Synthesis of fluorescein aromatic esters in the presence of P 2O5/SiO2 as catalyst and their hydrolysis studies in the presence of lipase

Article abstract of DOI:10.1016/j.dyepig.2010.09.013

A series of fluorescein aryl esters were synthesized by the esterification of fluorescein with carboxylic acids in the presence of P₂O ₅/SiO₂ and their hydrolytic properties were investigated. The rate of hydrolysis in the presence or absence of lipase, due to the increase of fluorescein concentration, was measured by monitoring of fluorescence of the solution and correlated with enzyme activity. In addition, the substitute effect on the aromatic ring of fluorescein esters was studied. In contrast to fluorescein diacetate or dibutyrate, fluorescein dibenzoate and fluorescein bis(4-methylbenzoate) were found to be better substrates for the fluorometric assay of lipase with the higher rate of hydrolysis and better K_m value, respectively. As little as 9.3 ng mL^-1 of lipase can be detected with fluorescein bis(4-methylbenzoate).

Full text of DOI:10.1016/j.dyepig.2010.09.013

Dyes and Pigments 89 (2011) 120e126
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Synthesis of fluorescein aromatic esters in the presence of P2O5/SiO2 as catalyst
and their hydrolysis studies in the presence of lipase
*
Hossein Eshghi , Narges Mirzaie, Ahmad Asoodeh
Department of Chemistry, School of Sciences, Ferdowsi University of Mashhad, Mashhad 91775-1436, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of fluorescein aryl esters were synthesized by the esterification of fluorescein with carboxylic acids
in the presence of P2O5/SiO2 and their hydrolytic properties were investigated. The rate of hydrolysis in the
presence or absence of lipase, due to the increase of fluorescein concentration, was measured by moni-
toring of fluorescence of the solution and correlated with enzyme activity. In addition, the substitute effect
on the aromatic ring of fluorescein esters was studied. In contrast to fluorescein diacetate or dibutyrate,
fluorescein dibenzoate and fluorescein bis(4-methylbenzoate) were found to be better substrates for the
fluorometric assay of lipase with the higher rate of hydrolysis and better Km value, respectively. As little as
9.3 ng mLꢀ1 of lipase can be detected with fluorescein bis(4-methylbenzoate).
Received 4 August 2010
Received in revised form
22 September 2010
Accepted 25 September 2010
Available online 25 October 2010
Keywords:
Fluorescein ester
P2O5/SiO2
Ó 2010 Elsevier Ltd. All rights reserved.
Lipase
Enzymatic hydrolysis
Esterification
Fluorometric assay
1. Introduction
esters with long chains have the stronger lipophilic ability. p-
Guanidinobenzoate esters of fluorescein which were designed and
Xanthene-based dyes such as rhodamines and fluorescein are
widely used for labeling and sensing biomolecules [1,2]. They have
high extinction coefficient and fluorescence quantum yield in
aqueous solution, and their excitation and emission wavelengths
are in the visible range. Brightness and biocompatibility of fluo-
resceins are interested to many researchers and many derivatives of
fluoresceins are reported [3]. However, fluorescein also has some
obvious disadvantages, particularly its hydrophilic nature limits its
applications for detection of enzymes [4e6]. Fluorescein esters due
to the locked spiro lactone structures are nonfluorescent [7], but
they could be easily cleaved by intracellular enzymes to yield
fluorescent products. Thus some lipophilic fluorescein esters have
been reported for this purpose [8]. Kramer and Guilbault [9]
successfully established the fluorometric method for the estima-
tion and detection of lipase using fluorescein dibutyrate ester. A
series of fluorescein esters with 2e18 carbon chains were examined
[8e10]. However, the spontaneous hydrolysis rate of fluorescein
esters was found to be decreased by the chain length of fluorescein
esters, whereas, the rate of the enzymatic hydrolysis increased with
the chain length of fluorescein esters. It is obvious that fluorescein
synthesized as active site titrant of serine protease [11] is the only
reported fluorescein aryl ester.
Fluorescein esters were prepared by either reaction of fluores-
cein with anhydride in the presence of a base [12,13] or with an
alcohol in the presence of diethyl azodicarboxylate/triphenyl-
phosphine or dicyclohexylcarbodiimide [14]. However, a few
anhydrides are available and other method needs to activation of
carboxylic acid. Considering the above reports, we wish to report
the synthesis of a series of aromatic esters of fluorescein from
carboxylic acids in the presence of P2O5/SiO2. In order to search for
a fluorogenic substrate for lipase, we evaluate the fluorogenic
behavior of fluorescein aryl esters by spontaneous and enzymatic
hydrolysis.
