Molecular design strategies for near-infrared ratiometric fluorescent probes based on the unique spectral properties of aminocyanines

Article abstract of DOI:10.1002/chem.200900035

In spite of the wide availability of various near-infrared (NIR) fluorophores as labeling reagents, there are few functional NIR fluorescent probes for which change in the absorption and/or fluorescence spectra upon specific reaction with biomolecules is

Full text of DOI:10.1002/chem.200900035

FULL PAPER
DOI: 10.1002/chem.200900035
Molecular Design Strategies for Near-Infrared Ratiometric Fluorescent
Probes Based on the Unique Spectral Properties of Aminocyanines
Kazuki Kiyose,[a, b] Sakiko Aizawa,[a, b] Eita Sasaki,[a, b] Hirotatsu Kojima,[b, d]
Kenjiro Hanaoka,[a, b] Takuya Terai,[a, b] Yasuteru Urano,[a, c] and Tetsuo Nagano*[a, b]
Abstract: In spite of the wide availabil-
ity of various near-infrared (NIR) fluo-
rophores as labeling reagents, there are
few functional NIR fluorescent probes
for which change in the absorption
and/or fluorescence spectra upon spe-
cific reaction with biomolecules is seen.
The widely used photoinduced elec-
tron-transfer mechanism is unsuitable
for NIR fluorophores, such as tricarbo-
cyanines, because their long excitation
wavelength results in a small singlet ex-
citation energy. We have reported the
unique spectral properties of amine-
substituted tricarbocyanines, which
were utilized to develop two design
strategies. One approach was based on
control of the absorption wavelength
by using the difference in electron-do-
nating ability before and after a specif-
ic reaction with a biomolecule, and the
other approach was based on control
of the fluorescence intensity by modu-
lating the Fçrster resonance energy-
transfer efficiency through a change in
the overlap integral that arises from
the change in absorption under acidic
conditions. These strategies were vali-
dated by obtaining tricarbocyanine-
based ratiometric NIR fluorescent
probes for esterase and for pH level.
Keywords: enzymes · esterase · flu-
orescent probes · IR spectroscopy ·
pH probes · sensors
Introduction
structure has been used as a scaffold for NIR fluorescent
probes. However, most of these probes are dyes for straight-
forward fluorescence labeling,[2–8] and very few show fluores-
cence intensity changes upon specific reaction with biomole-
cules.[9] Moreover, the rational design of NIR fluorescence
modulation is difficult, and few functional probes have been
reported. Recently, some NIR fluorescent probes in which
the fluorescence is modulated by the photoinduced electron-
transfer (PeT) mechanism have been reported.[9] However,
tricarbocyanine dyes have much longer excitation wave-
lengths than other dyes, and although this characteristic is
favorable for imaging in vivo, it makes modulation of the
fluorescence by means of PeT difficult. The major determi-
nant of PeT is DGPeT, and this value can be calculated from
the Rehm–Weller equation DGPeT =EoxꢀEredꢀDE00ꢀwp,
where DGPeT is the free energy change of a PeT, Eox and Ered
are the oxidation and reduction potentials of the electron
donor and acceptor, DE00 is the singlet excitation energy
and wp is the work term for the change separation state. The
DE00 value is especially influential in the case of the tricar-
bocyanine dye.[10,11] Tricarbocyanine is excited at around l=
750–800 nm, so its DE00 value is much smaller than those of
other dyes, which is disadvantageous for fluorescence con-
trol by PeT. To confirm this feature of tricarbocyanine, we
synthesized several tricarbocyanine derivatives (see Scheme
S1 in the Supporting Information), and investigated the cor-
Tricarbocyanine dyes are representative of the dyes that
have absorption and emission in the near-infrared (NIR)
region.[1] Because light in the NIR region (around l=650-
900 nm) is suitable for imaging in vivo, the tricarbocyanine
[a] K. Kiyose, S. Aizawa, E. Sasaki, Dr. K. Hanaoka, T. Terai,
Dr. Y. Urano, Prof. Dr. T. Nagano
Graduate School of Pharmaceutical Sciences
The University of Tokyo
7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
Fax : (+81)3-5841-4855
E-mail: tlong@mol.f.u-tokyo.ac.jp
[b] K. Kiyose, S. Aizawa, E. Sasaki, Dr. H. Kojima, Dr. K. Hanaoka,
T. Terai, Prof. Dr. T. Nagano
CREST, JST Agency
4-1-8, Honcho, Kawaguchi
Saitama 332-0012 (Japan)
[c] Dr. Y. Urano
PRESTO, JST Agency
4-1-8, Honcho, Kawaguchi
Saitama 332-0012 (Japan)
[d] Dr. H. Kojima
Chemical Biology Research Initiative
The University of Tokyo
7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200900035.
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relation between the calculated HOMO energy of the
phenol moiety and the fluorescence quantum yield of the
dyes. Furthermore, we compared these results with those for
Tokyo Green derivatives, which contain various benzene
moieties that have absorption and emission peaks in the
visible region. We found that benzene moieties with a much
higher HOMO energy level were needed for PeT-based
quenching of the fluorescence of tricarbocyanines relative to
Tokyo Green derivatives (Figure 1).[10] To overcome this lim-
itation of tricarbocyanine as a scaffold for functional NIR
fluorescent probes, a novel fluorescence modulating mecha-
nism, other than PeT, seems to be needed.
upon covalent protein binding.[14,15] This characteristic is de-
sirable in a labeling agent, but not in a functional probe.
However, very few studies of the spectral properties of NIR
dyes have been reported.[16,17] Therefore, we investigated the
spectral properties of tricarbocyanine derivatives and found
that amine-substituted tricarbocyanines (aminocyanines)
have very interesting properties that may make them suita-
ble for use as the scaffolds for functional fluorescent probes.
