Development of an ‘OFF-ON-OFF’ fluorescent pH sensor suitable for the study of intracellular pH

Article abstract of DOI:10.1016/j.tet.2016.06.065

Changes of pH in living organisms are closely related to various physiological functions, so fluorescent sensors of pH would be useful as experimental tools and possibly also for medical diagnosis. We previously reported a pH sensor with ‘OFF-ON-OFF’ -type fluorescence change, which would be potentially more useful than conventional sensors with ‘OFF-ON’ type change. It can be utilized as a sensor for a specific range of pH, however, the range with strong fluorescence is relatively wide, around pH 7–10, which renders it impractical for use in biological studies. So in the present work, we adjusted the pH detection range by the introduction of chloro groups at the ortho-positions to the phenolic hydroxyl groups, whose deprotonation could control fluorescence change, and obtained a sensor specifically responsive to pH around 6. This sensor was confirmed to be suitable for fluorescence imaging of changes of intracellular pH in living cells.

Full text of DOI:10.1016/j.tet.2016.06.065

Accepted Manuscript
Development of an “OFF-ON-OFF” fluorescent pH sensor suitable for the study of
intracellular pH
Tomoya Hirano, Yuki Noji, Takuya Shiraishi, Mari Ishigami-Yuasa, Hiroyuki
Kagechika
PII:
S0040-4020(16)30597-X
10.1016/j.tet.2016.06.065
TET 27879
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 31 May 2016
Revised Date: 22 June 2016
Accepted Date: 24 June 2016
Please cite this article as: Hirano T, Noji Y, Shiraishi T, Ishigami-Yuasa M, Kagechika H, Development of
an “OFF-ON-OFF” fluorescent pH sensor suitable for the study of intracellular pH, Tetrahedron (2016),
doi: 10.1016/j.tet.2016.06.065.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Development of an “OFF-ON-OFF” fluorescent pH sensor suitable for
the study of intracellular pH
Tomoya Hirano*, Yuki Noji, Takuya Shiraishi, Mari Ishigami-Yuasa and Hiroyuki Kagechika
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU),
2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
*Corresponding
author.
Tel.:
+81-3-5280-8128;
fax:
+81-3-5280-8127;
e-mail:
hira.chem@tmd.ac.jp
1. Introduction
Regulation of pH is important for most cellular processes, including metabolism, calcium
regulation and signal transduction pathways.1-3 Cytosolic pH is maintained at around pH 7, but
different values are maintained in some subcellular domains. For example, in the Golgi apparatus,
the pH is slightly acidic, while in mitochondria it is slightly basic; these values are influenced by
physiological processes such as metabolism and respiration.4 In the case of endocytosis, the
endosomal pH is around pH 6.0 - 6.5 at the early stage, whereas in the late stage and in lysosomes,
it is more acidic, around pH 5.0, since this is favorable for the activities of enzymes in the
degradation pathway.5-7 Cellular pH is altered in some pathological conditions.8-10 The extracellular
domain around cancer tissues becomes weakly acidic, and this might be utilized as a hallmark of
disease.
Various fluorescent sensors for pH, which enable spatio-temporal monitoring of pH in living
cells, tissues and organisms, have been developed.11-16 In most cases, a fluorescence signal
(intensity, the ratio of intensities at two wavelengths, or lifetime) is changed in an “OFF-ON”
manner above or below a specific value of pH (Fig. 1a). Such sensors could distinguish between
conditions “b” and “c” in Fig. 1a, but not between “a” and “b”. On the other hand, pH sensors that
show an “OFF-ON-OFF” type signal could selectively detect “b” (Fig. 1b).
Fig. 1. Schematic representation of two types of pH sensors.
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To date, a limited number of pH sensors with an “OFF-ON-OFF”-type fluorescence change have
been reported.17-23 One of them, compound 1a developed by our group, consists of a coumarin
scaffold with a hydroxyl group at the 7-position linked to a phenolic moiety via a triazole ring (Fig.
