Journal of Agricultural and Food Chemistry
Article
mL). The mixture was allowed to be stirred with a magnetic stirring bar
in a round-bottomed flask at 0 °C for 1 h and at room temperature for 24
h. Saturated aqueous NaHCO3 solution was added to the mixture, and
the aqueous solution was extracted with ethyl acetate (50 mL × 3). The
combined organic solution was washed with saturated aqueous NaCl
solution and dried over anhydrous MgSO4, and the solution was
concentrated using a rotary evaporator at 40 °C under reduced pressure.
The residue was purified by silica gel column chromatography using an
isocratic elution with hexane/ethyl acetate (3:1, v/v) to afford
compound 2.
Anhydrous pyridine (1.2 mL, 15 mmol) and trifluolomethanesulfonic
anhydride (0.82 mL, 5 mmol) were successively added to a solution of
compound 2 (0.68 g, 3.5 mmol) in anhydrous dichloromethane (5 mL),
and the mixture was allowed to be stirred at 0 °C for 1 h followed by 3 h
at room temperature. The reaction mixture was extracted with ethyl
acetate (50 mL × 3), and the combined organic solution was washed
with saturated aqueous NaCl solution and dried over anhydrous MgSO4.
The solvent was removed under reduced pressure, and the residue was
purified by silica gel column chromatography using an isocratic elution
with hexane/ethyl acetate (3:1, v/v) to afford the triflate 3.
ionization (ESI) interface with a detector voltage of 1.5 kV, from m/z
150 to 800 in the mass analyzer, and with an even time of 1.0 s. The ESI
parameters were as follows: source voltage, 2.50 kV; sheath gas flow rate,
50 Arb; aux/sweep gas flow rate, 10 arb; capillary voltage, −41.00 V; and
capillary temperature, 330 °C. ESI was operated in both negative-ion
(NI) and positive-ion (PI) modes.
Fourier Transform Infrared (FTIR) Spectroscopy Analysis of
Compound 5. A FTIR spectrum of the purified compound 5 was
recorded by a FTIR spectrophotometer (Tensor 27, Bruker, Germany)
using a KBr disk containing 1% finely ground samples.
HPLC Analysis of Compound 5. HPLC analysis of compound 5
was performed using an Agilent Technologies Series 1200 system
(Agilent, Santa Clara, CA) equipped with a G1322A automatic degasser,
a G1311A quaternary pump, a G1367 auto sampler, and an ultraviolet−
visible (UV−vis) diode array detector (DAD). A C18 column (250 mm
length, 4.6 mm inner diameter, and 5 μm particle size, Merck Germany,
Ltd., Germany) operated at 30 °C was used, and the sample was eluted
using an isocratic solvent system containg methanol/1% HAc (90:10),
at a flow rate of 1.0 mL/min. A total of 20 μL of sample was injected into
the system and detected at 310 nm by UV detection.
Tyrosinase Inhibitory Activity Assay. The mushroom tyrosinase
was obtained from Worthington Biochemical Corporation, Freehold,
NJ. L-3,4-Dihydroxyphenylalanine (L-DOPA) and kojic acid were
purchased from Sigma-Aldrich (St. Louis, MO). The tyrosinase assay
was performed with modification, as reported by Zhang et al.22 A total of
80 μL of 0.1 M phosphate-buffered saline (PBS) at pH 6.8, 20 μL of
mushroom tyrosinase diluted in the phosphate buffer (985 units/mL),
and various concentrations of different test samples dissolved in 50 μL of
90% methanol were inserted into 96-well plates for 5 min of pre-
incubation at 30 °C. A total of 50 μL of L-DOPA was then added to start
the enzymatic reaction, and absorbance at 492 nm was measured by a
Thermo Scientific Multiskan GO UV/vis microplate spectrophotom-
eter (Thermo Scientific, Waltham, MA) to observe dopachrome
formation for 20 min. All experiments were carried out at least in
triplicate. The percentage of inhibition was calculated as follows:
Synthesis of 2,2-Dimethyl-5-tridec-1-enyl-benzo[1,3]dioxin-4-one
(4′). A solution of 1-tridecene (124 mg, 0.68 mmol) in N,N-
dimethylformamide (DMF) was stirred with K2CO3 (94.8 mg, 0.68
mmol) at ambient temperature for 30 min prior to the addition of the
triflate 3 (200 mg, 0.62 mmol) and PdCl2(dppf) (16.9 mg, 0.02 mmol)
under a nitrogen atmosphere. The reaction mixture was heated for 12 h
at 75 °C, and the solvent was evaporated. The residue was partitioned
between Et2O and water (30 mL each), and the organic layer was
separated and dried (Na2SO4). The solvent was removed under reduced
pressure, and the crude product is purified by silica gel column
chromatography with hexane/ethyl acetate (20:1, v/v) as the eluent,
thus affording a product whose structure could be confirmed by mass
spectrometry (MS) and proton nuclear magnetic resonance (1H NMR).
