Rational design principle for modulating fluorescence properties of fluorescein-based probes by photoinduced electron transfer

Article abstract of DOI:10.1021/ja035282s

Fluorescence properties of fluorescein-based probes are shown to be finely controlled by the rate of photoinduced electron transfer from the benzoic acid moiety (electron donor) to the singlet excited state of the xanthene moiety (electron acceptor fluoro

Full text of DOI:10.1021/ja035282s

Published on Web 06/19/2003
Rational Design Principle for Modulating Fluorescence
Properties of Fluorescein-Based Probes by Photoinduced
Electron Transfer
Tetsuo Miura,† Yasuteru Urano,† Kumi Tanaka,† Tetsuo Nagano,*,†
Kei Ohkubo,‡ and Shunichi Fukuzumi*,‡
Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo, Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan, and Department of Material and Life Science,
Graduate School of Engineering, Osaka UniVersity, CREST, Japan Science and Technology
Corporation (JST), Yamada-oka, Suita, Osaka 565-0871, Japan
Received March 22, 2003; E-mail: tlong@mol.f.u-tokyo.ac.jp; fukuzumi@ap.chem.eng.osaka-u.ac.jp
Abstract: Fluorescence properties of fluorescein-based probes are shown to be finely controlled by the
rate of photoinduced electron transfer from the benzoic acid moiety (electron donor) to the singlet excited
state of the xanthene moiety (electron acceptor fluorophore). The occurrence of photoinduced electron
transfer is clearly evidenced by transient absorption spectra showing bands due to the radical cation of the
electron donor moiety and the radical anion of the xanthene moiety, observed in laser flash photolysis
experiments. The photoinduced electron transfer rates and the rates of back electron transfer follow the
Marcus parabolic dependence of electron transfer rate on the driving force. Such a dependence provides
for the first time a quantitative basis for a rational design principle which has high efficiency in modulating
fluorescence properties of fluorescein-based probes.
Fluorescence imaging is the most powerful technique cur-
rently available for continuous observation of the dynamic
intracellular processes of living cells. Fluorescein is widely
employed as the core of various fluorescence probes used in
imaging important biological effectors, such as nitric oxide and
calcium.1-5 Despite the extensive use of fluorescein derivatives
and the importance of the applications, the mechanism that
controls the quantum yield of fluorescence has not been fully
established. Without such mechanistic information there can be
no rational design strategy to develop new, practically useful
fluorescence probes. A plausible mechanism that might control
the fluorescence quantum yields of fluorescein derivatives has
been suggested to be photoinduced electron transfer from the
electron donor moiety to the singlet excited state of the xanthene
moiety.6 Since the fluorescence quantum yields in photoinduced
electron transfer reactions may be quantitatively predicted on
the basis of the Marcus theory of electron transfer,7-9 confirma-
tion of the above mechanism would provide a quantitative basis
for rational design of fluorescence probes. Further, the stability
of fluorescence probes could be increased by accelerating the
back electron transfer in the radical ion pair generated by
photoinduced electron transfer. The shorter the lifetime of the
highly reactive intermediate state, such as the radical ion pair,
the less will be the cell damage. However, the occurrence of
photoinduced electron transfer in fluorescein-based probes has
yet to be established.
We report herein the first definitive evidence for the occur-
rence of photoinduced electron transfer in fluorescein-based
probes in which the electron donor moiety is directly linked
with the xanthene moiety. Formation of the radical ion pair upon
photoirradiation of the fluorescein-based probes was detected
by means of laser flash photolysis experiments, which afforded
transient absorption spectra showing bands due to the radical
cation of the electron donor moiety and the xanthene radical
anion. The rates of photoinduced electron transfer and the back
electron transfer were determined and analyzed in terms of the
Marcus theory of electron transfer. The results provide for the
first time a quantitative basis for rational design of fluorescein-
based probes with high efficiency in fluorescence ON/OFF
switching, as well as high stability to repeated photoirradiation.
† The University of Tokyo.
‡ Osaka University.
(1) Minta, A.; Kao, J. P. Y.; Tsien, R. Y. J. Biol. Chem. 1989, 264, 8171-
8178.
(2) Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi,
H.; Hirata, Y.; Nagano, T. Anal. Chem. 1998, 70, 2446-2453.
(3) Umezawa, N.; Tanaka, K.; Urano, Y.; Kikuchi, K.; Higuchi, T.; Nagano,
T. Angew. Chem., Int. Ed. 1999, 38, 2899-2901.
Experimental Section
(4) Walkup, G. K.; Burdette, S. C.; Lippard, S. J.; Tsien, R. Y. J. Am. Chem.
Soc. 2000, 122, 5644-5645.
Materials and General Instrumentation. General chemicals were
of the best grade available, supplied by Tokyo Chemical Industries,
Wako Pure Chemical, or Aldrich Chemical Co., and were used without
further purification. Other chemicals used were dimethyl sulfoxide
(DMSO, fluorometric grade, Dojindo) and tetrabutylammonium per-
chlorate (TBAP, electrochemical grade, dried over P2O5 before use,
(5) Hirano, T.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Nagano, T. J. Am. Chem.
