Mild synthesis of asymmetric 2′-carboxyethyl-substituted fluoresceins

Article abstract of DOI:10.1021/jo702422v

(Chemical Equation Presented) Asymmetric fluoresceins bearing a carboxyethyl group in the chromophoric portion of the dyes were prepared by a reaction of substituted phthalic anhydride with a carboxyethyl substituted resorcinol analogue followed by a condensation with a second resorcinol analogue. In order to avoid an accumulation of symmetric side products, the second step was performed in two substeps: acid-catalyzed formation of a triphenylmethyl intermediate followed by base-catalyzed cyclization which furnished the desired dyes.

Full text of DOI:10.1021/jo702422v

SCHEME 1. Preparation of Fluorescent Conjugates from
2′-Carboxyethyl-Substitited Fluoresceins
Mild Synthesis of Asymmetric
2′-Carboxyethyl-Substituted Fluoresceins
Eugeny A. Lukhtanov* and Alexei V. Vorobiev
Nanogen Inc., 21720 23rd DriVe SE, Suite 150,
Bothell, Washington 98021
elukhtanoV@nanogen.com
ReceiVed NoVember 9, 2007
Asymmetric fluoresceins bearing a carboxyethyl group in
the chromophoric portion of the dyes were prepared by a
reaction of substituted phthalic anhydride with a carboxyethyl
substituted resorcinol analogue followed by a condensation
with a second resorcinol analogue. In order to avoid an
accumulation of symmetric side products, the second step
was performed in two substeps: acid-catalyzed formation
of a triphenylmethyl intermediate followed by base-catalyzed
cyclization which furnished the desired dyes.
reported for 5(6)-carboxyfluorescein,2a,b 4,7,2′,7′-tetrachloro-
5(6)-carboxyfluorescein,3 fluorescein-5(6)-sulfonic acid,4 5(6)-
bromofluorescein,5 and 2′,7′-dichloro-5(6)-carboxyfluorescein.6
In most cases dye mixtures should be transformed into the bis-
acylated lactone form before crystallization. As a result, this
procedure requires additional protection/deprotection steps and
is not universally applicable.
An alternative strategy for the preparation of functionalized
fluoresceins is based on a modification of the xanthenone
fragment of the dyes. Asymmetric 2′-carboxyethyl-substituted
rhodol dyes7,8 are examples of this approach. Mono-2′-carboxy-
ethyl substituted fluoresceins of structure 1 have also been sug-
gested.9 However, to our knowledge, no actual compounds have
been prepared. A useful attribute of these analogues is their
ability to form intramolecular fused lactones 2. Given that the
pKa of the phenolic group for most fluorescein analogues is
below 7, the activity of the lactones should be suitable for
selective reaction with amine containing nucleophiles (Scheme
1). The goal of this study was to develop a reliable chemical
route to monocarboxyethyl functionalized fluorescein analogues
of structure 1.
Fluoresceins bearing tethered functional groups are used in
the preparation of fluorescent conjugates as well as interme-
diates for more elaborate reagents such as dye phosphorami-
dites.1a-e The most commonly used activated fluoresceins are
derived from their amino- or carboxy-substituted analogues
with the substituents located in the carboxyphenyl portion of
the dyes.
Two approaches to the synthesis of asymmetric fluoresceins
have been previously reported. One is the direct condensation
of substituted phthalic anhydrides with a mixture of two
These compounds are typically prepared as mixtures of 5-
and 6-isomers by a reaction of 4-substituted phthalic anhydrides
(such as 1,2,4-benzenetricarboxylic anhydride) with appropriate
resorcinol analogues in the presence of strong acids or ZnCl2.
The isomers can be separated by fractional crystallization as
(2) (a) Rossi, F. M.; Kao, J. P. Y. Bioconjugate Chem. 1997, 8, 495-
497. (b) Ueno, Y.; Jiao, G.-S.; Burgess, K. Synthesis 2004, 2591-2593.
(3) Lyttle, M. H.; Carter, T. G.; Cook, R. M. Org. Process Res. DeV.
2001, 5, 45-49.
(4) Woodroofe, C. C.; Lim, M. H.; Bu, W.; Lippard, S. J. Tetrahedron
2005, 61, 3097-3105.
