Intramolecular fluorescence resonance-energy-transfer of fluorescein derivative; phenyl and styryl p-disubstituted fluorescein

Article abstract of DOI:10.1246/cl.2008.1232

We describe synthesis and fluorescence properties of phenyl and styryl p-disubstituted fluorescein. The compound was synthesized in three reaction steps from trans, trans, trans-1,6-diphenyl-l, 3,5-hexatriene. The fluorescence spectrum of the product in a

Full text of DOI:10.1246/cl.2008.1232

1232
Chemistry Letters Vol.37, No.12 (2008)
Intramolecular Fluorescence Resonance-energy-transfer of Fluorescein Derivative;
Phenyl and Styryl p-Disubstituted Fluorescein
Jun-ichi Kadokawa,Ã Masakazu Suenaga, and Yoshiro Kaneko
Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065
(Received September 5, 2008; CL-080845; E-mail: kadokawa@eng.kagoshima-u.ac.jp)
We describe synthesis and fluorescence properties of phenyl
and styryl p-disubstituted fluorescein. The compound was syn-
thesized in three reaction steps from trans,trans,trans-1,6-
diphenyl-1,3,5-hexatriene. The fluorescence spectrum of the
product in alkaline methanol solution suggested occurrence of
intramolecular FRET from the donor site of the benzene moiety
to the acceptor site of the xanthene moiety.
DDQ
+
m
Benzene
1,4 -Dioxane
O
m
O
O
1; m = 0
2; m = 1
O
O
O
3; m = 0
4; m = 1
2
HO
CH3SO3H
OH
m
m
1,2-Dichloroethane
HO
O
O
O
O
O
O
In recent years, considerable effort has been devoted to de-
veloping new fluorescence dye-based probes as excellent sensors
for biomolecules, being sensitive, fast-responding, and capable
of affording high spatial resolution via microscopic imaging.1
Among many fluorescent dyes, fluorescein is known to have a
high quantum yield of fluorescence in aqueous solution and to
be excitable at long wavelength.2 The fluorescein is appropriate-
ly divided into two parts, i.e., the benzene moiety and the xan-
thene moiety.3 The fluorescein can be prepared by the condensa-
tion reaction of phthalic anhydride with resorcinol under acidic
conditions.4 However, little has been reported to prepare fluores-
cein derivatives directly having p-substituents with ꢀ conjuga-
tion on the benzene moiety.
The fluorescence intensity of the probes is influenced by
many factors, such as the changes of environment around the
probe (pH, polarity, and temperature) and the changes in the con-
centration and the excitation intensity. To reduce the influence
of the factors, ratiometric measurements are utilized,5 in which
the fluorescence intensities are simultaneously recorded and
their ratio is calculated. This allows more precise measurement
than that at a single wavelength. To perform the ratiometric
measurement, the probes necessarily exhibit a large shift in their
emission or excitation spectrum after they react or bind with
the target molecules. A fluorescence resonance-energy-transfer
(FRET)6 technique has been used in the designed fluorescent
probes to obtain a large shift in the spectroscopic peak. FRET
is an interaction between the electronic excited states of two flu-
orophores, in which excitation energy is transferred from a donor
to an acceptor without emission of a photon.7 In previous studies,
intramolecular FRET compounds bearing a fluorescein acceptor
and a dye-based donor such as coumarin have been synthesized.8
In the compounds, the donors and acceptors are connected by
flexible linkers such as ethylene glycols and phosphates, and
the excitation energy of the coumarin donor is transferred to
the fluorescein acceptor.
5; m = 0
6; m = 1
OH
7; m = 0
8a; m = 1
Scheme 1. Synthesis of diphenyl p-disubstituted fluorescein 7
and phenyl and styryl p-disubstituted fluorescein 8a (8b is a re-
gioisomer of 8a).
Then, the reaction of 5 with resorcinol was conducted in the
presence of methanesulfonic acid in 1,2-dichloroethane, giving
rise to diphenyl p-disubstituted fluorescein 7. Both the polymer
and the analogous compound 7 have the ꢀ-conjugated structures
at the benzene moieties, and accordingly, the above synthetic
route can be considered to be useful for the preparation of vari-
ous fluorescein derivatives having ꢀ-conjugated structures at the
benzene moieties.
On the basis of the above viewpoints, in this letter, we report
the synthesis of a new fluorescein derivative, i.e., phenyl and
styryl p-disubstituted fluorescein 8 according to the synthetic
route of Scheme 1, which has longer ꢀ conjugation at the ben-
zene moiety than that of 7. Furthermore, we found interesting
fluorescence properties of 8, in which intramolecular FRET from
the benzene moiety to the xanthene moiety in the fluorescein
structure occurred, which is possibly applicable to the ratiomet-
ric measurement.
As the first step, we performed Dields–Alder reaction of a
starting compound, trans,trans,trans-1,6-diphenyl-1,3,5-hexa-
triene (2)11 with maleic anhydride to give 4.10,12 The H NMR
(CDCl3) and IR spectra of the isolated product fully supported
1
the structure of 4. Then, the aromatization of 4 was carried out
1
using DDQ at 60 ꢀC in 1,4-dioxane.13 The H NMR spectrum
(CDCl3) of the isolated product showed the disappearance of
the signals due to the cyclohexene group in 4, indicating the oc-
currence of perfect aromatization. The resulting 6 was converted
into the desired compound 8 by the reaction with resorcinol in
the presence of methanesulfonic acid in 1,2-dichloroethane at
40 ꢀC.3,4 After the reaction was performed for 375 h, the crude
product was purified by column chromatography on silica gel
to give a mixture of two regioisomers 8a and 8b (the structure
of 8b is shown in Figure S314). The structures were confirmed
Recently, we reported the synthesis of poly(p-phenylene)
having fluorescein moieties in the main chain and the analogous
compound 7 of its repeating unit structure.9 As shown in
Scheme 1 for the synthesis of 7, Diels–Alder reaction of trans,
trans-1,4-diphenyl-1,3-butadiene (1) with maleic anhydride,
followed by aromatization with 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ) gave 3,6-diphenylphthalic anhydride (5).10
by the H NMR spectrum (Figure S3,14 DMSO-d6 containing a
small amount of D2O). The molar ratio of 8a to 8b was calcu-
lated by the integrated ratio of the NMR signals to be 85:15.
1
Copyright ꢀ 2008 The Chemical Society of Japan

