Novel inhibitors of tyrosinase produced by the 4-substitution of TCT (П)

Article abstract of DOI:10.1016/j.ijbiomac.2017.05.036

Novel Tyrosinase Inhibitors of 4-functionalized Thiophene-2-carbaldehyde thiosemicarbazone (TCT) derivatives (1–8) had been synthesized and Spectrofluorimetry, ¹H and ¹³C NMR titration and Molecular docking had been used to investigate their inhibitory activities and mechanisms on tyrosinase. The results showed that the inter-molecular interactions or hydrogen bond formation by increasing length of carbon chain or introducing benzene ring to the 4-functionalized ester group promoted or stabilized formation of complexes between modifier and tyrosinase, and enhanced the inhibitory activity of modifiers. The inhibitory activity of 4-benzoy methoxy-TCT was much stronger than that of any other synthesized tested modifiers, which was well explained by molecular docking and further verified by spectrofluorimetry and NMR titration by assuming that there existed an inter-molecular interaction besides formation of hydrogen bonds between the amino acid residues ASN260, GLU256, HIS85 of enzyme and the modifier.We concluded that 4-benzoy methoxy substitution of TCT was a good route obtaining novel tyrosinase inhibitors and deserved further studies.

Full text of DOI:10.1016/j.ijbiomac.2017.05.036

Accepted Manuscript
Title: Novel Inhibitors of Tyrosinase Produced by the
4-substitution of TCT (П)
Authors: Jing Liu, Mengrong Li, Yanying Yu, Shuwen Cao
PII:
S0141-8130(17)30163-0
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.05.036
BIOMAC 7525
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
11-1-2017
2-5-2017
8-5-2017
Please cite this article as: Jing Liu, Mengrong Li, Yanying Yu,
Shuwen Cao, Novel Inhibitors of Tyrosinase Produced by the
4-substitution
of
TCT
(П),
International
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Research highlights
1. Intermolecular interactions between inhibitor and amino acid residues ASN81 of
enzyme
2. The 4-benzoy methoxy substitution of TCT produced new novel tyrosinase
inhibitors
3. The inhibitor introduced secondary and tertiary conformational changes in enzyme
1

Novel Inhibitors of Tyrosinase Produced by the
4-substitution of TCT (П)
Jing Liua, Mengrong Lia, Yanying Yu a, Shuwen Cao a,b*
aDepartment of Chemistry, Nanchang University, Nanchang 330031, PR China
bState Key Laboratory of Food Science and Technology, Nanchang University,
Nanchang 330047, PR China
*Corresponding author. Tel.: +86 0791 83969610. Fax: +86 0791 83969610.
E-mail address: caosw@ncu.edu.cn (S.-W.Cao).
Abstract
Novel Tyrosinase Inhibitors of 4-functionalized Thiophene-2-carbaldehyde
thiosemicarbazone (TCT) derivatives (1-8) had been synthesized and
1
13
Spectrofluorimetry, H and C NMR titration and Molecular docking had been used
to investigate their inhibitory activities and mechanisms on tyrosinase. The results
showed that the inter-molecular interactions or hydrogen bond formation by
increasing length of carbon chain or introducing benzene ring to the 4-functionalized
ester group promoted or stabilized formation of complexes between modifier and
tyrosinase, and enhanced the inhibitory activity of modifiers. The inhibitory activity
of 4-benzoy methoxy-TCT was much stronger than that of any other synthesized
tested modifiers, which was well explained by molecular docking and further verified
by spectrofluorimetry and NMR titration by assuming that there existed an
inter-molecular interaction besides formation of hydrogen bonds between the amino
2

acid residues ASN260, GLU256, HIS85 of enzyme and the modifier.We concluded
that 4-benzoy methoxy substitution of TCT was a good route obtaining novel
tyrosinase inhibitors and deserved further studies.
Keywords: Thiophene-2-carbaldehyde thiosemicarbazone (TCT) derivatives;
Tyrosinase inhibitors; Spectrofluorimetry; 1H and 13C NMR titration.
3

1. Introduction
Tyrosinase (EC 1.14.18.1), a kind of metalloenzyme with dinuclear copper ions,
is common in animals, insects and microorganisms [1]. Tyrosinase belongs to the
Type-3 copper protein family, there are two closely spaced copper ions at the active
site of the structures, each of them is coordinated by three His residues through
nitrogen atoms [2]. Generally, tyrosinase can catalyze the rate-limiting step in the
biosynthetic pathway of melanin pigments, is widely distributed in nature [3]. The
enzyme participates in several important reactions of host defence, wound healing and
sclerotization in insects and other arthropods [4]. Tyrosinase has also been found to be
responsible for undesired enzymatic browning of farm products, such as bruised or cut
fruits and vegetables, which subsequently leads to a significant decrease in their
nutritional and market values[5]. Moreover, pigmentation, melanoma and Parkinson's
syndrome are also closely related to it [6, 7].
To find naturally occurring or synthesize novel tyrosinase inhibitors is receiving
a considerable attention from scientists of food, medicine, chemistry and animal fields
[8-12]. As an outcome of these studies, a huge library of tyrosinase inhibitors has been
discovered from natural sources or synthesized in the laboratories. However, these
inhibitors such as kojic acid, arbutin, etc, have been troubled by the cost, effectiveness,
safety [13]. Although in our prior study of some novel tyrosinase inhibitors, TCT and
its modifier of 4-methoxyacetyl -TCT, we obtained a preliminary understanding of the
structure-activity relationships and inhibitory mechanisms between these modifiers
1
and target enzymes by using fluorescence, H NMR titration and molecular docking
4