Herein, we describe the utilization of these properties to de-
velop two design strategies: one based on control of the ab-
sorption wavelength by using the difference in electron-do-
nating ability before and after a specific reaction with a bio-
molecule and the other based on control of the fluorescence
intensity by modulating fluorescence resonance energy-
transfer (FRET) efficiency through a change in the overlap
integral that arises from the change in the absorption under
acidic conditions. We also validated these strategies by em-
ploying them to obtain tricarbocyanine-based ratiometric
NIR fluorescent probes for esterase and for pH level.
Tricarbocyanine dyes bearing chloro-substituted poly-
ACHTUNGTRENNUNGmethine linkers are suitable for use as scaffolds for fluores-
cent probes because the chlorine atom can be easily substi-
tuted by various nucleophiles.[12,13] Some probes based on
this strategy have been reported, and these dyes are useful
protein labeling agents because of their monofunctionality.
The spectral properties of these dyes are hardly changed
^
Figure 1. Change of fluorescence quantum efficiencies of tricarbocyanine derivatives (*) and Tokyo Green derivatives ( ) depending on the HOMO
energy level of their phenol moiety. A) Structures of tricarbocyanine derivatives of IR-783 and the corresponding phenol moieties. B) Correlation be-
tween the HOMO energy level of the phenol (or benzene) moiety and the fluorescence quantum efficiencies of tricarbocyanines and Tokyo Green deriv-
atives. Data for the Tokyo Green derivatives were collected from ref. [10]. The HOMO energy level was calculated by the DFT method by using
B3LYP/6-31G(d).
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The Development of Near-Infrared Ratiometric Fluorescent Probes
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Results and Discussion
from the present study that the characteristics of aminocya-
nines are primarily determined by ICT. Therefore, we fo-
cused simply on applying the characteristics of aminocya-
nines to developing a unique strategy for the design of func-
tional fluorescent probes.
Tricarbocyanines containing various substituents at the
center of methine linker: The chlorine atom of tricarbocya-
nines bearing a chloro-substituted methine linker can be
substituted by various nucleophiles, for example, alcohols,
amines, and thiols. First, we measured the absorption and
emission spectra of IR-780, IR-768, IR-792, and 1 and calcu-
lated the fluorescence quantum yield to investigate the sub-
stituent dependence of the spectral properties (Scheme 1
and Table 1). There are no great differences in the spectral
Investigation of spectral properties of aminocyanines: A
series of aminocyanines 2–6 was synthesized to investigate
their spectral properties (Scheme 2 and Table 2).[20] The re-
sults indicate that the lower the electron density on the
amine, the longer the wavelength of the absorption maxi-
mum, with little change in the
emission maximum. Next, we
synthesized 7 and 8 (Scheme 2),
and, as expected, the spectral
properties of 8 were similar to
those of IR-786 (see Figure 2
and Table S1 in the Supporting
Information). The attenuation
of the electron-donating ability
of the amine substituent caused
a drastic red shift of the absorp-
tion maximum without any
change of the emission maxi-
mum or fluorescence quantum
yield. These results provide a
basis for the rational molecular
design of novel ratiometric NIR
probes by utilizing the differ-
ence in the electron-donating
ability of the amine substituent
Scheme 1. Structures of nucleophile-substituted tricarbocyanine dyes.
before and after a reaction with
a biomolecule of interest.
Table 1. Spectral properties of IR-780 and its derivatives.[a]
Amax
[nm]
e
Emmax
[nm]
FF
Stokes
shift [nm]
NIR ratiometric esterase-activated probe: As described
above, a change in the electron-donating ability of amino-
cyanines at the amine functional group causes a shift in the
absorption maximum. This shift can be applied to the design
of functional fluorescent probes, that is, we considered that
the difference in the inductive effect between the carboxylic
acid and carboxylate ester could be utilized to detect ester-
ases. We synthesized IR-786-b-alanine and IR-786-b-alanine
ethyl ester (9 and 10, respectively) and compared their spec-
tral properties to test this hypothesis. Indeed, the absorption
maximum of 10 was at a wavelength dl=20 nm longer than
9, which indicated that the electron-donating ability was de-
creased by an increase in the inductive effect (Scheme 3 and
Table 3). These results suggest that 10 can act as an esterase
probe. Esterases are important enzymes that are involved in
various biological phenomena, and several fluorescent
probes for esterases have been reported.[21–23] We demon-
strated the application of 10 in the assay of porcine liver es-
terase (PLE), which is recognized as a substrate of esterase.
PLE was hydrolyzed, which resulted in a blue shift in the
absorption maximum of dl=20 nm and a change in the
fluorescence ratio at excitations of l=600 and 700 nm.
Thus, 10 could work as a ratiometric NIR esterase probe
[105 mꢀ1 cmꢀ1
]
IR-780
IR-768
IR-792
1
780
767
792
742
3.3
3.3
3.0
2.3
798
786
815
788
0.07
0.14
0.04
0.05
18
19
23
46
[a] All the samples were dissolved in methanol.
properties of IR-780, IR-768, and IR-792. The fluorescence
quantum yields are slightly different from each other, and
the absorption coefficient and the Stokes shifts are almost
the same. Namely, these compounds are not suitable as scaf-
folds for the functional probes. In contrast, however, the
spectral properties of 1, which is an amine-substituted tricar-
bocyanine (aminocyanine), are interesting. Compound 1 has
absorption maximum at a much shorter wavelength and the
Stokes shift is much larger than those of the other cyanines
(Table 1). Furthermore, the extinction coefficient was re-
markably low, and the absorption spectrum is much broader
than those of the other cyanines (see Table 1 and Figure S1
in the Supporting Information). Peng et al. suggested that
these characteristics might be caused by intramolecular
charge transfer (ICT).[18,19] However, we could not confirm
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T. Nagano et al.
Scheme 2. Synthesis of IR-786 derivatives. Reagents: a) corresponding amine, DMF; b) acetyl chloride, DIEA, dichloromethane. DIEA=N, N-diisopro-
pylethylamine.
