R. X. Tan et al.
sites (Tyr70, Trp84, Trp279, and Phe330 underlined in Table S3, Support-
ing Information) in a complex of Torpedo californica acetylcholinesterase
with its inhibitor Bw284c51. By superimposition of the three crystal struc-
tures, the results reveal the common sites maybe exist in Electrophorus
electricus acetylcholinesterase, namely Tyr72 and Trp286. And three
other sites, Trp86, Tyr124 and Tyr 337, also play a role during interaction
between acetylcholinesterase and its inhibitor. Thus, hopeahainol A (2)
was docked in a pocket surrounding by these sites mentioned above and
the interactional energy (including whole energy, electrostatic energy and
steric energy) was calculated and optimized. The docked conformations
(Figure 6) were then used to analyze the binding interactions.
Enzyme kinetic studies:[16] Kinetic studies were performed using AChE
from an electric eel. Enzyme actvities were determined at 258C using
five concentrations of substrate (50, 100, 200, 300 and 400 mm) in the
presence or absence of three concentrations of 2 (10, 20 and 40 mm)
against AChE. Data were plotted by the method of Lineweaver–Burk to
reveal the mechanism of inhibition. Plots of the slopes versus the inhibi-
tor concentrations gave estimates of Ki, the dissociation constant for in-
hibitor binding to AChE.
Experimental Section
General methods: Melting points were measured on an XT-4 apparatus.
Optical rotations were determined in MeOH on a WXG-4 disc polarime-
ter, and IR spectra in KBr disks on a Nexus 870 FT-IR spectrometer.
The UV spectra were recorded on a Hitachi U-3000 spectrophotometer.
ESI and HR-ESI mass spectra were obtained on a Mariner Mass 5304 in-
strument. All NMR experiments were performed on a Bruker DRX-500
NMR spectrometer and using solvent signal ([D6]acetone, dH: 2.05 ppm)
as an internal standard. Silica gel (200–300 mesh) for CC and GF254
(10–20 mm) for TLC were produced by Qingdao Marine Chemical Com-
pany, China. Sephadex LH-20 was purchased from Pharmacia Biotech,
Sweden. Electric-eel AChE (EC 3.1.1.7), acetylthiocholine iodide
(ATCh), 5,5’-dithiobis[2-nitrobenzoic acid] (DTNB) were purchased from
Sigma (St. Louis, MO, USA). All other chemicals used in the study were
of analytical grade.
Plant material: Hopea hainaninesis was collected on July 21, 2003 from
the Botanical Garden of South China University of Tropical Agriculture
with the specimen identified by Professor X. Q. Zheng (South China
University of Tropical Agriculture). A voucher specimen (no. IFB030721)
was deposited at Institute of Functional Biomolecules, Nanjing Universi-
ty, Nanjing, China.
Hopeahainol A (2): Red amorphous powder, m.p. 216–2178C; [a]2D0 =+
673.58 (c = 0.090, MeOH); UV/Vis (MeOH): lmaxACHTRE(UNG log e) = 218 (4.41),
307 (3.91), 450 nm (3.63); IR (KBr): n˜max = 3195, 2975, 1796, 1695, 1633,
1592, 1508, 1448, 1335, 1261, 1162 1076 cmÀ1; NMR: see Table S1, Sup-
porting Information; positive ESIMS: m/z: 481[ M+H]+, 503 [M+Na]+;
HR-ESIMS: m/z: calcd for C28H17O8: 481.0923; found: 481.0920 [M+H]+,
503.0749 [M+Na]+.
Extraction and isolation: All mother liquors left over by previous isola-
tions[6] were combined to give, after evaporation of solvent, a residue
(98.8 g) that was subsequently purified by chromatography over silica gel
column and eluted with CHCl3/MeOH gradients of growing polarity to
give eight fractions. And the second fraction (2.8 g) with bioactivity, af-
forded by elution with CHCl3/MeOH 20:1, was separated on silica gel
with CHCl3/MeOH 50:1, 30:1, 20:1, 10:1, 5:1, 0:100 to give six fractions
(fractions 1–6). Fraction 2 (110 mg) was further subjected to Sephadex
LH-20 column chromatography (MeOH) to give 1 (31mg). Fraction 3
(530 mg) was further purified on a silica gel column eluted with petrole-
um ether/acetone 3:1, 2:1, 1:1, 0:1 to give four fractions (fractions 3a–
3d). Compound 3 (54 mg) and 2 (73 mg) were obtained from fractions 3-
Hopeahainol B (3): Red amorphous powder, m.p. 227–2288C; [a]D20
+1105.68 (c 0.024, MeOH); UV/Vis (MeOH): lmax (log e) 214
(4.32), 306 nm (3.88); IR (KBr): n˜max = 3382, 3160, 2969, 2258, 1797,
1698, 1654, 1633, 1606, 1507, 1448, 1333, 1252, 1163 1076, 1003 cmÀ1
=
=
=
;
NMR: see Table S1, Supporting Information; positive ESIMS: m/z: 495
[M+H]+, 51 7 [M+Na]+; HR-ESIMS: m/z: calcd for C29H19O8: 495.1080;
found: 495.1075 [M+H]+.
