Indolequinone - rhodol conjugate as a fluorescent probe for hypoxic cells : enzymatic activation and fluorescence properties

Hirokazu Komatsu; Hiroshi Harada; Kazuhito Tanabe; Masahiro Hiraoka; Sei-ichi Nishimoto

doi:10.1039/C0MD00024H

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DOI: 10.1039/C0MD00024H (Concise Article) Med. Chem. Commun., 2010, 1, 50-53

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Indolequinone-rhodol conjugate as a fluorescent probe for hypoxic cells: enzymatic activation and fluorescence properties†

Hirokazu Komatsu ^ab, Hiroshi Harada ^c, Kazuhito Tanabe *^b, Masahiro Hiraoka ^d and Sei-ichi Nishimoto *^b
^aKyoto City Collaboration of Regional Entities for the Advancement of Technological Excellence, ASTEM, Kyoto, 615-8510, Japan
^bDepartment of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto, 615-8510, Japan. E-mail: tanabeka@scl.kyoto-u.ac.jp; nishimot@scl.kyoto-u.ac.jp; Fax: (+81)-75-383-2504; Tel: (+81)-75-383-2505
^cGroup of Radiation and Tumor Biology, Career-Path Promotion Unit for Young Life Scientists, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan
^dDepartment of Radiation Oncology and Image-applied Therapy, Graduate School of Medicine, Kyoto University, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan

Received 8th March 2010 , Accepted 7th May 2010

First published on 20th May 2010

Abstract

Hypoxia is an important feature of many diseases such as malignant solid tumors, inflammatory diseases and cardiac ischemia. We herein focused on the development of a novel hypoxia-sensitive fluorescent probe, IQ-R, consisting of an indolequinone unit and a rhodol fluorophore. IQ-R has good solubility in water and longer wavelength for absorption and emission, which are favorable for cellular bioimaging. While the fluorescence of rhodol in the IQ-R conjugate was quenched by the function of intramolecular indolequinone unit, it was restored under hypoxic conditions through the enzymatic one-electron reduction of IQ-R by NADPH:cytochrome P450 reductase to release the nonconjugated free rhodol. When administered to A549 cells, IQ-R was activated and reduced by endogenous reductase preferentially under hypoxic conditions, thereby visualizing hypoxic cancer cells by robust fluorescence.

Introduction

While appropriate oxygen supply from blood vessels maintains cellular activities, a dysregulation of oxygen homeostasis causes generation of pathological cells that are responsible for several diseases. In particular, hypoxic cells as generated by a supplied oxygen deficiency¹ are well-characterized to be related to malignant solid tumors,² inflammatory diseases³ and cardiac ischemia.⁴ Since hypoxic cells are identified as one of the most important features of diseases, there is increasing demand for the development of hypoxia-specific molecular probes to achieve the pathophysiological analysis of diseases.

Fluorescent molecular probes have recently attracted much attention to be widely used in the fields of biology, physiology and pharmacology, because of their wide applicability and high sensitivity for bioimaging.⁵ In the recent study, we proposed a new type of hypoxia-sensing fluorescence probe consisting of an indolequinone unit and a coumarin fluorophore (IQ-Cou),^6,7 in which the indolequinone unit has a quenching function of fluorescence⁶ as well as hypoxia-sensitive reduction reactivity.⁸ Consequently, while fluorescence emission of coumarin in the IQ-Cou conjugation was effectively suppressed, enzymatic treatment under hypoxic conditions resulted in an intense fluorescence in a hypoxia-selective manner as a result of a one-electron reductive bond dissociation of IQ-Cou to release coumarin fluorophore. Thus, IQ-Cou shows several unique properties that are favorable for hypoxia imaging. However, further application of IQ-Cou to cellular imaging has been limited due to its lower solubility in water⁹ and shorter wavelengths for the absorption and fluorescence emission of the coumarin unit.