Table 2. Photochemical properties of IR-786-related aminocyanines.
[a]
Compound
pKa
Amax
Emmax
FF
[nm]
[nm]
2
3
4
5
6
10.6
10.6
9.7
9.5
9.4
626
627
633
635
647
745
748
747
748
744
0.06
0.07
0.07
0.07
0.07
Scheme 3. Structures of 9 (R=COOꢀ) and 10 (R=COOEt).
Table 3. Spectral properties of 9 and 10.
[a] These pKa values are for the corresponding amines.
Compound
FF
Amax
Emax
Stokes
[nm]
[nm]
shift [nm]
9
10
0.03
0.03
620
640
755
756
135
116
based on aminocyanine. We also followed this reaction by
means of analytical HPLC and confirmed that 10 disap-
peared and 9 emerged (see Figure 3 and Figure S2 in the
Supporting Information). Thus, our design strategy appears
to work well for esterases.
Sensitivity of aminocyanines toward pH value and novel mo-
lecular design for NIR fluorescent probes: Generally, when
dyes with an amino or hydroxy group are protonated or de-
protonated, their spectral properties are expected to change
drastically, and some of them have been widely used as fluo-
rescent pH indicators for investigating biological phenom-
ena.[24–28] Recently, some NIR pH indicators have been re-
ported,[29–31] but all of them are probes that have an increase
in fluorescence intensity when they are protonated. Amino-
cyanine also has such an amino group, and it is expected
that the spectral properties, especially absorption wave-
length, will change drastically after protonation, so we con-
sidered that it might be applicable as a ratiometric pH
probe. Peng et al. previously investigated the pH sensitivity
of aminocyanine and concluded that the NIR emission of
aminocyanines disappeared because the amine is quater-
Figure 2. A) Normalized absorption and B) emission spectra of 7 (a), 8
(g), and IR-786 (c). All the data were measured in methanol. Com-
pounds IR-786 and 8 were excited at l=740 nm and 7 was excited at l=
594 nm.
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The Development of Near-Infrared Ratiometric Fluorescent Probes
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Figure 3. A) Excitation spectra of 10 in the presence of PLE (0.027 U).
The spectra were obtained every 5 min; all the samples were measured in
HEPES buffer (pH 7.4) containing 0.1% DMSO as a cosolvent; fluores-
cence wavelength was l=760 nm. B) Plot of the fluorescence ratio values
at l=760 nm for excitation at l=700 nm and excitation at l=600 nm
(n=3). ~: PLE (ꢀ); *: PLE (+).
nized under strongly acidic conditions and the ICT emission
disappears.[18] However, we felt that the effects of changing
the pH value might not be consistent with this explanation,
so we examined the temporal changes of fluorescence inten-
sity and absorption spectrum of 7 in weakly acidic sodium
phosphate buffer (pH 4.3). The NIR emission almost disap-
peared after 60 minutes and a drastic blue shift in the ab-
sorption spectrum was observed (Figure 4A,B). We mea-
sured the absorption spectrum of 7 at a concentration of
1 mm in weakly acidic buffer (pH 4.3; prepared 60 min
before) and of the same sample diluted tenfold with pH 9.4
buffer. The normalized absorption spectra are shown in Fig-
ure 4C. These results indicated that the protonation was ir-
reversible because 7 is comparatively stable at pH 9.4, and
no drastic spectral changes were observed (see Figure S3 in
the Supporting Information). Recently, Descalzo and
Rurack elegantly revealed the cause of these phenomena
and concluded that two protonation steps are involved.[32]
We thought that this property could be of great advantage
in designing pH probes with fluorescence that can be con-
trolled by the Fçrster resonance energy-transfer (FRET)
mechanism. FRET is associated with a large spectral shift
and is an interaction between the electronic excited states of
Figure 4. A) Absorption spectra of 7 in sodium phosphate buffer (pH 4.3,
1 mm). Data were obtained every 5 min (up to 60 min). B) Change in fluo-
rescence of 7 in sodium phosphate buffer (pH 4.3, 1 mm). The excitation
and fluorescence wavelength were l=594 and 760 nm, respectively.
C) Normalized absorption spectra of 7. c: Measured at pH 4.3; the
data were obtained 60 min after preparation. a: The same sample was
diluted to 0.1 mm with sodium phosphate buffer (pH 9.4) and spectra
were measured.
two fluorophores, in which the excitation energy of a donor
is transferred to an acceptor without emission of
a
photon.[33] The FRET efficiency can be altered either by
changing the distance between the FRET donor or by
changing the overlap integral between the emission spec-
trum of the FRET donor and the absorption spectrum of
the FRET acceptor. Indeed, ratiometric fluorescent probes
have been developed based on the former method, including
probes for calcium,[34] phosphodiesterase,[35] and b-lacta-
mase,[36] and on the latter method, including probes for pro-
tein tyrosine phosphatase[37] and hydrogen peroxide.[38] How-
ever, all of them work in the visible region, and little infor-
mation is available about NIR ratiometric probes based on
the FRET mechanism. Because the absorption of aminocya-
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T. Nagano et al.
and has two carboxyl groups, which should provide good
water solubility and can be used to provide a link to other
fluorophores (Figure 5 and Scheme 4).
We also investigated the spectral properties of three can-
didates for the FRET acceptor (Scheme 4). Compound 15
was optimal because it had the longest alkyl chain, which
should prevent intramolecular stacking, and had the largest
pKa value, which should be suitable for the acidic environ-
ment of organs (Table 4). We chose an aminocyanine con-
Table 4. Photochemical properties of 13–15.
