Facile Large-Scale Synthesis of 5- and 6-Carboxyfluoresceins: Application for the Preparation of New Fluorescent Dyes

Article abstract of DOI:10.1002/ejoc.201501013

A series of fluorescein dyes have been prepared from a common precursor through a very simple synthetic procedure, giving access to important precursors for fluorescent probes. The method has proven an efficient access to regioisomerically pure 5- and 6-carboxyfluoresceins on a large scale, in good yields, and with high regioisomeric purity. Furthermore, we have applied the method to the development of a new type of mixed fluorescein derivatives of 5-carboxyfluorescein. We have demonstrated the scope of the procedure by synthesizing a new type of double chromophore within the fluoro-Jade family.

Full text of DOI:10.1002/ejoc.201501013

FULL PAPER
DOI: 10.1002/ejoc.201501013
Facile Large-Scale Synthesis of 5- and 6-Carboxyfluoresceins: Application for
the Preparation of New Fluorescent Dyes
[a]
[b]
[a]
[b]
Peter Hammershøj,* E. K. Pramod Kumar, Pernille Harris, Thomas L. Andresen,
and Mads H. Clausen*[
a]
Keywords: Synthetic methods / Fluorescent probes / Chromophores / Polycycles
A series of fluorescein dyes have been prepared from a com-
mon precursor through a very simple synthetic procedure,
giving access to important precursors for fluorescent probes.
The method has proven an efficient access to regioisomer-
ically pure 5- and 6-carboxyfluoresceins on a large scale, in
good yields, and with high regioisomeric purity. Further-
more, we have applied the method to the development of a
new type of mixed fluorescein derivatives of 5-carboxy-
fluorescein. We have demonstrated the scope of the pro-
cedure by synthesizing a new type of double chromophore
within the fluoro-Jade family.
Introduction
desirable to separate the two isomers before they are uti-
lized in the production of fluorescent probes.
In recent years there has been increasing interest in a
diverse assortment of pH-sensitive fluorescent probes with
an array of absorption and emission maxima as well as pH
characteristics. For the last couple of decades, 5- and 6-
substituted fluorescein derivatives have been widely used as
We describe herein the synthesis of regioisomerically
pure 5- and 6-carboxyfluoresceins as precursors for the
large-scale synthesis of fluorescein-based probes. Further-
more, a route to new derivatives of known analogues of
carboxyfluorescein using benzophenone as a key intermedi-
ate is described. This method, which is applicable to many
fluorescein derivatives, directly produces analogues with
photophysical characteristics comparable to their closely re-
[
1–5]
fluorescent probes in bioimaging and nanotechnology.
- and 6-carboxyfluoresceins are well-known probes and
5
are usually used as regioisomeric mixtures. Burgess and co-
workers reported a procedure to separate the regioisomers
[
6]
lated dyes. The method has a very flexible design strategy,
starting from the key benzophenones 5 and 6 (see
Scheme 1), and has the possibility of yielding a large range
of fluorescent probes for bioimaging. Based on the results
of Nagano and co-workers, we focused on the synthesis of
by fractional crystallization on a multigram scale, achieving
8% regioisomeric purity of both isomers.[ Other re-
6]
9
gioisomeric fluorescein derivatives have also been sepa-
[
7–9]
rated.
To the best of our knowledge, a large-scale syn-
thesis of 100% regioisomeric pure 5- and 6-carboxyfluores-
cein without chromatographic purification has never been
reported. Carboxyfluoresceins are widely used as fluores-
cent-labeling reagents by esterification or amidation at the
5-carboxyfluorescein derivatives. By using our new pro-
cedure we have synthesized new types of seminaphtho-
[
12]
fluorescein (1, carboxy-SNAFL-1),
4Ј,5Ј-dichloro-2Ј,7Ј-
[
2,13,14]
dimethoxy-5(6)-carboxyfluorescein (2, carboxy-JOE),
and 2Ј,7Ј-difluoro-5(6)-carboxyfluorescein (3,
5- or 6-position. They are achieved in high yields, in a large
[
15]
carboxy-
range of solvent systems, and by using different rea-
oregon green) chromophores (Figure 1 and Scheme 1).
Additionally, we have developed a new type of dye bear-
ing two chromophoric groups by using the same synthetic
strategy. The new chromophores are difluorescein deriva-
[
1–3]
gents.
Nagano and co-workers found that upon esterifi-
[
5,10,11]
cation emission intensities are decreased.
This effect
is less pronounced for the tert-butyl 5-carboxyfluorescein
ester than with the tert-butyl 6-carboxyfluorescein ester (in-
tensity decrease of 16 and 59%, respectively). Thus, it is
[
16,17]
tives and are part of the fluoro-Jade family.
The di-
fluorescein derivatives have two identical chromophores in
close proximity linked by an out of plane benzene ring, ef-
fectively minimizing the conjugation between the two
fluorescein units. Furthermore, a mixed difluorescein
system with the same general structure only diverging by
having two different chromophores has been developed. A
feature of the new double chromophores is a broadening of
[
a] Department of Chemistry, Technical University of Denmark,
Building 206, 2800 Kgs. Lyngby, Denmark
E-mail: peham@kemi.dtu.dk
mhc@kemi.dtu.dk
http://www.kemi.dtu.dk/
[
b] Department of Micro- and Nanotechnology,
Technical University of Denmark, DTU Nanotech,
Building 423, 2800 Kgs. Lyngby, Denmark
Supporting Information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201501013.
the pK profile and an OFF/ON/OFF pH-dependent emis-
a
sion, which we believe will be useful for various applications
Eur. J. Org. Chem. 2015, 7301–7309
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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P. Hammershøj, M. H. Clausen et al.
FULL PAPER
Scheme 1. Synthetic route to regioisomerically pure 5- and 6-carboxyfluoresceins 7 and 8 and mixed fluorescein derivatives 9–11.
