Rational design of fluorescein-based fluorescence probes. Mechanism-based design of a maximum fluorescence probe for singlet oxygen

Article abstract of DOI:10.1021/ja0035708

Fluorescein is one of the best available fluorophores for biological applications, but the factors that control its fluorescence properties are not fully established. Thus, we initiated a study aimed at providing a strategy for rational design of functional fluorescence probes bearing fluorescein structure. We have synthesized various kinds of fluorescein derivatives and examined the relationship between their fluorescence properties and the highest occupied molecular orbital (HOMO) levels of their benzoic acid moieties obtained by semiempirical PM3 calculations. It was concluded that the fluorescence properties of fluorescein derivatives are controlled by a photoinduced electron transfer (PET) process from the benzoic acid moiety to the xanthene ring and that the threshold of fluorescence OFF/ON switching lies around -8.9 eV for the HOMO level of the benzoic acid moiety. This information provides the basis for a practical strategy for rational design of functional fluorescence probes to detect certain biomolecules. We used this approach to design and synthesize 9-[2-(3carboxy-9,10-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one (DMAX) as a singlet oxygen probe and confirmed that it is the most sensitive probe currently known for ¹O₂. This novel fluorescence probe has a 9,10-dimethylanthracene moiety as an extremely fast chemical trap of ¹O₂. As was expected from PM3 calculations, DMAX scarcely fluoresces, while DMAX endoperoxide (DMAX-EP) is strongly fluorescent. Further, DMAX reacts with ¹O₂ more rapidly, and its sensitivity is 53-fold higher than that of 9-[2-(3-carboxy-9,10-diphenyl)anthryl]-6-hydroxy-3H-xanthen-3-ones (DPAXs), which are a series of fluorescence probes for singlet oxygen that we recently developed. DMAX should be useful as a fluorescence probe for detecting ¹O₂ in a variety of biological systems.

Full text of DOI:10.1021/ja0035708

2530
J. Am. Chem. Soc. 2001, 123, 2530-2536
Rational Design of Fluorescein-Based Fluorescence Probes.
Mechanism-Based Design of a Maximum Fluorescence Probe for
Singlet Oxygen
Kumi Tanaka, Tetsuo Miura, Naoki Umezawa, Yasuteru Urano, Kazuya Kikuchi,
Tsunehiko Higuchi, and Tetsuo Nagano*
Contribution from the Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
ReceiVed October 2, 2000. ReVised Manuscript ReceiVed January 6, 2001
Abstract: Fluorescein is one of the best available fluorophores for biological applications, but the factors that
control its fluorescence properties are not fully established. Thus, we initiated a study aimed at providing a
strategy for rational design of functional fluorescence probes bearing fluorescein structure. We have synthesized
various kinds of fluorescein derivatives and examined the relationship between their fluorescence properties
and the highest occupied molecular orbital (HOMO) levels of their benzoic acid moieties obtained by
semiempirical PM3 calculations. It was concluded that the fluorescence properties of fluorescein derivatives
are controlled by a photoinduced electron transfer (PET) process from the benzoic acid moiety to the xanthene
ring and that the threshold of fluorescence OFF/ON switching lies around -8.9 eV for the HOMO level of the
benzoic acid moiety. This information provides the basis for a practical strategy for rational design of functional
fluorescence probes to detect certain biomolecules. We used this approach to design and synthesize 9-[2-(3-
carboxy-9,10-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one (DMAX) as a singlet oxygen probe and confirmed
1
that it is the most sensitive probe currently known for O2. This novel fluorescence probe has a 9,10-
dimethylanthracene moiety as an extremely fast chemical trap of 1O2. As was expected from PM3 calculations,
DMAX scarcely fluoresces, while DMAX endoperoxide (DMAX-EP) is strongly fluorescent. Further, DMAX
reacts with 1O2 more rapidly, and its sensitivity is 53-fold higher than that of 9-[2-(3-carboxy-9,10-diphenyl)-
anthryl]-6-hydroxy-3H-xanthen-3-ones (DPAXs), which are a series of fluorescence probes for singlet oxygen
that we recently developed. DMAX should be useful as a fluorescence probe for detecting 1O2 in a variety of
biological systems.
Introduction
density of aminofluorescein and its derivatives is the most
important factor determining the quantum efficiency of fluo-
rescence (φ); that is, aminofluorescein has a low φ value of
0.015, whereas its amide derivatives fluoresce strongly.5 How-
ever, to our knowledge, little more is known about the relation-
ship between the chemical structures of fluorescein derivatives
and their fluorescent properties. Thus, we initiated a study aimed
at providing a strategy for rational design of functional
fluorescence probes based on the fluorescein structure. Further,
using the developed strategy, we designed and synthesized the
Fluorescence probes are excellent sensors for biomolecules,
being sensitive, fast-responding, and capable of affording high
spatial resolution via microscopic imaging.1 Among many
fluorescent compounds, fluorescein is known to have a high
quantum yield of fluorescence (φ) in aqueous solution and to
be excitable at long wavelength,2 and it is the most widely used
fluorophore for labeling and sensing biomolecules.3 Long-
wavelength light does not cause severe cell damage and is free
from interference by autofluorescence of biological molecules.4
Further, excitation around 490 nm is convenient for fluorescence
confocal microscopy using an Ar/ion laser.
1
most sensitive fluorescence probe for O2 so far known.
Results and Discussion
Nevertheless, only limited empirical information is available
about the factors that control the fluorescence properties of
fluorescein. Munkholm et al. reported that the nitrogen electron
Photoinduced Electron Transfer6 as a Mechanism That
Controls the Fluorescence Properties of Fluorescein Deriva-
tives. Recently we reported a series of fluorescence probes for
1O2, 9-[2-(3-carboxy-9,10-diphenyl)anthryl]-6-hydroxy-3H-xan-
then-3-ones (DPAXs).7 DPAXs were the first chemical traps
for 1O2 that permit fluorescence detection. They react with 1O2
to produce DPAX endoperoxides (DPAX-EPs). Although
DPAXs are fluorescein derivatives, DPAXs themselves scarcely
fluoresce, while DPAX-EPs are strongly fluorescent. However,
the mechanisms accounting for the diminution of fluorescence
in DPAXs and its enhancement in DPAX-EPs remain unclear.
Our working hypothesis is that the fluorescence properties of
fluorescein derivatives are controlled by the photoinduced
(1) Bissell, R. A.; de Silva, A. P.; Gunarathe, H. Q. N.; Lynch, P. L.
