The first ratiometric fluorescent probes for aminopeptidase N cell imaging

Article abstract of DOI:10.1039/c2ob26564h

In the current paper, three activity-based colorimetric and ratiometric fluorescent probes based on a naphthalimide fluorophore were well designed and synthesized, which can be recognized and hydrolyzed by aminopeptidase N (APN) at both the enzymatic and cellular level by following the fluorescent emission wavelength change from blue to green light. As a result, these molecules were successfully identified as the first ratiometric fluorescent probes for APN cell imaging.

Full text of DOI:10.1039/c2ob26564h

Organic &
Biomolecular
Chemistry
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The first ratiometric fluorescent probes for
aminopeptidase N cell imaging†‡
Cite this: Org. Biomol. Chem., 2013, 11,
378
Laizhong Chen, Wei Sun, Jing Li, Zhenzhen Liu, Zhao Ma, Wei Zhang, Lupei Du,
Wenfang Xu, Hao Fang and Minyong Li*
In the current paper, three activity-based colorimetric and ratiometric fluorescent probes based on a
naphthalimide fluorophore were well designed and synthesized, which can be recognized and hydro-
lyzed by aminopeptidase N (APN) at both the enzymatic and cellular level by following the fluorescent
emission wavelength change from blue to green light. As a result, these molecules were successfully
identified as the first ratiometric fluorescent probes for APN cell imaging.
Received 7th August 2012,
Accepted 5th November 2012
DOI: 10.1039/c2ob26564h
www.rsc.org/obc
calibration for environmental effects.20,21 We have reported the
first ratiometric fluorescent probe, Ala-PABA-7HC, which could
Introduction
Aminopeptidase N (APN, EC 3.4.11.2), which belongs to the detect the activity of aminopeptidase N (APN) at both the enzy-
M1 family of the MA clan of peptidases, is a Zn2+ dependent matic and the cellular level with minimal environmental influ-
exopeptidase that degrades preferentially proteins and pep- ence.22 Nevertheless, Ala-PABA-7HC was not suitable to be
tides with an N-terminal neutral amino acid.1,2 It should be implemented in cell imaging considering that the maximum
underlined that APN, which is highly expressed in tumor cells, emission peaks of both Ala-PABA-7HC (390 nm) and the releas-
has been demonstrated to perform a vital role in tumor inva- ing fluorophore 7HC (450 nm) are in the wavelength range of
sion, metastasis and angiogenesis,3–7 and has become a diag- blue light. Herein, we would like to describe a novel series of
nostic or prognostic biomarker for cancers.8 Moreover, APN ratiometric fluorescent probes based on an intramolecular
had been demonstrated to hold a critical position as a bio- charge transfer (ICT) mechanism, which can be used to image
marker for cancer stem cells.9
APN in cancer cells.
Hitherto, many efforts have been undertaken to understand
the distribution of APN and enhancing the imaging of APN in
cancer tissues,8,10–18 of which, the fluorescence-based imaging
approach has exclusive advantages since it presents a real-time
examination, displays a reasonable sensitivity, and provides a
high spatiotemporal resolution. However, the reported fluor-
escent imaging probes for APN are all NGR (Asn-Gly-Arg)
based affinity probes,11–13,15,18 which can image APN in cancer
cells or in mice with a high background signal, in such a way
that it will significantly interfere with the imaging quality. By
contrast, activity-based fluorogenic probes, which are non-
fluorescent themselves and become fluorescent when
degraded by their targeting peptidases, can reduce the non-
specific background signal.19 Moreover, probes with a dual
emission are particularly viable because they enable a built-in
Result and discussion
To find an activity-based fluorescent probe for APN, we chose
1,8-naphthalimide, a prototype intramolecular charge transfer
(ICT) fluorophore as a reporter due to its advantageous optical
properties, such as strong absorption and emission in the
visible region, high photostability, large Stokes shift and insen-
sitivity to pH.23,24 Alanine and norvaline, which are favored by
APN’s activity site,25 were chosen as the recognition moieties.
