molecules Article
Biomimetic Synthesis of Resveratrol Trimers Catalyzed by Horseradish Peroxidase Jian-Qiao Zhang 1 , Gan-Peng Li 2 , Yu-Long Kang 1 , Bin-Hao Teng 1 and Chun-Suo Yao 1, * 1
2
*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China;
[email protected] (J.-Q.Z.);
[email protected] (Y.-L.K.);
[email protected] (B.-H.T.) Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu University, Kunming 650500, China;
[email protected] Correspondence:
[email protected]; Tel.: +86-10-6021-2110; Fax: +86-10-6301-7757
Academic Editor: Derek J. McPhee Received: 31 March 2017; Accepted: 11 May 2017; Published: 17 May 2017
Abstract: Biotransformation of trans-resveratrol and synthetic (±)-ε-viniferin in aqueous acetone using horseradish peroxidase and hydrogen peroxide as oxidants resulted in the isolation of two new resveratrol trimers (3 and 4), one new resveratrol derivative (5) with a dihydrobenzofuran skeleton, together with two known stilbene trimers (6 and 7), and six known stilbene dimers (8–13). Their structures and relative configurations were identified through spectral analysis and possible formation mechanisms were also discussed. Among these oligomers, trimers 6 and 7 were obtained for the first time through direct transformation from resveratrol. Results indicated that this reaction is suitable for the preparation of resveratrol oligomers with a complex structure. Keywords: horseradish peroxidase; ε-viniferin; resveratrol trimer; biotransformation; radical reaction
1. Introduction Stilbenes are a class of plant polyphenols that can be divided into two categories, namely, monomeric and oligomeric stilbenes. Resveratrol oligomers possess novel structures and exhibit various biological activities, such as anticarcinogenesis, anti-inflammation, and tyrosinase activity inhibition. These oligomers can be used to treat cancer, AIDS, bacterial infections, and other diseases. Some oligostilbenes exhibit more potent bioactivities than their monomers do [1–3]. Various resveratrol oligomers exist in nature, especially in grapevine. However, the structures of minor stilbene oligomers have yet to be elucidated because sufficient amounts of these minor components are difficult to isolate for subsequent structural characterization. In recent years, some researchers focused their attention on the synthesis of these oligomers and total synthetic routes of numerous resveratrol oligomers, including dimers, trimers, and tetramers, have been reported in literature [4–10], but long reaction steps render these approaches unsuitable for specific preparations of the complex oligomers. Therefore, biomimetic synthesis is still a concise and practical alternative for the preparation of oligostilbenes with intricate structures. The natural biotransformation of stilbene oligomers in nature can be simulated in vitro by transformation with biological enzymes, unorganized fermentation, metal oxidants, light, acids and alkali [2,11]. In combination with diverse separation methods, transformation can induce the accumulation of large amounts of minor compounds. Using this approach, a number of known natural stilbene oligomers and new stilbene oligomers have been obtained, and their structures have also been identified successfully [11]. The oxidative coupling of resveratrol, including its analogs, has been examined under different conditions since ε-viniferin was first isolated in 1977 by Langcake and Pryce [12]. Nevertheless, the in vitro biocatalyzed oxidation of stilbenes has rarely been explored, and studies on biocatalyzed Molecules 2017, 22, 819; doi:10.3390/molecules22050819
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enzymes have focused on horseradish peroxidase (HRP) laccase [13,14], which indicated that peroxidase (HRP) and laccase [13,14], which indicated thatand dimers, such as dihydrobenzofuran-like dimers, such as dihydrobenzofuran-like dimers, are the main products of reactions by these dimers, are the main products of reactions mediated by these enzymes. As reportedmediated in the literature enzymes. As reportedwhich in the literature ε-viniferin, which would synthesized [12,15], ε-viniferin, would be[12,15], synthesized enzymatically in be plant tissues, enzymatically seems to be in a plant tissues, seems to be a biogenetically important precursor of many oligostilbenes in plants, suchB, as biogenetically important precursor of many oligostilbenes in plants, such as davidiol A, davidiol davidiol A, davidiol B, hopeaphenol, ampelopsin E, and so on.ofSo, biotransformation of ε-viniferin hopeaphenol, ampelopsin E, and so on. So, biotransformation ε-viniferin is a promising way to is a promising way to prepare these oligomers. Wilkens et al. [16] reported the transformation of prepare these oligomers. Wilkens et al. [16] reported the transformation of resveratrol and (−)-ε-viniferin resveratrol and ( − )-ε-viniferin catalyzed by HRP produces different stilbene oligomers, including catalyzed by HRP produces different stilbene oligomers, including dimers, trimers, tetramers, and dimers, trimers, tetramers, and polymeric products. In addition trimeric obtained polymeric products. In addition to two trimeric stilbenes obtainedtointwo a pure form,stilbenes other oligomers in a pure form, other oligomers only have been detected through HPLC-PDA or HPLC-ESI-MS/MS. only have been detected through HPLC-PDA or HPLC-ESI-MS/MS. Many resveratrol trimers and Many resveratrol trimers and tetramers can be possibly obtained by further this tetramers can be possibly obtained by further investigating this reaction. As aninvestigating extension of the reaction. Aswork, an extension of demonstrated the preliminary this study demonstrated the biotransformation preliminary this study thework, biotransformation of trans-resveratrol (1) and synthetic of trans-resveratrol (1) and synthetic ( ± )-ε-viniferin (2) in aqueous acetone with HRP and hydrogen (±)-ε-viniferin (2) in aqueous acetone with HRP and hydrogen peroxide as oxidants. Thus, two new peroxide as oxidants. Thus, two new resveratrol trimers (3 and 4), one new resveratrol resveratrol trimers (3 and 4), one new resveratrol derivative (5) with a dihydrobenzofuranderivative skeleton, (5) with awith dihydrobenzofuran skeleton, together two7),known natural and 7), together two known natural stilbene trimerswith (6 and and six knownstilbene naturaltrimers stilbene(6dimers and six known natural stilbene dimers (8–13) were isolated and structurally identified (Figure (8–13) were isolated and structurally identified (Figure 1). Among these substances, trimers 6 and 1). 7 Among these substances, trimers and 7 were obtained for the firstfrom time through direct transformation were obtained for the first time6 through direct transformation resveratrol. Moreover, their from resveratrol. Moreover, their were potential potential formation mechanisms also formation discussed. mechanisms were also discussed. OH
HO
4a
HO
HO
13b
4b
B1
H
1b
H
7b
10c
HO
O
9b
H
C2
11c
7a 11a
13 a
OH
4a
4b 7b 4c
OH
12a
C2 11c
4a
7a
11b
HO
Davidiol B (6)
O
13a
1b
3c
1c 4c
O
4b
OH HO
7b
HO
O
OH HO OH
9c
OH
OH
OH
11c
HO
HO
Resveratrol-cis-dehydrodimer (9)
Resveratrol-trans-dehydrodimer (8)
rel-(7aS ,8aS,7bS ,8bS )-cis-Diptoindonesin B (7) HO
HO
4a
4a
OH 1a
OH
7a
HO
OH
OH
HO
9b
9a 7b 10a
OH
11b
8a
13a 8b
OH
1a
HO
OH
HO
13b 1b
OH
OH
OH
4b
OH Leachianol G (10)
HO
13b
8a
13a
9b
8b 11a
OH
OH
HO
4a
H
9a
13a
11b
OH
1b
HO
13b
7a
1a
9b
7b
OH
H O
O 7a
10b
9b
H
7b
10a
1b
OH 4b
OH Leachianol F (11)
12a 11a
13c
4
13b 9a
13c
12c
H H
13a
7c 1c
H
11c
5
OH
HO
HO
8c 9c
Parthenostilbenin B (12)
Figure Figure 1. 1. Structures Structures of of compounds compounds 3–13. 3–13.
