Enzyme-catalyzed Michael addition for the synthesis of warfarin and its determination via fluorescence quenching of L-tryptophan

Article abstract of DOI:10.1016/j.saa.2017.01.014

A sensitive fluorescence sensor for warfarin was proposed via quenching the fluorescence of L-tryptophan due to the interaction between warfarin and L-tryptophan. Warfarin, as one of the most effective anticoagulants, was designed and synthesized via lipase from porcine pancreas (PPL) as a biocatalyst to catalyze the Michael addition of 4-hydroxycoumarin to α, β-unsaturated enones in organic medium in the presence of water. Furthermore, the spectrofluorometry was used to detect the concentration of warfarin with a linear range and detection limit (3σ/k) of 0.04–12.0?μmol?L^??1 (R²?=?0.994) and 0.01?μmol?L^??1, respectively. Herein, this was the first application of bio-catalytic synthesis and fluorescence for the determination of warfarin. The proposed method was applied to determine warfarin of the drug in tablets with satisfactory results.

Full text of DOI:10.1016/j.saa.2017.01.014

Accepted Manuscript
Enzyme-catalyzed Michael addition for the synthesis of warfarin
and its determination via fluorescence quenching of l-tryptophan
Yusheng Yuan, Liu Yang, Shaopu Liu, Jidong Yang, Hui Zhang,
Jingjing Yan, Xiaoli Hu
PII:
DOI:
Reference:
S1386-1425(17)30016-1
doi: 10.1016/j.saa.2017.01.014
SAA 14867
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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Accepted date:
12 October 2016
30 December 2016
6 January 2017
Please cite this article as: Yusheng Yuan, Liu Yang, Shaopu Liu, Jidong Yang, Hui
Zhang, Jingjing Yan, Xiaoli Hu , Enzyme-catalyzed Michael addition for the synthesis of
warfarin and its determination via fluorescence quenching of l-tryptophan. The address
for the corresponding author was captured as affiliation for all authors. Please check if
appropriate. Saa(2017), doi: 10.1016/j.saa.2017.01.014
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ACCEPTED MANUSCRIPT
Enzyme-catalyzed Michael addition for the synthesis of warfarin and its
determination via fluorescence quenching of L-tryptophan
Yusheng Yuana, Liu Yanga, Shaopu Liua, Jidong Yangb, Hui zhanga, Jingjing Yana, Xiaoli Hua*
a: Key Laboratory of Luminescent and Real-Time analytical Chemistry (Southwest University),
ministry of Education, College of Chemistry and Chemical Engineering, Southwest University,
Chongqing 400715, China. E-mail: xiaolihu@swu.edu.cn
b: College of Chemical and Environmental Engineering, Chongqing Three Gorges University,
Wanzhou, Chongqing 404100, China.

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ABSTRACT
A sensitive fluorescence sensor for warfarin was proposed via quenching the
fluorescence of L-tryptophan due to the interaction between warfarin and L-
tryptophan. Warfarin, as one of the most effective anticoagulants, was designed and
synthesized via lipase from porcine pancreas (PPL) as a biocatalyst to catalyze the
Michael addition of 4-hydroxycoumarin to α, β-unsaturated enones in organic
medium in the presence of water. Furthermore, the spectrofluorometry was used to
detect the concentration of warfarin with a linear range and detection limit (3σ/k) of
0.04–12.0 μmol L−1(R2=0.994) and 0.01 μmol L−1, respectively. Herein, this was the
first application of bio-catalytic synthesis and fluorescence for the determination of
warfarin. The proposed method was applied to determine warfarin of the drug in
tablets with satisfactory results.
Keywords: bio-catalysis; Michael addition; porcine pancreas; warfarin; fluorescence

