FRET-based fluorescence probes for hydrolysis study and pig liver esterase activity

Article abstract of DOI:10.1016/j.tet.2008.06.051

New fluorescent probes based on simple organic synthesis were designed and synthesized, and their hydrolysis catalyzed via base and pig liver esterase (PLE) was studied using FRET (fluorescence resonant energy transfer), with 1-naphthylacetic group as a donor and dansyl group as an acceptor. By simultaneous recording of changes of the donor fluorescence intensities, kinetic parameters for base-catalyzed and PLE-catalyzed hydrolysis can be determined. The presented FRET assay is a convenient and simple method and both fluorescent probes are good real-time indicators for the analysis of ester hydrolysis such as PLE activities.

Full text of DOI:10.1016/j.tet.2008.06.051

Tetrahedron 64 (2008) 8947–8951
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
FRET-based fluorescence probes for hydrolysis study and pig liver
esterase activity
*
Long Yi, Li Cao, Liangliang Liu, Zhen Xi
State Key Laboratory of Elemento-Organic Chemistry, Department of Chemical Biology, Nankai University, Tianjin 300071, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 May 2008
Received in revised form 11 June 2008
Accepted 13 June 2008
Available online 19 June 2008
New fluorescent probes based on simple organic synthesis were designed and synthesized, and their
hydrolysis catalyzed via base and pig liver esterase (PLE) was studied using FRET (fluorescence resonant
energy transfer), with 1-naphthylacetic group as a donor and dansyl group as an acceptor. By simulta-
neous recording of changes of the donor fluorescence intensities, kinetic parameters for base-catalyzed
and PLE-catalyzed hydrolysis can be determined. The presented FRET assay is a convenient and simple
method and both fluorescent probes are good real-time indicators for the analysis of ester hydrolysis
such as PLE activities.
Keywords:
Carboxyl ester
FRET
Ó 2008 Elsevier Ltd. All rights reserved.
Hydrolysis
Kinetic study
Pig liver esterase
1. Introduction
spectra between donor and acceptor to allow independent mea-
surement of the fluorescence of each fluorophore.3 FRET has been
Fluorescence resonance energy transfer (FRET) as a physical
phenomenon was first described by Fo¨rster,1 to describe a non-
radiative process whereby an excited state fluorophore donor
transfers energy to a proximal ground state acceptor through long-
range dipole–dipole interactions. The rate of energy transfer is highly
dependentonmanyfactors, suchastheextentofspectraloverlap, the
relative orientation of the transition dipoles, and, most importantly,
the distance between the donor and acceptor moieties (usually
<6 nm).2 The general rules for selecting appropriate FRET pairs in-
clude sufficient separation in excitation spectra for selective stimu-
lation of the donor, a overlap (>30%) between the emission spectrum
of the donor and the excitation spectrum of the acceptor to obtain
efficient energy transfer, and reasonable separation in emission
used over the past four decades as a versatile method for measuring
noncovalent binding events in biochemical research such as struc-
tural elucidation of biological molecules and their interactions,4 in
vitro assays,5 in vivo monitoring in cellular research,6 nucleic acid
analysis,7 signal transduction,8 light harvesting, and nanomaterials.9
The FRET assay has some advantages including high-throughput
screening, real-time detection, and in vivo ratiometric probes due to
donor/acceptor fluorescence ratio change. However, the general in-
vestigation of the chemical bond formation and disassociation based
on the FRET technique in organic synthesis (Scheme 1) has only been
reported recently, such as in catalysts discovery10,11 and hydrolysis
reactions of phosphodiesters.12 In this work, we have designed and
synthesized a FRET pair with total separation of their emission
Scheme 1. General application of FRET in organic synthesis.
* Corresponding author. Fax: þ86 22 23504782.
