Nonpeptide-Based Small-Molecule Probe for Fluorogenic and Chromogenic Detection of Chymotrypsin

Article abstract of DOI:10.1021/acs.analchem.6b05115

We report herein a nonpeptide-based small-molecule probe for fluorogenic and chromogenic detection of chymotrypsin, as well as the primary application for this probe. This probe was rationally designed by mimicking the peptide substrate and optimized by a

Full text of DOI:10.1021/acs.analchem.6b05115

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Article
Non-Peptide-based Small-Molecule Probe for Fluorogenic
and Chromogenic Detection of Chymotrypsin
Lei Wu, Shu-Hou Yang, Hao Xiong, Jia-Qian Yang, Jun Guo, Wen-Chao Yang, and Guang-Fu Yang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b05115 • Publication Date (Web): 23 Feb 2017
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Non-Peptide-based Small-Molecule Probe for Fluorogenic and
Chromogenic Detection of Chymotrypsin
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Lei Wu,a † Shu-Hou Yang,a † Hao Xiong,a Jia-Qian Yang, a Jun Guo, a Wen-Chao Yang a * and Guang-Fu
Yanga, b *
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aKey Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan 430079, P.R. China;
bCollaborative Innovation Center of Chemical Science and Engineering, Tianjin 30071, P.R. China.
—————————
† These two authors made equal contributions.
* Wen-Chao Yang, e-mail: tomyang@mail.ccnu.edu.cn; Tel: 86-27-67867706; Fax: 86-27-67867141;
Guang-Fu Yang, e-mail: gfyang@mail.ccnu.edu.cn; Tel: 86-27-67867800; Fax: 86-27-67867141.
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ABSTRACT
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We report herein a non-peptide-based small molecule probe for fluorogenic and chromogenic
detection of chymotrypsin, and the primary application. This probe was rationally designed by
mimicking the peptide substrate and optimized by adjusting the recognization group. The
refined probe 2 exhibits good specificity toward chymotrypsin, producing about 25-fold
higher enhancement in both the fluorescence intensity and absorbance upon the catalysis by
chymotrypsin. Compared with the most widely used peptide substrate (AMC-FPAA-Suc) of
chymotrypsin, probe 2 shows about 5-fold higher binding affinity, and comparable catalytical
efficiency against chymotrypsin. Furthermore, it was successfully applied for the inhibitor
characterization. To the best of our knowledge, probe 2 is the first non-peptide-based
small-molecule probe for chymotrypsin, with the advantages of simple structure and high
sensitivity compared to the widely used peptide-based substrates. This small-molecule probe
is expected to be a useful molecular tool for drug discovery and chymotrypsin-related
diseases diagnosis.
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Keywords: Chymotrypsin, non-peptide substrates, drug discovery, sensors, kinetics
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INTRODUCTION
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Chymotrypsin (EC 3.4.21.1), one of the most common serine proteases, is involved in many
physiological processes, including digestion of dietary proteins, necrosis and apoptosis.1-6 In
addition, it has been implied in the pathogenesis of various diseases, such as pancreatic
fibrosis, maldigestion, diabetes mellitus, hypertension, inflammation, and many types of
cancer, particularly pancreatic cancer.7-14 Moreover, the chymotrypsin itself is a medicine for
reducing redness, swelling, or damages that associated with various situations likes infection
and surgery.15, 16 Therefore, the detection of chymotrypsin activity is highly important in drug
discovery and clinical diagnostics.
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To date, three major methods have been developed for assessing the activity of
chymotrypsin and screening chymotrypsin inhibitors: 1) UV/Vis assays with direct
chromogenic reaction catalyzed by chymotrypsin; 2) fluorometric assays with fluorogenic
peptide substrates that yield fluorescence enhancement after the catalysis; 3) enzyme-linked
immunosorbent assay (ELISA) and western blot methods using antibodies specific to
chymotrypsin.10, 17-26 Among them, ELISA and western blot methods are expensive and
time-consuming. Moreover, other than the concentration of the active form, the total
concentration of chymotrypsin was determined by this method. The colorimetric and
fluorometric assays both used peptide substrates (identified decades ago) that are prone to
degradation and thus require special storage. As far as we know, no small molecular probes
have been reported for the specific detection of chymotrypsin. Herein, we report the discovery
and the primary application of low-molecular weight non-peptide-based probes as substrates
for the fluorogenic and chromogenic detection of chymotrypsin.
