Highly Sensitive Hill-Type Small-Molecule pH Probe That Recognizes

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Article Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

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Highly Sensitive Hill-Type Small-Molecule pH Probe That Recognizes the Reversed pH Gradient of Cancer Cells Xiao Luo,‡ Haotian Yang,§ Haolu Wang,§ Zhiwei Ye,∥ Zhongneng Zhou,⊥ Luyan Gu,‡ Jinquan Chen,⊥ Yi Xiao,∥ Xiaowen Liang,*,§ Xuhong Qian,†,‡ and Youjun Yang*,†,‡ †

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China Shanghai Key Laboratory of Chemical Biology East China University of Science and Technology, Shanghai, 200237, China § Therapeutics Research Centre, The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Woolloongabba QLD 4102, Australia ∥ State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian, Liaoning 116024, China ⊥ State Key Laboratory of Precision Spectroscopy, East China Normal University, Shanghai 200062, China ‡

S Supporting Information *

ABSTRACT: A hallmark of cancer cells is a reversed transmembrane pH gradient, which could be exploited for robust and convenient intraoperative histopathological analysis. However, pathologically relevant pH changes are not significant enough for sensitive detection by conventional Henderson−Hasselbalch-type pH probes, exhibiting an acid− base transition width of 2 pH units. This challenge could potentially be addressed by a pH probe with a reduced acid−base transition width (i.e., Hill-type probe), appropriate pKa, and membrane permeability. Yet, a guideline to allow rational design of such small-molecule Hill-type pH probes is still lacking. We have devised a novel molecular mechanism, enabled sequential protonation with high positive homotropic cooperativity, and synthesized small-molecule pH probes (PHX1−3) with acid−base transition ranges of ca. 1 pH unit. Notably, PHX2 has a pKa of 6.9, matching the extracellular pH of cancer cells. Also, PHX2 is readily permeable to cell membrane and allowed direct mapping of both intra- and extracellular pH, hence the transmembrane pH gradient. PHX2 was successfully used for rapid and highcontrast distinction of fresh unprocessed biopsies of cancer cells from normal cells and therefore has broad potentials for intraoperative analysis of cancer surgery.

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iological processes are unanimously dependent on pH, and cells have evolved a delicate mechanism to tightly regulate the intracellular pH with buffering and ion-transporting.1−3 The dysregulated homeostasis of cellular pH affects gene expression, induces protein misfolding, disturbs enzyme activities, and promotes progression of cancers.4−7 Normal cells typically have a cytosolic pH of ∼7.2 and an extracellular pH of ∼7.4, while various cancer cells were reported to have a cytosolic pH of ∼7.4 and an extracellular pH of ∼6.9.8−12 This reversed transmembrane pH gradient is widely acknowledged to be a hallmark of cancer cells and therefore could be exploited as a viable target for cancer diagnosis and intraoperative histopathological analysis.12 Fluorescent probes are sensitive, versatile, and compatible with complex biological milieu and have become indispensable for biological and medical applications.13−17 A practical probe for detection of cancer cells should fulfill the following three criteria. First, the classic Henderson−Hasselbalch-type pH probes cannot sensitively distinguish the minute pH variation due to their broad pH responsive range of 2 pH units, in which range the molar ratio of the conjugative acid and base changes from 1/10 to 10/1 (Figure 1).18−21 In contrast, Hill-type pH probes, with a reduced acid/base transition width are preferred.22−25 Second, © XXXX American Chemical Society

Figure 1. pH responsive profiles of a HH-type probe (red) and a Hilltype probe (blue): Θ, fraction of the conjugate base; h, Hill coefficient.

a probe should exhibit good membrane permeability to allow simultaneous mapping of the intracellular and extracellular pH in order to establish the transmembrane pH gradient. Third, the pKa of the probe should match the extracellular pH of cancer cells for highest detection sensitivity. Positive cooperativity in ligand binding at two or more sites is the key to Hill-type dose−response behavior.24,25 Though this is commonplace in biological events, e.g., oxygen binding Received: January 15, 2018 Accepted: April 3, 2018

