The chemistry of macrocyclic polyamines has been received much attention due to their widespread applications in coordination chemistry,medicinal chemistry,biochemistry,and material chemistry ^{[1, 2, 3, 4]}. Because of their protonation property at physiological conditions,macrocyclic polyamines often act as positive-charged units in constructing DNA condensation agents and non-viral gene vectors ^{[5, 6, 7, 8]}. As a binding unit,macrocyclic polyamines have been frequently applied in the development of artificial nucleases ^{[9, 10, 11, 12]} and a number of chemical sensors to monitor biologically- and environmentally-relevant metal and anion ions ^{[13, 14, 15, 16]}. Our group has recently focused on the design and synthesis of various macrocyclic polyamine ^[12]aneN₃ compounds and their applications in artificial nucleases,DNA condensation agents,non-viral gene vectors,and chemical sensors ^{[17, 18, 19, 20, 21]}.

It is well known that Cu²⁺ ion is the third-most abundant transition metal in human body. It exists in numerous metalloenzymes such as superoxide dismutase,cytochrome oxidase and tyrosinase which are critical forlife ^{[22, 23]}. However,overloading of Cu²⁺ ions in the neuronal cytoplasm can lead to Alzheimer’s disease, Wilson’s disease,and Menke’s disease ^{[24, 25, 26]}. Furthermore,ADP is an important biological phosphate which is involved in the consumption and production of ATP metabolism,such as the glycolysis,Kreb’s cycle,transporting activities of proteins,modulating ion channels and activating signaling cascades,which are the fundamental activities of life ^{[27, 28, 29]}. Thus developing new sensors to selectively and sensitively monitor both Cu²⁺ ions and ADP phosphate will be highly desired.

With the above consideration and to further expand the application of macrocyclic polyamines,we have designed and synthesized a ^[12]aneN₃ modified boron dipyrromethene (BODIPY) compound 1,which can be successfully applied as highly selective and sensitive sensor for the sequential recognition of Cu²⁺ ions and ADP in aqueous solution and in living cells. 2. Experimental

¹H NMR and ¹³C NMR spectra were obtained on a Bruker Avance III 400 MHz spectrometer at 25℃. Chemical shifts were referenced on residual solvents peaks. The infrared spectra were taken on a Nicolet 380 spectrometer in the range of 4000-400 cm^-1. Mass spectra were acquired on a Waters Quattro Mocro spectrometer and high resolution mass spectra were acquired on a Waters LCT Premier XE spectrometer. Fluorescence spectra were measured on a Varian Cary Eclipse spectrometer. UV-vis spectra were measured on a Varian Cary 300 UV-vis spectrophotometer using solutions in 1.0 cm quartz cuvettes. Fluorescence quantum yield of samples were recorded on a Fluormax-4 spectrophotometer at room temperature with an integrating sphere system,and the machine was revaluated using standard sample before measurement. 2.1. Synthesis of 1

Sensor 1 was synthesized according to the route shown in Scheme 1. Boc-protected N-(3-azidopropyl) ^[12]aneN₃ (5) was obtained from the azide substitution reaction with Boc-protected N-(3-bromopropyl) ^[12]aneN₃ (4),which was prepared through the reaction of ^[12]aneN₃ precursor ^[30] (2) with 1,3-dibromopropane and further hydrolysis (3) and protection by (Boc)₂O. Another starting material,1,5-bis(acetynyl) BODIPY 6,was obtained according to literature method ^[31]. Reaction of 6 with azide 5 through copper (I) mediated click cycloaddition reaction resulted in compound 7. Target compound 1 was obtained by the deprotection of 7 under hydrogen chloride condition in ethyl acetate with yield of 70%. The detail of the syntheses can be found in the Supporting information.

Scheme 1

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Scheme 1.The synthesis of sensor 1.

Compound 5: ¹H NMR (400 MHz,CDCl₃): δ 3.37-3.26 (m,10H), 2.47 (t,2H,J = 7.0 Hz),2.44-2.36 (m,4H),1.90-1.80 (m,2H),1.80- 1.71 (m,4H),1.71-1.64 (m,2H),1.44 (s,18H). ¹³C NMR (101 MHz, CDCl₃): δ 156.42,79.47,50.51,50.03,49.81,45.15,43.96,28.63, 27.52,26.64,25.60; IR (KBr,cm^-1): 3114,2958,2096,1688,1460, 1398,1254,1158,873,804,613. ESI-MS (m/z) calcd. for C₂₂H₄₀N₆O₄ [M+H]⁺: 455.3,found: 455.8.

