Microbial Transformation of neo-Clerodane Diterpenoid, Scutebarbatine F, by Streptomyces sp. CPCC 205437

Biotransformation of the neo-clerodane diterpene, scutebarbatine F (1), by Streptomyces sp. CPCC 205437 was investigated for the first time, which led to the isolation of nine new metabolites, scutebarbatine F1–F9 (2–10). Their structures were determined by extensive high-resolution electrospray ionization mass spectrometry (HRESIMS) and NMR data analyses. The reactions that occurred included hydroxylation, acetylation, and deacetylation. Compounds 2–4 and 8–10 possess 18-OAc fragment, which were the first examples of 13-spiro neo-clerodanes with 18-OAc group. Compounds 7–10 were the first report of 13-spiro neo-clerodanes with 2-OH. Compounds 1–10 were biologically evaluated for the cytotoxic, antiviral, and antibacterial activities. Compounds 5, 7, and 9 exhibited cytotoxic activities against H460 cancer cell line with inhibitory ratios of 46.0, 42.2, and 51.1%, respectively, at 0.3 μM. Compound 5 displayed a significant anti-influenza A virus activity with inhibitory ratio of 54.8% at 20 μM, close to the positive control, ribavirin.


INTRODUCTION
Biotransformation of natural products has been proved to be an efficient and environmentally friendly approach to the preparation of new compounds, and biotransformation is a powerful tool for the regioselective and stereoselective introduction under mild conditions (Bianchini et al., 2015;Cao et al., 2015;Hegazy et al., 2015;Zafar et al., 2016;Khojasteh et al., 2019). Microbial biotransformation can be an attractive alternative to introduce hydroxyl and acetyl groups at specific positions, which were difficult to achieve by conventional synthesis methods. So biotransformation method has been widely used for structural modification of terpenoids (Silva et al., 2013;Bhatti and Khera, 2014;Rico-Martínez et al., 2014;de Sousa et al., 2018;Luchnikova et al., 2020).
Clerodane diterpenoids are a widespread class of natural products and widely distributed in medicinal herbs from various families, such as Lamiaceae, Euphorbiaceae, Compositae, and Salicaceae (Li et al., 2016). So far, over 1,300 clerodane diterpene derivatives have been reported, and they displayed a broad range of promising biological properties, such as cytotoxic, insect antifeedant, anti-inflammatory, antiprotozoal, and antibacterial activities (Li et al., 2016;Luan et al., 2019;Shen et al., 2021). Structurally, all of the natural clerodane diterpenes were classified as seven different types on the basis of the decalin ring and C-11-C-16 moiety (Supplementary Figure 1; Li et al., 2016). Although more than 1,300 clerodane diterpenoids have been reported, there are a few reports on biotransformation of neoclerodane diterpenes (Hoffmann and Fraga, 1993;Choudhary et al., 2013;Mafezoli et al., 2014;Kumar et al., 2016;Vasconcelos et al., 2017). To the best of our knowledge, there is no report about biotransformation of 13-spiro neo-clerodane diterpenes till now. Scutebarbatine F (1) with 13-spiro neo-clerodane skeleton was firstly isolated from Scutellaria barbata in 2006 by Dai group and showed significant cytotoxic activities against HONE-1 nasopharyngeal, KB oral epidermoid carcinoma, and HT29 colorectal carcinoma cell lines (Dai et al., 2006). Subsequently, the chemical structure of scutebarbatine F was revised by Wang et al. (2010). Furthermore, the compound with the same chemical structure was also reported to be names as barbatine C and scutebata F (Nguyen et al., 2009;Zhu et al., 2010). In an attempt to obtain new biologically active neo-clerodane diterpenoids. We performed the biotransformation of scutebarbatine F using the Streptomyces sp. CPCC 205437, which resulted in the formation of nine previously undescribed metabolites (2-10) (Figure 1). Biotransformation of 1 introduces hydroxyl groups at C-2 and C-18, followed by introducing acetyl moiety in 18-OH. Herein, we describe the isolation, structure elucidation, and biological activities of nine biotransformation products (2-10).

Substrate
Scutebarbatine F was isolated and purified from Scutellaria barbata by the Dai group. Its structure was elucidated by HRESIMS and 1D and 2D NMR analyses as previously reported (Dai et al., 2006), and the purity was > 95% by HPLC analysis. The substrate was dissolved in dimethylformamide (DMF) before being used for biotransformation.

Microorganism and Culture Medium
The strain was originally purchased from the BeNa Culture Collection (BNCC) and identified on the basis of 16s rRNA gene sequence analysis by China Pharmaceutical Culture Collection. The 16S rRNA sequence was strongly homologous to Streptomyces kronopolitis NEAU-ML8(T) (99.3%) and has been deposited in GenBank with accession number MW699542. The strain was deposited at the China Pharmaceutical Culture Collection (No. CPCC 205437). It was grown on potato dextrose agar (PDA) slant at 28 • C and stored at 4 • C. The medium of Streptomyces sp. was prepared on modified potato dextrose broth (PDB) medium (potato 200 g, glucose 20 g, KH 2 PO 4 3 g, MgSO 4 0.73 g, vitamin B 1 10 mg, distilled water 1 L).