studies [14,15]. However, we still have little understanding of how the
structure-activity relationships and inhibitory mechanisms affect the inhibitory
activities when modifying TCT by increasing length of carbon chain or introducing
ring of cyclocarbon or benzene to the 4-functionalized ester group aiming at changing
chemical micro-environment of hydrophobic domain of tyrosinase active center.
Generally, this type of structural modifications either increases or decreases the
inhibitory activity of the modifier. Based on our understanding of the
structure-activity relationship of 4-methoxyacetyl -TCT, we find it possible to
increase the inhibitory activity of a modifier by introducing an appropriate functional
group at the thiophene ring on TCT, assuming that the introduced group promotes the
formation of complexes between the sulfur atom from the thiourea of target modifiers
and the copper ion of the enzyme active center. It is necessary to find new novel
tyrosinase inhibitors with strong inhibitory activities to better our understanding of the
structure-activity relationship of TCT.
With the advancement of science and technology, it is clear that good inhibitory
activity, bearable and acceptable toxicity were fundamental requirements for a
compound taken as a reasonable precursor undertaken chemical modifying aimed at
finding potential novel tyrosinase inhibitors. Based on this standard, we had
investigated the related toxicity data of TCT and found that it was a reasonable and
appropriate precursor (https://chem.sis.nlm.nih.gov) for finding candidates for
potential tyrosinase inhibitors of food additives [15].
Novel Tyrosinase Inhibitors of 4-functionalized TCT derivatives (1-8) were
5

designed, synthesized and evaluated for their inhibitory and mechanisms on tyrosinase
1
13
by using Spectrofluorimetry, H and C NMR titration and Molecular docking. Our
study helped further clarify the relationship between modifiers and tyrosinase and
understand effects on inhibitory activity introduced by modifying TCT with
increasing length of carbon chain or introducing ring of cyclocarbon or benzene to the
4-functionalized ester group of TCT, which may provide a promising route to obtain
novel and highly potent tyrosinase inhibitors.
2. Materials and methods
2.1. Chemicals and reagents
Mushroom tyrosinase (EC 1.14.18.1) and L-3, 4-dihydroxyphenyl-alanine (L-DOPA)
were purchased from Sigma (St. Louis, MO, USA). 2-thiophenecarboxaldehyde and
thiosemicarbazide were obtained from J&K Chemical Co (Shanghai, China). All other
reagents were local and of analytical grade. The water used was re-distilled and
ion-free.
2.2. Synthesis
The method of synthesis of intermediates and target compounds is described in
Fig.1, as references with yield improvement of reactions of 4-chloromethyl- and
4-hydroxymethyl to 65% and 70% [16-19], respectively.
For the synthesis of all intermediates of acid chlorides described in Fig. 1, (B),
the strict control of the reaction conditions (n acid:nSOCl2=1.25:1) was needed. When
the reaction of synthesizing acid chloride was completed, the produced liquid was
6

dropped directly into the cold mixture of 4-hydroxymethyl-2-thiophene formaldehyde
with dichloromethane, and then the intermediate of Acylmethoxy-2-thiophene
carboxaldehyde was received. Finally, the target compounds 1-8 were produced by the
reaction depicted in Fig. 1, (B) with the yield of about 90% [20].
ESI–MS data were obtained on a Agilent 6545 Q-TOF LC/MS (USA), and NMR
data were acquired on a 400 MHz NMR spectrometer (DD2-400) from Agilent
(USA).The IR spectra were measured with KBr pellets on a Nicolet 5700 FT-IR
spectrometer(USA).
4-Formyl methoxy-2-Thiophenealdehyde thiosemicarbazone (1): yellow crystals,
1
yield 23.4%, IR (KBr) 3155.34, 1721.70, 1598.55, 1528.20. H NMR (400 MHz,
DMSO-d6) δ 11.43 (s, 1H, NH), 8.28 (s, 1H, -CO-H), 8.21 (s, 1H, NH-H), 8.18 (s, 1H,
NH-H), 7.62 (s, 1H, thiophene-H), 7.57 (s, 1H, CH), 7.41 (s, 1H, thiophene-H), 5.09
(s, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ177.99 (1C, C=S), 162.34 (1C, C= O),
139.68 (1C, C= N), 137.63 (1C, 2-thiophene), 137.47 (1C, 4 -thiophene), 131.03 (1C,
3-thiophene), 128.00 (1C, 5-thiophene), 60.43 (1C, CH2). MS (ESI): m/z
244.0208[M+H] +.
4-Acetyl methoxy-2-Thiophenealdehyde thiosemicarbazone(2) [15]: synthesized as
the contrast.
4-Propionyl methoxy-2-Thiophenealdehyde thiosemicarbazone(3): yellow crystals,
yield 33.6%, IR(KBr) 3154.38, 1729.59, 1596.17, 1534.91.1H NMR (400 MHz,
7

