Two-Photon Probe for Cu2+ with an Internal Reference: Quantitative

Apr 21, 2014 - Department of Internal Medicine, Korea University College of Medicine, 73, Inchon-ro ... general method of designing TP probes for quan...
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Two-Photon Probe for Cu2+ with an Internal Reference: Quantitative Estimation of Cu2+ in Human Tissues by Two-Photon Microscopy Dong Eun Kang,† Chang Su Lim,† Ji Yeon Kim,† Eun Sun Kim,‡ Hoon Jai Chun,‡ and Bong Rae Cho*,† †

Department of Chemistry, Korea University, 145, Anam-ro, Sungbuk-gu, Seoul 136-713, Korea Department of Internal Medicine, Korea University College of Medicine, 73, Inchon-ro, Seoul 136-705, Korea

‡

S Supporting Information *

ABSTRACT: Copper ions play a crucial role in living systems as cofactors of numerous metalloenzymes. To quantitatively estimate the Cu2+ concentration in human tissue, we have developed a two-photon (TP) probe with an internal reference (ACCu2) that shows significant TP action cross-section and high selectivity for Cu2+ and can quantitatively estimate the Cu2+ concentration in human colon tissues by dual-color two-photon microscopy (TPM) imaging with minimum interference from other competing metal ions or pH and minimum cytotoxicity and photostability problems. The Cu2+ concentrations in human normal colon, polyp, and colon cancer tissues were found to be 8.3 ± 0.3, 13 ± 2, and 22 ± 3 μM, respectively. This result suggests that ACCu2 may be useful for the diagnosis of human colon cancer.

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with a ratiometric two-photon (TP) probe for Cu2+, TPM can quantitatively estimate the Cu2+ concentration ([Cu2+]) in living tissue. However, the design of a ratiometric TP probe is not straightforward. We therefore sought to develop a more general method of designing TP probes for quantitative measurements. We predicted that a TP turn-off probe linked to an internal reference through a spacer would be suitable. With this idea in mind, we have now developed a TP probe for Cu2+ with an internal reference (ACCu2) (Scheme 1). ACCu2

opper ions play crucial roles in living systems as cofactors of numerous metalloenzymes that generate cellular energy, reduce molecular oxygen, and induce signal transduction.1−7 While the disruption of copper ion homeostasis is related to various diseases, such as Menkes, Wilson, Alzheimer’s, and prion diseases,1−5 exposure to a high level of copper can cause gastrointestinal disorder and liver or kidney damage.6 Of the two copper ions, only the reduced Cu+ can be internalized to the cell, although Cu2+ ions are frequently found in cells under oxidative environments.5−7 There are two reports on the quantitative measurement of copper ion concentration in normal and malignant tissues.8,9 The average copper ion concentration estimated by the total-reflection X-ray fluorescence (TRXRF) method was almost the same in colorectal cancer tissue than in polyp tissue (3.87 vs 3.94 ppm).8 Similarly, the copper ion level determined by atomic absorption spectroscopy (AAS) was slightly higher in the carcinoma of the gallbladder group than the cholelithiasis group (2.00 vs 1.70 ppm).9 Unfortunately, neither of these methods could distinguish the oxidation state of the copper ions. Recently, a few fluorescent probes that can quantitatively estimate Cu2+ in the cells have been reported.10−13 However, there is no reliable method for the quantitative measurement of the copper ion concentration in live tissues. Therefore, tools to quantitatively estimate copper ion concentrations in living tissue would be of broad utility. An attractive approach for such quantification is two-photon microscopy (TPM). TPM, which utilizes two near-infrared photons as the excitation source, is an indispensable tool in biomedical research owing to its capability of imaging deep inside a living tissue for a long period of time with better spatial resolution than confocal microscopy.14−16 When combined © 2014 American Chemical Society

Scheme 1a

a

Reaction conditions: (a) methyl 6-(aminomethyl)nicotinate, DCC, DMAP, CH2Cl2, rt; (b) KOH, THF:MeOH:H2O (1:1:1), rt, 3 h; (c) DCC, DMAP, tert-butyl piperazine-1-carboxylate, CH2Cl2, rt; (d) TFA, CH2Cl2, rt, 3h; (e) EDCI, DMAP, CH2Cl2, rt. Received: January 23, 2014 Accepted: April 21, 2014 Published: April 21, 2014 5353

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was derived from an extended coumarin17 having a 2picolylmethylamide moiety at the 2-position as the fluorophore and a Cu2+ chelator (FL, red emission),18 and a chromene derivative19 as an internal reference (IR, blue emission), which were linked by a piperazine spacer. We anticipated that the intensity of the red emission, but not the blue emission, would respond to changes in [Cu2+]. Although the blue emission would be expected to decrease by Förster resonance energy transfer (FRET) from IR to FL,20−22 it should be detectable if the FRET efficiency is appreciably smaller than 100%. The combined effects allow the quantitative estimation of [Cu2+] by measuring the red/blue emission intensity ratios. We here report that ACCu2 can be used to quantitatively estimate [Cu2+] in normal colon, polyp, and colon cancer tissues by dual-color TPM imaging.

