Novel inhibitors of tyrosinase produced by the 4-substitution of TCT

Article abstract of DOI:10.1016/j.foodchem.2016.10.140

We synthesized a series of 4- or 5-functionalized TCT derivatives (1?12) and investigated their inhibitory activities and mechanisms on tyrosinase by using Spectrofluorimetry, 1H and 13C NMR titration and IR spectra. The results of the fluorescence spectr

Full text of DOI:10.1016/j.foodchem.2016.10.140

Food Chemistry xxx (2016) xxx–xxx
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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Novel inhibitors of tyrosinase produced by the 4-substitution of TCT
Jian Xu a, Jing Liu a, Xinqi Zhu a, Yanying Yu a, Shuwen Cao a,b,
⇑
a Department of Chemistry, Nanchang University, Nanchang 330031, PR China
b State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized a series of 4- or 5-functionalized TCT derivatives (1ꢀ12) and investigated their inhibitory
activities and mechanisms on tyrosinase by using Spectrofluorimetry, 1H and 13C NMR titration and IR
spectra. The results of the fluorescence spectra and NMR titrations showed that the thiosemicarbazone
moiety formed complexes with copper ions in the active center of the enzyme and played an important
role in inhibiting the activities of the target compounds. The 5-functionalization decreased the inhibitory
activity, but the 4-functionalization with a methoxyacetyl group enhanced the inhibitory activity, in
which a strong auxiliary vicinity synergistic effect from the methoxyacetyl group strengthens and
promotes the formation of complexes between the sulfur atoms of the inhibitor and the dicopper nucleus
of tyrosinase. We concluded that the appropriate 4-functionalization improved the inhibitory activity of
the modifier, and that 4-methoxyacetyl -TCT is promising for the inhibition of tyrosinase.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 26 February 2016
Received in revised form 10 October 2016
Accepted 28 October 2016
Available online xxxx
Keywords:
Thiophene-2-carbaldehyde
thiosemicarbazone (TCT) derivatives
Tyrosinase inhibitors
Fluorescence spectrum
1H and 13C NMR titration
1. Introduction
Before this study, hundreds of tyrosinase inhibitors with
good inhibition activities have been separated from nature or
Tyrosinase (EC 1.14.18.1), a kind of metalloenzyme with dinu-
clear copper ions, is common in animals, insects and microorgan-
isms (Song et al., 2006). Structurally, tyrosinase belongs to the
Type-3 copper protein family, with two copper ions bonded in a
coordinate and a distinct set of three histidine residues within the
active site (Motabo, Kumagai, Yamamoto, Yoshitsu, & Sugiyama,
2006). Generally, tyrosinase is known as the rate-limiting enzyme
synthesized in laboratories (Chang, 2009; Mendes, Perry,
Francisco, 2014; Sabudak, Demirkiron, Ozturk, & Topcu, 2013),
but only few were studied for their structure-activity
&
a
relationships, inhibitory mechanisms and structural modifica-
tions. Focusing on these particular properties of tyrosinase
inhibitors could lead to the development of new and better
inhibitors, although very few would be practical because of their
toxicity, side effects, and low stability and solubility. In our prior
study of a novel tyrosinase inhibitor, thiophene-2- carbaldehyde
thiosemicarbazone(TCT), we obtained a preliminary understand-
ing of the structure-activity relationships and inhibitory
mechanisms between precursors and target enzymes by using
fluorescence, 1H NMR titration and molecular docking studies
(Xie, Dong, Yu, & Cao, 2016). However, we still have little
understanding of how the structure-activity relationships and
inhibitory mechanisms affect the inhibitory activities when
modifying TCT using the 4- and 5-functionalization of a thiophene
ring on TCT. Generally, these types of structural modifications
either increase or decrease the inhibitory activity of the modifier.
Based on our understanding of the structure-activity relationship
of 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 center. It is necessary to find new tyrosinase inhibitors
involved in two-step oxidation for the transformation of L-tyrosine
into dopaquinone, which is crucial for melanin biosynthesis (Seo,
Sharma, & Sharma, 2003). Excessively active tyrosinase, which
results in an over-accumulation of melanin in the human body,
can cause a series of common skin diseases from freckles to malig-
nant melanoma (Choi, Park, & Jee, 2015). Abnormal tyrosinase activ-
ity is related to Parkinson’s disease (Pan, Li, & Jankovic, 2011). In
addition, tyrosinase promotes cuticle formation in insects, which
causes the browning of fruits and vegetables (Yin et al., 2015), and
their subsequent loss of quality (Shi, Chen, Wang, Song, & Qiu,
2005). Preventing this unfavorable browning reaction is still a chal-
lenge in food science (Arung et al., 2005; Chen, Xing, Wang, Zheng, &
Wang, 2015). Therefore, tyrosinase inhibitors with excellent inhibi-
tory activities and lower side effects have promising applications in
the fields of medicine, agriculture, food sciences and cosmetics.
⇑
Corresponding author at: Department of Chemistry, Nanchang University,
Nanchang 330031, PR China.