DMSO-d6) δ 11.44 (s, 1H, NH), 8.21, 8.17 (d, 2H, NH2), 7.58 (s, 2H, thiophene-H,
CH), 7.39 (s, 1H, thiophene-H), 5.00 (s, 2H, CH2), 2.34 (q, J = 7.5 Hz, 2H, CH2), 1.01
13
(t, J = 7.5 Hz, 3H, CH3). C NMR (100 MHz, DMSO-d6) δ177.99 (1C, C=S),
173.90(1C, C=O), 139.58 (1C, C=N), 138.07(1C, 2-thiophene), 137.69(1C,
4-thiophene), 131.01(1C, 3-thiophene), 127.55(1C, 5-thiophene), 61.00(1C, CH2). MS
(ESI): m/z 272.0520[M+H] +.
4-Butyryl methoxy-2-Thiophenealdehyde thiosemicarbazone(4): brown crystals, yield
32.7%, IR(KBr) 3153.14，1730.54, 1599.75, 1530.33. 1H NMR (400 MHz, DMSO-d6)
δ 11.42 (s, 1H, NH), 8.19 (s, 1H, NH2), 8.17 (s, 1H, NH2), 7.57 (s, 1H, thiophene-H),
7.55 (s, 1H, CH), 7.38 (s, 1H, thiophene-H), 5.00 (s, 2H, CH2), 2.29 (t, J=7.2 Hz, 2H,
13
CH2), 1.53 (dd, J = 14.7, 7.3 Hz, 2H, CH2), 0.85 (t, J = 7.4 Hz, 3H, CH3). C NMR
(100 MHz, DMSO-d6) δ 178.01(1C, C=S),173.05 (1C, C=O), 139.59(1C, C=N),
138.07(1C, 2-thiophene), 137.70(1C,4-thiophene), 131.02(1C, 3-thiophene), 127.60
(1C, 5-thiophene), 60.93(1C, CH2), 35.69(1C, CH2), 18.36(1C, CH2), 13.86(1C, CH3).
MS (ESI): m/z 286.0677[M+H] +.
4-Pentanoyl methoxy-2-Thiophenealdehyde thiosemicarbazone (5) yellow crystals,
1
yield 33.2%, IR (KBr) 3153.13, 1733.73, 1599.75, 1529.78. H NMR (400 MHz,
DMSO-d6) δ 11.42 (s, 1H, NH), 8.19 (s, 1H, NH2), 8.17 (s, 1H, NH2), 7.57 (s, 1H,
thiophene-H), 7.56–7.50 (m, 1H, thiophene-H, CH), 7.38 (s, 1H, thiophene-H), 5.03
(d, J = 23.2 Hz, 2H, CH2), 2.31 (t, J = 7.4 Hz, 2H, CH2), 1.57 – 1.42 (m, 2H, CH2),
8

13
1.33 – 1.18 (m, 2H, CH2), 0.83 (t, J = 7.3 Hz, 3H, CH3) C NMR (100 MHz,
DMSO-d6) 178.00(1C, C= S), 173.18(1C, C=O), 139.57(1C, C=N), 138.06(1C,
2-thiophene), 137.68(1C, 4-thiophene), 131.01(1C, 3-thiophene), 127.58 (1C,
5-thiophene), 60.94(1C, CH2), 33.55(1C, CH2), 26.96(1C, CH2), 22.03(1C, CH2),
14.04(1C, CH2). MS (ESI): m/z 300.0833[M+H] +.
4-Cyclopentanecarbonyl
methoxy-2-Thiophenealdehyde
thiosemicarbazone(6):
yellow crystals, yield 34.8%, IR(KBr) 3152.59, 1728.83, 1598.85, 1529.80.1H NMR
(400 MHz, DMSO-d6) δ 11.42 (s, 1H, NH), 8.20 (s, 1H, NH2), 8.17 (s, 1H, NH2), 7.56
(s, 2H, thiophene-H, CH), 7.38 (s, 1H, thiophene-H), 5.00 (s, 2H, CH2), 2.76 (p, J =
7.8 Hz, 1H, CH), 1.80 (s, 2H, CH-H), 1.66 (dd, J = 16.2, 10.8 Hz, 2H, CH-H), 1.61 –
1.43 (m, 4H, CH2, CH2). 13C NMR (100 MHz, DMSO-d6) δ178.02(1C, C=S),
175.95(1C, C=O), 139.61(1C, C=N), 138.15(1C, 2-thiophene), 137.70 (1C,
4-thiophene), 130.94(1C, 3-thiophene), 127.48(1C, 5-thiophene), 61.10(1C, CH2),
43.37(1C, CH2), 29.90(2C, CH2), 25.80(2C, CH2). MS (ESI): m/z 312.0833[M+H] +.
4-Cyclohexaformyl methoxy-2-Thiophenealdehyde thiosemicarbazone(7): yellow
crystals, yield 34.0%, IR(KBr) 3155.40, 1726.98, 1598.87, 1530.58.1H NMR (400
MHz, DMSO-d6) δ 11.43 (s, 1H, NH), 8.20 (s, 1H, NH-H), 8.17 (s, 1H, NH-H), 7.56
(s, 2H, thiophene-H, CH), 7.38 (s, 1H, thiophene-H), 4.99 (s, 2H, CH2), 2.32 (t, J =
10.8 Hz, 1H, CH), 1.80 (d, J = 12.1 Hz, 2H, CH-H), 1.63 (d, J = 12.3 Hz, 2H, CH-H),
1.55 (d, J = 11.2 Hz, 1H, CH-H), 1.39–1.11 (m, 6H, Cyclohexane-H). 13C NMR (100
9