Comprehensive characterization of the new chromo-
phores has been undertaken, including the determination of
their absorption, emission, and pK profiles.
a
Results and Discussion
We used a known and high-yielding synthesis to obtain
a crude mixture of 5- and 6-carboxyfluoresceins (8 and 7)
on a large scale starting from trimellitic anhydride (4) and
[
6]
resorcinol in methanesulfonic acid (Scheme 1). The re-
sulting crude mixture was precipitated from boiling EtOH/
H O and then filtered and dried in vacuo. Subsequent
2
hydrolysis under strong alkaline conditions followed by
acidification yielded the desired benzophenones 5 and 6.[
The key step to pure regioisomers of 5- and 6-carboxy-
fluoresceins (8 and 7) is the separation of benzophenones 5
and 6. Benzophenone 6 forms a 4:1 arrangement with
MeOH in the crystal structure after crystallization from
7]
Figure 1. Novel carboxyfluorescein derivatives 1–3. Compound 1
is shown with 5Ј and 6Ј for illustrative purposes. Compounds 2 and
3
have the same numbering.
MeOH/H
Thus, crystallization occurs faster for benzophenone 6 than
in which fluorescent detection reagents are used for bio- for 5. Taking advantage of this we separated 5 and 6 by
2
O (see Figure S2 in the Supporting Information).
imaging and cellular investigations.[
1,4,18]
crystallization in MeOH/H O to achieve 97 and 100% re-
2
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Large-Scale Synthesis of Carboxyfluoresceins
gioisomeric purity, respectively. Large-scale fractional a new key intermediate (Scheme 2). We subjected 13 to the
crystallization gave benzophenones 5 and 6 in isolated same reaction conditions with the same resorcinol deriva-
yields of 26 and 36%, respectively. Benzophenone 5 was tives as in Scheme 1. We obtained difluoresceins 15, 16, and
further recrystallized from H O to obtain 100% purity, 18, with the same competing reaction. In the first case, the
2
hence the lower yield. Pure isomers of 5- and 6-carboxy- less desired 5-carboxyfluorescein (8) was formed; alongside
fluoresceins (8 and 7) were then obtained by the reaction difluoresceins 15 and 18, the highly desired analogues of
of resorcinol and benzophenone 5 or 6 in methanesulfonic difluoresceins 14 and 17, respectively, were also obtained.
acid at room temperature in yields of more than 95% (see However, we were not successful in separating difluorescein
Figure 2). Carboxyfluoresceins 8 and 7 were purified by 14 and 15 despite numerous attempts. When dibenzo-
simple precipitation without column chromatography. The phenone 13 was allowed to react with an excess of 2-chloro-
overall yields starting from trimellitic anhydride (4) and 4-methoxybenzene-1,3-diol for 3 d at room temperature, di-
resorcinol were 25% for 6-carboxyfluorescein (7) and 35% fluoresceins 17 and 18 were obtained in yields of 23 and
for 5-carboxyfluorescein (8).
21%, respectively. As anticipated, NMR analysis of di-
To further examine the scope of the synthetic route we fluorescein 18 showed two peaks originating from the meth-
1
treated benzophenone 6 with 2,4-difluorobenzene-1,3- oxy groups. They were detected in the H NMR spectrum
[
15]
[2]
diol, 2-chloro-4-methoxybenzene-1,3-diol, and naphth- in a 2:1 ratio. They arise from the syn and anti isomers (as
alene-1,3-diol. Using the same reaction conditions, only shown for the mixture of difluoresceins 16 syn and 16 anti
varying the reaction time and purification technique, com- in Scheme 2, see below) and originate from the locked con-
pounds 9–11 were obtained in yields of 79, 60, and 28%, formation at room temperature. The condensation reaction
respectively. To our surprise, in each of these reaction mix- between naphthalene-1,3-diol and dibenzophenone 13 gave
tures 5-carboxyfluorescein (8) was detected as a side-prod- crude difluorescein 16 in 86% yield. LC–MS of the reaction
uct. It was not isolated during work-up; however, it was mixture shows two peaks, both with the same mass ion
+
+
detected by LC–MS and TLC analysis. We contemplate that peak at m/z = 687.1 (calcd. for C H O 687.1[M + H ])
42
23 10
it is formed in a competing side-reaction between two mol- in a 1:1 ratio. Based on these data and the NMR analysis,
ecules of benzophenone 6. This is confirmed by the decreas- we assigned the structures to compounds 16 syn and 16
ing yields achieved on going from 9 to 11, in line with the anti. Separation of the two isomers is not within the scope
decreasing nucleophilicity of the corresponding resorcinols. of this study.
These findings encouraged us to further explore the
For further confirmation of the structures of benzo-
chemistry shown in Scheme 1. Using only resorcinol and phenones 5 and 6 as well as dibenzophenone 13, X-ray
pyromellitic dianhydride (12) we obtained the only structures were obtained (Figure 3, Figures S1–S3 in the
known derivative described of this type in the literature: Supporting Information, and X-ray data given in ref.[ ).
Difluorescein (one of the components of Fluoro- Crystals suitable for single-crystal X-ray diffraction analysis
20]
[
8,17,19]
Jade B).
This was obtained via dibenzophenone 13 as were obtained for 6 and 13 by slow evaporation of a solu-
1
Figure 2. Aromatic H NMR (1 mm NaOD in D
2
O) spectral regions of a) 5-carboxyfluorescein (8) and b) 6-carboxyfluorescein (7).
Eur. J. Org. Chem. 2015, 7301–7309
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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P. Hammershøj, M. H. Clausen et al.
