Enantioselective inhibitory abilities of enantiomers of notoamides against RANKL-induced formation of multinuclear osteoclasts.

The marine-derived Aspergillus protuberus MF297-2 and the terrestrial A. amoenus NRRL 35600 produce enantiomeric prenylated indole alkaloids. Investigation of biological activities of the natural and synthetic derivatives revealed that (-)-enantiomers of notoamides A and B, 6-epi-notoamide T, and stephacidin A inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenic differentiation of murine RAW264 cells more strongly than their respective (+)-enantiomers. Among them, (-)-6-epi-notoamide T was the most potent inhibitor with an IC50 value of 1.7µM.

Applications of Ring Closing Metathesis. Total Synthesis of (±)-Pseudotabersonine.

A novel approach to the Aspidosperma family of alkaloids was developed and applied to a concise total synthesis of (±)-pseudotabersonine that was accomplished in 11 steps. Key transformations include a stepwise variant of a Mannich-like multicomponent assembly process, a double ring-closing metathesis sequence, and a one-pot deprotection/cyclization reaction.

Bioconversion of 6-epi-Notoamide T Produces Metabolites of Unprecedented Structures in a Marine-derived Aspergillus sp.

We previously described the bioconversion of Notoamide T into (+)-Stephacidin A and (-)-Notoamide B, which suggested that Versicolamide B (8) is biosynthesized from 6-epi-Notoamide T (10) via 6-epi-Stephacidin A. Here we report that [13C]2-10 was incorporated into isotopically enriched 8 and seven new metabolites, which were not produced under normal culture conditions. The results suggest that the addition of excess precursor activated the expression of dormant tailoring genes giving rise to these structurally unprecedented metabolites.

Biochemical characterization of NotB as an FAD-dependent oxidase in the biosynthesis of notoamide indole alkaloids.

Notoamides produced by Aspergillus spp. bearing the bicyclo[2.2.2]diazaoctane core structure with unusual structural diversity represent a compelling system to understand the biosynthesis of fungal prenylated indole alkaloids. Herein, we report the in vitro characterization of NotB, which catalyzes the indole 2,3-oxidation of notoamide E (13), leading to notoamides C (11) and D (12) through an apparent pinacol-like rearrangement. This unique enzymatic reaction with high substrate specificity, together with the information derived from precursor incorporation experiments using [(13)C](2)-[(15)N](2) quadruply labeled notoamide S (10), demonstrates 10 as a pivotal branching point in notoamide biosynthesis.

Studies on the Biosynthesis of the Stephacidin and Notoamide Natural Products: A Stereochemical and Genetic Conundrum.

The stephacidin and notoamide natural products belong to a group of prenylated indole alkaloids containing a bicyclo[2.2.2]diazaoctane core. Biosynthetically, this bicyclic core is believed to be the product of an intermolecular Diels- Alder (IMDA) cycloaddition of an achiral azadiene. Since all of the natural products in this family have been isolated in enantiomerically pure form to date, it is believed that an elusive Diels-Alderase enzyme mediates the IMDA reaction. Adding further intrigue to this biosynthetic puzzle is the fact that several related Aspergillus fungi produce a number of metabolites with the opposite absolute configuration, implying that these fungi have evolved enantiomerically distinct Diels-Alderases. We have undertaken a program to identify every step in the biogenesis of the stephacidins and notoamides, and by combining the techniques of chemical synthesis and biochemical analysis we have been able to identify the two prenyltransferases involved in the early stages of the stephacidin and notoamide biosyntheses. This has allowed us to propose a modified biosynthesis for stephacidin A, and has brought us closer to our goal of finding evidence for, or against, the presence of a Diels-Alderase in this biosynthetic pathway.

Genome-based characterization of two prenylation steps in the assembly of the stephacidin and notoamide anticancer agents in a marine-derived Aspergillus sp.