These previous results stimulated us to improve a molecular system so as to be applicable to visualization of hypoxic cells. We conjugated a water-soluble fluorophore of rhodol¹⁰ with the indolequinone unit to form a new hypoxia imaging probe (IQ-R). The rhodol has a good solubility in water, long excitation and emission wavelengths (around 550 nm), intense fluorescence emission with the quantum yield (Φ_F of 0.20)¹¹ and a favorable on-off switching property of fluorescence emission regulated by the hypoxia-sensitive reactivity of indolequinone. According to the density functional calculations of IQ-R at the B3LYP/6-31G(d) level,¹² the orbital of indolequinone and rhodol units was orthogonal. The energy level of the lowest unoccupied molecular orbital (LUMO = −3.03 eV) localized at the indolequinone unit was lower than the LUMO + 1 (= −2.16 eV) localized at the rhodol unit. This suggests that intramolecular electron transfer from the rhodol unit in the excited state to the indolequinone unit in the ground state is thermodynamically feasible.¹³ It is therefore predicted that the excited fluorescent state of rhodol in the IQ-R conjugate undergoes effective intramolecular quenching by the neighboring indolequinone as in the case of IQ-Cou, while the intense fluorescence would be restored upon enzymatic one-electron reduction under hypoxic conditions to release a nonconjugated free rhodol. We report herein enzymatic activation, fluorescence properties and application to cellular imaging of the IQ-R probe.

Result and discussion

Synthesis and one-electron reduction characteristics of IQ-R

IQ-R was synthesized by coupling 3-chloromethyl-5-methoxy-1,2-dimethylindole-4,7-dione^8d with rhodol (ESI, Scheme S1†). We initially confirmed better water solubility up to 120 μM of IQ-R in comparison with a conventional IQ-Cou. We characterized the fluorescence properties of IQ-R in aqueous solution during enzymatic one-electron reduction with NADPH:cytochrome P450 reductase (Fig. 1), which catalyzes the one-electron reduction of indolequinone derivatives under hypoxic conditions.⁶ Before incubation with reductase, IQ-R showed extremely weak fluorescence, indicating that the fluorescence of the rhodol fluorophore is quenched intramolecularly by the neighboring indolequinone unit: the fluorescence quantum yield (Φ_F) of IQ-R was 0.0098. A similar weak fluorescence was observed for the sample incubated with reductase under aerobic conditions. In contrast, it is striking that hypoxic incubation of IQ-R resulted in an intense fluorescence emission at around 550 nm: the fluorescence emission was about 11 times stronger than that of the original IQ-R, while the absorption spectra of the sample before and after incubation were quite similar (see ESI, Fig. S1†). As predicted, enzymatic activation to enhance the fluorescence emission occurs substantially under hypoxic conditions.


	Fig. 1 (A) Chemical structure of IQ-R. (B) Fluorescence spectra of IQ-R after enzymatic reduction: IQ-R (10 μM) was incubated with NADPH:cytochrome P450 reductase (20 μg mL⁻¹) and β-NADPH (2 mM) at 37 °C in phosphate buffer (pH 7.4); incubated for 30 min under hypoxic conditions (); incubated for 30 min under aerobic conditions (); and before incubation (⋯). The fluorescence spectra were measured with excitation at 485 nm.

HPLC analysis of enzymatic reduction of IQ-R

In a separate experiment, we monitored the course of the enzymatic reduction of IQ-R by reversed-phase HPLC. Fig. 2 shows a representative reaction profile of IQ-R under treatment with NADPH:cytochrome P450 reductase. The appearance of a single new peak in Fig. 2C was attributed to the formation of a corresponding fluorophore rhodol upon hypoxic treatment, as confirmed by the overlapped injection of authentic sample in the HPLC analysis. In contrast, the enzymatic decomposition of IQ-R was dramatically suppressed under aerobic conditions, leading to the formation of a small amount of rhodol (Fig. 2B). The steady-state kinetic parameters, V_max and K_m, for the release of rhodol were derived from a Lineweaver–Burk plot (Table 1).¹⁴ These results indicate that the change in rhodol fluorophore concentration on treatment with reductase is responsible for the change in fluorescence intensity.