2).24 It showed “OFF-ON-OFF” fluorescence change due to successive deprotonations of the two
hydroxyl groups: deprotonation of the 7-hydroxyl on going from an acidic to a neutral pH enhances
the fluorescence, while deprotonation of the phenolic group in going from weakly basic to strongly
basic pH decreases the fluorescence. Thus, 1a can be utilized as a sensor for a specific range of pH.
However, the pH range over which the dye shows strong fluorescence is relatively wide, around pH
7 - 10. Other reported sensors of the same type also have a similar problem, which renders them
impractical for use in biological studies. Therefore, in this work, we set out to optimize the pH
detection range of our sensor, and especially to obtain a sensor for the pH region around 6, which
would be potentially useful as a tool for studies of endocytosis and possibly also for diagnosis of
cancer.
Fig. 2. Fluorescent sensor 1a for a specific range of pH around 7 to 10
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2. Results and Discussions
2.1. Design and synthesis of derivatives of 1a
The range of pH at which the sensor shows strong fluorescence would be determined by the pKa
values of two hydroxyl groups. So, we designed compounds 1 – 3, which have chloro group(s) at
ortho position(s) with respect to the hydroxyl groups in order to shift the pKa values, and synthesize
them from 3-azido-7-hydroxycoumarins (4) and 4-ethynylphenols (5) (Fig. 3).
Fig. 3. Structures and synthetic strategy of 1 – 3
Compounds 4a was prepared according to a reported method,25 and 4b and 4c were similarly
prepared (Scheme S1). Compounds 5 were prepared according to a reported method.26,27 By
combinatorial conjugation of these compounds via Huisgen 1,3-cycloaddition, 1a, 1b, 2a, 2b, 3a
and 3b were obtained in moderate yields (Scheme 1). On the other hand, 1c, 2c and 3c were
obtained in low yields, so we prepared methoxymethyl (MOM)-protected 5d (Scheme S2).
Conjugation of 5d with 3-azidocoumarins afforded MOM-protected compounds 1d, 2d and 3d,
which were deprotected to obtain 1c, 2c and 3c, respectively.
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Scheme 1. Synthesis of compounds 1 – 3.
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2.2. Fluorescent properties of the compounds
The pH dependence of the fluorescence properties of the synthesized compounds was examined
(Fig. 4). All the compounds showed ”OFF-ON-OFF” fluorescence change in response to increase of
pH. Compounds 1 without chlorine substitution on the coumarin ring (R1, R2 = H) showed similar
increases of fluorescence intensity at around pH 7 as the pH was changed from strongly acidic,
while the subsequent decrease of fluorescence occurred at different pH values, at around pH 10 for
1a (R3 = H, R4 = H), at pH 9 for 1b (R3 = Cl, R4 = H) and at pH 7.5 for 1c (R3 = Cl, R4 = Cl) (Fig.
4a). Under these conditions (pH 3.0 - 12.0), triazole ring would not be correlated to the fluorescent
change, as indicated reported pKa values of triazole derivatives, i.e., those of 1,4-diphenyl-1,2,3-
triazole and 1-methyl-1,2,3-triazole are reported to be -0.4 and 1.4, respectively.28 So the observed
decreases of fluorescence and increase of absorbance around 280 nm (Fig. S1, S2) were thought to
be due to the deprotonation of the phenolic hydroxyl group, and the effect of chloro groups at its
ortho positions (R3, R4) on its pKa value was also observed, that is, the occurrence of decrease of
fluorescence under less basic condition by the introduction of more chloro groups. Similar
differences were observed in the cases of compounds 2 bearing a 6-chloro group (R1 = Cl, R2 = H)
and compounds 3 bearing 6,8-dichloro substituents (R1, R2 = Cl) (Fig. 4b, 4c, S3 – S8). In focusing
on the effects of chloro groups at 6- and 8-positons of coumarin moiety (R1, R2), the increase of
fluorescence occurred at around pH 7 for 1b (R1 = H, R2 = H), around pH 6 for 2b (R1 = Cl, R2 = H)
and around pH 5 for 3b (R1 = Cl, R2 = Cl), as well as the increase of absorption around 390 nm (Fig.