E-mail address: caosw@ncu.edu.cn (S. Cao).
http://dx.doi.org/10.1016/j.foodchem.2016.10.140
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Xu, J., et al. Novel inhibitors of tyrosinase produced by the 4-substitution of TCT. Food Chemistry (2016), http://dx.doi.org/
10.1016/j.foodchem.2016.10.140

2
J. Xu et al. / Food Chemistry xxx (2016) xxx–xxx
with strong inhibitory activities to better our understanding of
the structure-activity relationship of TCT.
carboxaldehyde were obtained from J&K Chemical Co
(Shanghai, China). 5-Nitro-2-thiophenecarboxaldehyde and 5-
Phenylthiophene-2-carbaldehyde were obtained from Zhengzhou
Xipaike Chemical Co (Henan, China) and Accela ChemBio Co
(Shanghai, China), respectively. All other reagents were local and
of analytical grade. The water used was re-distilled and ion-free.
In general excellent safety of precursor is the base and key to
get a promising tyrosinase inhibitor used as food additive after
structure modification. In order to evaluate and determine the
safety of TCT as precursor of promising tyrosinase inhibitor, we
obtained the toxicity data of TCT from https://chem.sis.nlm.
nih.gov. The toxicity for mice (oral) is LD50 226 mg/kg (Guo et al.,
1989), and for rats (oral) is LD 500 mg/kg. Further, a toxicity test
of TCT was conducted before we began this study according to
guiding principles of the drug acute toxicity test (CDER & FDA,
1996; Cordier, 1991). This test showed that the LD50 of TCT was
808.83 mg/kg bw (orally given to female mice) and 912.48 mg/kg
bw (orally given to male mice), which was of low toxicity and com-
parable to that commonly used in food additives in accord with F.
D.A. guidelines of TBHQ (Tert-Butylhydroqinone, synthetic food
additive, LD50 700–1000 mg/kg bw, orally given to rat), BHA (Butyl
Hydroxy Anisol, synthetic food additive, LD50 1100 mg/kg bw,
orally given to male mouse; 1300 mg/kg bw, orally given to female
mouse). Therefore, from the toxicity and safety point of view, TCT
was a good precursor for finding candidates for potential tyrosi-
nase inhibitors of food additives.
2.2. Synthesis
The method of synthesis of target compounds is described in
Fig. 1, as reference (Zhu et al., 2009) described. The products are
purified by recrystallization from ethanol and identified by ESI-
MS, 1H NMR and 13C NMR analyses. ESI-MS data were obtained
on a Bruker ESQUIRE-LC(Germany), 1H NMR and 13C 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.
4-Hydroxymethyl-thiophene-2-carbaldehyde
bazone (1): yellow solid, yield 85.2%, IR(KBr) 3421, 3151, 1601,
1532 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d 11.37 (s, 1H, NH),
thiosemicar-
;
8.17 (s, 2H, NH2), 7.50 (s, 1H, CH), 7.36 (s, 1H, thiophene-H), 7.33
(s, 1H, thiophene-H), 5.13 (s, H, OH), 4.40 (s, 2H, CH2). 13C NMR
(400 MHz, DMSO-d6) d 177.85(1C, C@S), 144.97(1C, C@N), 138.95
A series of 4-functionalized and 5-functionalized TCT deriva-
tives were designed, synthesized and evaluated for their inhibitory
effects on tyrosinase. Spectrofluorimetry, 1H and 13C NMR titration,
and IR spectra were used to investigate the interactions between
these modifiers and tyrosinase. Our study helped clarify the
relationship between modifiers and tyrosinase and provides a
promising route to obtain novel tyrosinase inhibitors and highly
potent tyrosinase inhibitors.
(1C,
thiophene-C),
138.08(1C,
thiophene-C),
130.59(1C,
thiophene-C), 124.25(1C, thiophene-C), 59.14(1C, CH2). MS (ESI):
m/z 214.0 [M-H]-.
4-Methoxymethyl-thiophene-2-carbaldehyde
bazone (2): greyish yellow crystals, yield 76.4%, IR(KBr) 3160,
1609, 1525 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d 11.40 (s, 1H,
thiosemicar-
;
NH), 8.17 (s, 2H, NH2), 7.54 (s, 1H, CH), 7.47 (s, 1H, thiophene-H),
7.36 (s, 1H, thiophene-H), 4.32(s, 2H, CH2), 3.24 (s, 3H, CH3). 13C
NMR (400 MHz, DMSO-d6) d 177.90(1C, C = S), 140.40(1C, C@N),
139.25(1C, thiophene-C), 137.86(1C, thiophene-C), 130.94(1C,
thiophene-C), 126.35(1C, thiophene-C), 69.21(1C, CH2), 57.84(1C,
CH3). MS (ESI): m/z 228.1 [M-H]-.
2. Materials and methods
2.1. Materials
Mushroom tyrosinase (EC 1.14.18.1) and L-3,4-dihydroxy
4-Ethoxymethyl-thiophene-2-carbaldehyde thiosemicarbazone
phenyl-alanine (
L
-DOPA) were purchased from Sigma (St. Louis,
(3): yellow solid, yield 74.3%, IR(KBr) 3149, 1597, 1522 cmꢀ1 1H
;
MO, USA). 2-thiophenecarboxaldehyde and 5-Methyl-2-thiophene
NMR (400 MHz, DMSO-d6) d 11.39 (s, 1H, NH), 8.17 (s, 2H, NH2),
Fig. 1. Synthesis of 4-functionalized and 5-functionalized thiophene-2-carbaldehyde thiosemicarbazone derivatives 1–12; synthesis method (a): chloromethylation reaction;
(b) etherification reaction; (c) esterification reaction; (d): hydrolysis reaction; and (e): methanol, acetic acid, reflux 2 h.
Please cite this article in press as: Xu, J., et al. Novel inhibitors of tyrosinase produced by the 4-substitution of TCT. Food Chemistry (2016), http://dx.doi.org/
10.1016/j.foodchem.2016.10.140

J. Xu et al. / Food Chemistry xxx (2016) xxx–xxx
3
7.53 (s, 1H, CH), 7.45 (s, 1H, thiophene-H), 7.35 (s, 1H, thiophene-
H), 4.35 (s, H, OH), 3.43 (m, 2H, CH2), 1.1(t, 3H, CH3). 13C NMR
(400 MHz, DMSO-d6) d 177.89(1C, C = S), 140.79(1C, C@N), 139.21
H), 2.50 (s, 3H, CH3). 13C NMR (400 MHz, DMSO-d6) d 177.70(1C,
C@S), 143.22(1C, C@N), 138.24(1C, thiophene-C), 136.81(1C,
thiophene-C), 131.48(1C, thiophene-C), 126.84(1C, thiophene-C),
15.80(1C, CH3). MS (ESI): m/z 200.0 [M + H]+.
(1C,
thiophene-C),
137.89(1C,
thiophene-C),
130.97(1C,
thiophene-C), 126.13(1C, thiophene-C), 67.28(1C, CH2), 65.34(1C,
5-Nitro-thiophene-2-carbaldehyde thiosemicarbazone (11):
yellow powder, yield 84.2%, IR(KBr) 3470, 1608, 1540,
CH2), 57.84(1C, CH3). MS (ESI): m/z 242.1 [M-H]-.
4-(2-Hydroxy-ethoxymethyl)-thiophene-2-carbaldehyde
thiosemicarbazone (4): pale yellow crystals, yield 84.1%, IR(KBr)
1335 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d 11.77 (s, 1H, NH),
;
8.41, 8.17 (d, 2H, NH2), 8.06 (d,1H, thiophene-H), 8.00(s, 1H, CH),
7.51 (d,1H, thiophene-H). 13C NMR (400 MHz, DMSO-d6) d 178.55
(1C, C@S), 151.50(1C, C@N), 147.19(1C, thiophene-C), 135.67(1C,
thiophene-C), 130.88(1C, thiophene-C), 129.66(1C, thiophene-C).
MS (ESI): m/z 229.1 [M-H]-.
3441, 3178, 1608, 1528 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d
;
11.39 (s, 1H, NH), 8.17 (s, 2H, NH2), 7.52 (s, 1H, CH), 7.47 (s,1H,
thiophene-H), 7.37 (s,1H, thiophene-H), 4.60 (s, H, OH), 4.40 (s,
2H, CH2), 3.48(t, 2H, CH2), 3.42(t, 2H, CH2). 13C NMR (400 MHz,
DMSO-d6)
d
177.88(1C, C@S), 140.76(1C, C@N), 139.17(1C,
5-Phenyl-thiophene-2-carbaldehyde thiosemicarbazone (12):
thiophene-C), 137.90(1C, thiophene-C), 131.07(1C, thiophene-C),
126.18(1C, thiophene-C), 72.03(1C, CH2), 67.71(1C, CH2), 60.62
(1C, CH2). MS (ESI): m/z 258.1 [M-H]-.
5-Hydroxymethyl-thiophene-2-carbaldehyde
bazone (5): pale yellow acicular crystal, yield 74.9%, IR(KBr)
3439, 3166, 1589, 1549 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d
11.36 (s, 1H, NH), 8.13 (d, 2H, NH2), 7.46 (s, 1H, CH), 7.25 (d, 1H,
thiophene-H), 6.90 (d, 1H, thiophene-H), 5.53 (s, H, OH), 4.59 (s,
2H, CH2). 13C NMR (400 MHz, DMSO-d6) d 177.77(1C, C@S),
150.21(1C, C@N), 138.25(1C, thiophene-C), 137.62(1C, thiophene-
C), 130.89(1C, thiophene-C), 124.74(1C, thiophene-C), 58.98(1C,
CH2). MS (ESI): m/z 214.2 [M-H]-.
yellow powder, yield 83.7%, IR(KBr) 3164, 1589, 1521 cmꢀ1 1H
;
NMR (400 MHz, DMSO-d6) d 11.47 (s, 1H, NH), 8.19 (s, 2H, NH2),
7.67 (d,2H, Ph-H), 7.55(s, 1H, CH), 7.49 (d,1H, thiophene-H), 7.43–
7.39 (t, 2H, Ph-H; d, H, thiophene-H), 7.32 (t, H, Ph-H). 13C NMR
(400 MHz, DMSO-d6) d 177.86(1C, C@S), 145.74(1C, C@N), 138.39
(1C, thiophene-C), 137.76(1C, thiophene-C), 133.65(1C, Ph-C),
132.35(1C,Ph-C), 129.68(2C,Ph-C), 128.74(1C, thiophene-C), 125.88
(2C,Ph-C), 124.84(1C, thiophene-C). MS (ESI): m/z 260.1 [M-H]-.
thiosemicar-
;
2.3. Determination of water solubility and lipid solubility
The experiment was performed as reference (Bai, Yan, Hu,
Zhang, & Huang, 2006) described with minor modifications to
determine the water/lipid solubility of selected compounds
preliminarily. An excess of sample was dissolved in water/ethyl
acetate, sonic disruption for 1 h, and oscillation incubation for
6 h in room temperature, after centrifugation for 10 min, the upper
clear liquid was retained by filtration. 1 mL Filtrate was then
diluted with water/ethyl acetate into five concentrations from
saturated to low, then the absorbance values were recorded using
a Shimadzu UV-2450 spectrophotometer (Japan) at its maximum
absorption wavelength, and standard curve was determined.
