Development of a Series of Practical Fluorescent Chemical Tools to Measure pH Values in Living Samples

Article abstract of DOI:10.1021/jacs.8b00277

In biological systems, the pH in intracellular organelles or tissues is strictly regulated, and differences of pH are deeply related to key biological events such as protein degradation, intracellular trafficking, renal failure, and cancer. Ratiometric fluorescence imaging is useful for determination of precise pH values, but existing fluorescence probes have substantial limitations, such as inappropriate pK_a for imaging in the physiological pH range, inadequate photobleaching resistance, and insufficiently long excitation and emission wavelengths. Here we report a versatile scaffold for ratiometric fluorescence pH probes, based on asymmetric rhodamine. To demonstrate its usefulness for biological applications, we employed it to develop two probes. (1) SiRpH5 has suitable pK_a and water solubility for imaging in acidic intracellular compartments; by using transferrin tagged with SiRpH5, we achieved time-lapse imaging of pH in endocytic compartments during protein trafficking for the first time. (2) Me-pEPPR is a near-infrared (NIR) probe; by using dextrin tagged with Me-pEPPR, we were able to image extracellular pH of renal tubules and tumors in situ. These chemical tools should be useful for studying the influence of intra- and extracellular pH on biological processes, as well as for in vivo imaging.

Full text of DOI:10.1021/jacs.8b00277

Article
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
pubs.acs.org/JACS
Development of a Series of Practical Fluorescent Chemical Tools To
Measure pH Values in Living Samples
Shodai Takahashi, Yu Kagami, Kenjiro Hanaoka,* Takuya Terai,∥ Toru Komatsu,
†
,¶
†,¶
,†
†
†
†,⊥
§
§
Tasuku Ueno, Masanobu Uchiyama,
Ikuko Koyama-Honda, Noboru Mizushima,
Tomohiko Taguchi,† Hiroyuki Arai, Tetsuo Nagano, and Yasuteru Urano*
†
‡
,†,§,#
†
‡
§
Graduate School of Pharmaceutical Sciences, Drug Discovery Initiative, and Graduate School of Medicine, The University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
∥
Graduate School of Science and Engineering, Saitama University, 255 Okubo, Sakura-ku, Saitama-shi, Saitama 338-8570, Japan
⊥
Cluster of Pioneering Research (CPR), Advanced Elements Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198,
Japan
#
CREST (Japan) Agency for Medical Research and Development (AMED), 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan
S Supporting Information
*
ABSTRACT: In biological systems, the pH in intracellular
organelles or tissues is strictly regulated, and differences of pH
are deeply related to key biological events such as protein
degradation, intracellular trafficking, renal failure, and cancer.
Ratiometric fluorescence imaging is useful for determination of
precise pH values, but existing fluorescence probes have
substantial limitations, such as inappropriate pK for imaging in
a
the physiological pH range, inadequate photobleaching
resistance, and insufficiently long excitation and emission
wavelengths. Here we report a versatile scaffold for ratiometric
fluorescence pH probes, based on asymmetric rhodamine. To demonstrate its usefulness for biological applications, we employed
it to develop two probes. (1) SiRpH5 has suitable pK and water solubility for imaging in acidic intracellular compartments; by
a
using transferrin tagged with SiRpH5, we achieved time-lapse imaging of pH in endocytic compartments during protein
trafficking for the first time. (2) Me-pEPPR is a near-infrared (NIR) probe; by using dextrin tagged with Me-pEPPR, we were
able to image extracellular pH of renal tubules and tumors in situ. These chemical tools should be useful for studying the
influence of intra- and extracellular pH on biological processes, as well as for in vivo imaging.
INTRODUCTION
and high photobleaching resistance (essential for long-term
imaging). For example, BCECF and SNARF-1, which are a
fluorescein derivative and a seminaphthorhodafluor respec-
tively, are conventionally used ratiometric fluorescence probes
■
7
Control of pH is critical for a variety of biological events,
including synthesis and degradation of proteins and trafficking
1
of cell components. To perform these reactions efficiently,
for pH; however, they possess neutral pK values derived from
a
intracellular organelles are maintained at specific pH values
appropriate for each process.
intracellular pH, but also extracellular pH is important; for
example, renal failure causes systemic acidosis, acidic tumors
2
,3
their phenolic group and are also vulnerable to photobleaching.
Recently, we have established synthetic scheme for
Furthermore, not only
12
4
asymmetric silicon-substituted rhodamines (SiRs). Among
them, we discovered a compound that shows an absorption
wavelength change of about 80 nm in response to pH change
are reported to have higher metastasis ability or proliferation
5
6
rate, and osteoclasts are activated under acidic conditions.
(
Figure S1a−c). Rhodamines are often used for fluorescence
Fluorescence imaging is a useful technique for pH measure-
ment, offering high spatiotemporal resolution, high sensitivity,
and multichannel imaging capacity. In particular, ratiometric
fluorescence imaging is an invaluable method for determination
of pH, and so far, ratiometric fluorescence probes for pH
imaging have been developed.
reported fluorescence probes for pH imaging in vivo except for
two photon excitation fluorescence probe, which is not
applicable to image wide range of the tissue. One reason for this
is the lack of suitable ratiometric fluorescence probes with both
imaging because of their high resistance to photobleaching,
high water solubility, and high fluorescence quantum yields.