[a]
Compound
Amax (acidic)
Amax (neutral)
Emax (neutral)
pKa
[nm]
[nm]
[nm]
13
14
15
501
512
511
628
622
620
760
760
760
3.7
4.0
4.4
[a] The pKa value is irreversible.
taining 6-amino-n-caproic acid as a FRET acceptor and syn-
thesized a more lipophilic aminocyanine 17, which had
almost the same structure as 15, with the aim of improving
the cell-membrane permeability of the probes (Scheme 5).
We investigated the spectral properties of 17 and found that
they resembled 15 (Figure 6). We calculated the overlap in-
tegral and Fçrster distance from the fluorescence spectrum
of donor 12 and the absorption spectrum of acceptor 17
under neutral and acidic conditions (see Table S2 and Fig-
ure S4 in the Supporting Information). Under neutral condi-
tions, aminocyanine has strong absorption in the dicarbocya-
nine emission region, and so acceptor fluorescence due to
FRET was expected to be observed. On the other hand,
under acidic conditions, the NIR absorption of aminocya-
nine disappears, so there is almost no overlap integral be-
tween the dicarbocyanine emission and aminocyanine ab-
sorption. Thus, aminocyanine cannot accept the excitation
energy of the donor, and so donor emission is expected to
be observed. These results indicated that 12 and 17 would
Figure 5. A) Absorption and B) emission spectra of 12. All the spectra
were measured in sodium phosphate buffer and acetonitrile (v/v=50:50,
100 mm) containing 0.1% DMSO as a cosolvent. Excitation wavelength
was l=640 nm.
nines completely disappeared upon protonation, and this
spectral property is favorable for a FRET acceptor, we hy-
pothesized that if aminocyanine was bonded to a pH-insen-
sitive cyanine group through an appropriate linker, it would
work as an NIR ratiometric pH probe. We chose 12 as a
FRET donor to test this idea because it is pH insensitive
Scheme 4. Synthesis of 12–15. Reagents: a) 3-iodopropionic acid, ortho-dichlorobenzene; b) 1)Ac2O, DIEA, dichloromethane; 2) 11, AcONa; c) b-ala-
nine (13), 4-aminobutyric acid (14), 6-amino-n-caproic acid (15), DMF.
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The Development of Near-Infrared Ratiometric Fluorescent Probes
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Scheme 5. Synthesis of 17. Reagents: a) 6-amino-n-caproic acid (15), DMF; b) 1) HOBT·H2O, HBTU, DIEA, DMF; 2) cyclohexylamine. HBTU=
(ortho-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate, HOBT=hydroxybenzotriazole.
be good candidates for the FRET donor and acceptor. We
therefore designed a ratiometric NIR fluorescent pH probe
19 that consisted of a pH-insensitive dicarbocyanine unit as
the FRET donor, cyclohexane as a rigid linker, and a pH-
sensitive aminocyanine group as the FRET acceptor.
We synthesized 19 as a candidate for a NIR ratiometric
pH probe and investigated its spectral properties in aqueous
buffers of various pH values (Scheme 6 and Figure 7). The
fluorescence ratios at l=750 and 660 nm derived from the
acceptor and donor moieties, respectively, changed as ex-
pected under acidic conditions. This result supports the con-
cept that the NIR fluorescence is controlled by changes in
the overlap integral that arises from the blue shift in the ab-
sorption of aminocyanine under acidic conditions. Finally,
we calculated the FRET efficiency E based on the fluores-
cence intensity at l=660 nm under neutral and acidic condi-
tions (pH 6.8 and 2.1, respectively) and found E=85 and
8.1%, respectively. As expected, FRET occurred efficiently
under neutral conditions but inefficiently under acidic condi-
tions. Therefore, 19 could work as a NIR ratiometric pH
probe utilizing the FRET mechanism based on the pH-de-
pendent spectral properties. The apparent pKa value of 19
(pKa =4.9) was determined by plotting the fluorescence
ratios at l=750 and 660 nm (see Figure S5 in the Supporting
Information). This modest pKa value is suitable for detecting
changes in the acidic environment in living organisms, for
Figure 6. A) Absorption and B) emission spectra of 17. All the spectra
example, detecting acidification of tumor tissues,[39] and vari-
were measured in sodium phosphate buffer and acetonitrile (v/v=50:50,
ous bioanalytical applications can be expected. This report
is the first of a NIR ratiometric pH probe based on carbo-
100 mm) containing 0.1% DMSO as a cosolvent. Excitation wavelength
was l=640 nm.
Scheme 6. Synthesis of ratiometric NIR fluorescent pH probe 19. Reagents: a) 1) HOBT·H2O, HBTU, DIEA, DMF; 2) trans-1,4-cyclohexanediamine;
b) 1) HOBT·H2O, HBTU, DIEA, DMF; 2) 12.
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T. Nagano et al.
ing that few methods are available for the control of NIR
fluorescence, these methods are expected to be powerful
tools for designing functional NIR fluorescent probes, espe-
cially probes for dual-wavelength ratiometric measurements.
Further studies on biological applications are now in prog-
ress.
Experimental Section
General procedures: All reagents, solvents, and compounds IR-768, IR-
780, IR-783, IR-786, and IR-792 were purchased from commercial sour-
ces. The reactions were monitored by TLC analysis with visual observa-
tion of the dye spots and mass-spectrometric analysis. The products were
purified by column chromatography on silica gel and semipreparative
HPLC.
Instruments: The 1H and 13C NMR spectra were recorded on a JEOL
JNM-LA300 spectrometer at 300 and 75 MHz and on a JMN-LA400
spectrometer at 400 and 100 MHz. The mass spectra were measured on a
JEOL JMS-T100 LC mass spectrometer (ESI+, ESIꢀ). HPLC purification
and analyses were performed on a reverse-phase column (GL Sciences,
Tokyo, Japan); Inertsil ODS-3 (10ꢁ250 mm) for purification and Inertsil
ODS-3 (4.6ꢁ250 mm) for analyses. All the absorption spectra were ob-
tained on a UV-1600 UV/Vis spectrometer (Shimadzu). Fluorescence
spectroscopic studies were performed on
a Hitachi F4500 machine
(Tokyo, Japan). The slit-width was 10 nm for both excitation and emis-
sion measurement. The photomultiplier voltage was 400 V.
