Development of a ratiometric fluorescent zinc ion probe in near-infrared region, based on tricarbocyanine chromophore

Article abstract of DOI:10.1021/ja060399c

Novel ratiometric fluorescent probes for Zn²⁺ in the near-infrared region, based on a tricarbocyanine chromophore, have been designed, synthesized, and evaluated. Upon addition of Zn²⁺, a 44 nm red shift of the absorption maximum was observed, which indicates that this probe could work as a ratiometric probe for Zn²⁺. This change is due to the difference in the electron-donating ability of the amine substituent before and after reaction with Zn²⁺. This fluorescence modulation of amine-substituted tricarbocyanines should be applicable to dual-wavelength measurement of various biomolecules or enzyme activities. Copyright

Full text of DOI:10.1021/ja060399c

Published on Web 04/29/2006
Development of a Ratiometric Fluorescent Zinc Ion Probe in Near-Infrared
Region, Based on Tricarbocyanine Chromophore
Kazuki Kiyose,†,‡ Hirotatsu Kojima,†,‡ Yasuteru Urano,†,§ and Tetsuo Nagano*,†,‡
Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan, and CREST and PRESTO, JST Agency, Kawaguchi, Saitama, Japan
Received January 18, 2006; E-mail: tlong@mol.f.u-tokyo.ac.jp
Scheme 1
Cyanine dyes have been widely used in various fields and have
been employed as fluorescent labels in fluorescence imaging studies
of biological mechanisms. In particular, tricarbocyanine dyes have
the advantage that light at their emission and absorption maxima
in the near-infrared (NIR) region around 650-900 nm is relatively
poorly absorbed by biomolecules, and so it can penetrate deeply
Table 1. Correlation between the pKa Values of Substituted
Amines12 and Photochemical Properties13
into tissues. There is also less autofluorescence in this region, and
so the characteristics of these dyes are favorable for in vivo imaging.
In addition to the cyanine dyes for straightforward fluorescence
labeling,1-6 two types of tricarbocyanine dyes, whose fluorescence
intensity changes upon specific reaction with biomolecules, have
recently been developed. One is an NIR fluorescent probe for Ca2+
reported by Akkaya et al.7 The other is a class of NIR fluorescent
probes for nitric oxide (NO), DACs, developed by us, which can
visualize NO in isolated rat kidneys.8 Fluorescence modulation of
DACs is controlled by photoinduced electron transfer (PeT), which
causes a change of fluorescence intensity without affecting the
emission and excitation wavelengths. Consequently, although DACs
are useful as NO probes with off/on switching of fluorescence
intensity in the NIR region, the fluorescence of these dyes tends to
be influenced by the dye concentration, as well as by photobleaching
and cellular environmental factors, such as pH and hydrophobicity.
Moreover, the longer the wavelength of excitation, the more difficult
it is to modulate fluorescence through the PeT mechanism because
of the small value of ∆E00 in the Rehm-Weller equation.9 To
overcome these limitations, ratiometric fluorescent probes which
exhibit a change of emission or excitation wavelength in the
presence of biomolecules of interest are required. The utility of
NIR fluorescence ratio imaging was demonstrated by Kircher et
al.10
Peng et al. reported that amine-substituted tricarbocyanines have
shorter wavelength of absorption, larger Stokes’ shift, and stronger
fluorescence intensity than nonsubstituted tricarbocyanines, owing
to intramolecular charge transfer (ICT).11 We synthesized a series
of IR-786 derivatives in order to examine the relationship between
the nature of the amine substituent and the photochemical properties
(Scheme 1 and Table 1). We found that the lower the electron
density of the amine, the longer the wavelength of the absorption
maximum, with little change of the emission maximum. The results
provide a rationale for the molecular design of novel ratiometric
NIR probes, based on the difference in the electron-donating ability
of the amine substituent before and after reaction with a biomolecule
of interest.
R
pKa
absmax (nm)
emmax (nm)
Φfl
hexyl
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
propyl
allyl
methoxyethyl
benzyl
Scheme 2
acts as a metal chelator. When the dipicolylamine coordinates to a
metal ion, the electron density of the DPEN moiety will be
decreased, lowering the electron-donating ability of the amine in
the fluorophore. The IR-783 chromophore (Scheme S1), bearing
highly water-soluble sulfonates, would be suitable as a mother
structure for detection of metal ions in aqueous solution.
