Shaobin Fu*ab,
Qingfeng Mengc,
Junshan Yanga,
Jiajia Tub and
Di-An Sun*a
aInstitute of Medical Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193, China. E-mail: diansun@sina.com; fushb@126.com; Fax: +86-0851-28609493; Tel: +86-10-57833298 Tel: +86-0851-28642513
bPharmacy School of Zunyi Medical University, Zunyi 563000, China
cDepartment of Public Health, Zunyi Medical University, Zunyi 563000, China
First published on 3rd May 2018
Biocatalysis of ursolic acid (UA 1) by Gliocladium roseum CGMCC 3.3657 was investigated. Baeyer–Villiger oxidation was found to occur during the reaction. Four metabolites were isolated from the cultures and their structures were identified as 21-oxo,A-homo-3a-oxa-urs-12-en-3-one-28-oic acid (2), 21-oxo-3,4-seco-ursan-4(23),12-dien-3,28-dioic acid (3), 21β-hydroxyl-A-homo-3a-oxa-urs-12-en-3-one-28-oic acid (4) and 21β-hydroxyl-3,4-seco-ursan-4(23),12-dien-3,28-dioic acid (5), based on their NMR and MS spectral data. All of the four metabolites were new and their anti-HCV activity was tested. Their biotransformation pathway was also proposed.
Structural modification of UA was carried out to improve its solubility and biological activity.10–12 Since UA does not have a lot of active sites for traditional organic modification, biocatalysis was investigated as a very good method to modify UA to obtain new metabolites.13–15
The hepatitis C virus (HCV) infects 170 million people around the world and is mostly transmitted via parenteral routes. The increased risk of HCV development in HCV-infected patients arises from the development of liver fibrosis and cirrhosis as a result of chronic inflammation.16 HCV entry inhibitors could satisfy a tandem mechanism for use with other inhibitors of viral replication, ultimately leading to a multifaceted approach for the eradication of the HCV infection. Recently, the inhibitory activity on HCV entry of EA and a series of derivatives was investigated, and EA and some of the derivatives showed potency for anti-HCV entry activity.17–19
This paper reports the microbial transformation of UA and also pentacyclic triterpene acid by filamentous fungus G. roseum CGMCC 3.3657. Hydroxyl and ketone groups were introduced in a regio- and stereo-selective way at C-21 which is difficult for organic synthesis. We suggest that ring A may be catalyzed by Baeyer–Villiger (BV) enzymes to form a seven-membered ring lactone and thus ring A can be cleaved. Compounds 2 and 3 exhibited better anti-HCV activity than 1, 4 and 5. This result indicates that the ketone group at C-21 may help to block HCV entry. Furthermore, the biotransformation pathway was also proposed.
Metabolite 2 was more polar than the substrate. The HR-ESI-MS data ([M−H]−: 483.3104) of 2 (Appendix 2-1†) suggested a molecular formula of C30H44O5. There were two more oxygen atoms than the substrate (1). The 1H-NMR spectrum (Appendix 2-2 and 2-3†) revealed the presence of seven methyl groups, five with a singlet and two with a double peak. The signal at δH 3.46 assigned to H-3α in the substrate disappeared in product 2. The carbon signals (Appendix 2-4†) changed significantly, indicating the presence of two carboxyl groups (δC 174.2 and δC 175.7). Analysis of the HMBC data (Fig. 2) revealed that the carbonyl group (δC 209.5) was at C-21 based on the correlation between C-21 and H-22 (δH 2.65 and δH 2.39) and the correlation between C-21 and H-30 (δH 0.93, d and J = 6.6 Hz). The carboxyl group δC 174.2 was assigned to C-28 based on the HMBC data (Fig. 2) due to the correlation between C-28 and H-22. The signal at δC 85.4, correlated with both H-23 (δH 1.38) and H-24 (δH 1.33) in the HMBC spectrum (Fig. 2), may be attributed to C-3 or C-4 (Appendix 2-6 and 2-7†). This was further identified as C-4 and the signal at δC 175.4 was confirmed to be C-3 with reference to the data of a known compound (A-homo-3a-oxa-olean-12-en-3-one-28-oic acid),20 which is structurally similar to product 2. Based on the above evidence, product 2 was elucidated as 21-oxo,A-homo-3a-oxa-urs-12-en-3-one-28-oic acid.