Preparation of 5-Tridecyl-2,2-dimethylbenzo[1,3]dioxin-4-one (4).
Reduction of the double bond in the long unsaturated chain substituent
of compound 4′ was conducted to afford compound 4. A solution of
compound 4′ (111.5 mg, 0.31 mmol) in EtOAc in a round-bottomed
flask was added with Pd/C (5%) (99 mg, 0.047 mmol) under a nitrogen
atmosphere, followed by exclusion of N2 through a three-way valve using
vacuum pump and injection of H2 with a hydrogen balloon. After 12 h of
reaction at ambient temperature, H2 in the round-bottomed flask was
excluded. The mixture was filtered to remove Pd−C, and the organic
phase was washed with water. The organic layer was separated and dried
(Na2SO4), and the solvent was removed under reduced pressure to
afford compound 4.
percentage of inhibition
= [(A − B) − (C − D)]/(A − B) × 100
where A is OD at 492 nm with tyrosinase but without the test sample, B
is OD at 492 nm without the test sample and tyrosinase, C is OD at 492
nm with the test sample and tyrosinase, and D is OD at 492 nm with the
test sample but without tyrosinase. Kojic acid was tested as a positive
control.
Preparation of GA (13:0) (5). The hydrolysis of compound 4 was
conducted to furnish the GA (13:0) 5. A solution of compound 4 (17
mg, 0.048 mmol), in aqueous KOH (50%, 0.2 mL) and dimethyl
sulfoxide (DMSO) (0.5 mL) was heated at 80 °C for 1 h. The reaction
mixture was cooled to ambient temperature and diluted with water (20
mL), followed by the acidification with HCl (1 mol/L). The
acidification was terminated when pH of the mixture reached 2. This
mixture was then extracted with ethyl acetate (50 mL), and the organic
layer was separated and dried over Na2SO4. The solvent was removed
under reduced pressure, and the crude product was subjected to column
chromatography on silica gel using an isocratic elusion with hexane/
ethyl acetate/HAc (70:29:1, v/v/v) to afford compound 5.
RESULTS AND DISCUSSION
■
Synthesis of Trifluoromethanesulfonic Acid 2,2-Di-
methyl-4-oxo-4H-1,3-benzodioxin-5-yl Ester (3). Prepara-
tion of compound 2 commenced with ketalization of 2,6-
dihydroxybenzoic acid (1) according to a procedure described by
Hadfield et al.23 The silica gel thin-layer chromatography (TLC)
using a mixed solvent system of hexanes and ethyl acetate (2:1)
was applied to monitor the progress of the reaction. Visualization
of TLC with a UV irradiation at 254 nm revealed that the
addition amount of acetone was critical for the conversion rate of
the reaction. Initially, acetone was added at the molar ratio of
2.6:1 to 2,6-dihydroxybenzoic acid, and the reaction did not
proceeded well. After several attempts, the addition amount of
acetone was finally optimized to a molar ratio of 10:1 to 2,6-
dihydroxybenzoic acid and a good yield of 67% was obtained
(Figure 2a).
The remaining hydroxyl group of compound 2 was readily
converted to the triflate 3 as a colorless solid in a good yield of
73% by treatment with trifluoromethanesulfonic anhydride and
pyridine in anhydrous dichloromethane following the procedure
established by Uchiyama et al.24 (Figure 2a). The structure of
synthesized compound 3 was confirmed by 1H NMR spectros-
NMR Analysis of Compounds 3 and 5. 1H NMR analysis of the
above purified compounds 3, 4′, and 5 was carried out to confirm their
structure. NMR spectra of compounds 3, 4′, and 5 were recorded on a
Bruker 500 MHz NMR spectrometer at room temperature in CDCl3,
with the solvent residual peak as the internal reference. Chemical shifts
were expressed in δ values.
MS Analysis of Compounds 4′, 4, 5, and 5′. The purity of
compound 5 was confirmed by high-performance liquid chromatog-
raphy (HPLC), and the identities of compounds 4′, 4, and 5 were
confirmed by a Thermo Finnigan LTQ-DECA-XP-MAX linear ion trap
mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Thermo
Finnigan Xcalibur software (version 2.1) was used for data acquisition
and processing. The analysis was monitored by an electrospray
B
dx.doi.org/10.1021/jf4012642 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX