Ratiometric detection of Cu+ in aqueous buffered solutions and in live cells using fluorescent peptidyl probe to mimic the binding site of the metalloprotein for Cu+

Accepted Manuscript Title: Ratiometric Detection of Cu+ in Aqueous Buffered Solutions and in Live Cells Using Fluorescent Peptidyl Probe to Mimic the Binding Site of the Metalloprotein for Cu+ Authors: Pramod Kumar Mehta, Eun-Taex Oh, Heon Joo Park, Keun-Hyeung Lee PII: DOI: Reference:

S0925-4005(17)31989-5 https://doi.org/10.1016/j.snb.2017.10.087 SNB 23391

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

3-7-2017 12-10-2017 16-10-2017

Please cite this article as: Pramod Kumar Mehta, Eun-Taex Oh, Heon Joo Park, KeunHyeung Lee, Ratiometric Detection of Cu+ in Aqueous Buffered Solutions and in Live Cells Using Fluorescent Peptidyl Probe to Mimic the Binding Site of the Metalloprotein for Cu+, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.10.087 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ratiometric Detection of Cu+ in Aqueous Buffered Solutions and in Live Cells Using Fluorescent Peptidyl Probe to Mimic the Binding Site of the Metalloprotein for Cu+ Pramod Kumar Mehta1, Eun-Taex Oh2,3, Heon Joo Park3,4,*, Keun-Hyeung Lee1,*

1

Bioorganic Chemistry Laboratory, Center for Design and Applications of Molecular Catalysts,

Department of Chemistry and Chemical Engineering, Inha University, Incheon 402-751, South Korea. 2

Department of Biomedical Sciences, College of Medicine, Inha University, Incheon 22212, South Korea.

3

Hypoxia-related Disease Research Center, College of Medicine, Inha University, Incheon 22212, South

Korea. 4Department of Microbiology, College of Medicine, Inha University, Incheon, 22212, South Korea.

Author information Corresponding Author * E-mail: [email protected] (Keun-Hyeung Lee) and [email protected] (Heon Joo Park)

  

Highlights The fluorescent peptidyl probe for Cu(I) was easily synthesized in high yield (75 %). Highly sensitive and selective response to Cu(I) among various biological relevant metal ions. Ratiometric response to Cu(I) and the intensity ratio (I472/I396) at 472 and 396 nm increased by about 130



fold. Cell penetration and ratiometric detection of intracellular Cu(I) in the Golgi apparatus in live cells.

Ratiometric detection of Cu+ in aqueous buffered solutions and live cells is highly recomended. We synthesized a fluorescent probe (1) based on the peptide receptor to mimic the binding site of the metalloprotein (CusF) for Cu+. 1 sensitively and selectively detected Cu+ among various biological relevant metal ions in aqueous solutions at physiological pH through a ratiometric response. Job’s plot analysis indicated that 1 formed a 2:1 complex with Cu+ and the binding affinity of 1 for Cu+ was measured to be 5.73×10-21 M2 from a competition experiment with bathocuproine disulfonate. The probe showed significant ratiometric responses to Cu+ over a wide range of pH (6.5~10.5). The binding mode 1

study showed that the imidazole and indole groups of the peptide receptor played a critical role in the tight binding to Cu+. 1 penetrated successfully in living A549 cells and detected intracellular Cu+ ions in Golgi apparatus through ratiometric response. Giving the recent growing interests in fluorescent imaging of Cu+, the development of a fluorescent ratiometric probe (1) based on the peptide receptor to mimic the binding site of the metalloprotein for Cu+ will provide a potential tool for detection of intracellular metal ions in live cells. Keywords: Copper, Probe, Fluorescent, Ratiometric, Chemosensor, Cu(I).

