A highly selective and sensitive fluorescent turn-off probe for Cu2+ based on a guanidine derivative

Article abstract of DOI:10.3390/molecules22101741

A new highly selective and sensitive fluorescent probe for Cu²⁺, N-n-butyl-4-(10-cyclooctene-10,30,60-triazole)-1,8-naphthalimide (L), was synthesized and evaluated. The structure of compound L was characterized via IR, ¹H-NMR, ¹³C-NMR and HRMS. The fluorescent probe was quenched by Cu²⁺ with a 1:1 binding ratio and behaved as a "turn-off" sensor. An efficient and sensitive spectrofluorometric method was developed for detecting and estimating trace levels of Cu²⁺ in EtOH/H₂O. The ligand exhibited excitation and emission maxima at 447 and 518 nm, respectively. The equilibrium binding constant of the ligand with Cu²⁺ was 1.57 × 10 ⁴ M ^-1, as calculated using the Stern-Volmer equation. Ligand L is stable and can be used to detect Cu²⁺ in the range of pH from 7 to 12. The sensor responded to Cu²⁺ rapidly and a large number of coexisting ions showed almost no obvious interference with the detection.

Full text of DOI:10.3390/molecules22101741

molecules
Article
A Highly Selective and Sensitive Fluorescent
2+
Turn-Off Probe for Cu Based on a
Guanidine Derivative
ID
Fei Ye, Qiong Chai, Xiao-Min Liang, Ming-Qiang Li, Zhi-Qiang Wang and Ying Fu *
Department of Applied Chemistry, College of Science, Northeast Agricultural University, Harbin 150030, China;
yefei@neau.edu.cn (F.Y.); chaiqiong01@163.com (Q.C.); abcliangxiaomin@163.com (X.-M.L.);
mqli@neau.edu.cn (M.-Q.L.); wzq19910101@163.com (Z.-Q.W.)
*
Correspondence: fuying@neau.edu.cn; Tel.: +86-451-5519-0070
Received: 16 September 2017; Accepted: 13 October 2017; Published: 16 October 2017
2
+
Abstract:
A
new highly selective and sensitive fluorescent probe for Cu ,
0
0
0
0
N-n-butyl-4-(1 -cyclooctene-1 ,3 ,6 -triazole)-1,8-naphthalimide (
The structure of compound
L
), was synthesized and evaluated.
was characterized via IR, H-NMR, C-NMR and HRMS.
The fluorescent probe was quenched by Cu with a 1:1 binding ratio and behaved as a “turn-off”
sensor. An efficient and sensitive spectrofluorometric method was developed for detecting and
estimating trace levels of Cu2 in EtOH/H O. The ligand exhibited excitation and emission maxima
at 447 and 518 nm, respectively. The equilibrium binding constant of the ligand with Cu was
is stable and can be used
to detect Cu2 in the range of pH from 7 to 12. The sensor responded to Cu rapidly and a large
1
13
L
2+
+
2
2+
4
−1
+
1
.57 × 10 M , as calculated using the Stern-Volmer equation. Ligand
L
2+
number of coexisting ions showed almost no obvious interference with the detection.
Keywords: fluorescence; naphthalimide; guanidine; octatomic ring; copper ion
1
. Introduction
Fluorescent sensors for chemical species used in biological and environmental detection are
currently an attractive field of research [
much attention because these metal ions not only play important roles in various physiological
processes but also have distinctly toxic impact on human health [ ]. It is highly desired to detect
these in relevant systems. The importance of metal ion sensing can never be overestimated because
of their widespread distribution in environmental systems and biological processes [ ]. Copper is
1–3]. In particular, pollution by heavy metal ions has attracted
4
5
a significant metal pollutant because of its widespread application and is also an essential trace
element in biological systems. Cu2 is present in various biological processes, as the third most
abundant transition metal ion in the human body. In addition, its homeostasis is critical for the
+
metabolism and development of living organisms [
a catalytic cofactor for a variety of metalloenzymes, including superoxide dismutase, cytochrome
oxidase and tyrosinase [
10]. The misregulation or excess accumulation of Cu2+ could cause serious
diseases [11]. Its concentration in the neuronal cytoplasm may contribute to the etiology of Alzheimer’s
6–8
]. For example, Cu2+ plays a critical role as
9,
0
2+
or ParkinsoN s disease [12–14]. However, exposure to high levels of Cu will cause gastrointestinal
2+
disturbance and even liver or kidney damage. The detection of Cu is meaningful for both disease
diagnosis and environmental monitoring [15]. The normal average concentration of Cu2+ in blood is
in the range of 15.7–23.6
in drinking water of 20
2
+
µ
M [16]. The U.S. Environmental Protection Agency (EPA) set a limit for Cu
M [17]. The detection of Cu2+ levels in water requires an efficient, selective
µ
and highly sensitive chemosensor [18,19].
Molecules 2017, 22, 1741; doi:10.3390/molecules22101741
www.mdpi.com/journal/molecules

