Enzyme-promoted regioselective coupling oligomerization of isorhapontigenin towards the first synthesis of (±)-gnetulin

Article abstract of DOI:10.1039/c3ob42456a

We report the first synthesis of a natural (±)-gnetulin and an unnatural analogue of (±)-gnemonol M by using the regioselective oxidative coupling reactions of 5-tert-butyl-isorhapontigenin as the key step. Both the effects of different enzyme-catalyzed s

Full text of DOI:10.1039/c3ob42456a

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Enzyme-promoted regioselective coupling
oligomerization of isorhapontigenin towards the
first synthesis of ( )-gnetulin†
Cite this: Org. Biomol. Chem., 2014,
12, 2273
Wenling Li,* Shixia Yang, Teng Lv and Yadong Yang
We report the first synthesis of a natural ( )-gnetulin and an unnatural analogue of ( )-gnemonol M by
using the regioselective oxidative coupling reactions of 5-tert-butyl-isorhapontigenin as the key step.
Both the effects of different enzyme-catalyzed systems on the structures of coupling products and struc-
tural transformations of coupling products in the presence of several Lewis acids were systematically
investigated.
Received 10th December 2013,
Accepted 3rd February 2014
DOI: 10.1039/c3ob42456a
www.rsc.org/obc
1. Introduction
Natural oligostilbenes, a class of polyphenols derived from
resveratrol (1) and isorhapontigenin (7), have attracted con-
siderable interest due to their novel architectures and diverse
bioactivities over the past three decades (Fig. 1).1 Many syn-
thetic chemists work on the synthesis of these oligomers,
especially resveratrol dimers such as 2–6, either through bio-
transformation strategies or chemically controlled approaches
to intensively investigate their structure–activity relationship.2
However, little attention has been given to the isorhaponti-
genin-based oligomers like 8–12. These have the same con-
struction patterns as 2–6, but differ in OMe groups at the
aromatic rings and few synthetic efforts towards them have
been reported to date. This is because the traditional biomi-
metic synthesis strategy was not specific for the isorhaponti-
genin oligomers owing to the lack of regioselective control in
the direct oxidative coupling of 7, which led to diverse oligo-
meric structures.3 Secondly, a variety of well-designed cascade
reactions directed to the chemically controlled syntheses of
resveratrol oligomers, reported by Snyder’s group for example,4
were not readily applicable to the syntheses of isorhaponti-
genin oligomers, because the phenolic hydroxyl groups of all
the common building blocks were generally protected by
methyl ethers before the crucial oligomeric skeletons were con-
structed. Thus, it was a significant challenge to develop
effective synthetic routes for the preparation of various oligo-
meric isorhapontigenins. However, it was at present practical
Fig. 1 Some representative natural oligostilbenes.
to produce these oligomers by improving the stereo-selectivity
in the biomimetic synthesis methods.
In 2006, Hou and coworkers succeeded in the first synthesis
of ( )-quadrangularin A (3) by utilizing the enzyme-catalyzed
regioselective oxidative coupling reaction of 2,5-di-tert-butyl-
resveratrol as the key step.5 To explore the validity of Hou’s
strategy in the syntheses of oligostilbenes, our group followed
School of Chemical and Biological Engineering, Lanzhou Jiaotong University,
Lanzhou, 730070, China. E-mail: liwl@mail.lzjtu.cn; Fax: +86 931 4956512;
Tel: +86 931 7661282
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ob42456a
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it and prepared several dimeric stilbenes including 3, 5, 6, 11 reaction system. However, isomers 16a and 16b with a 2 : 1
and 12 in the presence of different metallic oxidants.6 We then ratio were isolated from the coupling products in 63% yield
turned to a representative isorhapontigenin dimer—( )-gnetu- when 13 was subjected to HRP with higher activity (≥300 units
lin (9). To the best of our knowledge, no attempt at the syn- mg−1) in the acetone–water (2 : 1) system. The dimers’ struc-
thesis of 9 has been reported so far even though it was first tures were unaffected, but their yields obviously decreased by
isolated from the Gnetum species in early 1993, later in 2000 changing the ratio of acetone–water from 2 : 1, 1 : 1 to 1 : 2.
and 2001.7 Herein the oxidative coupling reactions of 5-tert- Additionally, acetone was replaced by methanol to survey the
butyl-isorhapontigenin (13) using different enzyme catalysts solvent effects on the coupling reaction as shown in entry 3, in
were studied to synthesize ( )-gnetulin (9).
which similar dimers 17a and 17b were obtained correspond-
ingly in 32% yield. To our delight, the desired coupling inter-
mediate 14 was relatively stable and isolated in 48% yield
when the coupling reaction of 13 was conducted under slightly
acidic conditions (pH = 6). Apart from HRP, laccase was also
2. Results and discussion
Our synthetic route commenced with the preparation of the employed to catalyze the oxidative coupling of 13 in the
coupling precursor 13 according to our previous procedure.6b neutral environment, and diastereoisomers 16a and 16b with a
Two commercially available enzymes with different activity 1 : 1.5 ratio were afforded in lower yield than in the HRP–H2O2
grades were selected as the catalysts under variable solvent system.