1. Introduction Copper is one of key trace metals to regulate biological processes such as cellular respiration, tissue formation, and antioxidant defense in cells. In particular, copper plays a critical role in cells as a redox cofactor of several cuproenzymes due to its potent redox activity [1]. However, its potent redox activity causes an intrinsic toxicity to cells through oxidative damage to DNA, proteins, and lipids [2]. To this end, intracellular trafficking systems in cells tightly regulate the level of labile Cu+ to minimize copper toxicity using several regulatory proteins [3-5]. In cells, copper is incorporated into the secreted and plasma membrane-targeted cuproenzymes in Golgi for normal copper homeostasis. In order to maintain copper homeostasis by reducing accumulation of toxic copper, the Golgi apparatus releases ATPases that transfer copper from the cytosol into the Golgi lumen [6]. Previous reports demonstrated that genetic alteration of proteins involved in intracellular copper trafficking systems might cause severe neurodegenerative diseases such as Menkes syndrome, Wilson’s disease, Alzheimer’s disease, and prion diseases [7-9]. Therefore, monitoring of the intracellular Cu+ in specific organelle of living cells is required to elucidate pathological mechanisms of copper-related diseases. As fluorescent imaging is regarded as an efficient tool for monitoring metal ions in live cells because of their simplicity, high sensitivity, and inexpensive instrumentation [10-12], several small fluorescent probes for Cu+ have been developed. On the other hand, relatively a few fluorescent probes for Cu+ have been reported in comparison to the fluorescent probes for other biologically relevant metal ions, such as Ca2+, Zn2+, Fe2+/Fe3+, and Cu2+ [13-18]. Furthermore, a few of them showed a successful detection of intracellular Cu+ in live cells because they should have sufficient membrane-permeable ability, relatively potent binding affinity for Cu+ compared to those of intracellular copper chelators, and high selectivity for Cu+ with respect to other biologically relevant metal ions. Since Yang et al. first reported their pioneer work on the detection of intracellular Cu+ in live cells using a small fluorescent probe based on a thioether-rich receptor [19], several fluorescent probes for Cu+ based on the different type of the receptors have been reported [13-15,19-32]. On the other hand, most of them have commonly shared an azatetrathiacrown receptor or a thioether-rich receptor for Cu+ [20-32]. Considering the various concentrations of Cu+ in specific organelles and endogenous Cu+ ligands within the cytoplasmic 2

environment of cells, it is highly advisable to develop fluorescent probes with different potent binding affinities for Cu+ based on the novel receptors. In addition, ratiometric detection for Cu+ ions are highly recommended in the detection of metal ions in live cells because the ratio between two emission bands could be used to confirm the concentration of the probe and provided a built-in correction for environmental effects for the emission intensity such as temperature, polarity of media, and pH [33,34]. However, to the best of the author’s knowledge, only two fluorescent ratiometric probes for Cu+ ions have been previously reported [22,31]. Furthermore, compared to the intensity change at one emission band, the emission intensity change by Cu+ ions at the other emission band was almost negligible or the emission intensity at the other band was small. Thus, the probes seemed to show turn on response to intracellular Cu+ in live cells rather than ratiometric response. Thus, it is quite challenging for the development of fluorescent probes for the detection of Cu+ by ratiometric response in aqueous solution as well as in live cells. In recent years, peptides have received attentions for the new receptors of fluorescent probes for the biologically relevant metal ions because of high solubility in water, cell penetrating ability, biological compatibility, targeting for specific organelles in cells, and potent binding affinities for the biological relevant metal ions [35-45]. In addition, there is an advantage that the binding affinity for target metal ions could be further tuned and optimized by changing the amino acid sequences of the peptide receptors. Our strategy for the ratiometric probes of cellular copper ion is based on the peptide receptor to resemble the binding site of metalloproteins for Cu+. Several research groups reported the binding sites of metalloproteins for Cu+ on the basis of X-ray crystallography and NMR spectroscopy [46-48]. Among them, CusF, a periplasmic Cu+ binding protein in Escherichia coli associated with Cu+ through the binding site consisting of one His, two Met, and one Trp, as shown in scheme 1 [47,48].

Interestingly, the Trp

+

residue of the binding site of CusF stabilized the metal coordination through Cu - interactions and might protects Cu+ from oxidation and coordination by other ligands. Keeping this into consideration, in the present study, we synthesized a fluorescent probe (1) based on the tripeptide receptor (TrpProHis) to resemble the half of the binding site of CusF for Cu+ and the property of the pyrene fluorophore. Pyrene fluorophore has an interesting unique property of the fluorescence emission; when two pyrene fluorophores will be close each other, excimer emission (enhanced and a red shifted emission) will occur [49, 50]. Interestingly, the fluorescent probe showed a sensitive and selective ratiometric response to Cu+ among the various biologically relevant metal ions in aqueous buffered solutions (10 mM HEPES, pH 7.4) containing 1% DMF. About 2 equiv of Cu+ was enough for the complete change of the ratiometric response. The peptidyl probe had a potent binding affinity (Kd = 5.73×10-21 M2) for Cu+ measured by a competition experiment with bathocuproine disulfonate. Furthermore, the peptidyl probe penetrated live cells and detected Cu+ in Golgi apparatus successfully by a ratiometric fluorescence response.