E-mail address: zhenxi@nankai.edu.cn (Z. Xi).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.06.051

8948
L. Yi et al. / Tetrahedron 64 (2008) 8947–8951
spectra to study base-catalyzed hydrolysis and pig liver esterase
(PLE) activity, the widely used esterase in industry.13
a variety of reactions. The hydrolysis reactions of carboxyl esters were
chosen in this work due to the importance of such reactions.13–15,17
Figure 1 shows the absorption and photoluminescence of two
pure hydrolysis products N-methyl-N-dansyl-2-aminoethylanol (1)
and 1-naphthylacetic acid. 1-Naphthylacetic acid has strong emis-
sion at 326 and 338 nm excited at 285 nm, while compound 1
displays broad absorption band in the range of 290–410 nm. FRET
efficiency is dependent on the spectral overlap integral between
the donor emission and acceptor absorption. There is a significant
spectral overlap between the two fluorophores, implying that they
are good FRET pairs in an aqueous solution. The fluorescence
quantum yields (Ff) of 1-naphthylacetic acid and 1-naphthylacetic
group as a donor in compound 2 were 0.070 and 0.006, re-
spectively, implying that the FRET efficiency in compound 2 is
larger than 90%.
PLE is a serine-type esterase with wide substrate tolerance.14
The reported kinetics of PLE were measured by applying the pH-
stat technique to unbuffered solution,14,15 which is not very con-
venient and sensitive. Furthermore, there is few effective method to
monitor PLE activity in real-time with high sensitivity. In order to
conveniently probe this widely used enzyme, herein a sensitive
ratiometric fluorescent probe, namely, observation of changes in
the ratio of the fluorescent intensities of the emission at two
wavelengths, were developed for quantitative detection of PLE ac-
tivity. A commercial fluorescence reader is enough for the mea-
surements and the hydrolysis of a carboxyl ester catalyzed by base
and PLE was studied based on the FRET assay.
2. Results and discussion
1-Naphthylacetic moiety was selected as the fluorescent donor
and a dansyl group as the acceptor.16 Both donor and acceptor are
located on opposite sides in a single molecule with ester bond as
a linker, as shown in Scheme 2. For the FRET pair, the fluorescence
from 1-naphthylacetic group is quenched by dansyl group with
increased fluorescence from dansyl. If the ester bond is cleaved, no
FRET occurs and the emission of donor increases. The changes of
the ratio of the emission intensities at both acceptor and donor’s
peak wavelengths can then be converted to concentrations. If we
follow the fluorescent data with time, kinetic parameters can be
determined.
Figure 1. Spectral overlap (shaded area) of the emission of 1-naphthylacetic acid and
the absorption of compound 1; emission spectra of 1-naphthylacetic acid and com-
pound 1 were excited at 285 and 338 nm, respectively; all spectra were in 3ꢁ10ꢀ5 M
aqueous solution containing 2% CH3CN. Left arrows represent fluorescence spectra;
right arrow represents UV–vis absorption spectra of compound 1.
The emission spectrum of compound 2 in aqueous solution
excited at 285 nm exhibits that the donor at around 338 nm was
strongly quenched and the acceptor at around 500 nm was ob-
served. This emission spectrum demonstrates that energy transfer
from the donor to the acceptor can proceed efficiently. Addition of
compound 2 into a 0.1 M aqueous NaOH solution resulted in a rapid
decrease in the acceptor fluorescence at around 500 nm and a rapid
increase in the donor fluorescence at around 338 nm (Fig. 2). With
enough reaction time, the emission intensity of the reaction solu-
tion nearly approached that of equimolar mixture of compound 1
and 1-naphthylacetic acid. Compound 2 displays a large shift (from
500 to 338 nm) in its emission spectrum after the base-catalyzed
cleavage of ester bond within two fluorophores.
The reaction rate can be determined by relying on either the
donor fluorescence or acceptor fluorescence only. Since the donor
fluorescence is a ‘turn-on’ signal and is linearly proportional to the
product formed, its change may be a better indicator of the initial
reaction rate. As shown in Figure 3, the donor fluorescence has
better linear correction (correlation coefficient R¼0.9979) in the
standard curve than that of the acceptor fluorescence (R¼0.9779).
Therefore, we used the donor fluorescence to determine kinetic
parameters.
Scheme 2. The FRET-based detection mechanism.