conditions to conduct the single-electron-transfer mediated
With these coupling products in hands, we hoped to syn-
oxidative coupling reactions of 13. As illustrated in Scheme 1 thesize several natural oligomers including 9 and 10 through
and Table 1, stilbene 13 was first treated with horseradish per- the respective deprotection reactions of 14–17. Nevertheless,
oxidase (HRP) with lower activity (250 units mg−1) in the none of them succeeded under our standard debutylation con-
acetone–water system and the coupling reaction oxidized by ditions (6 equiv. AlCl3, 50 °C) due to the complicated products
H2O2 proceeded slowly. Surprisingly, the expected 8–8 coup- that resulted. Inspired by Hou’s work,5 the protonation
ling intermediate 14 was very unstable and only detected rearrangement of 14 by treating with activated neutral Al2O3 to
initially by thin layer chromatography (TLC). Two diastereoiso- afford the key precursor 18 was attempted, but failed unexpect-
meric trimers 15a and 15b were formed gradually as major edly after numerous trials. Species 14 was almost unchanged
products when dimer 14 was attacked by monomer 13 in the with a small quantity of Al2O3, while only trace amounts of 18
Scheme 1
Table 1 Treatment of 13 with different enzyme-catalyzed conditions
Entry Enzyme oxidants Solvent systems (volume ratio) Time (hour) Coupling products (molar ratio) Conversion (%) Recovered 13 (%)
a
1
2
3
4
5
HRP–H2O2 b
HRP–H2O2 b
HRP–H2O2 b
HRP–H2O2
Laccasec
Acetone–H2O (3 : 1)
Acetone–H2O (2 : 1)
Methanol–H2O (4 : 1)
Acetone–pH 6.0 buffer (2 : 1)
Acetone–H2O (2 : 1)
60
48
48
60
60
15a : 15b (1.5 : 1)
16a : 16b (2 : 1)
17a : 17b (1 : 1)
14
44
63
32
48
37
40
11
12
27
21
16a : 16b (1 : 1.5)
a RZ ≈ 3, activity = 250 units mg−1
.
b RZ > 3, activity ≥ 300 units mg−1 c From Trametes versicolor, activity ≥ 0.5 unit mg−1
. .
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reached the acceptable values (3 : 1 and 71%) when 0.5 equi-
valent of AlCl3 was used for 20 hours as shown in entry 8.
Finally, the debutylation reaction of 19 was conducted with
20 equivalents of AlCl3 at 50 °C for an hour and produced
the unnatural dimeric isorhapontigenin 20, an analogue of
gnemonol M (12), in 82% yield (Scheme 3). Crucially, a mixture
of the desired products ( )-gnetulin (9) and 20 (1 : 1) was obtained
in 55% yield in a one-pot reaction of 16a, in which 0.5 equi-
valent of AlCl3 was used first in toluene at room temperature
for 12 hours and 20 equivalents of AlCl3 were added at 60 °C
for another hour. The spectrum of 9 was in good agreement
with the values reported in the literature.7 The formation of 20
in this process can be explained as that the unreacted 16a at
the first stage underwent intramolecular Friedel–Crafts alkyl-
ation and subsequent debutylation at the second stage. It
should be mentioned that ( )-9 was difficult to purify from the
mixture of 9 and 20 using the conventional chromatographic
methods because of their totally identical polarity.
In addition, we also synthesized 17a or 17b through the
reaction of 16a or 16b with HBr in MeOH solution at room
temperature in 82%–86% yield, respectively (Scheme 4). The
unexpected dimer 21 was obtained in 84% yield when the reac-
tion mixture of 16a was heated to 40 °C accidently. Interest-
ingly, when 16a reacted with 2 equivalents of BF3·Et2O for
8 hours, a small quantity of 21 was isolated from the products
including 18 and 19. The existence of 21 should be attributable
Scheme 2
and unconverted 14 were detected in reaction mixtures if 14
was absorbed onto excess Al2O3. This was unfavorable for the
accumulation of 18. Thus, we had to revise our preliminary
strategy for the synthesis of 9. As depicted in Scheme 2 and
Table 2, several Lewis acids were selected to eliminate H2O or
MeOH from 16 or 17 to produce 18.
The initial attempt to convert 16 to 18 through the dehy-
dration with p-TsOH resulted in complex products. Then
BF3·Et2O was selected to catalyze the dehydration of 16a as
shown in entries 2 and 3.8 Surprisingly, no new spots appeared
on the TLC plates in these two reactions. However, the 1H
NMR spectrum of the recovered starting materials showed the
mixture of the desired 18 and the unreacted 16a (1 : 2), or the
bridged structure 19. The formation of 19 may be explained as
a result of further intramolecular Friedel–Crafts alkylation of
16a and 18, in which the electron-rich C-2′ underwent an elec-
trophilic attack by the carbocation at C-7 formed in the pres-
ence of an excess amount of BF3·Et2O. It is worth noting that
the dimers 16, 18 and 19 possessed the same polarity and were
difficult to distinguish on TLC, except on the 1H NMR spec-
trum. The treatment of 17 with BF3·Et2O (5 equiv.) also pro-
vided 19 as the single product in high yield.