MHz, DMSO-d6) δ 178.00(1C, C=S), 175.17(1C, C=O), 139.60(1C, C=N), 138.13(1C,
2-thiophene), 137.70(1C, 4-thiophene), 130.86(1C, 3-thiophene), 127.34 (1C,
5-thiophene), 60.98(1C, CH2), 42.51(1C, CH2), 28.98(2C, CH2), 25.71(1C, CH2),
25.20(2C, CH2). MS (ESI): m/z 326.0991[M+H] +.
4-Benzoy methoxy-2-Thiophenealdehyde thiosemicarbazone (8): yellow crystals,
1
yield 35.6%, IR (KBr) 3153.18, 1714.51, 1598.58, 1526.30. H NMR (400 MHz,
DMSO-d6) δ 11.44 (s, 1H, NH), 8.19 (s, 2H, NH2), 7.99 (s, 1H, thiophene-H), 7.97 (d,
J = 1.3 Hz, 1H, CH), 7.70 (s, 1H, thiophene-H), 7.65 (t, J = 7.4 Hz, 1H, benzene-H),
13
7.57 (s, 1H, benzene-H), 7.55 – 7.47 (m, 3H, benzene-H), 5.27 (s, 2H, CH2). C
NMR (100 MHz, DMSO-d6) δ 178.01(1C, C=S), 165.98(1C, C=O), 139.75(1C, C=
N), 137.83(1C, 2-thiophene), 137.71(1C,4-thiophene), 133.95(1C, benzene),
131.05(1C, 3-thiophene), 129.92(1C, benzene), 129.73(2C, benzene), 129.27(2C,
benzene), 127.83(1C, 5-thiophene), 61.97(1C, CH2). MS (ESI): m/z 320.0518[M+H]+.
2.3. Enzyme activity assay
The assay of inhibition of target compounds on the diphenolase activity of
mushroom tyrosinase was performed by our reported procedure with minor
modifications [14]. L-DOPA was used as substrate for the enzyme activity assay. The
reaction media (3 mL) for activity assay contained 2.8 mL 0.5 mM L-DOPA in 50
mM Na2HPO4-NaH2PO4 buffer (pH 6.8) and 0.1 mL of different concentrations of
inhibitor (dissolved in DMSO previously). 0.1 mL of the aqueous solution of
10

mushroom tyrosinase was added to the mixture. The solution was immediately
monitored by measuring the linear increase in optical density at 475 nm of formation
of the DOPA chrome for 150s using a Shimadzu UV-2450 spectrophotometer (Japan).
The extent of inhibition by the addition of the sample was expressed as the percentage
necessary for 50% inhibition (IC50), calculated by SPSS19. The value of relative
enzymatic activity can be calculated by the following equation:
Relative enzymatic activity (%) =△OD1/△OD2×100%
Where △OD1 and △OD2 are the enzyme activity value of the reaction system
with inhibitor and reagent blank,respectively. Kojic acid was used as reference
standard inhibitors for comparison.
2.4. Determination of the inhibition mechanism and the inhibition type
Determination of inhibition mechanism was assayed by maintaining the
concentration of L-DOPA (0.5 mM) and changing the concentration of the enzyme
(16, 12, 8, 4 g /mL) in reaction medium. The enzyme activity was measured for
different concentrations of inhibitor. Determination of inhibition type and inhibition
constants was assayed by maintaining the concentration of enzyme (10 g/mL) and
changing the concentration of the L-DOPA (0.5, 1, 1.5, 2 mM) in reaction medium
[15]. The inhibition type was assayed by the Lineweaver–Burk plot, and inhibition
constants (KI or KIS) were determined by the second plots of the apparent Ks /Vm or
1/Vm versus the concentration of inhibitor [21], the equation can be written as:
[ ]
[ ]
퐼
1
퐾푆
퐼
1
=
(1 + ) + (1 +
)
[ ]
푉 푆
푉
퐾퐼
푉
푚
퐾퐼푆
푚
The V is the reaction velocity; KS is the Substrate constant; Vm is the maximal
11

velocity; [I] is the concentration of inhibitor; [S] is the concentration of substrate; KI is
the constants for the inhibitor binding with the free enzyme and KIS is the constants
for the inhibitor binding with enzyme substrate complex, They were obtained from the
slope or the vertical intercept versus the inhibitor concentration, respectively.
2.5. Fluorescence assay
The fluorescence quenching experiment was performed with reference [22]. The
reaction media (2 mL) contained 1.8 mL Na2HPO4-NaH2PO4 buffer (pH = 6.8, 50mM)
and 0.2 mL of the aqueous solution of mushroom tyrosinase(30 g/mL), and then
titrated by successive addition of compounds 1, 6, 8 and 6-Cu2+ complex (the
constituent ratio of ligand and Cu2+ was 2:1, the chelation ratio has been determined
by continuous variation method) [23] ,solution using a pipette (the concentrations
ranging from 0 to 94.12 μM, while the concentrations of compound 8 just half of it ).
Fluorescence
intensities
were
recorded
using
a
Hitachi
F-4600
0
spectrofluorophotometer (Japan) at three different temperatures (30, 35 and 40 C)
with an excitation wavelength of 280 nm, while excitation and emission slit widths
are 10 nm and 5 nm respectively.
To eliminate the probability of re-absorption and inner filter effects in UV
absorption, all of the fluorescence data were corrected for absorption of exciting light
and emitted light on the basis of the following relationship [24]:
⁄
2
= 퐹표푏푠푒(퐴 +퐴 ) 2
1
퐹
푐표푟푟
Fcorr and Fobs are the corrected and observed fluorescence intensities, respectively.
A1 and A2 are the absorbances of inhibitor at the excitation and emission wavelengths,
respectively. Fluorescence quenching was described by the well-known Stern-Volmer
12