Determination of fluorescence quantum efficiency: Optical properties of
the dyes were examined in sodium phosphate buffer (pH 7.4, 0.1m) or
methanol containing 0.1% (v/v) dimethyl sulfoxide (DMSO) as a cosol-
vent (for 16–18, 10% DMSO was used) on a Shimadzu UV-1600 UV/Vis
spectrophotometer and Hitachi F4500 fluorescence spectrophotometer.
For determination of the quantum efficiency of fluorescence FF, indocya-
nine green in DMSO (FF =0.13 for IR-780, IR-768, IR-792, IR-786, 1,
and 8),[40] cresyl violet in methanol (FF =0.54 for 2–7, 9, and 10)[41] were
used as standards and the FF value was calculated according to Equa-
tion (1).
Figure 7. A) Absorption and B) emission spectra of 19. All the samples
were measured in sodium phosphate buffer and acetonitrile (v/v=50:50,
100 mm) containing 0.1% DMSO as a cosolvent. Excitation wavelength
was l=640 nm.
cyanine that has a fluorescence controlled by the FRET
mechanism.
2
Fx=Fst ¼ ½Ast=Axꢁ½nx2=nst ꢁ½Dx=Dstꢁ
ð1Þ
Conclusions
where st refers to the standard, x refers to the sample, A is the absorb-
ance at the excitation wavelength, and n is the refractive index.
Synthesis and characterization: D1–D6 were synthesized according to
ref. [39] (details are given in the Supporting Information). The instrumen-
tal data of D1 and D6 are presented in ref. [42].
To develop a design strategy for ratiometric NIR fluorescent
probes, we focused on tricarbocyanine dyes containing a
polymethine linker substituted with various nucleophiles,
specifically amine-substituted dyes. These tricarbocyanine
dyes showed a broader absorption spectrum, a much shorter
absorption maximum, and a much larger Stokes shift than
other cyanines. We prepared a range of aminocyanine deriv-
atives, characterized their spectral properties, and developed
two methods for modulating NIR fluorescence. One method
controlled the absorption wavelength by using the difference
in the electron-donating ability of the amine substituent
before and after a reaction with a biomolecule of interest.
This approach was used in our previous work to develop a
NIR zinc ion probe, and herein we confirmed the utility of
this method by developing a ratiometric NIR esterase
probe. The other method controlled the fluorescence inten-
sity by modulating the FRET efficiency through a change in
the overlap integral based on the change in the absorption
of aminocyanine under acidic conditions. This method was
employed to develop a ratiometric NIR pH probe. Consider-
Synthesis of 1: Aniline (93 mg, 1 mmol) was added by syringe to IR-780
iodide (67 mg, 0.1 mmol) dissolved in anhydrous DMF (10 mL) under
argon. The reaction mixture was stirred at 858C overnight under argon.
The solvent was removed under reduced pressure, the crude product was
purified by column chromatography on silica gel (dichloromethane/meth-
anol 100:0 to 90:10) and treated with 2-propanol to afford a dark green
solid (2 mg, 16%). 1H NMR (300 MHz, CD3OD): d=0.89 (t, 6H, J=
7.3 Hz), 1.24 (s, 12H), 1.66 (br, 4H), 1.84 (br, 2H), 2.52 (br, 4H), 2.74
(br, 2H), 3.76 (br, 4H), 5.69 (br, 2H), 6.69–7.15 (m, 13H), 7.69 ppm (br
2H); 13C NMR (100 MHz, CDCl3): d=11.6, 19.8, 21.9, 25.3, 26.2, 28.4,
44.3, 93.1, 106.7, 119.5, 120.7, 121.6, 127.6, 129.4, 136.3, 139.7, 140.7,
144.0, 146.8, 186.4 ppm; HRMS (ESI+): m/z: calcd for C42H50IN3:
596.4004 [MꢀIꢀ]; found: 596.3963.
Synthesis of 7: Methylamine (30% in methanol; 34 mL, 0.4 mmol) was
added to IR-786 perchlorate (58.3 mg, 0.1 mmol) dissolved in anhydrous
DMF (10 mL). The reaction mixture was stirred at 858C for 3 h under
argon. The solvent was removed under reduced pressure and the crude
product was purified by column chromatography on silica gel with di-
chloromethane to afford a glossy blue solid (54.8 mg, 95%). 1H NMR
(400 MHz, [D6]DMSO): d=1.53 (s, 12H), 1.64–1.67 (m, 2H), 2.45 (t, 4H,
J=6.8 Hz), 3.26–3.30 (m, 9H), 5.62 (d 2H, J=12.7 Hz), 6.96 (t, 2H, J=
9198
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Chem. Eur. J. 2009, 15, 9191 – 9200

The Development of Near-Infrared Ratiometric Fluorescent Probes
FULL PAPER
7.3 Hz), 7.04 (d, 2H, J=7.8 Hz), 7.21 (t, 2H, J=7.8 Hz), 7.36 (d, 2H, J=
7.3 Hz), 7.49 (d, 2H, J=12.7 Hz), 9.20 ppm (d, 1H, J=4.4 Hz); 13C NMR
(100 MHz, [D6]DMSO): d=20.7, 25.6, 28.1, 29.7, 36.7, 46.7, 93.4, 108.5,
119.1, 121.8, 121.9, 127.9, 135.8, 139.5, 143.6, 166.3, 169.5 ppm; HRMS
(ESI+): m/z: calcd for C33H40ClN3O4: 478.3222 [MꢀClO4ꢀ]; found:
478.3241.
brown powder (180 mg, 29%). 1H NMR (300 MHz, CD3OD): d=1.72 (s,
12H), 2.80 (t, 4H, J=7.0 Hz), 4.40 (t, 4H, J=7.0 Hz,), 6.35 (d, 2H, J=
13.1 Hz), 6.64 (t, 1H, J=13.1 Hz), 7.23–7.49 (m, 8H), 8.28 ppm (t, 2H,
J=13.1 Hz); 13C NMR (100 MHz, CD3OD): d=27.9, 32.7, 41.0, 50.6,
104.8, 112.1, 123.4, 126.3, 127.3, 129.7, 142.6, 143.2, 155.9, 173.9,
174.9 ppm; HRMS (ESI+): m/z: calcd for C31H35IN2O4: 499.2597 [MꢀIꢀ];
found: 499.2552.