2. Experimental section 3

2.1. Reagents Fmoc-Trp(Boc)-OH, Fmoc-His(Trt)-OH Fmoc-Pro-OH, 1-Hydroxybenzotriazole (HOBt) and Rink Amide MBHA resin (100–200 mesh, 0.43 mmol/g) were purchased from BeadTech. N,N’diisopropylcarbodiimide (DIC) was acquired from TCI. N, N-dimethylformamide (DMF), trifluoroacetic acid (TFA) and triisopropylsilane (TIS) were supplied by Acros Organics. The other reagents for solid phase synthesis, including diethyl ether, triethylamine (TEA) and piperidine, were purchased from Sigma Aldrich. 1–pyrene acetic acid, tetrakis(acetonitrile)copper(I) hexafluorophosphate as a Cu+ source and all perchlorate salts of metal ions were purchased from Sigma Aldrich. A549 cells were purchased from ATCC (Manassas, VA, USA)

2.2. Synthesis and characterization of 1 1 was synthesized by solid phase peptide synthesis using Fmoc chemistry (Scheme S1). The coupling of 1–pyrene acetic acid was performed by applying the following procedure. 1-Pyreneacetic acid (78 mg, 0.3 mmol), HOBt (40 mg, 0.3 mmol) and DIPC (47 μL, 0.3 mmol) in DMF (3 mL) were added into the resin and the resulting solution was stirred for 4 h at room temperature. After the coupling reaction was complete, deprotection and cleavage from the resin were accomplished by a treatment with a mixture of TFA/TIS/H2O (95:2.5:2.5, v/v/v) at room temperature for 4h. After removing the excess TFA by N2, the crude product was precipitated by the addition of cold ether. The crude product was purified further by semi-preparative HPLC using water (0.1% TFA)/acetonitrile (0.1% TFA) gradient to give the final product with a 75% yield. 1 was characterized by ESI-TOF HRMS (Compact, Bruker) and NMR spectrometer (JNM-ECZ400S/L1). The high purity (>98%) of 1 was confirmed using an analytical HPLC on a C18 column. Characterization data for 1 : White solid; m.p 201 oC; 1H NMR (400 MHz, DMSO-d6, 25 oC) δ 8.98 (d, J = 8.2 Hz, 2H), 8.82 (d, J = 8.4 Hz, 1H), 8.34 (d, J = 7.2 Hz, 2H), 8.28 (d, J = 7.2 Hz, 2H), 8.21 (d, J = 7.2 Hz, 3H), 8.18-8.12 (m, 2H), 8.07 (t, J = 8.0 Hz, 2H), 7.89 (d, 8.2 Hz, 2H), 7.44 (s, 1H), 7.39 (s, 1H), 7.27 (s, 2H), 4.93-4.88 (m, 1H), 4.55-4.45 (m, 2H), 4.35-4.25 (m, 1H), 4.20 (s, 2H), 3.53-3.52 (m, 2H), 3.2-3.1 (m, 2H), 3.1-3.05 (m, 2H), 1.95-2.05 (m, 2H), 1.85-1.65 (m, 1H);

13

C NMR (100 MHz, DMF-d6, 25 oC) δ

181.4, 179.8, 178.7, 138.9, 138.3, 137.1, 136.9, 136.0, 134.7, 134.4, 127.1, 126.6, 69.7, 61.2, 59.4, 49.2, 49.0, 38.9, 36.3, 34.1; HRMS (m/z): [M + Na+]+ calculated for C40H37N7NaO4: 702.2797, observed: 702.2799; IR (KBr): 3285 (br s), 3044, 1672, 1440, 1202, 1136 cm−1. 2.3. General procedure for sample preparation A stock solution (1 mM) of 1 was prepared by dissolving 1 in degassed DMF/H2O (1:1, v/v). The stock solution was diluted by aqueous buffered solution to prepare sample in aqueous buffered solutions containing 1% DMF (10 mM HEPES, pH = 7.4) for fluorescent measurements or for cell imagining. The concentration of 1 was confirmed by measuring the absorbance at 342 nm for the pyrene group. The Cu+

4

solution was prepared by dissolving tetrakis(acetonitrile)copper(I) hexafluorophosphate in degassed acetonitrile.

