The arginine detection and cytotoxicity of fluorescent probes based on naphthalene derivatives

Article abstract of DOI:10.1002/hc.21449

From a biological point of view, Schiff base probes containing fluorescent groups are chemically modified to provide an effective method for the detection of amino acids. According to this method, three molecular probes containing Schiff base and hydroxyl group have been designed and synthesized. UV-Vis and fluorescent data indicated that compound 2 showed strong sensitivity and high selectivity for arginine (Arg) among normal twenty kinds of amino acids. In addition, compound 2 displayed high combining ability with Arg and low cytotoxicity in MCF-7 cell from 0 to 150?μg/mL. The above results showed the synthesized probes also can be used a biosensor for the Arg detection in vivo.

Full text of DOI:10.1002/hc.21449

Received: 18 July 2018
DOI: 10.1002/hc.21449
Revised: 15 October 2018
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R E S E A R C H A R T I C L E
The arginine detection and cytotoxicity of fluorescent probes
based on naphthalene derivatives
1
Congshu Li1
Jie Li1
Yanmei Chen1
Hongli Chen2
Xuefang Shang
Tianyun Wang3
|
|
|
|
|
1Key Laboratory of Medical Molecular
Probes, School of Basic Medical
Sciences, Xinxiang Medical University,
Xinxiang, Henan, China
2School of Life Sciences and Technology,
Xinxiang Medical University, Xinxiang,
Henan, China
3Department of Biochemistry, Xinxiang
Abstract
From a biological point of view, Schiff base probes containing fluorescent groups are
chemically modified to provide an effective method for the detection of amino acids.
According to this method, three molecular probes containing Schiff base and hy-
droxyl group have been designed and synthesized. UV-Vis and fluorescent data indi-
cated that compound 2 showed strong sensitivity and high selectivity for arginine
(Arg) among normal twenty kinds of amino acids. In addition, compound 2 displayed
high combining ability with Arg and low cytotoxicity in MCF-7 cell from 0 to
150 μg/mL. The above results showed the synthesized probes also can be used a bio-
sensor for the Arg detection in vivo.
Medical University, Xinxiang, Henan,
China
Correspondence
Xuefang Shang, Key Laboratory of Medical
Molecular Probes, School of Basic Medical
Sciences, Xinxiang Medical University,
Xinxiang, Henan, China.
Email: xuefangshang@126.com
Funding information
Fluorescence Probe and Biomedical
Detection Research Team of Xinxiang
City, Grant/Award Number: CXTD16001;
Science & Technology Innovation Talents
in Universities of Henan Province, Grant/
Award Number: 15HASTIT039; Scientific
and Technological Research Projects of
Henan Province, Grant/Award Number:
1
82102311124
1
|
INTRODUCTION
organisms. In particular, biosensors, as tools for detecting
[4,5]
amino acids, have gained more and more attentions.
So,
[
6,7]
In recent years, more and more attentions have been paid
to the research and application field of host–guest chem-
istry, which boosted the rapid development of molecular
the detection of amino acids is very vital in organisms.
Arginine (Arg) has an important physiological role in cell
division, gene regulation, wound healing, hormone release, and
[
1–3]
[8–10]
recognition.
Amino acids are important components of
immune system function.
In addition, Arg is also clinically
Contract grant sponsor: Fluorescence Probe and Biomedical Detection Research Team of Xinxiang City.
Contract grant number: CXTD16001.
Contract grant sponsor: Science & Technology Innovation Talents in Universities of Henan Province.
Contract grant number: 15HASTIT039.
Contract grant sponsor: Scientific and Technological Research Projects of Henan Province.
Contract grant number: 182102311124.
Heteroatom Chemistry. 2018;29:e21449.
https://doi.org/10.1002/hc.21449
wileyonlinelibrary.com/journal/hc
© 2018 Wiley Periodicals, Inc.
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SHANG et Al.
