Synthesis and antityrosinase activities of alkyl 3,4-dihydroxybenzoates

Article abstract of DOI:10.1021/jf200990g

In insects, tyrosinase plays important roles in normal developmental processes, such as cuticular tanning, scleration, wound healing, production of opsonins, encapsulation and nodule formation for defense against foreign pathogens. Thus, tyrosinase may be regarded as a potential candidate for novel bioinsecticide development. A family of alkyl 3,4-dihydroxybenzoates (C ₆-C₉), new tyrosinsase inhibitors, were synthesized. Their inhibitory effects on the activity of tyrosinase have been investigated. The results showed all of them could inhibit the activity of tyrosianse effectively. The order of potency was nonyl 3,4-dihydroxybenzoate (C₉DB) > octyl 3,4-dihydroxybenzoate(C₈DB) > heptyl 3,4- dihydroxybenzoate(C₇DB) > hexyl 3,4-dihydroxybenzoate (C ₆DB). The kinetic analysis of these four compounds on tyrosinase was taken to expound their inhibitory mechanism. The research of the control of insects in agriculture was taken as C₆DB for example. C₆DB could inhibit the development and molting of Plutella xylostella effectively. To clarify its insecticidal mechanism, we researched the expression of tyrosinase in the P. xylostella treated with C₆DB by real-time quantitative PCR. The results showed C₆DB could inhibit the expression of tyrosinase in the P. xylostella as expected.

Full text of DOI:10.1021/jf200990g

ARTICLE
pubs.acs.org/JAFC
Synthesis and Antityrosinase Activities of Alkyl 3,4-
Dihydroxybenzoates
Zhi-Zhen Pan,†,‡ Hua-Liang Li,†,‡ Xiao-Jie Yu,† Qi-Xuan Zuo,† Guo-Xing Zheng,§ Yan Shi,† Xuan Liu,†
||
Yi-Ming Lin,† Ge Liang,† Qin Wang,*,† and Qing-Xi Chen*,†,
†Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Science, Xiamen University,
Xiamen 361005, China
§Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas 77030, United States
Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005, China
ABSTRACT: In insects, tyrosinase plays important roles in normal developmental processes, such as cuticular tanning, scleration,
wound healing, production of opsonins, encapsulation and nodule formation for defense against foreign pathogens. Thus, tyrosinase
may be regarded as a potential candidate for novel bioinsecticide development. A family of alkyl 3,4-dihydroxybenzoates (C6ꢀC9),
new tyrosinsase inhibitors, were synthesized. Their inhibitory effects on the activity of tyrosinase have been investigated. The results
showed all of them could inhibit the activity of tyrosianse effectively. The order of potency was nonyl 3,4-dihydroxybenzoate
(C9DB) > octyl 3,4-dihydroxybenzoate(C8DB) > heptyl 3,4-dihydroxybenzoate(C7DB) > hexyl 3,4-dihydroxybenzoate (C6DB).
The kinetic analysis of these four compounds on tyrosinase was taken to expound their inhibitory mechanism. The research of the
control of insects in agriculture was taken as C6DB for example. C6DB could inhibit the development and molting of Plutella
xylostella effectively. To clarify its insecticidal mechanism, we researched the expression of tyrosinase in the P. xylostella treated with
C6DB by real-time quantitative PCR. The results showed C6DB could inhibit the expression of tyrosinase in the P. xylostella as
expected.
KEYWORDS: synthesize, tyrosinase, inhibition kinetics, Plutella xylostella, real-time quantitative PCR
’ INTRODUCTION
4-dihydroxybenzoates (C6ꢀC9), new tyrosinsase inhibitors,
after the research of the structure activity relationship of tyrosi-
nase inhibitors and the safety considerations. We studied the
inhibition of alkyl 3,4-dihydroxybenzoates on mushroom tyrosi-
nase and their effects on the expression of tyrosinase in Plutella
xylostella. It may provide the basis for developing novel
insecticides.
Tyrosinase (EC 1.14.18.1), a copper containing enzyme, is
ubiquitously distributed in microorganisms, animals, insects and
plants.1,2 It is essential for the formation of melanin and various
other functions. It can catalyze both the hydroxylation of
monophenol to o-diphenol (monophenolase activity) and the
oxidation of diphenol to o-quinones (diphenolase activity) by
using molecular oxygen followed by a series of nonenzymatic
steps resulting in the formation of melanin.3ꢀ5 In the bacterium,
melanin can protect the bacterial cells and spores against UV
radiation.6ꢀ8 Meanwhile melanin can bind heavy metals that are
toxic to the cells;8 it is important to bacterial protection
mechanisms. In insects, tyrosinase plays important roles in
normal developmental processes, such as cuticular tanning,
scleration, wound healing, production of opsonins, encapsulation
and nodule formation for defense against foreign pathogens.9
The inhibition of activity of tyrosinase could lead to abrogation of
insect defense mechanisms or abnormal body softening, both of
which could lead to pest control. Tyrosinase may be regarded as a
potential candidate for novel bioinsecticide development.