equation:
퐹0
퐹
[ ]
= 1 + 퐾푞휏0 푄 = 1 + 퐾푠푣
[ ]
푄
Where F0 and F are the fluorescence intensities before and after the addition of
the quencher, respectively, Kq is the bimolecular quenching constant, τ0 (10−8 s) is the
lifetime of the fluorophore in the absence of the quencher, [Q] is the concentration of
the quencher, and KSV is the Stern–Volmer quenching constant.
For the static quenching interaction, if it is assumed that there are similar and
independent sites in the biomolecule, the apparent binding constant (Ka) and the
number of binding sites (n) can be calculated by the following equation [25]:
퐹0 − 퐹
log
= 푙표푔퐾푎 + 푛푙표푔[푄]
퐹
Synchronous fluorescence spectra were recorded by setting the excitation and
emission wavelength interval (Δλ) at 15 and 60 nm over a wavelength range of
270-320 nm and 250-320 nm, respectively [26].
The three-dimensional fluorescence spectra were measured under the following
conditions: the emission wavelength was recorded between 200 and 450 nm, and the
initial excitation wavelength was set at 200 nm with an increment of 5 nm.
2.6. 1H NMR and 13C NMR titration
1H NMR titration studies were performed to investigate the interaction between
the compounds 1, 6, 8 and tyrosinase. A 0.5 mL of 21 mM solution of compounds 1, 6,
8 in DMSO-d6 was prepared and titrated with tyrosinase solution by using a
micropipette (to final concentrations ranging from 0, 0.098 to 0.341 mg/mL). While
13C NMR titration of compound 8 (62.6 mM) in DMSO-d6 was prepared and titrated
13

with tyrosinase solution from 0 to 0.406 mg/mL. After each addition of tyrosinase, the
1H NMR and 13C NMR spectra were recorded and the changes in the chemical shift of
the protons were noted.
2.7. Molecular docking study
Molecular docking is an application wherein molecular modeling techniques are
used to predict how a protein (enzyme) interacts with small molecules (ligands)
[27-29]. The active site was docked with inhibitors using the dock suite of Accelrys
Discovery Studio 2.0 software (Accelrys, Inc.). The 3D structure of compound 8 was
generated in Chem3D Ultra 8.0, and the X-ray crystal structure of Agaricus bisporus
tyrosinase (PDB ID: 2Y9W) was retrieved from the RCSB Protein Data Bank
(http://www.rcsb.org/pdb). All crystallographic water molecules, solvent molecules
and ions were removed from the protein structure. The forcefield CHARMm function
was used to roughly search the conformations when compounds docked into
tyrosinase, and then the modified tyrosinase was selected as the binding site for the
study and expanded to make the cavity. Subsequently, the conformations of
compounds were optimized using the same forcefield function, followed by flexibly
being docked in a stepwise manner with the protocol of Dock ligands. The docked
conformation, which had the highest score, was selected to analyze the mode of
binding. The outputs were further rendered with Pymol to obtain visible combination
models.
3. Results and discussion
3.1. Effect and inhibition mechanism of thiosemicarbazone derivatives on the
14

diphenolase activity of mushroom tyrosinase
The IC50 values of the compounds 1-8 were listed in Table 1, Kojic acid, TCT
and 4-acetyl methoxy-TCT (compound 2) was used as comparative reference standard
inhibitors. In previous work we found that TCT (thiophene 2-carboxaldehyde
thiosemicarbazone) have stronger inhibitory effect for tyrosinase (IC50 =0.43 M),
this is mainly attributed to an auxiliary vicinity synergistic effect of the thiophene ring
[14]. Further research revealed that it was beneficial to the improvement of the
activity that the 4- position of thiophene rings was substituted by acetylmethoxy
group (IC50 = 0.34 M). We assumed that this favorable effect may root from the
much stronger auxiliary vicinity synergistic effect from the 4- acetylmethoxy
substitution group of TCT than that from TCT itself. In order to better understand
effects on inhibitory activity introduced by modifying TCT with increasing length of
carbon chain or introducing ring of cyclocarbon or benzene to the 4-functionalized
ester group of TCT, we synthesized a series of new 4-functionalized TCT derivatives
(1-8).
As shown in Table 1 we can found that the inhibition activity of modifier was
well enhanced as the increasing of length of the alkyl carbon chain or introducing
cyclocarbon ring to the 4-functionalized ester group of thiophene ring of TCT.
Interestingly, the improvement of inhibitory activity of modifier was greatly enhanced
by introducing benzene ring to the same position as cyclocarbon ring of TCT (IC50
=0.068 M), in which the inhibitory activity was increased 68.52% compared to that
of 4-acetyl methoxy-TCT (compound 2). The reasonable explanation for the
15