4b
OH
OH Ampelopsin B (13)
OH
H 9a
O
1a
11a
7b
OH
HO
3
9b 8b
H
HO
14a
OH 13c
1b
2b 6b
9a
H
1a
7a 8a
10b
3b
1b
A2
1b
O
4b
7a
O
11b
13b
4b
8a
10a
HO
OH
12b
HO
HO
3b
O
1a
6a
B1
C1
2a
A1
9c
OH
8c
HO
OH
4a
2a
9b
3c
1c
8c
4c
HO
9a
13b
13a
C1
OH
A2 HO
1c
13c
B2
1 1a
OH
7c
O 11b
1a
HO
OH
H A2
H H
8b 8c
A1
A1
1a
11b 7a 8a
B2
OH
3a
4a
OH 4c
OH
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2. 2. Results Results and and Discussion Discussion 2.1. Synthesis of 22 with with Resveratrol Resveratrol as as aa Starting Starting Material and HRP and Hydrogen Peroxide-Catalyzed Biotransformation of 1 and 2 In this study, ±)-ε-viniferin (2) (2) under under the the procedure procedure shown in Figure 2 was used study, semisynthetic semisynthetic ((±)-ε-viniferin as the starting material for biotransformation. According to the method reported in our previous paper [17], trans-resveratrol was subjected to an oxidative coupling reaction in aqueous methanol 6H22OOas as an an oxidant, oxidant, and and this this procedure procedure was was performed performed in combination with column using FeCl3··6H chromatography on silica gel. Thus, 2 with 13.5% yield was produced. HO HO
OH
FeCl3 6H2O
O
OH
HO
OH OH 1
OH 2
Figure 2. Semi-synthetic 2. Figure 2. Semi-synthetic route route of of compound compound 2.
Successively, biotransformation of 1 and 2 in aqueous acetone catalyzed by horseradish peroxidase Successively, biotransformation of 1 and 2 in aqueous acetone catalyzed by horseradish peroxidase and hydrogen peroxide generated a major product peak 8 and a complicated mixture (Figure S30 in and hydrogen peroxide generated a major product peak 8 and a complicated mixture (Figure S30 in Supplementary Materials), which resulted in the isolation and identification of four resveratrol trimers Supplementary Materials), which resulted in the isolation and identification of four resveratrol trimers (3, 4, 6, and 7, where 3 and 4 are new ones), one new resveratrol derivative 5, and six known dimers (3, 4, 6, and 7, where 3 and 4 are new ones), one new resveratrol derivative 5, and six known dimers (8–13) (Figure 1). Their structures and stereochemistry were elucidated by analyzing spectroscopic data. (8–13) (Figure 1). Their structures and stereochemistry were elucidated by analyzing spectroscopic data. 2.2. Products 2.2. Structure Structure Elucidation Elucidation of of Biotransformation Biotransformation Products Compound was obtained obtained as as aa brown brown amorphous amorphous powder. powder. The Thenegative negativeion ionpeak peakat atm/z m/z 697.2076 Compound 33 was 697.2076 − [M − H] (cacld. for C 42H33O10, 697.2079) in its HR-ESI-MS (Figure S7) corresponded to the molecular − [M − H] (cacld. for C42 H33 O10 , 697.2079) in its HR-ESI-MS (Figure S7) corresponded to the molecular formula of C C42H 34O10 and indicated that 3 could be a resveratrol trimer. The IR spectrum (Figure S9) formula of 42 H34 O10 and indicated that 3 could be a resveratrol trimer. The IR spectrum (Figure S9) = 3395 and 3187 cm−1) cm and−1aromatic rings (νmax = 1600, 1513, and revealed the presence (νmax revealed the presenceofofhydroxyls hydroxyls (νmax = 3395 and 3187 ) and aromatic rings (νmax = 1600, −1 1469 cm Absorption were also observed λmaxobserved (log ε) 203at(5.14), and 283 (3.92) nm in 1513, and). 1469 cm−1 ).bands Absorption bands wereatalso λmax231 (log(4.66), ε) 203 (5.14), 231 (4.66), 1H-NMR spectrum (Figure S1, Table 1) showed three sets of A2B2 the UV spectrum (Figure S8). The 1 and 283 (3.92) nm in the UV spectrum (Figure S8). The H-NMR spectrum (Figure S1, Table 1) showed systems rings A1, B1, and C1 at δH of 6.83 (2H, d, J = 8.4 Hz, H-3(5)a), 7.24 (2H, d, J = 8.4 Hz, three setsfor of A 2 B2 systems for rings A1, B1, and C1 at δH of 6.83 (2H, d, J = 8.4 Hz, H-3(5)a), 7.24 (2H, d, H-2(6)a), (2H, d,6.54 J = 8.4 (2H, d,6.34 J = 8.4 Hz, 6.68 (2H, d,6.68 J = 8.4 Hz, J = 8.4 Hz,6.54 H-2(6)a), (2H,Hz, d, H-3(5)b), J = 8.4 Hz,6.34 H-3(5)b), (2H, d, H-2(6)b); J = 8.4 Hz, H-2(6)b); (2H, d, H-3(5)c), and 6.85 (2H, d, J = 8.4 Hz, H-2(6)c); one set of AB 2 system for ring A2 at δH of 6.36 (1H, t, J = J = 8.4 Hz, H-3(5)c), and 6.85 (2H, d, J = 8.4 Hz, H-2(6)c); one set of AB2 system for ring A2 at δH of 2.4 and 6.20 (2H, and d, J =6.20 2.4 (2H, Hz, H-10(14)a); sets of meta-coupled aromatic protons for 6.36Hz, (1H,H-12a) t, J = 2.4 Hz, H-12a) d, J = 2.4 Hz,two H-10(14)a); two sets of meta-coupled aromatic rings B2 and C2 at δ H of 5.98 (1H, d, J = 2.4 Hz, H-14b) and 6.19 (1H, d, J = 2.4 Hz, H-12b), and δH 5.17 protons for rings B2 and C2 at δH of 5.98 (1H, d, J = 2.4 Hz, H-14b) and 6.19 (1H, d, J = 2.4 Hz, H-12b), (1H, J = 1.8 Hz, H-14c) and 6.24 (1H, d, J = 1.8 Hz, H-12c). The 1H-NMR spectrum also displayed 1 and δd, H 5.17 (1H, d, J = 1.8 Hz, H-14c) and 6.24 (1H, d, J = 1.8 Hz, H-12c). The H-NMR spectrum the of the twopresence mutuallyofcoupled benzyl coupled methine benzyl protonsmethine at δH of 5.33 (1H,atd,δJ =of 3.05.33 Hz,(1H, H-7a) alsopresence displayed two mutually protons d, H and 4.83 (1H, d, J = 3.0 Hz, H-8a) and a sequence of successively coupled benzyl methine protons at δH J = 3.0 Hz, H-7a) and 4.83 (1H, d, J = 3.0 Hz, H-8a) and a sequence of successively coupled benzyl of 4.30 (1H, brs, H-7b), 3.76 (1H, brs, H-8b), 4.02 (1H, dd,brs, J = H-8b), 4.2, 3.0 4.02 Hz, (1H, H-7c),dd, and m, H-8c). methine protons at δH of 4.30 (1H, brs, H-7b), 3.76 (1H, J =2.96 4.2,(1H, 3.0 Hz, H-7c), Moreover, them, HSQC spectrum (Figure S4) supplied the complete assignment of all protonated carbon, and 2.96 (1H, H-8c). Moreover, the HSQC spectrum (Figure S4) supplied the complete assignment as shown in Table 1. Theas13shown C-NMR (Figure S2, Table 1) of(Figure 3 revealed the presence of six 13 C-NMR of all protonated carbon, in spectrum Table 1. The spectrum S2, Table 1) of 3 revealed 54.45, 65.07, 77.10, 54.73, and 93.7877.10, ppm and 36and aromatic aliphatic carbons at aliphatic δC of 52.64, the presence of six carbons at δC of 52.64, 54.45, 65.07, 54.73, 93.78 carbons. ppm andThe 36 aliphatic carbon at δ C of 77.10 ppm was due to an alcohol carbon. The carbon signals at δC of 54.73 and aromatic carbons. The aliphatic carbon at δC of 77.10 ppm was due to an alcohol carbon. The carbon 93.78 ppm proton signals at δH of 5.33 and 4.83 suggested the presence of dihydroxybenzofuran ring. signals at δand C of 54.73 and 93.78 ppm and proton signals at δH of 5.33 and 4.83 suggested the presence The three remaining aliphatic and three carbon atomsprotons indicated presence an indane ring. of dihydroxybenzofuran ring.protons The three remaining aliphatic andthe three carbonofatoms indicated This group of evidence demonstrated that 3 possessed a skeleton similar to that of davidiol B [18,19]. In addition, compared with those of davidiol B, the downfield shifts of H-2(6)a, H-7a, H-8a, H-10(14)a,