Subsequently, 16a was subjected to 8 equivalents of AlCl3 at
room temperature to give the mixture of 18 and 16a as well as
a few big polar unidentified products. By decreasing the
amount of AlCl3 and prolonging the reaction time, the molar
ratio of 18 and 16a and their yields increased significantly and
Scheme 3
Table 2 Reactions of coupling products with several Lewis acids
Entry
Coupling products
Lewis acid (equiv.)
Solvent
Time (hour)
Products (molar ratio)
Isolated yield (%)
1
2
3
4
5
6
7
8
16a (or 16b)
16a
16a (or 16b)
17a (or 17b)
16a
16a
16a
p-TsOH (1)
BF3·Et2O (2)
BF3·Et2O (4)
BF3·Et2O (5)
AlCl3 (8)
AlCl3 (4)
AlCl3 (1.5)
AlCl3 (0.5)
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
Toluene
Toluene
Toluene
Toluene
0.5
1
1
1
1.5
2
Complex
—
27
90
92
15
22
43
71
18 : 16a (1 : 2)a
19
19
18 : 16a (1 : 3)a
18 : 16a (1 : 2)a
18 : 16a (2 : 1)a
18 : 16a (3 : 1)a
4
20
16a
a Determined by a crude 1H NMR spectrum.
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peroxidase (13 mg, RZ ≈ 3, activity = 250 units mg−1) in
acetone (24 mL) and water (8 mL) was treated with hydrogen
peroxide (3%, 10 mL) at room temperature under an argon
atmosphere and continuously stirred for 60 hours. Acetone
was removed and the aqueous reaction mixture was extracted
with EtOAc (60 mL), washed with saturated brine and then
dried over MgSO4. The solvent was removed under reduced
pressure and the residue was purified on a silica gel (CH2Cl2–
MeOH = 20 : 1) to give unchanged 13 (260 mg), and two
trimers 15a (90 mg, 26.5%) and 15b (60 mg, 17.6%).
15a: Rf = 0.25 (CH2Cl2–MeOH = 15 : 1); yellow amorphous
powder; 1H NMR (CD3COCD3, 400 MHz) δ: 1.36 (s, 9H), 1.38 (s,
9H), 1.44 (s, 9H), 3.52 (t, J = 3.0 Hz, 1H), 3.60 (dd, J = 8.1,
3.0 Hz, 1H), 3.71 (s, 3H), 3.76 (s, 3H), 3.87 (s, 3H), 3.84 (d, J =
3.0 Hz, 1H), 5.04 (d, J = 8.1 Hz, 1H), 5.69 (brs, 1H), 6.26 (brs,
5H), 6.32 (d, J = 1.8 Hz, 1H), 6.35 (brs, 1H), 6.48 (s, 1H), 6.60
(d, J = 1.2 Hz, 1H), 6.66 (d, J = 1.2 Hz, 1H), 6.68 (s, 1H), 6.77 (d,
J = 14.4 Hz, 1H), 6.82 (d, J = 14.4 Hz, 1H), 6.91 (s, 1H), 6.98 (d,
J = 1.8 Hz, 1H), 7.11 (s, 1H), 7.13 (s, 1H); 13C NMR (CD3COCD3,
100 MHz) δ: 30.5 (9C), 35.2 (3C), 56.4 (3C), 59.3, 61.4, 84.3,
101.3, 102.5, 103.9, 105.2, 106.1 (3C), 107.1, 107.2, 108.3,
109.4, 118.0, 119.4, 120.4, 123.0, 126.4, 128.7, 129.9, 130.4,
134.9, 135.2, 135.8, 136.7, 140.3, 143.6, 145.0, 145.6, 145.9,
147.9, 148.1, 148.4, 150.7, 155.0, 158.4, 159.1, 159.3 (3C),
160.6; HRMS (ESI): m/z calcd for [M − H]+: 939.4325, found:
939.4341.