M.; McCoy, C. P.; Maguire, G. E. M.; Sandanayake, K. R. A. S. In
Fluorescent Chemosensors for Ion and Molecule Recognition; Czarnik, A.
W., Ed.; ACS Symposium Series 538; American Chemical Society:
Washington, DC, 1993; Chapter 9.
(2) Sun, W.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P. J. Org. Chem.
1997, 62, 6469-6475.
(3) (a) Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino,
Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Anal. Chem. 1998, 70, 2446-
2453. (b) Kojima, H.; Urano, Y.; Kikuchi, K.; Higuchi, T.; Nagano, T.
Angew. Chem., Int. Ed. 1999, 38, 2899-2901. (c) Mizukami, S.; Kikuchi,
K.; Higuchi, T.; Urano, Y.; Mashima, T.; Tsuruo, T.; Nagano, T. FEBS
Lett. 1999, 453, 356-360.
(4) Tsien, R. Y.; Waggoner, A. In Handbook of Biological Confocal
Microscopy; Pawley, J. B., Ed.; Plenum Press: New York, 1995; pp 267-
279.
(5) Munkholm, C.; Parkinso, D. R.; Walt, D. R. J. Am. Chem. Soc. 1990,
112, 2608-2612.
10.1021/ja0035708 CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/27/2001

Design of Fluorescein-Based Fluorescence Probes
J. Am. Chem. Soc., Vol. 123, No. 11, 2001 2531
level of the benzoic acid moiety is high enough for electron
transfer to the excited xanthene ring, the φ value will be small.
In other words, fluorescein derivatives with high φ values must
have benzoic moieties with low HOMO energy levels. The
HOMO energy levels of 3-aminobenzoic acid, 3-benzamido-
benzoic acid, 9,10-diphenylanthracene-2-carboxylic acid (DPA-
COOH), and 9,10-diphenylanthracene-9,10-endoperoxide-2-
carboxylic acid (DPA-EP-COOH), which are the benzoic
moieties of aminofluorescein, benzamidofluorescein, DPAX, and
DPAX-EP, respectively, were estimated by semiempirical (PM3)
calculations. DPA-COOH and aminobenzoic acid, which are
the benzoic acid moieties of weakly fluorescent fluorescein
derivatives, have relatively higher HOMO levels than DPA-
EP-COOH and amidobenzoic acid (Table 1). These results were
in accordance with our hypothesis. Further, to confirm this
hypothesis, we synthesized 9-[2-(3-carboxy)naphthyl]-6-hy-
droxy-3H-xanthen-3-one (NX) and 9-[2-(3-carboxy)anthryl]-6-
hydroxy-3H-xanthen-3-one (AX). It is well known that expand-
Figure 1. Frontier orbital energy diagram. (A) Illustration of the
thermodynamic situation of the fluorescence OFF/ON switch including
the PET process. (B) HOMO levels of 6-hydroxy-3H-xanthen-3-one
(fluorophore) and benzoic acid moieties (fluorescence switch) in various
fluorescein derivatives. These values were obtained from semiempirical
PM3 calculations.
ing the size of aromatic conjugates makes their HOMO levels
higher, as does introducing electron-donating substituents. So
we anticipated that naphthoic acid and anthracene-2-carboxylic
acid would have higher HOMO levels than benzoic acid and
that the fluorescence properties of NX and AX would differ
from those of fluorescein.
Their absorbance and fluorescence properties as well as the
HOMO levels of their benzoic acid moieties are summarized
in Table 1. The excitation maximum (Exmax) and emission
maximum (Emmax) were not much altered among these fluo-
rescein derivatives. However, the φ values were greatly altered:
NX is highly fluorescent, whereas AX is almost nonfluorescent.
Thus, a small change in the size of conjugated aromatics, namely
from naphthalene to anthracene, causes a great alteration of
fluorescence properties. When HOMO levels of benzoic acid
moieties were compared, we found that HOMO levels of benzoic
acid and naphthoic acid, which are present in highly fluorescent
fluorescein and NX, are lower than that of the xanthene ring,
while the HOMO level of anthracenecarboxylic acid, which is
present in the scarcely fluorescent AX, is higher than that of
the xanthene ring (Figure 1B). These results are consistent with
the idea that a PET process controls the fluorescence properties
of fluorescein derivatives and that these properties can be
predicted from the HOMO level of the benzoic acid moiety,
with a threshold around -8.9 eV. This, in turn, provides a basis
for developing novel fluorescence probes with fluorescein-
derived structure.
Figure 2. Fluorescein structure divided into two parts, the fluorescence
switch and the fluorophore.
electron transfer (PET) process from the benzoic acid moiety
to the fluorophore, the xanthene ring (Figure 1A). PET is a
widely accepted mechanism for fluorescence quenching, in
which electron transfer from the PET donor to the excited
fluorophore diminishes the fluorescence of the fluorophore. We
consider that it is appropriate to divide the fluorescein structure
into two parts, i.e., the benzoic acid moiety as the PET donor
and the xanthene ring as the fluorophore (Figure 2), because
only small alterations in absorbance were observed among
fluorescein and its derivatives and the dihedral angle between
the benzoic acid moiety and the xanthene ring is almost 90°, as
found by X-ray analysis,8 which suggests that there is little
ground-state interaction between these two parts. Our hypothesis
is that if the highest occupied molecular orbital (HOMO) energy
(6) (a) de Silva, A. P.; Gunarathe, H. Q. N.; Gunnlaugsson, T.; Huxley,
A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997,
97, 1515-1566. (b) Bissell, R. A.; de Silva, A. P.; Gunarathe, H. Q. N.;
Lynch, P. L. M.; McCoy, C. P.; Maguire, G. E. M.; Sandanayake, K. R. A.
S. In Fluorescent Chemosensors for Ion and Molecule Recognition; Czarnik,
A. W., Ed.; ACS Symposium Series 538; American Chemical Society:
Washington DC, 1993; Chapter 4. (c) Bissell, R. A.; de Silva, A. P.;
Gunarathe, H. Q. N.; Lynch, P. L. M.; Maguire, G. E. M.; Sandanayake,
K. R. A. S. Chem. Soc. ReV. 1992, 21, 187-195. (d) Hirano, T.; Kikuchi,
K.; Urano, Y.; Higuchi, T.; Nagano, T. Angew. Chem., Int. Ed. 2000, 39,
1052-1054.