FULL PAPER
Scheme 2. Synthetic route to difluorescein derivatives 14–18.
tion of the corresponding compound in a mixture of H O has the same, almost perpendicular, angles between the
2
and methanol at room temperature. Benzophenone 5 was plane of the inner aromatic ring and the planes of the outer
crystallized from H O. Selected crystallographic data are aromatic rings of 82.6 and 82.2°. It adopts a conformation
2
shown in Table S1 in the Supporting Information. In the with a dihedral angle of 0.5° between the planes of the two
crystal structures of benzophenones 5 and 6, the aromatic outer aromatic rings.
rings are essentially planar and lie almost perpendicular to
each other with an angle between the two planes of 84.4 Table 1. Spectroscopic and photophysical data of mixed fluorescein
and 85.9°, respectively. Thus, the structures adopt a confor- derivatives 9–11[ and mixed dual fluorescein derivatives 16–18.
a]
[a]
mation equivalent to that of 2-(2,4-dihydroxybenzoyl)-
benzoic acid, reported by Li et al. (almost perpendicular
with an angle of 86.8°).[ In addition, dibenzophenone 13
abs[b]
em[c]
[d]
fl[e] [%] Bleaching[f] [%]
λ
max
[nm]
λ
max
[nm]
pK
a
Φ
2[
3
8
9
1
g]
522
492
492
508
507
502
550
517
516
531
530
521
–
–
–
9
17
5
1
1
–
–
–
–
21]
[h]
4.8
6.4
6.9
6.3
5.6
3.9, 6.9
3.9, 7.3
2.7, 4.5
2.7, 4.3
2.9, 6.0
2.9, 6.5
92
92
11
73
63
0.5
0.1
0.4
0.3
[h]
0
1
6
1
1
452[
i]
517
537
[i]
[i]
5
05
[
i]
[i]
[i]
1
7
449
93
518
524
4
[i]
[i]
[i]
1
8
451
04
516
533
0.2
0.1
–
5
–
a
[a] Determined in phosphate buffer at pH 9.0, except for pK data
or unless otherwise stated. [b] Absorption maximum. [c] Fluores-
cence maximum. [d] Data obtained from the double-reciprocal plot
of pH vs. emission intensity titration curve. [e] Quantum yield de-
termined relative fluorescein in 0.1 m NaOH (Φfl = 90) as standard.
[f] Percentage of fluorescence lost after 40 min of irradiation in a
spectrofluorimeter at the wavelength of maximum excitation using
450 W Xe arc lamp. [g] Data obtained for 6-carboxy-4Ј,5Ј-dichloro-
Figure 3. X-ray structures of 2-(2,4-dihydroxybenzoyl)terephthalic
acid (5; left), 4-(2,4-dihydroxybenzoyl)isophthalic acid (6; middle)
and 2,5-bis(2,4-dihydroxybenzoyl)terephthalic acid (13; right). El-
lipsoids drawn at the 50% probability level. All hydrogen atoms
and coordinating solvent molecules have been omitted for clarity.
[22]
2Ј,7Ј-dimethoxyfluorescein, succinimidyl ester from ref. [h] Data
obtained from ref.[ [i] Determined at pH 5.3.
15]
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Large-Scale Synthesis of Carboxyfluoresceins
Figure 4. Top: Normalized absorption and emission spectra of compounds 9–11. Conditions: phosphate buffer at pH 9.0. Bottom: pH
vs. fluorescence emission intensity titration curves in phosphate buffer at pH values between 1 and 9 showing the pH dependence of 9
(left), 10 (middle), and 11 (right).
We next examined the photophysical properties of the
synthesized compounds. The maximum wavelengths of ab-
abs
em
sorption (λmax ) and fluorescence (λmax ), pKa values,
fluorescence quantum yields (Φ ), and photostabilities are
fl
summarized in Table 1 for compounds 9–11 and 16–18. For
comparison, the photophysical properties of carboxy-JOE
(
2), carboxy-oregon green (3), and 5-carboxyfluorescein (8)
[
15,22]
are also included.
Data for carboxy-SNAFL-1 (1) have
not been included due to a large difference in structure
compared with fluorescein, and hence incomparable prop-
erties. The absorption and fluorescence spectra of 9–11 re-
corded at pH 9 are shown in Figure 4 together with their
pK profiles.
a
In general, transforming the xanthene framework of 5-
carboxyfluorescein (8) into compounds 9–11 results in a
bathochromic shift of both the absorption and emission
maxima. A bathochromic shift is also observed for com-
pound 11 in comparison with the known carboxy-oregon
green (3), whereas a hypsochromic shift is observed for
compound 10 in comparison with the known carboxy-JOE
(
2).
The absorption and emission spectra of difluoresceins
6–18 at various pH values are shown in Figure 5 and their
1
pK profiles in Figure 6. The difluorescein derivatives show
a
a bathochromic shift of the absorption maxima with in-
creasing pH and double pK profiles that are irradiation-
a
dependent. The pH-dependent emission starts at low pH
abs
values, an OFF state, when irradiated at λmax = 450 nm,
a maximum fluorescence is observed at around pH 5 for 16,
pH 3 for 17, and pH 4 for 18, the ON state, and at high pH
values there is a drop in emission, another OFF state. The emission spectra recorded at λmax = 450 nm at various pH values.
Figure 5. Absorption spectra of difluorescein derivatives 16 (top),
7 (middle), and 18 (bottom) and the corresponding fluorescence
1
abs
Eur. J. Org. Chem. 2015, 7301–7309
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P. Hammershøj, M. H. Clausen et al.
FULL PAPER
Figure 6. pH dependence of difluorescein derivatives 16 (left), 17 (middle), and 18 (right). pH vs. fluorescence emission intensity titration
curves in a phosphate buffer at various pH values and excitation wavelengths. Top: 450 nm; bottom: 504 nm for 16 and 18, 490 nm for
17.
pH-dependent emission changes after irradiation at λmaxabs When [D
.50 ppm for H NMR and δ = 39.43 ppm for C NMR were used.
When 1 mm NaOD in D O was used as solvent, the residual peak
]DMSO was used as solvent, reference values of δ =
6
1
13
2
=
490 nm for 16 and 18, with the ON state at pH Ͼ 8 (with
2
a small increase starting at pH Ͼ 3.5), whereas 17 has the
same pH profile after irradiation at both λmax = 450 and
1
abs
was used as internal reference: δ = 4.79 ppm for H NMR spectra.
Melting points were measured with a Buch & Holm melting point
apparatus. TLC was performed on Merck aluminium sheets pre-
coated with silica gel 60 F254. Gravity-feed column chromatog-
raphy was performed on Merck kieselgel 60 (0.040–0.063 mm).
490 nm. The OFF/ON/OFF pH-dependent emission could
be suitable for many applications, however, the quantum
yields of the derivatives are very low, which indicates high
intramolecular self-quenching.