Chemistry Letters Vol.37, No.12 (2008)
1233
between the benzene and xanthene moieties in these compounds.
Generally, FRET takes place through the resonance interaction
between the donor and acceptor rather than their strong electron-
ic coupling interaction, which affects the absorption spectrum.
The absorption spectral pattern of 8 in 0.50 mmol/L NaOH/
methanol (Figure S5a14) is similar as the superimposed pattern
of the absorption spectra of 6 and fluorescein in the same solvent
(Figures S5b and 5c14), supporting no strong electronic interac-
tion between the benzene and xanthene moieties in 8.
In conclusion, we have synthesized phenyl and styryl p-
disubstituted fluorescein 8 according to Scheme 1. The 1H NMR
spectrum of the isolated product indicated that it was a mixture
of the regioisomers 8a and 8b. The fluorescence spectrum of 8
excited at 320 nm in 0.50 mmol/L NaOH/methanol indicated
occurrence of the intramolecular FRET between the benzene
and xanthene moieties. This fluorescence property has a possi-
bility for developing a novel ratiometric fluorescent probe as a
pH sensor.
Figure 1. Fluorescence spectra of 8 (—) and fluorescein (- - -)
(0.50 mmol/L); excited at 320 nm in 0.50 mmol/L HCl/metha-
nol (a) and excited at 320 nm in 0.50 mmol/L NaOH/methanol
(b).
Excitation
COOH
FRET
O
HO
O
References and Notes
Emission
1
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´
The emission intensities at 520 nm in the fluorescein spectra
of the standard fluorescein (Figure 1b) and 7 (data not shown)
excited at 320 nm in 0.50 mmol/L NaOH/methanol were much
lower than those of 8, indicating that no obvious FRET happened
14 Supporting Information is also available electronically on
the CSJ-Journal Web site, http://www.csj.jp/journals/chem-
lett/index.html.
Published on the web (Advance View) November 8, 2008; doi:10.1246/cl.2008.1232