One-pot synthesis of tetrafluoro- and tetrachlorofluorescein derivatives and their stabilization by β-cyclodextrin

Article abstract of DOI:10.1002/cjoc.201300073

A highly efficient one-pot procedure for the preparation of fluorescein-based dyes with relatively high yield was investigated. Significantly, introduction of these fluoro and chloro functional groups and forming host-guest complexes with cyclodextrins partially enhanced the photostability of these dyes. A highly efficient one-pot synthesis of fluorescein-based halogenated dyes with relatively high yield was reported. Introduction of fluoro and chloro functional groups and forming host-guest complexes with cyclodextrins enhanced their photostability. Copyright

Full text of DOI:10.1002/cjoc.201300073

FULL PAPER
DOI: 10.1002/cjoc.201300073
One-Pot Synthesis of Tetrafluoro- and Tetrachlorofluorescein
Derivatives and Their Stabilization by β-Cyclodextrin
Li-Li Tan, Ying-Wei Yang,* Yi-Pu Liu, and Sean Xiao-An Zhang*
State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry,
Jilin University, Changchun, Jilin 130012, China
A highly efficient one-pot procedure for the preparation of fluorescein-based dyes with relatively high yield was
investigated. Significantly, introduction of these fluoro and chloro functional groups and forming host-guest com-
plexes with cyclodextrins partially enhanced the photostability of these dyes.
Keywords fluorescein, cyclodextrin, photostability, cucurbit[7]uril, one-pot synthesis, supramolecular chemistry
Introduction
1,3-dione, β-CD were reagent grade and purchased from
Aladdin Co. Ltd. Deionized water was used in all ex-
periments. 1H NMR spectra were collected at 25 ℃ on
a Varian-300 MHz NMR spectrometer. 13C NMR spec-
tra were recorded at 25 ℃ on a Varian 75 MHz NMR
spectrometer and Varian 151 MHz NMR spectrometer.
19F NMR spectra were recorded at 25 ℃ on a Varian
565 MHz NMR spectrometer. Mass spectra were re-
corded on Liquid ChroMatography. Mass Spectrometry
Instrument (Agilent1290-micrOTOF Q II). UV-vis
spectra were recorded on a Shimadzu UV-2550 instru-
ment and all fluoresceins are in dianion form. The pKa2
of fluorescein is 6.5, the pKa2 of compound 1 is 6.1, and
the pKa2 of compound 2 is 5.97, when β-CD was added
the pKa2 of these dyes changed to 6.35, 6.67 and 7.40
(see the supporting information).[5] The NMR titration
experiments were performed in basic condition with 2
mmol/L of K2CO3 in D2O, which pH is measured to be
10.82.
Since the first synthesis in 1871 by von Bayer,[1]
fluoresceins in dianion form, due to their brightness,
excellent fluorescence quantum yields, low-energy ex-
citation and emission wavelengths, and good biocom-
patibility, have shown wide-spread applications in many
research fields, such as single molecule detection, fluo-
rescence labeling, dye lasers, conversion and storage of
solar energy, fluorescence-based assays, and cell stain-
ing and antitumor agents.[2] However, facile synthesis of
new analogues of fluoresceins is still in urgent need in
order to further improve their properties and overcome
the disadvantages of current dyes, such as the instability
of fluorescein conjugates, irreversible photo-bleaching
by a powerful laser, and strong pH dependence of fluo-
rescence in aqueous solutions.[3]
Cyclodextrins (CDs) and cucurbit[n]urils (CB[n]s)
are two kinds of functional macrocyclic compounds,
which can selectively form inclusion complexes with
organic guests.[4] Recently, Nau et al.[2a] reported that
rhodamine 6G could be made ultrastable upon com-
plexation with CB[7]. Herein, we wish to describe a
new method to obtain photostable fluoresceins. We not
only discussed the contribution of functional groups to
the stability of the dyes in dianion form, but also ex-
plored the influence of the formation of inclusion com-
plexes upon binding with CDs or CB[n]s.
4,5,6,7-Tetrafluorofluorescein (1)
In a 100 mL round-bottom flask, 4,5,6,7-tetrafluoro-
isobenzofuran-1,3-dione (4.76 g, 20 mmol) was dis-
solved in MeSO3H (21 mL) at 80 ℃ before concen-
trated H2SO4 (6 mL) was added. The reaction mixture
was heated at 90 ℃ under vigorous stirring after resor-
cinol (5.51 g, 50 mmol) was added in three portions.
After 2 h, the reaction mixture was heated up to 120 ℃
under vigorous stirring, and was allowed to react for
another 4 h. The reaction mixture was cooled down to
room temperature and quenched with H2O (100 mL).