Soc. 2000, 122, 12399-12400.
(6) Tanaka, K.; Miura, T.; Umezawa, N.; Urano, Y.; Kikuchi, K.; Higuchi,
T.; Nagano, T. J. Am. Chem. Soc. 2001, 123, 2530-2536.
(7) Marcus, R. A. Annu. ReV. Phys. Chem. 1964, 15, 155-196.
(8) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265-322.
(9) Marcus, R. A. Angew. Chem., Int. Ed. Engl. 1993, 32, 1111-1121.
9
8666
J. AM. CHEM. SOC. 2003, 125, 8666-8671
10.1021/ja035282s CCC: $25.00 © 2003 American Chemical Society

Rational Design of Fluorescein-Based Probes
A R T I C L E S
Fluka). Acetonitrile, acetone, N,N-dimethylformamide (DMF), tetrahy-
drofuran (THF), methanol, and ethanol were used after appropriate
distillation or purification. NMR spectra were recorded on a JNM-
9-[1-(2-Methoxycarbonyl-4-acetamido)phenyl]-6-methoxy-3H-
xanthen-3-one (4). 1H NMR (300 MHz, CDCl3): δ 2.28 (s, 3H), 3.61
(s, 3H), 3.92 (s, 3H), 6.46 (d, 1H, J ) 1.9 Hz), 6.50 (dd, 1H, J ) 9.7,
2.0 Hz), 6.74 (dd, 1H, J ) 8.8, 2.3 Hz), 6.85 (d, 1H, J ) 9.7 Hz), 6.93
(d, 1H, J ) 9.7 Hz), 6.95 (d, 1H, J ) 2.3 Hz), 8.00 (s, 1H), 8.01 (dd,
1H, J ) 8.8, 2.4 Hz), 8.31 (d, 1H, J ) 2.3 Hz). MS (EI+): m/z 417
(M+). Mp: 233 °C.
1
LA300 (JEOL) instrument at 300 MHz for H NMR and at 75 MHz
for 13C NMR. Mass spectra (MS) were measured with a JMS-DX300
(JEOL) for EI and a SX-102A (JEOL) for FAB. All experiments were
carried out at 298 K, unless otherwise specified.
9-[1-(2-Methoxycarbonyl-4-methoxy)phenyl]-6-methoxy-3H-xan-
then-3-one (5). 1H NMR (300 MHz, CDCl3): δ 3.61 (s, 3H), 3.92 (s,
3H), 3.96 (s, 3H), 6.44 (d, 1H, J ) 1.8 Hz), 6.53 (dd, 1H, J ) 9.7, 1.8
Hz), 6.73 (dd, 1H, J ) 9.0, 2.6 Hz), 6.88-6.95 (m, 3H), 7.18-7.24
(m, 2H), 7.73 (d, 1H, J ) 2.6 Hz). MS (EI+): m/z 390 (M+). Mp: 215
°C.
Preparation of 9-[1-(2-Carboxy-4-hydroxy)phenyl]-6-hydroxy-
3H-xanthen-3-one (4-Hydroxyfluorescein). 4-Aminofluorescein (350
mg) was dissolved in 1.5 mL of sulfuric acid and 4 mL of water. To
this solution was added 240 mg of sodium nitrite. After stirring at 0
°C for 2 h, 15 mg of urea was added. Sulfuric acid (0.5 mL) was diluted
with 10 mL of water, and to this was added dropwise the prepared
diazonium ion solution at 100 °C. The reaction mixture was refluxed
for 2 h, and the resulting solid was collected by filtration. The crude
product was purified by silica gel chromatography to afford 340 mg
9-[2-(3-Methoxycarbonyl)naphthyl]-6-methoxy-3H-xanthen-3-
1
one (6). H NMR (300 MHz, CDCl3): δ 3.63 (s, 3H), 3.90 (s, 3H),
6.26 (d, 1H, J ) 2.0 Hz), 6.35 (dd, 1H, J ) 9.5, 1.9 Hz), 6.82-6.92
(m, 3H), 7.24 (d, 1H, J ) 2.6 Hz), 7.75-7.81 (m, 2H), 8.05-8.11 (m,
2H), 8.26-8.30 (m, 1H), 8.90 (s, 1H). MS (EI+): m/z 410 (M+). Mp:
248 °C.
9-[2-(3-Methoxycarbonyl)anthryl]-6-methoxy-3H-xanthen-3-
one (7). 1H NMR (300 MHz, DMSO-d6): δ 3.66 (s, 3H), 3.91 (s, 3H),
6.26 (d, 1H, J ) 1.8 Hz), 6.37 (dd, 1H, J ) 9.7, 1.8 Hz), 6.86 (dd, 1H,
J ) 8.8, 2.4 Hz), 6.99 (d, 1H, J ) 4.7 Hz), 7.02 (d, 1H, J ) 5.5 Hz),
7.25 (d, 1H, J ) 2.3 Hz), 7.64-7.68 (m, 2H), 8.13-8.23 (m, 3H),
8.73 (s, 1H), 9.01 (s, 1H), 9.09 (s, 1H). MS (EI+): m/z 460 (M+). Mp:
290 °C.