Design of DMAX and Its Fluorescence Properties. Singlet
oxygen (1O2) is one of the active species for oxidation in
biological systems, together with superoxide ion, hydroxyl
radical, hydrogen peroxide, etc. Many researchers have inves-
tigated the reactivity of 1O2 toward organic substrates,9 as well
as its possible role in biological systems.10 However, some
results are still controversial, mainly because of the lack of a
1
reliable detection method. O2 has high reactivity and a short
(7) Umezawa, N.; Tanaka, K.; Urano, Y.; Kikuchi, K.; Higuchi, T.;
Nagano, T. Angew. Chem., Int. Ed. 1999, 38, 2899-2901.
(8) Yamaguchi, K.; Tamura, Z.; Maeda, M. Acta Crystallogr. 1997, C53,
284.
(9) (a)Frimer. A. A., Ed. Singlet Oxygen; CRC Press: Boca Raton, FL,
1997. (b) Prein, M.; Adam, W. Angew. Chem., Int. Ed. 1996, 35, 477-
494.

2532 J. Am. Chem. Soc., Vol. 123, No. 11, 2001
Tanaka et al.
Table 1. Absorbance and Fluorescence Properties of Fluorescein Derivatives, DMAX and DMAX-EP
absorbance
maximum
(nm)
molar absorption
coefficient
emission
maximum
(nm)
calculated HOMO
level of its benzoic
acid moiety (eV)
quantum
efficiency
(×104 M-1 cm-1
)
fluoresceina,d
4-benzamidofluoresceinb
DPAX-1-EPc,d
NXd
492
490
494
492
492
8.0
nde
7.9
6.9
7.1
517
nd
515
512
515
0.85
0.79
0.53
0.83
0.81
-10.1
-9.29
-9.81
-9.14
-9.83
DMAX-EPd
4-aminofluoresceinb
DPAX-1c,d
AXd
490
493
492
492
nd
nd
516
510
517
0.015
0.007
0.003
0.015
-8.90
-8.38
-8.51
-8.26
6.1
6.5
6.0
DMAXd
a References 2 and 4. b Reference 5. Data were measured in 0.1 M phosphate buffer, pH 7.5. c Reference 7. d Data were measured in 0.1 M
NaOH. e nd, not determined.
Scheme 1. Reaction of DMAX with Singlet Oxygen
Scheme 2. Synthetic Scheme of DMAX
lifetime (2 µs in water),11 and the only tools currently available
1
for the real-time bioimaging assay of O2 production in living
cells are DPAXs. Although DPAXs are useful for detection of
1O2, they have some limitations in application to biological
systems, especially with regard to their sensitivity.
We utilized our above-mentioned hypothesis to design a novel
1
fluorescence probe with much higher sensitivity for O2, with
a faster rate of formation of endoperoxide upon reaction with
1O2, namely 9-[2-(3-carboxy-9,10-dimethyl)anthryl]-6-hydroxy-
3H-xanthen-3-one (DMAX). We predicted that DMAX should
react rapidly with 1O2 and that DMAX endoperoxide (DMAX-
EP) should be strongly fluorescent, because dimethylanthracene-
2-carboxylic acid (DMA-COOH) and DMA-COOH endoper-
oxide (DMA-EP-COOH) were indicated to be suitable for OFF/
ON fluorescence switching by semiempirical PM3 calculation
(Table 1, Scheme 1). DMA is known to react very rapidly (k )
1
9.1 × 108 M-1 s-1 in water) with O2 specifically to give the
corresponding 9,10-endoperoxide, DMA-EP.11 This reaction rate
is much faster than that of DPA (for example, 1.0 × 106 M-1
s-1 in benzene; no data available in water),12 so we expected
DMAX would show much greater sensitivity than DPAXs for
1O2. With regard to the reaction rate, the classic singlet oxygen
trap 1,3-diphenylisobenzofuran (DPBF) has a comparable rate
constant (9.6 × 108 M-1 s-1 in water), but DPBF reacts with
other reactive oxygen species, i.e., hydroxyl radical and hypo-
chlorite, to yield the same product. Since we required a specific
probe for singlet oxygen, we chose DMA as the singlet oxygen
trap. Furthermore, the hydrophobicity of DMAX is less than
that of DPAXs, which is important for use in biological samples.
DMAX was prepared according to usual methods with an
excess of resorcinol and 9,10-dimethylanthracene-2,3-dicar-
boxylic anhydride,2,13 as shown in Scheme 2. DMAX-EP was
prepared from DMAX with the Mo6+-H2O2 system.14 Values
of Exmax, Emmax, φ, and ꢀ are summarized in Table 1. Exmax, ꢀ,
and Emmax were similar for DMAX and DMAX-EP, but, as
was expected from the PM3 calculations, DMAX itself is almost
nonfluorescent and DMAX-EP is highly fluorescent. The
quantum yield of DMAX-EP is almost 1.5 times higher than
that of DPAX-1-EP, which means that the sensitivity of DMAX
is greater than that of DPAX-1, apart from any consideration
of reaction rates.
1O2 Detection in Aqueous Media. Next we tried to detect
1O2 in aqueous media under neutral conditions (pH 7.4). 3-(1,4-
Dihydro-1,4-epidioxy-4-methyl-1-naphthyl)propionic acid (EP-
1) was used as a 1O2 source.14,15 EP-1 is a water-soluble
compound, producing 1O2 and 3-(4-methyl-1-naphthyl)propionic
acid (N-1) thermally. Various concentrations of EP-1 were added
to a buffer solution of DMAX or DPAX-1 (10 µM, 0.1%
(10) (a) Brivida, K.; Klotz, L. O.; Sies, H. J. Biol. Chem. 1997, 378,
1259-1265. (b) Scharffetter-Kochanek, K.; Wlaschek, M.; Brivida, K.; Sies,
H. FEBS Lett. 1993, 331, 304-306. (c) Grether-Beck, S.; Olaizola-Horn,
S.; Schmitt, H.; Grewe, M.; Jahnke, A.; Johnson, J. P.; Brivida, K.; Sies,
H.; Krutmann, J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14586-14591.
(11) Usui, Y.; Tsukada, M.; Nakamura, H. Bull. Chem. Soc. Jpn. 1978,
51, 379-384.
(13) Confalone, P. N. J. Heterocycl. Chem. 1997, 27, 31.