13
7
In addition to these advantages, silicon-substituted rhodamines
have longer absorption and emission wavelengths, which enable
multicolor imaging with widely used fluorescent proteins such
7
−10
However, there has not been
1
4−16
as GFP and YFP.
So, we considered that silicon-
11
substituted rhodamine could be an excellent scaffold for
Received: January 9, 2018
an appropriate pK for imaging in the physiological pH range
a
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.8b00277
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Article
Figure 1. Photophysical properties of silicon-substituted rhodamine-based probes for pH. (a) Two methyl groups were introduced at the 2′ and 6′
positions to prevent attack of nucleophiles on the 9 position of the xanthene moiety. In order to optimize the pK value, substituent groups were
a
1
5
introduced at R . In order to enable conjugation of probes to other molecules such as macromolecules, a carboxyl group was introduced at R for
SiRpH4 and SiRpH5. (b) Absorption, fluorescence, and excitation spectra of 2 μM SiRpH1 measured in 0.1 M sodium phosphate buffer of various
pH values, with 1% DMSO as a cosolvent. Ex = 600 nm (fluorescence spectra); Em = 675 nm (excitation spectra). (c) Ratio of fluorescence intensity
at 675 nm excited at 580 nm and that excited at 663 nm is plotted against pH. pK values are given in parentheses beside the graph. (d) Fluorescence
a
intensity changes of fluorescent dyes upon light irradiation at pH 7.4. Ex/Em = 610 nm/690 nm (SiRpH5), 475 nm/529 nm (BCECF), or 514 nm/
627 nm (SNARF).
developing fluorescence probes for ratiometric pH measure-
ments, because modification of the pKa value and the
absorption and emission wavelengths, as well as introduction
of functional groups, can be easily performed. Here we report
the development of two fluorescence probes based on this
scaffold (SiRpH5 and Me-pEPPR), one for imaging of
intracellular organelle pH, and the other for imaging of
extracellular pH. We confirmed the practical value of these
probes by using transferrin tagged with SiRpH5 for time-lapse
imaging of pH in endocytic compartments involved in protein
recycling, and by using dextrin tagged with Me-pEPPR to
image extracellular pH of renal tubules and tumors in situ.
RESULTS AND DISCUSSION
■
Development of a Fluorescence Probe for Ratiomet-
ric pH Imaging, SiRpH5. An asymmetric silicon-substituted
rhodamine 8, which bears a piperazine ring on its xanthene
moiety, was discovered to show an absorption shift of about 80
nm to shorter wavelength in acidic aqueous solutions (Figure
S1a−c). This spectral shift is comparable to that of conven-
tionally used ratiometric fluorescence probes such as BCECF
7
and SNARF-1. However, it showed a gradual absorption
increase at pH 7.0, probably due to dissociation of hydroxide
ion from the 9 position of the xanthene ring (Figure S1d,e). To
overcome this instability, methyl groups were introduced at
both the 2′ position and the 6′ position of the benzene ring in
the design of SiRpH1 (Figure 1a). As we expected, SiRpH1
B
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retained the same pH-responsive change of absorption
wavelength as compound 8, but did not show the time-
dependent gradual absorption increase (Figure 1b). Further-
more, both the protonated and deprotonated forms of SiRpH1
showed high fluorescence intensity. This photophysical
property is important for accurate ratiometric imaging, and is
in contrast to the case of BCECF, a conventional ratiometric
pH probe, which lacks this feature. A plot of the fluorescence
intensity ratio of SiRpH1 at 675 nm excited at 580 nm and at
responses as those of SiRpH3 and SiRpH5, respectively
(Figures S3b−d, S4b−d), and thus, they work as pH probes
even when conjugated to dextran.
In order to investigate whether these dextran conjugates can
image lysosomal pH values selectively, SiRpH3-Dex or
SiRpH5-Dex was incubated with cells and fluorescence
microscopic imaging was performed. We found that SiRpH5-
Dex stained lysosomes specifically (showing good colocaliza-
21
tion with lysosome marker, Vamp7 ), while SiRpH3-Dex
6
63 nm showed that the ratio is pH-dependent, with pK = 6.7
stained not only lysosomes, but also mitochondria (Figure 2a,
a
(Figure 1c). This result confirmed that the asymmetric silicon-
substituted rhodamine scaffold with a piperazine ring on the
xanthene moiety is a promising scaffold for ratiometric
fluorescent pH probes.
