Jiang et al.
FULL PAPER
Experimental
Theoretical calculations
Theoretical calculation was performed using the
Gaussian 03 program.33 The geometries were first opti-
mized by the semi-empirical AM1 method, and then by
B3LYP density functional method together with
6-31G(d) basis set. Atomic charges were calculated us-
ing Mulliken population analysis as implemented in the
Gaussian 03 program.
Apparatus and reagents
HPLC was conducted on Varian ProStar system
consisting of a ProStar 330 PDA UV detector with
Prontosil Eurobond C18 stationary phases for analysis
and Chromatorex C18 stationary phases for separation.
pH (pD) value was recorded by METLER TOLEDO
(made in Switzerland) pH meter and calibrated in
aqueous standard buffer before measurement.
The D2O (99.9% atom% D) was purchased from
J&K CHEMICAL LTD, CD3OD (99.8% atom% D)
from Cambridge Isotope Laboratories, Inc., Na2HPO4•
12H2O, NaH2PO4•2H2O, KH2PO4 and NaCl (all in ana-
lytical grade) from Shanghai Sinopharm Chemical Re-
agent Co. Ltd.
Phosphate salt was dissolved in pure D2O to obtain
intended pH buffer stock solution: pD= 7.6 buffer stock
solution was prepared by adding 0.2 mol/L NaH2PO4
(1.9 mL) to 0.2 mol/L Na2HPO4 (8.1 mL); pD= 9.0
buffer stock solution was prepared by adding 0.2 mol/L
KH2PO4 (0.05 mL) to 0.2 mol/L Na2HPO4 (9.95 mL).
Results and discussion
1
Figure 1a—1d showed expanded H spectra for the
compounds I, II, III and IV, respectively. The assign-
ments labeled in the figure were performed by conven-
tional NMR methods and confirmed mainly by
J-couplings and two-dimensional C-H correlation ex-
periment (data not shown).
The exchange dynamics of compounds I and II was
1
investigated using H spectra recorded at different time
intervals. Both compounds show H/D exchange at H-2,6
and H-4 positions. The data fitting of the relative inte-
grations of these proton resonances follows first-order
kinetics as reported.30 The exchange rate constants (k)
deduced in Figure 2 show an obvious order of H-2,6>
H-4 for each isomer. Besides, the exchange rates of
trans-isomer are much larger than those of cis-isomer,
which means the former undergoes faster H/D ex-
change.
Compounds III and IV, the homologous analogues
of compounds I and II with O-β-D-glucoside substitu-
tion on C-2, were also studied. The H/D exchange oc-
curs at H-6 and H-4 sites, which are chemical equivalent
positions of H-2,6 and H-4 in compounds I and II, re-
spectively. The exchange reaction fits the first-order
kinetics very well as illustrated in Figure 3, and the ex-
change rates of these active sites give the same reactiv-
ity order as those of compounds I and II, that is, H-2,6
> H-4 and trans-isomer> cis-isomer.
Sample preparation
trans-Resveratrol (I) was purchased from a com-
mercial source in 99% purity, and the structure and pu-
rity was verified by NMR. trans-Resveratrol was dis-
solved in 50% methanol/H2O and subjected to 365 nm
UV light for 2 h to produce its isomer, cis-resveratrol
(II)32. The two isomers were separated using HPLC.
Compound III was extracted from air-dried Radix Po-
lygoni Multiflori. Compound III was dissolved in H2O
and subjected to 365 nm UV light for 2 h to produce its
isomer, compound IV.32 The two isomers were sepa-
rated using HPLC. The samples I, II, III and IV were,
respectively, dissolved in the phosphate buffers for pH
(pD= 7.6, 9.0) dependent study and in D2O containing
20 mmol/L NaCl to obtain a native pH condition. The
time was recorded (t= 0) when the sample was added in
the solution.
The H/D exchange reactions of four compounds
were investigated at various pH conditions (pD 5.5, pD
7.6 and pD 9.0). The corresponding exchange rates are
listed in Table 1, and are obviously pH dependent. In
our measurements, the exchange rates are increased
from pD 5.5 to pD 9.0.
NMR spectroscopy
1H spectra were obtained at Bruker AVANCE 500
instrument at 500.13 MHz, at 298 K. Chemical shifts
were calibrated to the residual solvent signal of HDO (δ
4.70) and given on the δ scale. Single pulse 1H spectrum
was acquired into 65536 data points covering a spectral
width of 4000 Hz and with a recovery delay of 5 s, 4
dummy scans and 60 transients, which results in an ex-
It is interesting that, the O-β-D-glucoside substitu-
tion of C-2 site significantly accelerates the exchange
rates. Compound III experiences much faster H/D ex-
changing at its native condition (pD 5.5) than other
compounds. The left part of Figure 3 shows the
1
1
perimental time of about 10 min. All H spectra were
time-dependent H spectra of compound III at pD 5.5.
processed with Bruker’s TOPSPIN 2.0 software. An
exponential apodization function of 0.3 Hz was applied
on the data before Fourier transformation. After baseline
and phase correction, peaks of the interested protons
were integrated, the peak integrated area (denoted as A)
at different time intervals were recorded. The stable
H-2',6' proton resonances of B ring were used as inter-
nal integral reference and set to 2.
The intensity of H-6 resonance has attenuated signifi-
cantly in D2O after a 3-day storage. This may not be
awared if the sample dissolved in D2O is not tested im-
mediately and might cause mis-assignment in the struc-
ture determination.
It is well known that, the mechanism of H/D ex-
change reaction occurring at aromatic protons follows a
two-step process as show in Figure 4:30,34 firstly, the
2282
© 2010 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2010, 28, 2281— 2286