6
Tetrahedron
instrument with an Alltech 3300 ELSD detector (Columbia,
aforementioned, and analyzed by LCMS, too. The retention time
ACCEPTED MANUSCRIPT
MD, USA) using a Waters Sunfire RP C18, 5 ꢁm, 30 × 150 mm
column. Semipreparative HPLC was performed on a Waters
2690 instrument (Milford, MD, USA) coupled with a 996
photodiode array detector using a YMC-pack RP C18, 5 ꢁm, 10
× 250 mm column. Optical rotations were measured on a
Rudolph Autopol VIAutomatic polarimeter (Hackettstown, NJ,
USA). IR spectra were recorded on a Nicolet Magna FT-IR 750
spectrophotometer (Waltham, MA, USA) using KBr disks. ECD
spectra were recorded on a JASCO J-810 spectrometer. ESIMS
and HRESIMS data were recorded on Waters 2695-3100 LC-MS
and Agilent G6520 Q-TOF mass spectrometers (Santa Clara, CA,
USA), respectively. NMR spectra were recorded on a Bruker
Avance III (Bruker, Zurich, Switzerland) for 500 and 600 M
NMR spectrometer with TMS as internal standard. The chemical
shift (δ) values were given in ppm and coupling constants (J) are
in Hz. All solvents used for CC were of at least analytical grade
(Shanghai Chemical Reagents Co., Ltd., Shanghai, China), and
solvents used for HPLC were of HPLC grade (Merck KGaA,
Darmstadt, Germany).
of the derivatives of compound 1, D-glucose and L-glucose were
13.13, 13.17 and 12.42 min, respectively. Therefore, the sugar
moiety of compound 1 was determined as D-glucose (details see
Supporting Information).
4.3.2. Compound characteristics
Compound 1
yellow amorphous powder; [α]D20 –69.7 (c = 1.5, MeOH); UV
(MeOH) λmax (log ε) 324 (4.06), 219 (4.15) nm; ECD (MeOH)
(ꢀε) 244 (–2.16) nm; IR (KBr, cm–1): νmax 3430, 1732, 1636,
1528 cm–1; H and 13C NMR data, see Tables 1 and 2; ESIMS
1
m/z 569 [M – H]–; HRESIMS m/z 593.1494 [M + Na]+ (calcd.
For C25H30NaO15, 593.1477).
Compound 2
20
yellow amorphous powder; [α]D –72.5 (c = 0.13, MeOH);
UV (MeOH) λmax (log ε) 342 (2.80), 315 (2.73), 290 (2.66), 230
(3.25) nm; ECD (MeOH) (ꢀε) 348 (–2.30), 315 (0.93), 293 (–
0.79), 240 (–5.85), nm; IR (KBr, cm–1): νmax 3428, 1730, 1640,
1526 cm–1; H and 13C NMR data, see Tables 1 and 2; ESIMS
1
m/z 1137 [M – H]–; HRESIMS m/z 1137.2955 [M – H]– (calcd.
4.2. Plant material
for C50H57O30, 1137.2940).
The leaves of V. bracteatum were collected in Jiangsu
Province, China, in 2015, and identified by Professor Jin-Gui
Shen from Shanghai Institute of Materia Medica. A voucher
specimen (No. 20150926) was deposited at the Herbarium of the
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences.
Compound 3
20
yellow amorphous powder; [α]D +11.4 (c = 0.098, MeOH);
UV (MeOH) λmax (log ε) 344 (2.64), 312 (2.62), 292 (2.57), 230
(3.07) nm; ECD (MeOH) (ꢀε) 348 (2.21), 314 (–0.52), 293
(1.00), 247 (–5.74) nm; IR (KBr, cm–1): νmax 3424, 1726, 1638,
1
1527 cm–1; H and 13C NMR data, see Tables 1 and 2; ESIMS
4.3. Extraction and isolation
m/z 1137.6 [M – H]–; HRESIMS m/z 1137.2948 [M – H]– (calcd
for C50H57O30, 1137.2940).