Synthesis of 8: DIEA (13 mg, 0.1 mmol) was added to
7 (58 mg,
Synthesis of 13: DIEA (13 mg, 0.10 mmol) was added to IR-783 (76 mg,
0.10 mmol) and 3-aminopropionic acid (42 mg, 0.47 mmol) dissolved in
anhydrous DMF (10 mL). The reaction mixture was stirred at 808C for
8 h under argon. The solvent was removed under reduced pressure and
the crude product was purified by reversed-phase ODS chromatography
(H2O/methanol) to afford a glossy blue solid (26 mg, 33%). 1H NMR
(300 MHz, CD3OD): d=1.65 (s, 12H), 1.92 (br, 14H), 2.54 (t, 4H, J=
6.1 Hz), 2.86 (br, 4H), 3.98 (br, 4H), 5.87 (d, 2H, J=13.1 Hz), 7.05–7.35
(m, 8H), 7.75 ppm (d, 2H, J=13.1 Hz); 13C NMR (100 MHz, CD3OD):
d=22.9, 23.7, 25.9, 26.9, 29.1, 32.0, 35.9, 43.8, 47.3, 52.0, 96.2, 110.3, 122.0,
123.0, 124.0, 129.4, 140.5, 141.5, 144.4, 169.5, 170.6, 175.1 ppm; HRMS
(ESI+): m/z: calcd for C41H52N3NaO8S2: 780.3353 [MꢀNa+ +2H+];
found: 780.3371.
0.1 mmol) dissolved in dichloromethane (20 mL). Acetyl chloride (18 mg,
0.23 mmol) in dichloromethane (2 mL) was added dropwise to the solu-
tion on an ice bath. The solution was stirred at 08C to room temperature
for 30 min. Water was added, the solution was extracted with dichloro-
ACHTUNGTRENNUNGmethane, the organic extracts were washed with brine, dried over
Na2SO4, and evaporated to give a glossy green solid. The solid was re-
crystallized from isopropanol/hexane to give a golden powder (58 mg,
1
93%). H NMR (300 MHz, CDCl3): d=1.67 (s, 12H), 1.90–1.95 (m, 2H),
2.63 (t, 4H, J=5.9 Hz), 2.79 (d, 3H, J=6.0 Hz), 3.59 (s, 6H), 6.09 (d, 2H,
J=13.6 Hz), 7.05–7.38 (m, 8H), 7.95 ppm (d, 2H, J=13.6 Hz); 13C NMR
(75 MHz, CDCl3): d=20.8, 25.4, 26.5, 28.0, 31.2, 36.8, 49.0, 101.0, 109.9,
122.2, 124.8, 128.4, 132.4, 135.5, 141.0, 142.8, 152.7, 169.7, 172.1 ppm;
HRMS (ESI+): m/z: calcd for C35H42ClN3O5: 520.3328 [MꢀClO4ꢀ];
found: 520.3290.
Synthesis of 14: The same procedure as for the synthesis of 13 was fol-
1
lowed, except that 4-aminobutyric acid was used (34 mg, 41%). H NMR
Synthesis of 9: IR-786 perchlorate (29 mg, 0.05 mmol) and 3-aminopro-
pionic acid (b-alanine; 30 mg, 0.34 mmol) were dissolved in anhydrous
DMF (10 mL). The reaction mixture was stirred at 85 8C overnight under
argon. The solvent was removed under reduced pressure and the crude
product was purified by column chromatography on silica gel (dichloro-
methane/methanol 100:0 to 85:15) to afford a glossy blue solid (15 mg,
56%). 1H NMR (300 MHz, CD3OD): d=1.54 (s, 12H), 1.71–1.75 (m,
2H), 2.44 (t, 4H, J=6.3 Hz), 2.53 (t, 2H, J=6.0 Hz), 3.33 (s, 6H), 3.85 (t,
2H, J=6.0 Hz), 5.67 (d, 2H, J=13.0 Hz), 6.93–6.99 (m, 4H), 7.16–7.26
(m, 4H), 7.67 ppm (d, 2H, J=13.0 Hz); 13C NMR (75 MHz, CD3OD):
d=22.8, 25.9, 29.1, 30.6, 37.4, 47.9, 48.8, 95.6, 109.7, 121.5, 122.9, 123.9,
129.3, 134.4, 140.0, 141.4, 145.0, 170.0, 170.4 ppm; HRMS (ESI+): m/z:
calcd for C35H41N3O2: 536.3277 [M+H]; found: 536.3303.
(300 MHz, CD3OD): d=1.64 (s, 12H), 1.81–1.91 (m, 12H), 2.05 (t, 2H,
J=6.7 Hz), 2.54 (t, 4H, J=6.1 Hz), 2.86 (br, 4H), 3.82 (t, 2H, J=6.7 Hz),
3.97 (br, 4H), 5.84 (d, 2H, J=13.0 Hz), 7.03–7.34 (m, 8H), 7.74 ppm (d,
2H, J=13.0 Hz); 13C NMR (100 MHz, CD3OD): d=22.9, 23.7, 26.1, 26.7,
27.7, 29.1, 32.2, 40.2, 43.7, 51.1, 52.0, 95.9, 110.2, 121.8, 123.0, 123.9, 129.4,
140.1, 141.3, 144.5, 169.1, 171.0, 176.5 ppm; HRMS (ESI+): m/z: calcd for
C42H54N3NaO8S2: 794.3509 [MꢀNa+ +2H+]; found: 794.3516.