Fermentation and Biotransformation
The medium was distributed in 24 Erlenmeyer flasks of 1,000 ml containing 200 ml of modified PDB liquid medium and autoclaved at 121 • C. The mycelial agar plugs were transferred Frontiers in Microbiology | www.frontiersin.org into the flasks and incubated at 28 • C for 3 days. Compound 1 (240 mg) was dissolved in DMF, added to each, flask and incubated at 28 • C for an additional 6 days.

Cytotoxic Activity Assays
MTT assay was used to measure the cytotoxicity of compounds. Five human cancer cell lines (H460, HCT8, HT15, H1975, and MIA-PaCa-2) were obtained from American Type Culture Collection (ATCC). Cells (5 × 10 3 cells/well) were added to 96-well culture dishes and grown for 24 h followed by the addition of fresh medium (100 ml) and the test compound. After an additional 48 h, the medium was removed, and fresh medium with MTT solution was added. The cells were incubated for 1 h, and then the optical density at 450 nm was determined.

Anti-influenza A Virus Activity Assays
All the metabolites were evaluated for their anti-influenza A virus (anti-IAV) (H1N1) activities as in previously described methods (Gao et al., 2014).

Anti-HIV Activity Assays
All the metabolites were tested for their anti-HIV activities as in previously reported methods (Zhang et al., 2017).

Antibacterial Activity Assays
All the metabolites were tested for their antibacterial activities as in previously described methods (Zhang et al., 2017).
Metabolite 4 was obtained as a white amorphous powder. HRESIMS gave an ion peak at m/z 530.2386 [M + H] + , which refers to the molecular formula C 28 H 35 NO 9 (calcd for C 28 H 36 NO 9 , 530.2390), with 42 amu less than that of 3. This suggested that 4 lacked an acetoxy moiety compared with 3, which was further confirmed by the HMBCs from H-6 (δ H 3.63) to C-4, C-5, C-7, and C-19. The NOESY correlations of H-6/H-10; H-7/H-17, H-19, and H-20, along with the large coupling constant of 3 J H −6,H −7 (9.6 Hz), established the stereochemistry of 4 as the same as substrate 1. Accordingly, metabolite 4 was determined to be scutebarbatine F 3 . Metabolite 6 was obtained as a white amorphous powder. HRESIMS data of 6 showed an [M + H] + ion at m/z 488.2281, consistent with the molecular formula C 26 H 33 NO 8 , which is 42 amu less than that of 4, indicating the loss of an acetoxy moiety in 4. The HMBCs from H-6 to C-4, C-5, C-7, and C-19; from H-7 to C-5, C-6, C-8, and C-17; and from H-18 to C-3, C-4, and C-5 established the molecular structure of 6. The enhancement of H-10 was observed when H-6 was irradiated, and irradiation of H-7 enhanced H-17, H-19, and H-20 in the NOE experiments, indicating the relative configuration of 6. Thus, metabolite 6 was elucidated as scutebarbatine F 5 .
Metabolite 5 was obtained as a white amorphous powder and possessed the same molecular formula C 28 H 35 NO 9 with 4 as supported by the HRESIMS ion peak at m/z 530.2382 [M + H] + (calcd for C 28 H 36 NO 9 , 530.2390). The 1 H and 13 C NMR spectroscopic data of 5 resembled those of 2, except for the absence of an acetoxy substituent. The HMBC crosspeaks (Figure 2) from H-18 to C-3, C-4, and C-5 suggest that the C-18 acetoxy group disappeared in 5 compared with 2. In the NOE experiment, irradiation of H-6 resulted in the enhancement of H-10, together with the large coupling constant of 3 J H −6,H −7 (10.2 Hz), suggesting that H-6 and H-10 were syn-oriented. Therefore, metabolite 5 was established as scutebarbatine F 4 .
Metabolite 7 was isolated as a white amorphous powder, and its molecular formula of C 30 H 38 NO 10 was determined by the HRESIMS peak at m/z 572.2498 [M + H] + (calcd for C 30 H 38 NO 10 , 572.2496), with 16 amu higher than that of 1. The 1 H and 13 C NMR data of 7 were similar to those of 1, except for the presence of oxygenated methine proton at δ H 4.28 and oxygenated carbon at δ C 71.5, suggesting that a hydroxyl group might be introduced. The location of introduced OH group was attached to C-2 by the 1 H-1 H correlated spectroscopy (COSY) correlations of H-1/H-2/H-3 and the HMBC crosspeaks (Figure 2) from H-2 to C-1, C-3, and C-4. The NOESY correlation (Figure 3) between H-2 and H-10, together with the large coupling constant of 3 J H −1,H −2 (7.8 Hz), assigned the α-orientation of 2-OH. Therefore, metabolite 7 was determined to be scutebarbatine F 6 .
Metabolite 8 was obtained as a white amorphous powder. The HRESIMS spectrum displayed [M + H] + ion peak at m/z 588.2441 (calcd for C 30 H 38 NO 11 , 588.2445), suggesting the substitution of one additional OH moiety compared with 2. The 1 H NMR data of 8 were similar to those of 2 except that the methylene signals of H-2 at δ H 2.71 and 2.29 had disappeared, and an additional oxygen-bearing methine signal at δ H 4.34 was observed, indicating that the OH group may be introduced at C-2. It was further confirmed by the 1 H-1 H COSY correlations of H-1/H-2/H-3 and the HMBCs from H-2 to C-1, C-3, and C-4. The stereochemistry of 2-OH was determined as α-orientation on the basis of the large coupling constant of 3 J H −1,H −2 (7.8 Hz) and the NOE experiment, in which the integration value of H-10 when H-2 was irradiated. The integration value of H-10 was enhanced when H-6 was irradiated, indicating the α-configuration of 6-OH. Accordingly, metabolite 8 was elucidated to be scutebarbatine F 7 . Metabolite 9 was isolated as a white amorphous powder.
The molecular formula C 30 H 37 NO 11 was deduced from the HRESIMS peak at m/z 588.2440 [M + H] + , which is the same as that of 8. Comparison of the 1 H NMR data of 9 and 8 revealed an oxymethine signal at δ H 3.46 in 9 in place of a signal at δ H 3.72 in 8, indicating that changes happened in the C-6 and C-7 positions, and the HMBC cross-peaks from H-6 to C-4, C-5, C-7, C-19, and C-1 determined the acetoxy group at C-6. The NOESY correlations for H-2/H-10, H-7/H-19, and H-20 assigned the α-orientation of 2-OH and H-7. Thus, metabolite 9 was determined as scutebarbatine F 8 . Metabolite 10 was isolated as a white amorphous powder. HRESIMS spectrum of 10 provided a major ion peak at m/z 546.2342 [M + H] + (calcd for C 28 H 36 NO 10 , 546.2339), consistent with the molecular formula of C 28 H 35 NO 10 , suggesting that a hydroxyl group was introduced in comparison with 4. The 1 H and 13 C NMR spectroscopic data of 10 were similar to those of 4. The obvious difference was the presence of oxygenated methine signal at δ H 4.31 instead of methylene signals at δ H 2.67 and 2.27 in 4. The OH moiety was attached to C-2 according to the 1 H-1 H COSY correlations of H-1/H-2/H-3 and the HMBC cross-peaks from H-2 to C-1, C-3, and C-4. The NOESY correlations of H-2/H-10, H-7/H-19, and H-20, together with the large coupling constants of 3 J H −1,H −2 (7.8 Hz) and 3 J H −6,H −7 (9.6 Hz), established the relative configuration of C-2, C-6, and C-7. Therefore, metabolite 10 was established as scutebarbatine F 9 .