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DOI: 10.1021/acs.analchem.8b00218 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry by hemoglobin,26,27 the Hill-type host toward an analyte is very challenging to design, especially for ionic species because the electronic repulsion between the like charges poses a high energy barrier for the next binding event to occur.28−30 An early breakthrough was a pH-sensitive block copolymer with a transition width of 0.7 pH unit, albeit requiring a working temperature of 50 °C.31,32 In line with this strategy, novel micellar nanoparticles were devised from pH-sensitive block copolymers, which were operable at ambient temperature, and exhibited an even narrower acid/base transition width.33−36 Hill-type pH sensors based on DNA i-motif and fluorescent protein have been reported.37,38 Mechanistically, the first protonation to these macromolecular systems induces a structural change of the polymeric backbone, facilitating the subsequent protonation(s) to occur in an allosteric fashion, and therefore exhibiting positive cooperativity. For example, Gao et al. recently further showcased their potentials in tumor diagnosis.35 Albeit elegant, these polymeric systems are not readily membrane permeable. They typically rely on endocytosis to cross the cell membrane, are not suitable for sensing the cytosolic pH, and therefore cannot map the transmembrane gradient. Because of their high membrane permeability and readily engineerable cell localization specificity,39−49 small-molecule Hill-type pH probes are versatile for cell-based imaging applications and can complement the macromolecular congeners. Design of a small-molecule Hill-type pH probe is intrinsically more challenging than a macromolecular congener because less molecular surface is available to incorporate multiple protons, and hence the charge repulsion between protons will be larger due to closer proximity.22 Only a few probes with small Hill coefficients of 1.2−1.6 have been reported so far.50 A rational approach to access small-molecule pH probes with large Hill coefficients (>2) is desired. We have solved this longstanding challenge and report herein a series of novel small-molecule pH probes (PHX), with tunable pKas, high Hill coefficients (up to 7), and compatibility for in vitro and ex vivo studies.

PHX2 (128 mg) was obtained as a pink solid in 58% yield. H NMR (400 MHz, CDCl3): δ 7.24 (d, J = 8.9 Hz, 2H), 6.93 (d, J = 8.7 Hz, 1H), 6.70 (s, 1H), 6.67(d, J = 8.8 Hz, 1H), 6.41 (s, 2H), 6.37 (d, J = 8.9 Hz, 2H), 3.31−3.27 (m, 12H), 2.10 (s, 2H), 1.17−1.12 (m, 24H). 13C NMR (101 MHz, CDCl3): δ 152.7, 148.1, 147.1, 142.8, 131.8, 126.8, 117.7, 116.8, 114.4, 112.6, 107.5, 99.0, 72.2, 52.4, 45.5, 44.6, 32.0, 31.6, 12.8, 12.7. ESI-MS (m/z; [M + H]+): calcd for C35H48N3O2, 542.3747; found, 542.3743. PHX3 (107 mg) was obtained as a purple solid in 50% yield. 1 H NMR (400 MHz, CDCl3): δ 7.23 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.7 Hz, 1H), 6.77 (s, 1H), 6.74(d, J = 8.6 Hz, 1H), 6.42 (d, J = 7.7 Hz, 2H), 6.36 (dd, J = 8.8 Hz, 2.6 Hz, 2H), 3.85− 3.79 (m, 1H), 3.37−3.27 (m, 8H), 3.18 (q, J = 7.1 Hz, 2H), 2.09 (s, 2H), 1.17−1.12 (m, 27H). 13C NMR (101 MHz, CDCl3): δ 152.7, 148.1, 131.5, 126.8, 117.5, 116.9, 107.5, 99.0, 72.3, 53.6, 52.4, 44.6, 32.0, 31.6, 20.2, 12.7. ESI-MS (m/z; [M + H]+): calcd for C36H50N3O2, 556.3903; found, 556.3901. PHX4 (79 mg) was obtained as a red solid in 37% yield. 1H NMR (400 MHz, CDCl3): δ 8.28 (d, J = 2.7 Hz, 1H), 8.09 (dd, J = 9.0 Hz, 2.8 Hz, 1H), 7.12−7.10 (m, 3H), 6.46 (d, J = 2.5 Hz, 2H), 6.39 (dd, J = 8.8 Hz, 2.5 Hz, 2H), 3.35 (q, J = 7.0 Hz, 8H), 2.21 (s, 2H), 1.26 (s, 6H), 1.17 (t, J = 7.0 Hz, 12H). 13C NMR (101 MHz, CDCl3): δ 161.0, 152.7, 148.5, 141.6, 132.4, 126.2, 123.7, 123.7, 118.0, 114.6, 107.4, 98.8, 51.2, 44.5, 31.9, 31.8, 12.6. ESI-MS (m/z; [M + H]+): calcd for C31H38N3O4, 516.2862; found, 516.2864. pH-Dependent Intracellular Fluorescence Imaging. Hepa 1−6 liver cancer cells were purchased from ATCC (Manassas, VA, USA). The cells were cultured in Dubelcco’s modified Eagle’s medium (DMEM, Gibco) containing 10% (v/ v) fetal bovine serum (FBS, Hyclone), 1% (v/v) penicillin (100 units/mL), and streptomycin (100 μg/mL). Cultures were maintained at 37 °C in humidified air containing 5% CO2. For examining the pH response of PHX1−3 in live cells, 5 × 104 Hepa 1−6 per well were plated in 24-well plates and incubated overnight at 37 °C and 5% CO2. The medium was then removed. The cells were then exposed to 15 μM PHX1−3 (5 μM pHrodo Red AM, purchased from Invitrogen) in growth medium for 15 min in the same conditions. After that, the medium was removed and the cells were treated with nigericin (5 μg/mL) in PBS for another 10 min. Before imaging, the dye loaded cells were rinsed three times and incubated with highconcentration K+ buffer at various pH values (pH = 7.1, 6.9, 6.7, 6.5, 6.1, 5.8, 5.5, 5.2, and 5.0), respectively. Images were recorded with 20× objectives using inverted epi-fluorescence microscope (Olympus). The excitation wavelength was set to 559 nm for PHX1−3/pHrodo Red AM with emission signal range of 565−640 nm. The intracellular fluorescence intensities were determined using a total of 18 cells at each pH tested. Ex Vivo Mouse Tumor Imaging. Male 8 week old BALB/ c nude mice were purchased from the Animal Resource Centre (Perth, Western Australia). All animal procedures were approved by the Animal Ethics Committee of the University of Queensland. Hepatocellular carcinoma was induced by intrahepatic implantation of 5 × 106 Hepa 1−6 cells into BALB/c nude mice, and imaging procedures were performed after 20 days. The fresh and unprocessed biopsies of tumor and normal liver tissue were sectioned using surgical scalpel blade (Swann-Morton Ltd.) fixed by handle, The biopsies were fresh and without fixation and positioned in a cutting board; then they were sectioned to about 0.5−1 mm thickness by surgical scalpel blade. Sections were then exposed to 15 μM PHX2 in 1