(14) Aubry, J. M.; Cazin, B.; Duprat, F. J. Org. Chem. 1989, 54, 726-
728.
(15) (a) Saito, I.; Matsuura, T.; Inoue, K. J. Am. Chem. Soc. 1983, 105,
3200-3206. (b) Saito, I.; Matsuura, T.; Inoue, K. J. Am. Chem. Soc. 1981,
103, 188-190.
(12) Wilkinson, F.; Brummer, J. G. J. Phys. Ref. Data 1981, 10, 809-
999.

Design of Fluorescein-Based Fluorescence Probes
J. Am. Chem. Soc., Vol. 123, No. 11, 2001 2533
Figure 3. Reaction of DMAX and DPAX-1 with singlet oxygen. (A)
Time course of the fluorescence intensity (Ex ) 491 nm, Em ) 520
nm) in the reaction of DMAX (full line) and DPAX-1 (broken line)
1
with O2 generated from EP-1. Various concentrations of EP-1 were
added at the point indicated by the arrow. (B) V0 indicates the initial
rates of the fluorescence increase of DMAX and DPAX-1. The reaction
was performed at 37 °C in 0.1 M sodium phosphate buffer at pH 7.4.
Figure 4. HPLC chromatogram of DMAX with singlet oxygen.
DMAX was exposed to 1O2 generated from the H2O2/MoO42- system.
(A) Before the addition of H2O2, (B) After the addition of H2O2.
Column: Inertsil ODS-3 (4.6 × 250 mm). Eluent: CH3CN/100 mM
sodium phosphate buffer (pH 7.4) ) 2/5; flow rate ) 1.0 mL/min;
detection wavelength ) 492 nm (absorption).
DMSO), and the reactions were performed at 37 °C in 0.1 M
sodium phosphate buffer at pH 7.4. Fluorescence intensity (Ex
) 491 nm, Em ) 520 nm) was monitored as a function of time.
As shown in Figure 3 A, fluorescence intensity was increased
depending on the concentration of EP-1, and DMAX was clearly
more sensitive than DPAX-1. The fluorescence increase paral-
leled DMAX-EP and DPAX-1-EP formation, as confirmed by
HPLC (Figure 4). To measure the rate constants of DMAX-EP
and DPAX-1-EP formation and to elucidate whether DMAX
can detect 1O2 quantitatively or not, we examined the correlation
between the concentration of EP-1 and the initial rate of
fluorescence augmentation. Figure 3B shows a good linear
relationship between these two parameters, which suggests that
1O2 can be detected quantitatively with DMAX. The slopes were
1.0 and 1.9 × 10-2 (arbitrary unit) s-1 [EP-1 (mM)]-1 for
DMAX and DPAX-1, respectively. These results indicate that
DMAX is 53-fold more sensitive than DPAX-1. From the
fluorescence intensity-concentration relationships of DMAX-
Conclusion
We synthesized various kinds of fluorescein derivatives,
including NX and AX, and examined the relationship between
their fluorescence properties and the HOMO levels of their
benzoic acid moieties obtained by semiempirical PM3 calcula-
tions. The results are consistent with our hypothesis that the
fluorescence properties of fluorescein derivatives are determined
by a PET process from the benzoic acid moiety. This provides
a practical strategy for rational design of functional fluorescence
probes to detect certain biomolecules; that is, the threshold level
for the HOMO level of the benzoic acid moiety calculated with
the PM3 method is around -8.9 eV. The validity of our strategy
was confirmed by using it to develop a novel fluorescence probe,
1
1
DMAX, for O2. DMAX reacts with O2 more rapidly, and its
sensitivity is 53-fold higher than that of DPAXs. These results
indicate that DMAX is an excellent fluorescence probe for 1O2,
and we suggest that it should be widely useful in biological
systems. Our strategy should be applicable to develop fluorescein-
derived functional indicators for a wide range of applications.
1
1
EP and DPAX-1-EP, the O2 lifetime, and the O2 formation
rate from EP-1, we determined that the reaction rate constants
1
of DMAX and DPAX-1 with O2 were 2.5 × 107 and 8.1 ×
105 M-1 s-1, respectively. The reaction rate constants of DMAX
and DPAX-1 are somewhat smaller than those of DMA and
DPA. This might be due to the effect of the electron-
withdrawing carboxylic acid attached to the anthracene ring.
However, DMAX has a 31-fold faster rate constant and is 53-
fold more sensitive than DPAX-1. To our knowledge, there are
no chemical traps for 1O2 which have a faster rate constant and
better selectivity for 1O2 than DMA. In addition, the fluorescence
intensity of DMAX did not change upon reaction with 1.0 mM
H2O2, 0.1 mM nitric oxide, and 0.2 mM superoxide, confirming
Experimental Section
Materials. o-Xylene, 2,3-naphthalenecarboxylic anhydride, and
methanesulfonic acid were purchased from Tokyo Chemical Industries
Co., Ltd. 1,2-Dibromobenzene and phthalic anhydride were purchased
from Wako Pure Chemical Industries Ltd. Methylmagnesium chloride
was purchased from Aldrich Chemical Co. Inc. Except for o-xylene,
all of them were used without further purification. Benzene, o-xylene,
methanol, THF, and DMF were used after distillation. Other materials
were of the best grade available and were used without further
purification.
1
the specificity of DMAX for O2. We consider that DMAX is
Instruments. NMR spectra were recorded on a JEOL JNM-LA300
the best fluorescence reagent for 1O2 detection currently
available.
1
instrument at 300 MHz for H NMR, and at 75 MHz for 13C NMR.