The air-dried leaves of V. bracteatum (20 kg) were ground
into powder, and then extracted three times with 95% ethanol at
room temperature to afford a crude extract (1.5 kg). The extract
was further partitioned in water, and then extracted with
petroleum ether (PE) and EtOAc, successively, to give a PE, an
EtOAc, and a water soluble fraction. The water soluble fraction
(Fr. A) was then fractionated by a column chromatography (CC)
over macroporous resin AB-8 gel (EtOH/H2O, from 30 to 95%),
yieling fractions A1−A3. Fraction A1 (200 g) was then separated
on polyamide (MeOH/H2O, from 20 to 95%) to give four
subfractions (A1A-A1D). Then subfraction A1A was subjected
to CC over octadecyl silane (ODS) (MeOH/H2O, from 10 to
45%) to afford fractions A1A1-A1A10. Fraction A1A1 (180.0
mg) was applied to preparative HPLC (MeOH/H2O, from 5 to
20%, containing 0.2% formic acid; 0-120 min, 25 mL/min) and
then semipreparative HPLC (MeCN/H2O, from 9 to 14%,
containing 0.2% formic acid; 0-60 min, 3 mL/min) to afford
compounds 2 (3.2 mg) and 3 (2.4 mg) as trace components.
Fraction A1A4 (2.0 g) was chromatographed on Sephadex LH-20
(MeOH) to yield subfractions A1A4C (800 mg). Finally,
compound 1 (115 mg) was obtained from fraction A1A4C by
preparative HPLC (MeCN/H2O, from 10 to 28%, containing
0.2% formic acid; 0-120 min, 25 mL/min).
4.4. Computational section
Mixed torsional/low-frequencymode conformational searches
were carried out by means of the Macromodel 10.8.011 software
using the Merck Molecular Force Field (MMFF) with an implicit
solvent model for CHCl3.37 Geometry reoptimizations were
carried out at the B3LYP/6-31G(d) level in vacuo, the B3LYP/6-
31+G(d,p) level in vacuo, the B97D/TZVP30,31 and the
CAMB3LYP/TZVP32,33 levels with the PCM solvent model for
MeOH. DFT optimized geometries were clustered for all non-
hydrogen atoms. TDDFT ECD calculations were run with
various functionals (B3LYP, BH&HLYP, CAM-B3LYP, PBE0)
and the TZVP basis set as implemented in the Gaussian 09
package with the same or no solvent model as in the preceding
DFT optimization step.38 NMR calculations were performed at
the mPW1PW91/6-311+G(2d,p) level.34 ECD spectra were
generated as sums of Gaussians with 2400 and 3000 cm−1 widths
at half-height (corresponding to ca. 15 and 19 nm at 250 nm),
using dipole-velocity-computed rotational strength values.39
Computed NMR data were corrected with I = 185.4855 and S = -
1.0306.40,41 Boltzmann distributions were estimated from the
ZPVE-corrected B3LYP/6-31G(d) energies in the B3LYP/6-
31G(d) gas-phase calculations, and from the uncorrected
B3LYP/6-31+G(d,p), B97D/TZVP and CAM-B3LYP/TZVP
energies in the other cases. The MOLEKEL software package
was used for visualization of the results.42
4.3.1. Determination of sugar configuration
Compound 1 (11.4 mg) and cellulase (11.4 mg) were
dissolved in HOAc – NaOAc buffered solution (PH = 4.5, 2 mL)
and stirred at room temperature for a week. The reaction mixture
was evaporated by rotary evaporator, and then dissolved in
pyridine (2 mL) containing L-cysteine methyl ester
hydrochloride (2 mg) and heated at 60 °C for 60 min. Then o-
tolyl isothiocyanate (5 ꢁL) was added to the mixture and heated
at 60 °C for 60 min. After evaporation of the solvent, the residue
was dissolved in methanol, and then analyzed by LCMS. The
Acknowledgments
Financial support from the National Science and Technology
Major Project “Key New Drug Creation and Manufacturing
Program” (No. 2012ZX09301001-001, 2015ZX09103002), and
the National Natural Science Funds of China (No. 81302657,
81473112, 81573305) are gratefully acknowledged. A.M. and
authentic D/L-glucose samples were treated with the same method