2.4. General UV/Vis and fluorescent measurements UV/Vis absorption spectra of the samples in a 10 mm path length cuvette were measured using a PerkinElmer UV-Vis spectrophotometer (model Lambda 45). The fluorescence emission spectra of the samples in a 10 mm path length cuvette were measured using a Perkin Elmer luminescence spectrophotometer (model LS 55) with excitation at 342 nm and the slit widths for excitation and emission were described in the figure legends. The absorbance and fluorescence measurements were carried out in aqueous buffered solutions containing 1% DMF (10 mM HEPES, pH = 7.4). 2.5. Determination of the dissociation constant of 1 for Cu+ The dissociation constant (Kd) of 1 for Cu+ was determined by a competition experiment with bathocuproine disulfonate (BCS), which is commonly used to determine the potent binding affinity of copper-binding probes [38]. BCS formed a stable red complex [Cu(BCS)2]3- that displayed an absorption band in the visible region (λmax = 483 nm, ε = 13,300 M−1 cm−1); its association constant is 1019.8 M [38]. Cu+ + 2BCS2- ⇆ [Cu(BCS)2] 3-

[

β =[

( ][

)

] ]

The competition of Py-WPH with BCS is expressed by following equation. [2(1)−Cu+]+ + 2BCS2- ⇆ 2(1) + [Cu(BCS)2]3=

[ ] [Cu(BCS) ] = [2( ) − Cu ][BCS ]

×β

The equilibrium competition constant (Kcomp) was calculated based on the absorbance at 483 nm. The total concentration of [Cu(BCS)2]3- was determined by measuring the absorbance at 483 nm because 1 and its complex with Cu+ did not absorb near 483 nm. 2.6. Confocal laser scanning microscopy to assess cellular uptake of 1 and intracellular Cu+ in live cells A549 human lung cancer cells were purchased from ATCC (Manassas, VA, USA). The cells were maintained in 75-cm2 plastic tissue culture flasks with Dulbecco’s modified Eagle’s medium (DMEM, Hyclone Laboratories Inc., Logan, Utah, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone Laboratories Inc.) and 1% penicillin/streptomycin (P/S, Hyclone Laboratories Inc.) in a humidified 5% CO2/95% air incubator at 37 °C. To assess cellular uptake of 1 and the intracellular Cu+ in live cells, A549 cells were seeded in an 8-well chamber slide (Nunc, Roskilde, Denmark). The cells were treated with 1 for 2 h at 37oC in aqueous buffered solution containing 1% DMF and washed three times with PBS solution. Fluorescence imaging of the cells was performed with a Nikon C1-Plus laser scanning TE2000E confocal microscope (Nikon, Tokyo, Japan) inverted device for 30 min after the addition of Cu+ ions into the cells. Single-plane confocal image sequences were taken every 1 min for the confocal stacks. After this, A549 5

live cells loaded with 1 were incubated with Cu+ for 30 min, washed with PBS solution for three times and then confocal images were taken. Finally, the above A549 live cells were incubated with Cu+ chelator, neocuprione (1 mM) for 30 min, washed with PBS solution for three times followed by confocal images. Intracellular localization of 1 and the accumulation of Cu+ in Golgi apparatus were assessed microscopically in A549 cells. The cells were seeded into an 8-well chamber slide and treated with 1 for 2 hr. The cells were then washed three times with PBS and incubated 5 μM of BODIPY TR Ceramide (Life Technologies, Grand Island, NY, USA). After 30 min of treatment, the cells were washed with PBS and incubated with Cu+ for 30 min. The fluorescence intensities of 1 and BODIPY TR-stained Golgi apparatus were analyzed using a laser-scanning TE2000E confocal microscope (Nikon, Tokyo, Japan).

2.7. Cell Viability The cytotoxicity of 1 was assessed using a MTS solution assay (Promega, Madison, WI, USA). The A549 cells were seeded into a 96-well plate at 1 × 104/well in 150 μL of DMEM with 10% FBS and 1% P/S and incubated for 16 hr. The cells were treated with or without 1 for 24 hr. The cells were then washed three times with PBS and incubated with or without Cu+ for 30 min. After treatment, the supernatants were removed, and the cells were incubated with 100 μL of fresh DMEM media containing 20 μL of a MTS solution for a further 1 hr. Finally, the absorbance at 490 nm was measured using a microplate reader. Untreated cells were used as a control and incubated under the same conditions for the same time.

3. Results and discussion 3.1. Design and synthesis of 1 The fluorescent probe (1) based on the tripeptide receptor (TrpProHis) was designed to mimic the half of the binding site of CusF, a periplasmic Cu+ binding protein for the ratiometric detection of Cu+. Pyrene fluorophore was conjugated into the N-terminal of the peptide receptor for the ratiometric response because this fluorophore has high quantum yield, pH insensitivity, and unique monomer and excimer emissions depending on the proximity between the pyrene fluorophores [49-51]. 1 was easily synthesized in high yield (75 %) using solid phase synthesis. The tripeptide-based probe was characterized by HRMS, 1