The solid precipitate was suction filtered and washed
with H2O, then dried in vacuum to give 1 (6.65 g, yield:
Experimental
Unless otherwise noted, commercial reagents were
used as received and all reactions were carried out under
ambient condition. Fluorescein, 4,5,6,7-tetrafluoroiso-
benzofuran-1,3-dione, 4,5,6,7-tetrachloroisobenzofuran-
1
92%). H NMR (300 MHz, CD3SOCD3, 25 ℃) δ:
10.27 (s, 2H), 7.01 (d, J＝8.7 Hz, 2H), 6.70 (d, J＝2.0
*
E-mail: ywyang@jlu.edu.cn, seanzhang04@yahoo.com; Tel.: 0086-0431-85153810; Fax: 0086-0431-85153812
Received January 29, 2013; accepted April 7, 2013; published online XXXX, 2013.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201300073 or from the author.
Chin. J. Chem. 2013, XX, 1—5
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Tan et al.
FULL PAPER
Hz, 2H), 6.60 (dd, J＝8.7, 2.2 Hz, 2H); 13C NMR (151
MHz, CD3SOCD3, 25 ℃) δ: 170.38, 162.58, 160.37,
160.29, 156.30, 155.65, 152.18, 151.82, 151.33, 145.97,
145.86, 145.76, 144.56, 144.48, 144.23, 144.13, 144.03,
142.82, 142.73, 142.40, 141.58, 140.81, 140.72, 139.96,
133.10, 133.02, 128.99, 128.78, 126.71, 126.14, 122.42,
113.15, 112.90, 111.40, 111.32, 107.26, 107.00, 103.62,
102.50, 102.31, 81.68, 80.75, 59.79, 20.77, 14.10; 19F
NMR (565 MHz, CD3SOCD3, 25 ℃) δ: −140.12 (d,
J＝15.8 Hz, 1F), −144.04 (t, J＝15.7 Hz, 1F), −144.21
(t, J＝19.2 Hz, 1F), −151.97 (t, J＝20.1 Hz, 1F). m.p.＞
300 ℃. ESI-HRMS calcd for C20H9F4O5 405.0381,
found 405.0395.
acidity strength, and gradually increase of reaction
temperature effectively avoided the sublimation. The
yields of the obtained fluorescein derivatives (1 and 2)
were highly improved and the whole synthetic protocol
and purification progress were very straightforward.
Although fluorescein and its derivatives share a
common, xanthene-based skeleton, different substitu-
ents can be made to cause marked differences in ab-
sorbance and fluorescence emission wavelengths. Se-
lective substitution of aromatic hydrogen of fluoresceins
with chlorine has been seen to increase fluorescence
efficiency and to narrow emission and absorbance
maxima.[8] The absorption peak of the chlorinated fluo-
rescein (2) was found to be red-shifted by 25 nm (Figure
1), which may be due to the electron-withdrawing abil-
ity of the chlorine substituted group.[9]
The substitution of hydrogen atoms by fluorine at-
oms in organic compounds often results in profound
changes in their properties, largely due to the highly
electro-negative nature and small van der Waals radius
of the fluorine atom.[10] Unlike other halogenated com-
pounds, in which an expected pattern derived from the
substitution effect could be inferred, fluorination of or-
ganic compounds often results in products with unex-
pected properties. These halogenated derivatives un-
dergo significant intersystem crossing to the triplet state
after light absorption.[11] In our research for the fluori-
nation of fluorescein (1) to improve its properties, we
sought to retain the favorable characteristics of fluo-
rescein, especially high absorbance and absorption at
long wavelength, which were very useful as molecular
probes. Fluorination of fluorescein permits us to exam-
ine the influence of fluorine on this widely used fluo-
rescent molecule.[5a]
Photo-bleaching is a dynamic process by which a
fluorophore undergoes a photo-induced chemical de-
struction upon exposure to light and thus loses its ability
to fluoresce.[12] The photo-bleaching mechanism for a
fluorophore present in biological systems is a compli-
cated process on which many studies have been focused.
The photo-bleaching behavior of free and bound fluo-
rescein has also been investigated by experimental
methods. Both the theoretical simulation and experi-
mental data show that photo-bleaching of fluorescein
involved three steps of reactions that could lead to irre-
versible bleaching photoproducts,[5a] (a) the reactions of
two triplet dyes to form semireduced (R) and semioxi-
dized (X) form of dyes; (b) the reaction of triplet dye
with ground-state dye to form R and X; (c) the reaction
of triplet-state dye with oxygen to form X and HO2 (or
O－2 ). The first two reactions represent the occurrence of
an electron-transfer process; the third reaction is
chemical quenching by oxygen.