Similarly as other serine proteases, chymotrypsin uses its catalytic triad (consisted of the
amino acid residues His-57, Asp-102, and Ser-195, human source) to cleave the peptide bond
in peptide or protein substrate, in which the Ser-195 serves as the nucleophilic attacker.4, 27
The chymotrypsin is specific for hydrolyzing the peptide or esters that are adjacent to
aromatic residues (Phe, Tyr and Trp). Based on this mechanism, we made our effort in
designing small organic molecules by mimicking the peptide substrate, in which a central
ester bond flanked by a pair of fluorophore and quencher (Scheme 1, PDB entry is 2P8O).28-30
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The desired probes were expected to show very weak absorbance background and exhibit
very low fluorescence based on the mechanism of photoinduced electron transfer (PET), but
upon cleavage of the ester bond by chymotrypsin, should produce a high level of optical
signal. Accordingly, an “off-on” optical sensing of chymotrypsin activity was established.
Recently, a peptide-based substrate with hydroxymethyl rhodamine green was designed as the
fluorescent probe for chymotrypsin detection and enabled the good and rapid identification of
chymotrypsin in pancreatic juice.10 Since methylfluorescein shows very similar structure
(compared with hydroxymethyl rhodamine green) and good optical properties, we chose
methylfluorescein as the fluorophore. After structure and sensing property study, we finally
identified a non-peptide-based small molecule as a specific chymotrypsin probe. To our
delight, the new probe demonstrates good properties of specificity, sensitivity, binding affinity
and catalytic efficiency against chymotrypsin. In addition, the small-molecule probe may be
applied to kinetically characterize the chymotrypsin inhibitors for drug discovery.
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Scheme 1. Illustration of the one step reaction to assess chymotrypsin activity with designed
probes.
EXPERIMENTAL SECTION
Organic Synthesis
Unless otherwise noted, all chemical reagents were commercially available and treated
with standard methods before use. Silica gel column chromatography (CC): silica gel
(200-300 mesh); Qingdao Makall Group Co., Ltd (Qingdao; China). Solvents were dried in a
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routine way and redistilled. H NMR and C NMR spectra were recorded in DMSO-d6 on a
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Varian Mercury 400 MHz, 500 MHz and 600 MHz spectrometer and resonances (δ) are given
in ppm relative to tetramethylsilane (TMS). The following abbreviations were used to
designate chemical shift mutiplicities: s = singlet, d = doublet, t = triplet, m = multiplet, br =
broad, q = quartet. High resolution Mass Spectra (HRMS) were acquired in positive mode on
a WATERS MALDI SYNAPT G2 HDMS (MA, USA). Melting points were taken on a Buchi
B-545 melting point apparatus and uncorrected.
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Synthesis of the intermediates and probe 1 (Scheme 2)
The synthesis of M2 was according to the literature as shown in Scheme 2. The
synthesis of M1: CH3I (2.84 g, 20 mmol) was added to the mixture of fluorescein (3.32 g, 10
mmol) and Cs2CO3 (2.77 g, 20 mmol) in DMF (20 mL) at room temperature. After stirring for
12 hours, the reaction mixture was filtered and washed with ethyl acetate. After extraction
with water, the organic phase was dried over anhydrous Na2SO4 and then concentrated under
reduced pressure. The residue was subjected to silica gel chromatography with
CH2Cl2/CH3OH (V:V = 50:1) to give M1 as a yellow solid (yield: 90 %), which was used for
next synthesis without further purification.
Sheme 2. The synthetic route of probes 1-3.
