One-step synthesis of a fluorescein derivative and mechanistic studies

Article abstract of DOI:10.1016/j.tetlet.2012.07.084

We report a convenient method for the one-step synthesis of a fluorescein derivative under acidic conditions. Mechanistic studies indicate that the acid-promoted condensation of o-tolualdehyde and 4-chlororesorcinol to form the fluorescein derivative proceeds through a cyclization-oxidation pathway while an alternative oxidation-cyclization pathway remains possible.

Full text of DOI:10.1016/j.tetlet.2012.07.084

Tetrahedron Letters 53 (2012) 5284–5286
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Tetrahedron Letters
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One-step synthesis of a fluorescein derivative and mechanistic studies
⇑
Michael P. Cook, Shin Ando, Kazunori Koide
Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA 15260, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We report a convenient method for the one-step synthesis of a fluorescein derivative under acidic con-
ditions. Mechanistic studies indicate that the acid-promoted condensation of o-tolualdehyde and 4-chlor-
oresorcinol to form the fluorescein derivative proceeds through a cyclization-oxidation pathway while an
alternative oxidation–cyclization pathway remains possible.
Received 4 May 2012
Revised 13 July 2012
Accepted 19 July 2012
Available online 27 July 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Condensation
Fluorescein
Mechanism
Oxidation
Fluorescein and its derivatives such as dichlorofluorescein (DCF)
and Oregon Green1 (Fig. 1) are widely used fluorescent dyes in
chemistry, biology, and medicine.2 Historically, fluoresceins with a
carboxy group at the 2-position are the dominant species due to
their synthetic accessibility. However, this functional group, nega-
tively charged under physiological conditions, is not crucial for fluo-
rescence emission because the benzoate group is orthogonal to the
xanthene ring.3 Moreover, the negative charge may be detrimental
to cell permeability.4 For these two reasons, the carboxy group has
been replaced by other functional groups with more desirable prop-
erties. 2-Me Tokyo Green is the first of its class in which the carboxy
group is replaced by an alkyl group with significant fluorescence
intensity.5 Later, Pennsylvania Green was introduced and shown
to be superior to Tokyo Green for biological applications due to
the lower pKa value of the phenolic hydroxy group.6 We and others
recently reported the synthesis and applications of Pittsburgh Green
and its derivatives in chemistry, biology, geology, and environmen-
tal and pharmaceutical sciences.4,7 Pittsburgh Green was found to
be more permeable than DCF in a biological system.4 However, in
our attempts to image the presence of ozone in live cells, an O-
butenylated Pittsburgh Green was ineffective.7c This failure
prompted us to prepare the new DCF analog 1a and its O-butenylat-
ed derivative.7c Herein, we report both a convenient synthesis of the
Pittsburgh Green derivative 1a (‘Pittsburgh Green II’) in one step
from commercially available compounds as well as mechanistic
insights.
acid to form tetraol 2a in 93% yield according to the protocol from
the Van Vranken laboratory.8 The purification of this tetraol (45 g)
only required recrystallization in organic solvents, which should be
amenable to a large-scale synthesis. The treatment of tetraol 2a
with p-TsOH (8 equiv) in toluene at 110 °C produced mixture X.
Subsequently, this mixture was subjected to DDQ8 to form xanthe-
none 1a. This sequence was inconvenient for a gram-scale synthe-
sis because it was difficult to separate the desired product 1a from
DDQ and its reduced derivative DHQ. Perhaps for the same reason,
similar compounds were purified by HPLC.8 Nonetheless, similar
protocols were used by others to prepare related compounds.9 In
order to circumvent the tedious purification process associated
with the use of DDQ, the Nagano group performed an oxidation
reaction without DDQ, resulting in a very low yield.10 The Peterson
Fluorescein: X = H
2
CO2H
DCF: X = Cl
X
X
Oregon Green: X = F
HO
O
O
OH
Cl
X
X
Cl
As Scheme 1 shows, the condensation of o-tolualdehyde and
4-chlororesorcinol proceeded in the presence of methanesulfonic
HO
O
O
HO
O
O
2-Me Tokyo Green: X = H
Pittsburgh Green
Pennsylvania Green: X = F
1a: X = Cl
⇑
Corresponding author.
E-mail address: koide@pitt.edu (K. Koide).
Figure 1. Structures of fluorescein and its derivatives.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.07.084