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Numerous methods to detect metal ions have been developed, such as atomic absorption
spectrometry [20], inductively coupled plasma atomic emission spectroscopy [21], visual
detection [22,23], chemiluminescence detection [24] and electrochemical techniques [25,26]. However,
these methods have disadvantages, such as expensive instrumentation, complicated sample
preparation and tedious operating procedures, resulting in the need for other sensitive, simple detection
methods for copper ions. Fluorescent molecules offer many advantages over other systems in this area
as a result of their emissive behavior, easy signal detection, sensitivity, selectivity and low cost [27,28].
The development of the fluorescence method has been demonstrated to be promising because of
its advantages of wide applicability, ease of manipulation, high selectivity, sensitivity, rapidity,
nondestructive methodology and direct visual perception [29,30]. Thus, a number of high-performance
2+
sensors have been developed to detect metal ions [31]. Cu is a typical ion with a pronounced
quenching effect on fluorophores via mechanisms that are inherent to the paramagnetic species [32 35].
–
2+
Many excellent chemosensors have been reported for Cu in the past few years. However,
many of these sensors only exhibit sensitive and selective Cu2+ detection in pure organic
systems (Table 1) [36
–
38]. Hence, we designed and synthesized a new fluorescent sensor,
0
0
0
0
N-n-butyl-4-(1 -cyclooctene-1 ,3 ,6 -triazole)-1,8-naphthalimide, based on the transformation of
thiourea into guanidine derivatives, as shown in Scheme 1. This novel compound has a highly
sensitive fluorescent response toward Cu2+ in mixed aqueous solutions.
Table 1. Comparison of the recently reported sensors for the determination of Cu2+ ions.
Operation Mode
Solvent
CH CN
λex/λem (nm) Interference References
Turn-off
Turn-on
Turn-off
Turn-off
364/420
365/475
480/518
437/518
None
Zn(II)
None
None
[36]
[37]
[38]
3
CH CN
3
MeOH
EtOH/H O
This work
2
Scheme 1. Thiourea transformation into guanidine.
2
. Results and Discussion
2
.1. Synthesis and Characterization
Compound was synthesized through the route outlined in Scheme 2. The structure modification
was focused on the C-4 position. A dihydroxyethylamino moiety was introduced to replace the
Br atom, and the subsequent reaction modified the -OH group to N ; compound was obtained
L
4
3
in a 67% yield from the amination. Thiourea is easily transformed into guanidine; however,
the conversion from thiourea to guanidine has attracted little attention as a potential fluorogenic
reaction because most thiourea derivatives have the same color or fluorescence as their guanidine
analogues [39]. Initially, we envisioned that a similar molecular recognition/reactivity motif might be
incorporated into fluorophores to serve as a ratiometric fluorescent chemodosimeter for the selective
2+
detection of Hg , as reported in the literature [40], because compound
The expected thiourea derivative was not achieved; however, the product transformed into the inner
guanidine ring because compound contained a primary amine subunit. Compound was obtained
4 has two free amino groups.
4
L
through intramolecular cyclization with a recognition/reactivity motif that could be incorporated into
fluorophores so that metal ion addition led to a variation of the fluorescence.
Compound
L
was obtained with a yield of 57% in the final reaction, and its structure was
1
13
characterized by H-NMR, C-NMR and HRMS. As evidenced by the signals at 6.57–8.42 ppm in