Metabolite 3 showed an [M−H]− m/z peak at 483.3105 on the HR-ESI-MS spectrum (Appendix 3-1†), corresponding to the molecular formula of C30H44O5. In the 1H-NMR spectrum (Appendix 3-2†), two more olefinic proton signals at δH 4.65 and δH 4.86 with respective singlet peaks appeared besides H-12 at δH 5.40 (t, J = 3.6 Hz), and only six methyl group signals were observed. Correspondingly, the 13C-NMR spectrum (Appendix 3-3†) revealed two new olefinic carbon signals at δC 113.7 and δC 147.2. The protons at δH 4.65 and δH 4.86 were directly attached to the carbon at δC 113.7. Additional olefinic carbon signals at δC 127.6 and δC 137.2 were identified as C-12 and C-13, compatible with the substrate. This implied that compound 3 could be a product with ring-cleavage. From the HMBC data (Fig. 2 and Appendix 3-5 and 3-6†), HMQC data (Appendix 3-4†) and previous literature,20,21 we can conclude that ring-A was cleaved between C-3 and C-4, C-3 was oxygenated to become a carboxyl group and a new double bond was formed between C-23 and C-4. Like for metabolite 2, the carbon signal at δC 209.5 was a carbonyl group and assigned to C-21 based on the HMBC spectrum. Therefore, product 3 was confirmed as 21-oxo-3,4-seco-ursan-4(23),12-dien-3,28-dioic acid.
Metabolite 4 had the molecular formula of C30H46O5, as evidenced by the HR-ESI-MS data ([M−H]−: m/z 485.3263, shown in Appendix 4-1†). According to the 1H-NMR spectrum (Appendix 4-2†) and 13C-NMR spectrum (Appendix 4-3†), the structure of compound 4 was similar to that of compound 2. All of the methyl groups were unchanged compared to those of the substrate. In addition, ring-A also formed a seven-membered ring lactone. The double bond with a chemical shift of δC 126.4 and δC 137.5 occurred between C12 and C13 which was consistent with the substrate. The carbon signal at δC 71.2, which correlated with the proton signal at δH 3.43, was confirmed to be C-21 from the HMBC data (Fig. 2 and Appendix 4-5†) whereas C-21 was a carbonyl group in metabolite 2. Additionally, the orientation of the hydroxyl group at C-21 was deduced as the β-position from the coupling constants and splitting pattern of H-21 (δH 3.43, td, J = 4.2, 10.8 Hz) in the 1H-NMR spectrum which was at its axial (α) position. Ultimately, metabolite 4 was determined as 21β-hydroxyl-A-homo-3a-oxa-urs-12-en-3-one-28-oic acid.
The molecular formula of metabolite 5 was determined as C30H46O5 from its HR-ESI-MS spectrum ([M−H]−: 485.3215, shown in Appendix 5-1†). In the 1H-NMR spectrum (Appendix 5-2†), there were proton signals at δH 4.65 and δH 4.84 which were similar to those of compound 3. Correspondingly, the olefinic carbon signals at δ 113.4 and δ 147.1 in the 13C-NMR spectrum (Appendix 5-3†) were also consistent with metabolite 3. It was deduced that metabolite 5 was also a product with ring-A cleavage. The new double bond was assigned between C-4 and C-23, identical to compound 3. The two carboxyl groups were attributed to C-3 and C-28, with chemical shifts at 174.7 ppm and 177.4 ppm respectively. Moreover, the secondary hydroxyl group was assigned to C-21, based on the proton signal at δH 3.19 which correlated with the carbon signal at δC 69.0 in the HMQC spectrum (Appendix 5-4†). The orientation of the –OH group was determined to be its β-position from the coupling constants and splitting pattern of H-21 (td, J = 4.2, 10.8 Hz). Based on the above evidence, metabolite 5 was identified as 21β-hydroxyl-3,4-seco-ursan-4(23),12-dien-3,28-dioic acid.
