Rational design of ratiometric near-infrared fluorescent pH probes with various pKa values, based on aminocyanine

Article abstract of DOI:10.1021/ja1063058

Novel ratiometric, near-infrared fluorescent pH probes with various pK _a values have been designed and synthesized on the basis of aminocyanine bearing a diamine moiety, and their photochemical properties were evaluated. Under acidic conditions, these pH probes showed a 46- to 83-nm red shift of the absorption maximum. This change is sufficiently large to permit their use as ratiometric pH probes, and is reversible, whereas monoamine-substituted aminocyanines showed irreversible changes because of their instability under acidic conditions. Furthermore, the pK_a values of these probes can be predicted from the calculated pK_a values of the diamine moieties, obtained from the SciFinder database. This design strategy is very simple and flexible, and should be applicable to develop NIR pH probes for various applications.

Full text of DOI:10.1021/ja1063058

ARTICLE
pubs.acs.org/JACS
Rational Design of Ratiometric Near-Infrared Fluorescent pH Probes
with Various pKa Values, Based on Aminocyanine
Takuya Myochin,†,‡,|| Kazuki Kiyose,†,‡,|| Kenjiro Hanaoka,†,‡ Hirotatsu Kojima,‡,§ Takuya Terai,†,‡ and
Tetsuo Nagano*,†,‡
†Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
‡CREST, JST, Sanbancho-blg, 5 Sanbancho, Chiyoda-ku, Tokyo, 102-0075, Japan
§Chemical Biology Research Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
S Supporting Information
b
ABSTRACT: Novel ratiometric, near-infrared fluorescent pH
probes with various pKa values have been designed and synthe-
sized on the basis of aminocyanine bearing a diamine moiety,
and their photochemical properties were evaluated. Under
acidic conditions, these pH probes showed a 46- to 83-nm
red shift of the absorption maximum. This change is sufficiently
large to permit their use as ratiometric pH probes, and is
reversible, whereas monoamine-substituted aminocyanines showed irreversible changes because of their instability under acidic
conditions. Furthermore, the pKa values of these probes can be predicted from the calculated pKa values of the diamine moieties,
obtained from the SciFinder database. This design strategy is very simple and flexible, and should be applicable to develop NIR pH
probes for various applications.
’ INTRODUCTION
phenomenon to develop a ratiometric NIR probe for Zn2þ
ion.14 Furthermore, we also developed a ratiometric NIR fluor-
escent pH probe using this pH response of aminocyanines.15
However, the fluorescence change of this pH probe is irrever-
sible, which can be problematic.16 For example, due to this
irreversibility, aminocyanines containing a monoamine moiety
cannot monitor reversible pH changes in living organs. There-
fore, for the development of reversible ratiometric pH probes,
aminocyanines that are stable even in acidic environments are
required.
Intracellular or extracellular pH is influenced by diverse
physiological and pathological processes, so quantitatively mea-
suring pH is useful for cellular analysis or diagnosis. For example,
an acidic environment may be associated with inflammation or
tumor.1 So far, many pH probes have been developed, and some
are commercially available.2-4 However, most of them consist of
fluorophores that absorb and emit in the visible region, and there
are few near-infrared (NIR) pH probes.5 Some reviews mention
that the range of NIR light especially suitable for In Vivo imaging
is 650-900 or 700-1000 nm,6-8 while the visible light region is
generally 400-750 nm.9 NIR fluorophores, such as tricarbocya-
nine dyes, have the advantage that light in the NIR region
(around 650-900 nm) is relatively poorly absorbed by biomo-
lecules, so it can penetrate deeply into tissues. There is also less
autofluorescence in this region, and the characteristics of these
dyes are favorable for In Vivo imaging.6,7 Thus, NIR pH probes
based on tricarbocyanine are expected to be suitable for mon-
itoring pH change In Vivo.
Some NIR pH probes, which can detect acidic environments,
have been reported so far. Most of them are based on nordi-
carbocyanine or nortricarbocyanine, and they become highly
fluorescent when the protonatable amino group within the
fluorophore is protonated.10-13 Recently, we reported that the
excitation wavelength of amine-substituted tricarbocyanine
could be modulated by controlling the electron-donating ability
of the amine substituent, and we further utilized this
’ RESULTS AND DISCUSSION
pH Dependency of Aminocyanines. We hypothesized that
when a proton is delocalized at two nitrogen atoms of amino-
cyanines bearing a diamino moiety under acidic conditions, such
aminocyanines would be stable (Scheme 1). We previously
reported that amidation of aminocyanine induced a drastic red-
shift in absorption,15 and this suggests that when protonation
occurs at the two nitrogen atoms of such aminocyanines, it would
lead to a red-shift of the absorption peak owing to the decrease of
the electron-donating ability of the amine. Therefore, we focused
on aminocyanines containing ethylenediamine or piperazine
moiety as a diamine moiety. Indeed, we designed and synthesized
Received: August 16, 2010
Published: February 22, 2011
r
2011 American Chemical Society
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Scheme 1. Mechanism of pH Response by Protonation and Deprotonation of Aminocyanine Derivatives
Figure 1. Structures of aminocyanines derivatives 1, 2, and 3.