2-(2,4-Dihydroxybenzoyl)terephthalic Acid (5) and 4-(2,4-Dihydrox-
ybenzoyl)isophthalic Acid (6): 1,3-Dioxo-1,3-dihydroisobenzofuran-
Conclusions
5
0
2
-carboxylic acid (4; 20 g, 0.104 mol) and resorcinol (23 g,
.208 mol) in methanesulfonic acid (100 mL) were placed in a
50 mL conical flask equipped with a reflux condenser and a mag-
We have developed a new, simple, and highly efficient
route for the large-scale synthesis of regioisomerically pure
netic stirrer bar. The reaction mixture was stirred at 80 °C over-
night and then added to ice–water (500 mL) with stirring and fil-
tered. The solid residue was heated at reflux in EtOH (200 mL),
5- and 6-carboxyfluoresceins. By using fractional crystalli-
zation, we were able to separate two regioisomers of a key
intermediate. These were then converted in high yield into H O was added until precipitation, and the mixture cooled to room
2
the corresponding carboxyfluoresceins. Further extending temperature, filtered, and dried in vacuo to yield 36.5 g (93%) of
the scope of the method, we used one of the regioisomers crude 5(6)-carboxyfluorescein as an orange powder (only com-
pound detected on LC–MS). The crude compound was used with-
out further purification. H O (200 g) and NaOH (200 g) were
2
to synthesize new asymmetrical 5- or 6-carboxyfluoresceins
that are otherwise difficult to prepare by general methods.
In addition, our method provides useful access to difluores-
ceins, which were demonstrated to exhibit unique character-
istics, for example, highly pH-dependent fluorescence, al-
though their quantum yields were poor.
placed in a 500 mL conical flask equipped with a reflux condenser
and a magnetic stirrer bar under heat evolution. Crude 5(6)-
carboxyfluorescein was added to the warm mixture, which was then
stirred at 80 °C overnight after which time the solution had become
clear and almost colorless. The solution was added to ice (300 g)
and cooled with further ice. A 12 m HCl aqueous solution was
added slowly with stirring until a white compound precipitated
Experimental Section
(
pH 1–2). The mixture was left at 5 °C overnight, filtered, and dried
General: Unless otherwise stated, all starting materials were ob-
tained from commercial suppliers and used as received. Solvents
were of analytical-grade and used as received. High-resolution
mass spectra were recorded with an Ultimate 3000 Dionex UHPLC
instrument and a Bruker Maxis 3G QTOF ESI-MS. Reversed
phase analytical LC–MS was carried out with a Waters Acquity
Ultra Performance LC–MS instrument. NMR spectra were re-
corded by using a Varian Mercury 300 MHz spectrometer or a
Bruker 400 MHz spectrometer. Chemical shifts are given in ppm
and coupling constants in Hz; the field is indicated in each case.
in vacuo to yield a crude mixture of isomers (ca. 1:1 ratio) as an
off-white solid. The mixture was fractionally crystallized by dissolv-
ing the mixture in MeOH (100 mL) and subsequently adding H O
2
(3 L). Small crystals started to form on the surface of the solution
overnight, which was left to stand at room temp. for 1 week in an
open Erlenmeyer flask. The crystals were collected and the mother
liquor was extracted with diethyl ether. The ether phase was evapo-
rated to dryness and crystallized by using the same procedure as
2
before in MeOH/H O. The combined solids (benzophenone 6) were
recrystallized two to three times, each time combining the mother
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Large-Scale Synthesis of Carboxyfluoresceins
liquor (containing mostly benzophenone 5) to yield 11.5 g (36%)
of benzophenone 6. Benzophenone 5 was isolated by combining the
evaporated ether phases and crystallizing them four to five times in
6.47 (dd, J = 7.5, 2.2 Hz, 2 H), 6.33 (s, 1 H) ppm. 13C NMR
[101 MHz, D O (NaOD)]: δ = 180.42, 176.93, 174.50, 173.99,
162.98, 155.72, 155.05, 138.58, 138.12, 137.14, 131.55, 130.83,
2
H
2
O to yield 8.3 g (26%) of 5.
130.25, 129.84, 129.51, 129.43, 128.57, 126.71, 126.26, 124.66,
+
1
C
C
21.20, 111.73, 109.80, 103.14, 101.30 ppm. MS (ESI ): calcd. for
2-(2,4-Dihydroxybenzoyl)terephthalic Acid (5): M.p. 271–274 °C
+
+
1
25
H
H
15
O
O
7
427.1 [M + H] ; found 427.1. HRMS (ESI): calcd. for
(decomp.). H NMR (400 MHz, [D
6
]DMSO): δ = 13.54 (s, 2 H),
+
+
25
15
7
427.0812 [M + H] ; found 427.0835.
12.01 (s, 1 H), 10.75 (s, 1 H), 8.16 (dd, J = 8.1, 1.7 Hz, 1 H), 8.09
(
1
d, J = 8.0 Hz, 1 H), 7.85 (d, J = 1.4 Hz, 1 H), 6.99 (d, J = 8.7 Hz,
H), 6.34–6.29 (m, 2 H) ppm. 13C NMR (101 MHz, [D
]DMSO):
δ = 199.41, 166.74, 166.48, 165.58, 164.72, 140.70, 135.16, 134.30,
4
-(5-Chloro-6-hydroxy-7-methoxy-3-oxo-3H-xanthen-9-yl)iso-
6
phthalic Acid (10): The compound was prepared as in the case of
compound 7, starting from 4-(2,4-dihydroxybenzoyl)isophthalic
acid (6; 300 mg, 0.99 mmol), 2-chloro-4-methoxybenzene-1,3-diol
1
33.81, 130.91, 130.84, 128.38, 113.61, 108.91, 103.02 ppm. MS
+
+
+
(ESI ): calcd. for C15
H
11
O
7
303.0 [M + H] ; found 302.9. HRMS
(
200 mg, 1.15 mmol), and methanesulfonic acid (10 mL). The crude
ESI): [M + H]+ calcd. for C15
H
O
+
303.0499; found 303.0503.