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used as a treatment for diseases caused by endocrine and high
concentrations of probes solution were added to the cells and
further incubated for 24 hours. 96-well plates were tested for
absorbance at 490 nm using a microplate reader (Thermo
Multiscan MK3, Thermo Fisher Scientific, MA, USA). Cell
viability was calculated from the following formula: Cell
viability (%) = (average absorbance of drug group − aver-
age absorbance of blank) − (average absorbance of control
group − average absorbance of blank).[
[
11,12]
ammonia reactions.
Lacking of Arg will also lead to an au-
tosomal recessive inherited disease that is the final step in enzy-
[13–15]
matic urea cycling.
Therefore, the selective recognition of
Arg is crucial in biochemical and clinical applications.
A variety of detection procedures for Arg have been de-
veloped. More traditional detection methods, such as high-
performance liquid, ion exchange, gas chromatography, have
25]
[16–18]
been reported on the detection of Arg.
However, the tra-
Compounds 1-3 were synthesized according to the route
shown in Scheme 1.
ditional detection methods have some shortcomings, such as
complex and expensive instruments, inconvenient operation,
poor selectivity, precision, sensitivity, and high repeatabil-
Compound 1: o-aminophenol (1.0910 g, 10 mmol)
was added to absolute ethanol (20 mL) and the mixture
was stirred. Then, 2-hydroxy-1-naphthaldehyde (1.7220 g,
1 mmol) in the ethanol solution (20 mL) was added to the
above solution. After the mixture was heated for 6 hours,
yellow precipitate was obtained by hot filter, then washed
[19]
ity.
The design of artificial receptors becomes attractive
due to its high selectivity and sensitivity. And the fluores-
cence probe has been reflected in this regard as a convenient
[
20,21]
detection method.
In recent years, more reports on flu-
orescent molecular probes are based on their advantages in
the biological environment and their clinical potential appli-
three times by hot ethanol, dried in vacuum. Yield: 80%.
M.p.: 261~263°C. HNMR (400 MHz, DMSO-d , 298 K) δ
15.70 (t, J = 8.8 Hz, 1H), 10.34 (s, 1H), 9.46 (dd, J = 28.9,
23.2 Hz, 1H), 8.35 (t, J = 25.3 Hz, 1H), 8.02-7.86 (m, 1H),
7.78 (t, J = 19.3 Hz, 1H), 7.72 (s, 1H), 7.46 (m, 1H), 7.25 (dt,
J = 21.0, 11.4 Hz, 1H), 7.20 (s, 1H), 7.07-6.84 (m, 2H), 6.84
1
6
[22–24]
cations.
However, there are few reports based on the de-
tection and application of fluorescent probes in amino acids,
especially in Arg.
In addition, Arg has a certain basicity due to its guanidine
moiety, which can form hydrogen bonds. Based on the above
consideration, we have rationally designed and synthesized a
series of Schiff base derivatives obtained by the reaction of
aldehyde groups and amino groups. By detecting the reac-
tion of compounds with different amino acids, it was found
that the synthesized compounds showed the highest selec-
tivity and the highest specific binding ability to Arg among
amino acids tested. The changes of fluorescence intensity
were observed after the Arg was added to the synthesized
compounds.
+
(s, 1H). ESI-HRMS (m/z): 264.1017 (M + H) .
Compound 2: o-phenylenediamine (0.3460 g, 3.2 mmol)
was dissolved in anhydrous methanol at room tempera-
ture. The hot methanol solution (10 mL) containing
2-hydroxy-1-naphthaldehyde (1.0331 g, 6.0 mmol) was
slowly added to the above solution followed by heating and
stirring, the color of the mixture rapidly changed from color-
less to orange. The mixed solution was refluxed for 3 hours
at 65°C, then stood and cooled, orange powder was precipi-
tated. The powder was washed twice by CH Cl and CH OH
3
3
(
1:1, v:v) and recrystallized to give an orange needle-like
1
solid (0.9964 g). Yield: 79.8%. M.p. 235~236°C, HNMR
2
MATERIAL AND METHODS
(400 MHz, DMSO-d , 298 K) δ 15.13 (d, J = 4.0 Hz, 1H),
|
6
9
.71 (d, J = 3.9 Hz, 1H), 8.56 (d, J = 8.4 Hz, 1H), 7.98 (d,
Some of the materials were commercially available and the
reagents and solvents used are analytical. All amino acids
were from Aladdin Chemical Co. Ltd. (Shanghai, China)
and stored in a desiccator for use without purification.
J = 9.2 Hz, 1H), 7.84 (m, 2H), 7.56 (s, 1H), 7.46 (dd, J = 6.0,
Dimethyl sulfoxide (DMSO) was dried over CaH , vacuum
2
distilled and stored for use. C, H, and N elemental analyzes
1
were performed using a Vanio-EL instrument. H NMR
1
3
and C NMR spectra were determined using a Unity Plus-
4
00-MHz spectrometer. ESI-HRMS was measured using a
Mariner instrument. UV-Vis titrations were measured using
a Shimadzu UV2600. Fluorescence spectra were measured
using an Eclipse fluorescence spectrophotometer (Agilent,
USA). The binding constant K was fitted by a nonlinear least
s
square method. All experiments were conducted on the basis
of pH = 7.4.
The cultured cells were seeded in 96-well plates at a
4
density of 2.0 × 10 cells/well and cultured. Different
SCHEME 1 Synthesis route for compounds

SHANG et Al.
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FIGURE 1 UV-Vis spectral changes
of compound 1(A), 2(C, D), 3(B) upon
the addition of Arg (A, B, C) and Lys
−
5
(
D). [compounds] = 4.0 × 10 mol/L;
−
3
[
Arg] = 0-1.6 × 10 mol/L; [Lys] = 0-
−
3
1
.4 × 10 mol/L
3
.4 Hz, 1H), 7.38 (s, 1H), 7.08 (d, J = 9.2 Hz, 1H). ESI-
intensity of absorption peak at 360 nm decreased gradually
and formed a trough at 345 nm. At the same time, a new
absorption peak at about 420 nm appeared and the intensity
increased. As a result, the red-shift phenomenon of com-
pound 2 appeared interaction with Arg. A clear isosbestic
point appeared at 398 nm, indicating that a stable complex
(2-Arg) had formed between host and guest. After the addi-
tion of lysine (Lys), the similar spectral change was observed
compared with Arg (Figure 1C) which showed compound 2
also interacted with Lys (Figure 1D). In addition, red-shift
phenomenon occurred at different degree, and one clear isos-
bestic point appeared which might be related to the space
+
HRMS (m/z): 439.1415 (M + Na) .
Compound 3: Diethylenetriamine (0.3301 g, 3.2 mmol)
was dissolved in anhydrous methanol (20 mL) and then
added slowly to the methanol solution (10 mL) containing
2
-hydroxy-1-naphthaldehyde (1.0503 g, 6.1 mmol). The
mixture was heated and stirred magnetically until completely
resolving. After the reaction was carried out for 45 minutes,
the color of the mixture solution changed from colorless to
orange. Then, the mixture solution was refluxing for 3 hours.
The mixture was stood and cooled at room temperature.
The yellow solid was filtered, washed twice with anhydrous
ethanol, and recrystallized from trichloromethane. The yel-
lowish solid (0.6769 g) was obtained. Yield: 51.9%. M.p.:
1
2
73~275°C. H NMR (400 MHz, DMSO-d , 298K) δ 13.98
6
(
s, 1H), 9.08 (d, J = 10.6 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H),
.70 (d, J = 9.4 Hz, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.38 (t,
7
J = 7.6 Hz, 1H), 7.16 (t, J = 7.4 Hz, 1H), 6.70 (d, J = 9.4 Hz,
1
H), 3.71 (dd, J = 10.9, 5.4 Hz, 2H), 3.37 (s, 1H), 2.90 (t,
+
J = 5.9 Hz, 2H). ESI-HRMS (m/z): 434.1843 (M + Na) .
3
3
RESULTS AND DISCUSSION
UV-Vis titration
|
.1
|
The binding abilities of three Schiff base compounds with
amino acids were determined by UV-Vis absorption spec-
troscopy at 298 K. The UV-Vis spectral of compound 2 with
Arg was shown in Figure 1. In the absence of Arg, the probe
FIGURE 2 UV-Vis spectral changes of compound 2
−
5
−5
2
(4.0 × 10 mol/L in DMSO-H O (1:1, v/v)) exhibited
2
(4 × 10 mol/L) upon the additions of 10 equiv of various amino acids
(2 × 10− mol/L) at the wavelength of 420 nm
2
an obvious peak at 360 nm. With the addition of Arg, the

4
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SHANG et Al.
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geometries of host–guest. Other amino acids were similarly
studied. However, the additions of histidine, asparagine,
threonine, tryptophan, proline, leucine, phenylalanine, ala-
nine, glycine, valine, methionine, aspartic acid, glutamic
acid, isoleucine, serine, cysteine, tyrosine, and glutamine
induced very weak spectral responses which indicating com-
pound 2 did not show any effect on these amino acids or
the binding abilities were very weak and could be ignored
3
.2
Fluorescent response
|
The photophysical responses of three compounds to the added
amino acids were also surveyed in DMSO-H2O (1:1, v/v) so-
lution. With the increasing of Arg, the fluorescence intensity
of compound 1 with 575 nm increased (Figure 3A). Similarly,
the fluorescence intensity of compound 3 centered at 460 nm
also strengthened upon the addition of Arg (Figure 3C). After
other amino acids (leucine, phenylalanine, alanine, glycine,
valine, methionine, histidine, tryptophan, aspartic acid, glu-
tamic acid, cysteine, proline, isoleucine, serine, threonine,
glutamine, tyrosine, asparagine, and lysine) were added, the
fluorescence responses were very small which indicated that
compounds (1 and 3) showed almost no binding abilities to-
ward these amino acids (Figure 4). Nevertheless, the fluores-
cence intensity of compound 2 was unchanged after Arg was
added (Figure 3B). That is to say, upon the addition of Arg,
(
Figure 2).
In order to research the difference of binding abilities
induced by the different substituents, the structures of this
kind of compounds can be tuned by changing the electron
property of the substituents on the –C=N– and the near
phenolic hydroxyl group position. Therefore, compounds 1
and 3 were also synthesized. UV-Vis spectra showed that
compounds 1 and 3 changed little or almost did not change
(
Figure 1A,B).
FIGURE 3 Fluorescence
response of compound 1(A), 2(B), 3(C)
4.0 × 10− mol/L) with the addition of Arg
5
(
(
0-2.0 × 10− mol/L)
3
FIGURE 4 Changes in fluorescence
intensity of compounds 1 (A) and 3 (B)
4.0 × 10− mol/L) in presence of 10 equiv
5
(
of amino acids tested

SHANG et Al.
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TABLE 1 The binding constant of the interaction between the
host and the guest
Amino acid
Ks (1) M−1
Ks (2) M−1
Ks (3) M−1
6
3
Arg
Lys
Leu
Phe
Ala
Gly
Val
Met
His
Try
Asp
Glu
Pro
Iso
<10
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
<10
ND
ND
ND
ND
ND
ND
(3.90 ± 0.38) × 10
(5.73 ± 0.32) × 10
ND
<10
<10
ND
ND
<10
<10
ND
ND
ND
<10
ND
ND
ND
<10
ND
<10
ND
<10
<10
ND
<10
<10
<10
<10
ND
ND
ND
ND
ND
ND
ND
ND
<10
FIGURE 5 Cell viability value (%) is compared with the
concentration. MCF-7 was incubated at 37°C for 24 h with a probe
at 0-150 μg/mL. In the absence of probes, cell viability (in%) was
calculated in view of 100% growth
only Arg, Lys, and His belong to basic amino acids, and their
basic chemical groups are guanidyl, amino, and imidazolyl.
Guanidyl has a relatively strong basicity and the imidazolyl
group has the smallest basicity. And other amino acids are
acidic amino acids and neutral amino acids; they are not easy
to react with compounds (1, 2, 3). So, the binding ability of the
synthesized compounds to other amino acids was very weak.
Ser
Thr
Gln
Cys
ND: the spectral changes were very small and could not be determined.
1
Data analysis of UV-Vis, fluorescence, H NMR titrations
the UV-Vis of compound 2 changed; however, there was no
and binding constants showed that compounds 1 and 3 gave
negligible response to Arg. Despite significant response was
observed in the corresponding fluorescent titrations, com-
pound 2 had the strongest binding capacity for Arg.
fluorescence response of it. While the fluorescence spectra of
1
and 3 changed, their UV-Vis spectra did not change.
The fluorescence changes of compounds may be related
[
26]
to free energy. In the structure of the compounds, the re-
ceptor was separated from the fluorophore by two interacting
site –OH spacers. Therefore, the electron may be transferred
from –OH to –C=N– after interaction between compounds
3
.4
Cytotoxicity assay
|
Studies have shown that glutathione peroxidase (GPx1) and
selenium binding protein (SBP1) are two important seleno-
protein that are expressed in most tissues. However, GPx1
is not expressed in human breast cancer cells (MCF-7), so a
1
and Arg. The evidence came from H NMR titrations of
compounds with Arg. When Arg was added to compounds,
+
the loss of proton (H ) in compound 1 was derived from the
[
31]
phenolic hydroxyl group on the benzene, and the loss of pro-
special cell line MCF-7 was chosen. The MTT assay was
+
ton (H ) in the compound 2 was came from the phenolic hy-
used to evaluate the cytotoxicity of drugs on human breast
1
[32]
droxyl group on the naphthalene. But, the H NMR spectrum
cancer cell lines (MCF-7 cells).
When the concentration
of compound 3 did not change after Arg was added.
of probes was in the range (0-150 μg/mL), the proliferation
of cells did not appear much difference (Figure 5). Probes
(
<150 μg/mL) were found to be 80% viable after the incuba-
3
.3
Binding constant
|
tion was 24 hours. Based on the above analysis, compound 2
showed high binding ability and low cytotoxicity, and could
be used for in vivo detection of Arg.
The job-plot analysis showed that the ratios between com-
pounds 1, 2, 3, and Arg were 1:1, 1:2, and 2:3, respectively
(
Figure S10). The obtained binding constants were listed in
Table 1. The nonlinear least square method was fitted accord-
[27–29]
ing to UV-Vis data and fluorescence data.
From Table 1,
4
CONCLUSION
|
the probes showed the strongest binding ability with Arg
among all tested amino acids. The reason may be that based
on the C=N and –OH, the probes can interact with the guani-
In conclusion, the three naphthalene-based derivatives herein
showed high selectivity and sensitivity to Arg among natural
amino acids. Compound 2 demonstrated the strongest binding
[30]
dine group of Arg. However, among twenty amino acids,

6
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SHANG et Al.
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capacity for Arg and low cytotoxicity to MCF-7 cells, so it
can be used to detect Arg in vivo. This work broadens the
application fields of probes and provides the basis for clinical
application.
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ACKNOWLEDGMENTS
This work was supported by the Fund of Science &
Technology Innovation Talents in Universities of Henan
Province (15HASTIT039), Fluorescence Probe and
Biomedical Detection Research Team of Xinxiang City
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How to cite this article: Shang X, Li C, Li J, Chen Y,
Chen H, Wang T. The arginine detection and
cytotoxicity of fluorescent probes based on
naphthalene derivatives. Heteroatom Chem.
2
5
2
018;29:e21449. https://doi.org/10.1002/hc.21449
2