Mass spectra (MS) were measured with a JEOL JMS-DX300 for EI

2534 J. Am. Chem. Soc., Vol. 123, No. 11, 2001
Tanaka et al.
and a JEOL SX-102A for FAB. UV-visible spectra were obtained on
a Shimadzu UV-1600. Fluorescence spectroscopic studies were per-
formed on a Hitachi F4500. Determinations of reaction rate constants
were performed on a Perkin-Elmer LS-50B.
concentrated sulfuric acid (20 mL). The solution was warmed gradually
to 125 °C over 1 h, kept at this temperature for 30 min, and then poured
onto ice. The precipitated light gray powder was collected by filtration,
washed with water, dried, and purified by silica gel chromatography
(CH2Cl2/ n-hexane, 2:1) to afford a light yellow powder (1.38 g, yield
Preparation of 2-(3′,4′-Dimethylbenzoyl)benzoic Acid. Alumi-
num(III) chloride (15 g) was added to a suspension of phthalic
anhydride (7.5 g, 50.6 mmol) in o-xylene (60 mL). The mixture was
refluxed for 10 min, and 50 g of ice was added. The resulting mixture
was treated with concentrated HCl, taken up in ether, and extracted
with 2 M NaOH. The aqueous solution was washed with ether and
acidified to pH ∼2.5 with cold 6 M HCl. The resulting mixture was
extracted with ether. The organic layer was washed with brine, dried
over MgSO4, filtered, and evaporated to afford a light yellow powder
(4.78 g, yield 37%). 1H NMR (DMSO-d6): δ 2.23 (s, 3H), 7.22-7.30
(m, 2H), 7.35 (d, 1H, J ) 7.3 Hz), 7.46 (s, 1H), 7.72-7.30 (m, 2H),
7.96 (d, 1H, J ) 7.3 Hz). MS (EI+): m/z 254 (M+).
1
25%). H NMR (CDCl3): δ 7.83-7.86 (m, 2H), 8.30-8.33 (m, 2H),
8.53 (s, 2H). MS (EI+): m/z 364:366:368 ) 1:2:1 (M+). Mp: >300
°C.
Synthesis of 2,3-Dibromo-9,10-dihydroxy-9,10-dimethyl-9,10-
dihydroanthracene (4). Methylmagnesium chloride (3 M in THF, 4.5
mL) was gradually added to a solution of 3 (1.22 g, 3.33 mmol) in dry
THF (150 mL). The mixture was refluxed for 4 h under an Ar
atmosphere. Aqueous ammonium chloride was added, and the whole
was evaporated to a small volume. The resulting mixture was extracted
with CH2Cl2. The organic layer was washed with brine, dried over
Na2SO4, filtered and evaporated. The crude compound was purified
by silica gel chromatography (CH2Cl2) to afford a light yellow powder
Preparation of 2,3-Dimethylanthraquinone.16 2-(3′,4′-Dimethyl-
benzoyl)benzoic acid (4.7 g, 18.5 mmol) dissolved in 25.5 mL of
concentrated sulfuric acid was warmed gradually to 125 °C over 1 h,
kept at this temperature for 30 min, and then poured onto ice. The
precipitated light gray powder was collected by filtration, washed with
water, dried, and purified by silica gel chromatography (CH2Cl2/n-
hexane, 1:1), to afford a light yellow powder containing a small amount
of 1,2-dimethylanthraquinone. Isomerically pure 2,3-dimethylanthra-
quinone was obtained by recrystallization. A solution containing boiling
acetic acid (12 mL per gram of the isomeric mixture) was filtered, and
then the filtrate was allowed to cool and kept cool for 1 h. The resultant
crystals were collected, washed with acetic acid, and dried in air oven
to afford yellow needles (2.0 g, yield 46%). 1H NMR (CDCl3): δ 2.25
(s, 6H), 7.76-7.79 (m, 2H), 8.06 (s, 2H), 8.28-8.31 (m, 2H). MS (EI+):
m/z 236 (M+). Mp: 185 °C.
Preparation of Anthraquinone-2,3-dicarboxylic Acid. 2,3-Di-
methylanthraquinone (3.3 g, 14.0 mmol) was added to a stirred solution
of 8.10 g of Na2Cr2O7 in 43 mL of water buffered with 5.33 g of NaH2-
PO4‚2H2O. The suspension thus obtained was quickly introduced into
a stainless steel autoclave. The vessel was then heated to 250 °C for
20 h under stirring. After cooling to room temperature, the reaction
mixture was treated with excess sodium sulfite and extracted with 2
M NaOH, and the aqueous layer was acidified to pH ∼2 with cold 6
M HCl. The precipitate was collected by filtration, dried, purified by
silica gel chromatography (CH2Cl2/methanol, 9:1), and recrystallized
from acetic acid to afford light yellow crystals (392 mg, yield 9.5%).
1H NMR (DMSO-d6): δ 7.97-8.00 (m, 2H), 8.31-8.34 (m, 2H), 8.58
(s, 2H). MS (EI+): m/z 278 (M+ - H2O).
1
(995 mg, yield 75%). H NMR (CDCl3): cis, δ 1.63 (s, 6H), 7.41-
7.45 (m, 2H), 7.79-7.83 (m, 2H), 8.11 (s, 2H); trans, δ 1.86 (s, 6H),
7.41-7.45 (m, 2H), 7.79-7.83 (m, 2H), 8.01 (s, 2H). MS (EI+): m/z
396:398:400 ) 1:2:1 (M+). Mp: 125 °C.
Synthesis of 2,3-Dibromo-9,10-dimethylanthracene (5).17 A mix-
ture of 4 (1.08 g, 2.71 mmol) in 28 mL of acetic acid, 12.8 g of
stannous(II) chloride dihydrate, and 12 mL of concentrated hydrochloric
acid was refluxed for 1 h under an Ar atmosphere. The cooled solution
was poured into 500 mL of water, and the solid product was collected.
The resulting solid was purified by silica gel chromatography (CH2Cl2/
hexane, 1:2) to afford a yellow powder (744 mg, yield 78%). 1H NMR
(CDCl3): δ 3.04 (s, 6H), 7.54-7.57 (m, 2H), 8.28-8.31 (m, 2H), 8.62
(s, 2H). MS (EI+): m/z 362:364:366 ) 1:2:1 (M+). Mp: 151 °C.
Synthesis of 9,10-Dimethylanthracene-2,3-dicarbonitrile (6). A
stirred mixture of 5 (730 mg 2.00 mmol) and cuprous(I) cyanide (694
mg 7.74 mmol) in dimethylformamide (45 mL) was refluxed for 9 h
under an Ar atmosphere. The reaction mixture was cooled to room
temperature, and 14% aqueous ammonium solution (50 mL) was added
to it. The precipitate was collected, washed with water, and dried. The
crude product was purified by silica gel chromatography (CH2Cl2/
hexane, 2:5) to afford a yellow powder (255 mg, yield 50%). 1H NMR
(CDCl3): δ 3.14 (s, 6H), 7.71-7.76 (m, 2H), 8.39-8.43 (m, 2H), 8.86
(s, 2H). MS (EI+): 256 (M+). Mp: 266 °C dec.