Molecules 2017, 22, 1741
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1
the H-NMR spectrum, the number of Ar-H is 10, which indicated that benzoyl isothiocyanate had
reacted with compound 4. The signals at δ = 9.02 ppm indicated the existence of benzamide, and the
signals at
δ
= 1.26 ppm showed that -NH was substituted. Furthermore, the molecular ion peak in
2
+
the HRMS-ESI ([M + H] , 484.2332 m/z) spectrum was consistent with the theoretically calculated
molecular mass of compound L.
Scheme 2. Synthetic route to compound L.
2
.2. Spectral Characteristics
The photophysical characteristics of compound
L in EtOH/H O (4:1 v/v) were investigated.
2
Figure 1 shows the excitation (
The Stokes shift ( A ), an important parameter indicating the difference in properties and structure
between the ground state (S ) and first excited state (S ), was calculated by Equation (1) to be 3062 cm
λ = 447 nm) and emission (λ = 518 nm) spectra of compound L.
υ
–υ
F
−
1
.
0
1
υ − υ ) = (1/λ − 1/λ ) × 10 7cm
−
−1
(1)
(
A
F
A
F
The ability of the molecules to emit absorbed light energy is characterized quantitatively by the
fluorescence quantum yield (ΦF). The quantum yields of fluorescence were calculated using coumarin
6
G (Φsample = 0.94) in EtOH as the standard according to Equation (2), where A , S , and n and
ref ref ref
Asample, Ssample, and nsample represent the absorbance at the exited wavelength, the integrated emission
band area and the solvent refractive index of the standard and the sample, respectively [41,42]. Thus,
the fluorescence quantum yield of probe L in EtOH/H O (4:1 v/v) solution was 0.06.
2
!
!
!
Ssample
Sre f
Are f
Asample
n2
sample
Φ = Φ
F
(2)
ref
n2
re f
The photophysical properties of compound
L
as a ligand in the presence of different metal cations
were investigated in EtOH/H O (4:1 v/v) to determine its potential application as a sensor for detecting
2
these metal ions. The binding behavior of compound
L toward different metal ions such as their
corresponding chlorides, acetates, sulfides or nitrates was studied using fluorescence spectroscopy.
The anions did not affect the spectroscopic results.

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Figure 1. Excitation and emission spectra of compound L (10 µM) in EtOH/H O (4:1 v/v).
2
2+
To evaluate the selectively of compound
addition of various metal ions under the same conditions were investigated. The fluorescence spectrum
of compound exhibited strong fluorescence emission when excited at 447 nm in EtOH/H O (4:1 v/v;
Figure 2). Under the same conditions as those used for adding the metal ions, significant fluorescence
L for Cu , changes in the fluorescence intensity upon the
L
2
2+
changes were observed in the presence of Cu . Fluorescence quenching was observed immediately after
2+
Cu was added. When other metal ions were added, the fluorescence exhibit few changes. As shown
in Figure 2, remarkable color changes ranging from yellow to colorless and yellow floccules floating
in the solution could be distinguished by the naked eye. In addition, the introduction of Cu2+ to the
mixture solution of compound
L led to evident fluorescence quenching, while the rest of the metal cations
exerted a negligible or very small influence under identical conditions, which was further evidenced by
the illumination taken under a 365 nm portable UV lamp. On the basis of the literature, the fluorescence
2+
quench of probe L might have been due to the coordination to paramagnetic Cu [43].
Figure 2. Fluorescence changes of compound
cations (50 µM) in EtOH/H O (4:1 v/v).
L (10 µM) in the absence (CK) and presence of metal
2