The simple two-step syntheses successfully gave FRET-based fluo-
rescence probes 2 and 4 (Scheme 3), which have a 1-naphthylacetic
moiety as the donor and a dansyl moiety as the acceptor. Both donor
and acceptor possess functional groups that are inert toward most
types of coupling reactions. So a large number of nucleophiles and
electrophiles could be attached to this FRET pair for investigation of
The concentration of compound 2 in the reaction mixture was
determined by changes of the fluorescence at 338 nm. A plot of
concentration of compound 2 versus time in the base-catalyzed
hydrolysis (0.1 M NaOH) is shown in Figure 4. Due to excessive OHꢀ
in the reaction, the hydrolysis reaction can be seen as pseudo-first-
order. The observed kinetic constant kobs, kinetic constant k2, and
Scheme 3. Synthesis of the fluorescent probes.

L. Yi et al. / Tetrahedron 64 (2008) 8947–8951
8949
Figure 2. The emission spectra of compound 2 (9 mM) in 0.1 M aqueous NaOH solution
at 20 ꢂC: (A) 0 min, (B) 2 min, (C) 4 min, (D) 6 min, (E) 8 min, (F) 10 min, (G) 12 min,
(H) 14 min, (I) 18 min, (G) 22 min, (K) 30 min, (L) 44 min, after addition of compound
2; (M) equimolar mixture of 1 and 1-naphthylacetic acid in 0.1 M aqueous NaOH.
Figure 4. The concentration C versus time for the hydrolysis reaction in Figure 2 and
ln(C) versus time (inset) of compound 2.
and Vmax for compounds 2 and 4 were determined to be 12.22ꢁ10ꢀ5
and 1.56ꢁ10ꢀ6 M/min/unit enzyme, 3.42ꢁ10ꢀ5 and 0.82ꢁ10ꢀ6 M/
min/unitenzyme, respectively. The Km value of 2 islarger thanthatof
4, which may be due to the steric effect of methyl group in 2 influ-
encing its affinity for PLE. The reported kinetic parameters of PLE
varied widelyaccording tothe various structures of substrates (Km of
4.14ꢁ10ꢀ5 to 5.3ꢁ10ꢀ3 M and Vmax of 1.40ꢁ10ꢀ6 to 11.8ꢁ10ꢀ6 M/
min/unit enzyme).14,15 Comparing the experimentally obtained ki-
netic parameters of 2 and 4 with the literature data, Km value of both
compounds is at lower end of reported reactions, implying that both
probes have relatively high affinity for PLE with a relative fast cat-
alytic rate for hydrolysis. These results suggest that compounds 2
and 4 should be good ratiometric fluorescent probes for PLE activity.
half life time T1/2 can be calculated by equations ln(C)¼ꢀkobstþB (B
is a constant), k2¼kobs/C1, and T1/2¼ln2/kobs, where C and C1 are the
concentration of compound 2 and OHꢀ, respectively. Curve fitting
of the plots gives kobs¼0.089 minꢀ1, k2¼0.89 Mꢀ1 minꢀ1, and T1/2
¼
7.79 min (Fig. 4).
Compounds 2 and 4 were further chosen as ratiometric fluo-
rescent probes for pig liver esterase activity. Without the enzyme,
the fluorescence intensities of both compounds were hardly af-
fected in the buffer (100 mM phosphate, pH 8.0 at 20 ꢂC) for more
than 48 h. Addition of PLE to an aqueous solution of these fluo-
rescent probes resulted in an increase in the donor fluorescence
and a decrease in the acceptor fluorescence, which is similar to the
hydrolysis reaction in aqueous NaOH solution. Standard curves of
the donor fluorescence were made at the same conditions with
enzyme-catalyzed hydrolysis in the absence of enzyme.
3. Conclusions
Varying substrate concentrations and simultaneous recording of
donor fluorescence intensity with time yield the kinetic data (Fig. 5).
The plot of inverse initial velocity (1/v) of hydrolysis versus inverse
substrate concentrations (1/[S]) was fitted by Lineweaver–Burk
equation: 1/v¼((Km/Vmax)/[S])þ1/Vmax, where Vmax is the apparent
maximum rate and Km is the apparent Michaelis constant. The Km
In conclusion, we have developed two new FRET-based fluo-
rescent probes that can be easily used in the study of ester hy-
drolysis. 1-Naphthylacetic group as donor and dansyl group as
acceptor were chosen as a FRET pair because (1) both possess
functional groups to tether various types of coupling reactions for
study; (2) they have good FRET efficiency; and especially (3) both
emission spectra are totally separated for precision ratiometric
measurements. Used this FRET pair, the hydrolysis of a carboxyl
ester catalyzed by base and PLE was studied and the kinetic pa-
rameters were obtained. We believe that this FRET pair can be used
for investigation of a variety of chemical reactions and both fluo-
rescence probes 2 and 4 are practically useful for the real-time
analysis of ester hydrolysis such as PLE activities. Since the study of
protein dynamics in living cells is critical to an understanding of
sophisticated process of life,18 our further work will focus on de-
veloping small-molecular FRET probe to monitor specific proteins
in vivo.
4. Experimental
4.1. General
Pig liver esterase (PLE, 27 unit/mg) was purchased from Sigma
(Beijin, China), lyophilized powder containing less than 5% buffer
salts, 24 unit/mg. One unit will hydrolyze 1.0 mol of ethyl butyrate
to butyric acid and ethanol per minute at pH 8.0 at 25 ꢂC. All
chemical reagents were commercially available and were used
Figure 3. Standard curves for base-catalyzed hydrolysis. Conditions: compound 2
varied from 0 to 9 mM, the total concentration of 2 and 1-naphthylacetic acid was 9 mM,
equimolar mixture of compound 1 and 1-naphthylacetic acid in 0.1 M aqueous NaOH.
The donor fluorescence is a turn-on signal recorded at 338 nm, while the acceptor
fluorescence is a turn-off signal recorded at 500 nm.

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L. Yi et al. / Tetrahedron 64 (2008) 8947–8951
Figure 5. Determination of kinetic constants of fluorescence probes 2 and 4 for PLE. Conditions: pig liver esterase (0.2 unit enzyme/ml) in 0.1 M sodium phosphate buffer (pH 8.0) at
20 ꢂC, compounds 2 or 4 with increased concentrations from 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0, 18.0, 20.0, 30.0, 40.0 to 50.0
M. (a) and (c) changes in the donor emission at
m
338 nm. (b) and (d) Lineweaver–Burk plot of enzyme-catalytic hydrolysis reactions measured as a function of the concentration of substrates; the solid lines represent the best
fitting results.
2
without further purification. Solvents were distilled from the ap-
Ff
¼
FrðArFS=ASFrÞðhS
=
hr
Þ
(1)
propriate drying agents before use. All organic reactions were
monitored by TLC on silica gel GF254 (0.5 mm). Spots were detected
under UV light. Flash column chromatography was carried out on
silica gel H (400 mesh, Qingdao, China) or silica gel (200–300 mesh,
Qingdao, China). Yields refer to chromatographically and spectro-
scopically homogeneous material. 1H NMR and 13C NMR spectra
were recorded on a Bruker 300 spectrometer at 300 and 75.5 MHz,
respectively. Chemical shifts are given in parts per million down-
field from tetramethylsilane (0.0 ppm) for spectra in CDCl3. The
following abbreviations are used to explain multiplicities: s¼single,
d¼doublet, t¼triplet, m¼multiplet, br¼broad. High-resolution
mass spectra (HRMS) were obtained on a Varian QFT-ESI mass
spectrometer. UV–vis spectra were recorded on a CARY 100 Bio
spectrophotometer (Varian, USA). Fluorescence study was carried
out using Varian Cary Eclipse spectrophotometer at 20 ꢂC. The slit
width was 5 nm for both excitation and emission and the data
recorded each minute in a fast mode. The photomultiplier voltage
was 600 V.
where As and Ar are the absorbance of the sample and reference
solutions, respectively, at the same excitation wavelength, Fs and Fr
are the corresponding relative integrated fluorescence intensities,
and
h is the refractive index of the solvent.
4.3. Kinetic analysis of hydrolysis reactions
Compounds 2 and 4 were dissolved in CH3CN to make a 2.1 mM
stock solution, which was diluted to the required concentration for
measurement. The base-catalyzed kinetic parameters were de-
termined in 0.1 M aqueous NaOH solution containing 9 mM com-
pound 2 at 20 ꢂC. Pig liver esterase activity was determined in 3 ml
sodium phosphate buffer (100 mM, pH 8.0) with different con-
centrations (2.0–50.0 mM) of substrates (compounds 2 or 4) and
0.2 unit/ml esterase at 20 ꢂC. The real-time concentration of 2 in the
reaction mixture was determined by simultaneous recording of
changes of the donor fluorescence intensities at 338 nm. From
Lineweaver–Burk equation, Km and Vmax were calculated.
4.2. Fluorescence quantum yield
4.4. Preparation of compounds 1–4
Fluorescence quantum yield (Ff) was determined in absolute
ethanol using optically matching solution of rhodamine B (Ff¼0.50)
as the standard at an excitation wavelength of 495 nm,19 and the
quantum yield was calculated using Eq. 1:
4.4.1. Syntheses of N-methyl-N-dansyl-2-aminoethylanol (1)
N-Methylmonoethanolamine (450
ml, 5.6 mmol) was added into
dansylchloride (50 mg, 0.18 mmol) in 4 ml dry CH2Cl2 with stirring

L. Yi et al. / Tetrahedron 64 (2008) 8947–8951
8951
at room temperature for 15 min. The mixture was washed with
saturated Na2CO3 solution (2ꢁ5 ml) and then dried over sodium
sulfate. After evaporation of the CH2Cl2, the residue was purified by
silica gel column chromatography using chloroform/methanol (5:1,
v/v) as eluent to afford 1 (52 mg, 90%). 1H NMR (300 MHz, CDCl3):
45.95, 45.41; HRMS (ESI): m/z¼486.1346 [MþNa]þ (calcd for
C
26H25NO5SNa, 486.1351).
Acknowledgements
d
2.24 (br, 1H), 2.88 (s, 6H), 2.94 (s, 3H), 3.34 (t, 2H, J¼5.3), 3.75 (t,
This work was supported by the National Key Project for Basic
Research of China (2003CB114403), National Natural Science
Foundation of China (20272029, 20572053, 20421202, 20432010),
Ministry of Education of China (104189, B06005), and Nankai Uni-
versity ISC. We also thank referees of this paper for many critical
discussions.
2H, J¼5.3), 7.18 (d, 1H, J¼7.5), 7.45–7.57 (m, 2H), 8.14–8.20 (m, 1H),
8.33 (d, 1H, J¼9.1), 8.55 (d, 1H, J¼9.1); 13C NMR (75 MHz, CDCl3):
d
151.83,133.82,130.56,130.20,130.11,129.88,128.22,123.16,119.37,
115.28, 60.10, 51.84, 45.39, 35.34; HRMS (ESI): m/z¼309.1264
[MþH]þ (calcd for C15H21N2O3S, 309.1273).
4.4.2. Syntheses of 1-naphthylacetic acid(N-methyl-N-dansyl-2-
amino)ethylester (2)
References and notes
1-Naphthylacetic acid (20 mg, 0.11 mmol), DCC (23 mg,
0.11 mmol), DMAP (14 mg, 0.11 mmol), and 1 (30 mg, 0.1 mmol) in
5 ml dry CH2Cl2 were stirred at room temperature for 5 h and then
cooled to 0 ꢂC. The reaction mixture was filtered and the filtrate was
purified by silica gel column chromatography using dichloro-
methane as eluent to afford 2 (42 mg, 89%). 1H NMR (300 MHz,
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Nagano, T. Angew. Chem., Int. Ed. 2000, 39, 3438–3440; (b) Takakusa, H.;
Kikuchi, K.; Urano, Y.; Sakamoto, S.; Yamaguchi, K.; Nagano, T. J. Am. Chem. Soc.
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Nagano, T. Chem.dEur. J. 2003, 9, 1479–1485.