Scheme 2. Synthetic Scheme for Compounds 2-6a
a Reagents and conditions: (a) benzyl bromide, dichloromethane, r.t., y. 37%; (b) corresponding amine (R), DMF: R = N,N,N0-trimethylethylene-
diamine (2), r.t., y. 95%; R=N-methylpiperazine (3), r.t., quant.;R=N,N-diethyl-N0-methylethylenediamine (4), 80°C, y. 97%; R=piperazine(5), r.t., y. 99%;
R = N-benzyl piperazine (6), r.t., y. 86%.
our previously reported methylamine-substituted aminocyanine
(1) as a representative of the monoamine type, and novel
aminocyanines containing N,N,N0-trimethyl ethylenediamine
(2) and N-methyl piperazine (3) as representatives of the
diamine type (Figure 1 and Scheme 2), and we examined the
pH dependence of their spectral characteristics. Compounds 2
and 3 showed red shifts of 53 and 80 nm of the absorption
maximum under acidic conditions, respectively (Figures 2 and
3), while the emission maximum and fluorescence quantum
efficiency hardly changed upon protonation or deprotonation
(Table 1). Further, we checked the effects of temperature, 10%
DMSO and ionic strength on the pH-sensing properties of 8,
which is water-soluble (Figures S1, S2, and S3, and Table S1
of the Supporting Information). The effects seemed to be
negligible. These characteristics are favorable for ratiometric
pH probes.
Reversibility of the Change in Absorption Spectrum. We
further investigated whether 3 is stable in an acidic environment,
compared to 1, which contains a monoamine receptor for Hþ
(Figure 4). In agreement with previous findings,15,16 the NIR
absorption of aminocyanine containing a monoamine moiety (1)
was irreversibly diminished under acidic conditions (Figure 4A,
B). This irreversible blue shift in the absorption spectrum was
observed during 60 min. However, the NIR absorption of 3
hardly decreased during 60 min, and its spectral change was
reversible (Figure 4C,D and 5). This result indicates that 3 is
stable even under acidic conditions. From these results, we
concluded that it is possible to develop reversible, ratiometric
pH probes based on diamine-bearing aminocyanines.
Correlation between Measured pKa Values of pH Probes
and Calculated pKa Values of Corresponding Diamines. We
further tried to develop pH probes with various pKa values. We
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Figure 2. (A) Absorption spectra of 2 μM 2. All samples were measured in sodium phosphate buffer at pH 2.5, 3.5, 4.3, 5.4, 6.2, 6.7, 7.1, 7.8, 8.3, 9.1, 11.2,
and 12.3 in the presence of 10% DMSO as a cosolvent. Emission wavelength is 760 nm. (B) Changes in fluorescence ratio of 2 (750-nm excitation/
670-nm excitation, 780-nm emission) in sodium phosphate buffer at the same pH values. The pKa value of 2 was 8.1.
Figure 3. (A) Absorption spectra of 3 μM 3. All samples were measured in sodium phosphate buffer at pH 2.5, 3.4, 4.3, 5.4, 6.2, 6.7, 7.1, 7.8, 8.3, 9.1, 11.2,
and 12.3 in the presence of 10% DMSO as a cosolvent. Emission wavelength is 760 nm. (B) Changes in fluorescence ratio of 3 (750-nm excitation/670-
nm excitation, 780-nm emission) in sodium phosphate buffer at the same pH values. The pKa value of 3 was 6.8.
Table 1. Photochemical Properties of pH Probes Based on the Aminocyanine Scaffold
compounda
absmax (nm)
Emmax (nm)
extinction coefficient (M-1cm-1
)
Q.E.
pKa
2 (protonated)
2 (deprotonated)
3 (protonated)
3 (deprotonated)
4 (protonated)
4 (deprotonated)
5 (protonated)
5 (deprotonated)
6 (protonated)
6 (deprotonated)
723
676
750
667
728
682
730
660
759
686
781
771
789
781
781
770
785
777
789
781
1.1 ꢀ 105
8.8 ꢀ 104
1.1 ꢀ 105
7.4 ꢀ 104
1.3 ꢀ 105
9.0 ꢀ 104
9.0 ꢀ 104
6.0 ꢀ 104
1.3 ꢀ 105
8.6 ꢀ 104
0.03
0.04
0.03
0.04
0.03
0.03
0.03
0.03
0.03
0.04
8.1
6.8
8.7
8.1
5.5
a Acidic condition was pH 3.6, and basic condition was pH 10.9 (compounds 2, 4, and 5) or pH 9.6 (compounds 3 and 6).