9-[2-(3-Methoxycarbonyl-9,10-diphenyl)anthryl]-6-methoxy-3H-
xanthen-3-one (8). 1H NMR (300 MHz, CDCl3): δ 3.53 (s, 3H), 3.89
(s, 3H), 6.42 (d, 1H, J ) 1.8 Hz), 6.50 (dd, 1H, J ) 9.7, 1.7 Hz), 6.69
(dd, 1H, J ) 9.0, 2.6 Hz), 6.92 (d, 1H, J ) 2.4 Hz), 6.95-7.00 (m,
2H), 7.41-7.82 (m, 15H), 8.66 (s, 1H). MS (EI+): m/z 612 (M+). Mp:
185 °C.
1
of 4-hydroxyfluorescein (yield 97.7%). H NMR (300 MHz, DMSO-
d6): δ 6.54 (dd, 2H, J ) 8.6, 2.2 Hz), 6.57 (d, 2H, J ) 8.5 Hz), 6.65
(d, 2H, J ) 2.1 Hz), 7.03 (d, 1H, J ) 7.9 Hz), 7.16 (dd, 1H, J ) 8.0,
2.4 Hz), 7.18 (d, 1H, J ) 2.4 Hz). MS (EI+): m/z 348 (M+).
Preparation of 9-[1-(2-Carboxy-4-acetamido)phenyl]-6-hydroxy-
3H-xanthen-3-one (4-Acetamidofluorescein). 4-Aminofluorescein (1.0
g) was suspended in 25 mL of acetic anhydride. After addition of 1
mL of pyridine, the reaction mixture was stirred at 140 °C for 1 h,
then cooled to room temperature. The precipitate was filtered off and
dried in vacuo to afford 1.27 g of 3′,6′-bis(acetoxy)-6-acetamidospiro-
[benzo[2,3-c]furan-1(3H),9′-[9H]-xanthen]-3-one. The obtained com-
pound (1.0 g) was resuspended in a mixture of methanol (50 mL), 25%
aqueous ammonium solution (15 mL), and water (10 mL). The whole
was stirred at room temperature for 10 min, then hydrochloric acid
was added. The organic solvent was removed under reduced pressure,
and the precipitate was collected by filtration and dried in vacuo.
1
Yield: 818 mg (quant.). H NMR (300 MHz, DMSO-d6): δ 2.11 (s,
Synthesis of 6-Methoxy-3H-xanthen-3-one (2). To a solution of
230 mg (1.01 mmol) of 3-hydroxy-6-methoxy-9H-xanthene, synthesized
from 2,2′,4,4′-tetrahydroxybenzophenone according to the procedure
reported by Shi et al.,10 in 9 mL of ethanol was added 2,3-dichloro-
5,6-dicyano-1,4-benzoquinone (560 mg, 2.47 mmol) at room temper-
ature. The reaction mixture was stirred for 8 h at room temperature,
then 10 mL of water was added, and the whole was extracted with
ethyl acetate. The organic layer was dried, then evaporated. The crude
product was purified by silica gel chromatography to afford 51 mg of
pure 2 (yield 22.3%). 1H NMR (300 MHz, DMSO-d6): δ 3.90 (s, 3H),
6.15 (s, 1H), 6.50 (dd, 1H, J ) 9.4, 2.4 Hz), 7.02 (dd, 1H, J ) 8.8, 2.5
Hz), 7.15 (d, 1H, J ) 2.4 Hz), 7.55 (d, 1H, J ) 9.5 Hz), 7.74 (d, 1H,
J ) 8.8 Hz), 8.24 (s, 1H). MS (EI+): m/z 226 (M+). Mp: 120 °C.
Syntheses of Benzene Moieties of Methylated Fluorescein De-
rivatives. The benzene moieties of methylated fluorescein derivatives,
i.e., substituted methyl benzoates, were synthesized by refluxing the
corresponding substituted benzoic acids in methanol with several drops
of sulfuric acid. All the benzoic acid derivatives are commercially
available, except 9,10-diphenyl-2-anthracenecarboxylic acid.
