# Design and Synthesis of a Dinuclear Copper(II) Probe for Selective Fluorescence Sensing of Pyrophosphate

Authors: Jinhe Xu; Jing Li; Chenxi Liu; Linlin Yang; Guangjie He; Tianjun Ni; Aiying Fan; Songjun Wang; Qingzhi Wang
Journal: Journal of Sensors
 (2019)

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## Abstract

A novel coumarin-based compound DPAC with two dipicolylamine (DPA) arms as the chelator sites was designed and synthesized. The compound DPAC exhibits a highly selective response to Cu2+ ions with a distinctly emission-quenching phenomenon. Moreover, the in situ formed complex DPAC-Cu2+ was used for the detection of pyrophosphate (PPi). The binding manner of probe DPAC-Cu2+ with PPi in 1 : 1 stoichiometry was supported by the Benesi-Hildebrand fitting, ESI-MS and HPLC analysis. The linear range of PPi concentration was 1-4 μM, and the detection limit was 0.53 μM. The competing experiments illustrated that the probe DPAC-Cu2+ had good sensitivity and selectivity for PPi than other anions, including ATP, ADP, AMP, and Pi in CH3CN : HEPES (3 : 2, v/v, pH=7.20) buffer. Further, cell fluorescence imaging experiments indicated that the probe DPAC-Cu2+ had a potential to be used to detect PPi in vivo.


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## Body

## 1. Introduction

Anions, widely spread in nature, play a fundamental role in biological, environmental, and chemical fields [1, 2]. Anions recognition and sensing have become one of the extremely promising topics in current research [3, 4]. Recently, considerable effort has been devoted to the design of probes that have the ability to selectively bind and recognize biologically essential anions with the output of spectra signals [5–8]. Biological phosphate anions, such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP), were essential to control the cellular movements and metabolic processes in the living organisms [9, 10]. The selective recognition of adenine-based nucleotides was considered to be meaningful as they were associated with the health status of biological cells. In particular, pyrophosphate (PPi), as the hydrolysis product of ATP, was formed by the combination of two phosphate units under cellular conditions [11]. The detection of PPi has successfully been used in a real-time DNA-sequencing method [12]. And the alteration of PPi concentrations in cells could also be employed to detect or diagnose many diseases. For instance, the high intracellular level of PPi in knee synovial fluid inhibited apatite deposition, which caused the calcium pyrophosphate dehydrate (CPPD) crystal deposition disease [13]. Recently, intracellular PPi in low concentration has become an important indicator for early cancer research [14, 15]. Therefore, the development of high-efficient probes for the detection and discrimination of PPi has become an attractive and challenging task in recent years [16–20].Among the variety of methods for detecting PPi, fluorescent chemosensors have attracted considerable attention because of their superiority in high sensitivity and selectivity, low cost, and fast detection [21]. The sensors, which selectively recognize the PPi anions, often rely on the interaction of hydrogen bonding, electrostatic, and coordination bonds (or combinations of these). In particular, the metal complexes based on strong coordination ability with anions were considered to be ideal candidates for PPi recognition [22]. Many of previously reported Cu(II)-based PPi sensors detected the PPi anions by releasing quenching copper(II) ion [23–27]; the probes based on PPi binding recognition mechanism were still rare [28–31].In recent years, a number of highly effective fluorescence probes based on coumarin derivatives have been developed due to their high fluorescence quantum yield and large stokes shift [32, 33]. And the spectra of coumarin derivatives could be easily modified to fall well within the visible range. Meanwhile, metal complexes based on dipicolylamine (DPA) units were known as good affinity groups to selectively bind multianionic phosphorylated biomolecules in the outer surfaces of cell membranes [34–36]. The configuration of dinuclear metal complexes is propitious to the binding action and display high affinity toward PPi, which could be a good candidate for PPi sensing [37–40]. Herein, we reported a new fluorescence probe (complex DPAC-Cu2+), a dinuclear copper ions complex for PPi, which was synthesized based on the coumarin and DPA moieties. This complex exhibited weak fluorescence, but the addition of 1 equiv. of PPi caused a significant recovery of the fluorescence intensity. The probe showed a high sensitivity and selectivity for PPi via fluorescence “turn-on” manner over other anions, including Pi, ATP, ADP, and AMP, and the recognition mechanism of DPAC-Cu2+ to PPi was also demonstrated. Furthermore, cell fluorescence imaging experiments indicated that the probe DPAC-Cu2+ had a potential to detect PPi in real-time imaging in living cells.


## 2. Experimental

### 2.1. Apparatus

1H NMR and 13C NMR spectra were performed on Bruker Ascend™ 400 spectrometer with chemical shifts reported as ppm with TMS as internal standard. Mass spectrometric data were carried on Bruker Microtof-QIII spectrometry. UV-vis absorption spectra were measured on Shimadzu UV2600 spectrophotometer. Fluorescence spectra were measured with Edinburgh FS-5 fluorescence spectrophotometer. Biological cell imaging was recorded with Leica DMI8 inverted fluorescence microscope.


### 2.2. Reagents and Chemicals

All the chemicals of analytical grade were obtained from commercial sources and used as supplied. Perchlorate solutions (2.0×10−2 M) of various metal ions (Al3+, K+, Na+, Mg2+, Ca2+, Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Zn2+, Cd2+, Hg2+, Ag+, Pb2+, and Cu2+) were prepared in aqueous solutions. The anions (PPi, Pi, ATP, ADP, AMP, S2-, F-, Cl-, Br-, I-, HSO3-, HCO3-, ClO4-, SO42-, CH3COO-, and CN-) were dissolved in aqueous solutions to prepare stock solutions (2.0×10−2 M).