Biological Activity
Compounds 1-10 were evaluated for cytotoxic activities against five human cancer cell lines including H460 (large cell lung carcinoma), HCT8 (colon carcinoma), HT15 (colon carcinoma), H1975 (non-small cell lung carcinoma), and MIA-PaCa-2 (pancreatic carcinoma). Compounds 5, 7, and 9 showed cytotoxic activities against H460 cancer cell line with inhibitory ratios of 46.0, 42.2, and 51.1%, respectively, at 0.3 µM (Figure 4 and Supplementary Table 1). Compounds 1-10 were tested for antiviral activities; compound 5 displayed a significant anti-IAV (H1N1) activity with inhibitory ratio of 54.8% at 20 µM, which is close to the positive control, ribavirin (inhibitory ratio of 49.5% at 20 µM); and none of them shows anti-HIV activities at the concentration of 20 µM (Table 4). Compounds 1-10 were also evaluated for antibacterial activities against Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922. All compounds showed no antibacterial activities with the minimal inhibitory concentration (MIC) values > 32 µg/ml.

CONCLUSION
In conclusion, the biotransformation of 13-spiro neo-clerodane diterpene was investigated for the first time. Nine previously undescribed neo-clerodane diterpenoids 2-10 were afforded from microbial transformation of scutebarbatine F (1) by Streptomyces sp. CPCC 205437. Three types of reactions were involved in the biotransformation, including hydroxylation, acetylation, and deacetylation, in which the hydroxylation follows region-and stereo-selective routes. Hydroxylation was performed at C-2 and C-18; acetylation was further catalyzed at 18-OH; and deacetylation was carried out at C-6 and C-7. Compounds 2-4 and 8-10 were the first examples of 13-spiro neo-clerodanes with 18-OAc group. Compounds 7-10 were the first report of 13-spiro neo-clerodanes with 2-OH. Compounds 5, 7, and 9 displayed good cytotoxic activities against H460 cancer cell line at 0.3 µM. Compound 5 exhibited a significant anti-IAV activity. Our findings not only enrich the structural diversity of neo-clerodane family but also provide the guidance to biotransformation of neo-clerodane diterpenoids.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
DZ and LY conceived and designed the experiments. DZ, XT, and GG contributed to fermentation, extraction, and isolation.
DZ and SD contributed to structure elucidation and wrote the manuscript. YW, WeZ, and WuZ contributed to bioactivities tests. YR and LY were the project leaders guiding the experiments and manuscript writing. All authors contributed to the article and approved the submitted version.