2-Methoxycarbonyl-9,10-diphenylanthracene. The carboxylic acid
moiety of anthraquinone-2-carboxylic acid was protected with the
oxazoline group according to the literature.11 After coupling of
2-substituted anthraquinone (303 mg, 1 mmol) with phenylmagnesium
bromide (1.0 mol L-1 THF solution, 5 mL, 5 mmol) via Grignard
reaction, the generated 2-substituted 9,10-dihydro-9,10-dihydroxyan-
thracene (209 mg, 0.45 mmol) was aromatized with an excess amount
of tin(II) chloride (3.0 g, 15.8 mmol) in a mixture of acetic acid (7.5
mL) and hydrochloric acid (3.0 mL). The oxazoline moiety was
removed by hydrolysis under acidic conditions. Then, to a solution of
9,10-diphenyl-2-anthracenecarboxylic acid (40 mg, 0.11 mmol) in
3H), 6.52 (dd, 2H, J ) 8.6, 2.2 Hz), 6.56 (d, 1H, J ) 8.6 Hz), 6.67 (d,
1H, J ) 2.2 Hz), 7.19 (d, 1H, J ) 8.4 Hz), 7.80 (dd, 1H, J ) 8.4, 1.9
Hz), 8.29 (d, 1H, J ) 1.9 Hz). MS (EI+): m/z 389 (M+).
Syntheses of Methylated Fluorescein Derivatives. Fluorescein and
4-aminofluorescein were purchased from Tokyo Chemical Industries.
4-Hydroxyfluorescein and 4-acetamidofluorescein were synthesized
according to the aforementioned schemes. DPAX (8),3 9-[2-(3-carboxy)-
naphthyl]-6-hydroxy-3H-xanthen-3-one (NX, 6), and 9-[2-(3-carboxy)-
anthryl]-6-hydroxy-3H-xanthen-3-one (AX, 7)6 were prepared according
to the literature. All methylated fluorescein derivatives were synthesized
by means of the following procedure: to a suspension of a fluorescein
derivative in methanol was added a few drops of sulfuric acid. The
reaction mixture was refluxed for 12 h, then poured onto ice, and
extracted with CH2Cl2. The organic layer was dried over Na2SO4 and
evaporated. The obtained methyl ester was dissolved in acetone. To
this solution, 2 (or 3 in the case of 4-hydroxyfluorescein) equiv of
iodomethane and cesium carbonate were added. The mixture was stirred
for 5 h at room temperature, water was added, and the whole was
extracted with CH2Cl2. The organic layer was dried and evaporated.
The crude product was purified by silica gel chromatography to afford
the pure methylated fluorescein derivative.
9-[1-(2-Methoxycarbonyl)phenyl]-6-methoxy-3H-xanthen-3-
1
one (1). H NMR (300 MHz, CDCl3): δ 3.57 (s, 3H), 3.90 (s, 3H),
6.23 (d, 1H, J ) 1.8 Hz), 6.38 (dd, 1H, J ) 9.7, 1.8 Hz), 6.78 (d, 1H,
J ) 9.7 Hz), 6.82 (d, 1H, J ) 8.8 Hz), 6.88 (dd, 1H, J ) 8.9, 2.2 Hz),
7.21 (d, 1H, J ) 2.2 Hz), 7.48 (d, 1H, J ) 7.7 Hz), 7.76 (t, 1H, J )
7.7 Hz), 7.86 (t, 1H, J ) 7.7 Hz), 8.20 (d, 1H, J ) 7.7 Hz). MS (EI+):
m/z 360 (M+). Mp: 206 °C.
9-[1-(2-Methoxycarbonyl-4-amino)phenyl]-6-methoxy-3H-xanthen-
1
3-one (3). H NMR (300 MHz, CDCl3): δ 3.57 (s, 3H), 3.91 (s, 3H),
(10) Shi, J. M.; Zhang, X. P.; Neckers, D. C. J. Org. Chem. 1992, 52, 4418-
4.16 (br, 2H), 6.43 (d, 1H, J ) 2.0 Hz), 6.55 (dd, 1H, J ) 9.7, 2.0
Hz), 6.73 (dd, 1H, J ) 9.0, 2.4 Hz), 6.93-7.05 (m, 5H), 7.48 (d, 1H,
J ) 2.4 Hz). MS (EI+): m/z 375 (M+). Mp: 209 °C.
4421.
(11) Meyers, A. I.; Temple, D. L.; Haidukew, D.; Mihelich, E. D. J. Org. Chem.
1974, 39, 2787-2793.
9
J. AM. CHEM. SOC. VOL. 125, NO. 28, 2003 8667

A R T I C L E S
Miura et al.
methanol was added a few drops of sulfuric acid. After refluxing for 6
h, the reaction mixture was poured onto ice. After extraction with CH2-
Cl2, the crude product was purified by silica gel chromatography to
afford 35 mg of pure 2-methoxycarbonyl-9,10-diphenylanthracene (yield
1
82.9%). H NMR (300 MHz, CDCl3): δ 3.79 (s, 3H), 7.46-7.70 (m,
15H), 7.82 (dd, 1H, J ) 9.2, 1.8 Hz), 8.35 (d, 1H, J ) 1.8 Hz). MS
(EI+): m/z 388 (M+). Mp: 123 °C.