found that the pKa value of 2 (8.1) was much larger than that of 3
(6.8) (Figure 2 and 3). We considered that this was due to the
difference of protonatable diamine, so we synthesized three more
aminocyanine derivatives containing various ethylenediamine or
piperazine moieties (compounds 4-6) (Figure 6). Further, we
measured their absorption spectra to determine the pKa values of
4-6 (Figure 7). We also searched the pKa values of the
corresponding diamine derivatives, calculated by using Advance
Chemistry Development (ACD/Laboratories) Software V8.14
for Solaris, from the SciFinder database. Interestingly, there was a
good correlation between the measured pKa values of the
corresponding aminocyanines and the calculated pKa values of
the diamine derivatives (Table 2 and Figure 8). The difference
between the measured pKa values of the corresponding amino-
cyanines and the calculated pKa values of the diamine derivatives
amounts to about 1.0 pH unit, and this may be derived from the
difference of chemical structures, i.e., the diamine moieties were
directly conjugated with the cyanine scaffold in the structures of
aminocyanines, and diamine derivatives obtained from the
SciFinder database do not include the cyanine scaffold. The
systematic shift of the measured pKa values of aminocyanines
relative to the calculated pKa values of diamines from the
database is probably due to this difference, i.e., the presence or
absence of the cyanine scaffold. Furthermore, the emission
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Figure 4. (A,C) Time-dependent changes of absorption of 10 μM 1 (A) or 3 (C) at pH 4.0 during 60 min. (B,D) Investigation of the reversibility of the
change for 1 (B) or 3 (D). The absorption spectrum was measured at 60 min after the stock solution of 1 (B) or 3 (D) was dissolved in the buffer (pH
4.0) (red line). After that, the solution was diluted with the buffer (pH 9.0), and the spectrum was measured (yellow line). The spectrum at pH 9.0 is also
shown (blue line). All samples were measured in sodium phosphate buffer in the presence of 10% DMSO as a cosolvent.
maximum of each probe hardly changed after protonation, while
the absorption maximum markedly changed after protonation
(46-83 nm) (Table 1). Namely, ratiometric NIR pH probes
with various pKa values could be rationally designed by referring
to the calculated pKa values of various diamines obtained from
the SciFinder database.
For example, if we need a pH probe for sensing an acidic
environment, then the pKa value of aminocyanine can be finely
tuned on the basis of the slightly different pKa values of various
diamines. This design strategy is very simple and flexible, and
should be applicable to a wide range of ratiometric pH probes for
various applications.
Figure 5. Reversible color changes of compound 3 (methanol
Application of pH Probe 7 as a Labeling Agent. For
solution).
biological and analytical applications, it is often effective to bind
a pH probe to macromolecules or carriers to detect certain
because even if the pKa value of the pH probe is influenced
by carriers or macromolecules, we can still tune the pKa value of
the carrier(or macromolecule)-pH probe conjugate by using
the approach described above. Further, we also checked the
stability of nano beads-probe 7 conjugate (Figure S4 and
Table S2 of the Supporting Information). The nano beads-
probe was stable, and the dye molecule was stably immobilized
on the nano beads without leakage from the surface for at least
a day.
Application of pH Probe 3 for Fluorescence Cellular
Imaging. We further investigated the application of 3 in cul-
tured cells (Figure 10). Cultured HeLa cells were incubated with
3 (1 μM) for 10 min at 37 °C. Compound 3 stained mitochon-
dria and lysosomes (Figure S6 of the Supporting Information),
and showed a difference of fluorescence ratio between these
analytes, which are difficult to detect with typical small sensor
molecules.17-19 Therefore, we synthesized an aminocyanine-
based pH probe with a carboxyl group (7), which is suitable for
labeling (Scheme 3). Its pKa value was expected to be close to
that of 6, because 7 has a phenyl methyl piperazine moiety. We
synthesized latex nano beads conjugated with 7, and the pKa
value of the nano beads-probe 7 conjugate was determined by
measurements of fluorescence intensities. The pH-sensitivity of
7 was retained, but unexpectedly, the pKa value of the probe in
the conjugate (4.2) was much lower than that of 6 (5.5)
(Figure 9). It is considered that aliphatic amino groups on the
surface of the nano beads might be easily protonated and the
resulting cations would influence the protonation efficiency of
aminocyanine, decreasing the pKa value. However, this unex-
pected pKa shift is not problematic for our design strategy,
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Figure 6. Structures of aminocyanine derivatives 4, 5, and 6.