(5) Jiao, G.-S.; Han, J. W.; Burgess, K. J. Org. Chem. 2003, 68, 8264-
8267.
(1) (a) Theisen, P.; McCollum, C.; Upadhya, K.; Jacobson, K.; Vu, H.;
Andrus, A. Tetahedron Lett. 1992, 33, 5033-5036. (b) Adamczyk, M.;
Chan, C. M.; Fino, J. R.; Mattingly, P. G. J. Org. Chem. 2000, 65, 596-
601. (c) Nelson, P. S.; Kent, M.; Muthini, S. Nucleic Acids Res. 1992, 20,
6253-6259. (d) Jadahav, V. R.; Barawkar, D. A.; Natu, A. A.; Ganesh, K.
N. Nucleosides Nucleotides 1997, 16, 107-114. (e) Su, S.-H.; Iyer, R. S.;
Aggarwal, S. K.; Kalra, K. L. Bioorg. Med. Chem. Lett. 1997, 7, 1639-
1644.
(6) Woodroofe, C. C.; Masalha, R.; Barnes, K. R.; Frederickson, C. J.;
Lippard, S. J. Chem. Biol. 2004, 11, 1659-1666.
(7) Whitaker, J. E.; Haugland, R. P.; Ryan, D.; Hewitt, P. C.; Haugland,
R. P.; Prendergast, F. G. Anal. Biochem. 1992, 207, 267-279.
(8) Smith, G. A.; Metcalfe, J. C.; Clarke, S. D. J. Chem. Soc., Perkin
Trans. 2 1993, 1195-1204.
(9) Khanna P.; Colvin W. U.S. Patent 4,439,356, 1984.
10.1021/jo702422v CCC: $40.75 © 2008 American Chemical Society
Published on Web 02/27/2008
2424
J. Org. Chem. 2008, 73, 2424-2427

SCHEME 2. Synthesis of Substituted Benzophenone Dye
Precursors
130 °C. All of those approaches require elevated (100-250 °C)
temperatures. Among those listed above, methanesulfonic acid
has become the most popular reagent16 since it is a fairly good
solvent and allows the reaction to be carried out at a moderately
low temperature (80-100 °C). Therefore, our initial dye prep-
arations were done in neat methanesulfonic acid. We later found
that methanesulfonic acid can be used in a mixture with tri-
fluoroacetic acid (TFA) to improve the solubility of the starting
materials and facilitate the conversion of the methyl ester to a
free carboxy group. With these initial conditions in our hands,
condensation reactions between benzophenones 9 and resorci-
nols 10 were carried out (Scheme 3, route A). Unexpectedly,
we found that, along with one main product, one or two addi-
tional side products were observed in many reactions. In the
case of the simplest analogue 1a, the side products were isolated
and shown to be unsubstituted fluorescein (11a) and bis-2′,7′-
(carboxyethyl)fluorescein17 (12a). The formation of the sym-
metric side products can be explained by the retro-Friedel-
Crafts fragmentation previously described for similar com-
pounds.14,18
The formation of asymmetric fluoresceins is likely to proceed
via an intermediate generation of a benzonium ion B (Scheme
419), which should exist in equilibrium with carbonium ion C,
followed by a cyclization (dehydration) step to furnish the
hydroxyxanthenone ring. Under the reaction conditions (TFA/
CH3SO3H, 80 °C) the intermediate B is able to reversibly
generate two types of biphenylcarbonyl fragments A eventually
leading to the formation of the symmetric side products 11 and
12. The nature of substituents in both resorcinol parts of the
triphenyl carbocation intermediate should influence which
benzophenone fragments are formed during retro-acylation.