(
11
7
compound was purified by silica gel dry-column vacuum
chromatography and was performed by dissolving the crude com-
pound in MeOH and 2 drops of a 12 m NaOH aqueous solution
4
-(2,4-Dihydroxybenzoyl)isophthalic Acid (6): M.p. 265–267 °C (de-
1
6
comp.). H NMR (400 MHz, [D ]DMSO): δ = 13.48 (s, 1 H), 12.00
(
s, 1 H), 10.76 (s, 1 H), 8.51 (d, J = 1.7 Hz, 1 H), 8.23 (dd, J = 7.9, followed by evaporation on Celite in vacuo using 2% AcOH in
1
6
1
1
(
(
.7 Hz, 1 H), 7.56 (d, J = 7.9 Hz, 1 H), 6.96 (d, J = 8.8 Hz, 1 H), CH
.45–6.17 (m, 1 H) ppm. 13C NMR (101 MHz, [D
]DMSO): δ =
was re-precipitated in NaOH/HCl, filtered, and dried in vacuo,
99.20, 166.03, 165.91, 165.12, 164.13, 143.83, 134.58, 132.84,
2
yield 265 mg, 60 %, m.p. Ͼ300 °C. H NMR [400 MHz, D O
2 2
Cl /MeOH in a gradient with 5% increments. The compound
6
1
31.79, 130.68, 129.72, 127.95, 113.10, 108.43, 102.49 ppm. MS (NaOD)]: δ = 8.21 (d, J = 1.6 Hz, 1 H), 8.04 (dd, J = 7.9, 1.7 Hz,
+
+
+
ESI ): calcd. for C15
ESI): calcd. for C15
H
11
O
7
303.0 [M + H] ; found 302.9. HRMS
1 H), 7.27 (d, J = 7.9 Hz, 1 H), 7.05 (d, J = 9.2 Hz, 1 H), 6.65 (d,
J = 2.2 Hz, 1 H), 6.59 (dd, J = 9.2, 2.2 Hz, 1 H), 6.31 (s, 1 H), 3.57
+
+
H
11
O
7
303.0499 [M + H] ; found 303.0503.
1
3
(
s, 3 H) ppm. C NMR [101 MHz, D
2
O (NaOD)]: δ = 178.91,
General Method for the Synthesis of Resorcinarenes Ϫ Synthesis of
-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)terephthalic Acid (7): 2-(2,4-
1
74.64, 174.41, 168.26, 157.27, 156.08, 150.98, 150.93, 139.71,
2
1
1
C
37.41, 134.05, 130.38, 130.27, 129.54, 128.57, 122.81, 111.86,
Dihydroxybenzoyl)terephthalic acid (5; 200 mg, 0.66 mmol) and
resorcinol (80 mg, 0.72 mmol) in methanesulfonic acid (5 mL) were
placed in a 20 mL conical flask. The reaction mixture was stirred
at room temp. overnight, added to ice–water (50 mL) whilst stir-
ring, and filtered. The solid residue was dissolved in a 2 m NaOH
aqueous solution (40 mL), precipitated with a 2 m HCl aqueous
solution, and filtered. The crude compound was re-precipitated
+
10.88, 107.82, 103.60, 55.34 ppm. MS (ESI ): calcd. for
+
+
22
H
14ClO
H
8
441.0 [M + H] ; found 441.0. HRMS (ESI): calcd.
for C22
14ClO +
8
441.0372 [M + H] ; found 441.0377.
+
4-(5,7-Difluoro-6-hydroxy-3-oxo-3H-xanthen-9-yl)isophthalic Acid
(11): The compound was prepared as in the case of compound 7,
starting from 4-(2,4-dihydroxybenzoyl)isophthalic acid (6; 600 mg,
1.98 mmol), 2,4-difluorobenzene-1,3-diol (440 mg, 3.9 mmol), and
methanesulfonic acid (10 mL). The crude compound was purified
first in EtOH/H
2
O and then in NaOH/HCl, filtered, and dried in
1
vacuo, yield 245 mg, 98%, m.p. Ͼ300 °C. H NMR [400 MHz,
D
2
O (NaOD)]: δ = 8.04 (dd, J = 8.0, 1.7 Hz, 1 H), 7.83 (d, J = by silica gel dry-column vacuum chromatography and was per-
.0 Hz, 1 H), 7.75 (d, J = 1.3 Hz, 1 H), 7.21–7.13 (m, 2 H), 6.64–
formed by dissolving the crude compound in MeOH and 2 drops
.54 (m, 4 H) ppm. 13C NMR [101 MHz, D
O (NaOD)]: δ = of a 12 m NaOH aqueous solution and evaporation on Celite in
8
6
1
1
2
80.61, 174.86, 174.35, 158.85, 158.65, 141.76, 137.05, 131.55,
31.30, 130.24, 129.72, 128.10, 122.87, 112.50, 103.52 ppm. MS
2 2
vacuo using 2% AcOH in CH Cl /MeOH in a gradient with 5%
increments. The compound was re-precipitated in NaOH/HCl, fil-
tered, and dried in vacuo, yield 237 mg, 28%, m.p. 259–265 °C (de-
+
+
+
(
(
ESI ): calcd. for C21
ESI): calcd. for C21
H
13
O
7
377.1 [M + H] ; found 377.0. HRMS
+
+
1
H
13
O
7
377.0655 [M + H] ; found 377.0676.
comp.). H NMR [400 MHz, D O (NaOD)]: δ = 8.16 (d, J =
2
1
7
6
1
.4 Hz, 1 H), 7.97 (d, J = 7.9 Hz, 1 H), 7.25 (d, J = 7.9 Hz, 1 H),
4
-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)isophthalic Acid (8): The
.09 (d, J = 9.4 Hz, 1 H), 6.74 (dd, J = 11.6, 1.3 Hz, 1 H), 6.56–
compound was prepared as in the case of compound 7, starting
from 4-(2,4-dihydroxybenzoyl)isophthalic acid (6; 0.5 g,
.65 mmol), resorcinol (0.2 g, 1.82 mmol), and methanesulfonic
1
3
.49 (m, 2 H) ppm. C NMR [101 MHz, D
2
O (NaOD)]: δ =
82.02, 174.44, 174.43, 158.49, 139.54, 137.67, 133.65, 131.55,
1
1
130.02, 129.64, 128.65, 124.10, 113.14, 108.53, 108.32, 103.71 ppm.