### 2.3. Synthetic Procedures

As shown in Scheme1, 7-(diethylamino)-2-oxo-2H-chromene-3-succinimidyl ester (compound D) and 4-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl) phenoxy)-butan-1-amine (compound E) were synthesized according to the literatures, respectively. The fluorescent sensing probe DPAC was synthesized by the condensation of compound D and E and characterized by 1H NMR, 13C NMR, and ESI-MS spectra (Figure S1, S2, and S3).Scheme 1
Synthesis procedures of compound DPAC.


#### 2.3.1. Synthesis of Compound C (7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxylic Acid) [41]

Diethyl malonate (6.40 g, 0.04 mol), 4-N, N-diethyl amino salicylic aldehyde (3.86 g, 0.02 mol), and 4 mL piperidine were dissolved in 60 mL dry ethanol and refluxed for 6 hours. After the solution was cooled to room temperature, 120 mL 10% NaOH solution was added and refluxed for 15 minutes to hydrolyze the product. The mixture was cooled to room temperature, and the solvent was removed by rotary evaporation. The residual product was acidified topH=2 with concentrated hydrochloric acid under ice cooling. The precipitate was collected by filtration, washed with cold ethanol, and dried in vacuo. Compound C was obtained as an orange solid in 79% yield: 1H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 7.48 (d, J=9.0Hz, 1H), 6.74 (dd, J=9.0, 2.4 Hz, 1H), 6.56 (d, J=2.3Hz, 1H), 3.65-3.43 (m, 4H), and 1.29 (t, J=7.1Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 165.65 (s), 164.64 (s), 158.21 (s), 154.05 (s), 150.22 (s), 131.98 (s), 110.98 (s), 108.96 (s), 105.39 (s), 96.74(s), 45.38 (s), and 12.40 (s); HRMS: m/z: 284.0891, [compound C+Na]+.


#### 2.3.2. Synthesis of Compound D (7-(Diethylamino)-2-Oxo-2H-Chromene-3-Succinimidyl Ester) [42]

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.3 g, 12 mmol) and N-hydroxysuccinimide (1.29 g, 11.2 mmol) were dissolved in 32 mL anhydrous DMF. Compound C (2.04 g, 7.84 mmol) in 32 mL anhydrous DMF was added dropwise to the above solution. The mixture was stirred at room temperature for 48 hours and poured into 500 mL ice water. The resulting precipitate was filtered to give compound D as a yellow solid that was taken forward in the synthesis without further purification:1H NMR (400 MHz, d6-DMSO) δ 8.80 (s, 1H), 7.73 (d, J=9.1Hz, 1H), 6.85 (d, J=9.0Hz, 1H), 6.60 (s, 1H), 3.53 (d, J=6.9Hz, 4H), 2.87 (s, 4H), and 1.16 (t, J=6.7Hz, 6H); 13C NMR (100 MHz, d6-DMSO) δ 171.04 (s), 159.52 (s), 156.94 (s), 154.67 (s), 151.86 (s), 133.35 (s), 111.03 (s), 107.88 (s), 100.98 (s), 96.40 (s), 45.25 (s), 25.95 (s), and 12.97 (s); HRMS: m/z: 381.1073, [compound D+Na]+.


#### 2.3.3. Synthesis of Compound DPAC

Compound D (0.18 g, 0.50 mmol) and compound E (4-(3,5-bis((bis(pyridine-2-ylmethyl)amino)methyl)phenoxy)-butan-1-amine) [43] (0.27 g, 0.46 mmol) were dissolved in 10 mL anhydrous DMF. The mixture was stirred for 24 h at room temperature. The product was poured into ice water and centrifuged. The obtained precipitate was dried and purified by flash column chromatography (silica gel, CH2Cl2:CH3OH=20:1) to afford compound DPAC (60% yield and 95% purity): 1H NMR (400 MHz, CDCl3) δ 8.89 (t, J=5.6Hz, 1H), 8.71 (s, 1H), 8.50 (d, J=4.8Hz, 4H), 7.67-7.60 (m, 8H), 7.43 (d, J=9.0Hz, 1H), 7.15-7.12 (m, 4H), 7.04 (s, 1H), 6.89 (s, 2H), 6.65 (dd, J=8.9, 2.4 Hz, 1H), 6.50 (d, J=2.2Hz, 1H), 3.99 (t, J=5.9Hz, 2H), 3.83 (s, 8H), 3.66 (s, 4H), 3.55-3.43 (m, 6H), 1.91-1.87 (m, 2H), 1.83-1.80 (m, 2H), and 1.24 (t, J=7.1Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 163.21 (s), 162.83 (s), 159.73 (s), 159.24 (s), 157.63 (s), 152.53 (s), 148.86 (s), 148.07 (s), 140.56 (s), 138.00 (s), 136.56 (s), 131.13 (s), 127.39 (s), 122.81 (s), 121.99 (s), 121.41 (s), 113.46 (s), 110.34 (s), 109.96 (s), 108.40 (s), 96.57 (s), 67.40 (s), 60.05 (s), 58.62 (s), 45.09 (s), 39.30 (s), 26.87 (s), 26.43 (s), and 12.44 (s); HRMS: m/z: 831.4341, [compound DPAC+H]+; 853.4159, [compound DPAC+Na]+.


#### 2.3.4. Synthesis of Complex DPAC-Cu2+

The corresponding DPAC-Cu2+ complex was prepared by mixing compound DPAC and Cu(ClO4)2·6H2O with the ratio of 1 : 2 in solution in situ, in which the DPA unit coordinated with Cu2+ ions to form the dinuclear complex DPAC-Cu2+.


### 2.4. General UV-Vis and Fluorescence Spectra Measurements

The stock solution of compound DPAC (1 mM) was prepared in DMSO. A portion of the stock solution (1 mL) of the probe DPAC was added to the CH3CN solution and adjusted to 100 mL CH3CN : HEPES (3 : 2, v/v, pH=7.20) solution with 0.1 M HEPES buffer. The test solution (10 μM) was formed and well-mixed.Each time a 3 mL test solution of compound DPAC or complex DPAC-Cu2+ was filled in a quartz cell of 1 cm optical path length, and stock solutions of different metal ions or anions were added into the quartz cell gradually by using a microsyringe. Excitation wavelength for probe DPAC and DPAC-Cu2+ was 423 nm with slit width as 1.2 nm.