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EXPERIMENTAL SECTION General Procedure for the Synthesis of PHX1−4. A solution of dilithium reagent 3 (1.3 equiv) in THF was added to a solution of compounds 4a−d (100 mg, 1 equiv) in THF (20 mL) at −78 °C via syringe. The reaction mixture was allowed to warm to room temperature within 30 min. Saturated NH4Cl solution was poured into the reaction flask, and the reaction mixture was extracted with CH2Cl2 repeatedly. The combined organic layer was dried with anhydrous MgSO4 powder, and all solid was removed with a suction filtration. Then, all CH2Cl2 was removed under reduced pressure to give a viscous residue, from which compounds PHX1−4 were obtained from a flash column over silica with a mixture of CH2Cl2 and MeOH [95:5, v/v]. PHX1 (121 mg) was obtained as a pink solid in 52% yield. 1 H NMR (400 MHz, CDCl3): δ 7.22 (d, J = 8.7 Hz, 2H), 6.95 (d, J = 8.7 Hz, 1H), 6.75 (d, J = 2.9 Hz, 1H), 6.71 (dd, J = 8.7 Hz, 3.0 Hz, 1H), 6.42 (d, J = 2.5 Hz, 2H), 6.37 (dd, J = 8.7 Hz, 2.6 Hz, 2H), 3.33 (q, J = 7.0 Hz, 8H), 2.92 (s, 6H), 2.12 (s, 2H), 1.19 (s, 6H), 1.15 (t, J = 7.0 Hz, 12H). 13C NMR (101 MHz, CDCl3): δ 152.7, 148.1, 147.6, 145.7, 131.9, 126.8, 117.7, 116.7, 114.1, 112.1, 107.5, 99.0, 72.4, 52.5, 44.6, 42.2, 32.0, 31.7, 12.7. ESI-MS (m/z; [M + H]+): calcd for C33H44N3O2, 514.3434; found, 514.3435. B

DOI: 10.1021/acs.analchem.8b00218 Anal. Chem. XXXX, XXX, XXX−XXX

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Figure 2. (A) Proposed pH-sensing mechanism of PHX, exhibiting positive cooperativity in protonation. (B) Two reference pH-sensitive compounds, their pH-sensitive sites, and pKas. (C) Synthesis of PHX1−4.

library of three analogues (PHX1−3) was constructed conveniently by condensation between a dilithium reagent 3 with lactone (4a−c) in moderate to good yields (50−58%). We also synthesized PHX4 with a nonprotonatable -NO2 (Figure 2C) and PHX5,-6 with -H or -OMe, respectively, (Supporting Information (SI)) as references to validate our design principle. Titrations were carried out with a solution of PHX1−3 (10 μM H2O with 0.5% DMSO), and the UV−vis absorption and fluorescence spectra at different pH were recorded (Figure 3). By lowering the solution pH values from 10 to 3, the absorption at 553 nm and the emission at 575 nm of PHX1−3 were enhanced (Figure 3A1−3,C1−3). By plotting the absorbance intensity at 553 nm and the emission intensity at

PBS for 15 min. Then sections were positioned on normal slides, and images were collected with laser scanning confocal microscope (FV1200, Olympus) using water-immersion 40× objectives. The fluorescence of PHX2 was excited at 559 nm, and emission was measured at 590−640 nm. All images were collected with 1024 × 1024 pixels resolution and zoom of 2.0. Figures were processed with FV10-ASW Viewer (4.2b, Olympus) and Adobe Photoshop CC 2015. Specimens from sites of confocal imaging were fixed in 4% buffered formalin and embedded in paraffin. Serial sections were obtained for hematoxylin and eosin (H&E) stain to evaluate histopathologic changes. The OlyVIA software 2.6 (Olympus, Münster, Germany) was used to visualize and scan the slides.