2. Experimental
2.1. General
Chemicals were either prepared in our laboratories or
purchased from Merck, Fluka and Aldrich Chemical Companies. All
yields refer to the isolated products. The products were character-
ized by comparison of their physical data with those of known
samples or by their reported spectral data. Melting points were
recorded on an Electro thermal type 9100 melting point apparatus.
* Corresponding author. Tel.: þ985118795457; fax: þ985118797964.
E-mail address: heshghi@ferdowsi.um.ac.ir (H. Eshghi).
0143-7208/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2010.09.013

Table 1
One-pot preparation of fluorescein esters from fluorescein and carboxylic acids by means of P2O5/SiO2 as catalysta.
Entry
Acid
Time (h)
Product
Yield (%)
O
O
O
O
O
O
OH
1
4
90
O
O
O
O
O
Cl
O
O
O
Cl
Cl
OH
2
4
93
O
O
Cl
H3C
F
Cl
CH3
F
O
O
O
O
OH
O
O
3
4
87
O
Cl
H3C
F
O
O
O
O
O
OH
O
O
4
4
89
O
O
O
O
O
O
OH
O
O
5
4
4
4
93
95
90
O
O
O
O
O
O
O2N
NO2
OH
O
O
6
O
NO2
O
O2N
NO2
O
O
O
O
O
O
O
O
O
O
OH
O
O
O
O
O
O
7
8
O
O
O
O2N
O
O
O
O
2
1
94
92
OH
O
Cl
Cl
Cl
9
OH
a
Isolated yield.

122
H. Eshghi et al. / Dyes and Pigments 89 (2011) 120e126
IR spectra were recorded on a ShimadzueIR 470 spectrophotom-
eter. The 1H NMR (100 MHz) spectra were recorded on a Bruker AC
100 spectrometer. Chemical shifts are reported in ppm downfield
from TMS as internal standard; coupling constance J are given in
Hertz. Elemental analyses were obtained on a Thermo Finnigan
Flash EA micro- analyzer. Silica gel 60 (230e400 mesh) was
purchased from Merck. P2O5/SiO2 was prepared according to
previously reported procedure [15,16].
Lipase: Sigma Company, activity 1302 units per mg.
Buffer: Tris(hydroxymethyl)aminomethane buffer, 0.1 M, pH 7.2,
was prepared by dissolving the pure compound in distilled water
and adjusting the pH with 0.1 M hydrochloric acid.
Working standard of fluorescein esters: fluorescein esters were,
respectively, dissolved in methylcellosolve as 10ꢀ3 M solution.
2.2. General procedure for the synthesis of compounds 3ae3i
Fig. 1. The spontaneous hydrolysis of fluorescein esters in pH 7.2 Tris buffer.
A mixture of fluorescein (1) (1.66 g, 5 mmol), carboxylic acid (2)
(10 mmol) and P2O5/SiO2 (0.02 mol) was grounded in a mortar for
10 min. and then, was heated at 60 ꢁC for 4 h. After the completion,
the reaction mixture was treated with CH2Cl2 (50 mL), stirred for
one hour, and the catalyst filtered off. The organic layer was washed
with water (25 mL), dried over Na2SO4 and concentrated to afford
crude product. The crystalline pure product was obtained by
further recrystallization in methanol (Table 1).
6.9 (m, 4H). IR (KBr disc)
n: 1770, 1745, 1615, 1595, 1480, 1460, 1290,
1280, 1245, 1190, 1155, 1110, 1050 cmꢀ1
.
2.3.6. Fluorescein di(3-nitrobenzoate) (3f)
Yield 95%; m.p. 168e169 ꢁC; 1H NMR: (CD3COCD3, ppm)
d: 8.9 (t,
2H, J ¼ 1.5 Hz), 8.60 (m, 4H), 7.7e8.1 (m, 5H), 7.45 (d, 2H, J ¼ 1.5 Hz),
7.4 (m, 1H), 7.2 (dd, 2H, J1 ¼ 8 Hz, J2 ¼ 1.5 Hz), 7.05 (d, 2H, J ¼ 8 Hz).