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The synthesis of M2: 10% aqueous solution of NaOH (10 mL) was added to the
solution of M1 (3.60 g, 10 mmol) in CH3OH (36 mL) at room temperature. After stirring for 3
hours, CH3OH was evaporated and the reaction mixture was diluted with H2O (100 mL). The
solution was acidified to pH = 5 with 1 M HCl, the resulting precipitate was filtered, washed
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with water and dried to give M2 as a yellow solid (yield: 82%). H NMR (400 MHz,
DMSO-d6): δ 10.19 (s, 1H), 8.01 (d, J = 4.0 Hz, 1H), 7.80 (t, J = 3.4 Hz, 1H), 7.73 (t, J = 4.0
Hz), 7.28 (d, J = 4.0 Hz, 1H), 6.94(s, 1H), 6.72-6.58 (m, 5H), 3.83 (s, 3H). HRMS calcd. for
[M + H]+: 347.0914. Found: 347.0911.
Synthesis of probe 1 as shown in Scheme 2: M2 (346 mg, 1.0 mmol) was dissolved in
dichloromethane, trimethylamine (151.5 mg, 1.5 mmol) was added, the resulting solution was
o
cooled to 0 C in ice bath. Then the solution of n-butyryl chloride (222 mg, 1.2 mmol) in
CH2Cl2 was added drop wise. The reaction mixture was stirred for 2 h at room temperature,
then diluted with H2O and extracted with CH2Cl2. The organic phase was dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The combined residue was
subjected to silica gel chromatography to give probe 1 as white solid (yield: 74%). m. p.
87-88 oC. 1H NMR (600 MHz, DMSO-d6): δ 8.05 (d, J = 7.2 Hz, 1H), 7.82 (t, J = 7.2 Hz, 1H),
7.75 (t, J = 6.6 Hz, 1H), 7.34 (d, J = 6.6 Hz, 1H), 7.24 (s, 1H), 6.96 (s, 1H), 6.94-6.86 (m, 2H),
6.75 (q, J = 9.2 Hz, 2H), 3.82 (s, 3H), 2.58 (t, J = 6.6 Hz, 2H), 1.66 (q, J = 7.2 Hz, 2H), 0.97 (t,
J = 6.9 Hz, 3H). 13C NMR (150 MHz, DMSO-d6): δ 170.59, 167.76, 160.43, 151.58, 151.41,
150.81, 150.31, 135.19, 129.73, 128.44, 128.39, 124.99, 124.23, 123.39, 117.71, 115.73,
111.71, 109.86, 109.71, 100.16, 81.12, 55.20, 34.76, 17.36, 12.97. HRMS calcd. for [M + H]+:
417.1333. Found: 417.1331.
The synthesis of probe 2 (Scheme 2)
Probe 2 was synthesized in the similar strategy as used in probe 1, and the yield is about
71 %. m. p. 198-199 oC. 1H NMR (600 MHz, DMSO-d6): δ 8.04 (d, J = 7.8 Hz, 1H), 7.68 (t, J
= 7.5 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.18 (d, J = 7.2 Hz, 1H), 7.08 (s, 1H), 6.82-6.78 (m,
3H), 6.70 (q, J = 16.4 Hz, 2H), 3.84 (s, 3H), 3.54 (t, J = 6.0 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H),
2.30 (quintuplet, J = 6.9 Hz, 2H). 13C NMR (150 MHz, DMSO-d6): δ 171.09, 168.96, 161.62,
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152.75, 152.22, 151.98, 151.46, 136.29, 130.82, 129.53, 129.47, 126.09, 125.33, 124.45,
118.74, 116.91, 112.76, 110.93, 110.80, 101.22, 82.11, 56.12, 34.31, 32.57, 27.86. HRMS
calcd. for [M + H]+: 495.0438. Found: 495.0437.
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The synthesis of probe 3 (Scheme 2)
The synthesis of probe 3 as shown in Scheme 2: M2 (346 mg, 1.0 mmol) was dissolved in
20 mL dichloromethane, (388 mg, 2.0 mmol) 2-methyl-3-phenylpropanoic acid was added.
The result solution was stirred in room temperature with (60 mg, 0.5 mmol) DMAP and (384
mg, 2 mmol) EDCI for 5 hours, then diluted with H2O and extracted with CH2Cl2. The
organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure.