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RESULTS AND DISCUSSION The cornerstone of PHX is p-dialkylaminophenol, a small structural fragment with two interacting pH-sensitive sites. To furnish PHX, this pH-sensing moiety was judiciously tethered to a rhodamine with a dimethyl-substituted ethylene linker, to translate the rich acid/base chemistry of the p-(diethylamino)phenol into an optical signal via the pH-modulated ring opening/closing of the rhodamine dye. The purpose of introducing two methyl groups onto the ethylene linker is to minimize the unwanted ring opening of PHX with steric compression.51 We expect the PHX to exhibit Hill-type pH sensitivity based on the following considerations (Figure 2A). The first protonation of PHX should occur to the bottom dialkylamino group to yield PHX_mp. This protonation converts the strongly electron-donating dialkylamino group into a strongly electron-withdrawing diethylammonium group, which stabilizes the lone pair of the p-oxygen atom and therefore facilitates the ring opening to generate PHX_mp_o. The phenoxide of PHX_mp_o is more basic than the bottom dialkylamino group of PHX, analogous to the higher pKa of trimethylammonium phenol at ∼8.052 and the lower pKa of p(dimethylamino)phenol at ∼5.953 (Figure 2B). Therefore, positive homotropic cooperativity in protonation and hence a Hill-type pH response are anticipated from PHX. A focused

Figure 3. (A) UV−vis absorption spectral changes of PHX1−4 solution with varying pH from 3 to 10. (B) pH-dependent absorbance intensity of PHX1−4 at 553 nm. (C) Fluorescence emission spectral changes of PHX1−4 solution with varying pH from 3 to 10. (D) pHdependent emission intensity of PHX1−4 at 575 nm. All data were obtained in H2O with 0.5% DMSO. C

DOI: 10.1021/acs.analchem.8b00218 Anal. Chem. XXXX, XXX, XXX−XXX

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Figure 4. Absorbance (A) and fluorescence (D) pH titration of PHX2 fitted to a biphasic Hill response equation, and their corresponding Hill component (B, E) and HH component (C, F). (G) PHX2 can turn on its absorbance or fluorescence signal via the Hill pathway (pink) of PHX2 to PHX2_mp to PHX2_mp_o to PHX2_bp_o or the HH pathway (blue) of PHX2 to PHX2_ o′ to PHX2_mp_o′ to PHX2_bp_o.

Figure 5. Fluorescence images of hepatocellular carcinoma Hepa 1−6 cells incubated with pHrodo Red AM (HH-type, row A, 5 μM), PHX1 (Hilltype, row B, 15 μM), PHX3 (Hill-type, row C, 15 μM), and PHX2 (Hill-type, row D, 15 μM) with nigericin (5 μg/mL) at pH = 7.2, 6.9, 6.7, 6.4, 6.1, 5.8, 5.5, 5.2, and 5.0, respectively. Scale bar: 50 μm. Images were collected at λex = 559 nm and λem = 565−640 nm. The fluorescence intensity was color coded, with yellow for high intensity and violet for low intensity. (n = 18 different cells from six representative image fields; error bars indicate s.d.).

575 nm against the solution pH, the pH titration curves of PHX1−3 were established (Figure 3B1−3,D1−3). A striking yet anticipated feature of the pH titration curves of PHX1−3 is their narrow acid/base transition width. Also, depending on the nature of the dialkylamino group, PHX1−3 have tunable apparent pKas, i.e., the inflection points of their pH titration curves. For example, PHX1 exhibits a pKa_abs of 6.21, with an acid/base transition width of 0.9 pH unit. PHX2 and PHX3 have pKa_abs values of 6.85 and 6.35 with an even narrower acid/base transition width of ∼0.8 or 0.7 pH unit, respectively. This suggests that the rational design proposed in Figure 2A is a viable approach to access the desired Hill-type pH responses. PHX4, the control compound bearing a nitro group instead of a dialkylamino group, did not exhibit a narrow acid/base transition width (Figure 3B4). The fluorescence titration curves of PHX1−4 were also examined. The acid/base transition width of PHX1 is relatively broader at 1.4 pH units, with an inflection point of 5.88. PHX2/-3 exhibit the desired narrow transition widths of 0.9 and 0.7 pH unit, respectively, and with inflection points of 6.85 and 6.49, respectively. The fluorescence titration curve of the control compound PHX4 has an acid/base transition width of 2 pH units, typical of an