### 2.5. Calculation Methods

The quenching constant (Ksv) was calculated by using the Stern-Volmer method [44]:
(1)F0F=1+KsvC,where F is the fluorescence intensity of compound DPAC in the presence of Cu2+, F0 is the fluorescence intensity of probe DPAC, Ksv is the quenching constant, and C is the concentration of Cu2+.The Benesi-Hildebrand equation was used as shown below [45]:
(2)logF−F0FL−F=logKa+nlogC,where Ka is the association constant; F and F0 represent the fluorescence intensity of DPAC-Cu2+ in the presence and absence of PPi, respectively. FL is the saturated fluorescence intensity, C is the concentration of PPi, and n is the stoichiometry for the binding of PPi.The detection limit is then calculated with the following equation [46]:
(3)LOD=K×δS,where δ is the standard deviation of blank measurements; S is the slope between intensity versus sample concentration.


## 2.1. Apparatus

1H NMR and 13C NMR spectra were performed on Bruker Ascend™ 400 spectrometer with chemical shifts reported as ppm with TMS as internal standard. Mass spectrometric data were carried on Bruker Microtof-QIII spectrometry. UV-vis absorption spectra were measured on Shimadzu UV2600 spectrophotometer. Fluorescence spectra were measured with Edinburgh FS-5 fluorescence spectrophotometer. Biological cell imaging was recorded with Leica DMI8 inverted fluorescence microscope.


## 2.2. Reagents and Chemicals

All the chemicals of analytical grade were obtained from commercial sources and used as supplied. Perchlorate solutions (2.0×10−2 M) of various metal ions (Al3+, K+, Na+, Mg2+, Ca2+, Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Zn2+, Cd2+, Hg2+, Ag+, Pb2+, and Cu2+) were prepared in aqueous solutions. The anions (PPi, Pi, ATP, ADP, AMP, S2-, F-, Cl-, Br-, I-, HSO3-, HCO3-, ClO4-, SO42-, CH3COO-, and CN-) were dissolved in aqueous solutions to prepare stock solutions (2.0×10−2 M).


## 2.3. Synthetic Procedures

As shown in Scheme1, 7-(diethylamino)-2-oxo-2H-chromene-3-succinimidyl ester (compound D) and 4-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl) phenoxy)-butan-1-amine (compound E) were synthesized according to the literatures, respectively. The fluorescent sensing probe DPAC was synthesized by the condensation of compound D and E and characterized by 1H NMR, 13C NMR, and ESI-MS spectra (Figure S1, S2, and S3).Scheme 1
Synthesis procedures of compound DPAC.


### 2.3.1. Synthesis of Compound C (7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxylic Acid) [41]

Diethyl malonate (6.40 g, 0.04 mol), 4-N, N-diethyl amino salicylic aldehyde (3.86 g, 0.02 mol), and 4 mL piperidine were dissolved in 60 mL dry ethanol and refluxed for 6 hours. After the solution was cooled to room temperature, 120 mL 10% NaOH solution was added and refluxed for 15 minutes to hydrolyze the product. The mixture was cooled to room temperature, and the solvent was removed by rotary evaporation. The residual product was acidified topH=2 with concentrated hydrochloric acid under ice cooling. The precipitate was collected by filtration, washed with cold ethanol, and dried in vacuo. Compound C was obtained as an orange solid in 79% yield: 1H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 7.48 (d, J=9.0Hz, 1H), 6.74 (dd, J=9.0, 2.4 Hz, 1H), 6.56 (d, J=2.3Hz, 1H), 3.65-3.43 (m, 4H), and 1.29 (t, J=7.1Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 165.65 (s), 164.64 (s), 158.21 (s), 154.05 (s), 150.22 (s), 131.98 (s), 110.98 (s), 108.96 (s), 105.39 (s), 96.74(s), 45.38 (s), and 12.40 (s); HRMS: m/z: 284.0891, [compound C+Na]+.


### 2.3.2. Synthesis of Compound D (7-(Diethylamino)-2-Oxo-2H-Chromene-3-Succinimidyl Ester) [42]

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.3 g, 12 mmol) and N-hydroxysuccinimide (1.29 g, 11.2 mmol) were dissolved in 32 mL anhydrous DMF. Compound C (2.04 g, 7.84 mmol) in 32 mL anhydrous DMF was added dropwise to the above solution. The mixture was stirred at room temperature for 48 hours and poured into 500 mL ice water. The resulting precipitate was filtered to give compound D as a yellow solid that was taken forward in the synthesis without further purification:1H NMR (400 MHz, d6-DMSO) δ 8.80 (s, 1H), 7.73 (d, J=9.1Hz, 1H), 6.85 (d, J=9.0Hz, 1H), 6.60 (s, 1H), 3.53 (d, J=6.9Hz, 4H), 2.87 (s, 4H), and 1.16 (t, J=6.7Hz, 6H); 13C NMR (100 MHz, d6-DMSO) δ 171.04 (s), 159.52 (s), 156.94 (s), 154.67 (s), 151.86 (s), 133.35 (s), 111.03 (s), 107.88 (s), 100.98 (s), 96.40 (s), 45.25 (s), 25.95 (s), and 12.97 (s); HRMS: m/z: 381.1073, [compound D+Na]+.