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The combined residue was subjected to silica gel chromatography to give 2 as brown oil. H
NMR (500 MHz, DMSO-d6): δ 8.06 (d, J = 7.5 Hz, 1H), 7.79 (t, J = 5.0 Hz, 1H), 7.74 (d, J =
6.0 Hz, 1H), 7.34-7.19 (m, 6H), 7.05 (s, 1H), 6.95 (d, J = 2.5 Hz, 1H), 6.86 (d, J = 9.0 Hz, 1H),
6.76-6.70 (m, 3H), 3.81 (s, 3H), 3.08-2.98 (m, 1H), 2.87-2.83 (m, 1H), 2.66-2.59 (m, 1H),
1.24 (d, J = 7.0 Hz, 3H). 13C NMR (125 MHz, DMSO-d6): δ 177.34, 174.18, 169.01, 161.68,
152.79, 152.22, 152.02, 151.51, 140.07, 139.23, 136.31, 130.85, 129.47 (2C), 129.34, 128.82
(2C), 128.65, 126.15, 125.37, 124.46, 118.57, 116.96, 112.81, 110.99, 101.27, 82.12, 56.14,
41.21, 41.11, 16.93. HRMS calcd. for [M + Na]+: 515.1521. Found: 515.1528.
Determination of Quantum Yield
The quantum yields for both probes and their fluorophores were determined with
rhodamine B as reference by the previously reported method31-33, using rhodamine B as the
reference. The quantum yields were estimated with the same equation showed below.
(1)
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Colormetric assay
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The chromogenic assay for chymotrypsin activity was measured with the following
protocol. Purified human chymotrypsin obtained from Sigma-Aldrich Company was
dissolved in 0.1 M HEPES (pH 8.0, 0.5% DMSO) and aliquots of them were frozen at -20 °C.
The reaction mixture in a total assay volume of 200 µL contained appropriate amounts of our
probe, 0.1 M HEPES buffer (pH 8.0, 0.5% DMSO), and chymotrypsin. Enzymatic hydrolysis
of the probe was monitored by microplate reader (BioTek) in the presence or absence of
various concentrations of inhibitor at 30 °C, in 0.1 M HEPES at different pH. Each
experiment was repeated at least three times and the values were averaged.
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Fluorometric assay
Fluorometric assay for chymotrypsin was performed as described in the following
procedures by employing probes 1-3 as substrates and the peptide substrate (AMC-FPAA-Suc)
according to PET mechanism. The weakly fluorescent substrate becomes highly fluorescent
once the chymotrypsin cleaves the ester bond or amide bond, and the fluorescence
enhancement could be observed and the fluorescence intensity is linearly correlated with the
rate of hydrolysis at the early stage of the enzymatic reaction. Although the probes for elastase
were designed based the mechanism of PET, the internal filter effect as well as other possible
factors that may affect fluorescent signal should be calibrated for accurate kinetic
measurement. The dose dependent fluorescence intensity of fluorophores was measured in the
absence and presence of various concentrations of substrate. The concentrations of probe 2
were set to be 0.2 µM, 0.5 µM, 1 µM, 2 µM, 5 µM, 10 µM, 20 µM, 30 µM, 40 µM, 50 µM
and 60 µM, while the concentrations of peptide substrate were set to be 5 µM, 10 µM, 50 µM,
100 µM, 200 µM, 300 µM and 400 µM. The slope obtained from the fluorescence
enhancement as the function of its concentration was converted as the correction factor of
fluorometric assay. For the inhibitory kinetic study, various concentrations of inhibitor, and
appropriate amounts of probe 2 or peptide substrate were preincubated in 100 mM HEPES
(pH 8.0, 0.5% DMSO) at 30 °C. Enzyme (50 μL) was then added to start the reaction and the
fluorescence signal was recorded at λem = 515 nm (λex = 450 nm) for our probe and λem = 445
nm (λex = 350 nm) for peptide substrate, with microplate reader (SpectraMax M5, Molecular
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Devices) using black microplate (96 wells). Each experiment was repeated at least three times
and the values were averaged.