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2
.3. Calculation of Binding Constants and Different Concentrations of Cu2+
A Job plot was generated by continuously varying the mole fraction of Cu from 0 to 1 in
2+
2+
a solution of [L-Cu ] with a total concentration of 50 µM to determine the stoichiometry of the
[L-Cu ] complex. The Job plot analysis revealed an approximate maximum at a 0.5 mole fraction,
which indicated a 1:1 stoichiometry of the ligand L and Cu complex (Figure 3).
2+
2+
Figure 3. Job plot to determine the stoichiometry of the [L-Cu2+] complex in solution.
To better understand the sensing process, changes in the fluorescence spectra of compound
L were
2+
measured along with the binding constant and different concentrations of Cu . The binding constant
was calculated using the Stern-Volmer equation, I /(I0 I) = 1/A + 1/KA 1/[Q], on the basis of a 1:1
stoichiometry, where I is the fluorescence intensity of the free ligand , I is the fluorescence intensity of
-Cu complex, Q is [Cu ], A is a constant and K is a binding constant [44]. When the reciprocal
of I /(I − I) was plotted as a function of the 1/[Q] concentration, a linear relationship was obtained,
−
·
0
L
0
2+
2+
the
L
0
0
(
y = A + Bx), y = I /(I − I), A = 1/A, B = 1/KA, x = 1/[Q], and K was calculated from A/B (Figure 4).
0
0
2+ 4
−1
10 M in the
Therefore, K, the binding association constant for Cu , was estimated to be 1.57
EtOH/H O (4:1 v/v) solution, as inferred from the fluorescence titration curves of compound
×
2+
L
with Cu .
2
Figure 4. Stern-Volmer plot for calculating the binding constant of the chemosensor with Cu2+
.

Molecules 2017, 22, 1741
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2+
Upon the addition of Cu , the fluorescence was quenched. The changes in the fluorescence
− −1
5
2+
emission intensities of compound
(
L
(1
×
10 mol L ) with different concentrations of Cu
−
4
−1
0–3 × 10 mol L ) are presented in Figure 5. A progressive decrease in the fluorescence intensity
2+
was observed. The plot of the fluorescence intensity versus externally added Cu (Figure 6) reaches
2+
saturation after a certain amount of externally added Cu . Curvilinearity was obtained for up to a
− −1
5
2+
value of 6 times (6
×
10 mol L ) the amount of externally added Cu . The results indicated that
compound L could detect Cu at a micromole level.
2+
Figure 5. Changes in the fluorescence spectra of compound
function of added [Cu2+].
L (10 µM) in EtOH/H O (4:1 v/v) as a
2
Figure 6. Fluorescence intensity vs Cu2+; inset shows that up to 6-fold Cu2+ curvilinearity was
sustained; λex: 437 nm; λem: 518 nm; slit width: 10 nm.
2
.4. Response Time
The response time is a significant practical parameter [45]. Time-dependence of probe
L
for
sensing Cu2+ was examined and the results are illustrated in Figure 7. Upon the addition of Cu ,
2+
the fluorescence intensity decreased to the minimum immediately and tended to be stable during the