H NMR,

13

C NMR and IR spectroscopies (Fig. S1~S5). The high purity (>98%) of the probe was

confirmed by HPLC with a C18 reverse column. As the peptidyl probe has a good solubility in aqueous solution, all of the photochemical experiments were carried out in aqueous buffered solutions (10 mM HEPES, pH 7.4) containing 1% DMF. 3.2. Fluorescent and UV/Vis titration experiment of 1 to Cu+ As shown in Fig. 1, 1 showed a significant monomer emission at around 370–410 nm with negligible excimer emission in aqueous buffered solution (10 mM HEPES, pH 7.4). Upon addition of increasing concentration of Cu+ into the solution containing 1, the monomer emission decreased significantly and the excimer emission at 472 nm increased significantly. This indicates that as we designed, the fluorescent 6

probe based on the peptide receptor to mimic the half of the binding site of CusF must dimerize in the presence of Cu+ ions. The intensity ratio (I472/I396) at 472 and 396 nm increased from 0.029 to 3.75 (about 130 fold) as the concentration of Cu+ increased. About 20 μM (2 equiv) of Cu+ was required to completely change the monomer and excimer emissions. We measured the quantum yield of 1 in the absence and presence of Cu+, respectively. The quantum yield of 1 was 0.0071 in the absence of Cu+, whereas the quantum yield was 0.0145 in the presence of Cu+. Upon addition of Cu+, the quantum yield increased by two times and the maximum emission was shifted from 396 nm to 472 nm. Thus, the probe showed a significant ratiometric response to Cu+. The low quantum yield of 1 was due to the fluorescent quenching effect of the Trp moiety including an indole group on the pyrene fluorophore [52]. The 2-fold enhancement of the quantum yield of 1 in the presence of Cu+ revealed that the chelation of Cu+ with the indole group of 1 might inhibit PET from the indole group to the pyrene fluorophore, resulting in the enhancement of the emission. UV−visible absorption spectra of 1 exhibited a typical pyrene absorption band at 343 nm in aqueous buffered solution (10 mM HEPES, pH 7.4). Upon addition of Cu+, the absorbance band at 343 nm corresponding to the pyrene decreased with a red shift (Fig. S6). The significant enhancement of the excimer emissions at 480 nm and the decrease of the absorption band at 343 nm in the presence of Cu+ indicate that two pyrene labelled probes dimerized in the presence of Cu+ and the pyrene fluorophores of the probes come close each other, resulting in the decrease of the absorbance at 343 nm [50,51].

3.3. Selectivity study of 1 The fluorescence emission spectra of 1 was measured in the presence of various biologically relevant metal ions (Na+, Mg2+, Al3+, K+, Ca2+, Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu+, Cu2+, and Zn2+) in aqueous buffered solutions at physiological pH. As shown in Fig. 2a, 1 showed a significant ratiometric response to Cu+ among the various biologically relevant metal ions. Even though the monomer emissions of 1 slightly decreased in the presence of Zn2+, Cu2+, Fe2+, and Fe3+, the excimer emissions did not increase considerably in the presence of these metal ions and the excimer emission increased significantly only in the presence of Cu+.

To examine the interference effect of the other biologically relevant metal ions on the detection of 1 for Cu+, the ratiometric response to Cu+ were measured in the presence of the other metal ions. As shown in Fig. 2b, the ratiometric response to Cu+ was not changed considerably in the presence of the other biologically relevant metal ions except Fe2+, Fe3+, Zn2+, and Cu2+. The decrease in ratiometric response of 1 to Cu+ in the presence of Fe2+ and Fe3+ can be explained by the quenching effects and/or the absorbance effect of the metal ions [53-56]. We carried out the fluorescent titration experiment of 1 with Cu+ in the presence of Zn2+ (2 equiv, 20 uM) because the concentration of Zn2+ in live cells is relatively high among the various biologically relevant metal ions. Upon addition of increasing concentration of Cu+, the monomer emissions at 396 nm decreased with concomitant increase in excimer emissions at 472 nm and about 2 equivalent of Cu+ was enough for the saturation of change in emission intensity ratio (I472/I396), as 7

shown in Fig. 3. We also carried out the fluorescent titration experiment of 1 with Cu+ in the presence of Cu2+ (2 equiv, 20 uM). Even though 1 showed a decreased monomer emission in the presence of Cu2+, the decrease in monomer emission at 396 nm and increase in excimer emission at 472 nm were observed upon gradual addition of Cu+ (Fig. S7). About 2 equivalent of Cu+ was sufficient for the complete change in the emission intensity ratio (I472/I396). Interestingly, 1 showed a sensitive ratiometric response to Cu+ in the presence of Zn2+ and Cu2+. These results suggested that the binding affinity of 1 for Cu+ was more potent than those for Cu2+ and Zn2+ and 1 could detect Cu+ by a ratiometric response in the presence of biologically relevant metal ions including Zn2+ and Cu2+.