15b: Rf = 0.24 (CH2Cl2–MeOH = 15 : 1); yellow amorphous
powder; 1H NMR (CD3COCD3, 400 MHz) δ: 1.31 (s, 9H), 1.33 (s,
9H), 1.43 (s, 9H), 2.89 (s, 1H), 3.50 (d, J = 8.4 Hz, 1H), 3.68 (s,
3H), 3.79 (s, 3H), 3.90 (s, 3H), 4.34 (s, 1H), 5.01 (d, J = 8.4 Hz,
1H), 5.84 (d, J = 1.8 Hz, 2H), 6.14 (t, J = 1.8 Hz, 1H), 6.26 (d, J =
1.5 Hz, 1H), 6.37 (d, J = 1.5 Hz, 1H), 6.49 (d, J = 1.8 Hz, 2H),
6.51(s, 2H), 6.56 (s, 1H), 6.63 (s, 1H), 6.80 (d, J = 16.8 Hz, 1H),
6.81 (d, J = 1.5 Hz, 1H), 6.86 (d, J = 16.8 Hz, 1H), 6.97 (s, 1H),
7.05 (s, 1H); 13C NMR (CD3COCD3, 100 MHz) δ: 29.8 (3C), 30.0
(6C), 35.2 (3C), 55.5, 56.4 (3C), 58.4, 61.3, 85.1, 101.0, 102.5,
104.0, 105.3, 105.5 (2C), 106.0, 107.0 (2C), 108.6, 109.3, 117.9,
119.2, 119.5, 122.1, 126.5, 128.7, 129.9, 131.3, 135.1, 135.4,
135.9, 136.8 (2C), 140.3, 143.9, 145.0, 145.6, 148.2, 148.1,
148.5, 149.0, 151.2, 155.1, 159.1 (3C), 160.9; HRMS (ESI): m/z
calcd for [M − H]+: 939.4325, found: 939.4315.
Scheme 4
to the further migration rearrangement of double bonds of 18
in an acidic environment, which clearly affirmed the instability
of 16 (or 18) under the acidic conditions.
3. Conclusions
In summary, the enzyme-catalyzed oxidative coupling reactions
of 5-tert-butyl-isorhapontigenin (13) in various solvents were
studied extensively and several 8–8 coupling oligomers includ-
ing 14–17 were prepared. The treatments of dimer 16 with
different Lewis acids led to 18 as the dehydrated product or 19
as the alkylated product, which depended on the type and the
amount of Lewis acid used. The first synthesis of ( )-gnetulin
(9) and unnatural dimeric isorhapontigenin (20) was success-
fully accomplished by the global removal of tert-butyl groups
in 18 and 19, respectively. Our synthetic strategy for this family
of interesting natural products is simple, rapid, and applicable
to the preparation of other analogues of 9, which is ongoing in
our laboratory.
4. Experimental
General methods
Method B: synthesis of coupling dimers 16a and 16b. (1) A
solution of compound 13 (0.43 g, 1.37 mmol) and horseradish
peroxidase (21 mg, RZ > 3, activity ≥ 300 units mg−1) in
acetone (12 mL) and water (6 mL) was treated with hydrogen
peroxide (3%, 4 mL) at room temperature under an argon
atmosphere and continuously stirred for 48 hours. Acetone
was removed and the aqueous reaction mixture was extracted
with EtOAc (50 mL), washed with saturated brine and then
dried over MgSO4. The solvent was removed under reduced
pressure and the residue was purified on a silica gel (25 : 1
CH2Cl2–MeOH) to give unchanged 13 (47 mg), and two dimers
16a (160 mg, 42%) and 16b (80 mg, 21%).
Structural determinations of the isolated compounds were
based on H, 13C NMR, NOESY, H–1H COSY, HMBC spectra,
and HRMS analysis. All NMR spectra were recorded on a
Varian Mercury 400 MHz instrument in a solvent as indicated.
HRMS spectra were measured on an Autostec-3090 mass
spectrometer. All solvents were freshly purified and dried by
standard techniques prior to use. Purification of products was
performed by column chromatography (CC) on silica gel
(200–300 mush), purchased from Qingdao Marine Chemical
Co. (Qingdao, China).
1
1
The enzyme-catalyzed coupling reaction of stilbene 13
(2) 24 mg laccase (from Trametes versicolor, activity ≥
Method A: synthesis of coupling trimers 15a and 15b. A 0.5 unit mg−1) was dissolved in water (2 mL) and added to the
solution of compound 13 (0.60 g, 1.9 mmol) and horseradish solution of compound 13 (0.10 g, 0.32 mmol) in acetone
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(4 mL) at room temperature under an argon atmosphere. The 8.17 (s, 1H); 13C NMR (CD3COCD3, 100 MHz) δ: 30.2 (6C), 35.0,
reaction mixture was continuously stirred for 60 hours. 35.2, 55.8, 56.3, 56.4, 56.7, 59.0, 60.8, 89.0, 101.0, 102.4, 105.6
Acetone was removed and the aqueous reaction mixture was (2C), 106.2, 108.9, 109.4, 118.2, 119.4, 122.1, 131.4, 134.8,
extracted with EtOAc (20 mL), washed with saturated brine and 135.3, 136.7, 143.7, 144.9, 147.9, 148.1, 149.7, 151.3, 155.0,
then dried over MgSO4. The solvent was removed under 159.0, 159.1 (2C); HRMS (ESI): m/z calcd for [M − H]+:
reduced pressure and the residue was purified on a silica gel 657.3069, found: 657.3075.
(CH2Cl2–MeOH = 25 : 1) to give unchanged 13 (21 mg), and
two minor trimers 16a (12 mg, 15%) and 16b (17 mg, 22%).
