Regiodivergent N-C and N-N aryl coupling reactions of indoloterpenes and cycloether formation mediated by a single bacterial flavoenzyme.

Indoloterpenoid natural products play an eminent role as drugs, and their significance for medicine has propelled a plethora of synthetic and biosynthetic studies. Interestingly, these alkaloids are a hallmark of plant and fungal metabolism, but virtually nothing was known about the corresponding bacterial pathways. Only recently, in the context of profiling the bacterial endophytes of widespread mangrove trees, we discovered the unprecedented bacterial indolosesquiterpenes xiamycin (1), indosespene (2), and sespenine (3 ; Figure 1). Together with oridamycin (4) and oxiamycin (5) these multicyclic alkaloids constitute a new family of bacterial indoloterpenes. The co-occurrence of these diverse hybrid metabolites in a single organism implies a biogenetic relationship. Thus, we and another research group have independently analyzed xiamycin (xia) biosynthesis gene clusters. Through mutational analyses and heterologous expression, we found that the pathway involves an unparalleled cyclization sequence to yield diverse ring systems. The heterologous expression of the entire gene cluster also led to the discovery of N C and N N coupled xiamycin dimers, which had been overlooked in the wild-type strain owing to their low production. These structurally intriguing bixiamycins represent the first examples of bacterial bisindolosesquiterpenes (BIST), and their biosynthesis has remained enigmatic. Herein, we report the discovery and full characterization of a series of highly regiodivergent, N C and N N aryl-coupled xiamycin dimers, and reveal their potent antibacterial activities. Furthermore, we show that a single flavoprotein not only mediates diverse aryl couplings, but also ether formation. Finally, we support a radical-based mechanism by a biomimetic synthesis of the xiamycin derivatives. To reveal the range of bixiamycins produced, we inspected the metabolic profile of Streptomyces albus carrying the entire xia biosynthesis gene cluster. HPLC-HRMS analyses indicated that the strain produces a number of compounds that likely result from the dimerization of xiamycin (1), as they have the same molecular formula (C46H48N2O6), but differ in retention times. To obtain sufficient amounts of these new compounds for a full structural characterization, the culture was scaled up. Both mycelia and culture filtrate of a scaled-up fermentation (50 L) were extracted with ethyl acetate, and the combined extracts were subjected to fractionation by flash chromatography, first through silica, then through a Sephadex LH-20 column. Final purification by preparative HPLC yielded various dimers, including the atroposiomeric pair of N N coupled bixiamycins (6a/6b) and other types of dimers (Figure 2) as pure compounds: 6a (10 mg), 6b (5 mg), 7a (3 mg), 7b (1.0 mg), and 8 (23.1 mg). NMR analysis of the new dimers revealed that they all possess the xiamycin backbone. Compounds 7a and 7b were obtained as a pair of atropodiastereomers. Their H NMR spectra show two sets of signals, thus indicating that the coupling sites of the two moieties of the molecule are not identical. As only a signal for H21 is visible, whereas the counterpart (H21’) is missing in the H NMR spectrum, a N C coupling between N1 and C21’ was proposed. COSY and HMBC correlations confirmed the proposed structure for the two halves of the dimer. Comparison of the experimental CD spectra with the TDCAMB3LYP/6-31G*//B97D/TZVP calculated ones revealed that 7a has a P configuration, whereas the minor atropodiastereomer 7b has an M configuration at the N C axis (Figure 3; see the Supporting Information for details). Figure 1. Structures of bacterial indolosesquiterpenes, xiamycin (1), indosespene (2), sespenine (3), oridamycin (4), and oxiamycin (5).