Zn2+ has a variety of essential physiological functions. Therefore,
several groups have been making efforts to create rather long-
wavelength fluorescent probes for Zn2+ 14-18
However, no probes
.
for Zn2+ in the NIR region have been developed. As shown in
Figure 1, the reaction of DIPCY with Zn2+ produced a remarkable
spectral change; the color of the solution changed from blue (Figure
1A) to teal (Figure 1B), and subsequent addition of an excess of
N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a strong
chelator of Zn2+, resulted in recovery of the original color (Figure
1C). The formation of a 1:1 complex of DIPCY-Zn2+ was
confirmed by ESI-MS spectrometry, and the absorption and
excitation spectra of DIPCY in the presence of various concentra-
tions of Zn2+ are shown in Figure 2A and 2B, respectively. At a
sufficiently high concentration of Zn2+, a 44 nm red shift of the
absorption maximum was observed, which indicates that DIPCY
Thus, we synthesized dipicolylcyanine (DIPCY, Scheme 2),
having an amine-substituted tricarbocyanine as the NIR fluorophore.
It has a high extinction coefficient of 7.0 × 104 M-1 cm-1 and a
large Stokes’ shift, and the dipicolylethylenediamine (DPEN) moiety
could work as a ratiometric probe for Zn2+
.
The apparent dissociation constant (Kd ) 98 ( 0.9 nM at pH
7.4 in HEPES buffer) for Zn2+ was determined by plotting the 760
nm fluorescence ratio for 627 and 671 nm excitation and was
confirmed by plotting the absorption ratio at 627 and 671 nm
† The University of Tokyo.
‡ CREST, JST Agency.
§ PRESTO, JST Agency.
9
6548
J. AM. CHEM. SOC. 2006, 128, 6548-6549
10.1021/ja060399c CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
fold excess of Co2+, a 39 nm red shift of the absorption maximum
was observed. This property is interesting because Co2+ usually
quenches fluorescence, like other heavy metals with partially filled
d-shells. A 20-fold excess of Cu2+ produced a 62 nm red shift of
the absorption maximum, while the fluorescence was completely
quenched. The shifted absorption also gradually disappeared. Even
if TPEN was added, the absorption did not recover, and the cyanine
chromophore was decomposed. Namely, the Cu2+ complex with
DIPCY is not stable. However, these free cations would have little
influence in vivo since they exist at very low concentrations.22
In summary, we have successfully modified a tricarbocyanine
to be a ratiometric fluorescent Zn2+ probe in the near-infrared
region. This fluorescence modulation of amine-substituted tricar-
bocyanines should be applicable to dual-wavelength measurement
of various biomolecules or enzyme activities. Studies along this
line are in progress.
Figure 1. Color change of DIPCY in the visible region. (A) DIPCY solution
in HEPES buffer: (B) and (A) +Zn2+; (C) and (B) +excess of TPEN.
Acknowledgment. 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, 16370071 to T.N., 17015011 and
17689006 to H.K.). H.K. was also supported by the Nissan Science
Foundation.
Figure 2. (A) Absorption spectra of 1 µM DIPCY at various Zn2+
concentrations (0, 0.01, 0.1, 0.8, 1.0, 2.0, 4.0, 10 µM) {in 100 mM HEPES
buffer, pH ) 7.4, I ) 0.1 (NaNO3)}. (B) Excitation spectra of 1 µM DIPCY
at various Zn2+ concentrations (0, 0.01, 0.1, 0.8, 1.0, 2.0, 4.0, 10 µM) {in
100 mM HEPES buffer, pH ) 7.4, I ) 0.1 (NaNO3)}. The emission was
corrected at 760 nm.
Supporting Information Available: Full experimental procedures,
characterization data for all compounds, and spectral properties of
DIPCY. This material is available free of charge via the Internet at
http://pubs.acs.org.
References
(1) Tung, C. H.; Lin, U.; Moon, W. K.; Weissleder, R. Chembiochem 2002,
8, 784-786.
(2) Bremer, C.; Tung, C. H.; Weissleder, R. Nat. Med. 2001, 7, 743-748.