Fig. 3 An anti-HCV activity test based on HCVpp and VSVG-pp entry assay: 1 is the substrate and 2–5 are products. |
Fraction A was further re-crystallized in petroleum ether/THF to afford metabolite 2 (25 mg, 6.25%). Fraction B was further purified by column chromatography on silica gel (300–400 mesh, 7 g) by eluting stepwise with CHCl3/petroleum ether (85:35) to afford metabolite 3 (4 mg, 1%). Fraction C was further re-crystallized in methanol to afford metabolite 4 (32 mg, 8%) and the mother liquor was further purified by column chromatography on silica gel (300–400 mesh, 9.5 g) by eluting stepwise with CHCl3/MeOH from 100:2 to 100:8 and with petroleum ether/acetone from 100:11 to 11:13 to afford metabolite 5 (6 mg, 1.5%).
Metabolite 2: white crystal, mp 263–265 °C, [α]D20 +54.4° (c = 1.23 × 10−4, EtOH), HR-ESI-MS: 483.3104 [M−H]− (calcd 483.3105). For 13C-NMR (150 MHz, DMSO-d6) and 1H-NMR (600 MHz, DMSO-d6) spectra see Tables 1 and 2.
No. | Product 2 | Product 3 | Product 4 | Product 5 |
---|---|---|---|---|
a Solvent DMSO-d6 was used for products 2 and 5; solvent CDCl3 was used for products 3 and 4. | ||||
1 | 37.8 | 34.1 | 38.3 | 33.9 |
2 | 31.7 | 28.5 | 32.1 | 28.2 |
3 | 174.2 | 178.5 | 175.2 | 174.1 |
4 | 85.4 | 147.1 | 86.2 | 147.1 |
5 | 53.4 | 51.0 | 54.7 | 49.3 |
6 | 25.1 | 24.5 | 23.7 | 24.0 |
7 | 31.7 | 31.8 | 32.4 | 31.3 |
8 | 39.1 | 39.6 | 39.6 | 38.7 |
9 | 46.5 | 38.2 | 47.4 | 37.1 |
10 | 39.2 | 39.5 | 39.8 | 38.7 |
11 | 23.3 | 24.0 | 25.2 | 23.0 |
12 | 126.0 | 127.6 | 126.4 | 124.8 |
13 | 137.2 | 137.1 | 137.5 | 138.1 |
14 | 41.6 | 42.5 | 42.2 | 42.1 |
15 | 27.4 | 28.1 | 28.0 | 27.6 |
16 | 25.6 | 26.0 | 22.9 | 24.8 |
17 | 50.1 | 51.4 | 48.7 | 47.8 |
18 | 51.7 | 52.7 | 52.2 | 52.2 |
19 | 40.6 | 41.8 | 38.0 | 37.5 |
20 | 50.1 | 51.3 | 46.6 | 46.4 |
21 | 209.5 | 209.5 | 71.2 | 69.0 |
22 | 49.8 | 50.7 | 44.6 | 45.2 |
23 | 31.8 | 113.7 | 32.4 | 113.4 |
24 | 30.4 | 23.6 | 25.8 | 23.5 |
25 | 16.8 | 19.5 | 17.1 | 19.3 |
26 | 16.6 | 17.3 | 16.9 | 17.0 |
27 | 23.2 | 23.9 | 23.3 | 23.0 |
28 | 175.7 | 179.5 | 179.6 | 177.4 |
29 | 18.1 | 18.7 | 17.1 | 17.3 |
30 | 12.5 | 12.6 | 15.6 | 15.8 |
No. | Product 2 | Product 3 | Product 4 | Product 5 |
---|---|---|---|---|
a Mult, multiplicity: s, singlet; d, doublet; t, triplet; o, overlap; solvent DMSO-d6 was used for products 2 and 5; solvent CDCl3 was used for products 3 and 4. | ||||