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Molecular Docking
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The three dimensional structures of the probes 1-3 was constructed and primarily
optimized by Sybyl 2.0 software. Then the structural optimizations for all the probes were
carried out in tripos force field. The optimized probes were docked into the crystal structures
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of chymotrypsin (PDB entry: 2P8O) with Autodock software, respectively. The gird size
was set to be 60×64×68 for both of the Autodock and Vina methods. The Lamarkian genetic
algorithm (LGA) was applied for the conformational search of the Autodock. The best poses
were selected for the binding model analysis for all the probes.
RESULTS AND DISCUSSION
Probe design and optimization
Considering the structural diversity of recognition group having different steric and
electronic effect, three acyl groups (butyryl, 4-bromobutyryl and 2-phenylbutyryl)
were selected as recognition site to establish the target probes (probes 1-3, Scheme 1).
In general, target compounds were prepared by straightforward synthetic routes shown
in Scheme 2. The chemical structures of all the three probes and their intermediates
were fully characterized (See Figures S1-S7). After structural validation, the
properties of the synthesized probes and the corresponding chromosphore
methylfluorescein were characterized by UV-Vis and fluorescence spectra,
respectively. To our delight, it was found that all three probes (Φ<0.01) exhibited
extremely low background signal on both the absorbance and the fluorescence. On the
contrary, the hydrolytic product is a good chromophore with maximum absorbance at
450 nm, and an excellent fluorophore (Φ= 0.43) with the maximum emission at 515
nm (see Figure S8). It is noteworthy that all probes showed good water solubility and
were very stable in aqueous solution and no significant change of color and the
fluorescence intensity (FI) was observed before the addition of chymotrypsin. We then
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performed the primary screening by measuring the UV-Vis and fluorescence spectra of
each probe in HEPES buffer (100 mM, pH 7.0, 0.5% DMSO) after the addition of
chymotrypsin, respectively. The most remarkable increase on the absorbance and the
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FI were identified for probe
2
and relatively lower response for probe
3, whereas no
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significant response for probe
1
. In our understanding, the bromo-substitution of probe 2
may make the nucleophilic attack of Ser much easier than that of probes 1 and 3. In addition,
we performed the molecular docking and obtained the simulated binding modes of probes 1-3
in the active site of chymotrypsin. As shown in Figure 1, both probes 1 and 2 formed H
bonds with Gly216 in the active site, respectively, whereas no H bond was observed in the
binding mode of probe 3. More importantly, the Ser195 showed the closest distance to the
ester bond of probe 2 than those of probes 1 and 3, which is expected to facilitate the
nucleophilic attack in the active site. Therefore, probe
studies.
2 was picked for the follow-up
Figure 1. Simulated binding models of probe 1 (A), probe 2 (B) and probe 3 (C) in the active
sites of chymotrypsin, respectively. The distances between the key residue Ser195 and the
carbonyl group of probes were showed with arrow line in blue, and the H bonds were
depicted in red dash line.
Since the pH value may affect the reactivity of probe, we therefore studied the
influence of pH on the chymotrypsin-catalyzed hydrolysis with probe
2. It revealed
that the fluorescence of probe in the presence of chymotrypsin significantly increased
2
as the pH increased from 6.0 to 9.0, and almost reached the maximum at 8.0 (see
Figure S9). Beside, previous report claimed that the optimum pH range for
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chymotrypsin is 7.8-8.0. As a whole, the most suitable pH for the reaction of probes
with chymotrypsin is 8.0, which was adopted in the following studies. We selected the
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moderate temperature 30 C as the temperature without further optimization. Under
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these conditions, the time-dependent increase on the absorbance (Figure 2A) and the
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FI (Figure 2B) were obtained for probe
2 in the presence of 10 µg/mL chymotrypsin in
HEPES buffer (100 mM, pH 8.0, 0.5% DMSO). Apparently, after incubation with
chymotrypsin for 35 min, the color of reaction mixture changed from colorless to green
and no fluorescence to strong green fluorescence was observed. The maximum
absorbance at 450 nm and the maximum FI at 515 nm jointly indicated around 25-fold
increase over the basal level (Figure 2). The data showed that probe
2 offered a
sensitive colormetric and fluorometric “off-on” system for sensing chymotrypsin
activity, which could be visualized by naked-eye and spectrophotometers.