Compound 7: ¹HNMR(400 MHz,CDCl₃): δ 8.45(s,2H),7.49(dd, 5H,J = 12.7,6.6 Hz),7.30 (d,2H,J = 4.4 Hz),6.86 (d,2H,J = 4.4 Hz), 4.43 (s,4H),3.27 (dt,16H,J = 13.0,6.5 Hz),2.47 (s,4H),2.40 (s,8H), 2.11(d,4H,J = 12.2 Hz),1.82-1.75(m,5H),1.69(s,9H),1.61(s,4H), 1.37 (s,36H). ¹³C NMR (101 MHz,CDCl₃): δ 155.37,146.89,138.45, 133.03,129.82,129.43,127.34,118.82,78.74,44.36,44.12,28.46, 28.33,27.62,27.44,21.66,13.10. IR (KBr,cm^-1): 3402,2960, 2920,2851,1746,1645,1582,1554,1467,1381,1249,1129,793, 655. ESI-MS (m/z) calcd. for C₆₃H₉₅BF₂N₁₄O₈ [M+H]⁺: 1225.8, found: 1225.9.

Compound 1: ¹H NMR (400 MHz,D₂O): δ 8.73 (s,2H),7.26- 6.89 (m,7H),6.45-6.20 (m,2H),4.60 (brs,4H),3.65-3.12 (m, 28H),2.45-2.31 (m,4H),2.30-1.89 (m,12H). ¹³C NMR (101 MHz, D₂O): δ 146.28,138.59,135.72,132.83,131.00,130.39,128.09, 126.56,119.39,50.71,48.11,47.69,42.66,41.67,24.27,20.47, 18.46,17.47,10.03. IR (KBr,cm^-1): 3421,2924,2852,1718, 1637,1579,1550,1467,1381,1325,1226,1141,1093,950,800, 752,721,597. HRMS (ES+) calcd. for C₄₃H₆₃BF₂N₁₄ (M+H)⁺: 825.5429,found 825.5503.

The characterization of the other new compounds and the spectra of ¹H NMR,¹³C NMR spectroscopy and MS of all new compounds can be found in the Supporting information. 2.3. Cell culture and fluorescent image assay

HepG2 cells were cultured in DMEM supplemented with 10% FBS in a humid atmosphere containing 5% CO₂ at 37℃. After 24-h incubation in the medium,for imaging studies,cells were plated in Glass Bottom Cell Culture Dish (Nest) containing 1 mL of complete DMEM and incubated at 37℃ under 5% CO₂ for 24 h. Bright field and fluorescence images were taken with a Zeiss Abserver A1 inverted fluorescence microscope equipped with an EM-CCD camera (Hamamatsu) and an X-Cite 120 metal halide lamp (EXFP). Bright field and fluorescence images were obtained using an 40× objective lens. 3. Results and discussion 3.1. Fluorescent signaling of Cu²⁺ ions

Sensor 1 displays a strong emission at 587 nm with fluorescent quantum yield of 0.041 in aqueous solution (10 μmol/L Tris-HCl, pH 7.4,25℃). The fluorescence spectra of 1 were investigated in the presence of respective fifteen metal ions including Li⁺,Ag⁺, Ca²⁺,Cd²⁺,Co²⁺,Cu²⁺,Fe³⁺,Fe²⁺,Hg²⁺,K⁺,Mg²⁺,Na⁺,Ni²⁺,Pb²⁺ and Zn²⁺ (Fig. 1). It can be seen that the fluorescence of 1 showed no obvious changes after the addition of metal ions except Cu²⁺ ion. However,the addition of Cu²⁺ to the solution of 1 resulted in a significant decrease (13-fold) in the fluorescence intensity at 587 nm. The quantum yieldof 1-Cu²⁺ complex was measured to be 0.0046. Obviously,compound 1 showed excellent selectivity for Cu²⁺ over other metal ions. The quench of fluorescence can be attributedtothephoto-inducedelectrontransfer(PET)mechanism caused by Cu²⁺ ions ^{[32, 33]}.

Fig. 1

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Fig. 1.Emission spectra of 1 (10 μmol/L) in the presence of various metal ions (5 equiv. of Ag+, Ca2+, Cd2+, Co2+, Fe2+, Fe3+, Hg2+, K+, Li+, Mg2+, Na+, Ni2+, Pb2+, Zn2+, Cu2+) in Tris-HCl buffer (1 mmol/L, pH 7.4, 25℃). λex = 540 nm. Inset: visual fluorescence changes of 1 upon addition of Cu2+ under illumination with a 365 nm UV lamp.

The titration profile based on the emission at 587 nm (Fig. 2a) suggested a 2:1 binding stoichiometry of Cu²⁺ to 1,which was further confirmed by a Job’s plot analysis (Fig. 2b).

Fig. 2

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Fig. 2.(a) Emission spectra of 1 (10 μmol/L) in the presence of various concentrations of Cu2+ (0–40 μmol/L). (b) Job’s plot of 1 and Cu2+. The total concentration of 1 and Cu2+ was kept constant at 10 μmol/L. Condition: λex = 540 nm, λem = 587 nm, 1 mmol/L Tris–HCl buffer, pH 7.4, 25℃.

