Important building blocks for the synthesis of drugs or natural products are found in Mannich bases and their derivatives. Modern variants of the Mannich reaction that expand the potential of the classical intermolecular reaction significantly and enable efficient control of the regioselectivity and stereoselectivity are therefore the topic of intensive research. Intramolecular reactions, in particular as part of domino reaction sequences, often afford astoundingly simple and elegant approaches to complex target compounds.

Multicomponent reactions (MCRs) are by far the most successful class of reactions leading to high structural diversity and molecular complexity through a single transformation. As part of the ongoing search for pharmacologically active lead structures, the obtained structural diversity allows for the fast exploration of a large chemical space. Not surprisingly, the development of MCRs, leading to new structural frameworks or serving as key transformations in the total synthesis of natural products, has expanded rapidly over the last few decades. To date a multitude of new three- and four-component reactions have already been described; however, examples of "higher-order" MCRs where five or even more components are combined in a single reaction vessel are remarkably scarce. This tutorial review aims to critically describe the developments achieved in recent years, charting the ideas, challenges, and milestone reactions that were essential for the progress of this field.

Fungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as the oxygen source. However, their heterologous production in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. We developed and applied a modular Golden Gate-based secretion system, allowing the first production of four active UPOs in yeast, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be universally applicable and consists of the three module types: i) signal peptides for secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid, modular yeast secretion workflow of UPOs yielding preparative scale enantioselective biotransformations.

Edited by Joseph M. Jez Nonhost resistance of Arabidopsis thaliana against Phytophthora infestans, a filamentous eukaryotic microbe and the causal agent of potato late blight, is based on a multilayered defense system. Arabidopsis thaliana controls pathogen entry through the penetration-resistance genes PEN2 and PEN3, encoding an atypical myrosinase and an ABC transporter, respectively, required for synthesis and export of unknown indole compounds. To identify pathogen-elicited leaf surface metabolites and further unravel nonhost resistance in Arabidopsis, we performed untargeted metabolite profiling by incubating a P. infestans zoospore suspension on leaves of WT or pen3 mutant Arabidopsis plants. Among the plant-secreted metabolites, 4-methoxyindol-3-yl-methanol and S-(4-methoxy-indol-3-ylmethyl) cysteine were detected in spore suspensions recollected from WT plants, but at reduced levels from the pen3 mutant plants. In both whole-cell and microsome-based assays, 4-methoxyindol-3-yl-methanol was transported in a PEN3-dependent manner, suggesting that this compound is a PEN3 substrate. The syntheses of both compounds were dependent on functional PEN2 and phytochelatin synthase 1. None of these compounds inhibited mycelial growth of P. infestans in vitro. Of note, exogenous application of 4-methoxyindol-3-yl methanol slightly elevated cytosolic Ca 2؉ levels and enhanced callose deposition in hydathodes of seedlings treated with a bacterial pathogenassociated molecular pattern (PAMP), flagellin (flg22). Loss of flg22-induced callose deposition in leaves of pen3 seedlings was partially reverted by the addition of 4-methoxyindol-3-yl methanol. In conclusion, we have identified a specific indole compound that is a substrate for PEN3 and contributes to the plant defense response against microbial pathogens. Phytophthora infestans is the causal agent of late blight, economically the most important potato disease. In an attempt to This work was supported by the DFG (SPP1212 Microbial Reprogramming of Plant Development, SCHE 235/14-3). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Tables S1 and S2 and Figs. S1-S3.

Inst. Mol. Mater., Radboud Univ. Nijmegen, NL-6525 ED Nijmegen, Neth.; Eng.) -Y. Steudel 46-232

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