17b: Rf = 0.35 (CH2Cl2–MeOH = 15 : 1); yellow amorphous
powder; 1H NMR (CD3COCD3, 400 MHz) δ: 1.29 (s, 9H), 1.31 (s,
16a: Rf = 0.33 (CH2Cl2–MeOH = 15 : 1); brown amorphous 9H), 2.99 (s, 3H), 3.34 (dd, J = 8.0, 3.6 Hz, 1H), 3.41 (t, J =
powder; 1H NMR (CD3COCD3, 400 MHz) δ: 1.33 (s, 9H), 1.34 (s, 3.6 Hz, 1H), 3.75 (s, 3H), 3.78 (s, 3H), 3.95 (d, J = 8.0 Hz, 1H),
9H), 3.34 (dd, J = 8.0, 3.6 Hz, 1H), 3.60 (t, J = 3.6 Hz, 1H), 3.76 4.25 (d, J = 3.6 Hz, 1H), 5.69 (brs, 1H), 6.16 (d, J = 2.0 Hz, 2H),
(s, 3H), 3.77 (s, 3H), 4.18 (d, J = 4.4 Hz, 1H), 4.27 (d, J = 3.6 Hz, 6.18 (d, J = 2.0 Hz, 1H), 6.23 (d, J = 2.0 Hz, 1H), 6.53 (d, J =
1H), 4.47 (dd, J = 8.0, 4.4 Hz, 1H), 5.66 (d, J = 2.0 Hz, 1H), 6.16 2.0 Hz, 1H), 6.55 (d, J = 2.0 Hz, 1H), 6.64 (d, J = 2.0 Hz, 1H),
(d, J = 2.0 Hz, 2H), 6.17 (d, J = 2.0 Hz, 1H), 6.23 (t, J = 2.0 Hz, 6.77 (d, J = 2.0 Hz, 1H), 7.12 (s, 1H), 7.27 (s, 2H), 7.90 (s, 1H),
1H), 6.58 (d, J = 2.0 Hz, 2), 6.66 (d, J = 2.0 Hz, 1H), 6.88 (d, J = 8.07 (s, 2H); 13C NMR (CD3COCD3, 100 MHz) δ: 30.2 (6C), 35.1,
2.0 Hz, 1H), 7.15(s, 1H), 7.21 (s, 1H), 7.35 (s, 1H), 7.93 (s, 1H), 35.2, 56.3, 56.4, 56.6, 59.2, 60.5, 61.1, 88.0, 101.2, 102.2, 106.2,
8.10 (s, 2H); 13C NMR (CD3COCD3, 100 MHz) δ: 30.1 (6C), 34.8, 106.3 (2C), 108.5, 109.2, 118.5, 120.6, 122.8, 130.6, 134.8,
34.9, 56.0, 56.1, 56.3, 58.7, 62.4, 77.7, 100.7, 101.8, 105.7, 105.9 135.2, 136.6, 143.6, 145.0, 146.8, 147.9, 148.1, 150.7, 155.0,
(2C), 107.8, 108.9, 118.2, 119.1, 122.6, 134.2, 134.3, 134.8, 158.5, 159.3 (2C); HRMS (ESI): m/z calcd for [M − H]+:
136.6, 143.2, 144.2, 147.0, 147.4, 147.6, 151.2, 154.6, 158.1, 657.3069, found: 657.3065.
158.9 (2C); HRMS (ESI): m/z calcd for [M − H]+: 643.2913,
found: 643.2919.
Method D: synthesis of coupling dimer 14. Horseradish per-
oxidase (5.2 mg, RZ > 3, activity ≥ 300 units mg−1) was dissolved
16b: Rf = 0.32 (CH2Cl2–MeOH = 15 : 1); brown amorphous in buffer solution (pH = 6), to which a solution of compound 13
powder; 1H NMR (CD3COCD3, 400 MHz) δ: 1.28 (s, 9H), 1.33 (s, (50 mg, 0.16 mmol) in acetone (6 mL) was added at room temp-
9H), 2.81 (dd, J = 3.2, 2.8 Hz, 1H), 3.33 (dd, J = 8.8, 3.2 Hz, 1H), erature under an argon atmosphere. After stirring for 10 min
3.66 (s, 3H), 3.75 (s, 3H), 4.08 (d, J = 4.8 Hz, 1H), 4.24 (d, J = the mixture was treated with hydrogen peroxide (3%, 0.5 mL)
2.8 Hz, 1H), 4.50 (dd, J = 8.8, 4.8 Hz, 1H), 5.85 (d, J = 2.4 Hz, and continuously stirred for 60 hours. The reaction mixture was
2H), 6.16 (d, J = 2.0 Hz, 2H), 6.12 (t, J = 2.4 Hz, 1H), 6.34 (d, J = quenched with water and was extracted with EtOAc (20 mL),
2.4 Hz, 1H), 6.46 (d, J = 1.6 Hz, 1H), 6.49 (d, J = 2.0 Hz, 1H), washed with saturated brine and then dried over MgSO4. The
6.54 (d, J = 2.0 Hz, 1H), 6.56 (d, J = 2.0 Hz, 1H), 7.14 (s, 1H), solvent was removed under reduced pressure and the residue
7.16 (s, 1H), 7.49 (s, 1H), 8.02 (s, 2H), 8.10 (s, 1H); 13C NMR was purified on a silica gel (CH2Cl2–MeOH = 35 : 1) to give
(CD3COCD3, 100 MHz) δ: 29.6 (6C), 34.7, 34.9, 55.8, 56.0, 56.1, unchanged 13 (13 mg), and dimer 14 (18 mg, 48%).