	Fig. 2 HPLC profiles of IQ-R (100 μM) upon treatment with NADPH:cytochrome P450 reductase (10 μg mL⁻¹) in the presence of β-NADPH (0.2 mM) at 37 °C for 0 h (A) and 6 h under aerobic (B) or hypoxic (C) conditions. The elution peaks were monitored at 315 nm wavelength.

Table 1 The steady-state kinetic parameters for enzymatic one-electron reduction of IQ-R under hypoxic or aerobic conditions.^a

	Hypoxic	Aerobic
a IQ-R was incubated with NADPH:cytochrome P450 reductase in the presence of β-NADPH. V_max and K_m were calculated from a Lineweaver–Burk plot.
V _max (pmol min⁻¹)	710	180
K _m (μM)	10	3.6

Hypoxic cancer cell imaging using IQ-R

In light of the behavior in the enzymatic activation, we applied IQ-R to a cellular imaging of a human lung adenocarcinoma cell line, A549, that expresses NADPH:cytochrome P450 reductase in high amount.¹⁵ A549 cells were cultured in the presence of IQ-R (50 μM) for 24 h at 37 °C under hypoxic or aerobic conditions.¹⁶ As shown in Fig. 3A, blight fluorescence was observed from the cytoplasm of cells incubated under hypoxic conditions. In contrast, there was substantially no fluorescence emission in the case of the similar-cultured cells under aerobic conditions (Fig. 3B). Considering the evidence that treatment of IQ-R with lysate of A549 cells resulted in an intense fluorescence emission in a hypoxia-dependent manner (Fig. 3C), it is reasonable to conclude that IQ-R penetrates into living cells wherein it is activated to release rhodol by intracellular reductase in a hypoxic environment to show an intense fluorescence.


	Fig. 3 (A,B) Fluorescent microscopic observation of A549 cells as incubated with 50 μM IQ-R for 24 h at 37 °C under hypoxic (A: 0.02% oxygen) or aerobic conditions (B: 20% oxygen) and then washed with PBS. (C) The fluorescent spectra of IQ-R (5 μM) upon treatment for 6 h with the cell lysate derived from A549 cells under hypoxic () or aerobic conditions (). The fluorescence spectra were measured with excitation at 525 nm.

Conclusion

In summary, we characterized the biological one-electron reduction and fluorescence properties of IQ-R. The fluorescence of IQ-R was significantly suppressed by the intramolecular quenching function of the indolequinone unit. On the other hand, hypoxic treatment with isolated or intracellular reductase induced the efficient decomposition of IQ-R and release of free fluorophore rhodol, resulting in the intense fluorescence emission. Thus, imaging of hypoxic cells by means of enzymatic reduction characteristics of indolequinone derivatives was successfully accomplished for the first time. IQ-R would be a promising candidate as a hypoxia-specific fluorescence probe. Application of IQ-R to in vivo optical imaging of tumor hypoxia is now in progress.