In order to image intracellular acidic organelles, the pK
should be adjusted to be around 5−6. Since the fluorescence
ratio change of SiRpH1 is induced by protonation of the amino
a
2
,3
group on the piperazine ring, we expected that the pK of
a
SiRpH1 could be modified by introducing various substituent
groups onto the N atom of the piperazine ring. We tried a
benzyl group (SiRpH2), a 3-fluorobenzyl group (SiRpH3), a 2-
sulfobenzyl group (SiRpH4), and a 2,4-disulfobenzyl group
(SiRpH5) (Figure 1a). We previously found that introduction
of a benzyl group in place of a methyl group lowers the pK of
a
17
an amino group by about 1.0 unit. Indeed, introduction of the
benzyl group lowered the pK of SiRpH2 to 5.9 (SiRpH1: 6.7)
a
(Figure 1c, Figure S2a). Introduction of the more electron-
withdrawing 3-fluorobenzyl group (SiRpH3) resulted in a more
Figure 2. Fluorescence imaging of pH in intracellular organelles using
SiRpH5-Dex. (a) Schematic illustration of endocytic delivery of
SiRpH5-Dex to lysosomes. (b) Protocol of lysosomal pH imaging. (c)
Fluorescence ratio images of the MEF cells before and after addition of
acidic pK of 5.5 (Figure 1c, Figure S2b). Though the 2-
a
sulfobenzyl group is a strong electron-withdrawing group, the
pK of SiRpH4 (6.6) was unexpectedly higher than that of
a
SiRpH2 (5.9) (Figure 1c, Figure S2c). On the other hand, the
NH Cl aq.
4
pK of SiRpH5 having a 2,4-disulfobenzyl group was 6.1, which
a
should be suitable for imaging acidic intracellular compartments
(
Figure 1c, Figure S2d). Furthermore, SiRpH5 showed higher
Figure S5). This nonspecific staining by SiRpH3-Dex might be
due to the hydrophobicity of SiRpH3, i.e., some SiRpH3 were
noncovalently attached to dextran. These results indicated that
SiRpH5 is suitable for specific imaging of lysosomes. Then, we
investigated whether SiRpH5-Dex can be applied to visualize
photostability than the widely used ratiometric pH probes
BCECF and SNARF-1 (Figure 1d). In addition, the excitation
and emission wavelengths of BCECF are shorter than those of
SiRpH5, and the pK values of BCECF and SNARF-1 are 7.0
and 7.5.
properties for multicolor imaging with GFP or YFP or for
imaging intracellular organelle pH. In contrast, our newly
developed ratiometric fluorescence probes have appropriate
1
8
a
1
8,19
Thus, BCECF and SNARF-1 do not have favorable
the change of lysosomal pH upon addition of NH Cl or not,
4
22
which is reported to neutralize lysosomal pH (Figure 2b).
Fluorescence ratio images were constructed by dividing the
fluorescence intensity at 690−750 nm excited at 580 nm by
that excited at 670 nm, and showed a clear increase of pH in
pK values for imaging intracellular acidic organelles, high
a
resistance to photobleaching, and long excitation and emission
wavelengths.
lysosomes just after the addition of NH Cl (at 56 s), suggesting
4
that neutralization of lysosomal pH by NH Cl occurs within
4
Fluorescence Imaging of pH in Intracellular Compart-
ments. In order to image luminal pH in intracellular
organelles, pH probes were linked to macromolecules that
accumulate in specific intracellular compartments. First, we
focused on dextran, which is a high-molecular weight
polysaccharide composed of glucose as a monomer. Though
dextran is basically cell-impermeable, it can be transferred into
cells by endocytosis, and is transported from early endosomes
seconds (Figure 2c, Supplementary Movie S1). Conversion of
the ratio values to pH by means of a calibration curve (Figure
S6) indicated that the lysosomal pH was 4.7 before addition of
NH Cl and 6.2 afterward (Figure 2c), in accordance with
4
22
reported values. Thought the error bars in the calibration
curve are large, it is not because of the lack of brightness of the
fluorescence probe (fluorescence quantum yield is comparable
to that of conventional fluorophores), and pH values were
determined with errors of 0.2−0.5 unit.
20
to late endosomes, and finally to lysosomes. Among the
probes synthesized above, SiRpH3 and SiRpH5, which have
Next, we sought to image endosomal pH changes during
3
+
sufficiently acidic pK values, were used for labeling dextran.
intracellular trafficking of transferrin (Tfn). Two Fe ions bind
23
a
Probes bearing succinimidyl ester were conjugated to amino
groups of 10 kDa aminodextran to yield SiRpH3-Dex and
SiRpH5-Dex (Figures S3a, S4a). In the case of SiRpH5-Dex, a
polyethylene glycol (PEG) linker was used. The degree of
labeling (DOL) was 1.8 for SiRpH3-Dex and 2.6 for SiRph5-
Dex. SiRpH3-Dex and SiRpH5-Dex retained the same pH
to Tfn to form holo-Tfn, which binds to Tfn receptors
(TfnR) on the plasma membrane, and is internalized by
3
+
endocytosis into early endosomes (EE), where Fe is
dissociated from Tfn by the acidic environment. Apo-Tfn/
TfnR is then transported to recycling endosomes (RE), and
finally exported back to the plasma membrane (Figure 3a).