Molecules 2017, 22, 1741
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test time. No changes in the fluorescence of probe
increased time. This showed that probe L exhibited a reliable rapid response to Cu .
L
in the absence of Cu2+ were detected with the
2+
Figure 7. Response time of 10
v/v); λex: 437 nm; λem: 518 nm; slit width: 10 nm.
µM probe L in the absence or presence of 50 µ
M Cu2+ in EtOH/H O (4:1
2
2
.5. Effect of pH
In order to investigate the application of probe
L
within complex media, the fluorescence spectra
−
5
−1
2+
response of probe
L
was measured in the absence or presence of 5
×
10 mol L Cu under
did not reveal
any clear differences over a pH range of 2–12, which was consistent with the goal of our experimental
different pH conditions. As shown in Figure 8, the fluorescence intensity of probe
L
2+
design. The fluorescence of the [
L-Cu ] solution could not remarkably decrease when the pH values
were lower than 5, possibly because the imino groups of probe
resulting in weak coordination with Cu . In addition, the fluorescence intensity of the [
L
were protonated at acid conditions,
2+
2+
L-Cu ]
solution showed a 5-fold change from pH 4 to pH 7, which suggested that it is sensitive to pH changes.
2+
These results indicated that probe L could be used to detect biological as well environmental Cu in
the range of pH from 7 to 12.
Figure 8. Effect of pH on the fluorescence intensity of 10
0 µM Cu2+
µM probe L in the absence or presence of
5
.

Molecules 2017, 22, 1741
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2
.6. Selectivity
The selectivity is clearly one of the most important characteristics of a cation-selective chemosensor.
2+
Thus, the influence of other metal ions on the fluorescence intensity of the proposed Cu chemosensor
was investigated (Figure 9). In this study, Ag , Na , K , Ba , Ca , Co , Mg , Mn , Ni , Pb , Sn ,
Zn , Al , Cu , Hg and Fe (100
+
+
+
2+
2+
2+
2+
2+
2+
2+
2+
2+
3+
+
2+
3+
2+
µM) were added to the compound L (10 µM)/Cu (50 µM) solution,
and the fluorescence was observed. The fluorescence emission intensity at 518 nm significantly decreased
2
+
2+
as a result of binding with Cu . The coexisting ions showed little influence on the detection of Cu .
Common anions, including oxalate, sulfate, nitrate and chloride, did not interfere with the fluorescence
2+
intensity of the ligand or [
L
-Cu ] complex. Compared to the other common cations, compound
exhibited good selectivity for Cu , indicating that
L
2
+
2+
L
has a good anti-interference ability in detecting Cu .
Figure 9. Interference of foreign cations. Solvents: EtOH/H O (4:1 v/v).
2
2
.7. Thermal Studies
The stability of probe
L
and its Cu2+ complex was studied by thermogravimetry to prove the binding
L with Cu ions, and the results are presented in Figures 10 and 11, respectively. The thermal
2+
of ligand
2+
◦
◦
stability of the Cu complex (up to 324.76 C) was better than that of the free ligand (up to 288.57 C).
Figure 10. Thermal studies of the fluorescence chemosensor.

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Figure 11. Thermal studies of the [L-Cu2+] complex with the fluorescent chemosensor.
2
.8. IR Spectra
The structural matching played an important role in selective recognition. According to the
examples in the literature, a similar mode of probes containing a tridentate structure binding with
2+
Cu has been reported [46
Cu , IR spectroscopy was employed to elucidate the possible coordination mode. The IR spectra
,
47]. To further study the possible binding mode of compound
L and
2+
2+
of compound
(Figure 12a) has two slender peaks appearing between 3350 and 3290 cm . One of the extremely
sharp peaks in this range vanished when the complex was formed (Figure 12b) and only appeared at
L and the [L-Cu ] complex are compared in Figure 12. The IR spectra of compound
−
1
L
−
1
3
325.72 cm . This result suggested that the aromatic amide was not involved in the coordination of
2+
[L-Cu ]. However, the extremely sharp peak in the IR spectra corresponding to the imine disappeared.
For fluorescence quenching, the N atom directly connected to naphthalene must be present in the
coordination of the complex. On the basis of the aforementioned fact, a plausible binding mode of the
complex was proposed, as shown in Scheme 3.
Figure 12. IR spectra of compound L before (a) and after (b) the addition of Cu2+
.

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Scheme 3. Proposed coordination of compound L combined with Cu2+
.
3
. Experimental
3
.1. Chemicals and Instruments
All of the solvents and reactants were commercially available and used without purification.
The IR spectra were recorded on a Bruker ALPHA-T spectrometer (KBr, BRUKER Inc., Beijing,
China). The melting points were determined using a Beijing Taike melting point apparatus (X-4)
1
13
and were uncorrected, and H-NMR and C-NMR spectra were recorded on a Bruker AVANVE 300 or
00 MHz nuclear magnetic resonance spectrometer (Energy Chemical, Shanghai, China) using CDCl3
4
or DMSO-d as the solvent and TMS as the internal standard. The high-resolution mass spectrometry
6
was recorded on a FT-ICR MS spectrometer (BRUKER Inc., Beijing, China). Fluorescence emission
spectra were obtained using a PerkinElmer LS55 fluorescence spectrometer (PerkinElmer, Liantrisant,
UK). The pH values were measured using a PHS-3C pH meter (Inesa, Shanghai, China). Thermal
analysis was performed using a 449F3 thermal analyzer (Netzsch, Bavaria, Germany). The spectrogram
data of the synthesized compounds could be found in the Supplementary Materials.
3
.2. Testing Methods
Compound
−
3+
3
2+
+
+
+
L
was dissolved in EtOH to form a 10 M stock solution. Cu , Ag , Na , K ,
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
3+
Ba , Ca , Co , Mg , Mn , Ni , Pb , Sn , Zn , Al , Hg and Fe sources were dissolved in
deionized water (H O) to obtain 10 M stock solutions. For the fluorescence measurement of Cu ,
−
2
2+
2
the mixed stock solutions were diluted to 10 mL with EtOH and H O to form EtOH/H O (4:1 v/v)
2
2
solutions. For the titration experiments, compound
L
stock solution (100
µ
L) was mixed with a certain
amount of Cu2 stock solution and diluted to 10 mL with EtOH and H O to form EtOH/H O (4:1
+
2
2
−
1
v/v) solutions. The wide pH range solutions were prepared by adjustment of 0.05 mol L Tris-HCl
solution with HCl or NaOH solution. Thermal studies of compound
2+
L and its [L-Cu ] complex were
2+
also performed to prove the binding of the ligand with Cu . All fluorescence spectra were recorded at
◦
2
5 C with the excitation wavelength set at 437 nm and the slit width set at 10 nm.
3
.3. Synthesis of N-n-Butyl-4-bromo-1,8-naphthalimide (1)
N-n-Butyl-4-bromo-1,8-naphthalimide was prepared according to the reported method [48].
-Bromo-1,8-naphthalic anhydride (16.1 g, 58 mmol) and n-butyl amine (60 mmol, 4.4 g) were heated
4
under reflux in EtOH (250 mL) with vigorous stirring for 12 h under N . Then, the mixture was cooled,
2
and the precipitate was filtered and recrystallized from ethanol to yield 13.5 g (84%) of a light-yellow
◦
1
product; m.p.: 105–106 C; H-NMR (DMSO-d , 300 MHz)
δ
(ppm): 8.46–8.52 (m, Ar-H, 2H), 8.27 (d,
6
J = 7.9 Hz, Ar-H, 1H), 8.16 (d, J = 7.9 Hz, Ar-H, 1H), 7.92–7.97 (m, Ar-H, 1H), 4.01 (t, J = 7.3 Hz, N-CH2,
2
H), 1.56–1.66 (m, CH , 2H), 1.31–1.39 (m, CH , 2H), and 0.93 (t, J = 7.3Hz, CH , 3H).
2 2 3
0 0
.4. Synthesis of N-n-Butyl-4-N ,N -dihydroxyethyl-1,8-naphthalimide (2)
3
0
0
N-n-butyl-4-(N ,N -dihydroxyethyl)amino-1,8-naphthalimide was obtained using a procedure
similar to that reported by Guo et al. [49]. N-n-butyl-4-bromine-1,8-naphthalimide (15 g, 45.2 mmol)
and diethanolamine (75 mL) were mixed in ethylene glycol monomethyl ether (100 mL). The mixture
was refluxed for 6 h. The crude product was purified by column chromatography using silica gel,
with EtOAc and light petroleum (1:4 v/v) as the eluent. Compound 2 was obtained as a yellow solid