Figure 7. (A,C,E) Absorption spectra of 2 μM 4 (A), 1 μM 5 (C), and 3 μM 6 (E). All samples were measured in sodium phosphate buffer at pH 3.7, 4.8,
6.0, 6.7, 7.8, 8.3, 9.2, 9.4, 10.0, and 12.3 (A), pH 3.7, 5.2, 5.6, 6.2, 6.8, 7.6, 7.8, 8.3, 8.6, and 10.3 (C) or pH 2.5, 3.7, 4.5, 5.0, 5.8, 6.1, 6.9, 7.6, 8.2, and 9.3 (E)
in the presence of 10% DMSO as a cosolvent. (B,D,F) Changes in fluorescence ratio of 4 (B), 5 (D), and 6 (F) (750-nm excitation/670-nm excitation,
780-nm emission) in sodium phosphate buffer at the above pH values.
organelles inside the cell (Figure 10B). Next, we added NH4Cl
aq. (final 10 mM) to the cell incubation buffer. It is reported that
the pH value of lysosomes changes from pH 4.7-4.8 to 6.2-6.4
inside cells upon addition of NH4Cl.20 The fluorescence ratio of
lysosomes changed at 1 min after the addition of NH4Cl, whereas
the fluorescence ratio of mitochondria did not change. This
result demonstrates that 3 can be used to monitor intracellular
pH changes.
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Table 2. Calculated pKa Values of Diamines and Measured pKa Values of Diamine-Substituted Aminocyanines
diamine
pKa of diaminea
pKa of diamine-substituted aminocyanine
N,N-diethyl-N0-methylethylenediamine (4)
N,N,N0-trimethylethylenediamine (2)
piperazine (5)
10.16
9.3
8.7
8.1
8.1
6.8
5.5
8.94
7.83
6.31
N-methylpiperazine (3)
N-benzylpiperazine (6)
a Calculated using Advanced Chemistry Development (ACD/Laboratories) Software V8.14 for Solaris (1994-2009 ACD/Laboratories); taken from
SciFinder Scholar.
performed with a Hitachi F4500 (Tokyo, Japan). The slit width was
10 nm for both excitation and emission. The photomultiplier voltage
was 400 V.
Optical Properties and Quantum Efficiency of Fluores-
cence. Optical properties of dyes were examined in 0.1 M sodium
phosphate buffer (pH 7.4) containing 0.1% (compound 1) or 10% (v/v)
(compounds 2-6) DMSO as a cosolvent, using a Shimadzu UV-1600
UV-vis spectrophotometer and Hitachi F4500 fluorescence spectro-
photometer. For determination of the quantum efficiency of fluores-
cence (Φfl), cresyl violet in methanol (Φfl = 0.54) was used as a
standard. Values were calculated according to the following equation.
2
2
Φx=Φst ¼ ½Ast=Axꢁ½nx =nst ꢁ½Dx=Dstꢁ
where st: standard x, sample; A, absorbance at the excitation wavelength;
n, refractive index; D, area under the fluorescence spectra on an
energy scale.
Calculation of pKa Values. pKa values of the compounds were
calculated by regression analysis of the fluorescence data to fit eq 1.21 R is
the ratio of emission intensity at two wavelengths. Rmax and Rmin are
maximum and minimum limiting values of R, and c is the slope (positive
for the basic forms of the dyes and negative for the acidic forms). Ia/Ib is
the ratio of the absorption intensity in acid to the absorption intensity in
base at the wavelength chosen for the denominator of R.
Figure 8. Correlation between pKa values of pH probes based on
aminocyanine (2-6) and calculated pKa values of the corresponding
diamines, calculated by using Advanced Chemistry Development
(ACD/Laboratories) Software V8.14 for Solaris (1994-2009 ACD/
Laboratories) as listed in SciFinder Scholar.
’ CONCLUSIONS
We have established a novel design strategy of pH-induced
fluorescence modulation for ratiometric NIR fluorescent pH
probes. This approach leads to drastic changes of the absorption
or excitation spectra, resulting in large changes of the fluores-
cence ratio. Furthermore, the pKa values of such probes can be
precisely tuned by referring to the calculated pKa value of the
diamine moiety. A pH probe library designed according to this
strategy should be applicable to ratiometric measurement of pH
changes over a wide range both in vitro and In Vivo. Synthesis
and application of such a library is in progress.
ꢀ
ꢁ
R - Rmin
Rmax - R
Ia
Ib
pH ¼ pKa þ c log
þ log
ð1Þ
Assessment of the Reversibility of the Change in Absorp-
tion Spectrum. Compound 1 or 3 was dissolved in 0.1 M sodium
phosphate buffer (pH 4.0) containing 10% DMSO as a cosolvent. The
absorption spectra of 1 μM 1 or 3 were measured every 10 min from 0 to
60 min (Figure 4). Next, the solvent was diluted to pH 9.0 with 0.1 M
sodium phosphate buffer (pH 9.0), and the absorption spectrum was
measured again.