acid (10 mL), yield 602 mg, 96%, m.p. Ͼ300 °C. H NMR
400 MHz, D O (NaOD)]: δ = 8.21 (d, J = 1.6 Hz, 1 H), 8.00 (dd,
J = 7.9, 1.7 Hz, 1 H), 7.24 (d, J = 7.9 Hz, 1 H), 7.13 (d, J = 9.2 Hz,
1
9
+
2
F NMR [282 MHz, D O (NaOD)]: δ = 127, 154 ppm. MS (ESI ):
[
2
+
+
calcd. for C21
H
11
F
H
2
O
7
413.0 [M + H] ; found 413.0. HRMS
+
+
(
ESI): calcd. for C21
11
F
2
O
7
413.0467 [M + H] ; found 413.0478.
2
H), 6.58 (dd, J = 9.2, 2.2 Hz, 2 H), 6.54 (d, J = 2.2 Hz, 2 H) ppm.
C NMR [101 MHz, D O (NaOD)]: δ = 180.59, 174.74, 174.49,
2
13
2
,5-Bis(2,4-dihydroxybenzoyl)terephthalic Acid (13): Benzo[1,2-
1
1
58.67, 139.59, 137.36, 134.08, 131.40, 130.05, 129.45, 128.43,
22.88, 112.19, 103.58 ppm. MS (ESI ): calcd. for C21
c:4,5-cЈ]difuran-1,3,5,7-tetraone (12; 5 g, 22.9 mmol) and resorcinol
10 g, 91.6 mmol) in methanesulfonic acid (60 mL) were placed in
+
+
H
13
O
7
377.1
(
+
+
[M + H] ; found 377.1. HRMS (ESI): calcd. for C21
H
13
O
7
a 100 mL conical flask equipped with a reflux condenser and a
magnetic stirrer bar. The reaction mixture was stirred at 80 °C over-
night, added to ice–water (500 mL) whilst stirring, and filtered. The
solid residue was heated at reflux in EtOH (200 mL), cooled to
room temperature, filtered, and dried in vacuo to yield 12 g of
crude 2,5-bis[3,6-dihydroxy-9-(methoxysulfonyl)-9H-xanthen-9-yl]
terephthalic acid as an orange powder. The crude compound was
+
377.0655 [M + H] ; found 377.0667.
4-(5-Hydroxy-9-oxo-9H-benzo[a]xanthen-12-yl)isophthalic Acid (9):
The compound was prepared as in the case of compound 7, starting
from 4-(2,4-dihydroxybenzoyl)isophthalic acid (6; 500 mg,
1.65 mmol), naphthalene-1,3-diol (500 mg, 3.12 mmol), and meth-
1
anesulfonic acid (10 mL), yield 557 mg, 79%, m.p. Ͼ300 °C.
NMR [400 MHz, D
H
2
2
O (NaOD)]: δ = 8.22 (d, J = 1.5 Hz, 1 H), used without further purification. H O (100 g) and NaOH (100 g)
8
7
.10 (dd, J = 8.1, 1.3 Hz, 1 H), 7.78 (dd, J = 7.9, 1.8 Hz, 1 H),
.37–7.28 (m, 1 H), 7.08 (ddd, J = 8.6, 7.1, 1.5 Hz, 1 H), 6.88 (d,
were placed in a 250 mL conical flask equipped with a reflux con-
denser and a magnetic stirrer bar under heat evolution. To the
J = 8.5 Hz, 1 H), 6.81–6.74 (m, 1 H), 6.71 (d, J = 7.9 Hz, 1 H), warm mixture was added crude 2,5-bis[3,6-dihydroxy-9-(meth-
Eur. J. Org. Chem. 2015, 7301–7309
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7307

P. Hammershøj, M. H. Clausen et al.
FULL PAPER
oxysulfonyl)-9H-xanthen-9-yl]terephthalic acid and the mixture
was stirred at 80 °C overnight after which time the solution had
become clear and almost colorless. The solution was added to ice
zene-1,3-diol (90 mg, 0.52 mmol), and methanesulfonic acid
1
(3 mL). Compound 17: Yield: 34 mg, 23%, m.p. Ͼ300 °C.
NMR [400 MHz, D
H
2
O (NaOD)]: δ = 8.04 (s, 1 H), 7.83 (s, 1 H),
(
200 g) and cooled with further ice. A 12 m HCl aqueous solution
7.35 (dd, J = 19.6, 9.2 Hz, 2 H), 7.26 (d, J = 9.2 Hz, 1 H), 6.80 (d,
J = 2.2 Hz, 1 H), 6.72–6.70 (m, 1 H), 6.70–6.68 (m, 2 H), 6.66 (d,
J = 2.3 Hz, 1 H), 6.65–6.64 (m, 2 H), 6.54 (s, 1 H), 3.74 (s, 3
H) ppm. C NMR [101 MHz, D O (NaOD)]: δ = 180.75, 179.11,
2
173.68, 173.51, 168.39, 158.90, 158.86, 157.84, 157.52, 155.45,
151.12, 151.08, 140.55, 140.25, 132.90, 132.80, 131.44, 130.51,
was added slowly until a white compound precipitated (pH 1–2).