### 2.3.3. Synthesis of Compound DPAC

Compound D (0.18 g, 0.50 mmol) and compound E (4-(3,5-bis((bis(pyridine-2-ylmethyl)amino)methyl)phenoxy)-butan-1-amine) [43] (0.27 g, 0.46 mmol) were dissolved in 10 mL anhydrous DMF. The mixture was stirred for 24 h at room temperature. The product was poured into ice water and centrifuged. The obtained precipitate was dried and purified by flash column chromatography (silica gel, CH2Cl2:CH3OH=20:1) to afford compound DPAC (60% yield and 95% purity): 1H NMR (400 MHz, CDCl3) δ 8.89 (t, J=5.6Hz, 1H), 8.71 (s, 1H), 8.50 (d, J=4.8Hz, 4H), 7.67-7.60 (m, 8H), 7.43 (d, J=9.0Hz, 1H), 7.15-7.12 (m, 4H), 7.04 (s, 1H), 6.89 (s, 2H), 6.65 (dd, J=8.9, 2.4 Hz, 1H), 6.50 (d, J=2.2Hz, 1H), 3.99 (t, J=5.9Hz, 2H), 3.83 (s, 8H), 3.66 (s, 4H), 3.55-3.43 (m, 6H), 1.91-1.87 (m, 2H), 1.83-1.80 (m, 2H), and 1.24 (t, J=7.1Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 163.21 (s), 162.83 (s), 159.73 (s), 159.24 (s), 157.63 (s), 152.53 (s), 148.86 (s), 148.07 (s), 140.56 (s), 138.00 (s), 136.56 (s), 131.13 (s), 127.39 (s), 122.81 (s), 121.99 (s), 121.41 (s), 113.46 (s), 110.34 (s), 109.96 (s), 108.40 (s), 96.57 (s), 67.40 (s), 60.05 (s), 58.62 (s), 45.09 (s), 39.30 (s), 26.87 (s), 26.43 (s), and 12.44 (s); HRMS: m/z: 831.4341, [compound DPAC+H]+; 853.4159, [compound DPAC+Na]+.


### 2.3.4. Synthesis of Complex DPAC-Cu2+

The corresponding DPAC-Cu2+ complex was prepared by mixing compound DPAC and Cu(ClO4)2·6H2O with the ratio of 1 : 2 in solution in situ, in which the DPA unit coordinated with Cu2+ ions to form the dinuclear complex DPAC-Cu2+.


## 2.3.1. Synthesis of Compound C (7-(Diethylamino)-2-Oxo-2H-Chromene-3-Carboxylic Acid) [41]

Diethyl malonate (6.40 g, 0.04 mol), 4-N, N-diethyl amino salicylic aldehyde (3.86 g, 0.02 mol), and 4 mL piperidine were dissolved in 60 mL dry ethanol and refluxed for 6 hours. After the solution was cooled to room temperature, 120 mL 10% NaOH solution was added and refluxed for 15 minutes to hydrolyze the product. The mixture was cooled to room temperature, and the solvent was removed by rotary evaporation. The residual product was acidified topH=2 with concentrated hydrochloric acid under ice cooling. The precipitate was collected by filtration, washed with cold ethanol, and dried in vacuo. Compound C was obtained as an orange solid in 79% yield: 1H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 7.48 (d, J=9.0Hz, 1H), 6.74 (dd, J=9.0, 2.4 Hz, 1H), 6.56 (d, J=2.3Hz, 1H), 3.65-3.43 (m, 4H), and 1.29 (t, J=7.1Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 165.65 (s), 164.64 (s), 158.21 (s), 154.05 (s), 150.22 (s), 131.98 (s), 110.98 (s), 108.96 (s), 105.39 (s), 96.74(s), 45.38 (s), and 12.40 (s); HRMS: m/z: 284.0891, [compound C+Na]+.


## 2.3.2. Synthesis of Compound D (7-(Diethylamino)-2-Oxo-2H-Chromene-3-Succinimidyl Ester) [42]

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.3 g, 12 mmol) and N-hydroxysuccinimide (1.29 g, 11.2 mmol) were dissolved in 32 mL anhydrous DMF. Compound C (2.04 g, 7.84 mmol) in 32 mL anhydrous DMF was added dropwise to the above solution. The mixture was stirred at room temperature for 48 hours and poured into 500 mL ice water. The resulting precipitate was filtered to give compound D as a yellow solid that was taken forward in the synthesis without further purification:1H NMR (400 MHz, d6-DMSO) δ 8.80 (s, 1H), 7.73 (d, J=9.1Hz, 1H), 6.85 (d, J=9.0Hz, 1H), 6.60 (s, 1H), 3.53 (d, J=6.9Hz, 4H), 2.87 (s, 4H), and 1.16 (t, J=6.7Hz, 6H); 13C NMR (100 MHz, d6-DMSO) δ 171.04 (s), 159.52 (s), 156.94 (s), 154.67 (s), 151.86 (s), 133.35 (s), 111.03 (s), 107.88 (s), 100.98 (s), 96.40 (s), 45.25 (s), 25.95 (s), and 12.97 (s); HRMS: m/z: 381.1073, [compound D+Na]+.


## 2.3.3. Synthesis of Compound DPAC

Compound D (0.18 g, 0.50 mmol) and compound E (4-(3,5-bis((bis(pyridine-2-ylmethyl)amino)methyl)phenoxy)-butan-1-amine) [43] (0.27 g, 0.46 mmol) were dissolved in 10 mL anhydrous DMF. The mixture was stirred for 24 h at room temperature. The product was poured into ice water and centrifuged. The obtained precipitate was dried and purified by flash column chromatography (silica gel, CH2Cl2:CH3OH=20:1) to afford compound DPAC (60% yield and 95% purity): 1H NMR (400 MHz, CDCl3) δ 8.89 (t, J=5.6Hz, 1H), 8.71 (s, 1H), 8.50 (d, J=4.8Hz, 4H), 7.67-7.60 (m, 8H), 7.43 (d, J=9.0Hz, 1H), 7.15-7.12 (m, 4H), 7.04 (s, 1H), 6.89 (s, 2H), 6.65 (dd, J=8.9, 2.4 Hz, 1H), 6.50 (d, J=2.2Hz, 1H), 3.99 (t, J=5.9Hz, 2H), 3.83 (s, 8H), 3.66 (s, 4H), 3.55-3.43 (m, 6H), 1.91-1.87 (m, 2H), 1.83-1.80 (m, 2H), and 1.24 (t, J=7.1Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 163.21 (s), 162.83 (s), 159.73 (s), 159.24 (s), 157.63 (s), 152.53 (s), 148.86 (s), 148.07 (s), 140.56 (s), 138.00 (s), 136.56 (s), 131.13 (s), 127.39 (s), 122.81 (s), 121.99 (s), 121.41 (s), 113.46 (s), 110.34 (s), 109.96 (s), 108.40 (s), 96.57 (s), 67.40 (s), 60.05 (s), 58.62 (s), 45.09 (s), 39.30 (s), 26.87 (s), 26.43 (s), and 12.44 (s); HRMS: m/z: 831.4341, [compound DPAC+H]+; 853.4159, [compound DPAC+Na]+.