Indeed, the same benzophenone 9b affords a much higher yield
of asymmetric dye when reacted with unsubstituted resorcinol
10a compared to chloro-substituted 10b and 10c (reactions 4,
7, and 10 in Table 1). This can be explained by preferential
formation of benzonium ion B with the positive charge located
in the most electron-rich resorcinol ring, which in the case of
the 9b + 10a combination is the non-chlorinated one. This will
consequently lead to retro-formation of the starting benzophe-
none 9b. The selectivity is lost when both resorcinol rings are
nearly equivalent as seen in reactions 7 (9b + 10b) and 10 (9b
+ 10c) (Table 1).
resorcinol analogues in the presence of a strong acid.10 This
method requires separation of several dyes and suffers from low
product yields. The second approach is based on preparing the
benzophenone precursor by reacting substituted phthalic anhy-
dride with 1 equiv of a resorcinol analogue in the presence of
aluminum chloride.8,9 This is followed by a condensation with
another resorcinol analogue to form the dye. The second method
became the focus of our synthetic strategy because it offered a
regiospecific way of dye assembly. The synthesis of the benzo-
phenone intermediates is shown in Scheme 2. 2-Substitued 1,3-
dimethoxybenzenes 4 were formylated using R,R-dichloro-
methylmethyl ether in the presence of TiCl4 to afford benzal-
dehydes 5. The Knoevenagel condensation with malonic acid
produced cinnamic acids 6. Catalytic reduction (H2/Pd-C) of
the double bond followed by demethylation of the phenolic
groups (HBr/AcOH/H2O or BBr3/CH2Cl2 for 7b) and esterifi-
cation of the carboxy group furnished 2-substituted methyl 1,3-
dihydroxyphenyl-4-propanoates 8 in 73-84% overall yields
(starting from 4). The Friedel-Crafts acylation with phthalic
anhydride (or its substituted analogues) in the presence of AlCl3
generated the desired benzophenone derivatives 9 in 60-80%
yields.
Effects of substituents in the carboxyphenyl ring are less clear.
Our results show that reaction of a pentachloro-substituted
benzophenone 9c with resorcinol 10b (reaction 13) affords 97%
of desired asymmetric dye 1e. In contrast, its trichloro-
substituted analogue 9b, which lacks the R2 chloro substituents
in the carboxyphenyl ring, generates only about 44% of the
(11) Ghatak, N. N; Dutt, S. J. Indian Chem. Soc. 1929, 6, 465-471.
(12) Graichen, C.; Molitor, J. C. J. Assoc. Off. Agric. Chem. 1959, 42,
149-160.
(13) Haugland, R. P.; Whitaker, J. U.S. Patent 4,945,171, 1990.
(14) Burdette, S. C.; Frederickson, C. J.; Bu, W.; Lippard, S. J. J. Am.
Chem. Soc. 2003, 125, 1778-1787.
A number of reaction conditions have been previously
reported for condensation of 2,4-dihydroxy-2′-carboxybenzo-
phenones with resorcinol analogues. They include the follow-
ing: heating neat components9 at 190 °C, sulfuric acid11 at 140
°C, p-toluenesulfonic acid12 at 103 °C, fusion with zinc
chloride9,13,14 at 150-250 °C or methanesulfonic acid15 at 120-
(15) Benson, S. C.; Menchen, S. M.; Theisen, P. D.; Upadhya, K. G.;
Hauser, J. D. U.S. Patent 6,008,379, 1999.
(16) Sun, W.-C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P. J. Org.
Chem. 1997, 62, 6469-6475.
(17) Khanna, P. L.; Ullman, E. F. European Patent 0025912 B1 1986.
(18) Bacci, J. P.; Kearney, A. M.; Van Vranken, D. L. J. Org. Chem.
2005, 70, 9051-9053.
(19) No substituents are shown for simplicity. Not all possible intermedi-
ates are shown for simplicity. The lactone structure of intermediates A, B,
D, and final dye may alternatively exist as a methanesulfonate adduct as
proposed in ref 2b.
(10) Benson, S. C.; Menchen, S. M.; Theisen, P. D.; Hennessey, K. M.;
Furniss, V. C.; Hauser, J. U.S. Patent 6,020,481, 2000.
J. Org. Chem, Vol. 73, No. 6, 2008 2425

SCHEME 3. Two Synthetic Routes to Asymmetric 2′-Carboxyethyl-Substituted Fluoresceins
SCHEME 4. Proposed Mechanism for Dye Formation by
Route A
in the excess of the resorcinol component had only a limited
effect on the amount of the side products (Table 1). We also
attempted to carry out the reactions at lower temperatures (<20
°C). The formation of the carbocation C (Scheme 4) (observed
as a dark-purple intermediate) was moderately fast and achiev-
able at the lower temperatures. However, elevated temperatures
were required to promote the cyclization. Unfortunately, these
conditions also facilitated the undesired retro-Friedel-Crafts
fragmentation and accumulation of the side products.