1 | 1.61 (o), 1.49 (o) | 1.61 (m), 1.58 (m) | 1.56 (m), 1.79 (m) | 1.45 (m) |
2 | 2.58 (o) | 2.39 (m), 2.56 (m) | 2.61 (m) | 2.05 (m) |
3 | — | — | — | — |
4 | — | — | — | — |
5 | 1.68 (o) | 1.92 (o) | 1.66 (d, 10.8) | 1.96 (dd, 1.8, 12.6) |
6 | 1.76 (m) | 1.37 (m) | 1.99 (m) | 1.30 (m) |
7 | 1.48 (m), 1.24 (m) | 1.47 (m), 1.26 (m) | 1.36 (m) | 1.21 (m), 1.47 (m) |
8 | — | — | — | — |
9 | 1.61 (o) | 1.68 (m) | 1.62 (m) | 1.86 (m) |
10 | — | — | — | — |
11 | 1.94 (o) | 1.93 (m) | 1.85 (m) | 1.04 (o) |
12 | 5.32 (t, 3.6) | 5.40 (t, 3.6) | 5.32 (t-like) | 5.16 (t, 3.6) |
13 | — | — | — | — |
14 | — | — | — | — |
15 | 1.77 (o), 1.03 (o) | 1.11 (m) | 1.11 (m), 1.84 (m) | 1.75 (m) |
16 | 1.67 (m), 1.48 (m) | 1.85 (m), 1.59 (m) | 1.52 (m) | 1.65 (m) |
17 | — | — | — | — |
18 | 2.64 (d, 10.8) | 2.68 (d, 11.4) | 2.249 (d, 11.4) | 2.11 (d, 10.8) |
19 | 1.69 (o) | 1.77 (m) | 1.44 (m) | 1.40 (m) |
20 | 2.23 (dd, 6.0, 10.8) | 2.14 (dd, 6.0, 10.8) | 0.98 (o) | 0.79 (m) |
21 | — | — | 3.43 (td, 4.2, 10.8) | 3.19 (td, 4.2, 10.8) |
22 | 2.14 (d, 13.2), 2.69 (d, 12.6) | 2.65 (d, 12.6), 2.39 (d, 12.6) | 1.57 (m), 2.41 (dd, 4.2, 12.6) | 1.80 (m), 1.35 (m) |
23 | 1.40 (s) | 4.86 (brs), 4.65 (brs) | 1.48 (s) | 4.65 (brs), 4.84 (brs) |
24 | 1.34 (s) | 1.72 (s) | 1.42 (s) | 1.71 (s) |
25 | 1.08 (s) | 0.92 (s) | 1.15 (s) | 0.86 (s) |
26 | 0.81 (s) | 0.82 (s) | 0.84 (s) | 0.80 (s) |
27 | 1.02 (s) | 1.05 (o) | 1.07 (s) | 1.05 (s) |
28 | — | — | — | — |
29 | 0.96 (d, 6.6) | 1.01 (d, 6.6) | 0.91 (d, 6.0) | 0.84 (d, 6.6) |
30 | 0.93 (d, 6.6) | 1.05 (d, 6.6) | 1.08 (d, 6.0) | 0.98 (d, 6.6) |
Metabolite 3: white crystal, mp 217–220 °C, [α]D20 +27.2° (c = 8.46 × 10−3, EtOH), HR-ESI-MS: 483.3105 [M−H]− (calcd 483.3105). For 13C-NMR (150 MHz, CDCl3) and 1H-NMR (600 MHz, CDCl3) spectra see Tables 1 and 2.
Metabolite 4: white solid, mp 204–206 °C, [α]D20 +63.6 (c = 6.92 × 10−3, EtOH), HR-ESI-MS: 485.3263 [M−H]− (calcd 485.3262). For 13C-NMR (150 MHz, CDCl3) and 1H-NMR (600 MHz, CDCl3) spectra see Tables 1 and 2.
Metabolite 5: white solid, mp 201–203 °C, [α]D20 +80.4° (c = 2.07 × 10−4, EtOH), HR-ESI-MS: 485.3215 [M−H]− (calcd 485.3262). For 13C-NMR (150 MHz, DMSO-d6) and 1H-NMR (600 MHz, DMSO-d6) spectra see Tables 1 and 2.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8ra01217b |
This journal is © The Royal Society of Chemistry 2018 |