Fluorescence Properties and Quantum Efficiency of Fluores-
cence. Steady-state fluorescence spectroscopic studies were performed
on an F4500 (Hitachi). UV-visible spectra were obtained on a UV-
1600 (Shimadzu), with 0.1 mol L-1 sodium phosphate buffer (pH 7.4)
as the solvent. Each solution contained up to 0.2% (v/v) DMSO as a
cosolvent. For determination of the quantum efficiency of fluorescence
(φf), fluorescein in 0.1 mol L-1 NaOH (φf 0.85) was used as a
fluorescence standard.12 The quantum efficiencies of fluorescence were
obtained from multiple measurements (N ) 3) with the following
equation (F denotes fluorescence intensity at each wavelength and ∑[F]
was calculated by summation of fluorescence intensity).
[F(sample)
[F(standard)
]
]
(standard) Abs(standard)
∑
∑
φ
(sample) ) φ
Abs(sample)
Figure 1. Structures of compounds used in this study: (A) methylated
form of fluorescein (1), 6-hydroxy-3H-xanthen-3-one (2), and their benzene
ring-substituted derivatives (3-8). (B) Fluorescein structure was divided
into two parts, the benzene moiety and the xanthene ring.
Fluorescence decay of 2 was recorded with a C4780 system
(Hamamatsu Photonics). The solution of 2 was prepared to be 5.0 ×
10-6 M in 0.1 mol L-1 sodium phosphate buffer (pH 7.4) containing
1.0% DMSO as a cosolvent. It was excited with an N2:Coumarin 307
pulse laser. The obtained data were appropriately deconvoluted and
fitted to monoexponential decay curves to determine the fluorescence
lifetime (τ0) of 2 as 3.6 ns.
Cyclic Voltammetry. Cyclic voltammetry was performed on a 600A
electrochemical analyzer (ALS). A three-electrode arrangement in a
single cell was used for the measurements: a Pt wire as the auxiliary
electrode, a Pt microelectrode (i.d. ) 25 µm) as the working electrode,
and an Ag/Ag+ electrode as the reference electrode. The sample
solutions contained 1.0 × 10-3 M sample and 0.1 M tetrabutylammo-
nium perchlorate (TBAP) as a supporting electrolyte in acetonitrile,
and argon was bubbled for 10 min before each measurement. Obtained
potentials (vs Ag/Ag+) were converted to those vs SCE by adding 0.29
V.
Laser Flash Photolysis. The measurements of transient absorption
spectra of 8 in acetonitrile were performed according to the following
procedures. The deaerated acetonitrile solution of 8 (5.0 × 10-5 M)
was excited by a Nd:YAG laser (GCR-130, Quanta-Ray) at 475 nm
with the power of 4.5 mJ. A pulsed xenon flash lamp (XF80-60, Tokyo
Instruments) was used for the probe beam. The output was recorded
with a digitizing oscilloscope (HP 54510B, 300 MHz).
ESR Measurements. The ESR measurements of photoexcited 8
were carried out with a JES-RE1XE X-band spectrometer (JEOL) to
detect the transient radical species in a solution of 8 (1.0 × 10-3 M) in
frozen acetonitrile at 143 K under irradiation with light from a high-
pressure mercury lamp (USH-1005D, Ushio). A quartz ESR tube
containing a deaerated acetonitrile solution of 8 was irradiated in the
cavity of the ESR spectrometer with the focused light of a 1000 W
high-pressure Hg lamp through an aqueous filter at low temperature.
The ESR spectra in frozen acetonitrile were measured at 143K using
an attached variable-temperature apparatus.
quantum yields.13 Various electron donors are directly linked
with the xanthene moiety. There is little interaction between
the benzoic acid moiety (hereafter called the benzene moiety)
and the xanthene moiety, since they are orthogonal to each
other.14 Therefore, it could be acceptable to regard the benzene
moiety as the electron donor and the xanthene moiety as the
fluorophore (Figure 1B).
A deoxygenated acetonitrile solution containing 8 (Figure 2A)
gives rise, upon a 475 nm laser pulse, to a transient absorption
spectrum with maxima at 380, 460, and 700 nm, as shown in
Figure 2B.
The absorption band at 700 nm is attributable to diphenyl-
anthracene-2-carboxylic acid (DPA) radical cation, since the
3+
one-electron oxidation of DPA by Ru(bpy)3 (bpy ) 2,2′-
bipyridine) yields the radical cation of DPA (DPA•+), which
exhibits the same absorption band at 700 nm (Figure 2B inset).15
The absorption band at 460 nm is assigned to the triplet excited
state of DPA by comparison with that of authentic 3DPA
produced by triplet energy transfer from excited anthracene, as
shown in Figure 2C.16,17 The remaining absorption band at 380
nm is ascribed to the xanthene radical anion (X•-), as shown in
Figure 2D.18 Decay of the absorbances at 380, 460, and 700
nm obeyed first-order kinetics with the same rate constant of
1.7 × 104 s-1, as shown in Figure 2E,F.
The first-order decay kinetics correspond to the back electron
transfer from the X•- to the DPA•+ moiety in the radical ion
(13) Chen, S.; Nakamura, H.; Tamura, Z. Chem. Pharm. Bull. 1979, 27, 475-
479.
(14) Yamaguchi, K.; Tamura, Z.; Maeda, M. Acta Crystallogr. C 1997, 53, 284-
285.