The electron-withdrawing amide group weakened the ICT
effect and resulted in a fluorescent emission wavelength blue
shift. After the cleavage of the amide bond between the amino
acids and the fluorophores by APN, 1,8-naphthalimide can be
released in a non-reversible manner to emit a strong bright
green fluorescence (Scheme 1). In this research, all of the three
probes were efficiently synthesized (Scheme 2) and well charac-
terized as depicted in the experimental and supplementary
parts.
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology of Natural
Products (MOE), School of Pharmacy, Shandong University, Jinan, Shandong
250012, China. E-mail: mli@sdu.edu.cn; Tel: +86-531-8838-2076
†This article is dedicated to Professor Wenfang Xu on the occasion of his 60th
After carefully examining the spectroscopic properties in
Tris-HCl buffer (100 mM, pH 7.5), the absorption spectra of
these three probes and their fluorophores were found to have
birthday.
‡Electronic supplementary information (ESI) available: Experimental pro-
cedures, 1H NMR, 13C NMR and HR-MS spectra. See DOI: 10.1039/c2ob26564h
378 | Org. Biomol. Chem., 2013, 11, 378–382
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Scheme 1 Plausible detection mechanism of probes.
Fig. 1 Absorption spectra of 6c, 6d, 6e, 2a and 2b (50 μM).
Fig. 2 Emission spectra of the probes 6c, 6d, 6e, 2a and 2b (250 μM, excitation
at 400 nm).
Scheme 2 Synthesis and structures of probes 6c, 6d and 6e.
an intersection at a wavelength of 400 nm (Fig. 1). This inter-
esting result envisions that they could be equally excited at
this wavelength. When excited at 400 nm, the maximum emis-
sion wavelength of 6c, 6d and 6e was at 460 nm, and that of 2a
and 2b was at 550 nm (Fig. 2). The 90 nm shift indicated that
these three molecules could be considered as candidates for
ratiometric fluorescent probes.
In the enzymatic experiments, upon addition of porcine
APN (0.2 IU) to the solution of 6e (200 μM in 100 mM Tris-HCl,
pH 7.5), the maximum emission peak experienced a red shift
from 460 nm to 550 nm, and the ratio of fluorescence intensi-
ties (I550/I460) (λex = 400 nm) meaningfully augmented from
0.45 to 4.67 (R = 10.4 fold) after 50 min (Fig. 3). Similar results
were obtained as well when adding APN to the solution 6c and
6d (Fig. S1‡). Kinetics assays showed that all of the Km values
of the three probes (752.7 μM, 487.7 μM and 107.5 μM for 6c,
6d and 6e, respectively) were lower than that of the commercial
Fig. 3 Emission spectra of 6e (200 μM) in the presence of APN (0.2 IU) in a
Tris-HCl buffer (100 mM, pH 7.5). Inset: ratios of fluorescent intensities at 550
and 460 nm as a function of time after adding APN (λex = 400 nm).
substrate L-Leu-p-nitroanilide (1180 μM), which indicates that
the three probes may bind more strongly to APN than L-Leu-p-
nitroanilide.25 Because APN is also used as a biomarker to
detect damage to the kidneys and is mainly located in the
small-intestinal and renal microvillar membranes, the fluor-
escence probes should be stable enough to pass the gastro-
intestinal environment, then to arrive on the small intestinal
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Fig. 4 Emission spectra of 6e with ES-2 and REH cells. Cells and 6e were sus-
pended in RPMI-1640 medium and incubated at 37 °C for 1 h (λex = 445 nm,
λem = 550 nm).
membrane to express their activities. The stability test results
(Fig. S3‡) disclosed that all of 6c, 6d and 6e were stable in Tris-
HCl buffer and artificial gastric juice. Moreover, there was no
significant hydrolysis evidence of 6c and 6e in the artificial intes-
tinal juice up to 5 h. All the above results confirmed that 6c, 6d
and 6e were successful ratiometric fluorescent probes for detect-
ing the activity of APN in a neutral or acidic environment, and
that 6c as well as 6e can also be used in artificial intestinal juice.