S1, S4 and S7). Those changes would be due to the deprotonation of 7-hydroxyl group of coumarin
moiety, as indicated reported pKa values of 7-hydroxycoumarins, i.e., those of
7-hydroxy-4-methylcoumarin,
6-chloro-7-hydroxy-4-methylcoumarin
and
6,8-dichloro-7-hydroxy-4-methylcoumarin are reported to be 8.0, 6.5 and 5.1, respectively.28 As a
result, the pH region where the fluorescence was strong varied for each compound. Among them, 3c
should be useful as fluorescence sensor for pH around 6, where the quantum yield reached 0.14, as
compared with 0.028 at pH 4 and 0.005 at pH 8.29
pH-dependent fluorescent change of these compounds is thought to be differences in the
efficiency of photo-induced electron transfer (PeT)30-32, one of quenching mechanisms of
fluorescence, between the coumarin moiety (presumably electron acceptor) and the phenolic group
(presumably electron donor). In each case, the most fluorescent form is considered to be the
structure in which deprotonated 7-hydroxyl group of coumarin (less electron accepting ability
compared with its non-deprotonated form), and non-deprotonated phenolic hydroxyl group (less
electron donating ability compared with its deprotonated from). Regardless of the number or
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positon of chloro group, such property was retained among the compounds 1 - 3, while those
maximum fluorescence intensities were varied. Calculated HOMO levels of the proposed PeT
donors, phenol (1a, 2a and 3a), 2-chlorophenol (1b, 2b and 3b) and 2,6-dichlorophenol (1c, 2c and
3c), are reported to be 0.292 eV, 0.029 eV and -0.247 eV, respectively.33 These values indicated that
introduction of chloro group(s) decreases the electron-donating ability, so the fluorescence is
expected to be stronger due to reduced quenching. In the compounds bearing
6,8-dichloro-7-hydroxycoumarin (3), the maximum fluorescence intensities were consistent with
this expectation, that is, the order was correlated with the number of chloro groups on the phenol
moiety, 3c > 3b > 3a. On the other hand, this trend was not observed in compounds based on
6-chloro-7-hydroxycoumarin (2) or 7-hydroxycoumarin (1), that is, 2b > 2c > 2a and 1b > 1a > 1c.
The apparent discordance can be explained at least partly in terms of the existence ratio of the most
fluorescent form. For example, in the case of 1c, the pKa values of the two hydroxyl groups are
quite close, so relatively little of the most fluorescent mono anion form might exist in any pH,
resulting in a weak maximum fluorescence intensity.
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Fig. 4. pH-profiles of fluorescence intensity. Fluorescence intensity (excitation at 360 nm; emission
at 475 nm) of (a) 1, (b) 2 and (c) 3 at each pH is shown. These data were obtained in 100 mM
sodium phosphate buffer (pH 3.0–12.0) containing 0.3% DMSO as a co-solvent. (d) Photographs of
each solution under UV tranilluminator (Ex. 365 nm).
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2.3. Biological applications of 3c
To confirm the suitability of 3c as a tool for biological studies, we applied it to fluorescence
imaging of pH in living cells. Cultured MCF7 cells were incubated with 3c in Krebs-Ringer buffer
for 20 min. The cells were washed, and buffer (pH 4, 6 or 8) containing a proton ionophore,
nigercin, was added to ensure that intracellular pH was equal to the buffer pH.34
Fig. 5. Bright-field and fluorescence images of MCF7 cells loaded with 3c. The cells were
incubated with 30 µM 3c in Krebs-Ringer buffer, and then washed. Krebs-Ringer buffer ((a, b) pH
4, (c, d) pH 6 and (e, f) pH 8)) containing 5 µM nigericin was added. (a, c, e) Bright-field and (b, d,
f) fluorescence images were collected. (g) Average values of fluorescence intensity of the
corresponding areas in (a), (c) and (e) are shown.