Results and Discussion
(15) Fukuzumi, S.; Nakanishi, I.; Tanaka, K. J. Phys. Chem. A 1999, 103,
11212-11220.
The structures of the fluorescein-based fluorescence probes
employed in this study (1-8) are shown in Figure 1A.
Both the carboxylic acid and the phenolic moiety of the
fluorescein derivatives are methylated to avoid complex pH-
dependent equilibria which would affect the fluorescence
(16) Chattopadhyay, S. K.; Kumar, C. V.; Das, P. K. Chem. Phys. Lett. 1983,
98, 250-254.
(17) 3DPA was generated by photoirradiation of anthracene (5.0 × 10-5 M)
with DPA (5.0 × 10-5 M) in deaerated acetonitrile at 298 K. T-T
absorption spectrum was taken at 50 µs after laser excitation at 355 nm
(18) Compound 2 (50 µM) was photoirraditated in acetonitrile in the presence
of p-phenylenediamine (1.0 mM) to give the xanthene radical anion (2•-).
The transient absorption spectrum was taken at 298 K at 4.0 µs after laser
excitation at 454 nm.
(12) Parker, C. A.; Rees, W. T. Analyst 1960, 85, 587-600.
9
8668 J. AM. CHEM. SOC. VOL. 125, NO. 28, 2003

Rational Design of Fluorescein-Based Probes
A R T I C L E S
Figure 2. (A) Structure of 8. (B) Transient absorption spectra of 8 (50 µM) in deaerated acetonitrile at 298 K taken at 14 µs after laser pulse excitation at
475 nm. The visible spectrum of DPA radical cation observed in the reaction of DPA (1.7 × 10-4 M) with Ru(bpy)3(PF6)3 (1.7 × 10-4 M) in acetonitrile
at 298 K is shown in the inset. (C) T-T absorption spectrum of DPA generated by photoirradiation of anthracene (5.0 × 10-5 M) with DPA (5.0 × 10-5
M) in deaerated acetonitrile at 298 K taken at 50 µs after laser excitation at 355 nm. (D) Transient absorption spectrum observed in the photoreaction of 2
(5.0 × 10-5 M) with p-phenylenediamine (1.0 × 10-3 M) in deaerated acetonitrile at 298 K taken at 4.0 µs after laser excitation at 454 nm. (E, F) Decay
profiles at (E) 460 nm and (F) 700 nm observed in the photoreaction of 8 after laser excitation at 475 nm. First-order plots are also shown as insets. (G) ESR
spectrum measured at 143 K of 8 (1.0 × 10-3 M) under irradiation with light from a high-pressure mercury lamp in frozen acetonitrile.
Table 1. Photochemical Properties of Fluorescein Derivatives
rate constant of electron transfer (kET, s-1
observed
calculated f
)
absorbance
maximum (nm)a
emission
maximum (nm)a,b
relative quantum
efficiency b,c
driving force
of electron transfer (−∆G°ET, eV)
derivative
1
2
3
4
5
6
7
8
454, 478
450, 472
454, 482
456, 480
454, 478
455, 479
454, 483
465, 494
515
500
510
517
516
513
518
516
0.27
0.37
0.008
0.21
0.27
0.26
0.01
0.009
n.d.e
d
d
d
d
d
0.91
0.19
0.19
0.25
0.69
0.75
1.69 × 1010
1.89 × 108
1.03 × 108
1.18 × 108
3.40 × 1010
1.11 × 1010
1.27 × 1010
1.38 × 108
1.38 × 108
3.22 × 108
1.18 × 1010
1.35 × 1010
a Measured in 0.1 M phosphate buffer (pH 7.4). b Excited at longer absorbance maximum. c Calculated by using fluorescein as a fluorescence standard
(φf 0.85). d Not determined. e Not detectable. f Values were generated from a fit to the Marcus equation. The rate constant of back electron transfer in 8 is
also calculated as 1.98 × 104 s-1 (observed rate was 1.70 × 104 s-1).
pair, which is in equilibrium with the triplet excited state of
DPA, to regenerate the original ground state, because none the
derivatives showed any detectable photodegradation, such as
dimerization of the anthracene moiety in the case of 8, under
the conditions used.
two characteristic signals, of which one is attributable to the
radical cation of the diphenylanthracene moiety (g ) 2.0029)
and the other to the radical anion of the fluorophore at a higher
g value (g ) 2.0040), since the authentic DPA•+ and X•- gave
similar ESR signals of g ) 2.0028 and 2.0038, respectively.
The rate constant of forward electron transfer (kET) was
calculated by applying eq 1, where φf and φf ° are the quantum
efficiencies of fluorescence of a fluorescein derivative and 2
used as the reference compound, respectively, and k0 ) 1/τ0,
where τ0 denotes the fluorescence lifetime of 2. The k0 value
was determined as 2.8 × 108 s-1 (τ0 ) 3.6 ns). The kET values
are listed in Table 1.
