On the Mechanisms of Hypohalous Acid Formation and Electrophilic Halogenation by Non-Native Halogenases.

Enzymatic electrophilic halogenation is a mild tool for functionalization of diverse organic compounds. Only a few groups of native halogenases are capable of catalyzing such a reaction. In this study, we used a mechanism-guided strategy to discover the electrophilic halogenation activity catalyzed by non-native halogenases. As the ability to form a hypohalous acid (HOX) is key for halogenation, flavin-dependent monooxygenases/oxidases capable of forming C4a-hydroperoxyflavin (FlC4a-OOH), such as dehalogenase, hydroxylases, luciferase and pyranose-2-oxidase (P2O), and flavin reductase capable of forming H2O2 were explored for their abilities to generate HOX in situ. Transient kinetic analyses using stopped-flow spectrophotometry/fluorometry and product analysis indicate that FlC4a-OOH in dehalogenases, selected hydroxylases and luciferases, but not in P2O can form HOX; however, the HOX generated from FlC4a-OOH cannot halogenate their substrates. Remarkably, in situ H2O2 generated by P2O can form HOI and also iodinate various compounds. Because not all enzymes capable of forming FlC4a-OOH can react with halides to form HOX, QM/MM calculations, site-directed mutagenesis and structural analysis were carried out to elucidate the mechanism underlying HOX formation and characterize the active site environment. Our findings shed light on identifying new halogenase scaffolds besides the currently known enzymes and have invoked a new mode of chemoenzymatic halogenation.

Furanocoumarin compounds isolated from Dorstenia foetida potentiate irinotecan anticancer activity against colorectal cancer cells.

Although the anticancer activity of Dorstenia foetida was already observed, the chemical entity responsible for this activity remained unidentified. In this study, the cytotoxic activity of two furanocoumarin compounds, i.e., 5-methoxy--3-(3-methyl-2,3-dihydroxybutyl)-psoralen (1) and 5-methoxy-3-(3-methyl-2,3-dihydroxybutyl)-psoralen diacetate (2) isolated from ethyl acetate fraction of D. foetida (whole plant) was investigated in several cancer cell lines including HN22, MDA-MB-231, HCT116, and HT29. The results revealed that compound 2 exhibited cytotoxic activity, particularly against colorectal cancer cell lines HCT116 and HT29. The interplay between compound 2 and irinotecan (Iri) showed synergism against HCT116, which was analyzed by CompuSyn software. The simulation revealed that, at the molar ratio of Iri:2 of 1:40, the concentration predicted to achieve a 90 % inhibitory effect when used in the combination would be ~28- and ~4-fold lower than the concentration of compound 2 and Iri, resp., when used individually. Finally, the percentage of apoptotic cells in the HCT116 line treated with the combination was markedly higher than in the cells treated with the individual agent (60 % apoptotic cells for the combination compared to 17 and 45 % for Iri and compound 2 monotherapy, resp). In conclusion, our results identified compound 2 as a plant-derived compound exhibiting anticancer properties that can act synergistically with Iri and warranted further research to assess the potential of this synergism for colorectal cancer treatment.

A new acylated triterpene glycoside and cytotoxic constituents from Dolichandrone serrulata (Wall. ex DC.) Seem.

In this study, a new acylated triterpene glycoside, 3α-O-stearoyl-28-[2'-stearoyl-α-l-arabinopyranosyl]-olean-12-en-28-oic acid (1), was isolated from the flowers of Dolichandrone serrulata. In addition to this compound, eleven known compounds were also isolated, including a related pentacyclic triterpenoid: ursolic acid (2), two cycloartane triterpenoids: 24-methylenecycloartanol (3) and 24-methylenecycloartane-3,28-diol (4), three cyclohexylethane derivatives: (-)-rengyolone (5), (-)-cleroindicin C (6) and (-)-cleroindicin D (7), an iridoid: 6-O-trans-feruloyl catalpol (8), two phenylethanoid glycosides: salidroside (9) and verbascoside (10), and two steroids: ß-sitosterol (11) and ß-sitosterol-3-O-ß-d-glucopyranoside (12). The chemical structures of these compounds were determined by analysing their HRMS and NMR spectroscopic data. Additionally, their cytotoxic activities against NH22, HCT116, MCF7, MDA-MB-231, and HeLa cell lines were evaluated for all the compounds. Ursolic acid exhibited moderate cytotoxic activity against all cancer cell lines tested, particularly against HN22, MDA-MB-231, MCF-7, and HCT116 cells with IC50 values of approximately 19-34 µM.