59.1, 61.8, 88.1, 100.7, 102.0, 105.5 (2C), 106.0, 108.2, 108.9,
14: Rf = 0.46 (CH2Cl2–MeOH = 15 : 1); yellow amorphous
1
118.0, 118.3, 121.9, 134.6, 134.7, 135.0, 136.3, 143.4, 144.0, powder; H NMR (CD3COCD3, 400 MHz): δ: 1.30 (s, 9H), 1.34
147.3, 147.7, 149.2, 151.0, 154.7, 158.5, 158.8 (2C); HRMS (s, 9H), 3.11 (dd, J = 9.0, 8.4 Hz, 1H), 4.36 (d, J = 8.4 Hz, 1H),
(ESI): m/z calcd for [M − H]+: 643.2913, found: 643.2907.
4.63 (dd, J = 9.2, 9.0 Hz, 1H), 6.17 (brs, 1H), 6.22 (brs, 1H), 6.27
Method C: synthesis of coupling dimers 17a and 17b. A (brs, 2H), 6.31 (brs, 1H), 6.49 (d, J = 9.2 Hz, 1 H), 6.61 (brs,
solution of compound 13 (95 mg, 0.30 mmol) and horseradish 1 H), 6.63 (brs, 1 H), 6.64 (brs, 1 H), 7.08 (s, 1H), 7.19 (s, 1H),
peroxidase (8.9 mg, RZ > 3, activity ≥ 300 units mg−1) in 7.21 (s, 1H), 8.17 (s, 2H), 8.21 (s, 1 H); 13C NMR (CD3COCD3,
methanol (4 mL) and water (1 mL) was treated with hydrogen 100 MHz): δ: 30.4 (6 C), 34.8, 34.9, 56.0, 56.1, 58.7, 60.2, 62.4,
peroxide (3%, 3 mL) at room temperature under an argon 100.7, 101.9, 105.7, 105.9 (2 C), 106.0, 108.2, 108.9, 118.2,
atmosphere and stirred continuously for 48 hours. Acetone 119.1, 122.6, 134.2, 134.8, 136.6, 143.2, 144.3, 147.1, 149.8,
was removed and the aqueous reaction mixture was extracted 151.0, 153.6, 154.6, 156.9, 158.2, 159.0 (2C); HRMS (ESI): m/z
with EtOAc (20 mL), washed with saturated brine and then calcd for [M − H]+: 625.2796, found: 625.2787.
dried over MgSO4. The solvent was removed under reduced
Synthesis of 18. AlCl3 (4.6 mg, 0.035 mmol) was added to a
pressure and the residue was purified on a silica gel (CH2Cl2– solution of compound 16a (45 mg, 0.07 mmol) in dry toluene
MeOH = 25 : 1) to give unchanged 13 (11 mg), and two dimers (3 mL) at room temperature. The reaction mixture was stirred
17a (13.4 mg, 16%) and 17b (13.6 mg, 16%).
for 20 hours and then quenched with ice-water, and extracted
17a: Rf = 0.36 (CH2Cl2–MeOH = 15 : 1); yellow amorphous with EtOAc (20 mL). The combined organic layer was washed
powder; 1H NMR (CD3COCD3, 400 MHz) δ: 1.28 (s, 9H), 1.34 (s, with saturated brine and then dried over MgSO4. The solvent
9H), 2.69 (brs, 1H), 3.05 (s, 3H), 3.34 (d, J = 8.0 Hz, 1H), 3.66 was removed under reduced pressure and the residue purified
(s, 3H), 3.78 (s, 3H), 3.93 (d, J = 8.0 Hz, 1H), 4.25 (brs, 1H), on a silica gel (CH2Cl2–MeOH = 25 : 1) to afford the mixture of
5.77 (d, J = 2.0 Hz, 2H), 6.09 (d, J = 2.0 Hz, 1H), 6.32 (d, J = unreacted 16a and 18 (3 : 1, 71%).