C
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30
low in this region. But, although several NIR fluorescence
probes for ratiometric fluorescence imaging have been
developed, they have inappropriate properties for in vivo
31
imaging of pH, i.e., low pK , low fluorescence quantum
a
1
7
9
yield, or insufficiently long excitation wavelength. Recently, it
has been reported that phosphorus-substituted xanthene
fluorophores have longer absorption and fluorescence wave-
32,33
lengths than silicon-substituted xanthene fluorophores.
Although SiRpH5 has NIR excitation and emission wave-
lengths in the deprotonated form, its excitation wavelength in
the protonated form is not long enough for NIR fluorescence
imaging (Figure S2). Therefore, we designed and synthesized a
series of phosphorus-substituted asymmetric rhodamines,
which we expected would show NIR excitation and emission
wavelengths not only in the deprotonated form, but also in the
protonated form.
Both of the initially synthesized compounds showed
absorption and fluorescence wavelengths in the NIR region
(
Figures 4a, S10, S11), and pipediEt-PPhR (pEPPR) showed a
higher fluorescence quantum yield than pipeindo-PPhR
Figures S10a, S11a). Therefore, we decided to utilize
(
pEPPR as a scaffold for NIR fluorescence pH probes.
Substitution of silicon to phosphorus at the xanthene 10
position did not affect the pH response of the absorption
spectra, and we confirmed that pEPPR can be used as a
ratiometric fluorescence pH probe (Figure S11c). We also
chose 650 and 705 nm as excitation wavelengths for measuring
the ratio values of pEPPR, because these are within the NIR
wavelength range. From the plot of fluorescence ratio against
Figure 3. Fluorescence imaging of pH in intracellular organelles using
SiRpH5-Tfn. (a) Schematic illustration of endocytosis of SiRpH5-Tfn
mediated by transferrin receptor (TfnR). pH values indicated in the
1
,23,24
figures are cited from the literature.
(b) Protocol of endosomal
pH imaging. (c) Time-lapse fluorescence ratio images of the COS-1
cells incubated at 37 °C. Scale bar: 10 μm.
pH, the pK of pEPPR was calculated to be 7.9, which is not
a
suitable for imaging of neutral to weakly acidic cellular
environments in organs and tumors (Figure 4c). The fitting
curve of pEPPR was also not completely matched with the pH
plots probably because of its water-solubility. Therefore, the
Although it is known that RE is more basic than EE, time-
lapse imaging of endosomal pH during the trafficking of Tfn
has not been achieved because of the lack of pH probes with
appropriate pK and resistance to photobleaching. First, we
pK
(methyl, ethyl, and i-propyl) (Figure 4a,b, Figure S12). The
methyl group lowered the pK of pEPPR from 7.9 to 6.8, which
a
of pEPPR was modified by introducing alkyl groups
a
conjugated SiRpH5 to holo-Tfn to yield SiRpH5-Tfn (DOL
was 7.3), and SiRpH5-Tfn was confirmed to have almost the
same pH response as SiRpH5 (Figure S7). COS-1 cells were
incubated with SiRpH5-Tfn for 20 min at 4 °C (endocytosis is
blocked at this temperature), and washed. The temperature was
then raised to 37 °C, and the time-lapse fluorescence imaging
was performed (Figure 3b). This protocol results in
synchronized endocytic trafficking of Tfn. Just after the start
of incubation at 37 °C, fluorescence of SiRpH5-Tfn was
a
is appropriate for the imaging of neutral to weakly acidic pH
(Figure 4c). Thus, Me-pEPPR was selected for ratiometric pH
imaging experiments.
Fluorescence Imaging of Organ pH in Vivo. While
intracellular pH is normally neutral (∼7.4), except for specific
organelles such as lysosomes and endosomes, extracellular pH
can easily be changed by various biological events, such as rapid
cell proliferation (e.g., cancer cells), and ion resorption in the
2
5−27
colocalized with EE marker GFP-3×FYVE
(Figure S8a),
4
,5
while after 10 min, it was colocalized with RE marker EGFP-
kidney. Therefore, imaging of extracellular pH is important
for monitoring biological events in vivo, and for this purpose
pH probes should be cell-impermeable. However, when small
fluorescent molecules are injected into animals, almost all of
them accumulate primarily in liver or kidney and are rapidly
2
4,28
Rab11
(Figure S8b). This result suggests that SiRpH5-Tfn
was transported from early endosomes to recycling endosomes
similarly to Tfn. Ratiometric fluorescence images showed that
endosomal pH was gradually basified (Figure 3c, Supplemen-
tary Movie S2). By converting the ratio values of fluorescence
intensities to pH values based on the calibration curve (Figure
S9), endosomal pH was measured to be 5.1 and 6.2 at 0 and 10
min after incubation at 37 °C, respectively (Figure 3c). Thus,
we have succeeded for the first time in time-lapse imaging of
the luminal pH of endosomes involved in recycling.
34
excreted, making it difficult to image other organs and tissues.
So, we decided to conjugate Me-pEPPR with dextran. Owing
to its large molecular weight, dextran is cell-impermeable, and it
35,36
is used for vascular imaging.