are the absorbances of the IR moiety and IR at 373 nm, and FIR and F′IR are the emission intensities of the IR moiety and IR upon excitation at 373 nm, respectively.20−22 The ETE was 93.4%. In addition, the antenna effect in the one-photon (OP) (or TP) mode was 3.0 (or 3.1), as calculated by dividing the area of emission from the FL moiety upon excitation of the IR moiety at 373 (or 750) nm by that collected from the direct excitation of FL at 461 (or 880) nm.20−22 Therefore, the decrease in Iblue can be attributed to the FRET from IR to FL with an energy transfer efficiency of 93.4%, and the increase in Ired can be attributed to an antenna effect.20−22 Moreover, the incomplete FRET allowed the measurement of Iblue, which is crucial for the quantitative measurements. The TP action cross-section (Φδ) was determined by a fluorescence method using Rhodamine 6G as the reference.27,28 The maximum Φδ values (Φδmax) of ACCu2 in EtOH/HEPES (9/1 v/v, pH 7.0) were 32 GM and 30 GM at 750 and 880 nm, respectively, while those for IR and FL were 46 GM at 750 nm and 32 GM at 880 nm (Figure 2a). The value decreased by 1.4-

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RESULTS AND DISCUSSION Synthesis. FL was prepared in 64% yield by the coupling of 1 with methyl 6-(aminomethyl)nicotinate. FL was then hydrolyzed using KOH to obtain 3 in 52% yield. IR was obtained in overall 49% yield by the coupling of 8-[2′-(2′methoxyethoxy)ethoxy]-2-oxo-2H-benzo[h]chromene-3-carboxylic acid with tert-butyl piperazine-1-carboxylate followed by the deprotection. ACCu2 was prepared in 36% yield by the coupling of IR with 3 as shown in Scheme 1. The detailed synthetic procedure is described in the Experimental Section. Photophysical Properties. The solubilities of ACCu2 in HEPES buffer ([HEPES] = 20 mM, pH 7.0) and EtOH/ HEPES (9/1 v/v, pH 7.0) as determined by the fluorescence method23 were 8.0 and 3.0 μM, respectively, (Supporting Information Figure S1), which were sufficient to stain the cells. The emission spectra of ACCu2 showed gradual red shifts with the solvent polarity (ENT )24 in the following order: 1,4-dioxane < DMF < EtOH < H2O (Supporting Information Figure S2 and Table S1). The large bathochromic shift in the emission spectra (∼69 nm) indicates the utility of ACCu2 as a polarity probe. In EtOH/HEPES (9/1 v/v, pH 7.0), which is a good model of an intracellular environment (see below), the absorption spectrum of ACCu2 was nearly identical to the sum of those for IR and FL (Figure 1a). While the emission spectrum showed

Figure 2. (a) Two-photon action spectra of IR, FL, and ACCu2 in the absence and presence of Cu2+ in EtOH/HEPES (9/1, v/v, pH = 7.0). (b) Two-photon excited fluorescence (TPEF) spectra of IR, FL, and ACCu2 measured in HeLa cells. The excitation wavelength was 740 nm. The TPEF spectrum of ACCu2 was fitted to two Gaussian functions (solid and dotted curves) centered at 470 and 582 nm, respectively.

fold from that of IR at 750 nm (Figure 2a), presumably because of the partial FRET from IR to FL,20−22 which would have reduced the blue emission while enhancing the red emission. However, only part of the transferred energy would have contributed to the red emission because the fluorescence quantum yield of FL is smaller than that of IR (Supporting Information Table S1). This would predict that TPEF intensity of ACCu2 should be weaker than that of IR when excited at 750 nm (Supporting Information Figure S3a), thereby resulting in a smaller Φδ value. On the other hand, the Φδ values of ACCu2 and FL were nearly identical at 880 nm. This is because the values were determined by exciting the molecules at 880 nm, where IR shows near zero Φδ value (Figure 2a). Under this condition, the IR moiety would emit little TPEF and cannot contribute to the Φδ value of ACCu2. These values allowed us to obtain bright TPM images of the cells and tissues labeled with ACCu2 (Figures 5−7). Detection Windows. Using a 750 nm TP excitation in scanning lambda mode, HeLa cells labeled with ACCu2 showed a broad spectrum (●), which could be dissected into two Gaussian functions with emission maxima at 470 (solid line) and 582 nm (dotted line) (Figure 2b). The two curves were similar to the TP-excited fluorescence (TPEF) spectra of IR and FL measured in the HeLa cells (Figure 2b) and the OPexcited fluorescence (OPEF) spectra of IR and FL measured in EtOH/HEPES (9/1 v/v, pH = 7.0) (Supporting Information

Figure 1. (a) Absorption and (b) fluorescence spectra of IR, FL, and ACCu2 in EtOH/HEPES (9/1, v/v, pH 7.0). The excitation wavelength was 373 nm.

two bands that could be attributed to the IR and FL moieties, the area of blue emission decreased by 9.2-fold, and that of the red emission increased by 3.4-fold from those of IR and FL, respectively (Figure 1b). A similar result was observed in a TP mode, with a 13-fold decrease in the blue emission and a 3.1fold increase in the red emission (Supporting Information Figure S3a). To assess the origin of the spectral changes, the energy transfer efficiency (ETE) was calculated by using the formula ETE = (1 − AIRFIR/AIR ′ FIR ′ ) × 100, where AIR and AIR ′ 5354