enhancement from the chemical modification by increasing length of substituted
group was that when the length of the alkyl carbon chain was increased or the
cyclocarbon ring was introduced to the 4-functionalized ester group of thiophene ring
of TCT, the formation of complexes between the inhibitor and tyrosinase was
promoted and enhanced due to improvement inter-molecular interaction between the
inhibitor and hydrophobic domain located in the active center of enzyme. The great
increase of inhibitory activity from the chemical modification by introducing benzene
ring to the 4-functionalized ester group of thiophene ring of TCT may partially
attribute to the much stronger inter-molecular interaction i. e. Vander Waals Force [30]
between introducing benzene ring and hydrophobic domain of enzyme active center,
which either increases the inhibitory activity of the modifier by disrupting both
tertiary and secondary structures of the tyrosinase or greatly promotes the formation
of complexes between the inhibitor and tyrosinase. These explanations were verified
by followed experiment data of spectrofluorimetry studies and well consistent with
the results of molecular docking depicted in Fig. 6.
Subsequently, the inhibitory mechanism of compounds 1-8 against tyrosinase for
the oxidation of L-DOPA had been studied. Fig.2A showed that the plots of the
enzymatic activity versus the concentrations of enzyme in the presence of different
concentrations of the inhibitors gave a family of straight lines that all passed through
the origin. A linear slope decrease with increasing concentration of the inhibitors
indicated that the inhibition caused by the inhibitors on tyrosinase was reversible [31,
32]. The inhibition type of inhibitors on tyrosinase was assayed using the
16

Lineweaver-Burk plots of the reciprocal of the reaction rate versus the reciprocal of
the substrate concentration. As shown in Fig. 2C, the double-reciprocal plots yielded a
group of straight lines intersecting at the second quadrant, which indicated that these
inhibitors most probably belonged to mixed-type inhibitors [21].
3.2 Spectrofluorimetry studies
3.2.1. Synchronous fluorescence
Synchronous fluorescence spectroscopy can provide information about the
micro-environmental alterations around fluorophores (tyrosine and tryptophan) of
tyrosinase, and understanding of conformation changes of enzyme.
As shown in Fig. 3 (A) and Fig. 3(B), the maximum emission wavelength of
tyrosine residues had a slight blue shift from 293.8→290.0 nm, while wavelength of
tryptophan residues had a slight red shift from 280.2→284.0 nm, which suggested that
the hydrophobicity and polarity around tyrosine and tryptophan residues of tyrosinase
were increased due to enhanced inter-molecular interaction (Van der Waals force)
between inhibitor and tyrosinase [26].
3.2.2. Three-dimensional fluorescence
The three-dimensional fluorescence spectroscopy has been recently employed as
a novel probe to monitor the secondary and tertiary conformation changes in proteins
[33]. As is well known, the peaks 1(λex = 225 nm, λem = 345 nm) and peak 2 (λex =
280 nm, λem = 360 nm) represented typical fluorescence peaks of protein. The peak 2
was related to the fluorescence spectral characteristics of Trp and Tyr residues due to
π→π* transition, showing changes in the tertiary structure [34], whereas peak 1
17

represented the fluorescence characteristics of the polypeptide backbone as a result of
n→π* transition, a probe for secondary structural changes [35].
Three-dimensional (3D) fluorescence spectra of tyrosinase in absence and
presence of compounds 1, 6 and 8 were performed. As shown in Fig. 3(a-d), we
observed different decreases of the fluorescence intensity of peak 1 and peak 2 for
tyrosinase under the same concentration of compounds 1, 6 and 8. Among them, the
largest decrease of the fluorescence intensity is tyrosinase in presence of compound 8
with decrease of 90.78% for peak 1 and 60.29% for peak 2, which indirectly
suggested that the interaction (Van der Waals force) between tyrosine and tryptophan
residues of tyrosinase and benzene ring moiety of compound 8 was the most powerful.
The decrease of the fluorescence intensity for the two peaks suggested that unfolding
of the polypeptide chain leads to the conformation changes and increases the exposure
of some buried sites (Trp/Tyr/peptide groups). These results suggested that the
disruption of both tertiary and secondary structures of the tyrosinase in
inhibitor-complex state [36] helped to enhance the inhibitory activity of inhibitor.
The calculations characteristic 3D parameters of other compounds are reported in
Table 2.
3.2.3. Fluorescence quenching of tyrosinase by inhibitors
The above studies suggested that the interaction between enzyme and inhibitors
may lead to change the surrounding chemical micro-environment of the active site of
tyrosinase. Therefore, in order to further understand reaction mechanism between
enzyme and inhibitors, we exploited fluorescence spectroscopy to characterize the
18

binding between small molecule and enzyme. The fluorescence emission spectra of
tyrosinase in the absence or in the presence of compound 6 excited at 280 nm are
shown in Fig. 4(A). Obviously, the enzyme displayed a strong emission peak at 367.4
nm, which resulted from the tryptophan and tyrosine residues of tyrosinase [37]. With
increasing the amounts of compound 6, the fluorescence emission peak of enzyme
decreased significantly and regularly with a latter shift of the emission peak’s position,
which indicated that inhibitors interacted with tyrosinase and quenched the intrinsic
fluorescence of enzyme. The plots of F0-F/F versus [Q] at three different temperatures
(303, 308, and 313 K) are shown in Fig. 4(A) inset, and the corresponding KSV values
for the complexation of small molecule with tyrosinase are given in Table 1. A high
linearity suggested that only one type of quenching process occurred, either static or
dynamic quenching [38]. The KSV values clearly displayed a decreasing tendency with
increasing temperature, and the calculated Kq values at 303, 308, and 313 K were
1.318×1012, 1.299×1012 and 1.113×1012 L·mol−1·s−1, respectively, which were
considerably greater than the maximum scatter collision quenching constant of
various quenchers with biopolymers, 2.0×1010 L·mol−1·s−1[39], which indicates that
the mechanism of the fluorescence quenching of tyrosinase was static and resulted
from the formation of a inhibitors-enzyme complex.
To further investigate the acting site between the inhibitor and enzyme, we
performed a fluorescence titration of tyrosinase with 6-Cu2+ complex. As illustrated in
Fig. 4(B), the increased concentration of the 6-Cu2+ complex led to a similar decrease
in the intensity of the emission band at 367.2 nm like compound 6, However, because
19