HH-type pH probe (Figure 3D4). The absorption and fluorescence titrations of PHX5,-6 were carried out and provided in the SI (Figures S5 and S6). Among the three Hill-type pH probes (PHX1−3), PHX2 is particularly promising for detection of the reversed pH gradient of cancer cells. PHX2 exhibits an apparent pKa of ∼6.9, matching the pH value of the extracellular fluid of cancer cells. And the acid/base transition width of PHX2 is in agreement with the dynamic pH range of the intracellular and extracellular fluids of cancer cells. Also, the pH response of these probes is rapid and reversible (Figures S9−S11). The UV−vis and fluorescence titration curves of PHX1−3 were nonlinearly fitted. Neither absorption nor emission curves fit to a simple dose−response equation. Instead, the curves fit well to a biphasic dose−response equation with an R2 of 0.999 (eq S1), revealing the presence of an additional HH-type component in addition to the expected Hill-type component (Table S2). The curve fitting results of PHX2 are discussed as an example (Figure 4). The absorption pH titration (Figure 4A) is the sum of a Hill component with an apparent pKa of 6.88 and a large Hill coefficient of 4.4 contributing 64% of the total signal enhancement (Figure 4B, pink), and an HH D

DOI: 10.1021/acs.analchem.8b00218 Anal. Chem. XXXX, XXX, XXX−XXX

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Figure 6. (A) Macropathological images of resected mouse liver bearing a tumor. H&E histopathological images of fixed biopsies of tumor (B) and normal liver tissue (C), respectively. Confocal fluorescence images of a section of ex vivo mouse tumor (D) and normal liver tissue (E), stained with PHX2 (15 μM) for 15 min. Average fluorescence intensity of the nuclear, cytoplasm, and extracellular regions of tumor (F) and normal tissue (G). Note: confocal images were collected with λex = 559 nm and λem = 590−640 nm. (n = 18 different ROIs from six representative image fields; error bars indicate s.d.).

the intra- and extracellular pH according to literature procedures56 before fluorescence images were collected (Figure 5). The average cellular fluorescence intensity of pHrodo is indeed lower at high pH and higher at low pH (Figure 5A). Upon lowering the pH from 7.2 to 5.0, the average cellular fluorescence intensity gradually increases by 2.63-fold. However, it should also be noted that enhancement of the cellular fluorescence intensity caused by lowering of the pH by 0.2−0.3 unit is not statistically significant. This suggests that pHrodo, as a typical HH-type probe, is not sensitive enough to distinguish a small pH variation, e.g., 0.2−0.3 unit. Yet, such a small pH variation in vivo may be significant enough to induce pathological outcome. As for PHX1−3, which are Hill-type probes with high Hill coefficients, the average cellular signal exhibited a much greater extent of fluorescence enhancement (∼30-fold), when pH decreased from 7.2 to ∼5.0 for PHX1 (Figure 5B) or from 7.2 to 5.8 for PHX3 (Figure 5C) and PHX2 (Figure 5D). In particular, most of the signal enhancement occurred in a narrow range near the pKa, facilitating sensitive detection of minor pH variation near the pKa. Intraoperative biopsies are often requested by oncological surgeons for diagnostic purposes. Conventionally, H&E stain has been routinely used for frozen or fixed samples and the histopathological images have been acquired for subsequent analysis. This is a slow and anxious process for both surgeons and patients, and a rapid cancer cell diagnosis is keenly desired. A fluorescent pH probe, which sensitively recognizes the reversed pH gradient of cancer cells, is such a promising solution. The intracellular pH of cancer cells ranges from 7.2 to 7.4, and the extracellular pH ranges from 6.5 to 6.9.57,58 This range of pH is in good agreement with the dynamic pH range of PHX2. Therefore, the potentials of PHX2 in recognizing the reversed pH gradient of cancer cells were tested, with an orthotopic hepatocellular carcinoma mouse model induced by intrahepatic implantation of Hepa 1−6 cells into BALB/c nude mice.59 The mouse liver bearing a solid tumor was resected, and its macropathological image was collected (Figure 6A). Confocal images of the fresh unprocessed mouse liver biopsies of tumor (Figure 6D) and normal tissue (Figure 6E) treated with PHX2 (15 μM) for 15 min were collected. The