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As shown in Fig. 5, the intracellular region was most fluorescent at pH 6, while almost no
fluorescence was observed at pH 8. At pH 4, moderate fluorescence was observed, partly because
hydrophobic non-deprotonated 3c, which was mainly present under these conditions, could more
easily penetrate through the cell membrane and accumulate in the intracellular region. Nevertheless,
the fluorescence was strongest at pH 6, as shown in Fig. 5g, suggesting that 3c could be utilized a
sensor of pH 6 microenvironment in living cells. Further modification of the sensor, e.g., by
conjugating it with an antibody to control its localization, or with a specific receptor ligand, should
be possible. Work in this direction is in progress.
3. Conclusion
We synthesized several compounds containing a coumarin scaffold with a hydroxyl group at the
7-position linked to a phenolic moiety via a triazole ring; in this system, the range of pH at which
the sensor shows strong fluorescence is determined by the pKa values of the two hydroxyl groups.
Among them, 3c showed a marked “OFF-ON-OFF” fluorescence change with increase of pH, with
the strongest fluorescence at around pH 6. We showed that this sensor can image changes of
intracellular pH in living cells. It should have potential as a tool for studying the role of pH change
in physiological functions, and may also be applicable for clinical diagnosis of cancer.
4. Experimental Section
4.1. General
All reagents were purchased from Sigma-Aldrich Chemical, Tokyo Kasei Kogyo, Wako Pure
Chemical Industries, and Kanto Kagaku. Silica gel for column chromatography was purchased from
Kanto Kagaku. NMR spectra were recorded on a Bruker Avance 400 or Bruker Avance 500
spectrometer. Mass spectral data was obtained on a Brucker Daltonics microTOF-2focus in the
positive and negative ion detection modes. Melting points were taken on a Yanagimoto micro
melting point apparatus and are uncorrected. UV spectra were recorded with JASCO V-550, and
fluorescence spectra were recorded with JASCO FP-6600. The imaging system was comprised of
an inverted fluorescence microscope (IX-73, Olympus), a cooled charge-coupled device (cooled
CCD) camera (CoolSNAP EZ, Photometrics) and an imaging system (MetaVue, Molecular
Devices). The microscope was equipped with a high-pressure mercury lamp (U-HGLGPS,
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Olympus), an objective lens (x20), an excitation filter (340 - 390 nm), a dichroic mirror (410 nm)
and an emission filter (420- nm).
4.2. Preparation of compounds 1 - 3
5b (43 mg, 0.28 mmol) was added to a solution of 4a (56 mg, 0.28 mmol) and
tris((4-ethoxycarbonylmethyl)-[1,2,3]triazol-1-ylmethyl)amine (14 mg, 0.030 mmol) in ethanol (3
mL). Then, solutions of CuSO4･5H2O (7.3 mg, 0.030 mmol) in water (1 mL) and sodium ascorbate
(18 mg, 0.090 mmol) in water (1 mL) were added successively. The mixture was stirred at room
temperature for 1 h, and then filtered to afford 1b (83 mg, 85%) as a brown solid, which was
recrystallized from methanol. Mp >300°C; 1H NMR (400 MHz, DMSO-d6)
δ 10.9 (s, 1H), 10.4 (s,
1H), 8.91 (s, 1H), 8.61 (s, 1H), 7.91 (d, J = 2.0 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.72 (dd, J = 8.4,
2.0 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.89 (dd, J = 8.4, 2.0 Hz, 1H), 6.87-6.84 (m, 1H);13C NMR
(125 MHz, DMSO-d6) δ 162.9, 156.7, 155.1, 153.5, 145.9, 137.1, 131.4, 127.2, 125.7, 122.7, 121.8,
120.6, 119.6, 117.5, 114.7, 110.7, 102.6; HRMS (ESI) calcd for C17H9ClN3O4 (M-H)- 354.0287.
Found 354.0277.
2a, 2b, 3a and 3b were similarly prepared from the corresponding 7-hydroxycoumarins (4a, 4b,
4c) and 4-ethynylphenol compounds (5a, 5b). Analytical data are given below.
1
2a: Mp 286.8 – 287.9°C; H NMR (500 MHz, DMSO-d6)
δ
9.66 (s, 1H), 8.81 (s, 1H), 8.60 (s,
13
1H), 8.02 (s, 1H), 7.75 (d, J = 8.5 Hz, 2H), 7.03 (s, 1H), 6.85 (d, J = 8.5 Hz, 2H); C NMR (125
MHz, DMSO-d6)
δ 157.6, 157.5, 156.0, 152.9, 146.9, 135.0, 129.8, 126.9, 121.0, 120.6, 120.3,
118.0, 115.8, 111.1, 103.3; HRMS (ESI) calcd for C17H9ClN3O4 (M-H)- 354.0287. Found 354.0283.
1
2b: Mp 288.2 – 289.5°C; H NMR (500 MHz, DMSO-d6)
δ 10.5 (s, 1H), 8.95 (s, 1H), 8.61 (s,
1H), 8.02 (s, 1H), 7.93 (d, J = 2.0 Hz, 1H), 7.75 (dd, J = 8.0, 2.0 Hz, 1H), 7.06 (d, J = 8.50 Hz, 1H),
7.03 (s, 1H); 13C NMR (125 MHz, DMSO-d6)
157.6, 155.9, 153.2, 153.0, 145.6, 135.1, 129.9,
δ
126.8, 125.3, 122.2, 121.3, 120.2, 120.1, 118.0, 117.1, 111.0, 103.3; HRMS (ESI) calcd for
C17H8Cl2N3O4 (M-H)- 387.9886. Found 387.9897.
1
3a: Mp >300°C; H NMR (500 MHz, DMSO-d6)
δ 9.64 (s, 1H), 8.80 (s, 1H), 8.59 (s, 1H), 8.00
(s, 1H), 7.75 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.4 Hz, 2H); HRMS (ESI) calcd for C17H8Cl2N3O4
(M-H)- 387.9897. Found 387.9892.
1
3b: Mp 272.7 – 273.2°C; H NMR (500 MHz, DMSO-d6)
δ 10.4 (s, 1H), 8.95 (s, 1H), 8.63 (s,
1H), 8.02 (s, 1H), 7.93 (d, J = 2.0 Hz, 1H), 7.75 (dd, J = 8.4, 2.0 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H);
HRMS (ESI) calcd for C17H7Cl3N3O4 (M-H)- 421.9508. Found 421.9500.
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1d, 2d and 3d were also prepared from 7-hydroxycoumarins (4a, 4b, 4c) and 5d. Those
compounds were used in the subsequent reaction without further purification.
1d: 1H NMR (400 MHz, DMSO-d6)
δ 9.19 (s, 1H), 8.64 (s, 1H), 8.11 (s, 2H), 7.76-7.71 (m, 1H),
6.90-6.84 (m, 1H), 6.83-6.78 (m, 1H), 5.21 (s, 2H), 3.60 (s, 3H).
1
2d: H NMR (400 MHz, DMSO-d6)
δ
δ
9.18 (s, 1H), 8.55 (s, 1H), 8.11 (s, 2H), 5.21 (s, 2H), 3.60
9.16 (s, 1H), 8.50 (s, 1H), 8.11 (s, 2H), 7.89 (s, 1H), 5.21
(s, 3H).
1
3d: H NMR (400 MHz, DMSO-d6)
(s, 2H), 3.60 (s, 3H).
1c was prepared by the deprotection of 1d as shown below.