Synthesis of 9,10-Dimethyl-2,3-anthracenedicarboxylic Acid (7).
A solution of 6 (330 mg, 1.29 mmol) in 50 mL of n-butanolic potassium
hydroxide (3 M) was refluxed for 10 h under an Ar atmosphere. The
solution was acidified with concentrated HCl and extracted with ether.
The organic layer was washed with brine, dried over Na2SO4, filtered,
and evaporated. The resulting solid was purified by silica gel chroma-
tography (CH2Cl2/ MeOH, 20:1) to afford a yellow powder (268 mg,
yield 71%). 1H NMR (DMSO-d6): δ 3.08 (s, 6H), 7.64-7.68 (m, 2H),
8.42-8.45 (m, 2H), 8.66 (s, 2H). MS (EI+): m/z 294 (M+). Mp: >300
°C.
Synthesis of 9,10-Dimethyl-2,3-anthracenedicarboxylic Anhy-
dride (8). A suspension of 7 (645 mg, 2.19 mmol) in acetic anhydride
(110 mL) was refluxed for 10 min. The clear solution was cooled to 4
°C to afford 8 as a red precipitate (367 mg, yield 61%). 1H NMR
(CDCl3): δ 3.23 (s 6H), 7.72-7.75 (m, 2H), 8.42-8.46 (m, 2H), 9.12
(s, 2H). MS (EI+): m/z 276 (M+). Mp: 278 °C dec.
Synthesis of 5,10-Dimethyl-3′,6′-bis(acetyloxy)spiro[anthra[2,3-
c]furan-1(3H),9′-[9H]-xanthen]-3-one (DMAX-diAc), 3′,6′-Bis(acetyl-
oxy)spiro[naphtho[2,3-c]furan-1(3H),9′-[9H]-xanthen]-3-one (NX-
diAc), and 3′,6′-Bis(acetyloxy)spiro[anthra[2,3-c]furan-1(3H),9′-
[9H]-xanthen]-3-one (AX-diAc). Compound 8 (367 mg, 1.33 mmol)
was added to a solution of resorcinol (681 mg, 6.18 mmol) in
methanesulfonic acid (6.6 mL). The resulting suspension was heated
under Ar at 85 °C for 24 h. The cooled mixture was poured into 7
volumes of ice water, followed by filtration. The filtrate was dried at
60 °C in vacuo, and the residue was diacetylated. The product, acetic
anhydride (8 mL), and pyridine (4 mL) were stirred for 10 min at room
temperature under an Ar atmosphere. The resulting mixture was poured
Preparation of 2,3-Anthracenedicarboxylic Acid. Anthraquinone-
2,3-dicarboxylic acid (392 mg, 1.31 mmol) was mixed with a large
excess of zinc dust (2.5 g), dissolved in 14% ammonia solution (40
mL), and refluxed for 2 h. 2,3-Anthracenedicarboxylic acid was
precipitated from the hot filtered solution by the addition of hydrochloric
1
acid (335 mg, yield 95%). H NMR (DMSO-d6): δ 7.58-7.61 (m,
2H), 8.13-8.16 (m, 2H), 8.47 (s, 2H), 8.77 (s, 2H). MS (FAB+): m/z
267 (M+ + 1).
Preparation of 2,3-Anthracenedicarboxylic Anhydride. A suspen-
sion of 2,3-anthracenedicarboxylic acid (32 mg, 0.12 mmol) in acetyl
chloride (1.2 mL) was refluxed for 1 h. The resulting clear solution
was cooled to 4 °C to afford 2,3-anthracenedicarboxylic anhydride as
1
a yellow precipitate (21 mg, yield 70%). H NMR (CDCl3): δ 7.72-
7.75 (m, 2H), 8.23-8.26 (m, 2H), 8.77 (s, 2H), 8.80 (s, 2H). MS
(FAB+): m/z 249 (M+ + 1). Mp: >300 °C.
Preparation of 2,3-Dibromoanthraquinone (3). Aluminum(III)
chloride (4.4 g, 33.0 mmol) was added to a suspension of phthalic
anhydride 2 (2.24 g, 15.1 mmol) in 2,3-dibromobenzene 1 (6 mL).
The resulting mixture was heated at 150 °C for 1 h and then treated
with 2 M HCl and extracted with benzene. The benzene layer was
extracted with 2 M NaOH. The aqueous layer was acidified to pH ∼2.5
with cold 6 M HCl. The resulting mixture was extracted with ether.
The organic layer was washed with brine, dried over MgSO4, filtered,
and evaporated to afford a crude product. This was dissolved in
(16) (a) Fairbourne, A. J. Chem. Soc. 1921, 119, 1573-1582. (b) Elbs,
K.; Eurich, H. Ber. Dtsch. Chem. Ges. 1887, 20, 1361-1363.
(17) Rimmer, R. W.; Christiansen, R. G.; Brown, R. K.; Sandin, R. B.
J. Am. Chem. Soc. 1950, 72, 2298-2299.

Design of Fluorescein-Based Fluorescence Probes
J. Am. Chem. Soc., Vol. 123, No. 11, 2001 2535
into 2% HCl at 0 °C and extracted with CH2Cl2. The organic layer
was washed with brine, dried over Na2SO4, filtered, and evaporated.
The residue was recrystallized from benzene to afford DMAX-diAc
EP), 9-[2-(3-Carboxy)naphthyl]-6-hydroxy-3H-xanthen-3-one (NX),
and 9-[2-(3-Carboxy)anthryl]-6-hydroxy-3H-xanthen-3-one (AX).