It is well retained inside the
body, and its fluorophore conjugate accumulates in tumors as a
result of the enhanced permeability and retention (EPR)
37,38
Development of a NIR Fluorescence Probe for
Ratiometric pH Imaging, Me-pEPPR. For in vivo
fluorescence imaging, the NIR wavelength range of 650−900
effect.
Therefore, we expected that conjugation of Me-
pEPPR to dextran would afford a probe with cell-imperme-
ability, high retention, and tumor accumulation capability. We
also expected that the conjugation with dextran improves the
water-solubility of the probe. For our purpose, amino groups of
40 kDa dextran were labeled with Me-pEPPR bearing
succinimidyl ester to afford dextran-Me-pEPPR conjugate,
29
nm, commonly referred to as the biological optical window, is
useful because it affords high tissue penetration compared to
visible light (light in the visible region is efficiently absorbed by
2
9
hemoglobin ) and autofluorescence of biomolecules is quite
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Figure 4. Photophysical properties of phosphorus-substituted rhodamine-based probes for pH. (a) In order to optimize pK , various alkyl groups
a
1
were introduced at R . (b) Absorption, fluorescence, and excitation spectra of 1 μM Me-pEPPR measured in 0.1 M sodium phosphate buffer at
various pH values, with 0.1% DMSO as a cosolvent. Ex = 705 nm (fluorescence spectra); Em = 730 nm (excitation spectra). (c) Ratio of
fluorescence intensity at 730 nm excited at 650 nm and that excited at 705 nm is plotted against pH. pK values are given in parentheses beside the
a
graph.
Dex-Me-pEPPR. Dex-Me-pEPPRs with various degrees of
labeling (DOL) were prepared, and their photophysical
properties and cell permeability were investigated. Conjugation
of Me-pEPPR to dextran hardly affected the pK value or
a
fluorescence quantum yield of the probe, though some
fluorescence quenching was observed when the DOL reached
5.7 (Figure S13). Fluorescence microscopy confirmed that
Dex-Me-pEPPR with any DOL was cell-impermeable, though
Me-pEPPR itself was cell-permeable (Figue 5). Thus, it was
expected that Dex-Me-pEPPR could be used to measure
extracellular pH selectively.
As proof of concept, we first attempted to visualize renal
Figure 5. Investigation of cell-permeability of Me-pEPPR and Dex-
Me-pEPPR. A549 cells were incubated with 1 μM Me-pEPPR or 5
μM Dex-Me-pEPPR for 30 min at 37 °C and imaged with a confocal
fluorescence microscope. Degree of labeling (DOL) of Me-pEPPR
conjugated to dextran is given above each image. Scale bar: 20 μm.
tubular pH; this is important, as it can provide insight into renal
3
9
function. In order to identify the part of the kidney to which
Dex-Me-pEPPR is distributed, Dex-Me-pEPPR was intra-
venously (i.v.) injected into mice. After 1 h the kidneys were
removed, and sections were imaged by fluorescence micros-
copy. NIR fluorescence was observed from the renal cortex,
especially from tubules (Figure S14), which is the same pattern
41
inhibitors such as acetazolamide. Therefore, after admin-
istration of acetazolamide or saline as a control to mice for a
week, the animals were injected with Dex-Me-pEPPR, followed
by fluorescence ratio imaging 1 h later (Figure 6a).
40
as reported previously. Thus, the probe can be used for pH
imaging at renal tubules. It is reported that renal tubular pH is
around 6.3−7.0, but is neutralized by carbonic anhydrase
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Figure 7. In vivo fluorescence imaging of pH at tumors using Dex-Me-
pEPPR. (a) Protocol of tumoral pH imaging. The area surrounded by
the red square was imaged in b. (b) Fluorescence ratio images of mice
with subcutaneous tumors. White dotted lines and characters in the
figure indicate organ or tissues, respectively. K: kidney, N: normal
tissue, T: tumor. (c) pH values at normal tissue and tumor measured
by fluorescence imaging. Error bars represent standard errors (n = 11).
Figure 6. In vivo fluorescence imaging of pH at renal tubules using
Dex-Me-pEPPR. (a) Protocol of renal tubular pH imaging. Saline was
administered as a control. The area surrounded by the red square was
imaged in b. AZM: acetazolamide. (b) Fluorescence ratio images of
mice treated with saline or AZM. Kidneys are indicated with white
dotted lines. (c) pH values at kidney and muscle measured by
fluorescence imaging. Error bars represent standard errors (n = 3). *p
*
p < 0.05.
<
0.05.
CONCLUSIONS
■
We have found that asymmetric rhodamines bearing a
piperazine ring show a large, pH-dependent blue shift of the
absorption spectra upon protonation of the piperazine ring.