IR (KBr disc)
1120 cmꢀ1
n: 1765, 1750, 1610, 1540, 1495, 1390, 1295, 1250, 1150,
.
2.3. Spectral data of the prepared compounds
2.3.7. Fluorescein di(4-nitrobenzoate) (3g)
2.3.1. Fluorescein dibenzoate (3a)
Yield 90%; m.p. 178e180 ꢁC; 1H NMR: (CD3COCD3, ppm)
d: 8.45
Yield 90%; m.p. 208e210 ꢁC; 1H NMR: (CD3COCD3, ppm)
d
: 8.2
(dd, 4H, J1 ¼ 7.0 Hz, J2 ¼ 2 Hz), 7.4e8.1 (m, 12H), 7.1 (dd, 2H,
J1 ¼ 8 Hz, J2 ¼ 2 Hz), 7.0 (d, 2H, J ¼ 8 Hz). IR (KBr disc) : 1770, 1750,
1610, 1450, 1425, 1255, 1240, 1155, 1110, 1055 cmꢀ1
(m, 8H), 7.95 (m, 1H), 7.8 (m, 2H), 7.5 (m, 2H), 7.2 (dd, 2H, J1 ¼ 8 Hz,
J2 ¼ 2 Hz), 7.1 (d, 2H, J ¼ 8 Hz). IR (KBr disc) : 1765, 1750,1615,1530,
1450, 1420, 1380, 1240, 1160 cmꢀ1
n
n
.
.
2.3.8. Fluorescein diacetate (3h)
Yield 94%; m.p. 202e203 ꢁC; 1H NMR: (CDCl3, ppm)
2.3.2. Fluorescein di(2-chlorobenzoate) (3b)
Yield 93%; m.p. 188e189 ꢁC; 1H NMR: (CDCl3, ppm)
(m, 3H), 7.7 (dt, 2H, J1 ¼ 7 Hz, J2 ¼ 1.5 Hz), 7.1e7.6 (m, 8H), 6.9 (m,
5H). IR (KBr disc) : 1775, 1750, 1620, 1470, 1275, 1255, 1230, 1150,
1075 cmꢀ1
d
: 8.00 (dd,
d
: 8.0e8.2
1H, J1 ¼ 7.5 Hz, J2 ¼ 1.5 Hz), 7.65 (m, 2H), 7.15 (dd, 1H, J1 ¼ 7.5 Hz,
J2 ¼ 1.5 Hz), 7.1 (d, 2H, J ¼ 1.5 Hz), 6.8 (m, 4H), 2.29 (s, 6H). IR (KBr
n
disc)
n: 1770, 1750, 1615, 1495, 1460, 1285, 1245, 1210, 1155, 1110,
.
1090 cmꢀ1
.
2.3.3. Fluorescein di(4-chlorobenzoate) (3c)
Yield 87%; m.p. 241e242 ꢁC; 1H NMR: (CDCl3, ppm)
d: 8.15 (dd,
2.3.9. Fluorescein di(2-chloroacetate) (3i)
Yield 92%; m.p. 193e194 ꢁC; 1H NMR: (CD3COCD3, ppm)
d: 8.10
1H, J1 ¼ 7.5 Hz, J2 ¼ 1.5 Hz), 8.05 (d, 4H, J ¼ 8 Hz), 7.55e7.8 (m, 2H),
7.5 (d, 4H, J ¼ 8 Hz), 7.25(m, 3H), 6.9(m, 4H). IR (KBr disc) : 3100,
1765, 1745, 1615, 1595, 1545, 1460, 1260, 1250, 1160, 1105, 1075,
1050 cmꢀ1
(dd, 1H, J1 ¼7.5 Hz, J2 ¼ 1.5 Hz), 7.8 (m, 2H), 7.40 (dd, 1H, J1 ¼7.5 Hz,
n
J2 ¼ 1.5 Hz), 7.30 (d, 2H, J ¼ 1.5 Hz), 7.0 (m, 4H), 4.6(s, 4H). IR (KBr
disc)
n: 2900, 1775, 1750, 1625, 1580, 1470, 1290, 1230, 1220, 1150,
.
1120, 1085 cmꢀ1
.