(3) Tung, C. H.; Bredow, S.; Weissleder, R. Bioconjugate Chem. 1999, 10,
892-896.
(4) Weissleder, R.; Tung, C, H.; Mahmood, U.; Bogdanov, A. Nat. Biotechnol.
1999, 17, 375-378.
(5) Weissleder, R. Nat. Biotechnol. 2001, 19, 316-317.
(6) Zaheer, A.; Lenkinski, R. E.; Mahmood, A.; Jones, A, G.; Cangley, L.
C.; Frangioni, J. V. Nat. Biotechnol. 2001, 19, 1148-1154.
(7) Ozmen, B.; Akkaya, E. U. Tetrahedron Lett. 2000, 41, 9185-9188.
(8) Sasaki, E.; Kojima, H.; Nisimatsu, H.; Urano, Y.; Kikuchi, K.; Hirata,
Y.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 3684-3685.
(9) Urano, Y.; Kamiya, M.; Kanda, K.; Ueno, T.; Hirose, K.; Nagano, T. J.
Am. Chem. Soc. 2005, 127, 4888-4894.
Figure 3. Metal ion selectivity of DIPCY. Bars indicate the fluorescence
ratio (671/627 nm excitation, 760 nm emission). DIPCY (1 µM) was added
to heavy metals (1 µM) and other agents (5 mM). All samples were
measured in 100 mM HEPES buffer, pH ) 7.4, I ) 0.1 (NaNO3). Colorless
bars: each cation was added. Dark bars: each cation and zinc ion were
added.
(10) Kircher, M. F.; Weissleder, R.; Josephson, L. Bioconjugate Chem. 2004,
15, 242-248.
(11) Peng, X.; Song, F.; Lu, E.; Wang, Y.; Zhou, W.; Fan, J.; Gao, Y. J. Am.
Chem. Soc. 2005, 127, 4170-4171.
(12) Dean, J. A. Lange’s Handbook of Chemistry, 14th ed.; McGraw-Hill: New
York, 1992; pp 8.19-8.71
(13) All samples were measured in methanol.
(14) Nolan, E. M.; Burdette, S. C.; Harvey, J. H.; Hilderbrand, S. A.; Lippard,
S. J. Inorg. Chem. 2004, 43, 2624-2635.
(Figure S1). The Kd values of ZnAFs with DPEN moieties, which
were previously reported fluorescent Zn2+ probes,19 are of sub-
nanomolar order. The Kd value of DIPCY is rather high, in
comparison to that of dipicolylamine (around 23 nM).20 This is
presumably due to the steric hindrance of the four methyl groups
of the fluorophore. This Kd value suggests that DIPCY would be
able to detect 10 nM to 1 µM Zn2+, which is a suitable range for
biological applications.21
(15) Gee, R. K.; Zhou, Z.-L.; Qian, W.-J.; Kennedy, R. J. Am. Chem. Soc.
2002, 124, 776-778.
(16) Nolan, E. M.; Jaworski, J.; Okamoto, K.; Hayashi, Y.; Sheng, M.; Lippard,
S. J. J. Am. Chem. Soc. 2005, 127, 16812-16823.
(17) Burdette, S. C.; Lippard, S. J. Inorg. Chem. 2002, 41, 6816-6823.
(18) Chang, C. J.; Jaworski, J.; Nolan, E. M.; Sheng, M.; Lippard, S. J. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 1129-1134.
(19) Hirano, T.; Kikuchi, K.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2002,
124, 6555-6562.
(20) Gruenwedel, D. W. Inorg. Chem. 1968, 7, 495-501.
(21) Komatsu, K.; Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T. J. Am.
Chem. Soc. 2005, 127, 10197-10204.
Dipicolylamine is a chelator of Mn2+, Fe2+, Co2+, Ni2+, Cu2+
,
Ag+, Cd2+, Hg2+, and Pb2+, in addition to Zn2+. The metal
selectivity of DIPCY is shown in Figure 3. We also found a
fluorescence change of DIPCY with Co2+. Upon addition of a 20-
(22) Maruyama, S.; Kikuchi, K.; Hirano, T.; Urano, Y.; Nagano, T. J. Am.
Chem. Soc. 2002, 124, 10650-10651.
JA060399C
9
J. AM. CHEM. SOC. VOL. 128, NO. 20, 2006 6549