Time-dependent density functional theory (TDDFT) calcu-
lations reproduced well the spectral changes, and the NIR
absorption bands observed for 8 and pEPPR can be assigned to
the HOMO → LUMO transitions (Figure S18). Figure S19
shows the frontier orbitals of these compounds. The HOMO
orbitals of the deprotonated forms spread widely from the
xanthene ring to the piperazine ring, and the HOMO energy
levels are significantly destabilized by the strong antibonding
interaction between the two rings, compared to those of the
protonated forms. The HOMO−LUMO gaps in the deproto-
nated forms are increased mainly because the connecting C−N
bond lengths in the protonated forms are longer than those in
the deprotonated forms, which effectively stabilizes the HOMO
energies due to the weaker antibonding interaction, while
stabilization of the LUMOs is modest. In addition, the
compounds were highly fluorescent in both protonated and
deprotonated forms. On the basis of these findings, we were
Fluorescence ratio was converted to pH according to a
calibration curve measured in 0.1 M sodium phosphate buffer
of various pH values (Figure S15). The results indicated that
the renal tubular pH of control mice was 6.9, while that of
acetazolamide-treated mice was 7.4 (Figure 6b,c). Thus, Dex-
Me-pEPPR could visualize pH in the kidney in vivo after i.v.
injection.
Finally, we examined pH imaging of tumors with Dex-Me-
pEPPR. Tumor cells show upregulated glycolysis, which causes
intracellular acidification due to overproduction of metabolic
42
acids such as lactate and CO2. In order to survive in this
acidic environment, cells overexpress various transporters or
43
enzymes and excrete excess intracellular acids. In conse-
quence, most tumors have an acidic extracellular pH, which
5
,44
may be as low as 6.5−7.0.
Current techniques for in vivo
imaging of tumor pH are based mainly on magnetic resonance
4
5−47
imaging (MRI),
but this requires expensive equipment as
well as a long measurement time owing to the low detection
29
sensitivity of MRI contrast agents. A fluorescence-based
method should enable relatively high-throughput imaging of
tumor pH in vivo. Here, fluorescence ratio imaging was
performed 1 h after i.v. injection of Dex-Me-pEPPR into mice
bearing a subcutaneous tumor (Figure 7a).
able to fine-tune the probe properties (i.e., pK , water solubility,
a
and fluorescence quantum yield) in order to obtain the desired
ratiometric fluorescence pH probes (SiRpH5 and Me-pEPPR).
SiRpH5 is a fluorophore whose excitation and emission
wavelengths lie in the far-red to NIR region. This provides a
new color window for multicolor pH imaging, and indeed, pH
imaging with GFP and Venus was achieved in this report. In
addition, SiRpH5 has a higher resistance to photobleaching
compared to conventional ratiometric probes, suggesting that
SiRpH5 would be a suitable probe for long-term tracking of
intracellular organelle pH. Conjugation of SiRpH5 to macro-
molecules was an effective strategy for specific imaging of
intracellular organelles, and SiRpH5 could be delivered to the
targeted intracellular organelles by choosing an appropriate
Although large fluorescence intensity change was hardly
detectable, significant change of fluorescence ratio can be
detected at tumor (Figure S16). The tumor pH was measured
to be 6.8, corresponding to weak acidification of tumors, while
the pH of normal tissue (muscle) was measured to be 7.4
(
Figure 7b,c). These pH values measured by ratiometric
fluorescence imaging showed good agreement with those
measured using a pH electrode (Figure S17). Thus, NIR
fluorescence imaging of tumor extracellular pH was successfully
achieved for the first time.
F
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Journal of the American Chemical Society
Article
macromolecule for conjugation. By using SiRpH5 conjugated
with Tfn, endosomal pH change during trafficking of Tfn was
imaged for the first time. Moreover, conjugation of the NIR
probe to dextran enabled imaging of extracellular pH in vivo.
Thus, i.v. injection of Dex-Me-pEPPR enabled NIR
fluorescence imaging of kidney and tumor pH for the first
time. It should be possible to image the pH of other organs,
such as gastrointestinal tract, similarly by conjugating the probe
to a suitable macromolecule to control the biodistribution.
Thus, the asymmetric rhodamine scaffold is useful for
developing functional ratiometric fluorescence probes. In
particular, the absorption and emission wavelengths can be
modified by changing the 10-position atom of the xanthene
moiety and the alkyl group on amino group of the xanthene
Labeling of Dextran with SiRpH3-SE. Solutions of 10 kDa
aminodextran (Invitrogen) (1.42 mg, 142 nmol) in 284 μL of 100 mM
borate buffer (pH 8.0) and 9 equiv of SiRpH3-SE in 57 μL of DMSO
were mixed and stirred at room temperature for 1.5 h. Reaction
mixtures were purified on size exclusion PD-10 columns (GE
Healthcare) using water as the mobile phase, and the eluate was
lyophilized to yield SiRpH3-Dextran.
Labeling of Dextran with SiRpH5-PEG -SE. Solutions of 10 kDa
6
aminodextran (1.30 mg, 130 nmol) in 460 μL of 100 mM NaHCO3
aq. (pH 8.4) and 3.8 equiv of SiRpH5-PEG -SE in 160 μL of DMSO
6
were mixed and stirred at room temperature for 2 h. Reaction mixtures
were purified on size exclusion PD-10 columns using PBS as the
mobile phase, and desalted with PD-10 column using water as the
mobile phase. The eluate was lyophilized to yield SiRpH5-Dex.