The binding constant for the 1-Cu²⁺ complex was calculated to be (1.89±0.03) × 10⁷ L/mol based on the fluorescence titration (Fig. S1,Supporting information),indicating a strong binding ability of 1 with Cu²⁺. At 10 μmol/L concentration of 1,a good linear relationship between the fluorescence response and the concentration of Cu²⁺ (0- 20 μmol/L) was found (Fig. S2 in Supporting information). The detection limit was estimated to be 0.04 μmol/L in aqueous solution, which is significantly lower than the typical concentration of blood copper(II) (11.8-23.6 μmol/L) in normal individuals ^[34] and the limit of copper(II) in drinking water (GB 160 μmol/L) set by the Department of Environmental Protection of China. 3.2. Fluorescent signaling of ADP by 1-Cu²⁺

Fluorescent sensing of biological anions has drawn much attention in recent years ^{[35, 36, 37]}. Thus 1-Cu²⁺ complex was prepared in situ by mixing 1 with Cu(ClO₄)₂ at a 1:3 ratio in aqueous solution,and its fluorescent emission in the presence of various anions was examined. As shown in Fig. 3,a significant increase in fluorescence intensity at 587 nm was observed after the addition of ADP,the emission enhancement was 15.7-fold in the presence of 15 equiv. of ADP,while the fluorescence enhancement of ATP at the same condition was 3.6-fold. There was no obvious change of the fluorescence emission upon the addition of other organic phosphate anions and inorganic anions. This demonstrated that 1-Cu²⁺ complex can be successfully applied in the selective recognition of ADP. ADP also caused a color change from purple to pink in 1-Cu²⁺ ensemble.

The emission spectra of 1-Cu²⁺ complex (1 mmol/L,Tris-HCl buffer,pH 7.4) were further investigated at various concentration of ADP. As shown in Fig. S5 in Supporting information,the maximum fluorescence intensity at 587 nm started to increase when the ADP concentration was accumulated to 5 equiv. and was linear-dependent in the range of 50-150 μmol/L of ADP (R² = 0.993),indicating that this approach was applicable for quantitative detection of ADP. No more intensity change was observed after the addition of more than 16 equiv. ADP. The limit of detection was calculated to be 0.1 μmol/L according to 3 sigma rules.

Fig. 3

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Fig. 3.(a) Emission spectra of 1–Cu2+ (10 μmol/L) toward each of various anions (15 equiv. of ATP, ADP, AMP, GTP, GDP, GMP, CTP, CDP, CMP, UTP, UDP, UMP, PPi) in Tris–HCl buffer (1 mmol/L, pH 7.4, 25℃). λex = 540 nm. (b) The color changes of 1–Cu2+-ADP and other biological molecules under the irradiation at 365 nm.

The applicability of 1 in cell imaging was then tested. It was found that HepG2 cells showed clear red fluorescence after the incubation with sensor 1 for 5 min,confirming that 1 was cellpermeable (Fig. 4a and d). Addition of Cu²⁺ to HepG2 cells and further incubation with compound 1 resulted in the quench of the red fluorescence emission resulted from 1 (Fig. 4b and e). With the addition of ADP to HepG2 cells,and further incubation with the 1-Cu²⁺ complex solution,it was found that the red fluorescence was dramatically recovered (Fig. 4c and f). These results clearly demonstrated that 1-Cu²⁺ complex was suitable for imaging of ADP in living cells.

Fig. 4

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Fig. 4.Confocal fluorescence and bright field images in HepG2 cells: (a) Bright field image and (d) fluorescence image of HepG2 cells incubated with 1 (5 μmol/L) for 5 min. (b) Bright field image and (e) fluorescence image of HepG2 cells incubated with 15 μmol/L Cu(ClO4)2 for 5 min and then incubated with 1 (5 μmol/L) for 10 min. (c) Bright field image and (f) fluorescence image of HepG2 cells incubated with 100 μmol/L ADP for 10 min and then incubated with 1–Cu2+ (5 μmol/L) for 10 min.

In summary,a novel fluorescent sensor 1 has been prepared through the click reaction between 1,5-bis(acetynyl)BODIPY and Boc-protected N-(3-azidopropyl) ^[12]aneN₃. The fluorescence of 1 was completely quenched upon the formation of Cu²⁺ complex, which provided highly selective and sensitive turn-off sensor to detect Cu²⁺ ions over a range of other metal ions. The detection limit was 0.04 μmol/L for Cu²⁺ in aqueous buffer solution. The quenched fluorescence of 1-Cu²⁺ complex can be selectively restored by the addition of ADP in aqueous solution and living cells. Results from this work have demonstrated that macrocyclic polyamine ^[12]aneN₃ derivatives can be used as good receptors for fluorescent sensing applications. Sensor 1 has combined the advantages of ^[12]aneN₃ (water soluble,strong coordinating ability) and BODIPY (longer wavelength emission and photostable),which are important for the biological applications. Further work to explore the detailed mechanism and application in metabolism process are being performed in our lab.

Acknowledgments

The authors gratefully acknowledge the financial assistances from the National Natural Science Foundation of China (Nos. 21372032 and 91227109),the Fundamental Research Funds for the Central Universities(2009SC-1),Beijing Municipal Commission of Education,the Program for Changjiang Scholars and Innovative Research Team in University.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2015.05.032.