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the presence of an indane ring. This group of evidence demonstrated that 3 possessed a skeleton similar to that of davidiol B [18,19]. In addition, compared with those of davidiol B, the downfield shifts of H-2(6)a, H-7a, H-8a, H-10(14)a, H-8b, and H-2(6)c in 3 (∆δH +0.24, +0.28, +1.72, +0.29, +0.95, and +0.50 ppm) caused by the anisotropic effect of the aromatic ring suggested that 3 could be a 7c-empimer of davidiol B [20]. In the HMBC spectrum (Figure S5 and Figure 3a) of 3, the correlations of H-2c, H-6c, H-14c, and C-7c, which were attached to the hydroxyl group, showed that C-7c was excluded from the additional ring, and ring C1 was attached at C-7c. The correlations between H-7b, H-8b, H-5b, and C-1b verified that ring B1 was connected at C-7b. Moreover, the correlations between H-7a and C-10(14)a and H-8a and C-2(6)a substantiated that ring A1 was linked at C-7a, and ring A2 was linked at C-8a. Comparison of the spectral data with those of davidiol B and the analysis of DEPT, HMBC, and HSQC (Figures S3~S5) correlations determined the planar structure of 3 (Figure 1). Table 1. 1 H- and 13 C-NMR spectroscopic data of compounds 3–5 *. No. 1a 2(6)a 3(5)a 4a 7a 8a 9a 10a 11a 12a 13a 14a 1b 2b 3b 4b 5b 6b 7b 8b 9b 10b 11b 12b 13b 14b 1c 2c 3c 4c 5c 6c 7c 8c 9c 10c 11c 12c 13c 14c
3 134.69s 128.25d 116.04d 158.11s 93.78d 54.73d 149.28s 107.13d 159.93s 101.82d 159.93s 107.13d 137.41s 129.35d 115.48d 155.85s 115.48d 129.35d 52.64d 54.45d 147.43s 119.71s 161.56s 95.45d 159.93s 104.82d 136.42s 129.22d 115.33d 157.34s 115.33d 129.22d 77.10d 65.07d 148.34s 123.66s 154.66s 102.22d 158.38s 106.66d
4
7.24 (d, 8.4) 6.83 (d, 8.4) 5.33 (d, 3.0) 4.83 (d, 3.0) 6.20 (d, 2.4) 6.36 (t, 2.4) 6.20 (d, 2.4) 6.34 (d, 8.4) 6.54 (d, 8.4) 6.54 (d, 8.4) 6.34 (d, 8.4) 4.30 (s, 1H) 3.76 (s, 1H)
6.19 (d, 2.4) 5.98 (d, 2.4) 6.85 (d, 8.4) 6.68(d, 8.4) 6.68 (d, 8.4) 6.85 (d, 8.4) 4.02 (dd, 4.2, 3.0) 2.96 (m)
6.24 (d, 1.8) 5.17 (d, 1.8)
133.85s 128.80d 116.36d 158.58s 95.00d 57.8d 147.28s 107.27d 159.56s 101.90d 159.56s 107.27d 131.87s 128.53d 116.30d 158.69s 116.30d 128.53d 94.86dd 58.78d 145.04s 120.40s 162.74s 96.83d 159.48s 109.40d 131.51s 127.36d 132.74s 160.43s 109.85d 130.09d 131.70d 127.04d 137.54s 107.71d 159.79s 102.54d 159.79s 107.71d
5
7.00 (d, 9.0) 6.80 (d, 9.0) 5.22 (d, 6.1) 3.85 (d, 6.1). 5.94 (d, 2.4) 6.09 (t, 2.4) 5.94 (d,2.4) 7.14 (d, 8.4) 6.75 (d, 8.4) 6.75 (d, 8.4) 7.14 (d, 8.4) 5.30 (d, 8.4) 4.30 (d, 8.4)
131.98s 127.59d 115.14d 157.36s 92.53d 57.66d 144.29s 106.51d 158.79s 101.34d 158.79 106.51d 153.02s 131.30s 112.10d 151.57s 109.08d 114.69d
7.20 (d, 8.5) 6.82 (d, 8.5) 5.34 (d, 8.3) 4.36 (d, 8.3) 6.16 (d, 2.1) 6.25 (t, 2.1) 6.16 (d, 2.1)
6.47 (d, 2.2) 6.67 (brs) 6.67 (brs)
6.23 (d, 2.4) 6.25 (d, 2.4) 6.76 (d, 2.4)
6.65 (d, 8.4) 6.98 (dd, 8.4, 2.4) 6.21 (d, 12.0) 6.05 (d, 12.0) 6.03 (d, 2.4) 6.17 (t, 2.4) 6.03 (d, 2.4)
* Data (δH ) were measured in MeOD for 1 H-NMR at 600 MHz and for 13 C-NMR at 150 MHz. The assignments were based on DEPT, 1 H-1 H COSY, HSQC, HMBC, and NOESY experiments, respectively.
attached to the hydroxyl group, showed that C-7c was excluded from the additional ring, and ring C1 was attached at C-7c. The correlations between H-7b, H-8b, H-5b, and C-1b verified that ring B1 was connected at C-7b. Moreover, the correlations between H-7a and C-10(14)a and H-8a and C-2(6)a substantiated that ring A1 was linked at C-7a, and ring A2 was linked at C-8a. Comparison of the spectral data22, with Molecules 2017, 819 those of davidiol B and the analysis of DEPT, HMBC, and HSQC (Figures S3~S5) 5 of 12 correlations determined the planar structure of 3 (Figure 1). OH HO
HO
O H
B2 B1
H
C2
HO
HO
O
OH
H
OH
H
H HO
A1
OH
H
OH
H
A2
H
H
H OH
HO
H OH
H
OH
C1 OH
OH
a
OH
b
OH
Figure 3. Important (b) interactions interactions of of 3. 3. Figure 3. Important HMBC HMBC (a) (a) and and NOESY NOESY (b)
The stereochemistry of 3 was determined by analyzing the NOESY spectrum (Figures S6 and 3b). The stereochemistry of 3 was determined by analyzing the NOESY spectrum (Figure S6 and The interactions among H-2(6)b and H-7b, H-8b suggested a trans orientation of rings B1 and B2. The Figure 3b). The interactions among H-2(6)b and H-7b, H-8b suggested a trans orientation of rings NOE interactions among H-7c and H-2(6)c, H-2(6)b suggested a cis orientation of rings C1 and B1. B1 and B2. The NOE interactions among H-7c and H-2(6)c, H-2(6)b suggested a cis orientation of The NOE interactions between H-8a and H-2(6)a and between H-7a and H-10(14)a indicated a trans rings C1 and B1. The NOE interactions between H-8a and H-2(6)a and between H-7a and H-10(14)a orientation between rings A1 and A2. Furthermore, the NOE interactions among H-14c and H-2(6)c, indicated a trans orientation between rings A1 and A2. Furthermore, the NOE interactions among H-8c revealed that ring C1 could be located near ring C2. The downfield shift of H-14c [6.68–6.65 (m, H-14c and H-2(6)c, H-8c revealed that ring C1 could be located near ring C2. The downfield shift 1H)] and upfield shift of H-8b [2.81 (s, 1H)] in 3 compared with those of davidiol B further substantiated of H-14c (6.68–6.65 (m, 1H)) and upfield shift of H-8b (2.81 (s, 1H)) in 3 compared with those of this observation. Therefore, the structure of 3 was determined as shown in Figure 1. davidiol B further substantiated this observation. Therefore, the structure of 3 was determined as Compound 4 was obtained as a light brown amorphous powder. Its corresponding negative shown in Figure 1. HR-ESI-MS (Figure