4,5,6,7-Tetrachlorofluorescein (2) 4,5,6,7-Tetra-
chloroisobenzofuran-1,3-dione (0.2859 g, 1 mmol) was
dissolved in MeSO3H (1.25 mL) at 90 ℃, and resorci-
nol (0.2753 g, 2.5 mmol) was added in three portions.
After 2 h of reaction, the mixture was stirred at 125 ℃
for 4 h, and then cooled down to room temperature and
quenched with H2O (12.5 mL). The solid precipitate
was suction filtered and washed with water, then dried
1
in vacuum to give 2 (0.2928 g, yield 82%). H NMR
(300 MHz, CD3SOCD3, 25 ℃) δ: 10.19 (s, 2H), 6.94 (d,
J＝8.7 Hz, 2H), 6.67 (d, J＝2.3 Hz, 2H), 6.55 (dd, J＝
8.7, 2.4 Hz, 2H); 13C NMR (75 MHz, CD3SOCD3, 25
℃) δ: 163.5, 159.9, 148.3, 134.8, 128.9, 124.1, 112.6,
106.6, 102.3, 81.6. m.p.＞300 ℃. ESI-HRMS calcd for
C20H9Cl4O5 468.9199, found 468.9216.
Results and Discussion
Fluorescein and its derivatives were synthesized by
condensation of appropriate resorcinol with various de-
rivatives of phthalic anhydride in the presence of vari-
ous acid catalysts, e.g., MeSO3H,[5] AcOH-HCl,[6] CsF[7]
and ZnCl2.[1] But these conventional methodologies for
the synthesis of fluorescein and derivatives suffer from
relatively high cost, low yield, hazard materials, high
temperature and long process. Herein, a novel method-
ology for preparing dyes and switchable dyes is shown
in Scheme 1. It is a technological break-through that
enables us to prepare conventional or special functional
dyes in a simple one-pot, low cost and highly efficient
process. MeSO3H served as both a suitable solvent and
a Lewis acid catalyst in the dye-forming reaction. On
the other hand, H2SO4 was introduced to increase the
Scheme 1 One-pot synthetic route to the fluorescent dyes (1
and 2) with MeSO3H-H2SO4 as catalyst
c
HO
O
OH
HO
OH
O
b
O
R
O
a
R
O
O
MeSO3H/H2SO4
R
R
A slower rate of bleaching for the introduction of
these two kinds of groups indicates that at least one of
the three reactions is affected assuming other conditions
remain the same (Figure 1). There are two possible ex-
R
R
R
R
1 R = F
2 R = Cl
2
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Chin. J. Chem. 2013, XX, 1—5

One-Pot Synthesis of Tetrafluoro- and Tetrachlorofluorescein Derivatives
eral advantageous effects, which include increased
quantum yields, increased photostability, high bright-
ness, spectral shifts, prevention of unspecific adsorption
and dye aggregation.[2a] While anionic centers result in
low binding,[15] expectedly, the anionic dye fluorescein
does not form a complex with CB[7]. We introduced
CB[7] into the solutions of fluorescein and its deriva-
tives (1 and 2), the administration of CB[7] does not
have any influence on improving the photo-bleaching of
the synthesized dyes as expected (see the supporting
information). Meanwhile, due to the solvation effect
caused by a higher concentration of CB[7], the absorp-
tion strength was reduced.
Figure 1 Photo-bleaching of fluorescein (4 μmol/L), 1 (4
μmol/L), and 2 (4 μmol/L) in a phosphate buffer solution (pH 8.0)
in the absence of β-CD and CB[7] followed through the increas-
ing time of a 250-W mercury arc lamp irradiation. The inset
shows the graph and correlation lines for the characteristic func-
tion f＝log([10A0−1]/[10A−1]) versus irradiation time for the de-
termination of the relative quantum yield of photo-bleaching.[13]
planations that account for the improved resistance to
photo-bleaching: (a) the introduction of group could
shorten the triplet lifetime, thus decreasing the probabil-
ity of its reaction with a quencher; (b) the incorporation
of these groups into fluorescein inhibits the chemical
reactions involving the triplet state, including the three
irreversible bleaching reactions described above. These
two possibilities could be present simultaneously with-
out contradicting each other. And, the degree of the sta-
bilization depends on electro-withdrawing ability of
these groups, i.e., the higher of the electro-withdrawing
ability of the substituents, the more photostable of the
fluorescein derivatives.
Figure 3 Photo-bleaching of 1 (4 μmol/l) in phosphate buffer
(pH 8.0) in the absence and presence of β-CD (2 mmol/L) with an
increasing time of irradiation by a 250 W mercury arc lamp.