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Selectivity of ACCu2 for Cu2+ and pH Dependence. ACCu2 showed excellent selectivity for Cu2+ over competing metal ions, as revealed by the unperturbed Ired/Iblue ratios in the presence of millimolar concentrations of alkali and alkaline earth metal ions (Na+, Mg2+, K+, Ca2+), 500 μM concentrations of transition metal ions (Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu+, Zn2+, Pd2+, Ag+, Cd2+, Hg2+), as well as the dramatic decrease in the Ired/Iblue ratio upon addition of 200 μM Cu2+ to the HEPES solutions containing ACCu2 and the competing ions (Figure 4a).

Figure S2 and Table S1), indicating that this solvent is indeed a good model for the intracellular environment (see above). Furthermore, the TPEF intensities of IR and FL moieties could be detected with minimum interference from each other by using detection windows at 400−450 nm (Ch1) and 550−650 nm (Ch2), respectively (Figure 2b and Supporting Information Figure S3b). Fluorescence Titration. When small increments of Cu2+ were added to ACCu2 in EtOH/HEPES (9/1 v/v, pH = 7.0), the OPEF intensity at Ch2 (Ired) decreased gradually without any appreciable change at Ch1 (Iblue) (Figures 3a). A similar

Figure 4. (a) Ired/Iblue ratios of ACCu2 (3 μM) in the presence of Na+, K+, Mg2+, Ca2+ (1 mM), and other cations (500 μM) (black bars) followed by the addition of Cu2+ (200 μM) (white bars) in EtOH/ HEPES (9/1 v/v, pH 7.0). (1) Na+, (2) Mg2+, (3) K+, (4) Ca2+, (5) Cr3+, (6) Mn2+, (7) Fe2+, (8) Fe3+, (9) Co2+, (10) Ni+, (11) Cu+, (12) Zn2+, (13) Pd2+, (14) Ag+, (15) Cd2+, (16) Hg2+, and (17) Cu2+. (b) Effect of pH on the fluorescence intensity ratio (Ired/Iblue) of ACCu2 (3 μM) in the presence of 0 (black square) and 200 μM (white circle) of Cu2+ in EtOH/HEPES (9/1 v/v). The excitation wavelength was 373 nm. The data are the average of 5 experiments.

Figure 3. (a) One photon fluorescence spectra of ACCu2 in EtOH/ HEPES (9/1 v/v, pH = 7.0) in the presence of Cu2+ (0−200 μM). (b) One-photon (●) and two-photon (○) titration curves for the complexation of ACCu2 with free Cu2+ (0−200 μM). The empty (○) and filled (●) circles are the experimental data and solid line is theoretically fitted curve. (Inset) Plot of Ired/Iblue vs [Cu2+].

result was observed in a TP mode (Supporting Information Figure S3b). This outcome could be attributed to the quenching of Ired by Cu2+ through energy- and electron-transfer processes,18,30−32 as well as the negligible influence of Cu2+ on the FRET from IR to FL.20−22 Notably, the relative emission intensity ratios (Ired/Iblue) measured by the OP and TP processes decreased by 10-fold in the presence of 200 μM Cu2+, indicating a high sensitivity of the probe to the change in TP [Cu2+]. The dissociation constants (KOP d and Kd ) of ACCu2 for the OP and TP processes were calculated from the fluorescence titration curves (Figure 3b).25,26 The titration curves were well fitted with a 1:1 binding model (Figure 3b). The Hill plots were linear with a slope of 1.0 (Supporting Information Figure S3c), and the Job plot exhibited a maximum at a molar fraction of 0.50 (Supporting Information Figure S3d), indicating a 1:1 complexation between the probe and Cu2+.25,26 The calculated values of 2+ TP KOP were 21 ± 3 μM and 22 ± 4 μM, d and Kd for Cu respectively. In addition, the plot of Ired/Iblue vs [Cu2+] was linear in the range of 0 to 25 μM (Figure 3b, inset). Hence, a quantitative measurement of [Cu2+] was possible in EtOH/ HEPES (9/1 v/v, pH = 7.0) in this concentration range. Moreover, the Ired/Iblue ratios determined using the TP mode were well fitted by the OP titration curve (Figure 3b). Similarly, the Ired/Iblue ratios measured in ACCu2-labeled HeLa cells showed a straight line except that the slope was smaller by about 10-fold (Supporting Information Figure S4h), a result that can be attributed to the poor internalization of Cu2+. Although it was not possible to calculate the Kd value in the cells because of the difficulty associated with the measurement of exact concentration of Cu2+ in the cells, the [Cu2+] estimated from the Kd value measured in the solution should provide quantitative information on the relative Cu2+ concentration in live cells and tissues. Furthermore, the in vitro detection limit for Cu2+ using ACCu2 by TPM was found to be 0.84 μM.