Hopeanol B (4): Light yellow amorphous powder, m.p. 198–1998C;
[a]2D0 =+146.08 (c = 0.373, MeOH); UV/Vis (MeOH): lmax (log e) = 207
(4.91), 225 (4.77), 3011 (3.32), 355 nm (3.41); IR (KBr): n˜max = 3200,
2923, 2882, 1718, 1660, 1609, 1608, 1516, 1461, 1336, 1263, 1160, 1113,
1082, 1056, 1017 cmÀ1; NMR: see Table S1, Supporting Information; posi-
tive ESIMS: m/z: 499 [M+H]+, 521[ M+Na]+; HR-ESIMS: m/z: calcd
for C29H19O9: 495.1029; found: 499.1033 [M+H]+.
b
and 3-d, respectively, after purification by Sephadex LH-20 CC
(MeOH). Compound 4 (17 mg) was obtained from fraction 5 after purifi-
cation by PTLC (EtOAc/CHCl3/MeOH/H2O 5:10:1:0.1) and Sephadex
LH-20 CC (MeOH).
AChE inhibition assay: The AChE inhibitory activities were measured by
a spectrophotometric method developed by Ellman et al.[12] The reaction
was run at 258C in a final volume of 200 mL of a 0.1m phosphate buffer
pH 8.0, 333 mm 5,5’-dithio-bis(2-nitrobenzoic acid), 0.035 unit per mL
AChE and 530 mm of acetylthiocholine iodide in 96-well microplates. Test
compounds were added to the assay solution and followed at 412 nm for
5 min with a plate reader (Sunrise, Tecan, Austria). Inhibition curves
were performed in triplicate by incubating with at least 10 concentrations
of each test compound. One triplicate sample without inhibitor was
always present to yield 100% of AChE activity. The reaction rates were
compared and the percent inhibition due to the presence of test com-
pounds was calculated. IC50 (concentration of drug producing 50% of
enzyme–activity inhibition) values were determined graphically from log
concentration–inhibition curves.
Methylation of 2: Hopeahainol A (2) (20 mg) was allowed to react with
K2CO3 (500 mg) and MeI (200 mg) in dry acetone under reflux for 6 h at
658C. The reaction was treated in the usual manner and the crude prod-
uct 25 mg was purified by Sephadex LH-20 CC (CHCl3/MeOH 1:1) to
give 2a (16 mg). A yellow rhombic crystal; positive ion ESIMS: m/z: 537
[M+H]À; 1H NMR (500 MHz, [D6]acetone, À308C): d= 7.46 (d, J
=
2.2 Hz, H-10a), 7.35 (dd, J = 10.3, 2.4 Hz, H-2a), 7.31 (d, J = 2.2 Hz, H-
12a), 7.21 (dd, J = 8.7, 1.7 Hz, H-6b), 7.04 (d, J = 2.3 Hz, H-14b), 6.77
(dd, J = 8.7, 2.1Hz, H-5b), 6.75 (d, J = 2.3 Hz, H-12b), 6.60 (dd, J =
8.6, 1.9 Hz, H-2b), 6.53 (dd, J = 8.6, 2.1Hz, H-3b), 6.52 (dd, J = 10.1,
2.4 Hz, H-6a), 6.15 (dd, J = 10.3, 1.8 Hz, H-3a), 5.95 (dd, J = 10.1,
1.8 Hz, H-5a), 3.97 (s, OMe), 3.93 (s, OMe), 3.74
OMe).
ACHTRE(UNG s, OMe), 3.54 ppm (s,
Molecular docking of hopeahainol A into Electrophorus electricus ace-
tylcholinesterase: Molecular docking was performed on a Silicon Graph-
ics Iris O2 (SGI Inc, Silicon, CA, USA) workstation using the DOCK
modules of the commercial software packages InsightII 2000 (MSI, St
Louis, MI, USA). Three high-resolution X-ray crystal structures of Elec-
trophorus electricus acetylcholinesterase (PDB code: 1C2B),[13] mouse
acetylcholinesterase (PDB code: 2 JGE)[14] and Torpedo californica ace-
tylcholinesterase (PDB code: 1E3Q)[15] were downloaded from protein
data bank. The results of sequence alignment by BLAST show there is
higher homologous property between Electrophorus electricus acetylcho-
linesterase and mouse acetylcholinesterase or Torpedo californica acetyl-
cholinesterase, the former identity up 100% _i540/540_jand the latter
similarity nearly 87.2% (471/540) (Table S3, Supporting Information).
There are three active sites (Tyr72, Tyr124, and Trp286 (underlined in
Table S3, Supporting Information) in mouse acetylcholinesterase in the
complex with organophosphorus compounds and four inhibitor binding
Acknowledgements
The work was co-financed by the key projects from the National Natural
Science Foundation (20432030 and 20533060) and the Ministry of Educa-
tion of China (104195).
[1] P. J. Whitehouse, D. L. Price, R. G. Struble, A. W. Clarke, J. T.
380
ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 376 – 381