S4) m/z 679.1973 [M − H]− (cacld. for C42H31O9, 679.1968) showed a molecular Compound 4 was obtained as a light brown amorphous powder. Its corresponding negative formula of C42H31O9, which implied that 4 could be a resveratrol trimer. The UV (λmax = 203.4 (4.99), HR-ESI-MS (Figure S4) m/z 679.1973 [M − H]− (cacld. for C42 H31 O9 , 679.1968) showed a molecular 228 (4.69), and 281 (4.22) nm) and IR (νmax = 3335, 1604, 1516, 1487, 1449, 1004, and 836 cm−1) spectra formula of C H31 O9 , which implied that 4 could be a resveratrol trimer. The UV (λmax = 203.4 (4.99), (Figures S18 42 and S19) of 4 displayed the presence of a phenolic oligostilbene containing a−cis olefinic 228 (4.69), and 281 (4.22)13nm) and IR (νmax = 3335, 1604, 1516, 1487, 1449, 1004, and 836 cm 1 ) spectra 1 bond [21]. The H- and C-NMR spectral data (Figures S10 and S11, Table 1) of 4, along with 1H-1H (Figures S18 and S19) of 4 displayed the presence of a phenolic oligostilbene containing a cis olefinic COSY, DEPT, HSQC, and HMBC spectra (Figures S12~S14 and S16), revealed resonances attributable to bond [21]. The 1 H- and 13 C-NMR spectral data (Figures S10 and S11, Table 1) of 4, along with 1 H-1 H two 4-hydroxyphenyl groups (rings A1 and B1), two 3,5-dihydroxyphenyl groups (rings A2 and C2), a COSY, DEPT, HSQC, and HMBC spectra (Figures S12~S14 and S16), revealed resonances attributable 3,5-dihydroxy-1,2-disubstituted benzene ring (ring B2), and a 4-hydroxy-1,3-disubstituted benzene ring to two 4-hydroxyphenyl groups (rings A1 and B1), two 3,5-dihydroxyphenyl groups (rings A2 and (ring C1). The 1H-NMR spectral data also suggested the presence of two sets of aliphatic signals at δH C2), a 3,5-dihydroxy-1,2-disubstituted benzene ring (ring B2), and a 4-hydroxy-1,3-disubstituted of 5.22 (1H, d, J = 6.1 Hz, H-7a) and 3.85 (1H, d, J = 6.1 Hz, H-8a). These signals are characteristic of a benzene ring (ring C1). The 1 H-NMR spectral data also suggested the presence of two sets of aliphatic 2,3-diaryldihydrobenzofuran moiety, except a cis-1,2-disubstituted olefinic bond at δH 6.21 (1H, d, J = signals at δH of 5.22 (1H, d, J = 6.1 Hz, H-7a) and 3.85 (1H, d, J = 6.1 Hz, H-8a). These signals are 12.0 Hz, H-7c) and 6.05 (1H, d, J = 12.0 Hz, H-8c). Our comparison revealed that the NMR spectroscopic characteristic of a 2,3-diaryldihydrobenzofuran moiety, except a cis-1,2-disubstituted olefinic bond at data of 4 were remarkably similar to those of cis-diptoindonesin B reported in the literature [21], which δH 6.21 (1H, d, J = 12.0 Hz, H-7c) and 6.05 (1H, d, J = 12.0 Hz, H-8c). Our comparison revealed that the suggested that 4 possessed the same planar structure as cis-diptoindonesin B. In the HMBC spectrum NMR spectroscopic data of 4 were remarkably similar to those of cis-diptoindonesin B reported in the (Figures S14 and 4a) of 4, the significant correlations of H-7a/C-2(6)a, C-9a, and C-11b; H-8a/C-10(14)a, literature [21], which suggested that 4 possessed the same planar structure as cis-diptoindonesin B. C-1a, and C-9b; H-7b/C-2(6)b, and C-9b; and H-8b/C-1b, C-2c, and C-4c; H-7c/C-5c; H-8c/C-2(6)c, in In the HMBC spectrum (Figure S14 and Figure 4a) of 4, the significant correlations of H-7a/C-2(6)a, combination with their shifts, further supported the planar structure of 4 as shown in Figure 1. C-9a, and C-11b; H-8a/C-10(14)a, C-1a, and C-9b; H-7b/C-2(6)b, and C-9b; and H-8b/C-1b, C-2c, The relative stereochemistry of H-7a/H-8a and H-7b/H-8b was determined by analyzing the and C-4c; H-7c/C-5c; H-8c/C-2(6)c, in combination with their shifts, further supported the planar NOESY spectrum of 4 (Figures S15 and 4b). The NOE interactions among H-7a and H-2(6)a, H-10(14)a structure of 4 as shown in Figure 1. and those among H-8a and H-2(6)a, H-10(14a) suggested a trans orientation between H-7a and H-8a. The relative stereochemistry of H-7a/H-8a and H-7b/H-8b was determined by analyzing the Similarly, the NOE interactions between H-8b and H-2(6)b also suggested a trans relationship of NOESY spectrum of 4 (Figure S15 and Figure 4b). The NOE interactions among H-7a and H-2(6)a, H-7b and H-8b. Despite that 4 exhibited a planar structure similar to that of cis-diptoindonesin B, H-10(14)a and those among H-8a and H-2(6)a, H-10(14a) suggested a trans orientation between H-7a their difference indicated that 4 could be a stereoisomer of cis-diptoindonesin B. The comparison of and H-8a. Similarly, the NOE interactions between H-8b and H-2(6)b also suggested a trans relationship the NMR data of 4 with cis-diptoindonesin B demonstrated that H-8a, H-8c, and H-10(14)c and C-8b, of H-7b and H-8b. Despite that 4 exhibited a planar structure similar to that of cis-diptoindonesin B, their difference indicated that 4 could be a stereoisomer of cis-diptoindonesin B. The comparison of the NMR data of 4 with cis-diptoindonesin B demonstrated that H-8a, H-8c, and H-10(14)c and C-8b, C-9b, C-8c, and C-9c in 4 were shifted by ∆δH of +0.36, −0.24, −0.19 and ∆δC of +3.3, +4.7, −2.5, and −3.5 ppm, respectively. This evidence demonstrated that the relative configurations of C-7b and C-8b in 4 could be 7bR and 8bR instead of 7bS and 8bS in cis-diptoindonesin B, respectively [21]. In contrast to the relative configuration of rel-(7aS, 8aS, 7bS, and 8bS) in cis-diptoindonesin B, the relative configurations of 4 for C-7a, C-8a, C-7b, and C-8b were determined as rel-(7aS, 8aS, 7bR, and 8bR), respectively
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(Figure 1). This conjecture was confirmed by the fact that H-8a and H-2(6)b were deshielded because of anisotropic effects induced by rings B2 and C1. On the contrary, H-7c, H-8c, and H-10(14)c were shielded due to anisotropic effects elicited by rings B2. Molecules 2017, 22, 819 6 of 11 Molecules 2017, 22, 819
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HO HO
A1 A1
HO HO
B1 B1
A2 A2 HO HO
C2 C2 HO HO
HO HO
O O OH OH
B2 B2
C1 C1
H H O O OH OH
HO HO OH OH
HO HO
H H H H
OH OH O H O H
O O OH OH
OH OH
aa
HO HO
b b
Figure 4. Significant Significant HMBC (a) (a) and NOESY NOESY (b) correlations correlations of compound compound 4. Figure Figure 4. 4. Significant HMBC HMBC (a) and and NOESY (b) (b) correlations of of compound 4. 4.