component with an apparent pKa of 6.72 contributing the remaining 36% signal enhancement (Figure 4C, purple). While the Hill component obviously corresponds to the signal turn on from the cooperative proton binding pathway (Figure 4G, pink), the HH-type component suggests the presence of an alternative absorbance turn-on pathway (Figure 4G, purple). The absorbance of the PHX2 solution at high pH is not zero. This indicates that PHX2 is in an equilibrium with its ringopened isomer (PHX2_o′). The solution is not fluorescent, though, due to fluorescence quenching of the ring-opened isomer by photoinduced electron transfer (PeT).54 The phenoxide of the ring-opened isomer can potentially be protonated to yield PHX2_mp_o′. Subsequently, the -NEt2 of PHX2_mp_o′ is protonated to PHX2_bp_o upon further lowering of the solution pH. Analogously, two components were also identified from the fluorescence titration curve (Figure 4D), i.e., a Hill component with an apparent pKa of 6.91 (Hill coefficient = 4.4, Figure 4E), and an HH component with an apparent pKa of 6.43 (Figure 4F). Though the pH response of PHX2 is already very steep (Hill coefficient = 4.4) and promising for cell-based application, data fitting analysis has clearly suggested that it would be a viable future direction to further narrow the pH-titration range, i.e., by prohibiting the HH process. This could potentially be realized by lowering the pKa of the HH component or raising the pKa of the Hill component. The nonlinear fitting results of PHX1, PHX3, and PHX4 are also provided (Table S2). Analogous to PHX2, a Hill and an HH component are identified. It is noteworthy that PHX3 exhibits the highest Hill coefficient of 7.03, among PHX1−3. On the contrary, titration curves of PHX4 were well fitted to the HH equation. PHX5 and PHX6 exhibited a small Hill coefficient of only ∼1.6. The potentials of PHX1−3 in in vitro pH sensing were studied with hepatocellular carcinoma cells (Hepa 1−6). Hepa 1−6 cells were incubated with PHX dyes for 6 h. Cell viability was higher than 90% with 10 μM probe (Figure S13). A commercial HH-type probe (pHrodo55) with a pKa of 6.5 was used for comparison purposes. A short incubation time of 15 min or less was sufficient for PHX1−3 (15 μM) to freely diffuse through the cell plasma membrane. The H+/K+ ionophore nigericin (5 μg/mL) was then added to equilibrate E

DOI: 10.1021/acs.analchem.8b00218 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry fluorescence intensity of three selected regions of interest (ROI) were calculated. PHX2 did not enter cell nuclei (ROI1), and this accounted for the low signal intensity of this region. The low fluorescence intensity of the cytosolic region (ROI2) and the high intensity of the extracellular region (ROI3) are exactly what the reversed transmembrane pH gradient of cancer cells would dictate (Figure 6D,F). As for normal cells, the cell nuclei (ROI1) were dark, the cytosolic region (ROI2) was dimly fluorescent, and notably the extracellular region (ROI3) was dark (Figure 6E,G). This is in sharp contrast with the cancer cells and verified that cancer cells could be readily distinguished from normal cells by PHX2, targeting at the reserved pH gradient. The histopathological images of both the fixed tumor (Figure 6B) and the normal tissues (Figure 6C) were also collected, respectively.

Youjun Yang: 0000-0001-7085-6048

CONCLUSION We have designed a novel and rational molecular mechanism to achieve high positive homotropic cooperativity in protonation and further synthesized a series of Hill-type small-molecule pH probes (PHX1−3) with high Hill coefficients (>4). PHX1−3 exhibit good membrane permeability. They are valuable for sensitive and accurate mapping of a small pH fluctuation and feasible for in vitro and ex vivo pH-monitoring applications. Such Hill-type probes are superior to the conventional HHtype pH probes (e.g., pHrodo) in terms of detection sensitivity as demonstrated by a side-by-side comparative study. The apparent pKa of ∼6.9, the narrow acid/base transition width of ∼0.9 pH unit, and the good membrane permeability together render PHX2 an ideal probe for imaging the reversed transmembrane gradient of cancer cells. PHX2 can light up the mildly acidic extracellular region of hepatocellular carcinoma to a much higher extent than the cytosolic region, in agreement with the reversed transmembrane pH gradient, the hallmark of cancer cells. This offers an alternative and convenient method for analysis of frozen sections and highlights the potentials of PHX in cancer diagnosis and fluorescence-guided surgery. A future direction is the development of a dual-channel Hill-type pH probe by tethering PHX1−3 to a second fluorophore, further extending their scope of applications to encompass sensitive and accurate pH measurements in in vitro or in vivo settings.