## 2.3.4. Synthesis of Complex DPAC-Cu2+

The corresponding DPAC-Cu2+ complex was prepared by mixing compound DPAC and Cu(ClO4)2·6H2O with the ratio of 1 : 2 in solution in situ, in which the DPA unit coordinated with Cu2+ ions to form the dinuclear complex DPAC-Cu2+.


## 2.4. General UV-Vis and Fluorescence Spectra Measurements

The stock solution of compound DPAC (1 mM) was prepared in DMSO. A portion of the stock solution (1 mL) of the probe DPAC was added to the CH3CN solution and adjusted to 100 mL CH3CN : HEPES (3 : 2, v/v, pH=7.20) solution with 0.1 M HEPES buffer. The test solution (10 μM) was formed and well-mixed.Each time a 3 mL test solution of compound DPAC or complex DPAC-Cu2+ was filled in a quartz cell of 1 cm optical path length, and stock solutions of different metal ions or anions were added into the quartz cell gradually by using a microsyringe. Excitation wavelength for probe DPAC and DPAC-Cu2+ was 423 nm with slit width as 1.2 nm.


## 2.5. Calculation Methods

The quenching constant (Ksv) was calculated by using the Stern-Volmer method [44]:
(1)F0F=1+KsvC,where F is the fluorescence intensity of compound DPAC in the presence of Cu2+, F0 is the fluorescence intensity of probe DPAC, Ksv is the quenching constant, and C is the concentration of Cu2+.The Benesi-Hildebrand equation was used as shown below [45]:
(2)logF−F0FL−F=logKa+nlogC,where Ka is the association constant; F and F0 represent the fluorescence intensity of DPAC-Cu2+ in the presence and absence of PPi, respectively. FL is the saturated fluorescence intensity, C is the concentration of PPi, and n is the stoichiometry for the binding of PPi.The detection limit is then calculated with the following equation [46]:
(3)LOD=K×δS,where δ is the standard deviation of blank measurements; S is the slope between intensity versus sample concentration.


## 3. Results and Discussion

### 3.1. The Absorption and Fluorescence Spectra Responses of Compound DPAC to Cu2+ Ions

The UV-vis absorption spectra of compound DPAC (10μM) upon the addition of various metal ions (2 equiv.) in the CH3CN : HEPES (3 : 2, v/v, pH=7.20) solutions were shown in Figure S4. The maximum absorption peaks of compound DPAC and the corresponding metal complexes were all at about 423 nm. Almost no obvious changes could be monitored with the addition of various metal ions. The fluorescence spectra of compound DPAC (10 μM) in the CH3CN : HEPES (3 : 2, v/v, pH=7.20) solution showed a strong emission peak at 470 nm when excited at 423 nm. To calculate the fluorescent response ability of compound DPAC, the fluorescence experiment was monitored by adding copper ions to the solution of compound DPAC (Figure 1(a)). The fluorescence intensity gradually decreased with the increasing concentration of Cu2+ ions. After the addition of 2 equiv. of Cu2+ ions, the fluorescence intensity nearly reached equilibrium, and the probe DPAC-Cu2+ was formed in situ. The low fluorescence intensity of the probe might be due to the quenching effect from the paramagnetism of copper ions [47]. The plot of fluorescence intensity showed a good linear relationship (R2=0.996) to the concentration of Cu2+ in the range of 1-10 μM (Figure 1(b)). The quenching constant of Cu2+ was calculated as 1.47×105 L mol-1 based on the Stern-Volmer fitting of the titration plots. The detection limit calculated following the S/N=3:1 was estimated to be about 0.36 μM, which is lower than the WHO guideline in the drinking water. The Benesi-Hildebrand fitting method indicated that the stoichiometric ratio of compound DPAC with Cu2+ was 1 : 2 in complex DPAC-Cu2+(Figure S5). The apparent association constant of DPAC and Cu2+ was estimated to be 1.67×105 M-2. The fluorescence selectivity of compound DPAC was carried out by additions of different metal ions to the solution of compound DPAC, and the results were displayed in Figure S6. However, Co2+ ions showed similar quenching phenomenon, whereas the other metal ions did not cause any detectable changes.Figure 1
(a) Fluorescence spectra of compound DPAC (10μM) upon the continuously addition of Cu2+ ions (0-3 equiv.) in the CH3CN : HEPES (3 : 2, v/v, pH=7.20) solution. Inset: fluorescence titration profile of DPAC at 470 nm upon the addition of Cu2+ ions. (b) The Stern-Volmer fitting of titration plots with the concentration of Cu2+ ions (1-10 μM).
(a)
(b)The complexation formation of DPAC with Cu2+ in the 1 : 2 ratio was further confirmed by ESI-MS spectra. As shown in Figure S7, upon the addition of 2 equiv. Cu2+metal ions to the solution of DPAC in CH3CN, the signals at m/z=352.3921, 578.0921, and 1255.1358 were observed. The peaks were assigned to the [Cu2(DPAC)(ClO4)]3+, [Cu2(DPAC)(ClO4)2]2+, and [Cu2(DPAC)(ClO4)3]+ species, respectively. The specific isotopic patterns fitted well with the simulated peaks calculated by IsoPro 3.0 program (Figure S8 and S9). These results were consistent with the stable presence of Cu2(DPAC) species in the solution. The coordinated ClO4- anion was easy to leave, and the quenching effect could be inhibited by other coordinated anions. All these phenomena indicated that the complex DPAC-Cu2+ could be used as a potential candidate chemosensor for biological anions [48].