Compound 5 was obtained as brown amorphous powder. The The corresponding corresponding positive ion Compound 5 was obtained as brown amorphous powder. The corresponding positive ion + (cacld. for for C20H H17 17O5, 337.1071) provided the HR-ESI-MS (Figure (Figure S27) S27) peak peak at at m/z m/z 337.1073 337.1073 [M [M ++ H] H] (cacld. HR-ESI-MS (Figure S27) peak at m/z 337.1073 [M + H]++ (cacld. for CC2020H 337.1071) provided the 17O55,, 337.1071) molecular formula of C C20 20H16O5. The IR spectrum (Figure S29) displayed the presence of hydroxyls O 5 . The IR spectrum (Figure S29) displayed the presence of hydroxyls hydroxyls molecular formula of C20H16 O . The IR spectrum (Figure S29) displayed 16 5 −1) and aromatic rings (1615, 1517, and 1464 cm−1). The 1H-NMR spectrum (Figure S20, Table 1) (3337 cm cm−1 − 1 − 1 1 −1 1 aromatic rings (1615, 1517, and and 14641464 cm ).cm The ).H-NMR spectrum (Figure S20, Table 1) (3337 cm ) and ) and aromatic rings (1615, 1517, The H-NMR spectrum (Figure S20, showed one one A2B2 A2B2 system system at δδHH of of 7.20 (2H, (2H, d, d, JJ == 8.5 8.5 Hz, Hz, H-3(5)a) H-3(5)a) and 6.82 6.82 (2H, d, d, J = 8.5 8.5 Hz, Hz, H-3(5)a), H-3(5)a), showed Table 1) showed one A2B2at system 7.20 at δH of 7.20 (2H, d, J = 8.5 Hz, and H-3(5)a) (2H, and 6.82J =(2H, d, J = 8.5 Hz, one AB2 AB2 system system at at δδHH of of 6.25 6.25 (1H, (1H, t, JJ == 2.1 2.1 Hz, H-12a) H-12a) and and 6.16 6.16 (2H, d, d, JJ == 2.1 2.1 Hz, H-10(14)a), H-10(14)a), one one ABX ABX one H-3(5)a), one AB2 system at δH of t, 6.25 (1H,Hz, t, J = 2.1 Hz, H-12a)(2H, and 6.16 (2H, Hz, d, J = 2.1 Hz, H-10(14)a), system at δ H of 6.47 (1H, d, J = 2.2 Hz, H-3b) and 6.67 (2H, brs, H-5b, H-6b), and two mutually coupled system δH of 6.47 d, J =(1H, 2.2 Hz, H-3b) andH-3b) 6.67 (2H, brs, (2H, H-5b,brs, H-6b), and two and mutually coupled one ABXatsystem at δ(1H, d, J = 2.2 Hz, and 6.67 H-5b, H-6b), two mutually H of 6.47 benzyl methine methine protons protons at at δδHH of of 5.34 5.34 (1H, (1H, d, d, JJ == 8.3 8.3 Hz, Hz, H-7a) and and 4.36 (1H, (1H, d, d, JJ == 8.3 8.3 Hz, H-7a). H-7a). In In benzyl coupled benzyl methine protons at δH of 5.34 (1H, d, J = 8.3H-7a) Hz, H-7a)4.36 and 4.36 (1H, d, J = Hz, 8.3 Hz, H-7a). 13C-NMR (Figure S21, Table 1), together addition, the carbon signals at δ C of 54.66 and 92.53 ppm in 13 addition, thethe carbon signals atatδCδC ofof54.66 C-NMR In addition, carbon signals 54.66and and92.53 92.53ppm ppminin 13 C-NMR(Figure (FigureS21, S21, Table Table 1), together with the the proton proton signals signals at at δδHH of of 5.34 5.34 and and 4.36 4.36 ppm, ppm, suggested suggested the the presence presence of of dihydroxybenzofuran dihydroxybenzofuran with the proton signals at δH of 5.34 and 4.36 ppm, suggested the presence of dihydroxybenzofuran ring. Accordingly, 55 was was identified as as a resveratrol derivative derivative with benzofuran benzofuran skeleton, skeleton, as as shown shown in in ring. Accordingly, Accordingly, 5 was identified identified asaaresveratrol resveratrol derivativewith with benzofuran skeleton, as shown Figure 1, which was confirmed by DEPT, HSQC, HMBC, COSY, and UV spectra (Figures S22~S26, Figure 1, 1, which was confirmed bybyDEPT, S22~S26, in Figure which was confirmed DEPT,HSQC, HSQC,HMBC, HMBC,COSY, COSY,and and UV UV spectra spectra (Figures (Figures S22~S26 and S28). S28). In In HMBC HMBC spectrum spectrum (Figures (Figures S24 S24 and and 5a), 5a), the the correlations correlations between between H-7a H-7a and and C-2(6)a, C-2(6)a, C-9a, C-9a, and S28). In HMBC spectrum (Figure S24 and Figure 5a), the correlations between H-7a and C-2(6)a, C-2b and H-8a and C-10(14)a, C-1a, C-6b suggested that ring A1 could be linked at C-7a, and ring A2 C-2b and and C-10(14)a, C-1a,C-1a, C-6bC-6b suggested that that ring ring A1 could be linked at C-7a, andand ringring A2 C-9a, C-2bH-8a and H-8a and C-10(14)a, suggested A1 could be linked at C-7a, could be linked at C-8a. In NOESY experiment (Figures S25 and 5b), the NOE enhancements between could be linked at C-8a. In NOESY experiment (Figures S25 and 5b), the NOE enhancements between A2 could be linked at C-8a. In NOESY experiment (Figure S25 and Figure 5b), the NOE enhancements H-7a and and H-2(6)a/H-10(14)a H-2(6)a/H-10(14)a and and H-8a H-8a and and H-10(14)a/H-2(6)a H-10(14)a/H-2(6)a suggested suggested aa trans trans orientation orientation of of rings rings H-7a between H-7a and H-2(6)a/H-10(14)a and H-8a and H-10(14)a/H-2(6)a suggested a trans orientation A1 and A2. Therefore, the structure of 5 was determined as shown in Figure 1. A1rings and A2. Therefore, the structure of 5 wasofdetermined as shown Figure of A1 and A2. Therefore, the structure 5 was determined asin shown in1.Figure 1.
HO HO
HO HO
O O OH OH
HO HO
HO HO OH OH a a
A1 A1
O O
OH OH
A2 A2 OH OH b b
Figure 5. Significant HMBC (a) and NOESY (b) correlations of compound 5. Figure 5. 5. Significant HMBC (a) (a) and and NOESY NOESY (b) (b) correlations correlations of of compound compound 5. 5. Figure Significant HMBC
In addition addition to to these these three three compounds, compounds, two two known known resveratrol resveratrol trimers, trimers, namely, namely, davidiol davidiol B B (6) (6) [18] [18] In In addition to these three compounds, two known resveratrol trimers, namely, davidiol B (6) [18] and rel-(7aS,8aS,7bS,8bS)-cis-diptoindonesin B (7) [21], and six known dimers, namely, (±)-resveratroland rel-(7aS,8aS,7bS,8bS)-cis-diptoindonesin B (7) [21], and six known dimers, namely, (±)-resveratroland rel-(7aS,8aS,7bS,8bS)-cis-diptoindonesin B (7) [21], and six known dimers,leachianol namely, (±G)-resveratroltrans-dehydrodimer (8) [22,23], [22,23], (±)-resveratrol-cis-dehydrodimer (±)-resveratrol-cis-dehydrodimer (9) [23,24], [23,24], (10) [25,26], [25,26], trans-dehydrodimer (8) (9) leachianol G (10) trans-dehydrodimer (8) [22,23], ( ± )-resveratrol-cis-dehydrodimer (9) [23,24], leachianol G (10) [25,26], leachianol F F (11) (11) [25,26], [25,26], parthenostilbenin parthenostilbenin B B (12) (12) [20], [20], and and ampelopsin ampelopsin B B (13) (13) [27,28], [27,28], were were identified identified leachianol by comparing comparing their their physical physical and and spectroscopic spectroscopic data data with with those those reported reported in in previous previous studies. studies. Among Among by them, trimers trimers 66 and and 7, 7, which which are are difficult difficult to to obtain obtain by by common common organic organic reactions, reactions, were were obtained obtained for for them, the first time by direct biotransformation from resveratrol. the first time by direct biotransformation from resveratrol.