2.3.4. Fluorescein di(4-methylbenzoate) (3d)
Yield 189%; m.p. 89e190 ꢁC; 1H NMR: (CDCl3, ppm)
d: 8.1 (d, 4H,
2.4. The spontaneous hydrolysis of fluorescein aryl esters
J ¼ 8 Hz), 8.05 (m,1H), 7.7 (m, 2H), 7.30 (d, 4H, J ¼ X Hz), 7.2 (m, 3H),
6.9 (m, 4H), 2.45 (s, 6H). IR (KBr disc) : 1765, 1745, 1610, 1585, 1500,
1470, 1260, 1245, 1155, 1110, 1055 cmꢀ1
n
In a 10 mL volumetric flask, place accurately measured volume
of various substrates (0.1 mL) and pH 7.2 Tris buffer (9.9 mL).
Measure the rate of change in the fluorescence with time (ΔF/min)
at lex of 456 nm and lem of 510 nm. The rate of spontaneous
hydrolysis could be obtained. The results of spontaneous hydrolysis
of fluorescein esters are shown in Fig. 1 and Table 2.
.
2.3.5. Fluorescein di(4- fluorobenzoate) (3e)
Yield 93%; m.p. 234e235 ꢁC; 1H NMR: (CDCl3, ppm)
d: 8.0 (ABq,
8H), 7.9 (dd, 1H, J1 ¼7.5 Hz, J2 ¼ 1.5 Hz), 7.7 (m, 3H), 7.2e7.6 (m, 2H),
Table 2
Comparison of the rates of enzymatic and spontaneous hydrolysis of fluorescein esters in pH 7.2 Tris buffer.
Compounds
3a
23.739
0.112
200
3b
3c
3d
14.630
0.13
112.5
3e
3f
3g
3h
21.572
0.146
150
Re(minꢀ1
)
13.794
0.368
35
22.139
0.298
74
35.971
0.945
40
9.137
1.236
7.5
25.202
6.764
4
Rs(minꢀ1
Re/Rs
)
Re: The rate of enzymatic hydrolysis; Rs: The rates of spontaneous hydrolysis.

H. Eshghi et al. / Dyes and Pigments 89 (2011) 120e126
123
a mixture of fluorescein (1), carboxylic acid (2) and P2O5/SiO2 was
grounded in a mortar for 10 min. and then, was heated in 60 ꢁC for
5 h. After a simple workup, fluorescein esters (3aeh) were obtained
in 87e95% yields. Absorption of the released water by P2O5/SiO2
plays a vital role in the completion of reaction (Scheme 1). In our
method fluorescein esters (3) were washed with CH2Cl2 from
reaction mixture and the catalyst regenerated by heating in an oven
for further re-use. Aliphatic and aromatic carboxylic acids were
reacted successfully in this condition and the corresponding fluo-
rescein esters were obtained in high yields (Table 1). However,
aliphatic carboxylic acids such as acetic acid and 2-chloroacetic acid
were reacted with fluorescein in shorter reaction times.
The products were characterized by 1H NMR, IR, and melting
points and in the cases of known compounds were compared with
authentic samples. The OH group’s signals were omitted in 1H NMR
and IR spectra and strong absorption bands were appeared in IR
spectra at 1765 and 1745 cmꢀ1 due to ester and lactone carbonyl
groups, respectively. In 1H NMR spectra of aromatic esters a signal
was appeared in 8e8.2 ppm corresponded to the ortho protons of
aromatic rings of esters.
Fig. 2. The hydrolysis of fluorescein esters in the presence of lipase in pH 7.2 Tris
buffer.
2.5. The hydrolysis of fluorescein aryl esters in the presence of lipase
Tris buffer (9.8 mL) of pH 7.2 and 0.1 mL of 10ꢀ3 M solution of
fluorescein esters were put in a 10 mL volumetric flask and the
instrument was adjusted to read zero. At the zero time, 0.1 mL of
lipase solution with certain concentration was added, and the
change in the fluorescence at lex of 456 nm with time, ΔF/min, was
recorded for over a period of 6 min. A plot of ΔF/min vs lipase
concentration was made. The enzymatic hydrolysis results of
fluorescein aryl esters in the presence of lipase are shown in Figs
2e8 and Tables 2 and 3.
3.2. The investigation of hydrolytic properties of fluorescein esters
Kramer and Guilbault [9] successfully established the fluoro-
metric method for the estimation and detection of lipase using
fluorescein dibutyrate. Recently, Ge et al. [10] have been reported
that fluorescein dilaurate is a better substrate for the lipase assay
owing to its higher rate of hydrolysis and better Km value, which
was correlated with the stronger lipophilicity of its longer chain.