### 3.2. The Fluorescence Spectra Response of the Probe (Complex DPAC-Cu2+) to PPi

Based on the moderate binding ability of the DPA group and high charge density of the complex DPAC-Cu2+, it was proposed to be used as an anion-selective fluorescence probe for PPi. The experimental data showed that the fluorescence intensity was gradually restored when PPi was added to the CH3CN : HEPES (3 : 2, v/v, pH=7.20) solution of the complex DPAC-Cu2+. To understand the detection mode of the complex DPAC-Cu2+ with PPi, the titration experiment was performed. As shown in Figure 2(a), after the addition of about 1 equiv. of PPi, the emission intensity was up to the maximum and reached to equilibrium in spite of further addition. It was deduced that the probe DPAC-Cu2+ bound PPi with 1 : 1 stoichiometry by the Benesi-Hildebrand method (Figure 2(b)). The detection limit was calculated as about 0.53 μM based on the equation of LOD=3δ/S (Figure S10).Figure 2
(a) Fluorescence alteration of compound DPAC-Cu2+ (10 μM) upon the addition of PPi with concentrations up to 20 μM in the CH3CN : HEPES (3 : 2, v/v, pH=7.20) solution. Inset: fluorescence titration profile of DPAC-Cu2+ at 470 nm upon the addition of PPi. (b) The Benesi-Hildebrand fitting of titration plots with the concentration of PPi in the range of 1-8 μM.
(a)
(b)


### 3.3. Detection Mechanism of DPAC-Cu2+ for PPi

The detection mechanism of DPAC-Cu2+ to PPi was further investigated. ESI-MS spectra analysis indicated that DPAC-Cu2+ and PPi formed a 1 : 1 complex as DPAC-Cu2+-PPi. As shown in Figure S11 and Figure S12, a peak at m/z 1133.2058 corresponding to [DPAC+2Cu+PPi+1H]+ was clearly observed when excess amount of PPi was added to the solution of DPAC-Cu2+. The ESI-MS spectra results fit well with the proposition that the probe DPAC-Cu2+ provided a platform to selectively detect PPi [49]. The further evidence of the binding mechanism came from HPLC analyses (Figure S13). Upon the addition of Cu2+ ions, the peak of DPAC (rt=7.3min) gradually disappeared along with the rise of the complex DPAC-Cu2+ peak (rt=0.9min). Interestingly, after adding 1 equiv. PPi, the peak of DPAC-Cu2+ finally vanished with the appearance of a new peak at 1.3 min, which was attributed to the formation of DPAC-Cu2+-PPi. These results confirmed that the probe DPAC-Cu2+ combined PPi to form the stable sandwiched complex DPAC-Cu2+-PPi as shown in Scheme 2 and thus inhibited the PET-quenching process along with fluorescence intensity enhancement. The specific structure was ascribed to the strong metal coordination interaction between the bis(2-pyridylmethyl)amine (DPA)-Cu2+ complex and PPi [50]. Therefore, this unique binding feature could be utilized to discriminatively detect PPi from other phosphate anions in the fluorescence “off-on” approach.Scheme 2
Schematic illustration of the proposed sensing mechanism of DPAC-Cu2+ toward PPi.For further evaluating the selectivity of the probe DPAC-Cu2+for PPi, the fluorescence emission intensity changes coexisted with the anions including PPi, F-, Cl-, Br-, I-, S2-, ATP, ADP, AMP, Pi, HSO3-, HCO3-, SO42-, CH3COO-, ClO4-, and CN- in CH3CN : HEPES (3 : 2, v/v, pH=7.20) buffer were investigated. As shown in Figure 3, among these anions, in the presence of PPi, the fluorescence intensity of the complex DPAC-Cu2+ (10 μM) increased. Nevertheless, no obvious fluorescence intensity changes was observed in the presence of the other anions including ATP, ADP, AMP, and Pi. Moreover, subsequently adding 2 equiv. of PPi to the other anion solutions gave rise to similar enhancement in accordance with the addition of equal amount of PPi alone, indicating that the PPi had specific response to the probe. The experiments above demonstrated that the complex DPAC-Cu2+ had good selectivity for PPi, which was not disturbed by the competitive anions. The selectivity for PPi over other phosphate anions was possibly due to the higher anionic charge density of the four O–P oxygen atoms involved in the complexation between the PPi and the two Cu2+ sites. Moreover, the hexacoordination of Cu2+ ions with PPi is clearly reflected in the extremely higher association constant than other phosphate anions. And the spatial configuration of the two Cu2+-DPA sites was benefited for the selective sensing of PPi.Figure 3
Fluorescence intensity of probe DPAC-Cu2+ (10 μM) upon the addition of various competitive anions (2 equiv.) (blue bars) and subsequent addition of PPi (2 equiv.) (red bars) in CH3CN : HEPES (3 : 2, v/v, pH=7.20) buffer. Anions: (1) PPi, (2) F-, (3) Cl-, (4) Br-, (5) I-, (6) S2-, (7) ATP, (8) ADP, (9) AMP, (10) Pi, (11) HSO3-, (12) HCO3-, (13) SO42-, (14) CH3COO-, (15) ClO4-, and (16) CN-.