The mixture was left overnight at 5 °C, filtered, and the solid was
dried in vacuo to yield a white powder, yield 5.5 g, 55%, m.p. 283–
1
3
1
2
86 (decomp.). H NMR (500 MHz, [D
6
]DMSO): δ = 13.70 (s, 2
H), 11.92 (s, 2 H), 10.91 (s, 2 H), 7.90 (s, 2 H), 7.17 (d, J = 8.7 Hz,
H), 6.49–6.15 (m, 4 H) ppm. 13C NMR (101 MHz, [D
]DMSO): 130.33, 130.09, 123.18, 123.13, 122.99, 112.45, 112.42, 112.07,
6
2
+
δ = 197.95, 165.61, 165.34, 164.12, 140.97, 134.82, 132.62, 128.79, 111.26, 107.90, 103.95, 103.67, 103.63, 55.57 ppm. MS (ESI ):
+
+
+
+
1
4
4
13.12, 108.55, 102.54 ppm. MS (ESI ): calcd. for C25
H
H
15
O
10
calcd. for C35
(ESI-TOF): calcd. for C35
Compound 18: Yield: 34 mg, 21%, m.p. Ͼ300 °C. H NMR
400 MHz, D O (NaOD)]: δ = 7.96 (s, 1 H), 7.34 (d, J = 9.2 Hz, 1
H), 6.84 (d, J = 2.2 Hz, 1 H), 6.72 (dd, J = 9.2, 2.2 Hz, 1 H), 6.57
H20ClO11 651.9 [M + H] ; found 651.0. HRMS
+
+
+
39.1 [M + H] ; found 439.0. HRMS (ESI): calcd. for C25
15
O
10
H20ClO11 651.068; found 651.0700.
+
1
39.0659 [M + H] ; found 439.0658.
[
2
General Method for the Synthesis of Difluoresceins 14–18 Ϫ Mix-
ture of 2-(5,7-Difluoro-6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-(6-
hydroxy-3-oxo-3H-xanthen-9-yl)terephthalic Acid (14) and 2,5-
Bis(5,7-difluoro-6-hydroxy-3-oxo-3H-xanthen-9-yl)terephthalic Acid
13
(
s, 1 H), 3.70 (s, 3 H) ppm. C NMR [101 MHz, D
2
O (NaOD)]: δ
=
179.10, 179.05, 173.58, 173.46, 168.33, 168.27, 157.46, 157.43,
1
1
5
55.43, 155.37, 151.03, 150.91, 140.55, 140.52, 132.90, 132.75,
30.53, 122.91, 111.97, 111.92, 111.20, 107.83, 107.78, 103.70,
(15): A mixture of 2,5-bis(2,4-dihydroxybenzoyl)terephthalic acid
(13; 100 mg, 0.23 mmol) and 2,4-difluorobenzene-1,3-diol (100 mg,
+
+
5.54, 55.00 ppm. MS (ESI ): calcd. for C36
H
21Cl
2
O
12 716.4 [M
0
.7 mmol) in methanesulfonic acid (20 mL) were placed in a 50 mL
+
+
+
H] ; found 715.0. HRMS (ESI-TOF): calcd. for C36
H
21Cl
2
O
12
conical flask equipped with a magnetic stirrer bar and the mixture
was heated at 50 °C overnight. 2,4-Difluorobenzene-1,3-diol
715.0405; found 715.0411.
(100 mg, 0.7 mmol) was added and the reaction mixture was stirred
for 2 d at 50 °C, added to ice–water (50 mL) whilst stirring, and
filtered. The solid residue was dissolved in a 2 m NaOH aqueous
solution (40 mL), precipitated with a 2 m HCl aqueous solution,
and filtered. The crude compound was purified by dry-column vac-
uum chromatography (5% AcOH in toluene to 40% EtOH in 5%
AcOH in toluene with 4% increments) to give a mixture of com-
Acknowledgments
This work was supported by Novo Nordisk Foundation, Biotech-
nology Based Synthesis and Production Research.
+
pounds 14 and 15. Data for 14: MS (ESI ): calcd. for
+
+
[1] J. Han, K. Burgess, Chem. Rev. 2010, 110, 2709–2728.
[2] D. A. Tsybulsky, M. V. Kvach, I. A. Stepanova, V. A. Korshun,
V. V. Shmanai, J. Org. Chem. 2012, 77, 977–984.
34 17 2
C H F O10 623.1 [M + H] ; found, 623.0. Data for 15: MS
(
+
+
+
ESI ): calcd. for C34
15 4
H F O10 659.1 [M + H] ; found 659.1.
[
3] M. Adamczyk, C. M. Chan, J. R. Fino, P. G. Mattingly, J. Org.
2
,5-Bis(5-hydroxy-9-oxo-9H-benzo[a]xanthen-12-yl)terephthalic
Chem. 2000, 65, 596–601.
Acid (16 anti and 16 syn): Compounds 16 were prepared as in the
case of compound 14, starting from 2,5-bis(2,4-dihydroxybenzoyl)-
terephthalic acid (13; 500 mg, 1.14 mmol), naphthalene-1,3-diol
[
[
4] M. Y. Berezin, S. Achilefu, Chem. Rev. 2010, 110, 2641–2684.
5] Y. Urano, M. Kamiya, K. Kanda, T. Ueno, K. Hirose, T. Na-
gano, J. Am. Chem. Soc. 2005, 127, 4888–4894.
(500 mg, 3.12 mmol), and methanesulfonic acid (10 mL). The reac-
[
6] Y. Ueno, G.-S. Jiao, K. Burgess, Synthesis 2004, 2591–2593.
7] C. C. Woodroofe, M. H. Lim, W. Bu, S. J. Lippard, Tetrahedron
2005, 61, 3097–3105.
tion mixture was stirred at 80 °C for 2 h. Purification was per-
formed by silica gel dry-column vacuum chromatography (2%
[
AcOH in CH
2
Cl
2
/MeOH in a gradient with 5% increments). Crude
[8] F. M. Rossi, J. P. Kao, Bioconjugate Chem. 1997, 8, 495–497.
[9] G.-S. Jiao, J. W. Han, K. Burgess, J. Org. Chem. 2003, 68, 8264–
1
yield: 665 mg, 85 %, m.p. Ͼ300 °C. H NMR [400 MHz, D
2
O
8267.
(
(
NaOD)]: δ = 8.35–8.25 (m, 2 H), 7.82 (d, J = 8.3 Hz, 0.75 H), 7.78
s, 1.25 H), 7.76 (s, 0.75 H), 7.60–7.53 (m, 2 H), 7.52–7.46 (m, 0.75
[
10] T. Ueno, Y. Urano, K. Setsukinai, H. Takakusa, H. Kojima,
K. Kikuchi, K. Ohkubo, S. Fukuzumi, T. Nagano, J. Am.
Chem. Soc. 2004, 126, 14079–14085.