In our previous research, a great many tyrosinase inhibitors
were screened from natural materials and synthetic methods. We
have reported some tyrosinase inhibitors, such as p-alkylbenzoic
acids;10 hinokitiol;11 fatty acids;12 2-phenylethanol, 2-phenyla-
cetaldehyde and 2-phenylacetic acid;13 R-cyano-4-hydroxycin-
namic acid;14 cefazolin and cefodizime;15 methyl trans-cinna-
mate;16 trans-cinnamaldehyde thiosemicarbazone;17 all of these
compounds showed inhibitory effects on the activity of tyrosi-
nase. In this present work, we synthesized a family of alkyl 3,
’ MATERIALS AND METHODS
Insects. Specimens of P. xylostella were obtained from Bio-Pesticide
Engineering Research Center of Hubei Province. Larvas were raised with
the feed from the Bio-Pesticide Engineering Research Center of Hubei
Province at 27 ( 2 °C, 70% humidity, over a 14:10 h light:dark
photoperiod.
Reagents. Tyrosinase (EC 1.14.18.1) from mushroom was the
product of Sigma Chemical Co (St. Louis, MO, USA). The specific
activity of the enzyme is 6680 U/mg. RNAiso Plus; PrimeScript RT
reagent Kit and SYBR Premix Ex Taq (Perfect Real Time) were
purchased from TaKaRa Co. L-3,4-Dihydroxyphenylalanine (L-DOPA),
4-dimethylaminopyridine (DMAP) and dimethyl sulfoxide (DMSO)
were purchased from Sigma-Aldrich (St. Louis, MO). The feed of P.
xylostella was purchased from Bio-Pesticide Engineering Research Cen-
ter of Hubei Province. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
Received: March 11, 2011
Accepted: May 19, 2011
Revised:
May 19, 2011
Published: May 19, 2011
r
2011 American Chemical Society
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Journal of Agricultural and Food Chemistry
ARTICLE
reaction) and one cycle at 85 °C for 5 s (denaturation of reverse
transcriptase).19 The primers used for PxPPO were 50-AGCA-
GATGGCTGACGAGG-30 and 50-CCGCAAAGTTGGGAATGG-30
for the forward and reverse respectively. GAPDH was used as the
reference to normalize the expression levels between the samples. The
primers used for real-time quantitative PCR were 50-CAGTGCC-
GATGCACCTATGTTC-30 (forward) and 50- AAGTTGTCGTT-
GAGGGAGATGCC-30 (reverse).20 Real-time quantitative PCR was
carried out using the SYBR Premix Ex Taq (Perfect Real Time)
(TaKaRa Code: DRR041A). PCR was performed in a Rotor-Gene
3000 (Corbett Research, Australia) according to the following protocol:
10 s at 95 °C, 1 cycle; 5 s at 95 °C and 20 s at 60 °C, 45 cycles.
Fluorescence was detected at the annealing stage of each cycle. A melting
curve was generated during the reactions to check for the possibility of
primerꢀdimer formation. The 2ꢀΔΔCt method was used to calculate the
relative mRNA level of each gene.21
Figure 1. Chemical structures of alkyl 3,4-dihydroxybenzoates.
hydrochloride (EDC HCl) and other reagents were local and of
3
analytical grade. The water used was redistilled and ion-free.
’ RESULTS AND DISCUSSION
Synthesis. A family of alkyl 3,4-dihydroxybenzoates (C6ꢀC9) (the
chemical structures are shown in Figure 1) were synthesized by
Synthesis of Alkyl 3,4-Dihydroxybenzoates. Hexyl 3,4-
dihydroxybenzoate (C6DB) obtained was a white powder. H
esterification reaction with EDC HCl and DMAP as catalysts. In brief,
1
3
a solution of 3,4-dihydroxybenzoic acid (1.0 mmol) and corresponding
NMR (600 MHz, dimethyl sulfoxide-d6): δ 9.55 (br s, 2H), 7.36
(d, J = 2.0 Hz, 1H), 7.32 (dd, J = 2.0, 8.3 Hz, 1H), 6.81 (d, J = 8.3
Hz, 1H), 4.17 (t, J = 6.5 Hz, 2H), 1,66 (m, 2H), 1.35 (m, 2H),
1.27 (m, 4H), 0.85 (m, 3H). 13C NMR (600 MHz, dimethyl
sulfoxide-d6): δ 166.17, 150.80, 145.50, 122.16, 121.28, 116.70,
115.72, 64.44, 31.35, 25.70, 25.64, 22.47, 14.29. ESI-MS: m/z
238.1 (M þ Naþ).