Known concentration of the sample solution(dissolved in water/
ethyl acetate) was configured, recorded the absorbance value using
a Shimadzu UV-2450 spectrophotometer (Japan) at its maximum
absorption wavelength. Since dilute solution conformed to
Lambert-Beer’s law, according to the obtained standard curve
and the absorbance value of known concentration, the sample’s
water solubility and lipid solubility can be calculated.
4-Methoxyacetyl-thiophene-2-carbaldehyde
bazone (6): pale yellow flaky crystals, yield 73.1%, IR(KBr) 3155,
1742, 1601, 1531 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d 11.40 (s,
1H, NH), 8.16 (s, 2H, NH2), 7.57 (s,1H, thiophene-H), 7.53 (s, 1H,
CH), 7.38 (s,1H, thiophene-H), 4.40 (s, 2H, CH2), 2.03(s, 3H, CH3).
13C NMR (400 MHz, DMSO-d6) d 177.99(1C, C@S), 170.64(1C,
C@O), 139.57(1C, C@N), 137.98(1C, thiophene-C), 137.69(1C,
thiophene-C), 131.04(1C, thiophene-C), 127.64(1C, thiophene-C),
61.04(1C, CH2), 21.13(1C, CH3). MS (ESI): m/z 256.0 [M-H]-.
thiosemicar-
;
5-Methoxymethyl-thiophene-2-carbaldehyde
bazone (7): yellow powder, yield 80.3%, IR(KBr) 3144, 1601,
1522 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d11.39 (s, 1H, NH),
thiosemicar-
;
8.15 (s, 2H, NH2), 7.50 (s, 1H, CH), 7.27 (d, 1H, thiophene-H), 6.98
(d, 1H, thiophene-H), 4.53 (s, 2H, CH2), 3.25(s, 3H, CH3). 13C NMR
(400 MHz, DMSO-d6) d 177.87(1C, C@S), 144.44(1C, C@N), 139.00
(1C,
thiophene-C),
137.93(1C,
thiophene-C),
130.67(1C,
thiophene-C), 127.23(1C, thiophene-C), 68.82(1C, CH2), 57.78(1C,
CH3). MS (ESI): m/z 228.0 [M-H]-.
5-(2-Hydroxy-ethoxymethyl)-thiophene-2-carbaldehyde
thiosemicarbazone (8): pale yellow powder, yield 72.2%, IR(KBr)
2.4. Tyrosinase activity assay
3162, 1628, 1545 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d 11.39 (s,
;
The assay of inhibition of target compounds on the diphenolase
activity of mushroom tyrosinase was performed by our reported
procedure with minor modifications (Xie et al., 2016). L-DOPA
was used as substrate for the assay. The reaction media (3 mL)
for activity assay contained 2.8 mL 0.5 mM l-DOPA in 50 mM Na2
HPO4-NaH2PO4 buffer (pH = 6.8) and 0.1 mL of different concentra-
tions of inhibitor (dissolved by DMSO previously), and then 0.1 mL
1H, NH), 8.16 (s, 2H, NH2), 7.50(s, 1H, CH), 7.27 (d,1H, thiophene-
H), 6.99 (d,1H, thiophene-H), 4.62 (s, 2H, CH2), 3.49(t, 2H, CH2),
3.45(t, 2H, CH2). 13C NMR (400 MHz, DMSO-d6) d 177.72(1C,
C@S), 145.04(1C, C@N), 138.90(1C, thiophene-C), 137.99(1C,
thiophene-C), 130.68(1C, thiophene-C), 127.10(1C, thiophene-C),
71.91(1C, CH2), 67.42(1C, CH2), 60.58(1C, CH2). MS (ESI): m/z
258.1 [M-H]-.
of the aqueous solution of mushroom tyrosinase(166.5 lg/mL) was
5-Methoxyacetyl-thiophene-2-carbaldehyde
bazone (9): pale yellow crystal, yield 81.1%, IR(KBr) 3174, 1723,
1611, 1536 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d 11.43 (s, 1H,
thiosemicar-
added to the mixture. The solution was immediately monitored by
measuring the linear increase in optical density at 475 nm of for-
mation of the DOPA chrome for 150 s using a Shimadzu UV-2450
spectrophotometer (Japan). The extent of inhibition by the addi-
tion of the sample was expressed as the percentage necessary for
50% inhibition (IC50), calculated by SPSS19. The value of inhibition
ratio of compounds on tyrosinase can be calculated by the follow-
ing equation:
;
NH), 8.16 (s, 2H, NH2), 7.54(s, 1H, CH), 7.29 (d,1H, thiophene-H),
7.10 (d,1H, thiophene-H), 5.20 (s, 2H, CH2), 2.03(s, 3H, CH3). 13C
NMR (400 MHz, DMSO-d6) d 177.95(1C, C@S), 170.13(1C, C@O),
141.07(1C, C@N), 140.11(1C, thiophene-C), 137.74(1C, thiophene-
C), 130.57(1C, thiophene-C), 129.25(1C, thiophene-C), 60.59(1C,
CH2), 21.06(1C, CH3). MS (ESI): m/z 258.0 [M + H]+.