To further confirm the practical feasibility of our ratio-
metric fluorescent probes, we applied 6e to the detection of
APN activity in living cancer cells in view of its superior solubi-
lity, stability and lower Km value. As demonstrated in Fig. 4,
the fluorescence intensities of the solution of 6e and APN high
expressed ES-2 cells increased significantly after incubating for
1 h at 37 °C. However, compared to ES-2 cells, APN low
expressed REH cells could hardly hydrolyze 6e in the same
conditions. This evidence indicated that 6e could be applied
in detecting APN activity in living cells, and may be used in
ratiometric fluorescence imaging of endogenous APN. ES-2
cells incubated with the probe 6e (5 μM) for 15 min disclosed
an intensely blue fluorescence in the nucleus (Fig. 5B). The
result revealed that the probe 6e could penetrate the cell mem-
brane and mainly label the nucleus. Moreover, a green intense
fluorescence generated in the whole cells (Fig. 5C) illustrated
that 6e could be hydrolyzed by endogenous APN. In compari-
son, ES-2 cells incubated with both 6e (5 μM) and the APN
inhibitor, Bestatin (4.9 mM), for 15 min unveiled similar blue
fluorescence in the nucleus but weak green fluorescence in the
whole cells (Fig. 5G). The significant differences in ratio fluor-
escence responses in Fig. 5D and 5H clearly demonstrated that
probe 6e can be employed for ratiometric fluorescent imaging
of endogenous APN in living cells (Fig. 5I).
Fig. 5 Fluorescence images of living ES-2 cells: top row, ES-2 cells incubated
with the probe 6e (5 μM) for 15 min: (A) bright-field, (B) blue channel, (C) green
channel, (D) ratio of (C) and (B); bottom row, ES-2 cells incubated with the
probe 6e (5 μM) and Bestatin (4.9 mM) for 5 min: (E) bright-field, (F) blue
channel, (G) green channel, (H) ratio of (G) and (F). (I) Bars represent the mean
ratio generated from the total integrated density from green channel over the
total integrated density from blue channel. Error bars represent standard error
measurement (s.e.m.).
and hydrolyzed by APN. These probes display a 90 nm red-shift
of fluorescence emission and the color changes from colorless
to olivinic upon addition of APN, and therefore can serve as
colorimetric and ratiometric fluorescent probes for APN detec-
tion. One of them, 6e, was also successfully applied in ratio-
metric fluorescent imaging of endogenous APN in living ES-2
cells. In view of the built-in correction ability for environ-
mental interference, 6e could be implemented as a useful
diagnostic probe for APN high expressed cancer lines.
Experimental
1. Materials and instruments
All reagents were purchased from Acros and Aldrich. Twice-dis-
tilled water was used throughout all experiments. 1H NMR and
13C NMR were recorded on Bruker 300 NMR, 400 NMR and
600 NMR spectrometers. Mass spectra were performed by the
analytical and the mass spectrometry facilities at Shandong
University. Specific rotation was tested using a Anton Paar
MCP200.
Conclusions
2. Synthesis of 6e
In summary, three amino acid-naphthalimide probes were well
6-BROMO-2-(2-(DIMETHYLAMINO)ETHYL)-1H-BENZO[DE]ISOQUINOLINE-1,3
designed and synthesized, and all of them could be recognized (2H)-DIONE (1B). The solution of 4-bromo-1,8-naphthalic
380 | Org. Biomol. Chem., 2013, 11, 378–382
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anhydride (13.8 g, 50 mmol) and N1,N1-dimethylethane-1,2- 8.56 (d, 1 H, J = 4.8 Hz), 8.41 (m, 3 H), 8.23 (d, 1 H, J = 8.4 Hz),
diamine (5.28 g, 60 mmol) in 500 mL 1,4-dioxane was refluxed 7.95 (t, 1 H, J = 8.7 Hz), 4.48 (m, 1 H), 4.40 (t, 2 H, J = 6.0 Hz),
at 120 °C for 4 h. Then the mixture was cooled and poured 3.46 (t, 2 H, J = 5.1 Hz), 2.90 (d, 6 H, J = 4.5 Hz), 1.61 (d, 3 H, J
into 500 mL water. The yielded yellowish sediments were col- = 6.6 Hz); 13C-NMR (DMSO, 150 MHz): δ 170.3, 164.5, 163.9,
lected by filtration and dried under 110 °C air atmosphere to 139.8, 131.9, 131.6, 130.3, 129.0, 127.2, 125.2, 123.0, 121.1,
obtain a pale-brown solid, 14.3 g, yield = 82.3%, mp: 119.3, 54.6, 49.5, 42.6, 35.4, 17.8; HRMS (ESI) m/z calcd for
142.5–143.5 °C.