Synthetic Protocol for Nano Beads-Probe Conjugate.
Nano beads; 20 nm ALIPHATIC AMINE LATEX, 2% W/V
(Spherotech)
1 Dilute 2.5 mL aliphatic amine latex microspheres with 10 mL MES
buffer (pH 6.0).
’ EXPERIMENTAL SECTION
General Procedures. Reagents and solvents were purchased from
commercial sources. Reactions were monitored by TLC with visual
observation of the dye spots, and by mass spectrometry. Products were
purified on a silica gel column, and by semipreparative HPLC. Products
were considered pure when a single HPLC peak was obtained.
2 Centrifuge the mixture to precipitate the particles; 3000 ꢀg for
20 min.
Instruments. 1H NMR spectra were recorded on a JEOL JNM-
LA300. Mass spectra were measured with a JEOL JMS-T100LC mass
spectrometer (ESIþ or ESI-). HPLC purification and analyses were
performed on a reverse-phase column (GL Sciences (Tokyo, Japan),
Inertsil ODS-3 10 ꢀ 250 mm for purification and Inertsil ODS-3 4.6 ꢀ
250 mm for analyses) fitted on a Jasco PU-1587 system, using eluent A
(0.1 M acetic acid/triethylamine buffer (pH 7.4)) and eluent B (CH3CN
with 20% H2O). The absorbance at 680 nm was monitored for
detection. Extinction coefficients were determined using a Shimadzu
UV-1600 (Tokyo, Japan). Fluorescence spectroscopic studies were
3 Remove supernatant and redisperse pellet in 10 mL MES buffer.
4 Centrifuge again, and remove the supernatant from the particles.
5 Resuspend pellet in 5 mL MES buffer, being sure to completely
suspend microsphere particles.
6 To 5 mL of pH probe 7 solution (10 μM) in MES buffer (10 μM),
add 2 mM of 50 mg/mL WSCD in MES buffer.
7 Incubate probe/WSCD mixture with gentle mixing at room
temperature for 20 min.
8 Take 5 mL of latex in MES buffer solution and add to 5 mL of probe
solution.
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Scheme 3. Synthetic Scheme for Compound 7a
a Reagents and conditions: (a) piperazine, DMF, 80 °C; (b) 4-(bromomethyl)phenylacetic acid, DMF, 60 °C.
Figure 9. (A) Changes in fluorescence ratio of nano beads-probe 7 conjugate (760-nm excitation/680-nm excitation, over 810-nm emission) in sodium
phosphate buffer at pH 1.6, 2.0, 3.0, 4.0, 4.6, 5.6, 5.8, 6.2, 7.0, 7.8, 8.1, and 9.0. Nano beads-probe 7 conjugate was diluted 10-fold with sodium phosphate
buffer, and the images were obtained with an imaging device. (B) Ratio images of nano beads-probe 7 conjugate in sodium phosphate buffer at pH.1.6,
2.0, 3.0, 4.0, 4.6, 5.6, 5.8, 6.2, 7.0, 7.8, and 8.1.
9 Incubate latex/probe mixture with gentle mixing at room tempera-
ture for 4 h.
Ratiometric Imaging of 3 in HeLa Cells. The cells were
incubated for 2 days before dye loading on an uncoated 35 mm diameter
glass-bottomed dish (D110100, Matsunami, Japan). Then, the cells were
rinsed with PBS, incubated with DMEM containing 1% FBS, 1 μM 3,
and 0.1% DMSO as a cosolvent for 10 min at 37 °C, washed with PBS
twice, and mounted on the microscope stage.
Ratiometric Imaging System. The ratiometric imaging system
comprised an inverted microscope (IX71; Olympus Corp., Tokyo,
Japan) and a cooled CCD camera (Cool Snap HQ; Roper Scientific,
Tucson, AZ). The microscope was equipped with a xenon lamp (AH2-
RX, Olympus), an objective lens (Uapo/340 40ꢀ/1.35, Olympus), a
dichroic mirror (DM735, Olympus), two excitation filters (IAD MC
660G6, MC 720FX, ASAHI SPECTRA, Japan), and an emission filter
(emission filter for Cy7, Olympus). The system was controlled with
Metafluor 7.3 software (Universal Imaging, Media, PA).
10 Remove supernatant, and redisperse pellet in 10 mL PBS.
11 Centrifuge again, and remove the supernatant from the particles
(procedure nos. 10 and 11 are repeated 3 times).
12 Resuspend the final latex in 5 mL Storage buffer (PBS; pH 7.4,
0.1% glycine).