of the overlap between the tyrosinase fluorescence and the 6-Cu2+ complex
absorbance, the decreased intensity was likely related to the quenching by the
fluorescence resonance energy transfer (FRET) [40]. The static quenching binding
constant (Ka) of the 6-Cu2+ complex was 102.255 L•mol-1•s-1 at 300C, less than the
static quenching of the binding constant (Ka) of compound 6 (104.022 L•mol-1•s-1). The
results showed that the copper ion may have occupied the inhibitor activity site after
the coordination of the inhibitor and copper, which led decreased quenching of
tyrosinase.
3.3. 1H NMR and 13C NMR titration studies
To investigate the bonding mode between the inhibitor and tyrosinase, we used
1H NMR and 13C NMR titrations to identify the bonding mode between different
molecules by determining the chemical shifts of hydrogen and carbon atoms [41]. The
interactions between compounds 1, 6, 8 and tyrosinase were further observed from 1H
NMR titrations in DMSO-d6. The titrations were carried out by addition of tyrosinase
to inhibitor in different concentrations and their NMR plots were collected (Fig. 5).
Upon the addition of 0.341 mg/mL (Final concentration) of tyrosinase to the target
compounds 1 or 6 or 8, in Fig. 5(A), the peaks at 11.45/11.45/11.47 ppm (of –CSNH–,
Ha) almost completely disappeared. This was caused by the tautomeric effect, in
which NH-C=S was transformed into N=C-SH. Then, the sulfur atom donated
hydrogen and formed a complex with enzymic Cu2+ and led to the disappearance of
the -CSNH- proton. The peaks at 8.22/8.23/8.23ppm(amino proton, –NH2, Hb)
gradually shifted to 8.14 / 8.12 / 8.13ppm with the dropwise addition of the enzyme,
20

which indicated that the increase of the electron density on amino proton due to
formation of hydrogen bond between -NH2 group proton and tyrosinase. The result
was consistent with molecular docking for compound 8, the Hb form hydrogen bonds
with GLU265. The peaks at 7.59/7.59 ppm (-CH=N-, Hc) for compounds 1 and 6
moved to 7.62/7.61ppm with the addition of enzyme solution, which suggested that
the original system (-CH=N-) translated the new big conjugate system (-CH=N-N=C-)
causing the electron cloud density of Hc is reduced, and the chemical shift value δ of
Hc is enhanced. The peak at 7.54 ppm (He, benzene-H) for the compound 8 split into
two peaks (7.58 ppm and 7.53 ppm) with the addition of enzyme solution. It suggests
that there exist some interactions between benzene-H atom of benzene ring and
enzyme changing the electric density and chemical micro-environment of benzene-H
atom differently. The results were well explained by assuming that there existed an
inter-molecular interaction besides formation of hydrogen bond and complexes
between the compound 8 and tyrosinase in the latter research of molecular docking
studies.
The interactions between compound 8 and tyrosinase were further observed from
13
the C NMR titrations in DMSO-d6 (Fig.5B). Upon the addition of 0.406 mg/mL of
tyrosinase to compound 8, the chemical shift values of C1( -C=S , 177.389ppm) and
C3 (-C=N-, 139.126ppm) moved upfield (177.011, 138.747 ppm), while C4 (2 -
thiophene) moved downfield from137.212 to 137.484 ppm, which indicated that the
electron density increased in the carbon atoms on -C=S and -C=N- group and
decreased in the carbon atoms on 2-thiophene.This occurred due to existing of big
21

conjugated system(thiophene-C=N-N=C-S-). Moreover, the chemical shift value of
C2(-C=O) of 4-functionalized ester group moved downfield from165.354 to 165.422
ppm, which may root from the formation of hydrogen bond between the oxygen atom
on C2 and amino acid residues of HIS85(molecular docking shown in Fig. 6A). The
chemical shift values of C5 (benzene ring) moved upfield from 129.887 to 128.944
ppm. This suggested that there existed an interaction between benzene ring group and
1
enzyme, which was in good conformity with results of the above H NMR titration
and well explained by the subsequent molecular docking studies.
3.4. Molecular docking study
The configuration of the binding complexes of tyrosinase with compound 8 by
using Accelry Discovery studio 2.0 was shown in Fig. 6. The relevant parameters of
molecular docking were -26.9452 kcal mol-1 (CDDOCKR-ENERGY) and -31.8851
kcal mol-1 (CDDOCKR-INTERACTION-ENERGY). From Fig. 6A , it was indicated
that amino acid residues of ASN206, GLU256, HIS85 may form hydrogen bonds with
-NH-, -NH2 and -C=O of compound 8 molecules, respectively. Obviously the
formation of these hydrogen bonds may greatly promote the formation of complexes
between inhibitor and tyrosinase and enhance the inhibitory activity of the tested
compound accordingly. Interestingly, it was observed that there was an additional
inter-molecular interaction shown in Fig. 6B between benzene ring group from
inhibitor and amino acid residues ASN81 from enzyme, which may lead the electronic
density and chemical micro-environment of hydrogen atoms of benzene ring to
change differently and split NMR peaks of hydrogen atoms of benzene ring into
22