2.0 Hz, 1H), 6.33 (d, J = 2.0 Hz, 1H), 6.36 (d, J = 2.0 Hz, 1H),
6.51 (d, J = 2.0 Hz, 1H), 6.56 (d, J = 2.0 Hz, 1H), 6.71 (d, J = powder; H NMR (CD3COCD3, 400 MHz): δ: 1.31 (s, 18H), 3.62
2.0 Hz, 1H), 7.24 (s, 1H), 7.31 (s, 1H), 7.71 (s, 1H), 8.05 (s, 2H), (s, 3H), 3.70 (s, 3H), 4.22 (brs, 1H), 4.29 (brs, 1H), 6.20 (d, J =
18: Rf = 0.33 (CH2Cl2–MeOH = 15 : 1); yellow amorphous
1
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2.0 Hz, 1H), 6.30 (d, J = 2.0 Hz, 1H), 6.35 (d, J = 2.0 Hz, 2H), and stirred for another 1 hour. The reaction was quenched
6.59 (d, J = 2.0 Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 6.81 (d, J = 2.0 with ice-water and extracted with EtOAc. The combined
Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 7.09 (s, 2H), 7.38 (brs, 1 H), organic layer was washed with saturated brine and then dried
7.89 (s, 1H), 8.21 (s, 2H), 8.26 (s, 1H); 13C NMR (CD3COCD3, over MgSO4. The solvent was removed under reduced pressure
100 MHz): δ: 30.4 (6C), 35.2 (2C), 56.3, 56.4, 58.1, 60.8, 98.5, and the residue purified on a silica gel (CH2Cl2–MeOH = 20 : 1)
101.4, 103.6, 106.5, 107.8, 108.7, 109.8, 118.1, 121.6, 122.4, to afford the mixture of ( )-9 and 20 (1 : 1, 55%) as a brown
123.9, 124.5, 128.9, 135.5, 135.6, 136.9, 142.8, 143.8, 144.8, amorphous powder.
147.4, 147.9, 149.2, 155.9, 159.6, 159.8 (2C); HRMS (ESI): m/z
( )-9: Rf
(CD3COCD3, 400 MHz): δ: 3.56 (s, 3H), 3.71 (s, 3H), 4.19 (brs,
= 0.28 (CH2Cl2–MeOH =
10 : 1); 1H NMR
calcd for [M − H]+: 625.2807, found: 625.2809.
Synthesis of 19. A solution of BF3·Et2O (4 equiv.) was added 1H), 4.27 (brs, 1H), 6.20 (t, J = 2.0 Hz, 1H), 6.30 (d, J = 2.0 Hz,
to a solution of compound 16a (45 mg, 0.06 mmol) in dry 1H), 6.33 (d, J = 2.0 Hz, 2H), 6.50 (dd, J = 8.0, 2.0 Hz, 1H), 6.64
dicholoromethane (5 mL). The reaction mixture was stirred for (d, J = 8.0 Hz, 1H), 6.69 (d, J = 8.0 Hz, 1H), 6.74 (d, J = 2.0 Hz,
1 hour at room temperature and then quenched with ice-water 1H), 6.78 (d, J = 2.0 Hz, 1H), 6.83 (dd, J = 8.0, 2.0 Hz, 1H), 6.89
(5 mL), and extracted with EtOAc. The combined organic layer (d, J = 2.0 Hz, 1H), 7.05 (s, 1H); 13C NMR (CD3COCD3,
was washed with saturated brine and then dried over MgSO4. 100 MHz): δ: 56.2 (2C), 58.0, 60.8, 98.5, 101.6, 103.9, 106.4
The solvent was removed under reduced pressure and the (2C), 112.2, 115.6 (2C), 120.1, 120.4, 123.3, 123.9, 124.6, 130.3,
residue purified on a silica gel (CH2Cl2–MeOH = 25 : 1) to 138.5, 142.9, 145.9, 146.7, 147.3, 148.1 (2C), 149.2, 156.0,
afford compound 19 (40 mg, 90%).
19: Rf = 0.33 (CH2Cl2–MeOH = 15 : 1); yellow amorphous 515.1700, found: 515.1705.
159.8, 159.9 (2C); HRMS (ESI): m/z calcd for [M + H]+:
1
powder; H NMR (CD3COCD3, 400 MHz): δ: 1.35 (s, 9H), 1.38
Synthesis of 17 from 16. Aqueous 40% HBr (2 mL) was
(s, 9H), 3.24 (s, 3H), 3.35 (brs, 1H), 3.72 (brs, 1H), 3.81 (s, 3H), added dropwise to the solution of 16a (or 16b) (90 mg,
4.23 (brs, 1H), 4.26 (brs, 1H), 6.00 (d, J = 2.0 Hz, 2H), 6.03 (d, 0.14 mmol) in methanol (12 mL). The reaction mixture was
J = 2.0 Hz, 2H), 6.47 (d, J = 2.0 Hz, 1H), 6.71 (d, J = 2.0 Hz, 1H), stirred continuously at room temperature for 2 hours and
6.83 (d, J = 2.0 Hz, 1H), 7.05 (s, 1H), 7.15 (s, 1 H), 7.22 (s, 1 H), quenched with aqueous NaHCO3. Methanol was removed and
7.88 (s, 1H), 7.89 (s, 1H), 7.99 (s, 2H); 13C NMR (CD3COCD3, the aqueous reaction mixture extracted with EtOAc (20 mL),
100 MHz): δ: 30.4 (6C), 35.1 (2C), 49.0, 52.0, 56.5, 57.7, 59.9, washed with saturated brine and then dried over MgSO4. The
60.5, 101.0, 101.9, 104.2, 105.8, 107.1 (2C), 109.8, 110.4, 119.5, solvent was removed under reduced pressure and the residue
119.6, 128.5, 135.0, 135.2, 136.5, 137.8, 143.5, 143.6, 147.2, purified on a silica gel (25 : 1 CH2Cl2–MeOH) to give 17a (or
147.9, 148.3, 152.8, 158.4, 158.8 (2C); HRMS (ESI): m/z calcd 17b) (82% or 86%) as a yellow amorphous powder.
for [M − H]+: 625.2796, found: 625.2789.