5-Methyl-thiophene-2-carbaldehyde thiosemicarbazone (10):
Inhibition ratioð%Þ ¼ ð1 ꢀ OD1=OD2Þ ꢁ 100%
yellow powder, yield 86.6%, IR(KBr) 3148, 1590, 1524 cmꢀ1 1H
;
NMR (400 MHz, DMSO-d6) d 11.37 (s, 1H, NH), 8.14 (s, 2H, NH2),
7.44(s, 1H, CH), 7.23 (d,1H, thiophene-H), 6.81 (d,1H, thiophene-
where OD1 is the slope of reaction kinetics equation obtained from
reaction with inhibitor; OD2 is the slope of reaction kinetics
Please cite this article in press as: Xu, J., et al. Novel inhibitors of tyrosinase produced by the 4-substitution of TCT. Food Chemistry (2016), http://dx.doi.org/
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J. Xu et al. / Food Chemistry xxx (2016) xxx–xxx
equation obtained from reaction with reagent blank. Kojic acid and
thiophene dicarboxaldehyde thiosemicarbazone were used as refer-
ence standard inhibitors for comparison, respectively.
binding constant (Ka) and the number of binding sites (n) can be
calculated by the following equation (Shahabadi, Maghsudi, &
Rouhani, 2012):
F0 ꢀ F
log
¼ log Ka þ n log½Qꢂ
2.5. Determination of inhibition mechanism, inhibition type and
inhibition constants
F
2.7. 1H NMR and 13C NMR titration
Determination of inhibition mechanism was assayed by main-
taining the concentration of
concentration of the enzyme (16, 12, 8, 4
ium. The enzyme activity was measured for different concentra-
tions of inhibitor.
Determination of inhibition type and inhibition constants was
assayed by maintaining the concentration of enzyme (10
and changing the concentration of the -DOPA(0.5, 1, 1.5, 2 mM)
in reaction medium. The inhibition type was assayed by the
L
-DOPA(0.5 mM) and changing the
1H NMR titration studies were performed to investigate the
interaction between the compounds 1, 5, 6 and tyrosinase, respec-
tively. A 0.5 mL 28 mM solution of target compound in DMSO-d6
was prepared and titrated with tyrosinase solution by using a
micropipette (to final concentrations ranging from 1.42 to
2.84 mg/mL for compounds 1, 5 and 6). After each addition of
tyrosinase, the 1H NMR spectrum was recorded and the changes
in the chemical shift of the protons were noted.
lg/mL) in reaction med-
lg/mL)
L
Lineweaver-Burk plot, the value of inhibition constants (KI, KIS)
For further investigate the interaction between the compound 6
1
m
can
Km
Vm½Sꢂ
be
obtained
1
by
the
following
equation:
¼
and tyrosinase, 13C NMR titration studies were performed. The
ꢀ
ꢁ
ꢀ
ꢁ
½Iꢂ
½Iꢂ
1 þ K þ V 1 þ K
where v is the reaction velocity; Km is
m
experiment was performed as 1H NMR titration.
A 0.5 mL
I
IS
the Michaelis constant; Vm is the maximal velocity; [I] is the con-
centration 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 inter-
cept versus the inhibitor concentration, respectively.
100 mM solution of compound 6 in DMSO-d6 was prepared and
titrated with tyrosinase solution by using a micropipette. After
each addition of tyrosinase, the 13C NMR spectrum was recorded.
2.8. IR and UV spectrum
In order to get further understanding manner and site of inter-
action between inhibitors and enzyme, complex (compound 6 and
Cu2+) was synthesized (Bisceglie et al., 2014) and its IR and UV
spectra were studied.
2.6. Fluorescence quenching titration
The experiment was performed as reference described with
minor modifications (Wang, Zhang, Yan, & Gong, 2014). The reac-
tion media (2 mL) contained 1.8 mL Na2HPO4-NaH2PO4 buffer
(pH = 6.8, 50 mM) and 0.2 mL of the aqueous solution of mush-
room tyrosinase, and then titrated by successive addition of com-
pounds 1, 2, 6 and 1-Cu2+ complex (the constituent ratio of
ligand and Cu2+ was 2:1, the chelation ratio has been determined
by continuous variation method) (Karikari, Mather, & Long, 2007)
solution using a pipette (to final concentrations ranging from 0
Compound 6 was dissolved in ethanol, and then copper sulfate
solution (40 mM) was slowly added, finally lots of flocculent pre-
cipitate was generated. The turbid liquid was centrifuged, and pre-
cipitate was separated. The precipitate was washed with ethanol
and water, and dried in a vacuum oven at 40 °C for 12 h. The IR
spectra were measured with KBr pellets on a Nicolet 5700 FT-IR
spectrometer in the range of 4000–400 cmꢀ1. The UV–vis absorp-
tion spectrum were examined on a UV-2450 spectrophotometer
(Japan) in the range 250 nm-500 nm using a 1.0 cm quartz cell at
room temperature in the ethanol medium.
to 105.6 lM). Fluorescence intensities were recorded using a
Hitachi F-4600 spectrofluorophotometer (Japan) at three different
temperatures (26, 31 and 36 °C) with an excitation wavelength of
280 nm, emission wavelength from 300 to 550 nm and excitation
and emission slit widths of 5 nm.