multiple peaks. The result was well coincident with the observed multiple peak (He,
benzene-H) finding of NMR titration. It was postulated that the inter-molecular
interaction (Van der Waals force including interactions of electrostatic, polarity and
dispersion) existing in inhibitor and hydrophobic domain of enzyme active center may
help to stabilize the complexes formed between inhibitor and enzyme, which surely
further promotes and enhances the inhibitory activity of the tested compound in
addition to the promoting effect of complexes formation. The amino residues of
HIS269, HIS244, ASN81 (the purple circles of 2D at Fig. 6A）may involve in the
interaction of electrostatic or polarity between inhibitor and enzyme, whereas the
HIS263, PHE90, VAL283 (the green circles of 2D at Fig. 6A）of enzyme involve in
interaction of dispersion with the benzene ring moiety of the compound 8
(explanation of Fig. 6A can be found in Tutorials of Discovery studio 2.0). The
promoting effect from inter-molecular interaction was verified by the
spectrofluorimetry studies.
4. Conclusion
Novel Tyrosinase inhibitors of 4-functionalized TCT derivatives (1-8) had been
synthesized and spectrofluorimetry, 1H and 13C NMR titration and molecular
docking had been used to investigate their inhibitory activities and mechanisms on
tyrosinase. We found that there existed an additional inter-molecular interaction
between benzene ring of compound 8 and amino acid residue of ASN81 in
hydrophobic domain of enzyme active center besides common formation of hydrogen
bonds and auxiliary vicinity synergistic effect, which helps to further stabilize the
23

complexes formation between the sulfur atom of the thiourea group in the inhibitor
and the dicopper ions of the enzyme active center and surely promotes the inhibitory
activity of the compound 8. All of these effects led a 68.52% enhancement of
inhibitory activity of compound 8 (IC50 =0.068 M) compared to that of its analog of
4-acetyl methoxy-TCT (compound 2). The finding was well explained by molecular
docking studies, verified by and consistent with experiments of spectrofluorimetry, 1H
13
and C NMR titration. The 4-benzoy methoxy substitution of the TCT successfully
produced novel inhibitors of tyrosinase in mushroom. Additional research including
toxicity is necessary to develop novel and highly potent tyrosinase inhibitors.
Acknowledgments
This work was supported by National Natural Sciences Foundation of China (No.
20962014) and the free searching foundation of State Key Laboratory of Food
Science and Technology (Nanchang University) in China (SKLF-TS-200914).
24

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Fig.1.Synthesis of 4-functionalized thiophene-2-carbaldehyde thiosemicarbazone
derivatives 1-8
.
Fig.2.(A) Effect of concentrations of mushroom tyrosinase on its activity for the
oxidation of L-DOPA at different concentrations of compound 1, 3-8. Concentrations
of compounds for cures 0-3 were 0, 0.04, 0.16, 0.64 M, respectively. (B) Relative
enzymatic activity of compounds 1-8. (C) Lineweaver-Burk plots of mushroom
tyrosinase on the presence of compound 1, 3-8 during the oxidation of L-DOPA.
Concentrations of compounds for curves 0-3 were 0, 0.04, 0.16 and 0.64 M.
Fig.3. Synchronous fluorescence spectra of tyrosinase in the absence and presence of
compound 6: (A) Δλ = 15 nm or (B) Δλ = 60 nm (pH 6.8, T = 298 K). c (tyrosinase) =
30 μg/ mL. c (compound 6) =0, 11.97, 23.88, 35.73, 47.52, 59.26 and 70.94 μM,
respectively. Three-dimensional fluorescence spectra of tyrosinase (a), the compound
1+tyrosinase system (b), the compound 6+tyrosinase system (c) and the compound
8+tyrosinase system (d), which the concentrations of compounds of system were
35.73 μM.
Fig.4. Fluorescence spectra of tyrosinase in the absence and presence compound 6 or
6-Cu2+ complex: (A) compound 6; (B) compound 6-Cu2+complex.Concentrations for
curves were 0, 5.99, 11.94, 17.87, 23.76, 29.63, 35.47, 41.28, and 47.06 μM,
respectively. The inset of (A) depicts Stern–Volmer plots for the fluorescence
quenching of tyrosinase by compound 6 at 30, 35 and 40 °C. The inset of (B) depicts
Stern–Volmer plots for the fluorescence quenching of tyrosinase by compound
6-Cu2+complex at 30 °C.
Fig.5. (A) 1H NMR titration studies of compounds 1, 6, 8 (21 mM) with tyrosinase in
DMSO-d6 solvent.The concentrations of tyrosinase ranging from 0, 0.098 to
13
0.341 mg/mL. (B) C NMR titration of compound 8 (62.6 mM) in DMSO-d6 was
prepared and titrated with tyrosinase from 0 to 0.406 mg/mL.
29