C19H23N4O3 ([M + H]+) 355.1765; found 355.1759.
6-AMINO-2-(2-(DIMETHYLAMINO)ETHYL)-1H-BENZO[DE]ISOQUINOLINE-1,3-
(2H)-DIONE (2B). The mixture of 1b (1.7 g, 5 mmol), conc.
ammonia (10 mL), copper power (0.032 g, 0.5 mmol) and a
3. Spectroscopic materials and methods
stirring bar were sealed in a 30 mL screwed tube and stirred in Buffer reagents were purchased from Aldrich and Acros and
a oil bath at 100 °C for 10 h. After cooling to r.t., the suspen- were used without purification. Water used for the fluor-
sion was extracted with 50 mL AcOEt, to the residue was added escence studies was doubly distilled and further purified with
50 mL water and extracted by another 50 mL AcOEt. The com- a Mill-Q filtration system. Absorption spectra were recorded
bination was washed with brine (100 mL × 1) and dried over using Shimadzu UV-1700 UV-visible spectrometer. Fluor-
anhydrous MgSO4 for 24 h. The solution was concentrated escence spectra were recorded using Varioskan microplate
under reduced pressure and then purified by combiflash to reader (Thermo Electron Corporation). Solutions of com-
yield yellow power. Yield = 31.2%, mp: 222.9–225.1 °C.
pounds 6c, 6d and 6e were prepared in 0.1 M Tris-HCl buffer
2-((((9H-FLUOREN-9-YL)METHOXY)CARBONYL)AMINO)PROPANOIC ACID (3C). at pH 7.5. Solutions of compounds 2a and 2b were prepared in
To the solution of Ala (3.5 g, 40 mmol) in 120 mL 10% Na2CO3 0.1 M Tris-HCl (contains 1% DMSO) buffer at pH 7.5
was added the solution of FmocOSu (13.5 g, 40 mmol) in
acetone (100 mL) at 0 °C. The mixture was stirred at r.t. over-
night. Acetone was evaporated under reduced pressure and the
4. Vmax and Km measurement
residue was washed by ether (100 mL × 3). The water phase Kinetic constants (Vmax and Km) were determined by following
was adjusted to pH 2 with conc. HCl and extracted with AcOEt the methods described in the literature.26 For 6c, 6d and 6e,
(100 mL × 3). The organic phase was dried over anhydrous an enzyme concentration of 0.2 IU mL−1 and [S] final between
MgSO4 for 24 h. The solution was concentrated under reduced 0.025 and 0.80 mM were used. For substrate L-Leu-p-nitroani-
pressure and then recrystallized with AcOEt–n-hexane to yield lide, an enzyme concentration of 0.05 IU mL−1 and [S] final
a white solid, 10 g, yield = 80.4%, mp: 138.2–139.7 °C.
between 0.025 and 1.6 mM were used. The assays were per-
(9H-FLUOREN-9-YL)METHYL(1-CHLORO-1-OXOPROPAN-2-YL)CARBAMATE (4C). formed in quadruplicate. Km and Vmax were calculated by the
To the solution of 3c (0.5 g, 1.6 mmol) in CH2Cl2 (8 mL) was Lineweaver–Burk plot by using SigmaPlot 12 (Fig. S2‡).
added SOCl2 (4 mL), the mixture was stirred at r.t. for 2 h. The
solution was concentrated under reduced pressure and recrys-
tallized with CH2Cl2–n-hexane to yield a white solid, 0.35 g,
5. Cell culture and fluorescent image assay
yield = 66.5%, mp: 94.5–96 °C.