In addition, the Ired/Iblue ratio of ACCu2 was pH-insensitive in the range of pH 3.0−9.0, while that of the Cu2+-bound probe showed a minimum at pH 6.0−7.0 (Figure 4b). The increase in the Ired/Iblue ratio at pH < 6 can be attributed to the protonation of the nitrogen atom in the pyridine moiety, which blocks the Cu2+ binding site (Scheme 2). Scheme 2. Possible Structure of Protonated ACCu2

Consistently, the Kd value increased gradually from 21 to 45 to 74 to 95 μM as the pH was decreased from 7.2 to 6.0 to 5.0 to 4.0, respectively (Supporting Information Figure S5b). On the other hand, the increase in the Ired/Iblue ratio at pH > 7.0 can be attributed to the reduced Cu2+ concentration due to the formation of Cu(OH)2.25,26 This outcome is different from that reported for the closely related OP probe, which showed pHindependent Ired/Iblue ratios of the apo- and Cu2+-bound probes over the entire pH range.18 The origin of this dichotomy is not clear. Nevertheless, ACCu2 was suitable to detect Cu2+ under physiological conditions. Quantitative Estimation of the Cu2+ Concentration in Live Cells. We then sought to utilize ACCu2 as a TP probe to detect Cu2+ in cellular environments. TPM images of HeLa cells labeled with 3 μM ACCu2 were obtained by collecting the TPEF intensities at Ch1 and Ch2 (Supporting Information Figure S6). The TPM images were bright presumably because of the good cell permeability and significant TP action cross5355

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section. The ratiometric images were constructed from the TPEF intensities collected in the two channels (Figure 5a-c).

Figure 6. (a, b) Ratiometric TPM images of a rat hippocampal slice labeled with ACCu2 (20 μM). Images were constructed from Ired/Iblue measured (a) before and (b) after addition of 500 μM Cu2+ and 200 μM PDTC to the imaging solution. (c) Ired/Iblue ratio and Cu2+ concentration in each sample. TPEF were collected using 750 nm excitation and emission windows at 400−450 nm (Iblue) and 550−650 nm (Ired), respectively. Ten ratiometric TPM images were accumulated along the z-direction at the depths of 90−190 μm with magnification at 10 ×. Scale bars: 300 μm.

Figure 5. (a−c) Ratiometric TPM images of HeLa cells labeled with 3 μM ACCu2. Images were constructed from Ired/Iblue (a) before and (b) after addition of 200 μM Cu2+ and 100 μM PDTC to the imaging solution, and (c) after addition of 100 μM EDTA to that shown in b. (d) Ired/Iblue ratio and Cu2+ concentration in each sample. Cells shown are representative images from replicate experiments (n = 15). TPEF were collected using 750 nm excitation and emission windows at 400− 450 nm (Iblue) and 550−650 nm (Ired), respectively. Scale bar: 30 μm.

concentration was estimated to be 2.5 μg/L (0.04 μM),34 the near zero [Cu2+] estimated in this study seems reasonable. The ratio decreased to 6.8 ± 0.6 upon treatment with Cu2+ (500 μM) and PDTC (200 μM) (Figure 6c). This ratio corresponds to 14 ± 1.5 μM Cu2+ (Figure 6c), a value similar to that estimated in the HeLa cells. Here again, the poor tissue permeability of Cu2+ is clearly evident. These results confirmed the capability of ACCu2 for measuring [Cu2+] in living tissue at depths of 90−190 μm using TPM (Supporting Information Figure S9). Quantitative Estimation of the Cu2+ Concentration in Human Colon Tissue. We then used ACCu2 to estimate [Cu2+] in human colon tissues. Ex vivo colon slices were obtained from outpatients who underwent elective colonoscopies at Korea University Medical Center Anam Hospital. We measured 1400, 1400, 1000, and 600 Ired/Iblue ratios in the EDTA-treated normal, normal, polyp, and cancer tissues, respectively, at the depths of 90−160 μm, which corresponded to the mucosal and submucosal layers of clinical importance (Figure 7b). The ratiometric TPM images of the ACCu2labeled ex vivo slices were constructed as described above (Figure 7a and Supporting Information Figure S10). The 3dimensional ratiometric TPM images revealed the overall distribution of the Cu2+, whereas the sectional images showed the distribution of the Cu2+ at different depth (Figure 7a). More importantly, the average Ired/Iblue ratios in the EDTAtreated normal, normal, polyp, and cancer tissues were 10.0 ± 0.3, 8.3 ± 0.4, 7.2 ± 0.5, and 5.2 ± 0.6, which corresponded to 0.0 ± 0.1, 8.2 ± 0.3, 13 ± 2, and 22 ± 3 μM Cu2+, respectively (Figure 7c). The Cu2+ concentration in the normal and malignant colon tissues are smaller than the 27−62 μM estimated by TRXF and AAS and larger than the near zero [Cu2+] estimated in rat brain tissue in the absence of external Cu2+. Considering that TRXRF and AAS measure the total copper ion concentration including free Cu+ and Cu2+, as well as the copper ions bound to intracellular chelators such as transporters and proteins, the smaller values measured in this experiment seem reasonable.8,9 Furthermore, a systematic increase in [Cu2+] is clearly evident as the normal tissue becomes increasingly more malignant. This outcome suggests the interesting possibility that ACCu2 may be useful for the diagnosis of colon cancer in its early stages. The combined results reveal that a TP turn-off probe with an internal reference could be a promising candidate for the quantitative detection of the biomedically important targets by