However, there is no report about fluorescein aryl esters to this day.
Actually, the longer chain fluorescein esters have the stronger lip-
ophilicity, but the chains longer than laurate cannot approach
easily with enzyme active site. We suggest that a small aryl group
may be approach easily to the active site in the enzyme. In order to
search for a fluorogenic substrate for lipase, we evaluated the flu-
orogenic behavior of fluorescein aryl esters (3ae3g) to the assay of
lipase and the results were compared with fluorescein diacetate
(3h) (Scheme 2).
3. Results and discussion
3.1. Direct synthesis of fluorescein esters
P2O5/SiO2 is a relatively new reagent which introduced firstly by
our research group for esterification of phenols [15] and alcohols
[16]. We decided to synthesis fluorescein esters from fluorescein
and carboxylic acids by means of P2O5/SiO2 as catalyst. Thus,
HO
O
OH
R
O
O
O
R
O
O
P2O5/SiO2
+
RCO2H
O
O
1) Grinding 10 min
o
2)
60 C , 4 h
O
O
2
1
3
a) R= C6H5
b) R= 2-Cl-C6H4
c) R= 4-Cl-C6H4
d) R= 4-Me-C6H4
e) R= 4-F-C6H4
f) R= 3-NO2-C6H4
g) R= 4-NO2-C6H4
h) R= CH3
i) R= CH2Cl
Scheme 1. Preparation of fluorescein aromatic esters in the presence of P2O5/SiO2.
R
O
O
O
R
O
O
OH
HO
O
OH
H2O, pH=7.2
O
O
a) Spontaneous
O
OH
O
O
b) Lipase-catalysed
O
O
3
4
1
Nonfluorescent
Nonfluorescent
Fluorescent
Scheme 2. Hydrolysis of fluorescein aryl esters in the presence or absence of lipase.

124
H. Eshghi et al. / Dyes and Pigments 89 (2011) 120e126
Fig. 3. The hydrolysis of fluorescein esters in the presence of different concentration of
lipase in pH 7.2 Tris buffer.
Fig. 5. The hydrolysis of different concentration of fluorescein bis(4-chlorobenzoate)
3c in the presence of certain concentration of lipase (10
g mLꢀ1) in pH 7.2 Tris buffer.
m
synthesis and the hydrolytic properties of a series of fluorescein
aryl esters in the presence of lipase.
3.2.1. The rate of spontaneous hydrolysis
The spontaneous hydrolysis rate of fluorescein esters in pH 7.2
Tris buffer was investigated by fluorometric method (Fig. 1), which
found to be in the following order:
As shown in Fig. 2, the order of the rate of enzymatic hydrolysis
is not similar to that of the spontaneous hydrolysis. The rate of the
enzymatic hydrolysis increases for all substituents and fluorescein
bis(4-fluorobenzoate) 3e has maximum rate and fluorescein bis(3-
nitrobenzoate) 3f has minimum rate. Thus the order of the
hydrolysis rate of fluorescein esters in the presence of lipase is
shown as: Fluorescein bis(4-fluorobenzoate) 3e >> fluorescein bis
(4-nitrobenzoate) 3g > fluorescein dibenzoate 3a > fluorescein bis
(4-chlorobenzoate) 3c > fluorescein diacetate 3h >> fluorescein
Fluorescein dibenzoate 3a < fluorescein bis(4-methylbenzoate)
3d < fluorescein diacetate 3h < fluorescein bis(4-chlorobenzoate)
3c < fluorescein bis(2-chlorobenzoate) 3b << fluorescein bis(4-flu-
orobenzoate) 3e < fluorescein bis(3-nitrobenzoate) 3f << flu
orescein bis(4-nitrobenzoate) 3g. The rate of the spontaneous
hydrolysis of fluorescein diesters increases in the presence of elec-
tron-withdrawing groups on the aryl rings. The spontaneous
hydrolysis of fluorescein dibenzoate 3a and fluorescein bis(4-meth-
ylbenzoate) 3d were as slow as fluorescein diacetate 3h and even
slower than that of fluorescein dibutyrate [9,10]. It seems to be
appropriate ester as model for the assay of enzymes. Furthermore,
fluorescein esters with electron-withdrawing groups such as 3ee3g,
proceeded more rapidly than fluorescein dibenzoate 3a. Fluorescein
dibenzoate 3a, fluorescein bis(4-methylbenzoate) 3d and fluorescein
bis(4-chlorobenzoate) 3c hydrolyzed very slowly, whereas, fluores-
cein bis(4-nitrobenzoate) 3g and fluorescein bis(2-chloroacetate) 3i
(not shown) hydrolyzed more rapidly. Ina comparisonof compounds
3ae3i, it can be seen that fluorescein dibenzoate 3a, fluorescein bis
(4-methylbenzoate) 3d and fluorescein bis(4-chlorobenzoate) 3c
which were hydrolyzed with more difficultly, were more stable in
aqueous solutions.