alcohol (1.2 mmol) was added to a solution of EDC HCl (1.2 mmol)
3
and DMAP. After being stirred overnight at room temperature, the
solvent was removed under reduced pressure. The residue was extracted
with citric acid and ethyl acetate several times and filtered. The organic
layer was dried over Na2SO4 and evaporated. The crude products were
purified by silica gel chromatography. The structures of these esters were
established by spectroscopic methods (MS and NMR).
Heptyl 3,4-dihydroxybenzoate(C7DB) was obtained as a
Enzyme Assay. The enzyme activity assay was performed as
reported by Chen et al.18 In this investigation, L-DOPA was used as a
substrate for the activity assay of tyrosinase. The reaction media (3 mL)
for activity assay contained 0.5 mM L-DOPA in 50 mM sodium
phosphate buffer (pH 6.8). The final concentration of mushroom
tyrosinase was 6.67 μg/mL for the o-diphenolase activity. The alkyl
3,4-dihydroxybenzoates were dissolved in DMSO and diluted to some
appropriate concentrations. The final concentration of DMSO in the
test solution was 3.3%. The controls, without inhibitors but containing
3.3% DMSO in the reaction media, were routinely carried out. The
reaction was carried out at a constant temperature of 30 °C. The
inhibitory effect of the alkyl 3,4-dihydroxybenzoates was expressed as
the concentration that inhibited 50% of the enzyme activity (IC50). The
inhibition type was assayed by the LineweaverꢀBurk plot, and the
inhibition constant was determined by the secondary plots of the
apparent Km/Vm or 1/Vm versus the concentration of the inhibitor. A
Beckman UV-650 spectrophotometer was used for absorbance and
kinetic parameters.
1
white powder. H NMR (600 MHz, dimethyl sulfoxide-d6): δ
9.70 (br s, 1H), 9.41 (br s, 1H), 7.35 (d, J = 1.9 Hz, 1H), 7.31 (dd,
J = 2.0, 8.2 Hz, 1H), 6.81 (d, J = 8.3 Hz, 1H), 4.17 (t, J = 6.5 Hz,
2H), 1,67 (m, 2H), 1.37 (m, 8H), 0.85 (m, 3H). 13C NMR (600
MHz, dimethyl sulfoxide-d6): δ 166.17, 150.81, 145.50, 122.16,
121.28, 116.71, 115.74, 64.45, 31.64, 28.80, 28.75, 25.95, 22.48,
14.35. ESI-MS: m/z 252.1 (M þ Naþ).
Octyl 3,4-dihydroxybenzoate(C8DB) was obtained as a white
1
powder. H NMR (600 MHz, dimethyl sulfoxide-d6): δ 9.76
(s, 1H), 9.35 (s, 1H), 7.34 (s, 1H), 7.30 (d, J = 8.3 Hz, 1H), 6.80
(d, J = 8.2 Hz, 1H), 4.18 (t, J = 6.4 Hz, 2H), 1,66 (m, 2H), 1.36
(m, 10H), 0.85 (m, 3H). 13C NMR (600 MHz, dimethyl
sulfoxide-d6): δ 166.17, 150.81, 145.50, 122.17, 121.27, 116.70,
115.76, 64.46, 31.66, 29.08, 29.05, 28.72, 25.98, 22.51, 14.39.
ESI-MS: m/z 266.2 (M þ Naþ).
Nonyl 3,4-dihydroxybenzoate (C9DB) was obtained as a white
1
powder. H NMR (600 MHz, dimethyl dulfoxide-d6): δ 9.77
The Expression of Tyrosinase in P. xylostella Treated with
C6DB by Real-Time Quantitative PCR. The C6DB was dissolved in
acetone and then diluted to five different concentrations (40, 20, 5, and
2.5 mg/mL). The feed was transferred to the 24-well plate, and then five
different concentrations of C6DB were spread well on the surface of the
feed, respectively. The newly molted second-instar P. xylostella were
gently removed to the feed after the acetone was completely evaporated.
Control was treated with the same volume of the acetone. P. xylostella
were placed in an air-conditioned room at 27.0 ( 2.0 °C and monitored
daily for 3 days. Measurement was performed after 72 h of treatment.