0.14
0.12
0.10
0.08
0.06
0.04
A
B
2800
2400
2000
1600
1200
800
35 min
0 min
35 min
0 min
400
0
480 500 520 540 560 580 600 620
375 400 425 450 475 500 525
Wavelength nm
（
）
Wavelength（nm）
Figure 2. The time-dependent absorbance (A) and fluorescence (λex = 455 nm, B) of probe 2
(10 μM) in HEPES buffer (100 mM, pH 8.0, 0.5% DMSO) in the presence of chymotrypsin
(10 μg/mL) at 30 °C; Left Insets show the photos of the corresponding reaction mixtures in
the absence and presence of chymotrypsin (0.02 U/mL) after 35 min incubation at 30 °C.
Photos in the right inset were taken under UV light (365 nm) illumination.
Since the sensitivity of fluorometric method is usually three orders of magnitude
higher than colormetric method, we mainly focus on the fluorometric “off-on” sensing
in the rest experiments. To further elucidate the applicability of the refined probe to
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quantitatively detect chymotrypsin, the fluorescence of the assay system was carried
out in the presence of various concentrations of chymotrypsin (0-40 µg/mL). As
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depicted in Figure S10, the FI of probe
increase of the concentration of chymotrypsin. Furthermore, a linear correlation was
obtained between the slope of the probe and the concentration of chymotrypsin from
2 increased notably and sharply with the
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2
0 µg/mL to 10 µg/mL. According to the limit of detection (LOD)=3σ/k method, in
which σ means the standard deviation of blank experiment and k means the slope of
the calibration curve, the LOD for this probe to detect chymotrypsin was determined to
be ~50 ng/mL based on three independent measurements.
To shed light on the reaction mechanism of chymotrypsin-catalyzed hydrolysis of the
probes, the reaction mixture (100 mM HEPES buffer at pH 8.0, 10 µg/mL
chymotrypsin) was analyzed by HRMS, with the similar reaction system containing no
chymotrypsin as the negative control. As shown in Figure 3, the peaks for the probe
2
alone and its pre-incubation with chymotrypsin in HEPES buffer (100 mM, pH 8.0,
0.5% DMSO) after 60 min were obtained. The molecular ion peak of m/z 495.0437
corresponds to the probe (2
+H+), while the molecular ion peak (m/z 347.0916)
correlates to the fluorophore that was released from cleavage of the ester bond of the
designed probe. Thus, the above observation indicated that the hydrolysis was caused
by the chymotrypsin-catalyzed cleavage at the ester bond.
5
7x105
6x105
5x105
4x105
3x105
2x105
B
A
1.8x10
1.6x105
1.4x105
1.2x105
1.0x105
8.0x104
6.0x104
4.0x104
495.0437
347.0916
1x105
2.0x104
0.0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
5
0
3
5
0
5
0
5
0
0
m/z45
3
4
4
5
5
3
3
4
5
5
m/z
Figure 3. HRMS spectrum of probe 2 (20 µM) without (A) and with (B) the addition of
chymotrypsin (10 μg/mL) and incubated for 1 h in HEPES buffer (100 mM, pH 8.0, 0.5%
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DMSO) at 30 °C.
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Probe Specificity
9
To disclose the specificity of probe 2, its hydrolysis was examined under the interference
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of various analytes, including metal ions (K+, Na+, Mg2+, Co2+, Mn2+, Fe3+), anions (HS-,
-
2-
-
2-
HSO3 , SO3 , HCO3 , HPO42- and CO3 ), amino acids and biothiols (Met, GSH, Phe, Lys,
Trp, Ala and His), as well as the common esterases or proteins (elastase, AChE, BChE,
trypsin, carboxypeptidase and BSA). The data presented in Figure 4A and Figure S11 shows
that this probe is highly selective for chymotrypsin over competing amino acids, metal ions,
and biothiols. More interestingly, the relative reaction rate of chymotrypsin-catalyzed
hydrolysis for probe 2 is much higher than that of other hydrolases and proteins (Figure 4B),
whereas none of those hydrolases and proteins showed significant interference against
chymotrypsin. The above study indicated that probe 2 is highly specific toward chymotrypsin.