11] T. Mineno, T. Ueno, Y. Urano, H. Kojima, T. Nagano, Org.
Lett. 2006, 8, 5963–5966.
12] E. Azuma, N. Nakamura, K. Kuramochi, T. Sasamori, N. Tok-
itoh, I. Sagami, K. Tsubaki, J. Org. Chem. 2012, 77, 3492–
5000.
H), 7.46–7.41 (m, 2.50 H), 7.29 (d, J = 9.1 Hz, 1.25 H), 7.08 (d, J
9.1 Hz, 0.75 H), 6.79 (dd, J = 9.1, 2.3 Hz, 1.25 H), 6.72 (d, J =
.3 Hz, 1.25 H), 6.71–6.69 (m, 1.125 H), 6.68 (d, J = 2.3 Hz, 0.375
=
[
2
1
3
H), 6.66 (s, 1.25 H), 6.63 (s, 0.75 H) ppm. C NMR [101 MHz,
O (NaOD)]: δ = 180.79, 180.65, 177.12, 176.96, 172.71, 172.64,
63.54, 163.48, 156.09, 156.01, 154.54, 154.39, 140.74, 140.70,
[
D
2
1
1
1
1
1
C
37.34, 137.23, 131.87, 131.81, 130.63, 130.60, 130.07, 129.85, [13] M. H. Lyttle, T. G. Carter, R. M. Cook, Org. Process Res. Dev.
29.50, 129.40, 129.36, 127.19, 127.01, 126.54, 126.48, 124.97,
24.92, 121.58, 121.53, 111.94, 111.71, 110.31, 110.27, 103.29,
03.22, 101.38, 101.35, 99.99 ppm. MS (ESI ): calcd. for
23
H O10 687.1 [M + H] ; found 687.1. HRMS (ESI-TOF):
2
001, 5, 45–49.
[
[
[
14] a) P. Khanna, E. F. Ullman, U.S. Patent 4,318,846, 1982; b) P.
Khanna, E. F. Ullman, Eur. Pat. Appl. 0050684, 1982.
15] W. Sun, K. R. Gee, D. H. Klaubert, R. P. Haugland, J. Org.
Chem. 1997, 62, 6469–6475.
+
+
+
42
+
calcd. for C42
23
H O10 687.1286; found 687.1289.
16] L. C. Schmued, C. C. Stowers, A. C. Scallet, L. Xu, Brain Res.
2
005, 1035, 24–31.
2
-(5-Chloro-6-hydroxy-7-methoxy-3-oxo-3H-xanthen-9-yl)-5-
[
[
17] J. A. Bishop, A. Wagner, J. Lumin. 1981, 26, 189–195.
18] S. Chen, Y. Hong, Y. Liu, J. Liu, C. W. T. Leung, M. Li,
R. T. K. Kwok, E. Zhao, J. W. Y. Lam, Y. Yu, B. Z. Tang, J.
Am. Chem. Soc. 2013, 4926–4929.
(
6-hydroxy-3-oxo-3H-xanthen-9-yl)terephthalic Acid (17) and 2,5-
Bis(5-chloro-6-hydroxy-7-methoxy-3-oxo-3H-xanthen-9-yl)tere-
phthalic Acid (18): The compounds were prepared as in the case of
compounds 14 and 15, starting from 2,5-bis(2,4-dihydroxybenzoyl)-
terephthalic acid (13; 100 mg, 0.23 mmol), 2-chloro-4-methoxyben-
[19] L. Xu, T. Heinze, A. Pogge, W. Slikker, L. Schmued, J. Liq.
Chromatogr. Relat. Technol. 2004, 27, 1627–1640.
7308
www.eurjoc.org
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 7301–7309

Large-Scale Synthesis of Carboxyfluoresceins
[
20] Compound 6: 4(C15
H
10
O
7
)·2H
2
O·CH
3
OH, M
r
= 1276.99, mo-
1 o o 2
R [F Ͼ4σ(F )] = 0.0431, wR [all data] = 0.1887. CCDC-
noclinic, space group C2/c, a = 31.481(2), b = 6.9194(4), c =
979564 (for 6), -979565 (for 5), and -979566 (for 13) contain
the supplementary crystallographic data for this paper. These
data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
3
2
7.107(2) Å, β = 111.838(1)°, V = 5480.8(6) Å , Z = 4, total
reflections: 29625, unique reflections: 6744, Rint = 0.1670, GOF
1.029, R [F Ͼ4σ(F )] = 0.0661, wR [all data] = 0.1984. Com-
pound 5: C15 ·H O, M = 320.25, monoclinic, space
group P2 /c, a = 7.0655(3), b = 20.806(1), c = 9.6255(5) Å, β [21]
104.174(5)°, V = 1371.9(1) Å , Z = 4, total reflections: 8324,
unique reflections: 2722, Rint = 0.1670, GOF = 1.137,
[F Ͼ4σ(F )] = 0.0426, wR [all data] = 0.1541. Compound
3: C22 10·2H O, M = 474.36, monoclinic, space group
C2/c, a = 13.2767(1), b = 13.7311(1), c = 11.0008(1) Å, β =
=
1
o
o
2
H
10
O
7
2
r
1
Y. Li, F.-Y. Ge, L.-G. Chen, C.-M. Dong, X.-L. Yan, E.-H.
Duan, T. Zeng, Y.-C. Zhang, G.-Y. Bai, Acta Crystallogr., Sect.
E Struct. Reports Online 2005, 61, o1769–o1770.
22] http://www.lifetechnologies.com/order/catalog/product/
C6171MP (12-09-2014).
3
=
R
1
1
o
o
2
[
H
18
O
2
r
Received: August 3, 2015
Published Online: October 16, 2015
3
93.2710(9)°, V = 2002.21(4) Å , Z = 4, total reflections: 17844,
unique reflections: 1997, Rint = 0.1670, GOF = 1.727,
Eur. J. Org. Chem. 2015, 7301–7309
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7309