The Journal of Organic Chemistry
Note
the solvent resonance as the internal standard (deuterochloroform: δ
7.27 for 1H and δ 77.0 for 13C).
activating transcription factors leading to deregulated inflamma-
tory, metabolic, and proliferative responses in the cells.20
In conclusion, this work offers for the first time complete
experimental evidence of the direct formation of THP after
photochemical isomerization and ring-closure reactions of
resveratrol. Because of the potential toxic effects of polycyclic
aromatic hydrocarbons, this method represents a good tool to
analyze the resveratrol derivative THP in biological, pharma-
ceutical, environmental, and food samples. This work also
represents an important reference for further in vivo investigation
of THP in view of its possible secondary biological effects on
human health.
A white solid, 14 mg (yield ∼7%). 1H NMR (400 MHz, CD3CN): δ
8.99 (d, J = 2.1 Hz, 1H), 8.04 (br s, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.59 (d,
J = 8.8 Hz, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.13 (br s, 2H), 7.06 (dd, J =
8.8, 2.4 Hz, 1H), 6.82 (d, J = 2.4 Hz, 1H), 6.67 (d, J = 2.1 Hz, 1H). 13C
NMR (100 MHz, CD3CN): δ 158.2, 156.7, 156.6, 137.5, 133.3, 130.6,
128.9, 126.7, 124.8, 115.6, 113.9, 112.9, 105.3, 103.5.
FT-IR. The infrared spectrum was measured on an infrared
spectrometer equipped with an ARK attenuated total reflectance device.
The internal reflection element was ZnSe. Spectra were recorded at 4
cm−1 resolution with a DTGS detector.
AUTHOR INFORMATION
Corresponding Author
*Fax: +39064440062. Tel: +390649910923. E-mail: antonio.
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EXPERIMENTAL SECTION
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Reagents. All reagents and solvents used for chromatography were
analytical- and HPLC-grade products.
Notes
UV Irradiation and HPLC Separation. trans-Resveratrol (200 mg)
was dissolved in 40 mL of isopropanol containing 5 mg of I2 in a glass
Petri dish. Irradiation was carried out in air for 30 h at a distance of 20 cm
from the irradiation source, a 14.7 W UV-B fluorescent tube emitting at
wavelengths of 270−320 nm with a peak at 313 nm. Chromatographic
analysis of the photoreaction products was performed by an HPLC
system consisting of a Waters apparatus equipped with a 600 pump and
pump controller, a Symmetry C18 column (reversed-phase, 3.9 mm ×
150 mm, 5 μm particle size, with a 10 mm guard column of the same
material), and a model 2996 UV−vis photodiode array detector. The
separation method was set up by modifying our previously described
HPLC method.21 Elution was performed at a flow rate of 1 mL/min in
isocratic mode with 80% solvent A (0.1% trifluoroacetic acid in water)
and 20% solvent B (acetonitrile). The putative THP peak was directly
analyzed via GC−MS and then collected and lyophilized for MS, NMR,
and FT-IR experiments.
GC−MS. A 0.5 mL aliquot of the purified HPLC peak was saturated
with NaCl and extracted with ethyl acetate (2 mL). The organic extract
was dried under reduced pressure and derivatized with N-tert-
butyldimethylsilyl-N-methyltrifluoroacetamide (70 °C, 0.5 h). GC−
MS analyses were performed with a gas chromatograph coupled to a
quadrupole mass-selective detector. Chromatographic analyses were
carried out with a fused-silica capillary column (30 m × 0.25 mm i.d.)
coated with 5%-phenyl/95%-dimethylpolysiloxane (film thickness 0.25
μm) as the stationary phase. Injection mode: splitless at a temperature of
260 °C. Column temperature program: 70 °C for 1 min, then to 300 °C
at a rate of 15 °C/min, and held at 300 °C for 5 min. The carrier gas was
helium at a constant flow rate of 1.0 mL/min. The spectra were obtained
in electron impact mode at an ionization energy of 70 eV with an ion
source temperature of 280 °C and an ion source vacuum of 10−5 Torr.
MS analysis was performed simultaneously in TIC mode (mass range
scan from m/z 50 to 650 at a rate of 0.42 scans s−1) and SIM mode
(selected ions: m/z 511, 568, and 381).
The authors declare no competing financial interest.
REFERENCES
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ESI-MS/MS and HRMS. The CID MS experiments were performed
on a linear ion trap mass spectrometer equipped with an ESI source and
a syringe pump. Operating conditions of the ESI source were as follows:
ion spray voltage = +4.0 kV; sheath gas = 5 (arbitrary scale); sweep gas =
5 (arbitrary scale); capillary temperature = 275 °C. Methanolic solutions
of THP (1 × 10−4 M) were infused via a syringe pump into the ESI
source at a flow rate of 5 μL/min. ESI of the solutions led to the
formation of appreciable amounts of the corresponding proton-bound
species [THP-H]+, which was isolated and submitted to CID
(normalized collision energy between 14% and 17%) by collisions
with He gas into the trap (nominal pressure, 1.4 × 10−5 Torr; activation
time = 30s; activation Q = 0.20). In each acquisition, the final spectrum
was the average of about 40 scans, each consisting of two microscans.
High-resolution MS spectra were obtained with an ESI-LTQ Orbitrap
mass spectrometer. ESI was employed at a flow rate of 10 μL/min. MS
(ESI): m/z 227 [M + H]+, high-resolution ESI-MS orbitrap, calculated
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(20) Pastore, S.; Lulli, D.; Pascarella, A.; Maurelli, R.; Dellambra, E.;
+
for C14H11O3 , 227.0698, 227.0703 found.
1H/13C NMR. THP NMR spectra were recorded at 400 and 100.6
MHz. Chemical shifts are reported in parts per million from TMS with
9384
dx.doi.org/10.1021/jo501405m | J. Org. Chem. 2014, 79, 9381−9384