80
B
A 60
60
40
20
0
50
40
30
20
10
0
Figure 4. (A) The specificity profile of probe 2 (10 µM) toward different metal ions (K+, Na+,
Mg2+, Co2+, Mn2+, Fe3+), amino acids and biothiols (Met, GSH, Phe, Lys, Trp, Ala and His).
All the analytes except chymotrypsin (10 μg/mL) are in the concentration of 1 mM. (B) The
specificity profile of probe 2 (10 µM) toward different esterases or proteins (elastase, AChE,
BChE, trypsin, BSA, carboxypeptidase) in HEPES buffer (100 mM, pH 8.0, 0.5% DMSO).
Shr refers the rate of spontaneous hydrolysis; AChE, BChE, and BSA are the abbreviations of
acetylcholinesterase, butyrylcholinesterase, and bovine serum albumin, respectively.
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Michaelis–Menten Kinetics
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To further elucidate the feasibility of the developed probe as a substrate for evaluating
the enzymatic mechanism of chymotrypsin, we performed the kinetic characterization. Here
we still only took the fluorogenic measurement, considering the higher sensitivity of
fluorometric assay compared to colorimetric method. Generally, the initial reaction rates for
the enzymatic hydrolysis of target probe were derived from the fluorescence response
depending on the concentration of probe 2 (0-50 µM), while the widely used method with the
peptide substrate (AMC-FPAA-Suc) as the reference. As anticipated, the kinetic curve
describing the velocity versus the concentration of substrate obeys the Michaelis–Menten
equation and the specific curves as shown in Figure 5A and 5B. On the basis of the above
kinetics, the Michaelis constant (Km) and the catalytic rate constant (kcat) of chymotrypsin for
probe 2 were determined to be 11.09 ± 2.08 µM and 3.45 ± 0.26 min-1, respectively. Thus, the
total catalytic efficiency (kcat/Km) of chymotrypsin in hydrolyzing probe 2 is about 0.22
µM-1•min-1, indicating the probe 2 is a suitable substrate for assessing the chymotrypsin
activity. For clear comparison, we also tested the Km (58.84 ± 6.47 µM) and kcat (210 ± 15
min−1) of chymotrypsin against the existing method with peptide substrate (AMC-FPAA-Suc)
under the same condition. In all, the binding affinity of probe 2 with chymotrypsin is ~5-fold
higher than that of AMC-FPAA-Suc, although the catalytic efficiency for probe 2 is relatively
lower. More importantly, the synthetic cost of probe 2 is ~40-50 ￥/g without further
optimization, whereas the price of AMC-FPAA-Suc is about 2500 ￥ per 5 mg according to
the Wuhan Fine Peptide Co., Ltd. Hence the cost of probe 2 is more than 10000-fold lower
than that of AMC-FPAA-Suc. The above analyses implied that probe 2 could be a suitable
substrate for chymotrypsin activity assessment.
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20
Probe 2 (
30
40
M)
50
60
0
100
200
300
M)
400
μ
AMC-FPAA-Suc (
μ
Figure 5. Michaelis-Menten curve of probe 2 (A) and AMC-FPAA-Suc (B) catalyzed by
chymotrypsin (5 µg /mL) in HEPES buffer (100 mM, pH 8.0, 0.5% DMSO) with fluorometric
method.
Application in Inhibitor Characterizing
One of the most important applications for the bioanalytical methods is the inhibitor
screening and characterization in drug discovery. Thus, the new fluorometric method with
probe 2 as the substrate was employed to study the inhibitory kinetics for the well-known
chymotrypsin inhibitors. Two typical chymotrypsin inhibitors, a covalent inhibitor
phenylmethanesulfonyl fluoride (PMSF, purchased from J&K Chemical Company) and a
reversible inhibitor chymostatin (CAS number 9076-44-2, purchased from Sigma-Aldrich),
were chosen as the two representatives for kinetic study with the newly developed method.