Molecules 2017, 22, 1741
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◦
1
(
3.28 g) in a 20% yield; m.p.: 129–130 C; H-NMR (400 MHz, CDCl )
δ
(ppm): 8.87 (d, J = 8.8 Hz,
3
Ar-H, 1H), 8.52 (d, J = 7.2 Hz, Ar-H, 2H), 7.66 (t, J = 8.0 Hz, Ar-H, 1H), 7.38 (t, J = 8.0 Hz, Ar-H, 1H),
4
.14 (t, J = 7.2 Hz, CH , 2H), 3.87 (t, J = 5.2 Hz, CH , 4H), 3.63 (t, J = 5.2 Hz, CH , 4H), 2.81 (s, OH,
2 2 2
13
2H), 1.68–1.69 (m, CH , 2H), 1.43–1.45 (m, CH , 2H), 0.97 (t, J = 7.2 Hz, CH , 3H); C-NMR (100 MHz,
2 2 3
CDCl3)
δ (ppm): 164.34, 163.91, 154.28, 131.18, 131.22, 130.85, 130.14, 127.24, 125.65, 122.92, 117.18,
1
17.05, 59.73, 59.73, 55.33, 55.33, 40.10, 30.20, 20.38, and 13.85.
0 0
.5. Synthesis of N-n-Butyl-4-N ,N -diaminoethyl-1,8-naphthalimide (4)
3
0
0
N-n-butyl-4-N ,N -diaminoethyl-1,8-naphthalimide was prepared according to the reported
procedure [50]. Compound (2.0 g, 5.6 mmol), PPh (4.41 g, 16.8 mmol) and NaN (1.82 g, 28.0 mmol)
were dissolved in anhydrous N,N-dimethylformamide (100 mL), the solution was cooled to 0 C, and
2
3
3
◦
CBr (5.58 g, 16.8 mmol) was added portionwise over 10 min. Then, the reaction temperature was
4
raised to room temperature, and the mixture was stirred for 12 h. The reaction mixture was diluted
with water and extracted with ethyl acetate (4
×
50 mL). The organic layer was dried over anhydrous
Na SO and concentrated in vacuo. The crude compound was separated through silica gel column
2
4
chromatography and afforded compound
8.1 mmol) were dissolved in 50 mL of pyridine and kept at room temperature for 1 h. Concentrated
ammonium hydroxide (10 mL) was added, and the mixture was allowed to stand for an additional
h. Pyridine was removed through reduced pressure. Then, CH Cl and 1 N HCl were added to the
3. The compound 3 (1.9 g, 4.67 mmol) and PPh (7.36 g,
3
2
2
2
2
reaction mixture. The organic layer was washed with additional 1 N HCl, and the aqueous layers were
combined, neutralized with KOH, and extracted with CH Cl . The combined organic layers were
2
2
washed with water and brine and then dried over anhydrous Na SO to afford compound
6
4
(1.11 g,
(ppm): 8.74 (d, J = 0.3 Hz,
Ar-H, 1H), 8.43 (d, J = 7.8 Hz, Ar-H, 1H), 8.27 (d, J = 8.4 Hz, Ar-H, 1H), 7.68 (t, J = 8.1 Hz, Ar-H, 1H),
.81 (d, J = 8.4 Hz, Ar-H, 1H), 4.01 (t, J = 7.8 Hz, CH , 2H), 3.44–3.48 (m, CH , 2H), 2.87 (t, J = 5.7 Hz,
2
4
◦
7%) as a yellow solid; m.p.: 99–101 C; H-NMR (DMSO-d , 300 MHz) δ
6
1
6
2
2
CH , 2H), 2.65–2.72 (m, CH , 4H), 1.84 (s, NH , 4H), 1.54–1.62 (m, CH , 2H), 1.30–1.37 (m, CH , 2H),
2
2
2
2
2
+
and 0.92 (t, J = 7.5 Hz, CH , 3H). HRMS-ESI m/z: found 355.2123 for [M + H] and calculated 354.2056
3
for C H N O .
20
26
4
2
0 0 0 0
.6. Synthesis of N-n-Butyl-4-(1 -cyclooctene-1 ,3 ,6 -triazole)-1,8-naphthalimide (L)