Human non-T, non-B acute lymphoblastic leukemia cells
(9H-FLUOREN-9-YL)METHYL(1-((2-(2-(DIMETHYLAMINO)ETHYL)-1,3-DIOXO- (REH) were grown in RPMI-1640 medium supplemented with
2,3-DIHYDRO-1H-BENZO[DE]ISOQUINOLIN-6-YL)AMINO)-1-OXOPROPAN-2-YL)- 10% (v/v) fetal bovine serum, 1.5 g L−1 NaHCO3, 2.5 g L−1
CARBAMATE HYDROCHLORIDE (5E). The suspension of 4c (0.96 g, glucose, 0.11 g L−1 sodium pyruvate in an atmosphere of 5%
3.0 mmol) and 2b (0.28 g, 1.0 mmol) in AcOH (20 mL) was CO2, 95% air at 37 °C. Human ovarian clear cell carcinoma
refluxed for 4 h. The mixture was cooled and concentrated to cells (ES-2) were grown in RPMI-1640 medium supplemented
5 mL, 40 mL AcOEt was added to the solution. The yield pre- with 10% (v/v) fetal bovine serum in an atmosphere of 5%
cipitation was filtered and washed with 20 mL AcOEt to gain a CO2, 95% air at 37 °C. For the cellular level APN activity assay,
deep yellow power. The deep yellow power was recrystallized ES-2 and REH cells were respectively centrifuged for 400 s at
with MeOH to yield a khaki powder, 0.15 g, yield = 26.0%, mp: 800 rpm, and then suspended with RPMI-1640 medium. The
156.7–158.1 °C.
mixture of 200 μL of the above RPMI-1640 medium suspension
2-AMINO-N-(2-(2-(DIMETHYLAMINO)ETHYL)-1,3-DIOXO-2,3-DIHYDRO-1H- with 6 × 105 or 3 × 105 cells and 10 μL 6e (1.4 mM) were incu-
BENZO[DE]ISOQUINOLIN-6-YL)PROPANAMIDE DIHYDROCHLORIDE (6E). The bated at 37 °C for 1 h. The fluorescence intensities were
mixture of 5e (0.10 g, 0.16 mmol) and 5 mL 20% piperidine– recorded using a Varioskan microplate reader. For the fluor-
DMF was stirred at r.t. for 30 min then concentrated under escent image assay, after the medium was removed, the cells
reduced pressure at 90 °C to remove excess piperidine and were carefully washed with RPMI-1640 medium, and then
DMF. 5 mL EtOAc solution saturated with HCl gas was added incubated at r.t. in the presence of probe 6e (5 μM) or the
and stirred at r.t. for 1 h. The yield precipitation was filtered mixture of probe 6e (5 μM) and Bestatin (4.9 mM) in
and recrystallized with AcOEt–MeOH to yield a light yellow RPMI-1640 medium for 5 min. Fluorescence imaging was per-
power, 30 mg, yield = 43.9%, mp: 255.3–257.8 °C, [α]2D0 = 16.09 formed using an OLYMPUS CK30-F200 fluorescence micro-
(c = 0.087, MeOH). 1H-NMR (DMSO, 300 MHz): δ 11.21 (s, 1 H), scope. Ratiometric pictures were achieved with ImageJ
9.60 (s, 1 H), 8.82 (d, 1 H, J = 8.1 Hz), 8.58 (d, 1 H, J = 3.0 Hz), software.27
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12 Z. Zhang, H. Harada, K. Tanabe, H. Hatta, M. Hiraoka and
Acknowledgements
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The present work was supported by the Program for New 13 A. Buehler, M. A. M. J. van Zandvoort, B. J. Stelt,
Century Excellent Talents in University (No. NCET-11-0306),
the Shandong Natural Science Foundation (No. JQ201019), the
Independent Innovation Foundation of Shandong University,
IIFSDU (No. 2010JQ005) and the Graduate Independent Inno-
vation Foundation of Shandong University, GIIFSDU (No.
yzc12096).
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