3.2.2. The rate of hydrolysis in the presence of the enzyme
Fig. 6. The hydrolysis of different concentration of fluorescein bis(4-methylbenzoate)
The importance of fluorescein esters are their applications as
fluorogenic probe of enzyme. As we all know, fluorescein dibuty-
rate has been successfully applied to test the activity of lipase and
elongation of ester chain increased this ability to the assay of lipase.
However, the synthesis and the application of fluorescein esters
with aryl groups are not reported. So we decided to study the
3d in the presence of certain concentration of lipase (10 m
g mLꢀ1) in pH 7.2 Tris buffer.
Fig. 7. Plot of rates of lipase catalyzed hydrolysis (ΔF/min) vs of fluorescein dibenzoate
3a, fluorescein bis(4-chlorobenzoate) 3c, and fluorescein bis(4-methylbenzoate) 3d
Fig. 4. The hydrolysis of different concentration of fluorescein dibenzoate 3a in the
concentrations (
pH 7.2 Tris buffer.
mM) in the presence of certain concentration of lipase (10 m
g mLꢀ1) in
presence of certain concentration of lipase (10 m
g mLꢀ1) in pH 7.2 Tris buffer.

H. Eshghi et al. / Dyes and Pigments 89 (2011) 120e126
125
lipophilic ability, these esters could easily and tightly bind to the
enzyme while esters with o- or m- substitution have less chance to
approach the enzyme. It may be explained that the o- or m-
substitution can add to the steric hindrance and therefore is dis-
favorable for the interaction between esters and enzyme, resulting
in the decreasing rate of enzymatic hydrolysis. Furthermore, the p-
substitution may have effect on the elasticity of fluorescein ester
which might perturb the approach of the substrate to the active site
in the enzyme. It is surprising that the rate of fluorescein diben-
zoate 3a is higher than that of fluorescein diacetate 3h and also
than fluorescein bis(4-methylbenzoate) 3d. The reason may be that
the phenyl rings have so much lipophilic ability and the rate of
enzymatic hydrolysis is increase seriously compared to the rate of
spontaneous hydrolysis.
In another approach, comparison of the rates of enzymatic and
spontaneous hydrolysis (Re/Rs) for these esters showed that lipase
hydrolysis acceleration are 150 for fluorescein diacetate 3h and
200 for fluorescein dibenzoate 3a (Table 2). This rate acceleration
decreases to 4 for fluorescein bis(4-nitrobenzoate) 3g. Fluorescein
bis(4-fluorobenzoate) 3e which have high rate of hydrolysis in the
presence of lipase, possess only a 40 times rate acceleration in
comparison to its spontaneous hydrolysis. Actually, the rate
acceleration of benzoate esters with electron-withdrawing groups
Fig. 8. LineweavereBurk plot of (1/V0) versus (1/[S]) of fluorescein dibenzoate 3a,
fluorescein bis(4-chlorobenzoate) 3c, and fluorescein bis(4-methylbenzoate) 3d.
bis(4-methylbenzoate) 3d
> fluorescein bis(2-chlorobenzoate)
3b >> fluorescein bis(3-nitrobenzoate) 3f. Accordingly, p-
substituted phenyl ring (H, Cl, Me, F, and NO2) have the high rates of
hydrolysis. 3b and 3f with substitution in o- or m-positions have
the lowest rates of hydrolysis. It is reasoned probably that fluo-
rescein esters with or without p-substitution have the stronger
Table 3
Comparison of various substrates for lipase assay.