(1) Roos, A.; Boron, W. F. Physiol. Rev. 1981, 61, 296−434. (2) Madshus, I. H. Biochem. J. 1988, 250, 1−8. (3) Casey, J. R.; Grinstein, S.; Orlowski, J. Nat. Rev. Mol. Cell Biol. 2010, 11, 50. (4) Matsuyama, S.; Reed, J. C. Cell Death Differ. 2000, 7, 1155−1165. (5) Wu, M. M.; Grabe, M.; Adams, S.; Tsien, R. Y.; Moore, H. H.; Machen, T. E. J. Biol. Chem. 2001, 276, 33027−33035. (6) Lagadic-Gossmann, D.; Huc, L.; Lecureur, V. Cell Death Differ. 2004, 11, 953−961. (7) Cardone, R. A.; Casavola, V.; Reshkin, S. J. Nat. Rev. Cancer 2005, 5, 786−795. (8) Tannock, I. F.; Rotin, D. Cancer Res. 1989, 49, 4373−4384. (9) Stüwe, L.; Müller, M.; Fabian, A.; Waning, J.; Mally, S.; Noël, J.; Schwab, A.; Stock, C. J. J. Physiol. 2007, 585, 351−360. (10) Gatenby, R. A.; Gillies, R. J. Nat. Rev. Cancer 2008, 8, 56−61. (11) Chiche, J.; Ilc, K.; Laferrière, J.; Trottier, E.; Dayan, F.; Mazure, N. M.; Brahimi-Horn, M. C.; Pouysségur, J. Cancer Res. 2009, 69, 358−368. (12) Webb, B. A.; Chimenti, M.; Jacobson, M. P.; Barber, D. L. Nat. Rev. Cancer 2011, 11, 671−677. (13) Llopis, J.; McCaffery, J. M.; Miyawaki, A.; Farquhar, M. G.; Tsien, R. Y. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 6803−6808. (14) Han, J. Y.; Burgess, K. Chem. Rev. 2010, 110, 2709−2728. (15) Li, X.; Gao, X.; Shi, W.; Ma, H. Chem. Rev. 2014, 114, 590−659. (16) Lee, M. H.; Park, N.; Yi, C.; Han, J. H.; Hong, J. H.; Kim, K. P.; Kang, D. H.; Sessler, J. L.; Kang, C.; Kim, J. S. J. Am. Chem. Soc. 2014, 136, 14136−14142. (17) Shi, W.; Li, X.; Ma, H. Methods Appl. Fluoresc. 2014, 2, 042001. (18) Henderson, L. J. Am. J. Physiol. 1908, 21, 173−179. (19) Hasselbalch, K. A. Biochem. Z. 1916, 78, 112−144. (20) Katchalsky, A.; Spitnik, P. J. J. Polym. Sci. 1947, 2, 432−446. (21) Po, H. N.; Senozan, N. M. J. Chem. Educ. 2001, 78, 1499−1503. (22) Onufriev, A.; Case, D. A.; Ullmann, G. M. Biochemistry 2001, 40, 3413−3419. (23) Klingen, A. R.; Bombarda, E.; Ullmann, G. M. Photochem. Photobio. Sci. 2006, 5, 588−596. (24) Vallée-Bélisle, A.; Ricci, F.; Plaxco, K. W. J. Am. Chem. Soc. 2012, 134, 2876−2879. (25) Porchetta, A.; Vallée-Bélisle, A.; Plaxco, K. W.; Ricci, F. J. Am. Chem. Soc. 2012, 134, 20601−20604. (26) Ogata, R. T.; McConnell, H. M. Proc. Natl. Acad. Sci. U. S. A. 1972, 69, 335−339. (27) Mihailescu, M.-R.; Russu, I. M. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 3773−3777. (28) Shinkai, S.; Ikeda, M.; Sugasaki, A.; Takeuchi, M. Acc. Chem. Res. 2001, 34, 494−503. (29) Badjić, J. D.; Nelson, A.; Cantrill, S. J.; Turnbull, W. B.; Stoddart, J. F. Acc. Chem. Res. 2005, 38, 723−732. (30) Mahadevi, A. S.; Sastry, G. N. Chem. Rev. 2016, 116, 2775− 2825. (31) Uchiyama, S.; Kawai, N.; de Silva, A. P.; Iwai, K. J. Am. Chem. Soc. 2004, 126, 3032−3033.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work is financially supported by the National Natural Science Foundation of China (Grant Nos. 21372080, 21572061, 21236002, and 11674101), the Fundamental Research Funds for the Central Universities (Grant Nos. WY1514053 and WY1516017), and the Australian National Health and Medical Research Council (Grant No. APP1122794).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b00218. General methods, nonlinear fitting procedures, synthesis, characterizations, additional spectral studies, MTT assay, photostability, detection selectivity, 1H/13C NMR spectra, and MS spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(Y.Y.) E-mail: [email protected]. Phone: +86-2164251781. *(X.L.) E-mail: [email protected]. ORCID