DMAX-diAc (30 mg, 65.1 µmol) was dissolved in THF (5 mL) and
MeOH (5 mL), and this solution was stirred with 15% aqueous
ammonia (1.4 mL) for 5 min in the dark. The reaction mixture was
poured into 60 mL of cold water, and the whole was acidified to pH
2 with 10% HCl and then evaporated to a small volume to remove
THF/MeOH. The resulting mixture was extracted with ether. The
organic layer was washed with brine, dried over MgSO4, filtered, and
1
(294 mg, yield 48%). H NMR (CDCl3): δ 2.31 (s, 6H, acetyl), 2.99
(s, 3H, CH3), 3.29 (s, 3H, CH3), 6.79 (dd, 2H, J ) 8.7, 2.2 Hz, H2′,
H7′), 6.92 (d, 2H, J ) 8.7 Hz, H1′, H8′), 7.14 (d, 2H, J ) 2.2 Hz, H4′,
H5′), 7.59-7.62 (m, 2H, H7, H8), 8.11 (s, 1H, H11), 8.30-8.42 (m, 2H,
H6, H9), 9.20 (s, 1H, H4). 13C NMR (CDCl3): δ 14.80 (CH3), 14.85
(CH3), 21.1 (CH3(Ac)), 82.3 (C1), 110.3 (C4′, C5′), 117.8 (C2′, C7′), 117.9
(C8a′, C9a′), 121.2 (C11), 123.1 (C3a), 125.45 (C4), 125.48 (C6/C9), 125.7
(C6/C9), 126.0 (C7/C8), 126.8 (C7/C8), 129.6 (C1′, C8′), 130.0 (C5/C10),
1
evaporated to yield DMAX (18 mg, yield 60%). H NMR (DMSO-
d6): δ 3.15 (s,3H), 3.17 (s, 3H), 6.49-6.52 (m, 2H), 6.65-6.69 (m,
4H), 7.63-7.67 (m, 2H), 8.14 (s, 1H), 8.35-8.50 (m, 2H), 9.09 (s,
1H). MS (FAB+): m/z 461 (M+ + 1). Mp: 236 °C dec.
DMAX-EP, NX, and AX were similarly prepared from DMAX-
EP-diAc, NX-diAc, and AX-diAc in 100, 100, and 83% yield,
respectively.
130.6, 131.9, 132.0, 132.5 (C4a, C5a, C9a, C10a), 144.4 (C11a), 151.6 (C4a′
,
C
10a′/C3′, C6′), 152.0 (C4a′, C10a′/C3′, C6′), 168.9 (CO(Ac)), 169.1 (C3).
MS (FAB+): m/z 461 (M+ + 1). Mp: 280 °C dec. Anal. Calcd for
C35H28O7: C, 74.99; H, 4.44; N, 0.00. Found: C, 74.82; H, 4.72; N,
0.00.
1
DMAX-EP. H NMR (DMSO-d6): δ 3.15 (s, 3H), 3.16 (s, 3H),
NX-diAc and AX-diAc were similarly prepared from 2,3-naphtha-
lenedicarboxylic anhydride and 2,3-anthracenedicarboxylic anhydride
in 32% and 25% yield, respectively.
6.42 (d, 2H, J ) 1.1 Hz), 6.60-6.68 (m, 4H), 7.32 (s, 3H), 7.37-7.56
(m, 4H), 8.02 (s, 1H). MS (FAB+): m/z 493 (M+ + 1). Mp: 172 °C
dec.
1
NX-diAc. H NMR (CDCl3): δ 2.31 (s, 6H, acetyl), 6.78 (dd, 2H,
1
NX. H NMR (DMSO-d6): δ 6.51 (dd, 2H, J ) 2.3, 8.6 Hz), 6.60
J ) 8.6, 2.2 Hz, H2′, H7′), 6.86 (d, 2H, J ) 8.6 Hz, H1′, H8′), 7.12 (d,
2H, J ) 2.2 Hz, H4′, H5′), 7.62-7.66 (m, 3H, H6, H7, H9), 7.82-7.85
(m, 1H, H5/H8), 8.12-8.14 (m, 1H, H5/H8), 8.61 (s, 1H, H4). 13C NMR
(d, 2H, J ) 8.6 Hz), 6.68 (d, 2H, J ) 2.2 Hz), 7.67-7.70 (m, 2H),
7.83 (s, 1H), 8.00-8.31 (m, 2H), 8.71 (s, 1H). MS (FAB+): m/z 383
(M+ + 1). Mp: 195 °C dec.
(CDCl3): δ 21.1 (CH3(Ac)), 81.9 (C1), 110.3 (C4′, C5′), 117.4 (C8a′
,
AX. 1H NMR (DMSO-d6): δ 6.52 (dd, 2H, J ) 2.4, 8.6 Hz), 6.66-
6.70 (m, 4H), 7.59-762 (m, 2H), 7.97 (s, 1H), 8.04-8.20 (m, 2H),
C9a′), 117.7 (C2′, C7′), 123.6 (C9), 124.0 (C3a), 126.7 (C4), 127.6 (C6/
C7), 128.7 (C5/C8), 129.3 (C1′, C8′), 129.4 (C6/C7), 130.0 (C5/C8), 133.5
(C4a/C8a), 136.8 (C4a/C8a), 147.3 (C9a), 151.5 (C4a′, C10a′/C3′, C6′), 152.0
8.67 (s, 1H) 8.93 (s, 1H) 9.03 (s, 1H). MS (FAB+): m/z 433 (M+
1). Mp: 195 °C dec.
+
(C4a′, C10a′/C3′, C6′), 168.0 (C3, CO(Ac)). MS (FAB+): m/z 467 (M+
1). Mp: 241 °C dec. Anal. Calcd for C28H18O7: C, 72.10; H, 3.89; N,
+
Computational Methods. Semiempirical PM3 calculations were
carried out using Spartan (version 5.0) on SGIO2. Several starting
geometries were used for the geometry optimization to ensure that the
optimized structure corresponds to a global minimum.
Fluorometric Analysis. The slit width was 2.5 nm for both excitation
and emission. The photon multiplier voltage was 950 V. Relative
quantum efficiencies of fluorescence of fluorescein derivatives were
obtained by comparing the area under the corrected spectrum of the
test sample excited at 492 nm in 0.1 M NaOH with that of a solution
of fluorescein, which has a quantum efficiency of 0.85 according to
the literature.18
In the experiment to measure the reaction rate constant, the slit width
was 7.5 nm for excitation and 3.5 nm for emission, and the photon
multiplier voltage was 750 V. DMAX and DPAX-1 were dissolved in
DMSO to obtain a 10 mM stock solution. A 3 µL aliquot of the stock
solution was added to 3 mL of sodium phosphate buffer (100 mM, pH
7.4) with stirring. After 5 min, 30 µL of DMSO solution in which
EP-1 was dissolved was added.
HPLC Analysis. HPLC analyses were performed on an Inertsil
ODS-3 (4.6 × 250 mm) column using an HPLC system composed of
a Jasco 880-PU pump and a Jasco 870-UV detector.