Km (m
M)a Vmax
Lowest detectable
Fluorescence
wavelengths
Rate of spontaneous
Lit
b
Substrate
concentrationc ng mLꢀ1
hydrolysis Ks (ΔF/min)d
O
O
O
O
O
e
O
7.04
83.3
12
lex ¼ 456 lem ¼ 510 0.112
O
3a
Cl
Cl
O
O
O
O
O
e
O
4.3
41.7
32
lex ¼ 456 lem ¼ 510 0.298
O
3c
Me
Me
O
O
O
O
O
e
O
1.1
50
9.3
lex ¼ 456 lem ¼ 510 0.13
O
3d
O
O
O
O
O
O
0.035
e
210
lex ¼ 492 lem ¼ 515 0.3
9,10
O
(Fluorescein dibutyrate)
a
Km is Michaelis constant for the enzymeesubstrate reaction. Km value was obtained by the method of LineweavereBurk.
Maximum rate of enzymatic hydrolysis expressed in Δ fluorescence units per minute.
The lowest detectable concentration of lipase reported was that concentration required to give an enzymatic rate twice that of the spontaneous rate.
Rate of spontaneous hydrolysis expressed in Δ fluorescence units per minute.
b
c
d
e
Present work.

126
H. Eshghi et al. / Dyes and Pigments 89 (2011) 120e126
in the presence of lipase is only about 4e40 times. Whereas, the
rate acceleration of fluorescein bis(4-chlorobenzoate) 3c and
fluorescein bis(4-methylbenzoate) 3d are 74 and 112 times,
respectively. The rate acceleration of fluorescein bis(4-chlor-
obenzoate) 3c rather than fluorescein bis(2-chlorobenzoate) 3b is
twofold which shows 3c easily and tightly binds to the enzyme
active site.
Thus, the order of the relative rate accelerations by lipase (Re/Rs)
are: Fluorescein dibenzoate 3a > fluorescein diacetate 3h >
fluorescein bis(4-methylbenzoate) 3d > fluorescein bis(4-chlor-
obenzoate) 3c > fluorescein bis(4-fluorobenzoate) 3e > fluorescein
bis(2-chlorobenzoate) 3b >> fluorescein bis(3-nitrobenzoate)
3f >> fluorescein bis(4-nitrobenzoate) 3g.
As shown in Figs 1 and 2, fluorescein dibenzoate 3a and fluo-
rescein bis(4-methylbenzoate) 3d were the best substrates in this
series of fluorescein esters with the highest lipase rate acceleration,
thus we decided to study 3a, 3c and 3d in details in comparison
with fluorescein dibutyrate which had been successfully applied to
test the activity of lipase [9,10].
electron-withdrawing group in aryl ester has an important effect on
the spontaneous hydrolysis. Although, lipase accelerates all
substrate hydrolysis, the rate acceleration is higher for 3a, 3d and 3c.
By comparison, fluorescein bis(4-methylbenzoate) 3d was found to
be the better substrate for the assay of lipase with the higher rate of
hydrolysis and better Km value. As little as 9.3 ng mLꢀ1 of lipase, may
be determined with this substrate. These results show that the
fluorescein aryl esters may be a very sensitive fluorogenic marker of
enzyme and have some latent applications in future.
Acknowledgements
We are grateful to Ferdowsi University of Mashhad Research
Council for their financial support of this work (Grant: P622:21-
09-88).
References
[1] Beija M, Afonso CAM, Martinho JMG. Synthesis and applications of rhodamine
derivatives as fluorescent probes. Chem Soc Rev 2009;38:2410e33.
[2] Mugherli L, Burchak ON, Chatelain F, Balakirev MY. Fluorogenic ester
substrates to assess proteolytic activity. Bioorg Med Chem Lett 2006;16:
4488e91.
[3] Duan Y, Liu M, Sun W, Wang M, Liu S, Li QX. Recent progress on synthesis of
fluorescein probes. Mini Rev Org Chem 2009;6:35e43.
[4] Song AM, Zhang JH, Zhang MH, Shen T, Tang JA. Spectral properties and
structure of fluorescein and its alkyl derivatives in micelles. Colloids Surf A
2000;167:253e60.