We discovered that this problem can be overcome if the dye
preparation is carried out in two separate steps (Scheme 3, route
B). The first step is done at 0 °C (TFA, CH3SO3H) for 10-15
h; the resulting lactones 13 are isolated by precipitation in cold
water as off-white or lightly colored solids (due to spontaneous
cyclization). The decrease in reaction temperature resulted in
significant reduction of the acid-catalyzed fragmentation. The
second step is carried out in a warm (20-100 °C) aqueous
solution at a pH of 7-10 followed by acidification to precipitate
the desired 2′-carboxyethyl-fluoresceins 1. Since the cyclization
step proceeds through an anionic form of 13, it is not subject
to retro-fragmentation even at elevated temperatures. The new
conditions increased the fraction of desired asymmetric dyes
up to 94-99% in all tested reactions.
In summary, we have developed a mild two-step synthesis
of asymmetric 2′-carboxyethyl-substituted fluoresceins including
ones that are not easily prepared using traditional approaches.
The developed procedure should also be applicable to the prep-
aration of a variety of other asymmetric fluorescein analogues.
The method is especially useful when harsh acidic treatments
are unacceptable due to either the instability of the intermediates
or undesired, acid-catalyzed chemical rearrangements.
target compound 1c and more than 50% of symmetric byproduct
11c (reaction 7). It is unlikely that such a dramatic change in
reaction specificity is solely due to the electronic effects of the
R2 chloro substituents. One possible explanation is that steric
effects attributable to the presence of the C-7 chloro atom in
the vicinity of the crowded central carbon destabilize the
nonplanar intermediate B, which is required for the fragmenta-
tion reaction. This conclusion is supported by published data
for an asymmetric fluorescein containing a single chloro group
in the C-7 position,20 which was synthesized with no evident
formation of symmetric side products.
Experimental Section
Representative Procedure for Dye Preparation (Route A).
2-[2-(2-Carboxyethyl)-4,7-dichloro-6-hydroxy-3-oxo-3H-xanthen-
9-yl]-3,4,5,6-tetrachlorobenzoic Acid (1e). A suspension of 9c
In order to reduce the formation of symmetric byproducts
we attempted to adjust the reaction conditions. A 2-fold decrease
(20) Lukhtanov, E.; Vorobiev, A. U.S. Patent application US 2006/
0199955 A1 2006.
2426 J. Org. Chem., Vol. 73, No. 6, 2008

TABLE 1. Effect of Reaction Conditions on Yields of 2′-Carboxyethylfluoresceins
distribution of productsa (%)
reaction no.
resorcinol
benzophenone
target product
route
10 to 9 ratio
1
11b
12b
<1
8.6
<0.1
<0.5
<0.5
<0.1
<0.5
19.4
<0.1
0.5
1
2
3
4
5
6
7
8
10a
10a
10a
10a
10a
10a
10b
10b
10b
10c
10c
10c
10b
10b
9a
9a
9a
9b
9b
9b
9b
9b
9b
9b
9b
9b
9c
9d
1a
1a
1a
1b
1b
1b
1c
1c
1c
1d
1d
1d
1e
1f
A
A
B
A
A
B
A
A
B
A
A
B
A
A
2
1
1.5
2
1
1.5
2
1
1.5
2
1
70.5
75.1
94.1
83.2
84.4
99.6
43.7
51.7
95.7
18.9
33.7
94.5
97.1
98.2
28.5
16.3
5.8
16.8
15
<0.5
56.2
28.9
4.3
80.6
47.5
4.5
9
10
11
12
13
14
18.8
<0.5
<0.5
<0.5
1.5
2
2
2.5
1.5
a Percent yields were calculated by analysis (260 nm) of reversed-phase HPLC profile of the reaction mixtures. b Except for 11a and 12a, structure
assignments were based on reversed-phase C18 HPLC mobilities with 12 being the most hydrophilic (shorter elution time) and 11 the most hydrophobic
(longer elution time) compared to corresponding asymmetric dye 1.