3.4. Binding stoichiometry and binding affinity of 1 The binding stoichiometry of the complex between 1 and Cu+ was investigated by Job’s plot analysis (Fig. S8). A Job’s plot, which exhibited a maximum at a 0.4 mole fraction indicated that 1 formed a 2:1 complex with Cu+. Considering the potent binding affinity for Cu+, the dissociation constant of 1 for Cu+ was determined by a competition experiment with bathocuproine disulfonate (BCS), which is commonly used to determine the potent binding affinity of copper-binding proteins and probes [38]. The addition of BCS to the solution containing 1 and Cu+ induced the characteristic absorption band of [Cu(BCS) 2]3- (Fig. S9). The absorbance at 483 nm increased with increasing concentration (0-10 M) of BCS, which indicates that BCS snatched Cu+ from 1 and formed a complex with Cu+. The dissociation constant of 1 for Cu+ was determined to be 5.73×10-21 M2 using the competition method (Table S1). Thus, 1 has a potent binding affinity towards Cu+. 3.5. pH Effect on the fluorescent response of 1 to Cu+ The ratiometric response to Cu+ was measured at various pH values to investigate the binding mode and working pH range (Fig. 4). As pH was lower than 6.0, 1 did not display a fluorescent response to Cu+ due to the protonation of the imidazole group (pKa = 6.04). This result confirmed that the imidazole group of 1 might play a critical role in the binding with Cu+. As pH increased over 6.0, the intensity ratio (I472/I396) gradually increased and became a maximum at pH 7.4. As pH increased over 7.4, the ratiometric response gradually decreased maybe due to the poor solubility of Cu+ at basic pH [57]. Overall result indicates that 1 showed significant ratiometric responses to Cu+ over a wide range of pH (6.5~9.5).

3.6. Binding mode of 1 with Cu+ We investigated the binding mode of 1 with Cu+ by using organic spectroscopy techniques such as ESI mass spectrometry and 1H-NMR spectroscopy. The complex between 1 and Cu+ was characterized by ESI mass spectrometry. For this, we investigated whether 1 showed a ratiometric response to Cu+ in the distilled water containing 50% acetonitrile and 1 mM ammonium formate that was a proper solvent system 8

for ESI mass spectrometry. 1 showed a ratiometric response to Cu+ in this solvent system. Thus, the solution containing 1 with and without Cu+ was analyzed by ESI mass spectrometry. When 1.0 equiv of Cu+ was added to the solution of 1, a new peak appeared at 1422.75 (m/z) corresponding to [2*1 + Cu+]+, as shown in Fig. 5a. The ESI mass spectrum indicated that that 1 formed a 2:1 complex with Cu+ in aqueous solutions and 1 had a potent binding affinity for Cu+. 1

H NMR titration experiments were carried out in methanol-D4/D2O (8:2;v/v) containing ammonium

formate (0.5 mM) because 1 showed a ratiometric response to Cu+ and 5 mM of 1 did not precipitate in the presence of Cu+ in this solvent system. Upon addition of Cu+, H(19) peak of the imidazole group was shifted downfield (∆ = 0.06 ppm) whereas H(21) peak of the imidazole group was shifted downfield and finally disappeared. This result clearly indicated that Cu+ coordinated with the imidazole group of 1. Similarly, H(9) peak of the indole group was shifted downfield and finally disappeared with increasing concentration of Cu+ . Furthermore, considerable downfield shifts (∆ = 0.025, 0.055, 0.02 and 0.061 ppm respectively) of the other protons (H11, H12, H13, and H14 respectively) of the indole ring were also observed. It may be due to the interaction between Cu+ and the indole group of 1. In addition, H(31) peak of the pyrene group was shifted down field (∆ = 0.035 ppm) and an another proton peak of the pyrene group, most preferably H(22) disappeared with gradual addition of Cu+, which may suggested that the pyrene fluorophore came closer to Cu+ after complexation of 1 with Cu+, as shown in scheme 1. Considering the results of mass spectrometry, NMR titration experiments, pH titration experiments, Job’s plot analysis, UV/vis titration experiments, and fluorescence experiments, the binding mode of 1 with Cu+ was proposed, as shown in Scheme 1. The imidazole group of 1 played a critical role in the binding to Cu+ and the indole group of 1 might chelate Cu+ maybe through cation-π interactions to form a stable complex between 1 and Cu+. The interactions of Cu+ with pi electron cloud of the indole group of the tryptophan residue were proposed in CusF [47,48]. Considering the ligands (imidazole and indole groups) of 1 and the binding mode of CusF for Cu+, 1 might form a 2:1 complex with Cu+. The 2:1 complexation induced the pyrene fluorophores came to closer, resulting in a ratiometric response with the decrease of monomer emission at 396 nm and increase of excimer emission at 472 nm. 3.7. Fluorescence imaging detection for Cu+ in live cells As the peptidyl probe tightly bound Cu+ and showed a ratiometric response to Cu+ in aqueous buffered solutions at physiological pH, this study investigated whether 1 could penetrate live cells and monitor the intracellular Cu+ via a ratiometric response. A549 cells were treated with 1 for 2 hr at 37 °C in aqueous buffered solution containing 1% DMF and washed with aqueous buffered solution. Then, the fluorescence images of 1 in cells were monitored by confocal laser scanning microscopy (Fig. 6). The blue color image of the cells reveals that 1 penetrated A549 cells successfully under these conditions. Cu+ ions were added into the medium containing the A549 cells and then fluorescence images of the A549 cells were monitored. After the addition of Cu+ into the probe loaded A549 cells, a strong green color and the decrease of blue color were observed, which indicated that 1 showed a sensitive ratiometric response to intracellular Cu+ in 9