M. P. Cook et al. / Tetrahedron Letters 53 (2012) 5284–5286
5285
byproducts and the starting material were coeluted with the de-
sired xanthenone 1a in column chromatography.
Cl
MeSO3H
93%
During these synthetic studies, we realized that both of the
steps (o-tolualdehyde+4-chlororesorcinol?2a and 2a?1a) were
promoted by acid. Therefore, it was hypothesized that these steps
could proceed in one step under acidic conditions. In effect, we
treated a solution of o-tolualdehyde and 4-chlororesorcinol with
MeSO3H at 110 °C, which produced xanthenone 1a in 26% isolated
yield. The purification did not require HPLC to obtain 1 in >95%
purity. A protocol devoid of column chromatography is also de-
scribed in the experimental section. These convenient protocols
may provide an easy access to fluorescein derivatives for others en-
gaged in the synthesis of this class of compounds.1,5,6,8,9,11,12
It should be noted that the mechanism for the conversion of tet-
raol 2a to xanthenone 1a is debatable. Specifically, as Scheme 2
indicates, the first step may be the acid-catalyzed cyclization to
form xanthene 3a or air-oxidation of the triarylmethane C–H to
form triol 4a. In a similar system, Gee et al. indicated that xanthene
3 was an intermediate en route to xanthenone 1.13 In contrast, Van
Vranken and co-workers implied that the intermediate might have
been triol 4 because tetraol 2 did not undergo cyclization to form
xanthene 3.8 The formation of triol 4 might be reasonable after
benzylic C–H oxidation followed by dehydration. To shed further
insight into this mechanism, we decided to closely examine the
conversion of tetraol 2a to xanthenone 1a.
During the conversion of tetraol 2a to xanthenone 1a, three
additional compounds were observed by TLC analysis. Attempts
to purify these compounds from mixture X were not fruitful, pos-
sibly due to air oxidation of xanthene 3a to xanthenone 1a and ra-
pid cyclization of triol 4a to xanthenone 1a among others. LC–MS
analysis of mixture X using reverse-phase chromatography did
not provide conclusive data due to poor separation of multiple
compounds. The 1H NMR spectrum of mixture X14 (Fig. S6, Supple-
mentary data) showed the presence of xanthene 3a, as indicated by
a singlet peak at 5.88 ppm that was absent in both tetraol 2a and
xanthenone 1a. The mass spectroscopic analysis of mixture X indi-
cated the presence of xanthene 3a and xanthenone 1a, but triol 4a
could not be observed. The structure of the air-sensitive xanthene
3a could be further corroborated after the treatment of mixture X
with MeI and NaH, which produced the more air-stable methyl
+ 2
Cl
Cl
HO
OH
CHO
HO
OH
HO OH
2a
MeSO3H,
110 °C;
26%
neutral alumina
9%
see text
see text
mixture X
Cl
Cl
O
low yield or
difficult
purification
HO
O
1a
Scheme 1. Synthesis of xanthenone 1a.
group converted an analogous intermediate to Pennsylvania Green
upon heating in the presence of p-TsOH, albeit with moderate
yields (37–38% yields).6b The low efficiency of these reactions
may be attributed to a retro-Friedel–Crafts process.11 Consistent
with these reports, heating mixture X with and without an acid
proved to be low yielding (<5%) and not reproducible in our labo-
ratory. These frustrations prompted us to consider an alternative
approach for the synthesis of 1a.
When we were following the literature protocol to cyclize tetra-
ol 2a to xanthenone 1a via mixture X, we noticed that a nonfluo-
rescent compound in mixture X became fluorescent after heating
the TLC plate. This could be due to the facile conversion of xan-
thene 3a to xanthenone 1a (Scheme 2) on a silica gel surface. To
test this hypothesis, tetraol 2a was dissolved in 1:1 MeOH/CH2Cl2
and added to silica gel and neutral alumina. After removing the sol-
vents under reduced pressure, the silica gel and alumina contain-
ing tetraol 2a were heated in an oven for 48 h at 140 °C
(CAUTION: The organic solvents must be thoroughly removed by a
high vacuum before placing the silica gel in the oven). The conversion
of tetraol 2a to xanthenone 1a was visually obvious because while
tetraol 2a and mixture X were white, xanthenone 1a was red. After
rinsing the red silica gel and alumina with 1:4 MeOH/CH2Cl2, xan-
thenone 1a was isolated in 9% yield from alumina and in a lower
yield from silica gel. Although this protocol was convenient, the
purification was not practical in a preparative scale because many
ether 5a. This ether derivative was semipurified using
a
preparative TLC and analyzed by 1H NMR spectroscopy and mass
spectroscopy (Figs. S7 and S8, Supplementary data). This result
Ar
X
X
MeO
O
5
OMe
MeI, Ag2O
Ar
oxidation
cyclization
X
X
H+
2a, 3a, 4a, 5a: X = Cl,
HO
O
3
OH
H2O
Ar
Ar
Ar =
X
X
X
X
HO
OH
HO
O
1
O
HO OH
2
Ar
H2O
cyclization
X
X
oxidation
HO
O
HO OH
4
Scheme 2. Two plausible pathways for the conversion of tetraol 2 to xanthenone 1.

5286
M. P. Cook et al. / Tetrahedron Letters 53 (2012) 5284–5286
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indicates that the cyclization–oxidation pathway (2a?3a?1a) ac-
counts for the formation of xanthenone 1a. A cyclization similar to
the conversion of 2a to 3a under the acidic conditions was previ-
ously observed,15 although the mechanism is not yet clear.
As for the alternative pathway (2a?4a?1a), it is noteworthy
that at least one fluorescent compound other than xanthenone
1a was observed transiently by TLC analysis during the conversion
of 2a to 1a, but this fluorescent compound could not be isolated.
This fluorescent intermediate might be triol 4a, which could cy-
clize to form xanthenone 1a. Therefore, the cyclization–oxidation
and oxidation–cyclization pathways might be concurrently
operating.
In summary, we developed a one-step protocol for the prepara-
tion of xanthenone 1a, and subsequent mechanistic studies sup-
ported the cyclization–oxidation pathway. It should be noted
that this study by no means excludes the oxidation–cyclization
pathway. Further mechanistic studies are warranted to improve
the efficiency of this important synthetic process for chemistry,
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Acknowledgment
We thank our former coworker Dr. Fengling Song for his preli-
minary studies. This work was supported by the US National Sci-
ence Foundation (CHE-0911092).
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Supplementary data
Supplementary data (experimental procedure and characteriza-
tion data of compounds 1a, 2a, 3a, and 5a) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.tetlet.2012.07.084.
14. The reaction mixture was washed with water then brine until the pH of the
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then solvents were removed in vacuo for this NMR analysis.
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