A rhodium-catalyzed cycloisomerization and oxidation of tethered dienynes for the synthesis of indanes is described. An auxiliary fragmentation/olefination method (also described herein) provides novel access to tethered alkyne-dienoate substrates. The reported method circumvents current limitations in and expands the scope of inverse-demand Diels-Alder-type cycloadditions. Traditional discovery substrates involving malonate-, ether-, and sulfonamide-based tethers are problematic in the current methodology, underscoring the unique virtue of neopentylene-tethered substrates for reaction discovery.

Strategic pairing of ring openings and cycloisomerization provides rapid and efficient "open and shut" entry into sparsely functionalized illudalanes, as exemplified here in the context of a six-step synthesis of alcyopterosin A. Key steps include a tandem ring-opening fragmentation/olefination process for preparing a neopentyl-tethered 1,6-enyne, ring-opening olefination telescoped with alkyne homologation, and Rh-catalyzed oxidative cycloisomerization.

Thiopeptides are a broad class of macrocyclic, heavily modified peptide natural products that are unified by the presence of a substituted, nitrogen-containing heterocycle core. Early work indicated that this core might be fashioned from two dehydroalanines by an enzyme-catalyzed aza-[4 + 2] cycloaddition to give a cyclic-hemiaminal intermediate. This common intermediate could then follow a reductive path toward a dehydropiperidine, as in the thiopeptide thiostrepton, or an aromatization path to yield the pyridine groups observed in many other thiopeptides. Although several of the enzymes proposed to perform this cycloaddition have been reconstituted, only pyridine products have been isolated and any hemiaminal intermediates have yet to be observed. Here, we identify the conditions and substrates that decouple the cycloaddition from subsequent steps and allow interception and characterization of this long hypothesized intermediate. Transition state modeling indicates that the key amide–iminol tautomerization is the major hurdle in an otherwise energetically favorable cycloaddition. An anionic model suggests that deprotonation and polarization of this amide bond by TbtD removes this barrier and provides a sufficient driving force for facile (stepwise) cycloaddition. This work provides evidence for a mechanistic link between disparate cyclases in thiopeptide biosynthesis.

Thiazole-containing pyritides (thiopeptides) are ribosomally synthesized and post-translationally modified peptides (RiPPs) that have attracted interest owing to their potent biological activities and structural complexity. The class-defining feature of a thiopeptide is a six-membered, nitrogenous heterocycle formed by an enzymatic [4 + 2]-cycloaddition. In rare cases, piperidine or dehydropiperidine (DHP) is present; however, the aromatized pyridine is considerably more common. Despite significant effort, the mechanism by which the central pyridine is formed remains poorly understood. Building on our recent observation of the Bycroft–Gowland intermediate (i.e., the direct product of the [4 + 2]-cycloaddition), we interrogated thiopeptide pyridine synthases using a combination of targeted mutagenesis, kinetic assays, substrate analogs, enzyme–substrate cross-linking, and chemical rescue experiments. Collectively, our data delineate roles for several conserved residues in thiopeptide pyridine synthases. A critical tyrosine facilitates the final aromatization step of pyridine formation. This work provides a foundation for further exploration of the [4 + 2]-cycloaddition reaction and future customization of pyridine-containing macrocyclic peptides.

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