3. Results and discussion
Considering the inner-filter effect of fluorescence, all of the flu-
orescence intensities were corrected for the absorption of exciting
light and reabsorption of emitted light in this study. The inner-
filter effect can be corrected by the following formula (Bi, Yan,
Wang, Pang, & Wang, 2012):
3.1. Effects of thiophene 2-carboxaldehyde thiosemicarbazone
derivatives on tyrosinase activities and the determination of water and
lipid solubility
The values of IC50 of compounds 1–12 are listed in Table 1. Kojic
acid and thiophene 2-carboxaldehyde thiosemicarbazone (TCT)
were used as comparative reference standard inhibitors. As shown
in Table 1, the inhibitory activity of compounds 1–12 were much
stronger than that of Kojic acid; however, compared to TCT, only
the inhibitory activity of compound 6 was better than that of its
precursor. In our previous study, we found that the excellent
tyrosinase inhibiting activity of the precursor was primarily based
on the formation of complexes between the sulfur atom from the
thiourea of the precursor and the copper ion of the enzyme center,
with an auxiliary vicinity synergistic effect of the thiophene ring
(Xie et al., 2016). From the experimental results, we found that
the 4- and 5-functionalization of the thiophene ring of the precur-
sor decreased the inhibitory activity of nearly every target modifier
with only one exception. Thus, the increased steric hindrance by
the 4- and 5-functionalization of the thiophene ring was crucial
to inhibit the formation of complexes between the sulfur atom
from the thiourea of the precursor and the copper ion of the
enzyme center, and decreased the inhibition of the modifier,
1þA2Þ=2
Fcorr ¼ FobseðA
where Fcor 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 equation:
F0 ꢀ F
log
¼ 1 þ Kqs0½Qꢂ ¼ 1 þ KSV½Qꢂ
F
where F0 and F are the fluorescence intensities before and after the
addition of the quencher, respectively; Kq and KSV are the bimolec-
ular quenching constant and the Stern-Volmer quenching constant,
respectively; s
0[10ꢀ8 s] (Xiao et al., 2007) is the average lifetime of
the fluorophore in the absence of the quencher, [Q] is the concentra-
tion of the quencher.
For the static quenching interaction, if it is assumed that there
are similar and independent sites in the biomolecule, the apparent
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J. Xu et al. / Food Chemistry xxx (2016) xxx–xxx
5
especially for the 5-functionalization modifiers. Moreover, we
found that 4-methoxyacetyl- thiophene-2-carbaldehyde
thiosemicarbazone had a 20.93% decrease of IC50 compared to
that of its precursor (IC50 = 0.43 M), which was not explained
l
successfully by its unfavourable effect of steric hindrance from
introducing 4-substituted groups on thiophene ring of the TCT
precursor. This may have occurred due to the presence of another
force with a stronger effect that promoted the formation of these
complexes and negated the inferior effect of steric hindrance. We
assumed that this favorable effect came from the carbonyl oxygen
atom of the methoxyacetyl group coordinating with the copper
ion in the complexes, which was verified by results of NMR titra-
tion and IR spectra, and much stronger than the auxiliary vicinity
synergistic effect of the thiophene ring on the precursor (Xie et al.,
2016).
We also studied the inhibitory mechanism by compounds 1, 2,
5, 6, 7 and 9 against the tyrosinase for the oxidation of L-DOPA.
The relationship between the enzymatic activity and the concen-
trations of the inhibitors was observed as groups of straight lines
passing through the origin (Fig. S1), indicating that the inhibition
was reversible. In addition, we investigated the inhibitory kinetics
of tyrosinase in mushrooms using compounds 1, 2, 5, 6, 7 and 9.
The double-reciprocal plots yielded a group of straight lines inter-
secting at the second quadrant (Fig. S2), indicating that they were
mixed-type inhibitors. The compounds were not only capable of
binding with the free enzymes, but also with enzyme-substrate
complexes. KI and KIS were obtained from the Lineweaver-Burk
plot, as shown in Table S1. The values of KIS were greater than
the values of KI, which indicated that the affinities of the com-
pounds bonding with the free enzyme were greater than those
of the compounds bonding with enzyme-substrate complexes.
Water solubility is a major factor in limiting drug absorption
(O’Driscoll & Griffin, 2008). In drug design, predicting lipid solu-
bility is important in identifying the proper excipients to solubi-
lize and formulate drugs in lipid formulations (Rane
&
Anderson, 2008). Here, the water and lipid solubility of the parent
compound and compounds 1, 2, 3 and 6 were preliminarily deter-
mined by UV Spectrophotometry. The values of the water and
lipid solubility are listed in Table 1. Compared to the water solu-
bility of the parent compound, the water solubility of compounds
1, 2, 3 and 6 improved slightly. The water solubility of all of com-
pounds was less than 0.5 g/L, which indicated that the introduc-
tion of these groups, even the hydroxymethyl group, had a
limited effect on improving water solubility. Compared to the
lipid solubility of the parent compound, the lipid solubility of
compounds 1 and 2 decreased significantly, while the lipid solu-
bility of compounds 3 and 6 increased significantly, which
showed that the extension of the hydrophobic chains improved
lipid solubility.