The rate constants of back electron transfer for other donor-
acceptor dyad systems range from 1.6 × 1010 to 9.1 × 103
s-1 19,20
so the value measured for 8 corresponds to a relatively
,
long-lived radical ion pair.
Formation of such a long-lived radical ion pair state of 8
enabled us to obtain the electron spin resonance (ESR) spectrum
of the radical ion pair under photoirradiation in acetonitrile at
143 K, as shown in Figure 2G. The ESR spectrum consists of
φf°
kET
k0
) 1 +
(1)
φf
(19) Fukuzumi, S.; Guldi, D. M. In Electron Transfer in Chemistry; Balzani,
V., Ed.; Wiley-VCH: Weinheim, 2001; Vol. 2, pp 270-337.
(20) Fukuzumi, S.; Ohkubo, K.; Imahori, H.; Shao, J.; Ou, Z.; Zheng, G.; Chen,
Y.; Pandey, R. K.; Fujitsuka, M.; Ito, O.; Kadish, K. M. J. Am. Chem.
Soc. 2001, 123, 10676-10683.
The driving force of the back electron transfer (-∆G°BET in
eV) can be calculated by means of the following equation and
9
J. AM. CHEM. SOC. VOL. 125, NO. 28, 2003 8669

A R T I C L E S
Miura et al.
Scheme 1
the Rehm-Weller equation (eq 3):
-∆G°BET ) ∆E00 + ∆G°ET
∆G°BET ) e[E°(D+/D) - E°(A/A-)] + wp
(2)
(3)
where e stands for the elementary charge, E°(D+/D) and E°(A/
A-) are the oxidation potential of the electron donor (methyl
benzoate derivative) and the reduction potential of the electron
acceptor (2, -1.28 V vs SCE), respectively, ∆E00 is the singlet
excitation energy of the fluorophore (2, 2.63 eV), and wp is the
electrostatic interaction energy in the radical ion pair, which
corresponds to the work term required to bring the product
radical ions to their mean separation in the radical ion pair for
the intermolecular electron transfer. If wp is neglected, then the
energy calculated for the charge separation state of 8 (2.60 eV)
is significantly higher than the triplet excited state energy of
DPA and X (1.81 and 1.94 eV, respectively).21,22 Since both
the radical ion pair and the triplet excited state of DPA are
observed in the transient absorption spectrum (Figure 2B), these
two states should lie at virtually the same energy levels. For
the contact radical ion pairs, the electrostatic interaction energy
can be evaluated as -e2/4r (r denotes the distance between
electron donor and acceptor), as reported by Suppan.23 The r
value was estimated as 5 Å by geometry optimization of 8 using
DFT calculation. With this value, the wp value of the radical
ion pair derived from 8 is calculated as 0.72 eV. Taking this
into account, the energy level of the radical ion pair is obtained
as 1.88 eV. This energy level is indeed comparable to that of
the triplet excited state of DPA, and the energy diagram of the
relaxation pathway of singlet excited compound 8 can be
schematically drawn as shown in Scheme 1, in which the charge
separation state is located at the lowest energy level, and this
firmly supports the aforementioned assignment of the observed
rate constant to that of the back electron transfer process.
To verify the importance of wp in determining the energy
level of a radical ion pair in close proximity, relative to the
triplet excited state, we synthesized compound 9, a derivative
of 8, which possesses a longer spacer module between DPA
and X (thereby having a larger r value), and examined its decay
pathway. The transient absorption spectrum obtained upon
irradiation of a solution of 9 in acetonitrile is shown in Figure
3B. The absorption band at 460 nm was attributed to that of
the DPA triplet. From the first-order plot of the decay profile
measured at 460 nm, the rate constant of the relaxation from
the triplet was determined to be 8.6 × 103 s-1 (Figure 3C).
Thus, we could observe only the absorption band of the triplet
excited state of DPA in the laser flash photolysis experiment
of 9.
Figure 3. (A) Structure of 9. (B) Transient absorption spectrum of 9 (5.0
× 10-5 M) in acetonitrile at 298 K taken at 20 µs after laser excitation at
454 nm. (C) Decay profile at 460 nm. Inset: first-order plot.
Scheme 2
In this case, wp was calculated from e2/ꢀr, where ꢀ is the
dielectric constant of the solvent and r is the distance between
the donor and acceptor. In compound 9, ꢀ ) 38 and r ) 13 Å,
and wp was obtained as 0.03 eV. From the Rehm-Weller
equation (eq 3), the driving force of the forward electron transfer
reaction in 9 was calculated as ∼0 eV, in contrast with the value
for directly connected 8 (0.75 eV). Thus, the energy level of
the radical ion pair state is much higher than that of the DPA
triplet excited state. This can explain the result of the laser flash
photolysis experiment for 9, in which only the triplet state of
DPA was observed. The energy diagram of the relaxation
pathway of the singlet excited state of 9 can be schematically
drawn as shown in Scheme 2.
