Stefan Diethelm

One‐Pot Enzymatic Synthesis of Merochlorin A and B

The polycycles merochlorin A and B are complex halogenated meroterpenoid natural products with significant antibacterial activities and are produced by the marine bacterium Streptomyces sp. strain CNH-189. Heterologously produced enzymes and chemical synthesis are employed herein to fully reconstitute the merochlorin biosynthesis in vitro. The interplay of a dedicated type III polyketide synthase, a prenyl diphosphate synthase, and an aromatic prenyltransferase allow formation of a highly unusual aromatic polyketide-terpene hybrid intermediate which features an unprecedented branched sesquiterpene moiety from isosesquilavandulyl diphosphate. As supported by in vivo experiments, this precursor is furthermore chlorinated and cyclized to merochlorin A and isomeric merochlorin B by a single vanadium-dependent haloperoxidase, thus completing the remarkably efficient pathway.

A Multitasking Vanadium-Dependent Chloroperoxidase as an Inspiration for the Chemical Synthesis of the Merochlorins†

The vanadium-dependent chloroperoxidase Mcl24 was discovered to mediate a complex series of unprecedented transformations in the biosynthesis of the merochlorin meroterpenoid antibiotics. In particular, a site-selective naphthol chlorination is followed by an oxidative dearomatization/terpene cyclization sequence to build up the stereochemically complex carbon framework of the merochlorins in one step. Inspired by the enzyme reactivity, a chemical chlorination protocol paralleling the biocatalytic process was developed. These chemical studies led to the identification of previously overlooked merochlorin natural products.

Total Synthesis of Gelsemoxonine through a Spirocyclopropane Isoxazolidine Ring Contraction

Plants of the species Gelsemium have found application in traditional Asian medicine for over a thousand years. Gelsemoxonine represents a novel constituent of this plant incorporating a highly functionalized azetidine at its core. We herein report a full account of our studies directed toward the total synthesis of gelsemoxonine that relies on a conceptually new approach for the construction of the central azacyclobutane. A spirocyclopropane isoxazolidine ring contraction was employed to access a key β-lactam intermediate, which could be further elaborated to the azetidine of the natural product. In the course of our studies, we have gained detailed insight into this intriguing transformation. Furthermore, we report on previously unnoticed oligomerization chemistry of gelsemoxonine. We also document an enantioselective synthesis of a key precursor en route to gelsemoxonine.

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase-thioesterase enzyme pair

Significance The majority of pharmaceuticals are inspired by natural product scaffolds that are functionalized by tailoring enzymes, such as halogenases. The degree of halogenation is an important determinant of natural product bioactivity, yet little is known regarding the molecular basis for the exquisite control exhibited by tailoring halogenases. Known pyrrole halogenases commonly perform up to two halogenations on the pyrrole. Our study of tetrabromopyrrole biosynthesis revealed a uniquely adapted halogenase–thioesterase enzyme pair that catalyzes an unprecedented series of halogenations on a pyrrole. Structural comparison of the pyrrole tetrahalogenase to a pyrrole dihalogenase revealed key residues involved in controlling the degree of halogenation. Our findings provide fundamental insights that might be applied in the rational design of biocatalysts toward directed biosynthesis of new chemicals. Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono- and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes.

Chemoenzymatic Synthesis of Acyl Coenzyme A Substrates Enables in Situ Labeling of Small Molecules and Proteins.

A chemoenzymatic approach to generate fully functional acyl coenzyme A molecules that are then used as substrates to drive in situ acyl transfer reactions is described. Mass spectrometry based assays to verify the identity of acyl coenzyme A enzymatic products are also illustrated. The approach is responsive to a diverse array of carboxylic acids that can be elaborated to their corresponding coenzyme A thioesters, with potential applications in wide-ranging chemical biology studies that utilize acyl coenzyme A substrates.

Mechanistic Insight into the Spirocyclopropane Isoxazolidine Ring Contraction

A mechanistic study of the ring contraction of spirocyclopropane isoxazolidines to form β-lactams is reported. Based on experimental and computational investigations, we propose a concerted mechanism that proceeds with retention of configuration during cyclopropane cleavage.

Amine-Selective Bioconjugation Using Arene Diazonium Salts

A novel bioconjugation strategy is presented that relies on the coupling of diazonium terephthalates with amines in proteins. The diazonium captures the amine while the vicinal ester locks it through cyclization, ensuring no reversibility. The reaction is highly efficient and proceeds under mild conditions and short reaction times. Densely functionalized, complex natural products were directly coupled to proteins using low concentrations of coupling partners.

A unifying paradigm for naphthoquinone-based meroterpenoid (bio)synthesis

Bacterial meroterpenoids constitute an important class of natural products with diverse biological properties and therapeutic potential. The biosynthetic logic for their production is unknown and defies explanation via classical biochemical paradigms. A large subgroup of naphthoquinone-based meroterpenoids exhibits a substitution pattern of the polyketide-derived aromatic core that seemingly contradicts the established reactivity pattern of polyketide phenol nucleophiles and terpene diphosphate electrophiles. We report the discovery of a hitherto unprecedented enzyme-promoted α-hydroxyketone rearrangement catalysed by vanadium-dependent haloperoxidases to account for these discrepancies in the merochlorin and napyradiomycin class of meroterpenoid antibiotics, and we demonstrate that the α-hydroxyketone rearrangement is potentially a conserved biosynthetic reaction in this molecular class. The biosynthetic α-hydroxyketone rearrangement was applied in a concise total synthesis of naphthomevalin, a prominent member of the napyradiomycin meroterpenes, and sheds further light on the mechanism of this unifying enzymatic transformation.

Total Synthesis of BE-43472B

Significance: The aromatic polyketide BE-43472B was isolated from a marine Streptomyces species and was shown to exhibit significant activity against several drug-resistant bacterial strains. Moreover, its unprecedented structure includes two anthraquinones linked through a highly hindered carbon–carbon bond as well as five contiguous stereocenters. The strategy reported by Suzuki and co-workers relies on a highly efficient pinacol rearrangement to form the key C–C bond between the two anthraquinone monomers. Comment: The synthesis starts with lithiation of bromonaphthalene B, followed by addition to ketone A. The resulting tertiary alcohol C was treated with triflic acid to induce a pinacol rearrangement to produce ketone D. Construction of the tetrahydrofuran ring proceeded via acetal E followed by methylation using Me3Al. Oxidation of F and subsequent quinone Diels–Alder reaction with diene G delivered anthraquinone H. This intermediate was converted into the natural product (±)-BE-43472B via epoxide J. O OH OH