Synthesis of 15: The same procedure as for the synthesis of 13 was fol-
lowed, except that 6-amino-n-caproic acid was used (195 Mg, 77%).
1H NMR (300 MHz, CD3OD): d=1.65 (s, 12H), 1.92 (br, 12H), 2.32 (t,
2H, J=7.2 Hz), 2.55 (t, 4H, J=6.2 Hz), 2.86 (t, 4H, J=6.6 Hz), 3.77 (t,
2H, J=6.8 Hz), 3.97 (br, 4H), 5.85 (d, 2H, J=13.0 Hz, g), 7.04–7.35 (m,
8H), 7.74 ppm (d, 2H, J=13.0 Hz); 13C NMR (100 MHz, CD3OD): d=
22.9, 23.6, 25.3, 26.8, 27.3, 28.1, 29.1, 32.1, 34.5, 40.5, 43.7, 51.4, 52.0, 95.7,
110.1, 121.7, 123.0, 123.8, 129.4, 139.9, 141.1, 144.3, 168.8, 170.8,
177.1 ppm; HRMS (ESI+): m/z: calcd for C44H55N3NaO8S2: 822.3822
[MꢀNa+ +2H+]; found: 822.3813.
Synthesis of 10: IR-786 perchlorate (29 mg, 0.05 mmol), ethyl 3-amino-
propionate (b-alanine ethyl ester; 40 mg, 0.34 mmol), and triethylamine
(34 mg, 0.34 mmol) were dissolved in anhydrous DMF (10 mL). The reac-
tion mixture was stirred at 85 8C for 2 days under argon. The solvent was
removed under reduced pressure, and the crude product was purified by
column chromatography on silica gel (dichloromethane/methanol 100:0
Synthesis of 16: The same procedure as for the synthesis of 15 was fol-
1
1
to 90:10) to afford a glossy blue solid (21 mg, 32%). H NMR (300 MHz,
lowed, except that IR-786 perchlorate was used (414 mg, 84%). H NMR
CD3OD): d=1.16 ( t, 3H, J=7.2 Hz), 1.54 (s, 12H), 1.72–1.76 (m, 2H),
2.45 (t, 4H, J=6.3 Hz), 2.69 (t, 4H, J=6.0 Hz), 3.35 (s, 6H), 3.91 (t, 2H,
J=6.0 Hz), 4.08 (q, 2H, J=7.2 Hz), 5.71 (d, 2H, J=13.0 Hz), 6.97–7.02
(m, 4H, m), 7.19–7.29 (m, 4H), 7.68 ppm (d, 2H, J=13.0 Hz); 13C NMR
(75 MHz, CDCl3): d=14.1, 21.4, 25.1, 25.6, 28.6, 35.1, 46.1, 47.7, 60.9,
94.7, 108.4, 120.5, 122.0, 122.9, 128.1, 138.3, 143.5, 158.5, 168.5,
172.4 ppm; HRMS (ESI+): m/z: calcd for C37H46ClN3O6: 564.3590
[MꢀClO4ꢀ]; found: 564.5393.
(300 MHz, CD3OD): d=1.31–1.73 (m, 20H), 2.09 (t, 2H, J=7.4 Hz), 2.41
(t, 4H, J=6.3 Hz), 3.28 (s, 6H), 3.64 (t, 2H, J=6.8 Hz), 5.63 (d, 2H, J=
13.0 Hz), 6.90–7.24 (m, 8H), 7.63 ppm (d, 2H, J=13.0 Hz); 13C NMR
(100 MHz, CD3OD): d=22.9, 26.0, 26.0, 27.2, 27.9, 29.0, 30.4, 32.2, 38.7,
51.7, 95.4, 109.7, 121.7, 123.0, 123.8, 129.3, 140.0, 141.2, 145.0, 169.7,
171.2, 182.0 ppm; HRMS (ESI+): m/z: calcd for C38H47N3O2: 578.3747
[M+H]; found: 578.3722.
Synthesis of 17: HOBT·H2O (60 mg, 0.39 mmol), HBTU (151 mg,
0.40 mmol), and DIEA (28 mg, 0.22 mmol) were added to 2-[7-(1,3,3-tri-
methyl-3H-indolin-2-ylidene)-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-
Synthesis of 11: This compound was prepared from 2,3,3-trimethyl-3H-
indole (7.17 g, 45 mmol) and 3-iodopropionic acid (12.5 g, 62.5 mmol) in
ortho-dichlorobenzene (70 mL) according to the procedure described in
yl]-1,3,3-trimethyl-3H-indolium[4-
(1-amino)]
hexanoate
(128 mg,
ref. [43].
A
white powder was obtained (10.7 g, 66%). 1H NMR
0.22 mmol) dissolved in anhydrous DMF (10 mL). Cyclohexylamine
(66 mg, 0.67 mmol) was added dropwise to the solution with stirring. The
reaction mixture was stirred at room temperature for 2 h under argon.
The solvent was removed under reduced pressure, and the crude product
was purified by column chromatography on silica gel (dichloromethane/
methanol 10:0 to 90:10) to afford a glossy blue solid (111 mg, 56%).