Similarly, the driving forces of photoinduced electron transfer
and back electron transfer were obtained from the one-electron
redox potentials of the electron donor and acceptor moieties
together with the excitation energy of the singlet excited state
of fluorescein derivatives and the wp values, as listed in Table
1.
Figure 4 reveals a striking parabolic dependence, that is, an
increase of the rate constant of photoinduced electron transfer
with increasing driving force, but a decrease in the rate constant
of back electron transfer in the highly exergonic region.
(21) Kikuchi, K.; Kokubun, H.; Koizumi, M. Bull. Chem. Soc. Jpn. 1970, 43,
2732-2739.
(22) Shen, T.; Zhao, Z. G.; Yu, Q.; Xu, H. J. J. Photochem. Photobiol. A 1989,
47, 203-212.
(23) Suppan, P. J. Chem. Soc., Faraday Trans. 1 1986, 82, 509-511.
9
8670 J. AM. CHEM. SOC. VOL. 125, NO. 28, 2003

Rational Design of Fluorescein-Based Probes
A R T I C L E S
intervening molecules of the medium, which would increase
the solvent reorganization energy.
Once the Marcus driving force dependence of the rate
constants of electron transfer is established, as shown in Figure
4, not only the fluorescence quantum yields of the fluorescein-
based fluorescence probes but also the stability of the probes
to photoirradiation can be readily predicted from the one-electron
oxidation potential of the electron donor moiety of the donor-
linked fluorescein molecule. The lower the one-electron oxida-
tion potential, the faster the photoinduced electron transfer in
the Marcus normal region in Figure 4, and the lower will be
the fluorescence quantum yield. On the other hand, the back
electron transfer process from the charge separation state in 8
to the ground state is located deeply in the Marcus inverted
region, where the rate constant of electron transfer decreases
with increasing driving force of electron transfer (-∆G°ET). This
caused a long-lifetime charge separation state (58 µs) derived
from 8, despite the very close orientation of the radical pair.
The faster the back electron transfer, the shorter the lifetime of
the reactive radical ion pair, so that less cell damage can be
expected, and the stability of the florescence probe may be
increased.
Figure 4. Parabolic dependence of the rate constant of electron transfer
(kET) (squares) and that of back electron transfer for 8 (circle) on the driving
force of electron transfer (-∆G°ET) as predicted from the Marcus theory.
The values of λ ) 0.81 eV and V ) 7.0 cm-1 are obtained from the best
fit.
To quantify the driving force dependence of the electron
transfer rate constants (kET), the Marcus equation (eq 4) was
employed, where V is the electronic coupling matrix element,
kB is the Boltzmann constant, h is the Planck constant, λ is the
reorganization energy of electron transfer, and T is the absolute
temperature.
Conclusion
In conclusion, the present study has provided for the first
time a quantitative basis for rational design of novel fluorescence
probes based on the fluorescein platform. Once the Marcus
driving force dependence of the rate constants of electron
transfer is established, the fluorescence quantum yields of the
fluorescein derivatives can be readily predicted, enabling us to
attain efficient fluorescence ON/OFF switching with the ap-
propriate reaction site of the probe. Further, the lifetime of the
radical ion pair can also be controlled by regulating the back
electron transfer rate to avoid undesirable effects on the cell
system and instability of the probe.
(∆G°ET + λ)2
1/2
4π3
h2λkBT
kET
)
V2 exp -
(4)
(
)
[
]
4λkBT
By fitting the data in Figure 4 with eq 4, the λ and V values are
obtained as λ ) 0.81 eV and V ) 7.0 cm-1 for these fluorescein
derivatives.
It should be noted here that the V value (7.0 cm-1) of these
fluorescein derivatives, covalently bonded donor-acceptor
systems, is relatively small as compared to those of reported
dyad systems (∼1-100 cm-1) even though the V term increases
exponentially with decreasing distance.19 This is considered to
be a consequence of the characteristic structure of fluorescein
derivatives: the carboxylic group of the benzene moiety prevents
free rotation around the carbon-carbon bond connecting the
benzene ring and the xanthene ring, which are orthogonal to
each other. Thus, the electronic coupling between the electron
donor (benzene ring) and acceptor (xanthene ring) is quite small,
even though they are connected directly. The relative small
reorganization energy (λ ) 0.81 eV) can also be explained by
the structural uniqueness of the fluorescein molecule. The direct
connection of the benzene ring and the xanthene ring excludes
Acknowledgment. This study was supported in part by the
Advanced and Innovational Research Program in Life Sciences
from the Ministry of Education, Culture, Sports, Science and
Technology, the Japanese Government, by Takeda Science
Foundation, and by Nagase Science and Technology Foundation,
by research grants from the Ministry of Education, Science,
Sports and Culture of Japan (Grant Nos. 12771349, 13557209,
and 14030023 to Y.U.), and by a Grant-in-Aid for Scientific
Research Priority Area from the Ministry of Education, Science,
Culture and Sports, Japan (No. 11228205 to S.F.).
JA035282S
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J. AM. CHEM. SOC. VOL. 125, NO. 28, 2003 8671