live cells.

To confirm that the significant enhancement of emissions for green fluorescence was due to the binding of 1 to Cu+, 1 was incubated in the medium containing Cu+-supplemented cells in the presence of a cellpermeable Cu+ chelator (neocuprione) and fluorescence images of the cells were then monitored. As shown in Fig. 6, the enhanced green fluorescent images were not observed whereas the blue fluorescent images of cells were observed. This result confirmed that the significant enhancement of the emissions for green fluorescence images of cells was due to the binding of 1 to Cu+ and 1 exhibited a ratiometric response to intracellular Cu+ in live cells. Cu+ is localized predominantly in the Golgi apparatus and the mitochondria under acute copper toxicity [58]. In particular, Golgi apparatus is a central organelle for cellular copper homeostasis [59]. Therefore, it is important to determine if 1 could detect Cu+ in the Golgi apparatus in cells. A co-localization experiment was carried out by co-staining cells with the Golgi apparatus marker, BODIPY TR Ceramide and 1. As shown in Fig. 7, the strong fluorescence (red color) from BODIPY TR Ceramide was well overlapped with the strong fluorescence (green color) from 1. This indicates that 1 could detect Cu+ in the Golgi apparatus of live cells. The cytotoxicity of 1 was measured using a MTS assay. 1 did not show any noticeable cytotoxicity to A549 cells (Fig. S10). This showed that the peptide-based probe was highly biocompatible and had a low cytotoxicity. As we discussed, the ratiometric detection for Cu+ ions is highly recommended in live cells because the ratio between two emission bands could be used to confirm the concentration of the probe and provides a built-in correction for environmental effects for the emission intensity [33,34]. Interestingly, the fluorescent probe (1) based on the peptide receptor to mimic the binding site of CusF showed several interesting properties including ratiometric detection of Cu+ such as high selectivity, high sensitivity, good solubility in aqueous solution, cell penetration ability, and localization in Golgi apparatus and cytosol. In comparison to the ratiometric response for Cu+ ions of the previously reported ratiometric probes [22,31], 1 detected intracellular Cu+ in live cells by significant intensity change of two emission bands with about 100 nm difference. Given the growing interest in fluorescent imaging of biological relevant metal ions in live cells, the fluorescent probe (1) based on the peptide receptor to mimic the binding site of the metalloprotein will provide the potential tool for monitoring the fluorescent imaging of biological relevant metal ions in live cells. Further work is currently under way to change the fluorophores with a longer excitation wavelength for cell images and to examine the localization of mitochondria in cells by conjugation with targeting peptide sequences for specific organelles. 4. Conclusion We synthesized a fluorescent probe (1) based on the peptide receptor to mimic the binding site of the metalloprotein (CusF) for Cu+. 1 selectively detected Cu+ among various biological relevant metal ions in 10

aqueous solutions at physiological pH. 1 sensitively detected Cu+ by a ratiometric response and the binding affinity for Cu+ was measured to be 5.73×10-21 M2. 1 showed significant ratiometric responses to Cu+ over a wide range of pH (6.5~10.5). The binding mode study showed that the imidazole group played a critical role in the tight binding to Cu+, and the pi-electrons of indole group of the probe might cooperate to form a stable complex between 1 and Cu+. The formation of a 2:1 complex between 1 and Cu+ might be important for the selective ratiometric response to Cu+. Furthermore, 1 penetrated successfully in live A549 cells and detected intracellular Cu+ ions in Golgi apparatus through ratiometric response. The newly designed fluorescent probe to mimic the binding site of the metalloprotein in this study showed a promising property for the detection of intracellular Cu+ ions as a new diagnostic tool. Notes The authors declare no competing financial interest.

