H.; Xu, K. H.; Liu, X. J.; Tang, B.
Chem. Sci. 2016, 7, 1051ꢀ1056.
(
13) Kong, F. P.; Liang, Z. Y.; Luan, D. R.; Liu, X. J.; Xu, K. H.; Tang,
B. Anal. Chem. 2016, 88, 6450ꢀ6456.
14) Li, X. H.; Gao, X. H.; Shi, W.; Ma, H. M. Chem. Rev. 2014, 114,
90ꢀ659.
(
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(15) Lukinavicius, G.; Reymond, L.; Umezawa, K.; Sallin, O.; D'Este,
E.; Gottfert, F.; Ta, H.; Hell, S. W.; Urano, Y.; Johnsson, K. J. Am.
Chem. Soc. 2016, 138, 9365ꢀ9368.
(
16) Gong, Y. J.; Zhang, X. B.; Mao, G. J.; Su, L.; Meng, H. M.; Tan,
W.; Feng, S.; Zhang, G. Chem. Sci. 2016, 7, 2275ꢀ2285.
17) Sakabe, M.; Asanuma, D.; Kamiya, M.; Iwatate, R. J.; Hanaoka,
K.; Terai, T.; Nagano, T.; Urano, Y. J. Am. Chem. Soc. 2013, 135, 409ꢀ
(25) He, X. Y.; Li, L. H.; Fang, Y.; Shi, W.; Li, X. H.; Ma, H. M.
Chem. Sci. 2017, 8, 3479ꢀ3483.
(
Near-infrared Fluorescent Probe with Remarkable Large Stokes Shift and Favorable Water Solubility for Real-time
Tracking Leucine Aminopeptidase In Living Cells and In Vivo
ACS Paragon Plus Environment