3
0
0
0
0
N-n-butyl-4-(1 -cyclooctene-1 ,3 ,6 -triazole)-1,8-naphthalimide was obtained using a procedure
similar to that reported in the literature [40]. Benzoyl isothiocyanate (0.92 g, 5.6 mmol) was added
0
0
dropwise to a solution of N-n-butyl-4-N ,N -diaminoethyl-1,8-naphthalimide (1.0 g, 2.8 mmol) in
0 mL of ketone and was stirred at 45 C. The reaction mixture was stirred continuously for 45 min.
◦
3
After cooling, the solution was filtered, and the filter cake was washed with EtOH. The crude product
◦
was purified by chromatography to afford compound
L
(0.77 g, 57%) as a green solid; m.p.: 165–167 C;
1
H-NMR (CDCl , 300 MHz)
δ (ppm): 9.02 (s, NH, 1H), 8.29–8.42 (m, Ar-H, 4H), 7.54 (d, J = 6.6 Hz,
3
Ar-H, 1H), 7.46 (t, J = 7.5 Hz, Ar-H, 3H), 6.57 (d, J = 8.7 Hz, Ar-H, 2H), 4.12 (t, J = 7.5 Hz, CH , 4H),
2
3
.60–3.86 (m, CH , 4H), 3.55–3.61 (m, CH , 2H), 1.63–1.70 (m, CH , 2H), 1.38–1.45 (m, CH , 2H); 1.26 (s,
2
2
2
2
13
CNHC, 1H), 0.95 (t, J = 7.5 Hz, CH , 3H). C-NMR (75 MHz, CDCl3)
δ
(ppm): 164.72, 164.26, 164.26,
3
150.97, 150.97, 134.24, 134.24, 131.02, 131.02, 129.54, 129.54, 128.35, 128.35, 128.35, 128.35, 124.25, 122.59,
1
4
10.26, 103.13, 103.13, 45.86, 41.31, 41.24, 41.05, 39.93, 30.32, 20.44 and 13.88. HRMS-ESI m/z: found
+
84.2332 for [M + H] and calculated 483.5616 for C H N O .
28
29
5
3
4
. Conclusions
In summary, a new, rapidly responsive, colorimetric and “turn-off” fluorescent Cu sensor,
2+
0
0
0
0
N-n-butyl-4-(1 -cyclooctene-1 ,3 ,6 -triazole)-1,8-naphthalimide, was successfully synthesized and
characterized. Probe
2+
L exhibited a markedly quenched fluorescence in the presence of Cu with
a simultaneous color change (yellow to colorless) over a range of metal cations. The detection was
highly sensitive and more selective than some reported probes and almost unaffected by the common

Molecules 2017, 22, 1741
12 of 14
2+
coexisting metal ions [15,51]. In addition, Probe L showed a 1:1 stoichiometry (sensor/Cu ) with a
high binding constant. This sensor should be valuable for copper analysis in environmental samples
and biological systems.
Supplementary Materials: Supplementary data associated with this article can be found in the online.
Acknowledgments: This work was supported by the Research Science Foundation in Technology Innovation of
Harbin (2015RAYXJ010).
Author Contributions: Y. Fu and M.Q. Li developed the concept of the work. Z.Q. Wang and X.M. Liang carried
out the synthetic work. Q. Chai conducted the fluorescence properties assay. Q. Chai and F. Ye wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors’ lab.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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