Zhiwei Ye: 0000-0002-4055-3676 Jinquan Chen: 0000-0003-0652-1379 F

REFERENCES

DOI: 10.1021/acs.analchem.8b00218 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry (32) Uchiyama, S.; Makino, Y. Chem. Commun. 2009, 19, 2646− 2648. (33) Zhou, K.; Wang, Y.; Huang, X.; Luby-Phelps, K.; Sumer, B. D.; Gao, J. Angew. Chem. 2011, 123, 6233−6238. (34) Zhou, K.; Liu, H.; Zhang, S.; Huang, X.; Wang, Y.; Huang, G.; Sumer, B. D.; Gao, J. J. Am. Chem. Soc. 2012, 134, 7803−7811. (35) Wang, Y.; Zhou, K.; Huang, G.; Hensley, C.; Huang, X.; Ma, X.; Zhao, T.; Sumer, B. D.; DeBerardinis, R. J.; Gao, J. Nat. Mater. 2014, 13, 204−212. (36) Zhao, T.; Huang, G.; Li, Y.; Yang, S.; Ramezani, S.; Lin, Z.; Wang, Y.; Ma, X.; Zeng, Z.; Luo, M.; de Boer, E.; Xie, X.; Thibodeaux, J.; Brekken, R. A.; Sun, X.; Sumer, B. D.; Gao, J. Nat. Biomed. Eng. 2016, 1, 0006. (37) Nesterova, I. V.; Nesterov, E. E. J. Am. Chem. Soc. 2014, 136, 8843−8846. (38) Shen, Y.; Rosendale, M.; Campbell, R. E.; Perrais, D. J. Cell Biol. 2014, 207, 419−432. (39) Kim, H. J.; Heo, C. H.; Kim, H. M. J. Am. Chem. Soc. 2013, 135, 17969−17977. (40) Yin, J.; Hu, Y.; Yoon, J. Chem. Soc. Rev. 2015, 44, 4619−4644. (41) Ling, J.; Naren, G.; Kelly, J.; Moody, T. S.; de Silva, A. P. J. Am. Chem. Soc. 2015, 137, 3763−3766. (42) Wallabregue, A.; Moreau, D.; Sherin, P.; Moneva Lorente, P.; Jarolímová, Z.; Bakker, E.; Vauthey, E.; Gruenberg, J.; Lacour, J. J. Am. Chem. Soc. 2016, 138, 1752−1755. (43) Grossi, M.; Morgunova, M.; Cheung, S.; Scholz, D.; Conroy, E.; Terrile, M.; Panarella, A.; Simpson, J. C.; Gallagher, W. M.; O’Shea, D. F. Nat. Commun. 2016, 7, 10855. (44) Anderson, M.; Moshnikova, A.; Engelman, D. M.; Reshetnyak, Y. K.; Andreev, O. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 8177−8181. (45) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13−21. (46) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48, 6974−6998. (47) Kalia, J.; Raines, R. T. Curr. Org. Chem. 2010, 14, 138−147. (48) Stephanopoulos, N.; Francis, M. B. Nat. Chem. Biol. 2011, 7, 876−884. (49) Gong, Y.; Pan, L. Tetrahedron Lett. 2015, 56, 2123−2132. (50) Grimm, J. B.; Brown, T. A.; Tkachuk, A. N.; Lavis, L. D. ACS Cent. Sci. 2017, 3, 975−985. (51) Anslyn, E. V.; Dougherty, D. A. Modern Physical Organic Chemistry; University Science Books: San Diego, 2006. (52) Williams, R. pKa Data compiled by R.W., can be found under: https://www.chem.wisc.edu/areas/reich/pkatable/pKa_compilation1-Williams.pdf. (53) Fickling, M. M.; Fischer, A.; Mann, B. R.; Packer, J.; Vaughan, J. J. Am. Chem. Soc. 1959, 81, 4226−4230. (54) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515−1566. (55) Ogawa, M.; Kosaka, N.; Regino, C. A. S.; Mitsunaga, M.; Choyke, P. L.; Kobayashi, H. Mol. BioSyst. 2010, 6, 888−893. (56) Thomas, J. A.; Buchsbaum, R. N.; Zimniak, A.; Racker, E. Biochemistry 1979, 18, 2210−2218. (57) van Sluis, R.; Bhujwalla, Z. M.; Raghunand, N.; Ballesteros, P.; Alvarez, J.; Cerdan, S.; Galons, J.-P.; Gillies, R. J. Magn. Reson. Med. 1999, 41, 743−750. (58) Busco, G.; Cardone, R. A.; Greco, M. R.; Bellizzi, A.; Colella, M.; Antelmi, E.; Mancini, M. T.; Dell’Aquila, M. E.; Casavola, V.; Paradiso, A.; Reshkin, S. J. FASEB J. 2010, 24, 3903−3915. (59) Wang, H.; Liang, X.; Mohammed, Y. H.; Thomas, J. A.; Bridle, K. R.; Thorling, C. A.; Grice, J. E.; Xu, Z. P.; Liu, X.; Crawford, D. H. G.; Roberts, M. S. Biomed. Opt. Express 2015, 6, 780−792.

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DOI: 10.1021/acs.analchem.8b00218 Anal. Chem. XXXX, XXX, XXX−XXX