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leachianol F (11) [25,26], parthenostilbenin B (12) [20], and ampelopsin B (13) [27,28], were identified by comparing their physical and spectroscopic data with those reported in previous studies. Among them,2017, trimers 6 and 7, which are difficult to obtain by common organic reactions, were obtained 7for Molecules 22, 819 of 11 the first time by direct biotransformation from resveratrol. 2017, 22, 819 Reaction Mechanisms 2.3Molecules Probable Coupling
2.3. Probable Coupling Molecules 2017, 22, 819
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Reaction Mechanisms
the obtained results, a mechanism on how different trimeric derivatives formed were 2.3Considering Probable Coupling Considering the Reaction obtainedMechanisms results, a mechanism on how different trimeric derivatives formed were 2.3 Probable Coupling Reaction Mechanisms proposed. HRP-catalyzed biotransformation isispresumed on basis of ofradical radicalreaction reaction[13,14,29]. [13,14,29]. proposed. HRP-catalyzed biotransformation presumed on the the basis Considering the peroxide, obtained results, a mechanism on how different trimeric derivativesand formed were Induced by hydrogen resveratrol, and (±)-ε-viniferin were dehydrogenated rearranged Induced by hydrogen peroxide, resveratrol, and ( ± )-ε-viniferin were dehydrogenated and rearranged Considering the obtained results, a mechanism on how different trimeric derivatives formed were proposed. HRP-catalyzed biotransformation is presumed on the basis of radical reaction [13,14,29]. to proposed. form different radicals (Figure 6). Afterward, these HRP-catalyzed radicals were combined to produce HRP-catalyzed biotransformation is presumed on the basis of radical reaction [13,14,29]. to form different radicals (Figure 6). Afterward, these HRP-catalyzed radicals were combined to Induced by hydrogen peroxide, resveratrol, and (±)-ε-viniferin were dehydrogenated and rearranged different dimers and trimers. The coupling of one radical Dwere and one radical A,and andsubsequent subsequent Induced by hydrogen peroxide, resveratrol, and (±)-ε-viniferin dehydrogenated rearranged produce different dimers and trimers. The coupling of one radical D and one radical A, to form different radicals (Figure 6). Afterward, these HRP-catalyzed radicals were combined to produce tautomeric rearrangement and nucleophilic attackto tothe theintermediate intermediate quinone yielded to form different radicals (Figure 6). Afterward, these were combined to produce tautomeric rearrangement andintramolecular intramolecular nucleophilic quinone yielded different dimers and trimers. The coupling of oneHRP-catalyzed radicalattack D andradicals one radical A, and subsequent thedifferent dihydrofuran trimers 4 4and 77(Figure 7). Consequently, the formation and can be easily dimers and trimers. The coupling one radical Dthe and one radical A, and subsequent the dihydrofuran trimers and (Figure 7).of Consequently, formation ofof33and 6 6can be easily tautomeric rearrangement and intramolecular nucleophilic attack to the intermediate quinone yielded explained by the coupling of one radical D with one radical B and subsequent addition of a water tautomeric rearrangement and intramolecular nucleophilic attack to the intermediate quinone yielded explained by the coupling of one radical D with radical B and subsequent addition of a water the dihydrofuran trimers 4 and 7 (Figure 7). Consequently, the formation of 3 and 6 can be easily molecule to to the intermediate quinone (Figure 8). Furthermore, may beformed formed through the the dihydrofuran trimers 4of and (Figure Consequently, formation of 3through and 6 can be easily molecule intermediate quinone (Figure 8). Furthermore, 5B5 may be the explained bythe the coupling one7 radical D7).with one radical the and subsequent addition ofoxidation aoxidation water explained by the coupling of one radical D with one radical B and subsequent addition of a water of (±)-ε-viniferin by O and HRP, but an appropriate account for the reactivity cannot be established of molecule (±)-ε-viniferin by H 2H O 2 and HRP, but an appropriate account the reactivity cannot be established 2 2 to the intermediate quinone (Figure 8). Furthermore, 5 may be formed through the oxidation molecule to the intermediate quinone (Figure 8). Furthermore, 5 may be formed through the oxidation with thisevidence. evidence. In addition, the of of thethe dimers obtained in this are theare with this the formation formationmechanisms mechanisms dimers obtained in work this work of (±)-ε-viniferin byIn Haddition, 2O2 and HRP, but an appropriate account for the reactivity cannot be established of (±)-ε-viniferin by H 2Oin 2 and HRP, but an appropriate account for the reactivity cannot be established same as those reported our previous paper [26,30,31]. thewith same as those reported in our previous paper [26,30,31]. this evidence. In addition, the formation mechanisms of the dimers obtained in this work are withsame this as evidence. In addition, formation mechanisms the those reported in ourthe previous paper [26,30,31]. of the dimers obtained in this work are HO HO the same as those reported in our OH previous paper [26,30,31]. HO HO OH HO HO
OH
HO
OH
HO
OH
OH O HO HRP/H 2 2
OH
HO
OH
HO
OH
HO
OH
HO
OH
HO HO
O
O
HO
OH 1
OH
O
OH
O
OH
O
HO HOHO HO HO
HO
HO
radical "A" radicalO"B" O radical "A" radical radical O "A" O "B" radical radical "A""A" radical "B" radical intermediates radical "A"
OH
O
HRP/H 2O2 OH HO HRP/H2O2 HO HRP/H2O2
O
HO HO
HRP/H2O2
1
O
O
HO
HRP/H2O2
OH
HO HO HO
O
radical O "D"
radical "C" O
OH
OH
HO
O
radicalO"C"
OH
O
HO
HO
OH
OH
HO
HO
2 2 2
OH
O
O radical "D"
1 Figure 6. Plausible from 1 and 2 by horseradish peroxidase and hydrogen radical "C" radical "D" peroxide. Figure 6. Plausible radical intermediates from 1 and 2 by horseradish peroxidase and hydrogen peroxide. Figure 6. Plausible radical intermediates from 1 and 2 by horseradish peroxidase and hydrogen peroxide. Figure 6. Plausible radical intermediates from 1 and 2 by horseradish peroxidase and hydrogen peroxide. HO HO O
HO
HO
O
HO HO
OH O
OH
O
OH
HO
HO
O
O
HO HO
O
OH OH
HO
HO HO
HO
OH
HO HO
O
radical "D" O radical "D" O radical "D"
HO
O
HO
OH OH
O
HO HO HO
HO HO
HO radical "A" OH radical "A" radical "A"
OH OH OH
H H H
O O
O O
HO HO
O
HO
HOHO
O
HO
O
HO
HO
O
OH
OH OH
O
HO
OH
HO
HO
OH OH
HO
OH
HO
OH OH
O
OH
O
O OH OH HO OH HO compounds 4 and 7 HO compounds 4 and 7
O
HO
HO
O O
O
HO HO
OH OH
HO HO HO
O
HO
O
HO
HO
compounds 4 and 7
Figure forcompounds compounds4 4and and7.7. Figure7.7.Proposed Proposedformation formation mechanisms mechanisms for Figure 7. 7. Proposed Proposed formation formation mechanisms mechanisms for for compounds compounds 44 and and 7. 7. Figure HO
HO HO
HO HO
O O
HO HO
O
OHOHHO HO OH HO
HO
OO
OH OH
OH
HO HO
O
OH
HO
HO
OH OH
HH HH OO H H O HO HO HO
OH
O O O
radical radical "D""D" radical "D"
OO O"B" radical"B" radical radical "B"
HO
HO
HO
HO
OH OH
OO
HO HO
O
HO
O O O
HO
OO
OH OH
HO HO
O
OH
HO
HO HO
OH
HO HO HO
OH
HO
HH H OO
HO HO
HO HO
HO
HO
O
HO HOHO OHOH HO OH
O O
OH OH
O
OH
HOHO
HOHOHO
OH OH OH
HO HOHO
OH OH
HO HO
OH
HOcompounds HO 3 and 6 3 and compounds
6
compounds 3 and 6
Figure8.8.Proposed Proposedformation formation mechanisms mechanisms for Figure forcompounds compounds3 3and and6.6. Figure 8. Proposed formation mechanisms for compounds 3 and 6. Figure 8. Proposed formation mechanisms for compounds 3 and 6.