Real-time quantitative PCR was performed to determine expression of P.
xylostella prophenoloxidase (PxPPO) after the treatment with C6DB.
Total RNA was extracted from P. xylostella using the RNAiso Plus
(TaKaRa Code: D9108A) following the manufacturer’s instructions and
then reverse transcribed to cDNA using the PrimeScript RT reagent Kit
(Perfect Real Time) (TaKaRa Code: DRR037S). The conditions for
reverse transcription include 37 °C for 15 min (reverse transcription
(s, 1H), 9.35 (s, 1H), 7.35 (d, J = 1.9 Hz, 1H), 7.30 (dd, J = 1.9,
8.2 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 4.17 (t, J = 6.5 Hz, 2H),
1,67 (m, 2H), 1.38 (m, 12H), 0.86 (m, 3H). 13C NMR (600
MHz, dimethyl sulfoxide-d6): δ 166.17, 150.81, 145.51, 122.16,
121.27, 116.70, 115.75, 64.45, 31.70, 29.35, 29.12, 29.08, 28.72,
25.97, 22.54, 14.39. ESI-MS: m/z 280.1 (M þ Naþ).
Concentration Effects of Alkyl 3,4-Dihydroxybenzoates
on the Activity of Mushroom Tyrosinase. The activity of the
mushroom tyrosinase was assayed using L-DOPA as substrate.
Alkyl 3,4-dihydroxybenzoates were tested for the inhibitory
effect on the oxidation of L-DOPA catalyzed by mushroom
tyrosinase. The enzyme activity was monitored by following
the increasing absorbance at 475 nm accompanying the oxidation
of L-DOPA. The progress curve of enzyme reaction was a line
passing through the origin without lag period. The value of the
slope of the line indicated the activity of tyrosinase. The activity
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Figure 2. Effects of alkyl 3,4-dihydroxybenzoates on the activity of
mushroom tyrosinase for the oxidation of L-DOPA. Curves a, b, c and d
denote the compounds C6DB, C7DB, C8DB and C9DB.
Table 1. Inhibition Constants of Alkyl 3,4-Dihydroxybenzo-
ates on Mushroom Tyrosinase
Figure 3. Determination of the inhibitory mechanism of alkyl 3,4-
dihydroxybenzoates on mushroom tyrosinase. Panels a, b, c and d
denote compounds C6DB, C7DB, C8DB and C9DB, respectively. The
concentrations of C6DB and for C7DB of lines 0ꢀ4 were 0, 50, 100, 150,
and 200 μM, respectively. The concentrations of C8DB for curves 0ꢀ4
were 0, 25, 50, 75, and 100 μM, respectively. The concentrations of
C9DB for curves 0ꢀ4 were 0, 20, 40, 60, and 80 μM, respectively.
IC50 (μM)
compd
tested
theor
mechanisma
type
UC
KIS (μM)
C6DB
C7DB
C8DB
C9DB
909
315
R
389
135
316
129
81
R
R/IRb
UC
UC
IR
not tested
a R: reversible inhibition. IR: irreversible inhibition. b Reversible at
[I] e 50 μM, irreversible at [I] g 75 μM.
of mushroom tyrosinase decreased with increasing concentra-
tions of alkyl 3,4-dihydroxybenzoates as shown in Figure 2. The
concentrations leading to 50% activity loss (IC50) obtained from
Figure 2 are summarized in Table 1 for comparison. The
inhibitory effects of C6DB, C7DB, C8DB and C9DB were in
the order C6DB < C7DB < C8DB < C9DB. The inhibitory effects
of these four compounds were potentiated with increasing length
of hydrocarbon chain.
Inhibitory Mechanism of Alkyl 3,4-Dihydroxybenzoates
on Mushroom Tyrosinase. Figure 3 shows the inhibition
mechanism of alkyl 3,4-dihydroxybenzoates on mushroom tyr-
osinase for the oxidation of L-DOPA. The plots of the remaining
enzyme activity versus the concentrations of enzyme in the
presence of different concentrations of compounds gave a family
of straight lines. If all straight lines passed through the origin, the
inhibition was reversible, but if they did not pass through the origin
and were parallel lines, the inhibition was irreversible. From Figure 3,
we found that C6DB and C7DB were reversible inhibitors and
C9DB was an irreversible inhibitor. When the concentration of
C8DB was lower than 50 μM, it showed a reversible inhibitory
mechanism, but when the concentration was above 75 μM, the
inhibitory mechanism of C8DB was irreversible. The kinetic
constants obtained are listed in Table 1.