Hydrochloric acid (10%, 0.05 mL) was added to a solution of 1d (35 mg, 0.080 mmol) in
methanol (30 mL). The mixture was refluxed at 80°C, then cooled to room temperature, and
evaporated under reduced pressure to afford 1c as a solid (34 mg, quant.), which was recrystallized
1
from methanol. Mp >300°C; H NMR (400 MHz, DMSO-d6)
δ 10.9 (s, 1H), 10.4 (s, 1H), 9.07 (s,
1H), 8.65 (s, 1H), 7.97 (s, 2H), 7.76 (d, J = 8.8 Hz, 1H), 6.91 (dd, J = 8.4, 2.0 Hz, 1H), 6.86 (d, J =
2.0 Hz, 1H); 13C NMR (125 MHz, DMSO-d6)
162.7, 156.2, 154.8, 149.4, 144.5, 136.8, 131.1,
δ
125.3, 123.0, 122.1, 119.1, 114.4, 110.3, 102.2; HRMS (ESI) calcd for C17H8Cl2N3O4 (M-H)-
387.9897. Found 387.9887.
2c and 3c were similarly prepared. Analytical data are given below.
2c: Mp >300°C; 1H NMR (400 MHz, DMSO-d6)
1H); 13C NMR (125 MHz, DMSO-d6)
157.7, 155.8, 153.0, 149.0, 144.4, 135.3, 129.9, 125.4,
δ 9.08 (s, 1H), 8.59 (s, 1H), 7.98 (s, 3H), 6.96 (s,
δ
123.1, 122.9, 122.2, 120.0, 118.1, 111.0, 103.3; HRMS (ESI) calcd for C17H7Cl3N3O4 (M-H)-
421.9508. Found 421.9497.
3c: Mp >300°C; 1H NMR (400 MHz, DMSO-d6)
2H), 7.97 (s, 1H); 13C NMR (125 MHz, DMSO-d6)
δ
10.4 (s, 1H), 9.09 (s, 1H), 8.60 (s, 3H), 7.99 (s,
155.2, 154.1, 149.0, 148.8, 144.5, 134.6, 127.6,
δ
125.4, 123.1, 122.9, 122.0, 120.4, 119.3, 111.3, 109.0; HRMS (ESI) calcd for C17H6Cl4N3O4 (M-H)-
455.9104. Found 455.9118.
4.3. Preparation of MCF7 cells
The human breast cancer cell line, MCF7 (RCB 1904) was provided by the RIKEN BRC through
the National Bio-Resource Project of the MEXT, Japan. MCF7 were maintained in MEM (GibcoTM)
medium containing 10% fetal bovine serum (SIGMA CR1211), 0.1 mM MEM Non-Essential
Amino Acids Solution (GibcoTM),
1
mM sodium pyruvate (GibcoTM) and 1%
Penicillin-Streptomycin Mixed Solution (Nakarai tesque) and incubated at 37 °C in a humidifies
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atmosphere of 5% CO2. Cells were passed 24 h before dye loading onto a 24 well plate at the
density of 1.0 x 105 cells/mL. The cells were rinsed with phosphate-buffered saline (PBS) three
times and incubated in Kreb-Ringer buffer containing 30 µM 3b for 20 min at room temperature.
The cells were washed with Kreb-Ringer buffer, and then Kreb-Ringer buffer with 5 µM nigericin
was added, and the cells were examined under a microscope.
Acknowledgments
The work described in this paper was partially supported by Grants-in-Aid for Scientific
Research from The Ministry of Education, Science, Sports and Culture, Japan (Grants No
15K08019 to TH, 26670052 to HK). TH also gratefully acknowledges funding from Terumo Life
Science Foundation. This work was also partially supported by Project for Development of
Innovative Research on Cancer Therapeutics, the Platform for Drug Discovery, Informatics, and
Structural Life Science (MEXT, Japan), and JSPS Core-to-Core Program A, Advanced Research
Networks.
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28. pKa values of the compounds are calculated using Advanced Chemistry Development
(ACD/Labs) Software V11.02 (1994-2014 ACD/Laboratories); taken from SciFinder
Scholar.
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29. Quantum yields of fluorescence were determined using that of quinine sulfate (0.577, in 0.1
M H2SO4) as a standard.
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Supplementary Material
Supplementary data (synthesis, absorption and fluorescence spectra of some
compounds) associated with this article is available and can be found in the online
version at http://xxxxx.
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