### 3.4. The Fluorescence Cell Imaging of the Probe (Complex DPAC-Cu2+) for PPi

The fluorescence imaging of the probe DPAC-Cu2+ for PPi was performed in lung cancer cells A549. Prior to fluorescence imaging, A549 cells were cultured in a 12-well cell plate for 24 hours and incubated with compound DPAC (1 μM) at 37°C, 5% CO2 for 30 min and then washed with PBS solution three times. Under Leica DMI8 inverted fluorescence microscope, the cell imaging results of A549 were shown in Figure 4. Excited with blue light, compound DPAC showed significant intracellular green fluorescence in A549 cells, indicating that compound DPAC had strong cell permeability. After the addition of excess 4 equiv. of copper ions, the probe DPAC-Cu2+ was prepared in situ, and the green fluorescence in A549 cells was significantly quenched. However, after the excess Cu2+ ions were washed away and 2 equiv. PPi were subsequently added, the fluorescence in the cells was recovered. These results demonstrated that the probe DPAC-Cu2+ could be used for imaging the PPi anions in living cells.Figure 4
The fluorescence images of A549 cells incubated with (a) compound DPAC (1μM), (b) probe DPAC-Cu2+, (c) DPAC-Cu2+-PPi, and (d-f) their corresponding bright-field images.
(a)
(b)
(c)
(d)
(e)
(f)


## 3.1. The Absorption and Fluorescence Spectra Responses of Compound DPAC to Cu2+ Ions

The UV-vis absorption spectra of compound DPAC (10μM) upon the addition of various metal ions (2 equiv.) in the CH3CN : HEPES (3 : 2, v/v, pH=7.20) solutions were shown in Figure S4. The maximum absorption peaks of compound DPAC and the corresponding metal complexes were all at about 423 nm. Almost no obvious changes could be monitored with the addition of various metal ions. The fluorescence spectra of compound DPAC (10 μM) in the CH3CN : HEPES (3 : 2, v/v, pH=7.20) solution showed a strong emission peak at 470 nm when excited at 423 nm. To calculate the fluorescent response ability of compound DPAC, the fluorescence experiment was monitored by adding copper ions to the solution of compound DPAC (Figure 1(a)). The fluorescence intensity gradually decreased with the increasing concentration of Cu2+ ions. After the addition of 2 equiv. of Cu2+ ions, the fluorescence intensity nearly reached equilibrium, and the probe DPAC-Cu2+ was formed in situ. The low fluorescence intensity of the probe might be due to the quenching effect from the paramagnetism of copper ions [47]. The plot of fluorescence intensity showed a good linear relationship (R2=0.996) to the concentration of Cu2+ in the range of 1-10 μM (Figure 1(b)). The quenching constant of Cu2+ was calculated as 1.47×105 L mol-1 based on the Stern-Volmer fitting of the titration plots. The detection limit calculated following the S/N=3:1 was estimated to be about 0.36 μM, which is lower than the WHO guideline in the drinking water. The Benesi-Hildebrand fitting method indicated that the stoichiometric ratio of compound DPAC with Cu2+ was 1 : 2 in complex DPAC-Cu2+(Figure S5). The apparent association constant of DPAC and Cu2+ was estimated to be 1.67×105 M-2. The fluorescence selectivity of compound DPAC was carried out by additions of different metal ions to the solution of compound DPAC, and the results were displayed in Figure S6. However, Co2+ ions showed similar quenching phenomenon, whereas the other metal ions did not cause any detectable changes.Figure 1
(a) Fluorescence spectra of compound DPAC (10μM) upon the continuously addition of Cu2+ ions (0-3 equiv.) in the CH3CN : HEPES (3 : 2, v/v, pH=7.20) solution. Inset: fluorescence titration profile of DPAC at 470 nm upon the addition of Cu2+ ions. (b) The Stern-Volmer fitting of titration plots with the concentration of Cu2+ ions (1-10 μM).
(a)
(b)The complexation formation of DPAC with Cu2+ in the 1 : 2 ratio was further confirmed by ESI-MS spectra. As shown in Figure S7, upon the addition of 2 equiv. Cu2+metal ions to the solution of DPAC in CH3CN, the signals at m/z=352.3921, 578.0921, and 1255.1358 were observed. The peaks were assigned to the [Cu2(DPAC)(ClO4)]3+, [Cu2(DPAC)(ClO4)2]2+, and [Cu2(DPAC)(ClO4)3]+ species, respectively. The specific isotopic patterns fitted well with the simulated peaks calculated by IsoPro 3.0 program (Figure S8 and S9). These results were consistent with the stable presence of Cu2(DPAC) species in the solution. The coordinated ClO4- anion was easy to leave, and the quenching effect could be inhibited by other coordinated anions. All these phenomena indicated that the complex DPAC-Cu2+ could be used as a potential candidate chemosensor for biological anions [48].


## 3.2. The Fluorescence Spectra Response of the Probe (Complex DPAC-Cu2+) to PPi

Based on the moderate binding ability of the DPA group and high charge density of the complex DPAC-Cu2+, it was proposed to be used as an anion-selective fluorescence probe for PPi. The experimental data showed that the fluorescence intensity was gradually restored when PPi was added to the CH3CN : HEPES (3 : 2, v/v, pH=7.20) solution of the complex DPAC-Cu2+. To understand the detection mode of the complex DPAC-Cu2+ with PPi, the titration experiment was performed. As shown in Figure 2(a), after the addition of about 1 equiv. of PPi, the emission intensity was up to the maximum and reached to equilibrium in spite of further addition. It was deduced that the probe DPAC-Cu2+ bound PPi with 1 : 1 stoichiometry by the Benesi-Hildebrand method (Figure 2(b)). The detection limit was calculated as about 0.53 μM based on the equation of LOD=3δ/S (Figure S10).Figure 2
(a) Fluorescence alteration of compound DPAC-Cu2+ (10 μM) upon the addition of PPi with concentrations up to 20 μM in the CH3CN : HEPES (3 : 2, v/v, pH=7.20) solution. Inset: fluorescence titration profile of DPAC-Cu2+ at 470 nm upon the addition of PPi. (b) The Benesi-Hildebrand fitting of titration plots with the concentration of PPi in the range of 1-8 μM.
(a)
(b)