Imaging of Nano Beads-Probe Conjugate. Fluorescence
images were captured with a Maestro In-vivo Imaging System (CRi
Inc., Woburn, MA), with an excitation filter of 680 and 760 nm or 661
and 704 nm, and emission filter of 810 nm long pass or 740 nm long pass,
and bright-field settings. Mean fluorescence intensity was measured with
Maestro 2.4.0 or 2.10.0.
Preparation of Cells. Hela cells (RIKEN BioResource Center,
Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
(Wako Pure Chemical Industries, Ltd., Osaka, Japan), supplemented
with 10% fetal bovine serum (Invitrogen Corp., Carlsbad, CA) and
penicillin (100 units/mL)-streptomycin (100 μg/mL) liquid
(Invitrogen Corp., Carlsbad, CA) at 37 °C in a humidified incubator
containing 5% CO2 in air.
Fluorescence Confocal Microscopy. Cells seeded on 35-mm
glass-bottomed dishes were washed with 1 mL PBS, then incubated in
DMEM containing 1% FBS, 1 μM 3, 5 nM lysotracker green or
mytotracker green and 0.2% (v/v) DMSO as a cosolvent for 10 min
at 37 °C, washed with 1 mL PBS twice, and mounted on the microscope
stage. Fluorescence images were captured using a Leica Application
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Figure 10. DIC and fluorescence ratiometric images of pH in HeLa cells. HeLa cells were incubated with 1 μM 3 for 10 min at 37 °C, and NH4Cl aq.
(final 10 mM) was added to the medium. (A) DIC image before NH4Cl aq. was added. (B) Fluorescence ratiometric images with 3 (730 ( 20 nm
excitation/660 ( 6 nm excitation, 750-800 nm emission) before NH4Cl aq. was added. (C) DIC image after NH4Cl aq. was added. (D) Fluorescence
ratiometric images with 3 (730 ( 20 nm excitation/660 ( 6 nm excitation, emission) after NH4Cl aq. was added.
Suite Advanced Fluorescence (LAS-AF) with a TCS SP5 and a 40ꢀ
objective lens. The light source was a white-light laser. The excitation
wavelength was 448 and 670 nm, and the emission wavelength was
500-530 nm (lysotracker green and mytotracker green), or 700-800
nm (dye 3), respectively. PMT; Gain: 800 V (lysotracker green and
mytotracker green), 550 V (dye 3) Offset 0%
stirred at room temperature for 3 h under an argon atmosphere. The
solvent was removed under reduced pressure, then the crude product
was purified by silica gel chromatography with dichloromethane and
methanol, and recrystallized from methanol and n-hexane to afford a
golden powder (66 mg, quant.). 1H NMR (300 MHz, CD3OD): δ 1.59
(s, 12H), 1.72-1.76 (m, 2H), 2.38 (s, 3H), 2.44 (t, 4H, J = 6.4 Hz, e),
2.66 (br, 4H), 3.42 (s, 6H), 3.67 (t, 4H, J = 4.8 Hz), 5.85 (d, 2H, J = 13.4
Hz), 7.04-7.08 (m, 4H), 7.23-7.35 (m, 4H), 7.69 (d, 2H, J = 13.4 Hz).
13C NMR (75 MHz, CDCl3): δ 21.6, 24.9, 28.8, 30.8, 46.1, 47.9, 54.9,
56.5, 96.2, 109.2, 121.8, 123.4, 124.4, 128.4, 139.9, 141.2, 143.2, 169.3,
173.6. HRMS (ESIþ) Calcd for [M - ClO4-]: 547.3800, found:
547.3815.
Synthesis of N-Benzylpiperazine. Piperazine (2.2 g, 26 mmol)
was dissolved in dichloromethane (20 mL). Benzyl bromide (3.4 g, 13
mmol) was added dropwise to the solution on ice bath. The mixture was
stirred at room temperature for 6 h. The solvent was removed under
reduced pressure, and then the crude product was purified by silica gel
chromatography with dichloromethane to afford a colorless solid (849
mg, 34%). 1H-NMR (300 MHz, CDCl3): δ 2.37 (br, 4H), 2.84 (t, 4H, J
= 4.95), 3.46 (s, 2H), 7.18-7.31 (m, 5H,). 13C NMR (75 MHz, CDCl3):
δ 45.7, 54.1, 63.3, 126.6, 127.8, 128.8, 137.8. LRMS (ESIþ): m/z 177 (M
þ Hþ).
