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Synthesis of fluorescein through a mechanism of Aromatic

Electrophilic Substitution (SEA)
Alejandra Garca-Daz, Ana P. Snchez-Navarrete and Genaro Reyes-Ros

Departamento de Qumica Orgnica, Facultad de Qumica, Universidad de Ixtlahuaca CUI, Carr. Ixtlahuaca-Jiquipilco Km. 1 Ixtlahuaca
s/n, Estado de Mxico, 50740, Mxico.
Abstract Fluorescein is one of the first derivatives synthetic dyes of xanthene, this dye was discovered in 1871 by Adolf Von Bayer 1, it is
used for its fluorescence in ophthalmic medicine and research in the area of microbiology. In this work we did the chemical synthesis of

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fluorescein, this was by a mechanism of aromatic electrophilic substitution (SEA) between two equivalents of resorcinol by one of phtalic
anhydride in acid. In addition to the synthesis of fluorescein, we analyzed its behavior at different pH solutions and also we identified the
functional groups of this compound in an infrared absorption spectrum. 2012 Elsevier Science. All rights reserved

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Introduction
Fluorescein can be prepared by reacting phthalic anhydride
with resorcinol using sulfuric acid through a mechanism of
Aromatic Electrophilic Substitution (SEA) (Scheme 1).
Fluorescent detection technique has played a significant
role on the advancement of modern medicine and
molecular biology and has achieved rapid development.
Fluorescein is one of the widely used fluorescent probes in
such applications. 2
The fluorescence of this molecule is very high, and
excitation occurs at 494 nm and emission at 521 nm.
Fluorescein has a pKa of 6.4, and its ionization equilibrium
leads to pH-dependent absorption and emission over the
range of 5 to 9. 3

Scheme 1: Synthesis of fluorescein from resorcinol and phthalic

anhydride.

The electronic structure of fluorescein is responsible for its

fluorescence. Ultraviolet light is able to excite an electron
from the highest occupied molecular orbital (HOMO) to the
lowest unoccupied molecular orbital (LUMO).The energy
difference between these two molecular orbital
configurations corresponds to the energy of the wave
exciting the electron. Fluorescence occurs when that
excited electron returns to the original HOMO causing the
emission radiation in the form of visible light (Figure 1).4

Figure 1: Fluorescein under UV illumination.

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to find an intense band at 1720cm-1 due to the presence of a

carboxyl group of fluorescein, but in the reference
spectrum, the band does not appear and the closest to this
value is band at 1600cm-1 characteristic of the stretching
vibration of the carbon-carbon double bond of the aromatic
rings. But for this case, normally expect to see four bands
of varying intensity a near 1600cm-1 and the other near
1500 cm-1 in the spectrum really no such behavior is
observed only two bands a little intense close to the
aforementioned regions, suggesting an overlapping of
signals of the C = C and C = O, and being a salt of
fluorescein (Figure 2).

This aromatic electrophilic substitution reaction is

catalyzed in an acid medium in order to the oxygen can be
protoned and can be formed a carbocation to be attacked by
the resorcinol (Scheme 3).
Phthalic anhydride is the electrophile, which is attacked by
the resorcinol ring. By joining the molecule, the OH of the
anhydride is protonated and loss a molecule of water
forming a carbocation again. Comes another molecule of
resorcinol and the carbocation attacks again. OH attack on
the adjacent carbon, removing water and forming a shaking
cycle. Then there is a COOH formation due to rupture of
95
the ester bond (Scheme 2).

997.01

90
85 of Fluorescein
Synthesis

417.31

917.20
2000

1500

612.13

760.15

796.80
883.79

970.53

1073.78

1106.36

1309.12

1439.07

1600.38

1695.47
2500

677.74

1356.67
1241.60

1503.78

2705.81

2852.89

2960.97

3063.61

3017.16

3378.31

%T

2572.60

80

We set a salt bath to a75 temperature between 180 and

200C.Then we added in70an Erlenmeyer flask 0.1630 g of
resorcinol and 0.1013 g65 of phthalic anhydride. To this
mixture we added 2 drops60 of H2SO4.
We placed the Erlenmeyer
flask in the preheated salt bath.
55
The reaction ran at a temperature between 180 and 200C.
50
The reaction ran for 30 minutes within this temperature
45
range. Once the reaction
time was up, we removed the
40
Erlenmeyer flask from the
oil bath and allow it to cool for
35
about 5 min.
30
We added 1.2780g of crushed
ice and stirred with a glass
rod to dislodge solid. The25 solid was filtered on filter paper.
20
Then we washed with cold distilled water. Was added
15
0.0056g of dry product10 obtained in 3ml of the NaOH
solution and 3ml of distilled
water, thereby obtaining green
5
fluorescent searched.
3500
3000

1000

Wavenumbers (cm-1)

Figure 2: Infrared absorption spectrum of fluorescein.

Conclusions
Sc
heme 3. Electrophile formation of phthalic anhydride.

In the infrared absorption spectrum for the fluorescein was

observed at approximately 3300cm-1 a broad band of
medium intensity characteristic of the phenyl hydroxyl
groups of both the carboxyl acid as part of the structure of
the compound. The decrease in the intensity of the band is
caused by the use of a liquid solvent for reading in the IR.
Between 3000 and 2800cm-1 is a characteristic intense band
stretching vibration of CH bonds sp2 hybridization, is also
shifted to the right by the resonance effect produced by
multiple double bonds in the molecule, because generally
in an aromatic hydrocarbon band is observed between
3100-3000cm-1.
Considering the structure of the compound, would expect

The percentage yield obtained was satisfactory; however

we established a new synthetic route for the purpose of
improvement. It was concluded that the fluorescein was
obtained from phthalic anhydride and resorcinol with
H2SO4.We determined that we cannot carry out the reaction
in fewer steps, as these are required for the synthesis of this
compound fluorescent. Heat was applied as the physical
properties of resorcinol and phthalic anhydride having a
melting point higher than 100 C.
Acknowledgments
Financial support from Universidad de Ixtlahuaca CUI is
gratefully acknowledged. The author would like to thank D.
Gonzalez-Calderon for the technical support.

500

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5
Supplementary data

2.

Supplementary data (including complete characterization,

spectral data and copies of IR spectra) associated with this
article can be found in the online version, at
http://www.photonicsolutions.co.uk/datasheets/exci/Fluores
cein%20548.

3.

References
1.

Luke D.R.; Thomas, Tuning the pka of fluorescein to

optimize binding assays 2007, 775.

4.

(a) Tremayne, M.; Kariuki, B. M.; Harris, K. D. M.

Angew. Chem. Int. Ed. Engl. 1997, 36, 770-772.
Green, F. J. The Sigma Aldrich Handbook of Stains
Dyes and Indicators; Aldrich Chemical Company Inc.:
Milwaukee 1990, 257-258, 314-316.
Vogel, S. Practical Organic Chemistry 1956, 985.