Figure 4. LineweaverꢀBurk plots for inhibition of C7DB on mush-
room tyrosinase for the catalysis of L-DOPA at 30 °C, pH 6.8. The
concentrations of C7DB for lines 1ꢀ5 were 0, 50, 100, 150, and 200 μM,
respectively. The inset represents the secondary plot of Km versus
concentration of C7DB to determine the inhibition constant KIS.
the plots of 1/v versus 1/[S] gave a family of parallel straight lines
with the same slopes. Accompanying the increase of the inhibitor
concentration, the values of both Km and Vm increased, but the
ratio of Km/Vm remained unchanged. The slopes were indepen-
dent of the concentration of C7DB, which indicated that C7DB
was an uncompetitive inhibitor on the enzyme. When the
concentration was lower than 50 μM, the inhibition type of
C6DB and C8DB was the same, belonging to be uncompetitive.
The results indicated that 3,4-dihydroxybenzoates could only
bind with the enzymeꢀsubstrate complex, not with the free
enzyme. The inhibition constant, KIS, was obtained from a plot of
the vertical intercept (1/Vm) versus the concentration of C7DB,
which is linear, as shown in the inset in Figure 4. The inhibition
constants are summarized in Table 1 for comparison.
Inhibition Type and Inhibition Constants of Alkyl 3,4-
Dihydroxybenzoates on Mushroom Tyrosinase. The kinetic
behaviors of the oxidation of L-DOPA, catalyzed by mushroom
tyrosinase at different concentrations of alky 3,4-dihydroxy-
benzoates, were studied. Figure 4 shows the double-reciprocal
plots of the enzyme inhibited by C7DB. The results showed that
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Funding Sources
The present investigation was supported by the National Foun-
dation for fostering talents of basic science (No. J1030626), the Natural
Science Foundation of China (No. 31071611, No. 31070522)
and Grant 2010N5013 of the Science and Technology Founda-
tion of Fujian Province.
’ ABBREVIATIONS USED
PPO, prophenoloxidase; PxPPO, Plutella xylostella prophenolox-
idase; EDC HCl, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
3
hydrochloride; DMAP, 4-dimethylaminopyridine; L-DOPA,
L-3,4-dihydroxyphenylalanine; DMSO, dimethyl sulfoxide; IC50,
the inhibitor concentration leading to 50% activity loss; KIS,
equilibrium constant of the inhibitor combining with the en-
zymeꢀsubstrate complex
Figure 5. Real-time quantitative PCR analysis of PxPPO2 transcripts
with C6DB treated. PxPP02 transcripts were determined from the whole
body at 72 h after treament with C6DB at concentrations of 2.5, 5, 20,
and 40 mg/mL. All the data were normalized relative to GAPDH.
The Expression of Tyrosinase in P. xylostella Treated with
C6DB by Real-Time Quantitative PCR. C6DB showed an
inhibitory effect against mushroom tyrosinase among these four
compounds with IC50 of 0.91 mM; it was a reversible and
uncompetitive inhibitor. The inhibition constant (KIS) was
determined to be 0.39 mM. We explored its insecticidal action
from the effect on the expression of tyrosinase in P. xylostella.
From the results, we found that it could effectively inhibit the
development and molting of P. xylostella. With the increasing of
the concentration of C6DB, the inhibitory effects become
stronger. The P. xylostella insects treated with C6DB in the
concentration of 40 mg/mL were 0.3 cm long on average after 3
days; with the control they were 0.95 cm on average. We analyzed
the expression of tyrosinase in P. xylostella treated with C6DB by
real-time quantitative PCR furthermore. The insect tyrosinase
was labile and easy to inactivate during purification. Its inactive
precursor, prophenoloxidase (PPO), was more stable. Tyrosi-
nase could be activated from PPO by specific proteolysis via a
serine protease cascade22 in P. xylostella, and the cDNA encoding
PPO from P. xylostella has been cloned. In the present work, we
investigate the influence of C6DB on the relative amount of
PxPPO transcripts by real-time quantitative PCR. As Figure 5
shows, PxPPO transcript levels were gradually decreased with
increasing concentration of C6DB as compared with control
samples. It indicated that C6DB could inhibit the expression of
tyrosinase in P. xylostella to inhibit the molting of P. xylostella. It
was a potent biological insecticide.
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’ AUTHOR INFORMATION
Corresponding Author
*Tel/fax: þ86 592 2185487. E-mail: chenqx@xmu.edu.cn
(Q.-X.C.).
Author Contributions
‡These authors contributed equally to this work.
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