We incubated all the reaction systems composing of probe 2 (10 µM), chymotrypsin (20 µg
/mL) and different amounts of PMSF (0.1 to 40 µM) and chymostatin (0 to 40 µM) in the
96-well plates at 30 °C and the FI at 515 nm was continually recorded for 5 min by microplate
reader. One thing to note is that the PMSF was preincubated with enzyme for 10 min due to
its irreversible binding manner. As expected, the enhancement of FI was retarded gradually
when the concentration of inhibitors increased, and the kinetic curves of the residual activity
against the concentration of inhibitors were obtained accordingly (See Figure 6A and 6B).
Then the IC50 values for PMSF and chymostatin inhibitor were calculated to be 22.89 ±
3.27 nM and 3.12 ± 0.34 µM, respectively. The above values are very close to those (16.65 ±
2.34 nM for chymostatin and 5.53 ± 1.49 µM) obtained with the peptide-based substrate
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(AMC-FPAA-Suc) under the same conditions. To further explore the selectivity of probe 2,
the fluorescence changes were studied in the presence of some other inhibitors, including the
acetylcholinesterase inhibitors (tacrine and donepezil), the elastase inhibitor (sivelestat) and
p-hydroxyphenylpyruvate dioxygenase inhibitor (sulcotrione). In general, no significant
fluorescence change was observed by non-chymotrypsin inhibitors against the probe 2-based
assay (see Figure S12). The above results demonstrated that probe 2 has a great potential to
be used as a reliable non-peptide substrate for discovering chymotrypsin inhibitors under mild
conditions.
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B
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With probe 2 as the substrate
0
10
20
30
40
0
10
20
30
40
PMSF (
μ
M)
PMSF (μM)
Figure 6. Plots of inhibitory efficiency of phenylmethanesulfonyl fluoride (PMSF) toward
the chymotrypsin with peptide (AMC-FPAA-Suc)-based method (A) and probe 2-based
method (B).
CONCLUSION
In conclusion, a non-peptide-based small-molecule probe (2), was discovered as a specific
chromogenic and fluorogenic substrate for chymotrypsin. Probe 2 showed very low
background in both absorbance and fluorescence, but exhibited remarkable enhancement in
bright green signal upon the catalysis of chymotrypsin. In addition, chymotrypsin showed
about 5-fold higher binding affinity against probe 2 than the most widely used peptide
substrate (AMC-FPAA-Suc), although the overall catalytical efficiency for probe 2 was
relatively lower. More interestingly, this refined probe exhibited excellent specificity for
chymotrypsin over other proteins or hydrolases. To our knowledge, probe 2 is the first-ever
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non-peptide-based chromogenic and fluorogenic substrate for chymotrypsin. Accordingly, a
turn-on colormetric and fluorescent assay was established for evaluating chymotrypsin
activity in aqueous solution, and subsequently applied in chymotrypsin inhibitors screening
and characterization. The current study demonstrated that the small-molecule probe 2 could
be potentially applied in future high-throughput studies in drug discovery and clinical
diagnosis of chymotrypsin-associated diseases.
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AUTHOR INFORMATION
† These authors contributed equally.
Corresponding authors
*Wen-Chao Yang, e-mail: tomyang@mail.ccnu.edu.cn; Tel: 86-27-67867706; Fax:
86-27-67867141;
*Guang-Fu Yang, e-mail: gfyang@mail.ccnu.edu.cn; Tel: 86-27-67867800; Fax:
86-27-67867141.
ACKNOWLEDEMENTS
We are grateful for the financial support from the National Natural Science Foundation of
China (Grant No. 21332004, 21372094 and 21672079).
SUPPORTING INFORMATION
The general synthesis and NMR and HRMS spectra for the newly synthesized compounds
and some figures for probes characterization. This material is available free of charge via the
Internet at http://pubs.acs.org.
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