Synthesis of 21. Aqueous 40% HBr (6 mL) was added drop-
Synthesis of 20. AlCl3 (92 mg, 0.69 mmol) was added to a wise to the solution of 16a (88 mg, 0.14 mmol) in methanol
solution of compound 19 (24 mg, 0.04 mmol) in dry toluene (12 mL). The reaction mixture was stirred at 40 °C for 1 hour
(8 mL) at room temperature. The mixture was heated to 50 °C and quenched with aqueous NaHCO3. Methanol was removed
and stirred for 2 hours. The reaction was quenched with ice- and the aqueous reaction mixture extracted with EtOAc
water and extracted with EtOAc. The combined organic layer (20 mL), washed with saturated brine and then dried over
was washed with saturated brine and then dried over MgSO4. MgSO4. The solvent was removed under reduced pressure and
The solvent was removed under reduced pressure and the the residue purified on a silica gel (25 : 1 CH2Cl2–MeOH) to
residue purified on a silica gel (CH2Cl2–MeOH = 20 : 1) to give 21 (77 mg, 84%).
afford compound 20 (16 mg, 82%).
21: Rf = 0.40 (CH2Cl2–MeOH = 15 : 1); yellow amorphous
1
20: Rf = 0.28 (CH2Cl2–MeOH = 10 : 1); brown amorphous powder; H NMR (CD3COCD3, 400 MHz): δ: 1.20 (s, 9H), 1.34
1
powder; H NMR (CD3COCD3, 400 MHz): δ: 3.23 (s, 3H), 3.33 (s, 9H), 3.62 (s, 3H), 3.73 (s, 3H), 3.88 (s, 2H), 4.91 (s, 1H), 6.17
(brs, 1H), 3.71 (brs, 1H), 3.82 (s, 3H), 4.22 (brs, 1H), 4.29 (brs, (d, J = 2.0 Hz, 1H), 6.20 (d, J = 2.0 Hz, 1H), 6.35 (d, J = 2.0 Hz,
1H), 5.98 (d, J = 2.0 Hz, 2H), 6.03 (d, J = 2.0 Hz, 2H), 6.60 (dd, 1H), 6.37 (d, J = 2.0 Hz, 2H), 6.53 (d, J = 2.0 Hz, 1H), 6.75 (brs,
J = 8.0, 2.0 Hz, 1H), 6.68 (d, J = 8.0 Hz, 1H), 6.76 (d, J = 8.0 Hz, 2H), 6.86 (brs, 2H), 7.01 (s, 1H), 7.19 (s, 1 H), 7.69 (brs, 1 H),
1H), 6.93 (d, J = 8.0 Hz, 1H), 6.98 (d, J = 2.0 Hz, 1H), 7.45 (brs, 8.00 (brs, 1H), 8.20 (brs, 2H); 13C NMR (CD3COCD3, 100 MHz):
1H), 7.77 (brs, 1 H), 7.88 (brs, 1 H), 7.94 (brs, 1H), 8.03 (brs, δ: 30.2 (6C), 32.6, 35.0, 35.2, 56.4 (2C), 56.5, 100.9, 101.0,
2H); 13C NMR (CD3COCD3, 100 MHz): δ: 48.2, 48.7, 51.5, 56.3, 102.0, 108.5, 108.6, 109.4, 109.9, 110.6, 119.5 (2C), 120.3,
57.7, 59.2, 100.9, 101.8, 104.1, 106.9, 107.0, 113.0, 115.0, 115.1, 129.9, 130.6, 134.8, 135.6, 137.2, 139.6, 143.6, 143.8, 148.0,
115.2, 121.5, 128.5, 130.5, 139.6, 139.9, 145.6, 146.8, 147.0, 148.8, 150.1, 153.9, 158.8, 159.0 (2C); HRMS (ESI): m/z calcd
148.0, 149.4, 152.6, 152.7, 158.4, 158.8, 158.9; HRMS (ESI): m/z for [M − H]+: 625.2796, found: 625.2805.
calcd for [M + H]+: 515.1700, found: 515.1704.
Synthesis of ( )-gnetulin (9). AlCl3 (5 mg, 0.037 mmol) was
added to a solution of compound 16a (51 mg, 0.079 mmol) in
dry toluene (5 mL) and was stirred for 24 hours at room temp-
Acknowledgements
erature. Then AlCl3 (210 mg, 1.58 mmol) and CH3NO2 (0.5 mL) This research work was financially supported by the National
were added to the reaction mixture, which was heated to 60 °C Natural Science Foundation of China (no. 21062010) and
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Paper
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