Preparation of Cells. HeLa cells and COS-1 cells were purchased
from American Type Culture Collection (ATCC). A cDNA encoding
moiety, while the pK value can be modified by changing the
21
a
mouse Vamp7 was obtained as described, and was inserted into
substituent group on the piperazine ring. Further, an
appropriate targeting moiety can be easily introduced because
conjugation does not affect the properties of the probes. We
believe ratiometric fluorescence probes based on this novel
scaffold will contribute to studies of various biological
phenomena associated with pH change, both in organelles of
living cells and in organs in situ.
pMRXIP (provided by S. Yamaoka, Tokyo Medical and Dental
University) together with Venus provided by A. Miyawaki (RIKEN).
4
8
MEFs stably expressing Venus-Vamp7 were generated using retrovirus
4
9
infection as previously described. Lung carcinoma cell line, A549
cells were purchased from RIKEN Bioresource Center cell bank
(Tsukuba, Japan). Murine colon carcinoma cell line, Colon-26 cells
were a gift from Dr. Yoshihiro Hayakawa (University of Toyama,
Institute of Natural Medicine). Cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) (Invitrogen Corp., Carlsbad, CA)
supplemented with 10% fetal bovine serum (Invitrogen), and 1%
penicillin streptomycin (Invitrogen). Cells were maintained at 37 °C
EXPERIMENTAL SECTION
■
Materials and General Information. Reagents and solvents were
of the best grade available, supplied by Tokyo Chemical Industries,
Wako Pure Chemical, Aldrich Chemical Co., Dojindo, Kanto
Chemical Co., Watanabe Chemical Industries, Matrix Scientific,
Thermo Fisher Scientific Inc. and Invitrogen, and were used without
further purification. NMR spectra were recorded on a JEOL JNM-
under an atmosphere of 5% CO in air.
2
Lysosomal pH Imaging in MEF Cells with SiRpH5-Dex. 1.6 ×
4
10 MEF cells stably expressing Vamp7-Venus were seeded on 8-
chamber plates (NUNCTM) and cultured for 1 day before imaging.
Cells were incubated in 200 μL of DMEM (10% FBS) containing 200
μg/mL SiRpH5-Dex for 2 h at 37 °C, then washed three times with
DMEM, further incubated in DMEM for 3 h at 37 °C, and then
imaged using a Leica Application Suite Advanced Fluorescence (LAS-
AF) instrument with a TCS SP5 and 63 × oil immersion objective
lens. The light source was a white-light laser SuperK (Leica). The
excitation and emission wavelengths were 510 nm/530−560 nm for
Vamp7-Venus and 580 nm, 670 nm/690−750 nm for SiRpH5-Dex.
1
13
LA300 instrument at 300 MHz for H NMR and at 75 MHz for
C
1
NMR, or a JEOL JNM-LA400 instrument at 400 MHz for H NMR
and at 100 MHz for 13C NMR. All chemical shifts (δ) reported in ppm
are relative to internal standard tetramethylsilane (δ = 0.0 ppm), or
1
relative to the signals of residual solvent CDCl (7.26 ppm for H,
3
1
13
7
7.16 ppm for 13C), CD OD (3.31 ppm for H, 49.00 ppm for C), or
CD CN (1.94 ppm for H, 1.32 ppm for C), and coupling constants
3
1
13
3
are given in Hz. Mass spectra (MS) were measured with a JEOL JMS-
T100LC AccuToF (ESI). HPLC analyses were performed on an
Inertsil ODS-3 (4.6 × 250 mm) column (GL Sciences Inc.) using an
HPLC system composed of a pump (PU-980, JASCO) and a detector
Labeling of Holo-transferrin with SiRpH5-PEG
of 80 kDa human holo-transferrin (9.04 mg, 113 nmol) in 1 mL of 100
mM borate buffer (pH 8.0) and 8 equiv of SiRpH5-PEG -SE in 1 mL
6
-SE. Solutions
6
of 100 mM borate buffer (pH 8.0) containing 20% DMSO were mixed
and stirred at room temperature for 1 h. Reaction mixtures were
purified by size exclusion PD-10 columns using PBS as the mobile
phase, desalted on a PD-10 column using water as the mobile phase,
and lyophilized to yield SiRpH5-Tfn.
(
MD-2015, JASCO). Preparative HPLC was performed on an Inertsil
ODS-3 (10.0 × 250 mm) column (GL Sciences Inc.) using an HPLC
system composed of a pump (PU-2080, JASCO) and a detector (MD-
2
015 or FP-2025, JASCO).