The rate of lipase catalyzed hydrolysisof the fluorescein esters 3a,
3c and 3d (ΔF/min) in different enzyme concentrations was studied
(Fig. 3). Also, the rates of hydrolysis of different concentrations of the
fluorescein esters in the presence of constant concentration of lipase
(10 m
g mLꢀ1) were shown in Figs. 4e6. As shown in Fig. 3, fluorescein
bis(4-methylbenzoate) 3d was the best substrate in these aryl ester
series with the highest lipase hydrolysis efficiency. Accordingly, as
little as 9.3 ng mLꢀ1 of lipase can be detected using this ester,
compared to 210 ng mLꢀ1 with fluorescein dibutyrate [9,10]. Also,
fluorescein dibenzoate 3a and fluorescein bis(4-chlorobenzoate) 3c
can be detected 12 and 32 ng mLꢀ1 of lipase, respectively. According
to Figs. 4e6, we was drawn the plot of V0 versus [S] and also, 1/V0
versus 1/[S] in Figs. 7 and 8. The results are shown in Table 3 and
compared with a well known lipophilic substrate, fluorescein
dibutyrate. Of the three substrates tested, fluorescein bis(4-meth-
ylbenzoate) 3d had the better Km value, and it showed the stronger
appetency which came into being between the alkyl-substituted
aryl ester and lipase. In addition, 3d was moꢀre1 sensitive in the
determination of lipase. As little as 9.3 ng mL of lipase can be
detected using this ester, compared to 210 ng mLꢀ1 with fluorescein
dibutyrate. Therefore, fluorescein diaryl esters are believed as an
ideal high affinity substrate for the assay of lipase by fluorescence.
The change in aryl rings or elongation of alkyl substitution are under
investigation in our laboratories.
[5] Hoefel D, Grooby WL, Monis PT, Andrews S, Saint CP. A comparative study
of carboxyfluorescein diacetate and carboxyfluorescein diacetate succini-
midyl ester as indicators of bacterial activity.
J Microbiol Meth 2003;
52:379e88.
[6] Rukavishnikov AV, Smith MP, Birrell GB, Keana JFW, Griffith OH. Synthesis of
a new fluorogenic substrate for the assay of phosphoinositide-specific phos-
pholipase C. Tetrahedron Lett 1998;39:6637e40.
[7] Patel BCM, Courtney JM, Evans JH, Paul JP. Biocompatibility assessment:
application of fluorescent probe response (FPR) technique. Biomaterials
1991;12:722e6.
[8] Guilbault GG, Hieserman J. Fluorometric substrate for sulfatase and lipase.
Anal Chem 1969;41:2006e9.
[9] Kramer DN, Guilbault GG. A substrate for the fluorometric determination of
lipase activity. Anal Chem 1963;35:588e9.
[10] Ge FY, Chen LG, Zhou XL, Pan HY, Yan FY, Bai GY, et al. Synthesis and study on
hydrolytic properties of fluorescein esters. Dyes Pigments 2007;72:322e6.
[11] Melhado LL, Peltz SW, Leytus SP, Mangel WF. p-Guanidinobenzoic acid esters
of fluorescein as active-site titrants of serine proteases. J Am Chem Soc
1982;104:7299e306.
[12] Woodroofe CC, Lim MH, Bu W, Lippard SJ. Synthesis of isomerically pure
carboxylate- and sulfonate-substituted xanthene fluorophores. Tetrahedron
Lett 2005;61:3097e105.
[13] Li X, Taylor JS. General strategy for the preparation of membrane permeable
fluorogenic peptide ester conjugates for in vivo studies of ester prodrug
stability. Bioorg Med Chem 2004;12:545e52.
[14] Falck JR, Krieger M, Goldstein JL, Brown MS. Preparation and spectral prop-
erties of lipophilic fluorescein derivatives: application to plasma low-density
lipoprotein. J Am Chem Soc 1981;103:7396e8.
4. Conclusions
A series of fluorescein aryl esters were synthesized, by the
esterification of fluoresceinwith the carboxylic acids in the presence
of P2O5/SiO2 under solvent-free conditions in high yields. The
hydrolytic properties of fluorescein aryl esters were investigated in
the presence or absence of lipase. It was seen that the presence of an
[15] Eshghi H, Rafei M, Karimi MH. P2O5/SiO2 as an efficient reagent for esterifi-
cation of phenols in dry media. Synth Commun 2001;31:771e4.
[16] Eshghi H, Shafieyoon P. P2O5/SiO2 as a mild and efficient reagent for acylation
of alcohols, phenols and amines under solvent-free conditions. J Chem Res;
2004:802e5.