Design and synthesis of the novel DNA topoisomerase II inhibitors: esterification and amination substituted 4'-demethylepipodophyllotoxin derivates exhibiting anti-tumor activity by activating ATM/ATR signaling pathways.

According to the structure-activity relationship, drug combination principle and bioisosterism, a series of the novel esterification and amination 4'-demethylepipodophyllotoxin derivates were rationally designed in order to discover the potential antitumor prodrug. And then these compounds were tested by the drug-topoisomerase II docking models for virtual screening. Thus, twelve target compounds were screened out and synthesized. Most of compounds exhibited promising in vitro anti-tumor activity, particularly 4-N-tris(hydroxymethyl)metylaminomethane-4-deoxy-4'-demethylepipodophyllotoxin (Compound 1). The anti-tumor activity of Compound 1 against the tumor cell lines BGC-823 (i.e., the IC50 value of 5.35 ± 0.77 µM), HeLa (i.e., the IC50 value of 160.48 ± 14.50 µM), and A549 (i.e., the IC50 value of 13.95 ± 5.41 µM) was significantly improved by 706%, 31% and 900% than that of etoposide (i.e., the IC50 values of 43.74 ± 5.13, 209.90 ± 13.42, and 139.54 ± 7.05 µM), respectively. Moreover, the IC50 value of Compound 1 against the normal human cell line HK-2 (i.e., 16.3 ± 3.77 µM) was 78% lower than that of etoposide (i.e., 9.17 ± 1.58 µM). Compound 1 could diminish the relaxation reaction topoisomerase II DNA decatenation at a concentration of 10 µM and induce BGC-823 apoptosis by breaking DNA double-strand and activating ATM/ATR signaling pathways.

Actinobacillus succinogenes ATCC 55618 fermentation medium optimization for the production of succinic acid by response surface methodology.

As a potential intermediary feedstock, succinic acid takes an important place in bulk chemical productions. For the first time, a method combining Plackett-Burman design (PBD), steepest ascent method (SA), and Box-Behnken design (BBD) was developed to optimize Actinobacillus succinogenes ATCC 55618 fermentation medium. First, glucose, yeast extract, and MgCO(3) were identified to be key medium components by PBD. Second, preliminary optimization was run by SA method to access the optimal region of the key medium components. Finally, the responses, that is, the production of succinic acid, were optimized simultaneously by using BBD, and the optimal concentration was located to be 84.6 g L(-1) of glucose, 14.5 g L(-1) of yeast extract, and 64.7 g L(-1) of MgCO(3). Verification experiment indicated that the maximal succinic acid production of 52.7 ± 0.8 g L(-1) was obtained under the identified optimal conditions. The result agreed with the predicted value well. Compared with that of the basic medium, the production of succinic acid and yield of succinic acid against glucose were enhanced by 67.3% and 111.1%, respectively. The results obtained in this study may be useful for the industrial commercial production of succinic acid.

Manipulation of heterogeneity product in 4'-demethylepipodophyllotoxin biotransformation process by using yeast extract as nitrogen source.

Manipulation of product heterogeneity was attempted by using yeast extract as nitrogen source in Alternaria alternata S-f6 transformation process of 4'-demethylepipodophyllotoxin. When the nitrogen source of NaNO(3) was replaced by yeast extract, the heterogeneity of biotransformation products was significantly varied from a single product (i.e., 4'-demethylpodophyllotoxone) to four podophyllum derivates. According to the kinetics of 4'-demethylepipodophyllotoxin biotransformation process by A. alternata S-f6, the starting substrate of 4'-demethylepipodophyllotoxin was preferentially transformed to produce 4'-demethylpodophyllotoxone (1) with an oxidation reaction. By the further comparison of products configuration, 4ß-caprinoyl-4'-demethylepipodophyllotoxin (3) was produced from 4'-demethylpodophyllotoxone (1) instead of 4'-demethylisopicropodophyllone (2), which might be produced from 4'-demethylpodophyllotoxone (1) with the isomerization of lactone. Finally, 4'-demethylisopicropodophyllone (2) was hydrolyzed to produce 3α-hydroxymethyl-(6, 7)-dioxol-4-one-naphthalene (4). This work shows new information on the 4'-demethylepipodophyllotoxin biotransformation process by A. alternata S-f6 and provides a foundation for further studies on the structural diversification of a bioactive natural lead compound.

Comparison of lipid content and fatty acid composition between Tuber fermentation mycelia and natural fruiting bodies.

A comparison of lipid content and fatty acid (FA) composition between Tuber fermentation mycelia and natural fruiting bodies indicates that the lipid content in Tuber fermentation mycelia is higher than that in fruiting bodies. Unsaturated FAs (particularly linoleic acid and oleic acid) were the predominant constituents in total FAs in both Tuber fermentation mycelia and fruiting bodies. A total of 23 FAs, including arachidonic, eicosapentaenoic, docosahexaenoic, and γ-linolenic acids, were first identified in the Tuber species. A hierarchical clustering analysis showed that the FA profile of fermentation mycelia was quite similar, regardless of Tuber species. However, the FA profile of the fruiting bodies was significantly influenced by its species and habitat environments. Interestingly, the FA profile of the Tuber indicum and Tuber aestivum fruiting bodies was nearly identical to that of the Tuber fermentation mycelia, which partially confirms the similarity between the Tuber fermentation mycelia and the fruiting bodies.

Determination of the nucleosides and nucleobases in Tuber samples by dispersive solid-phase extraction combined with liquid chromatography-mass spectrometry.

A simple, fast and inexpensive method based on dispersive solid phase extraction (DSPE) combined with LC-MS was developed for simultaneous determination of 7 nucleosides and nucleobases (i.e., adenine, hypoxanthine, uridine, adenosine, guanine, guanosine, and inosine) in Tuber fruiting-bodies and fermentation mycelia. The DSPE procedure was firstly introduced to remove the protein interference from sample solutions, and D3520 macroporous resin was chosen as the DSPE sorbent because of its high removal capability on protein interferences, but low adsorption rate on analytes. Besides, key parameters on DSPE procedure (i.e., macroporous resin type, macroporous resin amount, methanol concentration, and vortex time) were optimized, and the protein removal efficacy could achieve about 95% after the process optimization. Though the method validation test, the DSPE-LC-MS method was confirmed to be precise, accurate and sensitive, and the column blinding problem was solved successfully. By using this established method, the total amount of nucleosides and nucleobases in the fermentation mycelia was determined to range from 4881.5 to 12,592.9µgg⁻¹, which was about 2-25 times higher than the fruiting-bodies (from 498.1 to 2274.1µgg⁻¹). The formulation of nucleosides and nucleobases in the fermentation mycelia maintained relatively constant, while the formulation in Tuber fruiting-bodies varied significantly with their species. Hierarchical cluster analysis (HCA) showed the formulation similarity of nucleosides and nucleobases between Tuber fermentation mycelia and the fruiting-bodies of Tuber indicum and Tuber himalayense. From the viewpoint of nucleosides and nucleobases, this work confirms the potentiality of Tuber fermentation mycelia as the alternative resource for its fruiting-bodies.