UV−Vis Absorption and Fluorescence Spectroscopy. UV−
Endosomal pH Imaging in COS-1 Cells with SiRpH5-Tfn. 5.0
× 10 COS-1 cells were seeded on 35 mm poly-L-lysine-coated glass-
4
visible spectra were obtained on a Shimadzu UV-1650. Fluorescence
spectroscopic studies were performed on a Hitachi F4500. The slit
width was 5 nm for both excitation and emission. The photomultiplier
voltage was 700 V. Absolute quantum yields were measured with a
Hamamatsu Photonics Quantaurus QY. Relative fluorescence
quantum yields were obtained by comparing the area under the
emission spectra of the test samples with standard samples. They were
calculated according to the following equation.
bottomed dishes (Matsunami Glass Ind., Ltd.), transfected with GFP-
3×FYVE or EGFP-Rab11 by using Lipofectamine 2000 (Invitrogen)
and cultured for 1 day before imaging. Cells were incubated in 1 mL of
HBSS containing 25 μg/mL SiRpH5-Tfn for 20 min at 4 °C, then
washed twice with ice-cold HBSS and imaged at 37 °C using a Leica
Application Suite Advanced Fluorescence (LAS-AF) instrument with a
TCS SP5 and 63× oil immersion objective lens. The light source was a
white-light laser SuperK (Leica). The excitation and emission
wavelengths were 488 nm/500−530 nm for GFP-3×FYVE, and
EGFP-Rab11, and 580 nm, 660 nm/680−750 nm for SiRpH5-Tfn.
Labeling of Dextran with Me-pEPPR. Solutions of 40 kDa
aminodextran (Invitrogen) (2.50 mg, 62.5 nmol) in 250 μL of 100
2
2
Φ /Φ = [A /A ][n /n ][D /D ]
x
st
st
x
x
st
x
st
where st: standard; x: sample; A: absorbance at the excitation
wavelength; n: refractive index; D: area under the fluorescence spectra
on an energy scale.
mM NaHCO aq. (pH 8.4) and 2, 6, or 10 equiv of Me-pEPPR-SE in
3
Photobleaching Test. Fluorescence spectra of 1 μM BCECF,
SNARF-1 and SiRpH5 were measured in 0.1 mM sodium phosphate
buffer (pH 7.4) containing 0.1% DMSO as a cosolvent after light
irradiation (26 mW, 450−510 nm (BCECF), 26 mW, 515−569 nm
150 μL of 100 mM NaHCO aq. (pH 8.4) and 100 μL of DMSO were
3
mixed and stirred at room temperature for 2 h. Reaction mixtures were
purified on size exclusion PD-10 columns using PBS as the mobile
phase, desalted with PD-10 column using water as the mobile phase,
and then lyophilized to yield Dex-Me-pEPPR.
(
SNARF-1), or 26 mW, 615−695 nm (SiRpH5)) using a fiber optic
illuminator MAX-301 (Asahi Spectra) equipped with a 150 W xenon
lamp (Bunkoukeiki).
Evaluation of Cell Permeability of Me-pEPPR and Dex-Me-
5
pEPPR. 1.0 × 10 A549 cells were seeded on 35 mm poly-L-lysine-
G
DOI: 10.1021/jacs.8b00277
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
coated glass-bottomed dishes (Matsunami Glass Ind., Ltd.) and
cultured for 1 day before imaging. Cells were washed once with 1 mL
of PBS, incubated in probes in DMEM supplemented with 10% fetal
bovine serum (Invitrogen), 1% penicillin streptomycin (Invitrogen)
containing the probes at 37 °C for 30 min followed by fluorescence
imaging with a Leica Application Suite Advanced Fluorescence (LAS-
AF) with a TCS SP5 and a 63× oil immersion objective lens. The light
source was a white-light laser SuperK (Leica). The excitation and
emission wavelengths were 670 nm/700−780 nm.
ACKNOWLEDGMENTS
■
This work was supported in part by grants by JSPS KAKENHI
Grant Numbers 16H00823 and 16H05099 to K.H., 16H06574
to T.U. and KAKENHI (S) (No. 17H06173) (to M.U.) and
SENTAN, JST to K.H., who was also supported by a grant JSPS
Core-to-Core program, A. Advanced Research Networks, and
Mochida Memorial Foundation for Medical and Pharmaceutical
Research.
In Vivo Imaging of Renal Tubular pH. All procedures were
approved by the Animal Care and Use Committee of the University of
Tokyo. Jcl:ICR mice (female, 6-week-old) were purchased from CLEA
Japan, Inc. Acetazolamide (30 mg/kg/day) in saline or saline as a
control was orally administered for 7 days before imaging. Dex-Me-
pEPPR (DOL = 1.4) (100 μM in 140 μL of saline) was injected
intravenously 1 h before imaging. Mice were anesthetized with
isoflurane using a small animal anesthetizer MK-A110 (Muromachi
Kikai Co., Ltd., Tokyo, Japan), and skin on their back was excised.
Then, fluorescence images were captured with a Maestro In Vivo
Imaging System (PerkinElmer, Inc., MA, USA) equipped with an
excitation filter of 661 nm (641−681 nm) or 704 nm (684−729 nm)
and an emission long path filter of 745 nm. Fluorescence ratio was
converted to pH value using the standard curve (Figure S15).
In Vivo Imaging of Tumoral pH. All procedures were approved
by the Animal Care and Use Committee of the University of Tokyo.
BALB/cAJcl-nu-nu mice (male, 5−6-week-old) were purchased from
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