3.2. Fluorescence studies
Studies in inhibition kinetics found that compounds tend to
bond with free enzymes during catalysis, so that compounds form
complexes with enzymes. We used fluorescence measurements to
further investigate the interactions between compounds and
tyrosinase. The fluorescence emission spectra of tyrosinase in
the absence of and in the presence of compounds 1, 2 and 6
excited at 280 nm are shown in Fig. 2. Obviously, tyrosinase in
an aqueous solution exhibited a strong fluorescence emission
peak at 381 nm, and with the gradual addition of compounds 1,
2 and 6, the fluorescence intensity of tyrosinase decreased
remarkably without any significant peak changes, indicating that
compounds 1, 2 and 6 interacted with tyrosinase and quenched
its intrinsic fluorescence.
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J. Xu et al. / Food Chemistry xxx (2016) xxx–xxx
Fig. 2. Fluorescence spectra of tyrosinase in the presence of compounds 1, 2, 6 and the 1-Cu2+ complex at 26 °C. Concentrations for curves were 0, 12, 24, 36, 48, 59, 71, 83, 94
and 106
M. The inset depicts Stern-Volmer plots for the fluorescence quenching of tyrosinase by compounds 1, 2 and 6 at 26, 31 and 36 °C and the 1-Cu2+ complex at 26 °C.
l
The Stern-Volmer plots for the quenching of tyrosinase by com-
pounds 1, 2 and 6 (Fig. 2A–C) showed that they exhibited a linear
relationship. The corresponding Kq values (Table 1) at different
temperatures were much greater than the maximum scatter colli-
sion quenching constant of quenchers with biopolymers,
2.0 ꢁ 1010 Lꢃmolꢀ1ꢃsꢀ1 (Lakowicz, 2009), indicating that static
quenching was dominant in the quenching process. Moreover,
the Ka values (Table 1) appeared in a downward trend in the corre-
sponding temperature-changed experiments, which further indi-
cated that inhibitors quenched tyrosinase in a static process
(Wang et al., 2014). When the quenching originated from complex
formations between the quenchers and biopolymers, the forces
involved in the interactions determined whether higher tempera-
tures would increase or decrease the complex formation between
the quenchers and biopolymers (Joye, Davidov-Pardo, Ludescher,
& McClements, 2015). A significant decrease in the KSV and Ka
valves occurred when the temperature rose from 26 to 31 °C, indi-
cating that hydrogen bonding was dominant. This was expected
due to the weakening of hydrogen bridges at high temperatures.
When the temperature rose from 31 to 36 °C, the KSV values
appeared to be weakly temperature-dependent. The Ka value of
compound 6 increased slightly only because the hydrogen bridges
weakened at higher temperatures while the hydrophobic interac-
tions increased, which led additional complex formations from
the strengthening of the hydrophobic forces. (Joye et al., 2015).
The results from the quenching of tyrosinase by compounds 1–9
and the values of IC50 of compounds 1–9 are listed in Table.1,
and show that static quenching was dominant in the quenching
process of compounds 3, 4, 5, 7, 8 and 9, and that the Ka values
of compounds 1–9 were almost inversely proportional to the val-
To further investigate the acting site between the inhibitor and
enzyme, we performed a fluorescence titration of tyrosinase with
1-Cu2+ complex. As illustrated in (Fig. 2D), the increased concen-
tration of the 1-Cu2+ complex led to a greater decrease in the inten-
sity of the emission band at 381 nm, unlike that of compound 1.
However, because of the overlap between the tyrosinase fluores-
cence and the 1-Cu2+ complex absorbance (Fig. S3), the decreased
intensity was likely related to the quenching by the fluorescence
resonance energy transfer (FRET) (Joye et al., 2015). The static
quenching binding constant (Ka) of the 1-Cu2+ complex was
104.61 Lꢃmolꢀ1ꢃsꢀ1 at 26 °C, less than the static quenching of the
binding constant (Ka) of 1 (105.19 Lꢃmolꢀ1ꢃsꢀ1). 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. Bonding mode studies from 1H NMR, 13C NMR titration, IR and UV
of synthesized complex of compound 6 and Cu2+
3.3.1. Bonding mode studies from 1H NMR and 13C NMR titration
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 (Park, Sahoo, &
Choi, 2016).
The interactions between compounds 1, 5, 6 and tyrosinase
were observed from the 1H NMR titrations in DMSO-d6. After each
addition of tyrosinase to the inhibitor, the 1H NMR plot was
recorded (Fig. 3A). With the addition of 40 mg/mL of tyrosinase
to the target compounds, the peaks at 11.37/11.36/11.40 ppm
nearly disappeared (compounds 1, 5 and 6, of ANHACSA, Hc for
ues of IC50
.
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J. Xu et al. / Food Chemistry xxx (2016) xxx–xxx
7
Fig. 3. (A) 1H NMR titration of compounds 1, 5 and 6 (28 mM) with tyrosinase in the DMSO-d6 solvent. Concentrations of tyrosinase for curves a–c were 0, 1.42 and 2.84 mg/L.
The lines in the figure represent the major changes in NMR spectrum; and (B) 13C NMR titration of compound 6(100 mM) with tyrosinase in the DMSO-d6 solvent.
Concentrations of tyrosinase for curves a–b were 0 and 5.56 mg/L.
Fig. 4. (A) IR spectra of compound 6 and the complex (6-Cu2+); and (B) UV spectra of compound 6(a) and the complex (6-Cu2+)(b).