13. (a) Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic Synthesis: Regio and
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CDCl3):
d
2.67 (s, 3H), 2.86 (s, 6H), 3.43 (t, 2H, J¼5.3), 3.96 (s, 2H),
4.23 (t, 2H, J¼5.3), 7.17 (d, 1H, J¼7.5), 7.30–7.38 (m, 2H), 7.40–7.62
(m, 4H), 7.77 (d, 1H, J¼8.3), 7.78–7.83 (m, 2H), 7.90–7.95 (m, 2H),
8.14 (d, 1H, J¼7.5), 8.28 (d, 1H, J¼9.1), 8.54 (d, 1H, J¼9.1); 13C NMR
(75 MHz, CDCl3):
d 171.20, 151.83, 134.10, 133.79, 132.07, 130.54,
130.27, 130.16, 129.89, 128.70, 128.15, 128.10, 126.44, 125.82, 125.46,
123.77, 119.50, 115.28, 62.39, 48.27, 46.11, 45.40, 34.92; HRMS (ESI):
m/z¼477.1838 [MþH]þ (calcd for C27H29N2O4S, 477.1848).
4.4.3. Syntheses of dansyl-2-ol-ethylester (3)
Ethylene glycol (1 ml, 18 mmol) was added into dansylchloride
(100 mg, 0.36 mmol) in 8 ml dry CH2Cl2 with stirring at room
temperature for 15 min. The mixture was washed with saturated
Na2CO3 solution (2ꢁ5 ml) and then dried over sodium sulfate. After
evaporation of the CH2Cl2, the residue was purified by silica gel
column chromatography using ethyl ether/dichloromethane (1:1,
v/v) as eluent to afford 3 (37 mg, 35%). 1H NMR (300 MHz, CDCl3):
d
1.88 (br, 1H), 2.89 (s, 6H), 3.76 (t, 2H, J¼4.1), 4.11 (t, 2H, J¼4.6), 7.21
(d, 1H, J¼7.5), 7.53–7.62 (m, 2H), 8.24–8.29 (m, 2H), 8.61 (d, 1H,
J¼8.3); 13C NMR (75 MHz, CDCl3):
d 45.39, 60.75, 71.88, 115.63,
118.98,123.05, 128.94, 129.89, 130.67,130.98, 131.808,151.96; HRMS
(ESI): m/z¼318.0772 [MþNa]þ (calcd for C14H17NO4SNa, 318.0776).
4.4.4. Syntheses of 1-naphthylacetic acid(2-dansyloxy)-
ethylester (4)
1-Naphthylacetic acid (20 mg, 0.11 mmol), DCC (23 mg,
0.11 mmol), DMAP (14 mg, 0.11 mmol), and 3 (30 mg, 0.1 mmol) in
5 ml dry CH2Cl2 were stirred at room temperature for 5 h and then
cooled to 0 ꢂC. The reaction mixture was filtered and the filtrate was
purified by silica gel column chromatography using dichloro-
methane as eluent to afford 4 (37 mg, 78%). 1H NMR (300 MHz,
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CDCl3):
d
2.87 (s, 6H), 3.76 (s, 2H), 4.21 (s, 4H), 7.22 (t, 2H, J¼6.8),
17. (a) Goldsmith, H. A. Chem. Rev. 1943, 33, 257–349; (b) Fersht, A. R.; Kirby, A. J.
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18. Komatsu, T.; Kikuchi, K.; Takakusa, H.; Hanaoka, K.; Ueno, T.; Kamiya, M.; Urano,
Y.; Nagano, T. J. Am. Chem. Soc. 2006, 128, 15946–15947.
7.38 (t, 1H, J¼7.5), 7.46–7.64 (m, 4H), 7.76–7.86 (m, 3H), 8.29–8.33
(m, 2H), 8.65 (d, 1H, J¼9.1); 13C NMR (75 MHz, CDCl3):
d 170.85,
151.71, 133.78, 131.76, 130.62, 129.90, 128.77, 128.69, 128.15, 127.95,
126.42, 125.76, 125.39, 123.61, 123.14, 119.61, 115.74, 67.82, 61.85,
19. Karstens, T.; Kobs, K. J. Phys. Chem. 1980, 84, 1871–1872.