The Ired/Iblue ratio measured for the ACCu2-labeled HeLa cells was 9.9 ± 0.8 before addition of Cu2+ (Figure 5d and Supporting Information Figure S6e). The ratio decreased to 6.5 upon addition of Cu2+ (200 μM) and pyrrolidine dithiocarbamate (PDTC, 100 μM), a mediator that aids Cu 2+ accumulation inside the cell,29 and increased to 9.8 upon treatment with ethylenediamine tetraacetic acid (EDTA, 100 μM), a membrane-permeable metal ion chelator that effectively removes Cu2+ ions (Figure 5d and Supporting Information Figure S6e). Since Iblue remained constant, the Ired/Iblue ratio could be measured more conveniently than with a ratiometric probe which requires the measurements of the changes in both Iblue and Ired. The free Cu2+ concentrations in each sample calculated using the Ired/Iblue ratio and the titration curve shown in Figure 3b were 0.0 ± 0.7, 15 ± 2, and 0.0 ± 1.9 μM (Figure 5d), respectively. Because the intracellular free Cu+ ion concentration is less than one per cell,33 and copper ions are known to fluctuate between Cu+ and Cu2+,3 the near zero [Cu2+] estimated in the untreated cells is reasonable. This result also indicates the poor cell permeability of Cu2+. Moreover, ACCu2 showed negligible toxicity as estimated using a CCK-8 kit (Supporting Information Figure S7) and high photostability as revealed by the nearly constant TPEF intensity at a given spot on the ACCu2-labeled HeLa cells after continuous irradiation with femtosecond pulses for 60 min (Supporting Information Figure S8). These results confirmed that ACCu2 can quantitatively estimate [Cu2+] in live cells with minimum interference from competing metal ions and pH, minimum cytotoxicity, and good photostability. Quantitative Estimation of the Cu2+ Concentration in Rat Brain Tissue. We next investigated whether ACCu2 can be used to measure [Cu2+] in rat brain tissue. The hippocampus was isolated from a 2-day-old rat, and a slice was incubated with 20 μM ACCu2 for 1 h at 37 °C. Because it takes a relatively long amount of time to stain the tissue, excess ACCu2 was used to facilitate the staining. Because the structure of the brain tissue is heterogeneous, we acquired 10 TPM images in Ch1 and Ch2 at depths of 90−190 μm to visualize the overall Cu2+ distribution (Supporting Information Figure S9). The ratiometric TPM images were constructed from the TPEF intensities collected in the two channels (Figure 6a,b). The Ired/Iblue ratio in the rat brain tissue was 9.9 ± 0.5, which corresponded to 0.0 ± 1.1 μM free Cu2+ (Figure 6c). Because copper ions in brain tissues are expected to be transported from the cerebrospinal fluid (CSF), where the free copper ion 5356

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nicotinate,34 and 8-[2′-(2′-methoxyethoxy)ethoxy]-2-oxo-2Hbenzo[h]chromene-3-carboxylic acid (4)19 were prepared by modified literature methods. Synthesis of FL, compounds 3−5, IR, and ACCu2 is described below. FL. A mixture of 1 (0.20 g, 0.84 mmol), 6-(aminomethyl)nicotinate (0.21g, 1.3 mol),35 1,3-dicyclohexyl carbodiimide (DCC) (0.26 g, 1.3 mmol), and 4-dimethylaminopyridine (0.010 g, 0.084 mmol) in CH2Cl2 was stirred for 12 h under Ar. The resulting mixture was filtered and the filtrate was concentrated in vacuo. The crude product was purified by column chromatography (silica gel) using hexane/EtOAc (1:2) as the eluent. Yield: 0.20 g (64%); 1H NMR (300 MHz, CDCl3): δ 9.64 (1H, br), 9.20 (1H, d, J = 2.0 Hz), 8.96 (1H, s), 8.27 (1H, dd, J = 8.8, 2.0 Hz), 8.01 (1H, s), 7.89 (1H, d, J = 9.2 Hz), 7.46 (1H, s), 7.43 (1H, d, J = 8.8 Hz), 7.16 (1H, dd, J = 9.2, 2.5 Hz), 6.81(1H, d, J = 2.5 Hz), 4.88 (2H, d, J = 5.8 Hz), 3.95 (3H, s), 3.16 (6H, s). 13C NMR (100 MHz, CDCl3): δ 165.9, 162.9, 162.5, 162.1, 151.5, 150.9, 150.8, 149.3, 138.7, 138.0, 131.3, 130.6, 124.8, 123.9, 121.3, 116.4, 115.0, 114.8, 109.7, 103.9, 52.6, 45.6, 40.5 ppm. Compound 3. A solution of FL (100 mg, 0.23 mmol) in THF (5 mL) and MeOH (5 mL) was dissolved in water (5 mL) containing KOH (65 mg, 1.2 mmol) and was stirred 3h at RT. The contents of the flask were partially evaporated to remove organic solvent and adjusted the pH to 4−5 with diluted HCl. The residue was extracted with CH2Cl2 and recrystallized in CH2Cl2/MeOH to give orange crystal. Yield: 50 mg (52%). 1H NMR (300 MHz, DMSO-d6): δ 9.01 (1H, s), 8.96 (1H, s), 8.35 (1H, s), 8.22 (1H, d, J = 8.2 Hz), 7.87 (1H, d, J = 9.2 Hz), 7.57 (1H, s), 7.48 (1H, d, J = 8.2 Hz), 7.28 (1H, d, J = 9.2 Hz), 6.96 (1H, s), 4.72 (2H, d, J = 5.8 Hz), 3.10 (6H, s) ppm. Compound 5. A mixture of 4 (0.30 g, 0.75 mmol), tertbutyl piperazine-1-carboxylate (0.17 g, 0.89 mmol), 1,3dicyclohexyl carbodiimide (0.23 g, 1.1 mmol), and 4dimethylaminopyridine (9.0 mg, 0.075 mmol) in CH2Cl2 was stirred for 12 h under Ar. The resulting mixture was filtered and the filtrate was concentrated in vacuo. The crude product was purified by column chromatography (silica gel) using hexane/ EA (1:2 to pure EA) as the eluent to give colorless oily product. Yield: 0.28 g (65%). 1H NMR (300 MHz, CDCl3): δ 8.44 (1H, d, J = 9.3 Hz), 8.08 (1H, s), 7.58 (1H, d, J = 8.5 Hz), 7.44 (1H, d, J = 8.5 Hz), 7.33 (1H, dd, J = 9.3, 2.5 Hz), 7.19 (1H, d, J = 2.5 Hz), 4.30 (2H, m), 3.95 (2H, m), 3.78 (4H, m), 3.70 (2H, m), 3.65 (2H, m), 3.54 (6H, m), 3.40 (2H, m), 3.38 (3H, s), 1.47 (9H, s). 13C NMR (100 MHz, CDCl3): δ 164.3, 159.9, 158.3, 154.6, 152.4, 145.3, 137.5, 124.6, 124.4, 124.1, 122.5, 120.2, 117.8, 112.5, 107.6, 80.4, 72.0, 71.0, 70.8, 70.7, 69.7, 67.9, 59.1, 47.3, 42.3, 28.5 ppm. IR. To a solution of 5 (100 mg, 0.19 mmol) in CH2Cl2 (5 mL) was added CF3CO2H (1 mL), and the mixture was stirred for 12 h under Ar. The solvent was removed in vacuo. The crude product was purified by column chromatography (silica gel) using CHCl3/MeOH (20:1) to give colorless oily product. Yield: 67 mg (75%). 1H NMR (300 MHz, acetone-d6): δ 8.37 (1H, d, J = 9.0 Hz), 8.31 (1H, s), 7.67−7.79 (2H, m), 7.50 (1H, d, J = 3.0 Hz), 7.41 (1H, dd, J = 9.0, 3.0 Hz), 4.37 (2H, m), 4.10 (1H, br), 3.92−3.96 (4H, m), 3.69−3.74 (2H, m), 3.57−3.67 (6H, m), 3.43−3.51 (6H, m), 3.30 (3H, s). 13C NMR (125 MHz, acetone-d6): δ 164.6, 159.6, 158.2, 151.7, 145.7, 137.4, 124.9, 123.8, 123.4, 121.1, 119.7, 117.0, 112.4, 107.6, 71.5, 70.4, 70.1, 69.9, 69.3, 67.8, 58.1, 43.5, 43.2 ppm.

Figure 7. (a) (left) 3-Dimensional ratiometric TPM images of EDTAtreated normal, normal, polyp, and colon cancer tissues labeled with ACCu2 (20 μM) at a depth of 90−160 μm with magnification at 20×. This depth corresponds to the mucosal and submucosal layers of clinical importance. (right) Ratiometric TPM images of normal colon tissue at different depth. The images shown are representative images out of 50 sectional images obtained a depth of 90−160 μm. (b) A figure showing mucosa, muscularis mucosae, and submucosal layer. (c) Ired/Iblue ratios and Cu2+ concentration in each sample.

TPM. It easier to design and can reduce the errors involved in the measurement of the Ired/Iblue ratio compared with a ratiometric TP probe.

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CONCLUSIONS To conclude, we have developed a new TP probe for Cu2+ with an internal reference, which shows a significant TP crosssection, high selectivity for Cu2+, and can quantitatively estimate [Cu2+] in human tissues in the biologically relevant pH range by dual-color TPM imaging with minimum interference from other competing metal ions and minimal cytotoxicity and photostability problems. This probe may find use in the diagnosis of colon cancer. Moreover, such a TP probe with an internal reference could serve as a model for a new design strategy for TP probes for quantitative measurements.

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EXPERIMENTAL SECTION Synthesis of ACCu2. 8-Dimethylamino-2-oxo-2H-benzo[g]chromene-3-carboxylic acid (1),17 methyl 6-(aminomethyl)5357

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The intensities of the two-photon induced fluorescence spectra of the reference and sample emitted at the same excitation wavelength were determined. The TPA cross section was calculated by using δ = δr(SsΦrϕrcr)/(SrΦsϕscs): where the subscripts s and r stand for the sample and reference molecules. The intensity of the signal collected by a CCD detector was denoted as S. Φ is the fluorescence quantum yield. ϕ is the overall fluorescence collection efficiency of the experimental apparatus. The number density of the molecules in solution was denoted as c. δr is the TPA cross section of the reference molecule. Two-Photon Fluorescence Microscopy. Two-photon fluorescence microscopy images of probe-labeled HeLa cell sand tissues were obtained with spectral confocal and multiphoton microscopes (Leica TCS SP2) with ×100 oil and ×20 dry objective, numerical aperture (NA) = 1.30 and 0.50, respectively. The two-photon fluorescence microscopy images were obtained with a DM IRE2Microscope (Leica) by exciting the probes with a mode-locked titanium-sapphire laser source (Coherent Chameleon, 90 MHz, 200 fs) set at wavelength 750 nm and output power 1305 mW, which corresponded to approximately 2.41 × 108 mW/cm2 (20× ) and 1.44 × 109 mW/cm2 (100× ) power at the focal plane. To obtain images at 400−650 nm range, internal PMTs were used to collect the signals in an 8 bit unsigned 512 × 512 pixels at 400 Hz scan speed. Cell Culture and Imaging. HeLa human cervical carcinoma cells (ATCC, Manassas, VA, USA) were cultured in DMEM (WelGene Inc., Seoul, Korea) supplemented with 10% FBS (WelGene), penicillin (100 units/mL), and streptomycin (100 ug/mL). All the cells were maintained in a humidified atmosphere of 5/95 (v/v) of CO2/air at 37 °C. Two days before imaging, the cells were passed and plated on glass bottomed dishes (MatTek). For labeling, the growth medium was removed and replaced with DMEM without FBS. The cells were incubated with 3 μM ACCu2 for 20 min at 37 °C and were washed three times with DMEM without FBS and imaged. Cell Viability. To confirm that the probe could not affect the viability of HeLa cells in our incubation condition, we used CCK-8 kit (Cell Counting Kit-8, Dojindo, Japan) according to the manufacture’s protocol. The results are shown in Supporting Information Figure S7. Photostability. Photostability of ACCu2 was determined by monitoring the changes in TPEF intensity with time at three designated positions of ACCu2-labeled (3 μM) HeLa cells chosen without bias. The TPEF intensity remained nearly the same for 1 h, indicating high photostability (Supporting Information Figure S8). Preparation and Imaging of Mouse Brain Slices. Slices were prepared from the hippocampi and the hypothalmi of 2day-old rat (SD). Coronal slices were cut into 400 μm-thick using a vibrating-blade microtome in artificial cerebrospinal fluid (ACSF; 138.6 mM NaCl, 3.5 mM KCl, 21 mM NaHCO3, 0.6 mM NaH2PO4, 9.9 mM D-glucose, 1 mM CaCl2, and 3 mM MgCl2). Slices were incubated with 20 μM ACCu2 in ACSF bubbled with 95% O2 and 5% CO2 for 30 min at 37 °C. Slices were then washed three times with ACSF and transferred to glass-bottomed dishes (MatTek) and observed with a twophoton microscopy. Because it takes a relatively long amount of time to stain the tissue, excess ACCu2 was used to facilitate the staining. Because the structure of the brain tissue is heterogeneous, we acquired 10 TPM images in Ch1 and Ch2