## 3.3. Detection Mechanism of DPAC-Cu2+ for PPi

The detection mechanism of DPAC-Cu2+ to PPi was further investigated. ESI-MS spectra analysis indicated that DPAC-Cu2+ and PPi formed a 1 : 1 complex as DPAC-Cu2+-PPi. As shown in Figure S11 and Figure S12, a peak at m/z 1133.2058 corresponding to [DPAC+2Cu+PPi+1H]+ was clearly observed when excess amount of PPi was added to the solution of DPAC-Cu2+. The ESI-MS spectra results fit well with the proposition that the probe DPAC-Cu2+ provided a platform to selectively detect PPi [49]. The further evidence of the binding mechanism came from HPLC analyses (Figure S13). Upon the addition of Cu2+ ions, the peak of DPAC (rt=7.3min) gradually disappeared along with the rise of the complex DPAC-Cu2+ peak (rt=0.9min). Interestingly, after adding 1 equiv. PPi, the peak of DPAC-Cu2+ finally vanished with the appearance of a new peak at 1.3 min, which was attributed to the formation of DPAC-Cu2+-PPi. These results confirmed that the probe DPAC-Cu2+ combined PPi to form the stable sandwiched complex DPAC-Cu2+-PPi as shown in Scheme 2 and thus inhibited the PET-quenching process along with fluorescence intensity enhancement. The specific structure was ascribed to the strong metal coordination interaction between the bis(2-pyridylmethyl)amine (DPA)-Cu2+ complex and PPi [50]. Therefore, this unique binding feature could be utilized to discriminatively detect PPi from other phosphate anions in the fluorescence “off-on” approach.Scheme 2
Schematic illustration of the proposed sensing mechanism of DPAC-Cu2+ toward PPi.For further evaluating the selectivity of the probe DPAC-Cu2+for PPi, the fluorescence emission intensity changes coexisted with the anions including PPi, F-, Cl-, Br-, I-, S2-, ATP, ADP, AMP, Pi, HSO3-, HCO3-, SO42-, CH3COO-, ClO4-, and CN- in CH3CN : HEPES (3 : 2, v/v, pH=7.20) buffer were investigated. As shown in Figure 3, among these anions, in the presence of PPi, the fluorescence intensity of the complex DPAC-Cu2+ (10 μM) increased. Nevertheless, no obvious fluorescence intensity changes was observed in the presence of the other anions including ATP, ADP, AMP, and Pi. Moreover, subsequently adding 2 equiv. of PPi to the other anion solutions gave rise to similar enhancement in accordance with the addition of equal amount of PPi alone, indicating that the PPi had specific response to the probe. The experiments above demonstrated that the complex DPAC-Cu2+ had good selectivity for PPi, which was not disturbed by the competitive anions. The selectivity for PPi over other phosphate anions was possibly due to the higher anionic charge density of the four O–P oxygen atoms involved in the complexation between the PPi and the two Cu2+ sites. Moreover, the hexacoordination of Cu2+ ions with PPi is clearly reflected in the extremely higher association constant than other phosphate anions. And the spatial configuration of the two Cu2+-DPA sites was benefited for the selective sensing of PPi.Figure 3
Fluorescence intensity of probe DPAC-Cu2+ (10 μM) upon the addition of various competitive anions (2 equiv.) (blue bars) and subsequent addition of PPi (2 equiv.) (red bars) in CH3CN : HEPES (3 : 2, v/v, pH=7.20) buffer. Anions: (1) PPi, (2) F-, (3) Cl-, (4) Br-, (5) I-, (6) S2-, (7) ATP, (8) ADP, (9) AMP, (10) Pi, (11) HSO3-, (12) HCO3-, (13) SO42-, (14) CH3COO-, (15) ClO4-, and (16) CN-.


## 3.4. The Fluorescence Cell Imaging of the Probe (Complex DPAC-Cu2+) for PPi

The fluorescence imaging of the probe DPAC-Cu2+ for PPi was performed in lung cancer cells A549. Prior to fluorescence imaging, A549 cells were cultured in a 12-well cell plate for 24 hours and incubated with compound DPAC (1 μM) at 37°C, 5% CO2 for 30 min and then washed with PBS solution three times. Under Leica DMI8 inverted fluorescence microscope, the cell imaging results of A549 were shown in Figure 4. Excited with blue light, compound DPAC showed significant intracellular green fluorescence in A549 cells, indicating that compound DPAC had strong cell permeability. After the addition of excess 4 equiv. of copper ions, the probe DPAC-Cu2+ was prepared in situ, and the green fluorescence in A549 cells was significantly quenched. However, after the excess Cu2+ ions were washed away and 2 equiv. PPi were subsequently added, the fluorescence in the cells was recovered. These results demonstrated that the probe DPAC-Cu2+ could be used for imaging the PPi anions in living cells.Figure 4
The fluorescence images of A549 cells incubated with (a) compound DPAC (1μM), (b) probe DPAC-Cu2+, (c) DPAC-Cu2+-PPi, and (d-f) their corresponding bright-field images.
(a)
(b)
(c)
(d)
(e)
(f)


## 4. Conclusions

In summary, a coumarin-based dinuclear copper complex DPAC-Cu2+ was synthesized and investigated as a fluorescent probe for PPi. The Benesi-Hildebrand fitting analysis, ESI-MS and HPLC analyses, indicated a 1 : 1 stoichiometry of the complex DPAC-Cu2+ with PPi. The detection limit of PPi was estimated about 0.53 μM. The fluorescence spectra indicated that the probe had good selectivity and sensitivity for PPi in CH3CN : HEPES (3 : 2, v/v, pH=7.20) buffer. Ultimately, cell imaging results indicated that DPAC-Cu2+ could visually monitor the PPi anions in A549 cells.



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*Source: 5349124-2019-02-28.xml*