Experimental section

General procedures

All starting materials and reagents were purchased from Tokyo Kasei Kogyo (Tokyo, Japan), Wako (Tokyo, Japan) and Aldrich Chemical (Milwaukee, WI). All other solvents, purchased from Wako, were GR grade or dry grade and used without further purification. NADPH:cytochrome P450 reductase and β-NADPH were obtained from Oxford Bio-medical research and Oriental Yeast Co. (Japan), respectively. 3-Chloromethyl-5-methoxy-1,2-dimethyl-1H-indole-4,7-dione^8d and rhodol^10a were synthesized as described previously. The ¹H NMR spectra were recorded using a JOEL JNM-AL400 (400MHz) spectrometer in CDCl₃. Coupling constants are given in hertz. ¹³C NMR spectra were measured with JOEL JMN-AL-400 (100 MHz) spectrophotometer in CDCl₃. The FAB-MS spectra were recorded on a JOEL JMS-SX102A spectrometer, using nitrobenzyl alcohol as matrix. The organic reactions were carried out in oven-dried glassware under an argon atmosphere with magnetic stirring. All absorption spectra of IQ-R were recorded using a JASCO V-530 UV/Vis spectrophotometer with a 1 cm quartz cell. In a similar manner, fluorescence spectra in vitro were recorded on a SHIMADZU RF-5300PC spectrofluorophotometer with a 1 cm quartz cell. The slit widths were set to 5 nm for both excitation and emission. High-performance liquid chromatography (HPLC) was carried out with a Hitachi HPLC system (L-7455 Diode array detector, L-7300 column oven, L-7100 pump, D-7000 interface). Sample solutions were injected onto a reversed-phase column (Inertsil ODS-3, GL Science Inc.). The following program was used: of 70% B followed by 70–0% B (linear gradient over 70 min.; solution A: 100% acetonitrile, solution B: 0.1 M triethyl ammonium acetate (TEAA) buffer pH 7.

Synthesis of 2-(6-dimethylamino-3-oxo-3H-xanthen-9-yl)-benzoic acid 5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-ylmethyl ester (IQ-R)

Thionyl chloride (2 mL) was added to 3-hydroxymethyl-5-methoxy-1,2-dimethylindole-4,7-dione (20.9 mg, 0.888 mmol, 1 equiv.) and stirred for 30 min at ambient temperature. After the reaction, the solvent was removed under reduced pressure to give crude 3-chloromethyl-5-methoxy-1,2-dimethylindole-4,7-dione (22.5 mg). To the solution of crude product of 3-chloromethyl-5-methoxy-1,2-dimethylindole-4,7-dione (22.5 mg) in DMF (5 mL), rhodol (32.0 mg, 0.890 mmol) and K₂CO₃ (200 mg) were added, and then stirred for 5 h at 80 °C. After being cooled to room temperature, the reaction mixture was extracted with CHCl₃ two times, and the combined organic layer was washed with brine, dried over MgSO₄, filtered and concentrated by evaporation of the solvent. The crude product was purified by silica gel column chromatography (Chloroform/EtOAc = 1/1 to Chloroform/Methanol = 10/1) to afford 2-(6-Dimethylamino-3-oxo-3H-xanthen-9-yl)-benzoic acid 5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-ylmethyl ester (IQ-R) as a red solid (24.9 mg, 0.432 mmol, 48.5%): m.p. > 172 °C (decomposed), ¹H NMR (400MHz, CDCl₃) 1.71 (s, 3H), 3.00 (s, 6H), 3.69 (s, 3H), 3.80 (s, 3H), 4.88 (d, 2H, J = 11.7 Hz), 5.17 (d, 2H, J = 11.7 Hz), 5.48 (s, 1H), 6.10–6.40 (4H), 6.65–6.70 (2H), 7.05–7.10 (1H), 7.55–7.60 (2H), 8.20–8.25 (1H).;¹³C NMR(100MHz, CDCl₃) 8.7, 32.4, 40.3, 56.3, 57.0, 96.4, 104.3, 106.6, 110.9, 111.9, 113.5, 114.8, 120.9, 126.3, 128.9, 129.6, 130.0, 130.0, 130.1, 130.5, 131.5, 132.4, 133.9, 137.9, 154.6, 155.3, 158.5, 159.2, 165.2, 176,6, 178.4, 182.0, 182.0.; FABMS m/z 577 [(M + H) ⁺]; HRMS calced for C₃₄H₂₉O₇N₂⁺ [(M + H) ⁺] 577.1969, found 577.1989.

Bioreduction by NADPH-P450 reductase

To establish hypoxia, a solution of NADPH:cytochrome P450 reductase (final concentration: 20 μg mL⁻¹) and β-NADPH (final concentration: 2 mM) in 5 mM phosphate buffer (pH 7.4) was purged with argon for 10 min at 37 °C. To the resulting solution was added IQ-R (final concentration: 10 μM) and incubated at 37 °C for 30 min. Then, the fluorescence spectra were measured with excitation at 485 nm. A control aerobic sample solution was incubated and analyzed in a similar manner.