0.00. Found: C, 71.80; H, 3.89; N, 0.00.
1
AX-diAc. H NMR (CDCl3): δ 2.31 (s, 6H, acetyl), 6.80 (dd, 2H,
J ) 8.7, 2.2 Hz, H2′, H7′), 6.94 (d, 2H, J ) 8.7 Hz, H1′, H8′), 7.14 (d,
2H, J ) 2.2 Hz, H4′, H5′), 7.55-7.58 (m, 2H, H7, H8), 7.80 (s, 1H,
H11), 7.98-8.08 (m, 2H, H6, H9), 8.34 (s, 1H, H4/H5/H10) 8.42 (s, 1H,
H4/H5/H10) 8.76 (s, 1H, H4/H5/H10). 13C NMR (CDCl3): δ 21.1
(CH3(Ac)), 82.0 (C1), 110.3 (C4′, C5′), 117.76 (C2′, C7′), 117.83 (C8a′
,
C9a′), 123.8 (C3a), 123.9 (C11), 126.7 (C7/C8), 127.4 (C7/C8), 127.5 (C4/
C5/C10), 128.1 (C4/C5/C10), 128.2 (C6/C9), 128.5 (C6/C9), 129.4 (C1′,C8′),
129.6 (C4/C5/C10), 130.9, 132.4, 133.3, 133.6 (C4a, C5a, C9a, C10a), 145.4
(C11a), 151.5 (C4a′, C10a′/C3′, C6′), 152.0 (C4a′, C10a′/C3′, C6′), 168.7 (C3),
168.9 (CO(Ac)). MS (FAB+): m/z 517 (M+ + 1).
Synthesis of DMAX-9,10-endoperoxide-diAc (DMAX-EP-diAc).
A solution of DMAX (104 mg, 0.23 mmol) in DMSO (20 mL) was
added to an aqueous solution (180 mL) of NaOH (12.5 mM), NaHCO3
(6.5 mM), Na2CO3 (12.5 mM), and Na2MoO4‚2H2O (55.2 mM).
Throughout the reaction, the temperature was maintained at about 20
°C. Next, 30% H2O2 (5 mL) was added to the solution, and the mixture
was stirred for 15 min. Another 5 mL of H2O2 was allowed to react in
the same way, and the reaction was monitored by HPLC to check the
complete conversion of DMAX to DMAX-EP. The mixture was cooled
to 0 °C and was acidified to pH ∼2.5 with cold 2 M phosphoric acid.
The resulting mixture was extracted with ether. The organic layer was
washed with brine, dried over MgSO4, filtered, and evaporated. The
residue was then diacetylated by the same method as in the case of
DMAX-diAc. After evaporation, the crude compound was purified by
silica gel chromatography, eluted with CH2Cl2, to give DMAX-EP-
Determination of Reaction Rate Constant. The reaction scheme
for reaction of an acceptor (A), DMAX or DPAX-1, with 1O2 in solution
is expressed as follows:
1
diAc (59 mg, yield 45%). H NMR (CDCl3): δ 2.06 (s, 3H, CH3),
2.26 (s, 3H, CH3), 2.28 (s, 3H, acetyl), 2.33 (s, 3H, acetyl), 6.67-6.74
(m, 2H, H1′, H2′/H7′, H8′), 6.84-6.99 (m, 2H, H1′, H2′/H7′, H8′), 7.06-
7.13 (m, 2H, H4′, H5′), 7.14 (s, 1H, H11), 7.30-7.49 (m, 4H, H6, H7,
H8, H9), 8.01 (s, 1H, H4). 13C NMR (CDCl3): δ 13.9 (CH3), 14.0 (CH3),
21.10 (CH3(Ac)), 21.15 (CH3(Ac)), 79.5 (C5/C10), 79.7 (C5/C10), 81.8
(C1), 110.4 (C4′/ C5′), 110.5 (C4′/C 5′), 115.8 (C8a′/C9a′), 115.8 (C8a′/C9a′),
116.6 (C11), 117.3 (C4), 117.8 (C2′/C7′), 118.0 (C2′/C7′), 121.2 (C6/C9),
121.6 (C6/C9), 125.1 (C3a), 128.1 (C7/C8), 128.2 (C7/C8), 129.1 (C1′/
C8′), 129.4 (C1′/C8′), 139.1, 139.7, 143.5, 148.9, 152.6 (C4a, C5a, C9a,
C
10a, C11a), 151.4, 151.5, 152.1, 152.3 (C4a′/C10a′/C3′/C6′), 168.8 (C3),
1
r is the probability of the reaction of A with O2 ()krA/ksA), and γ is
168.9 (CO(Ac)), 170.0 (CO(Ac)). MS (FAB+): m/z 577 (M+ + 1).
Mp: 196 °C dec.
the probability of formation of 1O2 from EP-1 ()krO /ksO ). The values
2
2
of kd (5.00 × 105 s-1), ksO2 (4.9 × 10-4 s-1), and γ (0.82) are from the
Synthesis of 9-[2-(3-Carboxy-9,10-dimethyl)anthryl]-6-hydroxy-
3H-xanthen-3-one (DMAX), DMAX-9,10-endoperoxide (DMAX-
(18) Paeker, A.; Rees, W. T. Analyst 1960, 85, 587-600.

2536 J. Am. Chem. Soc., Vol. 123, No. 11, 2001
Tanaka et al.
literature.10,14b DMA and DPA are reported to be efficient acceptors,
so they can react without deactivation reaction of 1O2 (eq 5); thus, r is
close to 1. The data of time-dependent fluorescence increases of DMAX
and DPAX-1 upon reaction with various concentrations of EP-1 were
fitted to eq 6,
krA[A]
krA[A] + kd
R ) ksO [EP-1]γ
(8)
2
1
Thus, the rate constant for reaction of A with O2 is given by
R kd
F ) Fmax (1 - e-kt
)
(6)
krA
)
(9)
[A](ksO [EP-1]γ - R)
2
where F is the fluorescence intensity and Fmax is the maximum
fluorescence intensity. The initial reaction rate (R) is given by
Acknowledgment. This work was supported by research
grants from the Ministry of Education, Science, Sports and
Culture of Japan (Grant Nos. 11794026, 12470475, and
12557217 to T.N., 10771238 and 12771349 to Y.U.). We thank
Professor W. Nakanishi (Wakayama University) for helpful
discussion.
R ) kFmax/ꢀφ
(7)
where the value of ꢀφ is estimated from the relationship between the
fluorescence intensity of DMAX-EP or DPAX-1-EP and the concentra-
tion. Using the initial reaction rate, the following equation can be
written:
JA0035708