1H NMR (300 MHz, CD3OD): d=1.10–1.80 (m, 30H), 2.16 (t, 2H, J=
6.8 Hz), 2.51 (t, 4H, J=6.2 Hz), 3.39 (s, 6H), 3.58 (br, 1H), 3.74 (t, 2H,
J=6.8 Hz), 5.73 (d, 2H, J=13.0 Hz), 7.00–7.33 (m, 8H), 7.72 ppm (d,
2H, J=13.0 Hz); 13C NMR (75 MHz, CD3OD): d=22.9, 26.0, 26.2, 26.5,
26.7, 27.4, 29.1, 30.4, 32.1, 33.8, 34.6, 36.9, 51.5, 55.6, 95.5, 109.7, 110.0,
121.6, 123.0, 123.8, 129.4, 139.9, 141.1, 145.0, 169.7, 171.1, 174.7 ppm;
HRMS (ESI+): m/z: calcd for C44H59F6N4OP: 659.4689 [MꢀPF6ꢀ]; found:
659.4689.
(300 MHz, [D6]DMSO): d=1.53 (s, 6H), 2.89 (s, 3H), 2.99 (t, 2H, J=
6.8 Hz), 4.66 (t, 2H, J=6.8 Hz), 7.57–8.01 ppm (m, 4H); 13C NMR
(75 MHz, [D6]DMSO): d=15.0, 21.9, 31.2, 43.8, 54.3, 115.6, 123.6, 128.9,
129.3, 140.7, 141.7, 171.5, 197.8 ppm; HRMS (ESI+): m/z: calcd for
C14H18INO2: 232.1338 [MꢀIꢀ]; found: 232.1306.
Synthesis of 12: A solution of acetic anhydride (115 mg, 1 mmol) in di-
chloromethane (2 mL) was added dropwise to a cooled solution of malo-
naldehyde dianilide hydrochloride (260 mg, 1 mmol) and DIEA (268 mg,
2 mmol) in dichloromethane (4 mL). The reaction mixture was stirred at
08C to room temperature for 1 h. The solution was added dropwise to a
solution of 1-(2-carboxyethyl)-2,3,3-trimethyl-3H-indolium iodide
(815 mg, 2 mmol) and sodium acetate (321 mg, 4 mmol) in acetonitrile
(9.4 mL) and H2O (0.5 mL) at reflux. The mixture was heated to reflux
for a further 3 h. The precipitated crude product was collected by filtra-
tion and washed with CH3CN, 5% HCl, and diethyl ether to give a
Synthesis of 18: The same procedure as for the synthesis of 13 was fol-
lowed, except that trans-1,4-cyclohexanediamine was used (354 mg,
Chem. Eur. J. 2009, 15, 9191 – 9200
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T. Nagano et al.
60%). 1H NMR (300 MHz, CD3OD): d=1.18–1.94 (m, 29H), 2.17 (br,
2H), 2.54 (t, 4H, J=6.3 Hz), 3.43 (s, 6H), 3.61 (br, 1H), 3.76 (t, 2H, J=
7.0 Hz), 5.77 (d, 2H, J=13.0 Hz), 7.04–7.37 (m, 8H), 7.75 ppm (d, 2H,
J=13.0 Hz); 13C NMR (75 MHz, CD3OD): d=22.9, 26.0, 26.6, 27.4, 29.1,
30.6, 32.0, 33.2, 34.2, 36.9, 40.2, 50.6, 51.5, 54.8, 95.5, 109.7, 121.7, 122.9,
123.8, 129.3, 139.9, 141.2, 145.0, 169.8, 171.1, 175.0 ppm; HRMS (ESI+):
m/z: calcd for C44H60F6N5OP: 674.4798 [MꢀPF6ꢀ]; found: 674.4753.
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Synthesis of 19: Compound 18 (153 mg, 0.19 mmol) in anhydrous DMF
(20 mL) was added dropwise to 12 (253 mg, 0.4 mmol), HOBT·H2O
(94 mg, 0.6 mmol), HBTU (235 mg, 0.6 mmol), and DIEA (85 mg,
0.7 mmol) dissolved in anhydrous DMF (20 mL)with stirring. The reac-
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solvent was removed under reduced pressure and the crude product was
purified by column chromatography on silica gel (dichloromethane/meth-
anol 100:0 to 85:15) to afford a golden solid (35 mg, 15%). 1H NMR
(300 MHz, CD3OD): d=0.98–1.94 (m, 40H), 2.03 (t, 2H, J=8.0 Hz),
2.41–2.52 (m, 8H), 3.32 (s, 6H), 3.40 (br, 2H), 3.63 (t, 2H, J=6.8 Hz),
4.25 (br, 4H), 5.65 (d, 2H, J=13.0 Hz), 6.15, (d, 1H, J=13.3 Hz), 6.33
(d, 1H, J=13.3 Hz), 6.51 (t, 1H, J=13.3 Hz), 6.94–7.36 (m, 16H),
8.11 ppm (tt, 2H, J=13.2, 13.2 Hz); 13C NMR (75 MHz, CD3OD): d=
22.9, 25.0, 26.0, 26.6, 27.0, 27.4, 27.8, 28.1, 29.0, 30.5, 30.8, 32.2, 33.2, 35.1,
36.5, 36.8, 40.2, 41.6, 43.0, 48.9, 50.3, 50.8, 51.5, 54.8, 95.5, 104.2, 105.3,
109.8, 111.9, 112.5, 121.6, 123.0, 123.3, 123.9, 125.9, 126.5, 127.1, 129.4,
129.5, 129.8, 140.0, 141.1, 142.4, 142.9, 143.2, 143.4, 145.1, 155.0, 156.0,
169.8, 171.2, 171.6, 173.9, 175.0, 175.4, 177.5 ppm; HRMS (ESI+): m/z:
calcd for C75H92F6N7O4P: 1154.7211 [MꢀPF6ꢀ]; found: 1154.7243.
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Acknowledgements
This work was supported in part by the Ministry of Education, Culture,
Sports, Science and Technology of Japan (Grants for The Advanced and
Innovational Research Program in Life Sciences (grant numbers
16370071 (T.N.) and 17015011 and 17689006 (H.K.)). H.K. was also sup-
ported by the Nissan Science Foundation and K.K. was supported by a
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Received: January 7, 2009
Revised: June 2, 2009
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Published online: July 31, 2009
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