ACCu2. A mixture of 3 (50 mg, 0.12 mmol), IR (67 mg, 0.14 mmol), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDCI) (28 mg, 0.18 mmol), and 4-dimethylaminopyridine (2 mg, 0.012 mmol) in CH2Cl2 was stirred for 12 h under Ar. The resulting mixture was filtered, and the filtrate was concentrated in vacuo. The crude product was purified by column chromatography (silica gel) using ethyl acetate/acetone (3:1). It was further purified by recrystallization CH2Cl2/MeOH to give orange crystal. Yield: 38 mg (36%). IR (deposit from CH2Cl2 solution on a NaCl plate): 3340 (NH), 1707 (CO). 1 H NMR (500 MHz, CDCl3): δ 9.61 (1H, br), 8.94 (1H, s), 8.68 (1H, s), 8.43 (1H, br), 8.12 (1H, s), 7.99 (1H, s), 7.78 (2H, m), 7.58 (1H, d, J = 8.5 Hz), 7.46 (3H, m), 7.33 (1H, d, J = 8.5 Hz), 7.19 (1H, s), 7.16 (1H, dd, J = 9.3, 2.2 Hz), 6.80 (1H, d, J = 2.2 Hz), 4.86 (2H, d, J = 3.7 Hz), 4.29 (2H, m), 3.95 (2H, m), 3.76−3.80 (4H, m), 3.69−3.72 (4H, m), 3.64− 3.68 (4H, m), 3.53−3.57 (4H, m), 3.38 (3H, s), 3.16 (6H, s). 13 C NMR (100 MHz, CDCl3): δ 168.2, 164.5, 162.9, 162.5, 160.1, 158.5, 152.7, 151.6, 150.9, 149.4, 147.9, 138.7, 137.7, 136.2, 131.3, 130.7, 129.7, 128.8, 124.7, 124.6, 124.3, 123.9, 122.0, 121.7, 120.3, 117.9, 116.5, 115.1, 114.9, 113.1, 112.5, 109.8, 107.7, 104.0, 72.1, 71.1, 70.9, 70.8, 69.8, 67.9, 59.3, 45.5, 43.7, 40.5. HRMS(FAB+): m/z calcd for [C48H47N5O11+H+]: 870.3306, found 870.3350. Spectroscopic Measurements. Absorption spectra were recorded on a Hewlett-Packard 8453 diode array spectrophotometer, and fluorescence spectra were obtained with AmicoBowman series 2 luminescence spectrometer with a 1 cm standard quartz cell. The fluorescence quantum yield was determined by using Coumarin 307 and Rhodamine B as the reference by the literature method.36 The spectral data obtained under various conditions are summarized in Supporting Information Figure S2 and Table S1. Determination of Apparent Dissociation Constants. A stock solution of Cu2+ (2 mM) was prepared by dissolving Cu(ClO4)2·6H2O in EtOH/HEPES (9/1, v/v, pH = 7.0). A small increments of Cu2+ were added to 3 μM ACCu2 in EtOH/HEPES (9/1, v/v, pH = 7.0) and the fluorescence intensity was measured as a function of Cu2+ concentration. The apparent dissociation constant (Kd) was calculated by using the equation: I − Iinitial = [Cu2+](Ifinal − Iinitial)/(Kd + [Cu2+]), where I is the observed fluorescence intensity, Ifinal is the fluorescence intensity for the Cu2+-ACCu2 complex, and Iinitial is the fluorescence for the free ACCu2. The Kd value that best fits the titration curve with the above equation was calculated by using the Excel program as reported (Figure 3b).17 To determine the KdTP for the two-photon process, the TPEF intensity from the EtOH/HEPES (9/1, v/v, pH = 7.0) containing Cu2+ and ACCu2 were recorded in the range of 400−650 nm with a DM IRE2Microscope (Leica) excited by a mode-locked titanium-sapphire laser source (Coherent Chameleon, 90 MHz, 200 fs) set at wavelength 750 nm. The KdTP value was determined by the same method as described above. Detection limit was calculated by using (3 × standard deviation)/(slope of plot). Measurement of Two-Photon Cross Section. The twophoton cross section (δ) was determined by using femto second (fs) fluorescence measurement technique as described.18 ACCu2 (3.0 × 10−6 M) was dissolved in HEPES buffer solution ([HEPES] = 20 mM, pH 7.0) and the twophoton induced fluorescence intensity was measured at 740− 940 nm by using rhodamine 6G as the reference, whose twophoton property has been well characterized in the literature.19 5358

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Analytical Chemistry

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at the depths of 90−190 μm to visualize the overall Cu2+ distribution. Preparation and Imaging of Human Ex Vivo Colon Slices. The ex vivo colon slices were obtained from outpatients who underwent elective colonoscopy at Korea University Medical Center Anam hospital. The volunteers were recruited to participate in this study, which was approved by the hospital ethics committee, and all participating patients provided informed consents. Patients who had known or suspected bleeding disorders, an international normalized ratio of prothrombin time exceeding 1.4, a platelet count of 7.4. (26) Lide, D. R., Ed. The CRC Handbook of Chemistry and Physics; CRC Press: Boca Raton, FL, 1999. (27) Lee, S. K.; Yang, W. J.; Choi, J. J.; Kim, C. H.; Jeon, S. J.; Cho, B. R. Org. Lett. 2005, 7, 323−326. (28) Makarov, N. S.; Drobizhev, M.; Rebane, A. Opt. Express 2008, 16, 4029−4047. (29) Verhaegh, G. W.; Richard, M. J.; Hainaut, P. Mol. Cell. Biol. 1997, 17, 5699−5706. (30) Kavallieratos, K.; Rosenberg, J. M.; Chen, W. Z.; Ren, T. J. Am. Chem. Soc. 2005, 127, 6514−6515. (31) Zheng, Y.; Orbulescu, J.; Ji, X.; Andreopoulos, F. M.; Pham, S. M.; Leblanc, R. M. J. Am. Chem. Soc. 2003, 125, 2680−2686. (32) Quang, D. T.; Jung, H. S.; Yoon, J. H.; Lee, S.; Kim, J. S. Bull. Korean Chem. Soc. 2007, 28, 682−684. (33) Rae, T. D.; Schmidt, P. J.; Pufahl, R. A.; Culotta, V. C.; O’Halloran, T. V. Science 1999, 284, 805−808. (34) Joergstuerenburg, H.; Oechsner, M.; Schroeder, S.; Kunze, K. J. Neurol., Neurosurg. Psychiatry 1999, 67, 252−253. (35) Duric, S.; Tzschucke, C. C. Org. Lett. 2011, 13, 2310−2313. (36) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991−1024.

ASSOCIATED CONTENT

S Supporting Information *

Synthesis, additional methods, Table S1, and Figures S1−S20. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

D.E.K. and C.S.L. contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A111182), and the NRF grants (No. 2012007850 and 20100020209).

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REFERENCES

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