Synthesis of IR786-N,N-Diethyl-N0-Methylethylenediamine
(4). IR-786 perchlorate (60 mg, 0.1 mmol) was dissolved in anhydrous
DMF (5 mL), and N,N-diethyl-N0-methylethylenediamine (52 mg, 0.4
mmol) was added. The mixture was stirred at room temperature for 3 h
under an argon atmosphere. The solvent was removed under reduced
pressure, then the crude product was purified by silica gel chromatog-
raphy with dichloromethane and methanol, and washed with n-hexane
to afford a glossy blue solid (66 mg, 97%). 1H NMR (300 MHz, CDCl3):
δ 1.04 (t, 6H, J = 7.0 Hz), 1.67 (s, 12H,), 1.83-1.87 (m, 2H), 2.48 (t,
4H, J = 6.2 Hz), 2.58 (q, 4H, J = 7.0 Hz), 2.89 (br, 2H), 3.48 (s, 6H), 3.53
(s, 3H), 3.91 (t, 2H, J = 6.1 Hz), 5.73 (d, 2H, J = 13.4 Hz), 6.98 (d, 2H,
J = 7.9 Hz), 7.10 (t, 2H, J = 7.43 Hz), 7.29-7.34 (m, 4H), 7.58 (d, 2H,
J = 13.4 Hz). 13C NMR (75 MHz, CDCl3): δ 10.9, 21.7, 24.2, 29.1, 30.5,
44.8, 46.6, 47.7, 50.9, 55.2, 95.2, 108.8, 121.8, 123.0, 128.3, 139.9, 141.7,
143.3, 168.8, 175.8. HRMS (ESIþ) Calcd for [M - ClO4-]: 577.4270,
found: 577.4278.
Synthesis of IR786-Piperazine (5). IR-786 perchlorate (60 mg,
0.1 mmol) was dissolved in anhydrous DMF (5 mL), and piperazine (43
mg, 0.4 mmol) was added. The mixture was stirred at 80 °C for 3 h under
an argon atmosphere. The solvent was removed under reduced pressure,
then the crude product was recrystallized from 2-propanol to afford a
golden powder (62 mg, 99%). 1H NMR (300 MHz, CDCL3): δ 1.67 (s,
12H), 1.81-1.86 (m, 2H), 2.48 (t, 4H, J = 6.42 Hz), 3.27 (br, 4H), 3.50
(s, 6H), 3.90 (br, 4H), 5.74 (d, 2H, J = 13.20 Hz), 6.99 (d, 2H, J = 8.07 Hz),
Synthesis of IR786-Methylamine (1). This compound was
synthesized following ref 13.
Synthesis of IR786-N,N,N0-Trimethylethylenediamine (2).
IR-786 perchlorate (60 mg, 0.1 mmol) was dissolved in anhydrous DMF
(5 mL), and N,N,N0-trimethylethylenediamine (41 mg, 0.4 mmol) was
added. The mixture was stirred at room temperature for 3 h under an
argon atmosphere. The solvent was removed under reduced pressure,
then the crude product was purified by silica gel chromatography with
dichloromethane and methanol, and washed with n-hexane to afford a
glossy blue solid (62 mg, 95%). 1H NMR (300 MHz, CD3OD): δ 1.54
(s, 12H), 1.69-1.78 (m, 2H), 2.12 (s, 6H), 2.42 (t, 4H, J = 6.6 Hz), 2.64
(t, 2H, J = 6.1 Hz), 3.35 (s, 3H), 3.37 (s, 6H), 3.80 (t, 2H, J = 6.1 Hz),
5.77 (d, 2H, J = 13.4 Hz), 6.98-7.03 (m, 4H), 7.19-7.31 (m, 4H), 7.62
(d, 2H, J = 13.4 Hz). 13C NMR (75 MHz, CDCl3): δ 21.8, 24.1, 28.6,
28.9, 30.5, 44.4, 45.0, 47.7, 57.3, 95.1, 108.8, 121.8, 123.0, 123.2, 128.2,
139.9, 142.1, 143.3, 169.0, 176.1. HRMS (ESIþ) Calcd for [M -
ClO4-]: 549.3957, found: 549.3967.
Synthesis of IR786-N-Methylpiperazine (3). IR-786 perchlo-
rate (60 mg, 0.1 mmol) was dissolved in anhydrous DMF (5 mL), and
1-methylpiperazine (40 mg, 0.4 mmol) was added. The mixture was
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Journal of the American Chemical Society
ARTICLE
7.11 (t, 2H, J = 7.32 Hz), 7.28-7.33 (m, 4H), 7.65 (d, 2H, J = 13.20 Hz).
13C NMR (75 MHz, CDCL3): δ 21.6, 25.3, 28.8, 31.0, 47.0, 48.0, 55.1,
95.8, 109.1, 122.0, 123.4, 123.8, 128.3, 140.0, 140.9, 143.2, 158.0, 169.7.
HRMS (ESIþ) Calcd for [M - ClO4-]: 533.3644, found: 533.3680.