3. Materials andMethods Methods 3. Materials and 3. Materials and Methods Materials andInstrumentation Instrumentation 3.1.3.1. Materials and 3.1. Materials and Instrumentation Optical rotations were measured on P2000 polarimeter (JASCO, Tokyo, Japan). UV spectra were Optical rotations were P2000 (JASCO,Tokyo, Tokyo, Japan).UV UV spectra were Optical weremeasured measuredon on(JASCO). P2000 polarimeter polarimeter were obtained on arotations JASCOP650 spectrometer IR spectra(JASCO, were recorded Japan). on a Nicoletspectra 5700 FT-IR obtained onon a JASCOP650 spectrometer (JASCO). IR spectra spectra were were recordedon ona aNicolet Nicolet 5700 FT-IR obtained a JASCOP650 spectrometer (JASCO). IR 5700 FT-IR microscope instrument (FT-IR microscope transmission, Thermo recorded Electron Corporation, Madison, microscope instrument (FT-IR microscope transmission, Thermo Electron Corporation, Madison, microscope (FT-IR microscope transmission, Thermo Electron Madison, 1H andCorporation, WI, USA). 1Dinstrument and 2D NMR spectra were acquired at 500 or 600 MHz for 125 or 150 MHz for 13C,13 1H and 125 or 150 MHz for WI,WI, USA). 1D1D and 2D NMR spectra were acquired at 500 500 or or 600 600MHz MHzfor for1H USA). and 2D NMR spectra were acquired and 125 or 150 MHz for 13C, C, respectively, on Varian INOVA 500 MHz, or Bruker AVANCE III HD 600 MHz (Bruker Corporation, respectively, on Varian INOVA 500 MHz, or Bruker AVANCE III HD 600 MHz (Bruker Corporation, respectively,Germany), on Varianin INOVA 5006 MHz, or Bruker4, AVANCE III HD 600 as MHz (Bruker Corporation, Karlsruhe, acetone-d or methanol-d with solvent peaks references. ESI-MS and
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3. Materials and Methods 3.1. Materials and Instrumentation Optical rotations were measured on P2000 polarimeter (JASCO, Tokyo, Japan). UV spectra were obtained on a JASCOP650 spectrometer (JASCO). IR spectra were recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR microscope transmission, Thermo Electron Corporation, Madison, WI, USA). 1D and 2D NMR spectra were acquired at 500 or 600 MHz for 1 H and 125 or 150 MHz for 13 C, respectively, on Varian INOVA 500 MHz, or Bruker AVANCE III HD 600 MHz (Bruker Corporation, Karlsruhe, Germany), in acetone-d6 or methanol-d4 , with solvent peaks as references. ESI-MS and HR-ESI-MS data were measured using an AccuToFCS JMST100CS spectrometer (Agilent Technologies, Ltd., Santa Clara, CA, USA). Column chromatography (CC) was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China). HPLC separation was performed on an instrument consisting of a Waters 515 pump and a Waters 2487 dual λ absorbance detector (Waters Corporation, Milford, MA, USA) with a YMC semi-preparative column (250 × 10 mm i.d.) packed with C18 (5 µM). TLC was carried out with glass precoated silica gel GF254 plates (Qingdao Marine Chemical, Inc., Qingdao, China). Spots were visualized under UV light or by spraying with 7% H2 SO4 in 95% EtOH followed by heating. 3.2. Synthesis of Compoud 2 The solution of FeCl3 ·6H2 O (380 g, 1.43 mol) in H2 O (100 mL) was added to a solution of 1 (300 g, 1.32 mol) in methanol (500 mL) under stirring at room temperature, and the mixture was stirred for 60 h at room temperature. After removing of methanol in vacuo, water was added to the mixture, and the mixture was extracted with EtOAc. Subsequently, the obtained organic layer was washed with brine and water, dried over anhydrous Na2 SO4 for 24 h, then concentrated in vacuo to give a residue, which was further chromatographed on silica gel column with CHCl3 –MeOH (15:1, v/v) as eluent to provide unreacted resveratrol 130 g and product 2 (23 g, yield 13.5%). Compound 2: grey amorphous powder. 1 H-NMR(CD3 COCD3 , 500 MHz) δ: 8.41 (OH), 8.38 (OH), 8.33 (OH), 8.17 (2H, 2 × OH), 7.20 (2H, d, J = 9.0 Hz, H-2a, 6a), 7.16 (2H, d, J = 8.5 H, H-2b, 6b), 6.90 (1H, d, J = 16.0 Hz, H-7b), 6.82 (2H, d, J = 9.0 Hz, H-3a, 5a), 6.73 (2H, d, J = 8.5 Hz, H-3b, 5b), 6.72 (2H, d, J = 2.0 Hz, H-10a, 14a), 6.70 (1H, d, J = 16.0 Hz, H-8b), 6.32 (1H, d, J = 2.0 Hz, H-12b), 6.23 (2H, br s, H-12a, 14b), 5.41 (1H, d, J = 5.5 Hz, H-7a), 4.47 (1H, d, J = 5.5 Hz, H-8a); (+)-ESI m/z: 477 [M + Na]+ . 3.3. Treatment of 1 and 2 with Horseradish Peroxidase/Hydrogen Peroxide To a mixed solution of 1 (3000 mg, 13.2 mmol) and 2 (1500 mg, 3.3 mmol) in acetone (240 mL), 50 mL of water was added under stirring at room temperature. After that, a solution of HRP (10.0 mg) in water (30 mL) was added slowly. The reactant was stirred for 5 min, 30% H2 O2 (1.4 mL) was then added. The reactant was stirred for another 7 h at room temperature. Finally, the reaction mixture was suspended in water, and extracted with ethyl acetate. The organic layer was dried on anhydrous Na2 SO4 for 24 h, and concentrated in vacuo to yield a residue. The residue was subjected to column chromatography over ODS, eluting with a gradient of increasing methanol in water (15~100%) to provide nine fractions (R-I~R-IX) on the basis of HPLC analysis. R-VI (2.5 g) was further fractionated via silica gel column chromatography (CC), eluting with CHCl3 –MeOH (17:1, v/v) to yield 8 (0.493 g, 16.5%) and R-VI-2~R-VI-4. R-VI-3 was then separated by semi-preparative Rp-HPLC (column, Rp-18, 250 × 10 mm, 5 µm) eluted using methanol/water (56:44, v/v) to afford 9 (2 mg, 0.07%); 30 mg R-I (total 280 mg) was separated by semi-preparative Rp-HPLC using a mobile phase of methanol/water (16:84, v/v) to afford 10 (9 mg, 2.7%) and 11 (4 mg, 1.2%). R-II (30 mg) was further purified by semi-preparative Rp-HPLC eluted with a mobile phase of MeOH–H2 O (30:70, v/v) to afford 5 (1.7 mg). 300 mg R-III (total amount 600 mg) was