(0.259 g, 0.5 mmol) and 4-chlororesorcinol (10b) (0.145 g, 1 mmol)
in a mixture of TFA (1.25 mL) and methanesulfonic acid (1.25
mL) was heated with stirring at 80 °C for 2 h. The dark tan solution
was cooled and diluted with water to precipitate the product. The
solid was collected by filtration, washed with water, and dried. The
crude material was chromatographed on silica in a gradient (5-
20%) of MeOH in CH2Cl2 (+10% triethylamine). The pure product
fractions were concentrated. The resulting solid was suspended in
water, acidified with 1 N HCl to a pH of ∼2, filtered, and washed
with water. Drying under vacuum (over P2O5) afforded 0.25 g (82%)
of the title dye 1e as an orange solid. 1H NMR (300 MHz, CD3OD
+ 0.8% NaOD +1.2% D2O): δ 7.08 (s, 1H), 6.85 (s, 1H), 6.68 (s,
1H), 2.56 (m, 2H), 2.48 (m, 2H). 13C NMR (75.5 MHz, CD3OD +
0.8% NaOD +1.2% D2O): δ 182.3, 177.5, 175.2, 169.7, 157.6,
154.3, 148.0, 135.2, 132.6, 132.4, 130.2, 129.6, 128.8, 128.7, 127.2,
113.3, 110.7, 110.0, 105.1, 38.5, 29.4. HRMS (FTMS) (m/z) calcd
for C23H9Cl6Na2O7 (M - H + 2Na)+ 652.8269, found 652.8293.
Representative Procedure for the Preparation of Dyes (Route
B). 2-[2-(2-Carboxyethyl)-6-hydroxy-3-oxo-3H-xanthen-9-yl]-
benzoic Acid (1a). A solution of 9a (4.8 g, 14 mmol) and resorcinol
(10a) (2.3 g, 21 mmol) in 70 mL of TFA was prepared and cooled
to 0 °C using an ice/water bath. To this solution were slowly added
25 mL of methanesulfonic acid maintaining the temperature at 0-2
°C. The reaction was stirred at 0 °C for 15 h and poured onto 500
g of ice. Precipitated material was collected by filtration, resus-
pended in water (500 mL), and treated with triethylamine to a pH
of ∼10. The resultant dark-brown solution was heated to boiling
and slowly cooled to room temperature. It was then acidified with
concentrated hydrochloric acid to a pH of ∼2 and extracted with
ethyl acetate (5 × 100 mL). The extract was washed with brine
and dried over Na2SO4. Concentration gave a tan, viscous oil, which
solidified upon treatment with water (∼100 mL). Filtration and
drying over P2O5 in Vacuo afforded 5.1 g (90%) of 1a as an orange
solid. An analytical sample was purified by silica gel chromatog-
1
raphy as described for 1e. H NMR (300 MHz, CD3OD + 0.8%
NaOD +1.2% D2O): δ 8.03 (m, 1H), 7.75 (m 2H), 7.57 (m, lH),
6.99 (m, 2H), 6.6-6.4 (m, 3H), 2.74 (m, 2H), 2.33 (m, 2H). 13C
NMR (75.5 MHz, CD3OD + 0.8% NaOD +1.2% D2O): δ 182.7,
182.5, 180.7, 174.3, 160.0, 159.8, 159.3, 141.3, 137.2, 135.0, 131.8,
130.9, 130.2, 129.8, 129.7, 122.9, 113.5, 112.9, 104.3, 104.0, 38.6,
28.8. HRMS (FTMS) (m/z) calcd for C21H17O7 (M + H)+ 405.0945,
found 405.0956.
Acknowledgment. We thank N. Scarr for useful discussions
and help with compound characterization.
Supporting Information Available: Experimental details and
characterization data for compounds 1b, 1c, 1d, 1f, 5b, 6a, 6b, 7a,
7b, 8a-c, 9a-d, and 10c. This material is available free of charge
via the Internet at http://pubs.acs.org.
JO702422V
J. Org. Chem, Vol. 73, No. 6, 2008 2427