For the HPLC analysis, a solution of IQ-R (final concentration: 100 μM) in the presence of NADPH:cytochrome P450 reductase (final concentration: 10 μg mL⁻¹) and β-NADPH (final concentration: 0.2 mM) in 5 mM phosphate buffer (pH 7.4) and incubated at 37 °C for 6 h under hypoxic or aerobic conditions. After the reaction, the samples were analyzed.

Measurement of fluorescence quantum yield

The fluorescence quantum yield (Φ_F) was determined by using rhodol, with known Φ_F value of 0.20 in water, as in ref. 10a. The quantum yield was calculated according to eqn (1), in which Φ_F(S) and Φ_F(R) are the fluorescence quantum yields of the sample and the reference, respectively, the terms A_(S) and A_(R) are the area under the fluorescence spectra, (Abs)_(S) and (Abs)_(R) are the optical densities of the sample and reference solutions at the excitation wavelength, and n_(S) and n_(R) are the refractive indices of the solvents used for the sample and the reference.


Φ_F(S)/Φ_F(R) = A_(S)/A_(R) × (Abs)_(R)/(Abs)_(S) × n_(S)²/n_(R)²	(1)

Bioreductive activation of IQ-R by A549 cell lysate

A549 cells were cultured in 10 dishes (90% confluent in ϕ100 mm dishes) and washed twice with ice-cold PBS(−). The cell lysate was then harvested with 5 mL of ice-cold CelLytic M Cell Lysis Reagent (SIGMA-ALDRICH, Inc. St. Louis, MO), kept at ambient temperature for 15 min, and centrifuged at 14,000 rpm for 5 min to remove the cell debris. The resultant supernatant was pre-incubated in a well-oxygenized incubator (95% air and 5% CO₂ at 37 °C) for aerobic treatment or in Bactron II anaerobic environmental chamber (Sheldon Manufacturing, Cornelius, OR; 94% N₂, 5% CO₂ and 1% H₂ at 37 °C) for hypoxic treatment for 24 h. IQ-R (the final IQ-R concentrations 50 μM) was added to 600 μL of the aerobic or the hypoxic cell lysates and incubated under the same oxygen conditions as the pre-incubation for additional 6 h. The samples were quickly frozen until they were subjected to the spectral analysis. After a dilution (10 times by water) of the samples, fluorescence spectra were measured with excitation at 525 nm.

Cellular imaging of A549 cells

A549 cells (1 × 10⁶ cells/ϕ60 mm dish) were pre-cultured under aerobic or hypoxic conditions for 18 h and then treated with the aqueous solution of IQ-R (final concentration 50 μM) for 24 h under the same oxygen conditions as the pre-culture. After washing the cells twice with phosphate buffer, fluorescence images were observed with a fluorescent microscope (IX-71 Olympus) equipped with HQ type mirror unit (excitation filter: BP535-555HQ, emission filter: BA570-625HQ, and dichromatic mirror: DM565HQ) ideal for the wavelength chracteristics of a red fluorescent protein, DsRed2. Digital images were captured with a digital camera, DP-72 (Olympus).

Acknowledgements

This study is a part of joint researches, which are focusing on development of basis of technology for establishing COE of nanomedicine, carried out through Kyoto City Collaboration of Regional Entities for Advancement of Technology Excellence (CREATE) assigned by Japan Science and Technology Agency (JST), and is partly supported by the Program for Promotion of Fundamental Studies in Health Science of the National Institute of Biomedical Innovation (NIBIO), Japan.

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Fluorescence enhancement in hypoxic cells was also observed even in administration of 5 μM IQ-R (see ESI, Fig. S2†).

Footnote

† Electronic supplementary information (ESI) available: Synthesis, absorption spectra and fluorescence microscopy images. See DOI: 10.1039/c0md00024h

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