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1. Introduction
The coumarin derivative, warfarin, is widely used as the major drug for
anticoagulation in clinical applications due to the advantages of oral availability,
convenient application, inexpensive price, and lasting effect. Furthermore, it acts on
the liver to decrease the quantity of a few key proteins in blood that allow blood to
1
clot instead of affect the thickness or viscosity of blood.
Due to the narrow
therapeutic window of warfarin, variability in dose-response and interactions with
other compounds, careful monitoring of the effect of drug dosage must be performed.
2
Recently, the effect of various factors (e.g., disease, inflammation, genetic
polymorphisms) on cytochrome P450 (CYP) enzymes function and their subsequent
3,4
impact on warfarin dose requirements has been extensively studied.
Moreover, this
5
information is now used to optimize warfarin therapy.
Warfarin has been in clinical use as a race mate for more than half a century.
Different chemical strategies towards the synthesis of optically active warfarin have
6-8
been reported in recent years.
Recently, bio-catalysis highly attractive for the
enzyme catalytic activities with unnatural substrates and alternative chemical
9-10
transformations.
The importance of the bio-catalysis is noteworthy, since it not
only highlights the existing catalysts, but may provide novel and practical synthetic
11
path ways which are not currently available.
Bio-catalytic processes have often
proven to be economically feasible, ecologically advantageous and more sustainable
than current chemical technologies as they use inexpensive regenerable resources. 12,13
However, most metal or organocatalysts have to be synthesized through multiple steps

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and consume non-renewable fossil raw materials, which usually involve using some
toxic reagents or expensive materials and lots of organic solvents. Moreover
biocatalysts allow different chemical transformations to be carried out without the
need for tedious protection and deprotection steps, especially in compounds with
several functional groups. Herein, the lipase from porcine pancreas (PPL) as one of
the most widely used and well-known hydrolases catalyzed Michael addition of 4-
hydroxycoumarin and α,β-unsaturated enones was reported to prepare warfarin in
14,15
organic medium in the presence of water.
Therefore, various techniques have been utilized for sensing warfarin in the
biological samples such as high performance liquid chromatography (HPLC) with
16,17
18-20
ultraviolet or fluorescence detections,
HPLC-MS/MS,
capillary zone
21
22
electrophoresis (CZE),
cyclic voltammetry
and chiral capillary electrophoresis
23
with spectrophotometric. However, particular concerns for these large instrument-
based strategies are high energy and money consumption, a long working time,
tedious pretreatment, etc., which is especially unsuitable for fast therapeutic drug
monitoring. An alternative to the aforementioned techniques is spectrofluorometry,
which gains much attention owing to easy preparation, high sensitivity, low detection
limit simple and eco-friendly. In recent years, it has been used in widely applied to the
24,25
26,27
28,29
determination of metal ions,
organic compounds,
pharmaceuticals
and
30,31
macromolecules.
In this work, warfarin is synthesized by the hydrolases catalyzed Michael addition
of 4-hydroxycoumarin and α,β-unsaturated enones in organic medium in the presence

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of water, furthermore, a novel probe for the determination of warfarin is proposed by
quenching the fluorescence of L-tryptophan(L-try). As shown in Scheme. 1, the
fluorescence of tryptophan decreases when warfarin presences. Moreover, to the best
of our knowledge, this is the first application of bio-catalytic synthesis and
fluorescence for the determination of warfarin.
Scheme. 1. The simplified sketch for the fabrication process of warfarin and its interaction with
tryptophan.

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2. Experimental
2.1 Apparatus
The fluorescence spectrum was recorded with an F-2500 fluorescence
spectrophotometer (Hitachi, Tokyo, Japan) using a 1 cm path length. A UV-2450
spectrophotometer (Shimadzu, Japan) was used for acquiring absorption spectra and
measure absorbance. A FL-TCSPC Fluorolog-3 fluorescence spectrometer (Horiba
Jobin Yvon Inc., France) is used to measure fluorescence emission decay curves of
1
systems. H NMR spectra were recorded at 300 MHz, and 13C NMR spectra were
recorded at 150 MHz (Bruker Avance). Coupling constants (J) were given in Hz. All
reactions were monitored by thin-layer chromatography (TLC) with HaiyangGF254
silica gel plates. Reaction products were purified by flash column chromatography
using silica gel (100–200 mesh). HPLC analysis was performed on HPLC system
equipped with chiral stationary phase columns. A pHS-3D pH meter(Shanghai
Scientific Instruments Company, China) was used to measure the pH values.
2.2 Chemicals
Lipase from porcine pancreas (PPL, 300 u mg−1. One unit will hydrolyze 1.0
◦
microequivalent of fatty acid from triacetin in 1 hat pH 7.4 and 37 C) was purchased
from Shanghai Lanji Co. Ltd(Shanghai, China). 4-Hydroxycoumarin was purchased
from Aladdin (Shanghai, China) and α,β-unsaturated ketones were prepared according
32
to literature procedures.
Tryptophan was purchased from Aladdin (Shanghai,
China)and the other reagents were obtained from commercial suppliers and were used
without further purification. The Britton-Robinson (BR) buffer solutions at different

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pH values were prepared by mixing the mixed acid (composed of 2.71 mL 85%
H3PO4, 2.36 mL HAc and 2.47 g H3BO3) with 0.2 mol L-1 NaOH in different
proportions. The buffer solutions were used to control the acidity. All reagents were of
analytical reagent grade and ultrapure water was supplied by a Millipore System (18.2
MΩ cm)throughout the whole experiments.
3. Results and discussion
3.1 Characterization of warfarin
Warfarin was synthesized by bio-catalytic processes. 15 All the Michael adducts are
known compounds that exhibited spectroscopic data identical to those reported in the
34
literature . Relative and absolute configurations of the products were determined by
1
comparison with the known H NMR,13C NMR 7,8,33. The data has been sown in
supporting information.
3.2 Fluorescence spectra
As shown in Fig. 1, the fluorescence spectra of L-Try excited at 290 nm and had an
emission peak at 364 nm in pH 7.5 BR solution. However, it decreased when warfarin
was added in, which indicated that L-Try had been interacted with warfarin. When the
different concentrations of warfarin were added into the BR buffer solution (pH 7.5),
which contained L-Try, the fluorescence intensity was gradually decreased (Fig.
2).Therefore, a novel method for warfarin could be established.

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Fig. 1. Fluorescence excitation (a) and emission (b) spectra of L-Try in pH 7.5 BR solution.
Fig. 2. Changes of fluorescence spectra of L-Try upon increasing various concentration of
warfarin (μmol L-1): a to g (0; 2.0; 4.0; 6.0; 8.0; 10.0; 12.0) in pH 7.5 BR solution. The
concentration of L-Try was 4.0 × 10-5 mol L-1.
3.3 Optimum experimental conditions
3.3.1 Effects of pH
The effects of acidity on the fluorescence intensity of system were investigated

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according to adding different pH values of BR buffer solution. Fig. 3, where F0 and F
represent the fluorescence intensity of L-Try, L-Try–warfarin, respectively. From Fig.
3, it could be seen that the fluorescence intensity was steady in the pH range of 5.0–
7.7, while it increased when the pH value got bigger. Hence, the pH of 7.5 was chosen
to perform all the experiments as it was closed to physiological condition.
Fig. 3. Effects of pH on the fluorescence intensity. The concentration of L-Try, warfarin were 4.0
× 10-5 mol L-1;8.0 μmol L-1 respectively.
3.3.2 Effect of reaction time
The effect of the time on the fluorescence intensity of the system is also
investigated. Under the pH 7.5 BR buffer solution, the difference of F0 and F reaches
the maximum within 3 min and keep stable at least 2 h. Hence, the measurement is
carried out after 3 min.
3.3.3 Selectivity study
Therefore, in order to assess the selectivity of the new sensor, the interference of
foreign substances were evaluated with 8.0 μmol L−1 of warfarin. As shown in Table 1,

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about 500 times inorganics (NaCl, KCl, CaCl2, MgCl2, CdCl2, NH4Fe(SO4)2, ZnCl2),
300 times vitamins (ascorbic acid, pyridoxine), 200 times amine acids (glycine,
asparticacid, arginine), 100 times carbohydrates (glucose, sucrose, β-cyclodextrin), 50
times aspirin and hydrochlorothiazide were in a tolerable error within ± 5.0%.
Therefore, the method has a good selectivity.
Table 1. Effects of coexisting substances (c(warfarin)= 8.0 μmol L−1)
Foreign
substance
NaCl
Concentration
(μg mL-1)
233.6
Relative
error (%)
2.2
Foreign
substance
Sucrose
Concentration
(μg mL-1)
273.8
Relative
error (%)
2.9
NaNO3
ZnCl2
339.6
3.5
β-cyclodextrin
L-Tyrosine
L-Arginine
Methionine
Phenylalanine
Glycine
907.9
-4.6
-2.8
-2.7
2.7
545.2
-1.4
3.4
289.9
KCl
298.2
278.7
MgCl2
380.8
2.8
238.7
NH4Cl
214.0
1.4
264.3
-2.6
-2.3
-3.5
-2.1
-3.4
-1.3
3.4
NiSO4
262.8
-4.1
3.5
120.2
CaCl2
443.6
Lysine
233.9
CoSO4
NH4Fe (SO4)2
CdCl2
281.1
2.6
Valine
187.5
484.2
3.2
L-Aspartic acid
Vitamin C
Pyridoxine
ASP*
212.9
266.5
3.7
422.6
KNO3
404.4
1.2
406.0
MnSO4
Glucose
604.0
-2.9
2.6
72.1
4.1
144.2
HCT*
119.1
4.5
ASP*: Aspirin, HCT*: hydrochlorothiazide.
3.4 Work curve
A calibration curve was manufactured under the optimum conditions by conducting
a series similar experiment at various concentration of warfarin. As shown in Fig. 4,

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the fluorescence intensity of the L-Try decreased gradually with warfarin
concentration increasing and the quenching efficiency (ΔF=F0−F), however,
displayed a good linear relationship against the warfarin concentration over the range
of 0.04–12.0 μmol L−1(R2=0.994) with an excellent detection limit (3σ/k) of 0.01
μmol L−1, as shown in Fig. 4, the quenching efficiency can also be described by the
following equations: ΔF=139.41 c−28.57, (ΔF=F−F0), where F and F0 are the
fluorescence intensity of the L-Try in the presence and absence of warfarin,
respectively). which suggested that the sensitive response could be employed as a
probe for the quantification of warfarin. Furthermore, the fluorescence quenching
method is much more sensitive fast, facile than the previous works, which were
shown in Table 2.
1800
R2=0.994
F=139.41c - 28.57
1500
1200

900
600
300
2
4
6
8
10
12
c/mol L-1
Fig. 4. The linear correlations of fluorescent intensity toward warfarin concentrations. warfarin:
2.0; 4.0; 6.0; 8.0; 10.0; 12.0 μmol L−1 in pH 7.5 BR solution. The concentration of L-Try was 4.0 ×
10-5 mol L-1.

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Table 2. Comparison of the proposed method with some typical methods employed for warfarin
determination.
Method*
Linearity
(μmol L−1)
Detection limit
(μmol L−1)
0.06
Remarks
FL*35
Rapid and effective, but the sensitivity is lower
Low-cost and sensitive yet require much time and
0.32∼3.24
CPE*36
0.21
0.50∼1000.0
several complicated
functionalized electrodes
steps
to
fabricate
CdTe QDs*37
HPLC*17,38
0.08
complicated steps and the reagents are toxic
0.1∼160.0
NG*
NG*
0.01
Effective and selective, but need expensive
equipments; using toxic organic solvents
eco–friendly, fast, facile and sensitive
0.39∼0.97
0.32∼32.4
0.04∼12.0
this work
FL*: Fluorescence; CPE*:carbon paste electrode; CdTe QDs*: CdTe quantum dots; HPLC*: high
performance liquid chromatography. NG*: not given.
3.5 The reaction mechanism
As Scheme. 1 illustrated, the indolyl and amino could interact with warfarin by
forming the intermolecular hydrogen bond, which caused the fluorescence
quenching of L-Try. Furthermore, the fluorescence quenching mechanism was
studied by spectrofluorometry at two temperatures (283 K, 298 K) (Fig. 5) with the
39,40
Stern–Volmer equation
:
F0/F = 1 + Kqτ0[Q] = 1 + Ksv[Q]
where F and F0 represent the fluorescence intensity of L-Try in the presence and
absence of warfarin, respectively. Ksv is the Stern–Volmer quenching rate constant,
which is a quenching efficiency measurement, and [Q] is the concentration of
quencher. τ0 is the life time of fluorophore (taken as 10−8 s), Kq is quenching rate
constants, and it could be calculated by the equation Kq = Ksv/τ0.

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283K
2.5
298K
2.0
1.5
1.0
2
4
6
8
10
c/molL-1
Fig. 5. The Stern-Volmer curves of warfarin quenched L-Try at different temperatures. c(L-Trp):
4 ×10-5 mol L-1. c(warfarin): 2.0; 4.0; 6.0; 8.0; 10.0 μmol L−1.pH=7.5.
Fig. 5 shows the Stern–Volmer plots of F0/F versus [Q] at two different
temperatures, from which can be seen that with the rising of temperature, the
quenching constant decreases. It can be concluded that the quenching process is
probably static quenching resulting from the formation of hydrogen bonds between L-
Try and warfarin. However, in order to verify the real quenching process deeply, the
fluorescence lifetime and UV-visible absorption spectra are employed to give some
more evidences for the actual quenching process.
The powerful method to distinguish weather it is dynamic or static quenching
mechanism is fluorescence lifetime. If dynamic quenching, τ0/τ = F0/F, fluorescence
lifetime can be cut down by quenching medium; if static quenching, τ0/τ = 1, the
quenching medium can’t change fluorescence lifetime of excitation state fluorescence
41
molecule.
To further confirm the quenching mechanism, the time-resolved
fluorescence decays using a FL-TCSPC Fluorolog-3 fluorescence spectrometer to

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investigate the excited states of the L-Try and L-Try-warfarin. In comparison to the L-
Try alone, the L-Try-warfarin complex has a much shorter lifetime (1.04 ns versus
1.24 ns). These results shown in Fig. 6 provide powerful evidence in support of our
previously mentioned speculation. According to the above analysis, the fluorescence
quenching of the L-Try should be attributed to the static quenching process that arose
from the interaction between the L-Try and warfarin.
a
b
10000
8000
6000
4000
2000
0
a
b
0
100
200
300
400
Time/ns
Fig. 6. Fluorescence emission decay curves of L-Try (a) and L-Try-warfarin system (b). c(L-
Try): 4 ×10-5 mol L-1; c(warfarin)= 8.0 μmol L−1. pH=7.5.
Meanwhile, the UV-vis absorption spectra of L-Try in the absence and presence of
warfarin were recorded (Fig. 7). The maximum wavelength (λmax) of L-Try was
located at 278 nm and the warfarin had a wide peak between 300 nm to 310 nm.
-1
-1
While the molar absorption coefficient decreased from 1.4 × 105 L mol cm to 0.3 ×
-1
-1
105 L mol cm when warfarin was added in, then the absorption spectrum shape
changed, and a sharp valley appeared at 210 nm. This might caused by the binding
force and intermolecular forces between L-Try and warfarin. In short, the formation

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of these binary complexes could cause absorption spectral changes to different extents.
The absorption spectral change is an important symbol of static fluorescence
quenching. The absorption coefficient of L-Try decreased after it interacted with
warfarin. This resulted in a decrease in photon transition causing fluorescence
quenching. The fluorescence intensity decreased with decreasing the molar
absorptivity, thus, the formation of a low light absorption complex of L-Try–
42,43,44
warfarin is an important reason of fluorescence quenching.
Fig. 7 Absorption spectra of L-Try-warfarin system. a: L-Try–warfarin, b: L-Try, c: warfarin.
c(L-Try): 4 ×10-5 mol L-1; c(warfarin): 8.0 μmol L−1 pH=7.5.
3.6 Determination of warfarin in tablets
In order to evaluate the performance of the proposed method in practical analytical
work, quantity of warfarin in tablet was attempted in spiked samples using the
standard addition method under the optimized conditions. Four tablets of warfarin
were weighed and grinding. About 0.1 g of powder(contained 0.25 mg warfarin) was
dissolved in ethanol, then transferred into a 100 mL calibrated flask and diluted up to

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the mark with water. 1.0 mL warfarin solution of these tablets was pipetted into a 10
mL calibrated flask and warfarin was detected via the fluorescence quenching method
according to the general procedure. The obtained results are summarized in Table 3.
Obviously, the recovery of warfarin was found to be between 95.0 and 103.3% using
spectrofluorometry method that is comparable with those obtained with HPLC.
Furthermore, the results, which shown in Table 3, kept accordance with the HPLC
method. In addition, the relative standard deviation of the proposed method is less
than 2.4%, indicating the acceptable precision of the spectrofluorometry
determination of warfarin by quenching the fluorescence of L-Try.
Table 3. The determination results of warfarin in tablets.
Samples
spectrofluorometry method
HPLC* method
Found/mark
(mg/tablet)
0.23/0.25
0.22/0.25
0.26/0.25
0.27/0.25
Added
(mg/tablet)
0.1
Found
(mg/tablet)
0.33
Recovery
(%)
RSD Found/mark Added Found/mark Recovery RSD
(%)
(%)
(%)
(mg/tablet) (mg/tablet) (mg/tablet)
Tablet 1
Tablet 2
Tablet 3
Tablet 4
100.0
95.0
1.5
2.2
1.8
2.4
0.22/0.25
0.24/0.25
0.26/0.25
0.23/0.25
0.1
0.2
0.3
0.4
0.32
0.45
0.55
0.64
100.0
105.0
96.7
1.2
1.4
2.2
1.7
0.2
0.41
0.3
0.57
103.3
97.5
0.4
0.66
102.5
HPLC*: high performance liquid chromatography.
4. Conclusion
Warfarin have been successively synthesized by the hydrolases catalyzed Michael
addition. Similarly, a novel probe for the determination of warfarin is proposed with a
low level of limit of detection. This report is the first application of biocatalytic
synthesis and fluorescence for the determination of warfarin by quenching the
fluorescence of L-tryptophan. The proposed method is simple and cost effective and

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can be applied for the detection of the drug in tablets with satisfactory results.
Therefore, this asymmetric Michael addition activity of, for example, PPL is a
valuable case of enantioselective lipase catalytic promiscuity and further studies
focusing on the improvement of enantioselectivity of the PPL-catalyzed
transformation are currently under investigation.
Acknowledgments
The authors gratefully acknowledge financial support for thisstudy by grants from
the National Natural Science Foundationof China (Grant Nos. 21475014).
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Graphic Abstract
The fabrication process of warfarin and a specific fluorescence switch sensor for
warfarin.
Scheme 1. The simplified sketch for the fabrication process of warfarin and its interaction with
tryptophan.

ACCEPTED MANUSCRIPT
Highlights


This is the first application of bio-catalytic synthesis and
fluorescence for the determination of warfarin.
The validity was verified by comparing the results with other
methods and the detection limit is 0.01 μmol L-1, which is lower
than for most of the recently published methods.

The quenching mechanism, which is proved to be static quenching,
has been investigated systematically by the linear plots at varying
temperatures based on the Stern–Volmer equation, fluorescence
lifetime, UV-visible absorbance spectra.