Acknowledgement This work was supported by grants (2017R1A2B2006897) and (2015M2B2B1068599) from National Research Foundation of Korea. Appendix A. Supplementary data Experimental details, fluorescent spectra, HPLC, mass spectrum, NMR data. This material is available free of charge via the Internet at http://pubs.acs.org.

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Biographies Pramod Kumar Mehta received his M.Sc. degree in chemistry from Tribhuvan University, Nepal. He is doing Ph.D. in sensor and peptide chemistry under the supervision of Professor Keun-Hyeung Lee in Inha University in South Korea. Eun-Taex Oh has B.Sc. and M.Sc. degree in biological engineering from Inha University and Ph.D. in Medicine from Inha University. He works as an Assistant Professor in Inha University. His current research interests include Tumor microenvironment and Radiation Biology. Heon Joo Park has M.D. degree in Medicine from The Catholic University of Korea, M.Sc. degree in Neuroscience from University of Minnesota and Ph.D. degree in Radiation Oncology from University of Minnesota. She works as a professor in Inha University. Her current research interests include Tumor microenvironment and Radiation Biology. Keun-Hyeung Lee has a B.Sc. in applied chemistry from Seoul National University and Ph.D. in organic chemistry from University of Iowa. He works as a professor in Inha University. His current research interests include chemosensors and peptide chemistry.

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4

I472/I396

Intensity

350 300 250 200 150 100 50 0 360

3 2 1 0 0 5 10 15 20 25 30 + [Cu ] 

420

480

540

600

Wavelength (nm) Fig. 1. Fluorescence emission spectra of 1 (10 μM) with increasing concentration of Cu+ in aqueous buffered solution (10 mM HEPES, pH 7.4) containing 1% DMF.

Fig. 2. (a) Fluorescence spectra of 1 (10 μM) with various biologically relevant metal ions (2 equiv) and (b) emission intensity ratio (I472/I396) of 1 (10 μM) in the presence of Cu+ (2 equiv) and additional metal ions (2 equiv) in aqueous buffered solutions (10 mM HEPES, pH 7.4) containing 1% DMF.

16

I472/I396

Intensity

300 250 200 150 100 50 0 360

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0

5 10 15 20 25 30 +

[Cu ] 

420

480

540

600

Wavelength (nm) Fig. 3. Fluorescence spectra of 1 (10 μM) on gradual addition of Cu+ in presence of Zn2+ (2 equiv μM) in aqueous buffered solution (10 mM HEPES, pH 7.4) containing 1% DMF.



I472/I396

4



Cu

3 2 1 0 4

5

6

7

8

9 10 11 12

pH Fig. 4. Ratiometric response of 1 (10 µM) to Cu+ (2 equiv) in aqueous buffered solution containing 1 % DMF at different pH.

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Fig. 5. (a) ESI mass spectra of 1 (20 μM) in the presence of Cu+ (1 equiv) in 50% CH3CN/H2O and (b) Partial 1H NMR spectra of 1 (5 mM) in the presence of Cu+ in methanol-D4/D2O (8:2;v/v) containing ammonium formate (0.5 mM) at 25 ℃.

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Fig. 6. Confocal fluorescence images of live A549 cells incubated with 20 μM of 1 (a-e), A549 cells incubated with 1 in the presence of Cu+ (2 equiv) (f-j) and under copper-supplemented conditions in the presence of the Cu+ chelator, neocuprione (k-o). DAPI field (a, f, k), FITC field (b, g, i), bright field (c, h, m), merged images of DAPI and FITC field (d, I, n) and merged images of DAPI and FITC field and bright field (e, j, o). [Scale bar = 50 μm]

Fig.7. Confocal fluorescence images of live A549 cells stained with 1 (20 μM) and of BODIPYTR Ceramide (5 μM) for 30 min under copper-supplemented conditions. (a), (b) confocal fluorescence images, (c)bright field image, (d), (e) merged images. [Scale bar = 20 μm]

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(a)

The binding sites of metalloprotein (CusF) for Cu+

(b)

342 nm

1 342 nm

472 nm

396 nm

Scheme 1. (a) The binding site of the metalloprotein (CusF) for Cu+ and (b) the binding mode of 1 with Cu+

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