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resolved by Rp-MPLC with a gradient of increasing MeOH (18~30%) in water to give R-III-1~R-III-3. Among them, Fraction R-III-2 (34 mg) were subsequently separated by Rp-HPLC with a mobile phase of methanol/water (40:60, v/v) to afford 6 (5 mg, 0.43%) and 3 (3 mg, 0.26%); R-III-1 and R-III-3 were dealt with the same manner to provide 13 (13 mg, 0.87%), and 12 (7 mg, 0.44%), respectively. Furthermore, R-VII (600 mg) was subjected to Rp-MPLC eluting with methanol–water (49:51, v/v) to yield R-VII-1 and R-VII-2; subsequently, R-VII-1 was purified by semi-preparative Rp-HPLC using a mobile phase of methanol–water (46:54, v/v) to provide 7 (2 mg); R-VII-2 was dealt with methanol–water (54:46, v/v) by semi-preparative Rp-HPLC in the same manner to give 4 (6 mg). Compound 3: Brown amorphous powder. UV (MeOH) λmax (log ε): 203 (5.14), 231.2 (4.66), 283 (3.92) nm; IR (film) νmax : 3395, 3187, 2921, 2850, 1646, 1513, 1469, 1420, 1342, 1245, 1120, 1005, 834, 722, 648 cm−1 ; 1 H-NMR (600 MHz, MeOD) and 13 C-NMR (150 MHz, MeOD), see Table 1; (+)-ESI m/z: 721.0 [M + Na]+ , 736.9 [M + K]+ ; (−)-ESI m/z: 696.9 [M − H]− , 732.9 [M + Cl]− ; HR-ESI-MS m/z: 697.2076, [M − H]− (cacld. for C42 H33 O10 , 697.2079). Compound 4: Light brown amorphous powder. UV (MeOH) λmax (log ε): 203.4 (4.99), 228 (4.69), 281 (4.22) nm; IR (film) νmax : 3335, 2924, 2852, 1604, 1516, 1487, 1449, 1349, 1241, 1169, 1004, 836, 694 cm−1 ; 1 H-NMR (600 MHz, MeOD) and 13 C-NMR (150 MHz, MeOD), see Table 1; (+)-ESI m/z: 703.3 [M + Na]+ ; (−)-ESI m/z: 678.9 [M − H]− , 715.8 [M + Cl]− ; HR-ESI-MS m/z: 679.1973, [M − H]− (cacld. for C42 H31 O9 , 679.1968). Compound 5: Brown amorphous powder. UV (MeOH) λmax (log ε): 203.4 (4.73), 229.8 (4.29), 283 (3.70) nm; IR (film) νmax : 3337, 2921, 2852, 1615, 1517, 1488, 1464, 1351, 1235, 1198, 1157, 1105, 1000, 836, 778, 746, 691 cm−1 ; 1 H-NMR (600 MHz, acetone-d6 ) and 13 C-NMR (150 MHz, acetone-d6 ), see Table 1; HR-ESI-MS m/z: 337.1073 [M + H]+ (cacld. for C20 H17 O5 , 337.1071). Davidiol B (6): Brown amorphous powder. 1 H-NMR (600 MHz, acetone-d6 ), δ: 7.00 (2H, d, J = 8.5 Hz, H-2(6)a), 6.87 (2H, d, J = 8.5 tHz, H-3(5)a), 6.68–6.65 (1H, m, H-14c), 6.53 (2H, d, J = 8.6 Hz, H-3(5)b), 6.45 (2H, d, J = 8.5 Hz, H-3(5)c), 6.38 (1H, d, J = 2.1 Hz, H-12c), 6.35 (2H, d, J = 8.4 Hz, H-2(6)c), 6.31 (1H, t, J = 2.2 Hz, H-12a), 6.16 (1H, d, J = 2.1 Hz, H-12b), 6.13 (2H, d, J = 8.4 Hz, H-2(6)b), 5.99 (1H, d, J = 2.1 Hz, H-14b), 5.91 (2H, d, J = 2.0 Hz, H-10(14)a), 5.05 (1H, d, J = 2.3 Hz, H-7a), 4.10 (1H, s, H-7b), 3.95 (1H, d, J = 10.1 Hz, H-7c), 3.11 (1H, d, J = 2.4 Hz, H-8a), 2.90 (1H, d, J = 10.1 Hz, H-8c), 2.81 (1H, s, H-8b). 13 C-NMR (150 MHz, acetone-d6 ), δ: 161.74s (C-11b), 159.94s (C-11(13)a), 159.83s (C-13b), 158.71s (C-13c), 157.90s (C-4a), 157.48s (C-4c), 155.84s (C-4b), 154.98s (11c), 150.15s (C-9c), 148.81s (C-9a), 148.01s (C-9b), 136.55s (C-1b), 136.18s (C-1c), 134.35s (C-1a), 129.72d (C-2(6)b), 128.66d (C-2(6)c), 127.57d (C-2(6)a), 122.69s (C-10c), 118.50s (C-10b), 116.41d (C-3(5)c), 116.02d (C-3(5)a), 115.18d (C-3(5)b), 107.41d (C-14c), 106.90d (C-10(14)a), 104.52d (C-14b), 102.11d (C-12c), 101.70d (C-12a), 63.97d (C-8c), 54.57d (C-8b), 54.46d (C-8a), 53.24d (C-7b). (+)-ESI m/z: 721.0 [M + Na]+ , 736.9 [M + K]+ ; (−)-ESI m/z: 696.9 [M − H]− , 732.9 [M + Cl]− . cis-Diptoindonesin B (7): Brown amorphous powder. 1 H-NMR (600 MHz, methanol-d4 ), δ: 7.09 (1H, d, J = 8.6 Hz, H-6c), 6.99 (2H, d, J = 8.6 Hz, H-2(6)b(, 6.92 (2H, d, J = 8.5 Hz, H-2(6)a), 6.75 (2H, d, J = 8.6 Hz, H-3(5)a), 6.68 (2H, d, J = 8.5 Hz, H-3(5)b), 6.63 (1H, brs, H-2c), 6.60 (1H, d, J = 8.3 Hz, H-5c), 6.37 (1H, d, J = 12.2 Hz, H-7c), 6.31 (1H, d, J = 12.2 Hz, H-8c), 6.25 (1H, d, J = 2.1 Hz, H-12b), 6.22 (2H, d, J = 2.2 Hz, H-10(14)c), 6.19 (1H, d, J = 2.1 Hz, H-14b), 6.12 (1H, t, J = 2.2 Hz, H-12c), 6.10 (1H, t, J = 2.2 Hz, H-12a), 5.85 (2H, d, J = 2.2 Hz, H-10(14)a), 5.15 (1H, d, J = 10.0 Hz, H-7b), 5.14 (1H, d, J = 5.2 Hz, H-7a), 4.29 (1H, d, J = 10.0 Hz, H-8b), 3.49 (1H, d, J = 5.2 Hz, H-8a). 13 C-NMR (150 MHz, methanol-d4 ), δ: 162.61s (C-11b), 160.55s (C-4c), 160.25s (C-13b), 159.86s (C-11(13)a), 159.12s (C-11(13)c), 158.81s (C-4b), 158.41s (C-4a), 147.58s (C-9a), 140.93s (C-9c), 140.29s (C-9b), 133.87s (C-1a), 131.92s (C-1b), 131.73s (C-1c), 131.12s (C-3c), 130.32d (C-6c), 129.53d (C-8c), 128.94d (C-2(6)b), 128.17d (C-2(6)a), 127.23d (C-2c), 122.03s (C-10b), 116.36d (C-3(5)b), 116.28d (C-3(5)a), 109.68d (C-5c), 108.53d (C-10(14)c), 108.13d (C-14b), 107.09d (C-10(14)a), 102.56d (C-12c), 102.18d (C-12a), 96.83d (C-12b), 94.99d (C-7b),
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94.55d (C-7a), 56.84d (C-8a), 55.52d (C-8b). (+)-ESI-MS m/z: 681.0 [M + H]+ , 703.0 [M + Na]+ ; (−)-ESI m/z: 678.9 [M − H]− , 714.9 [M + Cl]− . Ampelopsin B (13): Brown amorphous powder. 1 H-NMR (500 MHz, methanol-d4 ), δ: 7.02 (2H, d, J = 8.6 Hz, H-2(6)b), 6.89 (2H, d, J = 7.9 Hz, H-2(6)a), 6.69 (2H, d, J = 8.6 Hz, H-3(5)b), 6.60 (2H, d, J = 8.6 Hz, H-3(5)a), 6.30 (1H, d, J = 2.2 Hz, H-12b), 6.27 (1H, d, J = 1.9 Hz, H-14a), 6.09 (1H, d, J = 1.8 Hz, H-14b), 6.01 (1H, d, J = 2.0 Hz, H-12a), 5.65 (1H, d, J = 11.4 Hz, H-7b), 5.14 (1H, t, J = 3.8 Hz, H-7a), 4.05 (1H, d, J = 11.4 Hz, H-8b), 3.53 (1H, dd, J = 17.4, 4.1 Hz, H-8aα), 3.17 (1H, brd, J = 17.4 Hz, H-8aβ). (+)-ESI m/z: 455.0 [M + H]+ , 477.0 [M + Na]+ ; (−)-ESI m/z: 453.0 [M − H]− , 489.0 [M + Cl]− . 4. Conclusions The HRP-catalyzed biotransformation of 1 and 2 produced various resveratrol stilbene oligomers, including dimers, trimers, and tetramers. In this reaction mixture, four resveratrol trimers (3, 4, 6, and 7), one new resveratrol derivative (5) with a dihydrofuran skeleton, and six dimers (8–13) were isolated and identified. Among these compounds, 3 and 4 were newly identified in our study. The raceme nature of the dimers was indicated by the zero values of their optical rotations, and this finding suggested that a radical mechanism was involved in HRP-catalyzed biotransformation. Our study favored the enzymatic biotransformation of stilbenes by HRP as a prominent method to produce oligomeric stilbenes for research activity. Considering that these new compounds may occur naturally as minor constituents, we observed that our reference data provided a basis for the detection of the presence of these stilbene oligomers in future investigations. Oligostilbenes were reported to show various activities [1,2]. Therefore, these products should be further examined, and results will be reported in our future research. Supplementary Materials: The following are available online. Figures S1~S9: 1 H-NMR, 13 C-NMR, DEPT, HSQC, HMBC, NOESY, HRESIMS, UV, and IR spectra in CD3 COCD3 of 3. Figures S10~S19: 1 H-NMR, 13 C-NMR, DEPT, HSQC, HMBC, NOESY, COSY, HRESIMS, UV, and IR spectra in CD3 COCD3 of 4. Figures S20~S29: 1 H-NMR, 13 C-NMR, DEPT, HSQC, HMBC, NOESY, COSY, HRESIMS, UV, and IR spectra in CD COCD of 5. Figure S30: 3 3 HPLC chromatogram of biotransformation products of 1 and 2. Acknowledgments: This study was conducted with grants from the Key Project of National Natural Science Funds of China (No. 81630094) and the CAMS Innovation Fund for Medical Sciences (No. 2016-I2M-2-002). We are grateful to the department of Instrumental Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences, and Peking Union Medical College for measuring the IR, NMR, and MS spectra. Author Contributions: C.-S.Y. and J.-Q.Z. conceived, designed the experiments and wrote the paper; J.-Q.Z., Y.-L.K. and B.-H.T. performed the experiments; C.-S.Y. and G.-P.L. analyzed the data. All authors read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