Synthesis of IR786-N-Benzylpiperazine (6). IR-786 perchlo-
rate (60 mg, 0.1 mmol) was dissolved in anhydrous DMF (5 mL), and
1-benzylpiperazine (70 mg, 0.4 mmol) was added. The mixture was
stirred at room temperature for 3 h under an argon atmosphere. The
solvent was removed under reduced pressure, then the crude product
was purified by silica gel chromatography with dichloromethane and
methanol, and washed with n-hexane to afford a glossy blue solid (62 mg,
86%). 1H NMR (300 MHz, CDCl3): δ 1.62 (s, 12H), 1.78-1.87 (m,
2H), 2.42 (br, 4H), 2.48 (t, 4H, J = 6.4 Hz, f), 3.49 (s, 6H), 3.74 (s, 2H),
3.78 (br, 4H), 5.76 (d, 2H, J = 13.4 Hz), 7.00 (d, 2H, J = 7.8 Hz), 7.12 (t,
2H, J = 7.5 Hz), 7.28-7.40 (m, 7H), 7.59 (d, 2H, J = 13.4 Hz). 13C NMR
(75 MHz, CDCl3): δ 21.6, 24.9, 28.8, 30.8, 47.8, 54.1, 55.0, 62.9, 96.0,
109.1, 121.8, 123.4, 124.4, 127.5, 128.4, 128.5, 129.4, 136.6, 139.9, 141.1,
143.2, 169.1, 173.7. HRMS (ESIþ): Calcd for [M - ClO4-]: 623.4139,
found: 623.4113.
Synthesis of pH Probe 7. IR-783 perchlorate (80 mg, 0.1 mmol)
was dissolved in anhydrous DMF (5 mL), and piperazine (43 mg, 0.4
mmol) was added. The mixture was stirred at 80 °C for 1.5 h under an
argon atmosphere. 4-Bromophenylacetic acid (275 mg, 1.2 mmol) was
added, and stirred at 60 °C under an argon atmosphere overnight. The
reaction mixture was poured into 200 mL of diethylether. The precipitate
was collected by filtration, and purified by HPLC to afford a blue solid
(13 mg, 14%). 1H NMR (400 MHz, CD3OD): δ 1.53 (s, 12H), 1.70-1.83
(m, 10H), 2.42 (t, 4H, J = 6.40 Hz), 2.72-2.78 (m, 8H), 3.52 (s, 2H),
3.67 (br, 4H), 3.71 (s, 2H), 3.94 (br, 4H), 5.89 (d, 2H, J = 13.20 Hz),
6.79-6.93 (m, 2H), 7.03-7.34 (m, 12H), 7.63 (d, 2H, J = 13.20 Hz).
13C NMR (100 MHz, CD3OD): δ 23.0, 23.6, 25.8, 26.9, 29.2, 43.0, 44.1,
44.8, 52.0, 55.2, 55.8, 63.4, 97.5, 110.9, 123.2, 124.7, 125.8, 129.6, 130.5,
131.0, 131.1, 135.6, 141.8, 143.0, 144.1, 170.7, 174.6. HRMS (ESIþ)
Calcd for [M þ Hþ]: 925.4243, found: 925.4195.
Technology Development Organization (NEDO) of Japan (to
T.T.). K.H. was also supported by Sankyo Foundation of Life
Science.
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’ ASSOCIATED CONTENT
S
Supporting Information. Detailed descriptions of syn-
b
thetic procedure for 8; data on the measurements of absorption
spectra of 8 with 0.1% or 10% DMSO as a cosolvent, and on the
measurements of absorption spectra of 8 in the presence of 500
mM NaCl; plot of the changes of absorbance intensities of 8;
relative fluorescence ratios of 8 at various temperatures; fluores-
cence intensities of nano beads-probe 7 conjugate after a day;
time-dependent changes of absorption spectra of 7; confocal
fluorescence images of live cells. This material is available free of
charge via the Internet at http://pubs.acs.org.
(21) Whitaker, J. E.; Haugland, R. P.; Prendergast, F. G. Anal.
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’ AUTHOR INFORMATION
Corresponding Author
tlong@mol.f.u-tokyo.ac.jp
Author Contributions
These authors contributed equally.
’ ACKNOWLEDGMENT
This work was supported in part by a Grant-in-Aid for JSPS
Fellows, by grants from the Ministry of Education, Culture,
Sports, Science and Technology of Japan [Grant Nos. 22000006
(Specially Promoted Research) to T.N., 20689001 and
21659204 to K.H.), and by the Industrial Technology Research
Grant Program in 2009 from the New Energy and Industrial
3409
dx.doi.org/10.1021/ja1063058 |J. Am. Chem. Soc. 2011, 133, 3401–3409