Figure 4 Photo-bleaching of 2 (4 μmol/L) in phosphate buffer
(pH 8.0) in the absence and presence of β-CD (2 mmol/L) with an
increasing time of irradiation by a 250 W mercury arc lamp.
β-CD and its various derivatives are well known,
possessing a truncated cone with hydrophilic outer sur-
face and hydrophobic internal cavity.[5c] Flamigni[16] and
Buvári et al.[17] reported the association constants of
β-CD and fluorescein at basic pH and acidic pH to be
(360±40) L•mol−1 and (1100±100) L•mol−1, respec-
tively. As is apparent from Figures 2－4, there is an
increase in the photostability upon addition of β-CD
(With longer time absorbing, photoproducts are formed,
which cause the curve to become nonlinear), and there
is no difference when we increase the concentration of
β-CD to 18 mmol/L (see the supporting information).
Apparently, β-CD selectively prevents the two-step
Figure 2 Photo-bleaching of fluorescein (4 μmol/L) in phos-
phate buffer (pH 8.0) in the absence and presence of β-CD (2
mmol/L) with an increasing time of irradiation by a 250 W mer-
cury arc lamp.
Supramolecular complexation of chromophoric
guest molecules by macrocyclic hosts can affect their
fluorescent properties as a consequence of an altered
microenviroment.[14] As reported, the addition of CB[7]
to aqueous solutions of some important cationic fluo-
rescent dyes has exposed a synergistic interplay of sev-
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Tan et al.
FULL PAPER
two-photon photolysis, and does not show any common
drawbacks. In another words, it does not act as a
quencher, does not absorb in the visible region, and
consequently displays non-fluorescence. These com-
bined properties distinguish β-CD from other stabilizing
additives.[18]
(637±70) L•mol−1 (Figure 6), respectively, which en-
sures the complete complexation between dyes and
β-CD in our photo-bleaching experiments (see the Sup-
porting Information).
β-CD has a stronger effect on the photostabilization
of 2, due to the binding interaction of β-CD and this
fluorescein derivative within the cavity. The structure of
guest is very important for host-guest inclusion com-
plexation. The shape and size of the guest must match
the cavity of β-CD, and its polarity should be smaller
than water. As compared with fluorescein and 1, the
hydrophobicity of 2 is relatively stronger, which made it
easier to enter the hydrophobic cavity of β-CD forming
a relatively stable inclusion complex.
Effort has been made to improve the photostabilizing
properties of organic dyes,[20] numerous additives have
been tested to date but showed in common limited pho-
tostabilizing properties on organic dyes, i.e., incompati-
bility with biologically or environmentally relevant ap-
plications, use in large scale, operable either by pre-
venting secondary photolytic steps or by quenching un-
desired reactive triplet states, etc. However, the addition
of β-CD provides an alternative, supramolecular ap-
proach to achieve increased photostabilization of fluo-
rescein-based dyes, as reported in this work. Signifi-
cantly, β-CD is transparent in the visible wavelength
range and does not act as fluorescence quencher for
dyes at a range of concentrations.
The photostablizing effect of β-CD is unquestiona-
bly related to a combination of factors, involving the
inert cavity, its low polarizability, the confinement of
the dye, and effective protection from water and oxygen.
As reported, glucose and alcohols were used to confirm
the formation of supramolecular complexes, and that the
improvement of the photostabilities is not caused by the
solvent effect but by a higher concentration of β-CD.[19]
1
Analysis on the chemical shifts of the H NMR signals
of the complex in relation to the signals from the pure
β-CD and 2 in dianion state with excess K2CO3, are the
main indication of the extent of the complex formation
(Figure 5). The association constants of β-CD with 1
and 2 were determined to be (122±15) L•mol−1 and
Conclusions
We have developed a new method for the synthesis
of fluorescein derivatives in high yields. Dye 1 has bet-
ter photophysical properties, i.e., high brightness, good
solubility in water, increased photostability, long ab-
sorption wavelength as compared with fluorescein and 1.
The addition of β-CD to form supramolecular inclusion
complex increased the photostabilities of fluorescein
and its derivatives, showing potential applications of
CDs in organic dye industry.
Figure 5 1H NMR spectra in D2O with excess K2CO3 (pH
10.82), the molar ratio of 2 and β-CD was 1∶1 to get the com-
plex: (a) 10 mmol/L 2; (b) 10 mmol/L 2 and 10 mmol/L β-CD; (c)
10 mmol/L β-CD.
Acknowledgement
This research was supported by the National Natural
Science Foundation of China (No. 21272093). The au-
thor also wish to thank the journal editor, Weifeng Ding,
and the reviewers who raised valuable points that help
improve the quality of the presentation of this article.
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