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J. Xu et al. / Food Chemistry xxx (2016) xxx–xxx
Fig. 5. (A) Plausible binding mode of compounds 1 and 5 with tyrosinase; and (B) plausible binding mode of compound 6 with tyrosinase.
compounds 1 and 5, Ha for compound 6). This was caused by the
tautomeric effect, in which NHAC@S was transformed into
N@CASH. Then, the sulfur atom donated hydrogen and formed a
complex with enzymic Cu2+ and led to the disappearance of the
ACSNHA proton. With the addition of tyrosinase to the target
compound 1 or 5 or 6, the peaks at 8.17/8.13/8.16 ppm (amino
proton, ANH2, Hd for compounds 1 and 5, Hb for compound 6)
split, and the chemical shifts of one proton moved upfield
(8.06/8.01/8.09 ppm), which indicated that the increased electron
density on the amino proton resulted from the formation of the
hydrogen bond between hydrogen atom of the -NH2 group and
tyrosinase.
assumed that compound 6 combined with the enzyme by forming
a complex between the thiocarbonyl/carbonyl group and the dinu-
clear copper ions in the active center of tyrosinase, which differed
from the interaction mode of compounds 1 and 5. This assumption
was reinforced by the experimental data of IR and UV spectrum
studies of the synthesized complex of the compound 6 and Cu2+
which was a mimic complex between compound 6 and enzyme.
,
3.3.2. Bonding mode studies from 1H NMR, 13C NMR titration of
inhibitors and enzymes, and the IR and UV of the synthesized complex
of compound 6 and Cu2+
Meanwhile, the peaks at 5.13/5.53 ppm for compounds 1 and 5
(Ha, CH2-OH) gradually shifted downfield (5.32/5.67 ppm), which
indicated that the decrease of the electron density on the hydroxyl
proton resulted from the formation of the hydrogen bond between
the oxygen atom of the AOH group and the amino residue of
tyrosinase.
To further verify the above assumption of the bonding mode
between compound 6 and the enzyme, a mimic complex of the inhi-
bitor and enzyme was synthesized and then the IR and UV spectra
were determined. As shown in (Fig. 4B), the UV spectra displayed a
redshift, which indicated that the formation of the complex led to
a d-d electronic transition. Further, by comparing the IR spectra
The interactions between compound 6 and tyrosinase were fur-
ther observed from the 13C NMR titrations in DMSO-d6 (Fig. 3B).
Upon the addition of 60 mg/mL of tyrosinase to compound 6, the
peaks at 177.99 ppm moved upfield (177.83 ppm), which indicated
that the electron density increased in the carbon atom on the
thiocarbonyl group. This occurred because the conjugated effect
increased the electron density in the carbon atom after the forma-
tion of the complex. The peaks at 170.64 ppm moved downfield
(170.94 ppm), which indicated that the electron density decreased
in the carbon atom on the carbonyl group. This may be explained
because the oxygen atom of the carbonyl group formed a coordina-
tion bond with the copper ions (C@O ? Cu2+) of the enzyme, which
caused the electron density of the oxygen atoms to decrease and
electronegativity to increase. Thus, the electron density of the adja-
cent carbon atom on the carbonyl group decreased by enhancing
the ϭ-effect of the carbonyl oxygen. From these results, we
(Fig. 4A) of compound 6 and the complex of compound 6 and Cu2+
,
we found that the intrinsic carbonyl stretch vibration changed from
1742.11 cmꢀ1 to 1723.75 cmꢀ1, which was introduced by the forma-
tion of complex bonding between the carbonyl oxygen of compound
6 and Cu2+, and led to a weaker double bond in the carbonyl group (Li
et al., 2014). Moreover, the stretching vibration of the carbon-
nitrogen double bond was practically unchanged (from
1600.53 cmꢀ1 to 1603.41 cmꢀ1). Amide II peaks (1535.15 cmꢀ1
)
(Guo, Kimura, & Furutani, 2013) nearly disappeared, which sug-
gested that when the thiocarbonyl group formed a coordinating
bond with the copper ions, the resulting conjugate structure
(N@CASH) led to disappearance of the ACSNHA proton by tau-
tomerization (Xie et al., 2016). The above results and IR spectra were
consistent with the results of the 1H NMR/13C NMR titrations.
Based on these results, we proposed a plausible binding mode
of compounds 1, 5 and 6 with tyrosinase in Fig. 5.
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J. Xu et al. / Food Chemistry xxx (2016) xxx–xxx
9
Guo, H., Kimura, T., & Furutani, Y. (2013). Distortion of the amide-I and -II bands of
an Å-helical membrane protein, pharaonis halorhodopsin, depends on thickness
of gold films utilized for surface-enhanced infrared absorption spectroscopy.
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4. Conclusion
We synthesized
a
series of 4-functionalized and 5-
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rooms using Spectrofluorimetry, 1H and 13C NMR titration and IR
spectra. We found that the strong auxiliary vicinity synergistic
effect from the 4-methoxyacetyl substitution group of 4-Methoxya
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carbaldehyde thiosemicarbazone successfully produced novel inhi-
bitors of tyrosinase in mushroom. Additional research is necessary
to develop novel and highly potent tyrosinase inhibitors.
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This work was supported by National Natural Sciences Founda-
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State Key Laboratory of Food Science and Technology (Nanchang
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