Stephacidin and notoamide natural products belong to a group of prenylated indole alkaloids containing a core bicyclo[2.2.2]diazaoctane ring system. These bioactive fungal secondary metabolites have a range of unusual structural and stereochemical features but their biosynthesis has remained uncharacterized. Herein, we report the first biosynthetic gene cluster for this class of fungal alkaloids based on whole genome sequencing of a marine-derived Aspergillus sp. Two central pathway enzymes catalyzing both normal and reverse prenyltransfer reactions were characterized in detail. Our results establish the early steps for creation of the prenylated indole alkaloid structure and suggest a scheme for the biosynthesis of stephacidin and notoamide metabolites. The work provides the first genetic and biochemical insights for understanding the structural diversity of this important family of fungal alkaloids.

Applications of multicomponent reactions to the synthesis of diverse heterocyclic scaffolds.

The sequencing of multicomponent reactions (MCRs) and subsequent cyclization reactions is a powerful stratagem for the rapid synthesis of diverse heterocyclic scaffolds. The optimal MCR is sufficiently flexible that it can be employed to generate adducts bearing a variety of functional groups that may then be selectively paired to enable different cyclization manifolds, thereby leading to a diverse collection of products. The growing interest in diversity-oriented synthesis has led to increased attention to this paradigm for library synthesis, which has inspired many advances in the design and implementation of MCRs for the construction of diverse heterocyclic scaffolds.

Synthesis of Diverse Heterocyclic Scaffolds via Tandem Additions to Imine Derivatives and Ring-Forming Reactions.

A novel strategy has been developed for the efficient syntheses of diverse arrays of heterocyclic compounds. The key elements of the approach comprise a Mannich-type, multicomponent coupling reaction in which functionalized amines, aromatic aldehydes, acylating agents, and π- and organometallic nucleophiles are combined to generate intermediates that are then further transformed into diverse heterocyclic scaffolds via a variety of cyclization manifolds. Significantly, many of these scaffolds bear functionality that may be exploited by further manipulation to create diverse collections of compounds having substructures found in biologically active natural products and clinically useful drugs. The practical utility of this strategy was exemplified by its application to the first, and extraordinarily concise synthesis of the isopavine alkaloid roelactamine.

Fungal indole alkaloid biogenesis through evolution of a bifunctional reductase/Diels-Alderase.

Prenylated indole alkaloids such as the calmodulin-inhibitory malbrancheamides and anthelmintic paraherquamides possess great structural diversity and pharmaceutical utility. Here, we report complete elucidation of the malbrancheamide biosynthetic pathway accomplished through complementary approaches. These include a biomimetic total synthesis to access the natural alkaloid and biosynthetic intermediates in racemic form and in vitro enzymatic reconstitution to provide access to the natural antipode (+)-malbrancheamide. Reductive cleavage of an L-Pro-L-Trp dipeptide from the MalG non-ribosomal peptide synthetase (NRPS) followed by reverse prenylation and a cascade of post-NRPS reactions culminates in an intramolecular [4+2] hetero-Diels-Alder (IMDA) cyclization to furnish the bicyclo[2.2.2]diazaoctane scaffold. Enzymatic assembly of optically pure (+)-premalbrancheamide involves an unexpected zwitterionic intermediate where MalC catalyses enantioselective cycloaddition as a bifunctional NADPH-dependent reductase/Diels-Alderase. The crystal structures of substrate and product complexes together with site-directed mutagenesis and molecular dynamics simulations demonstrate how MalC and PhqE (its homologue from the paraherquamide pathway) catalyse diastereo- and enantioselective cyclization in the construction of this important class of secondary metabolites.

Applications of multicomponent reactions for the synthesis of diverse heterocyclic scaffolds.

A four-component coupling process involving sequential reactions of aldehydes, primary amines, acid chlorides, and nucleophiles has been developed to prepare multifunctional substrates that may be employed in subsequent ring-forming reactions to generate a diverse array of functionalized heterocyclic scaffolds. This new approach to diversity-oriented synthesis was then applied to the first total synthesis of the isopavine alkaloid (+/-)-roelactamine.

Oxidation of carbon-silicon bonds: the dramatic advantage of strained siletanes.

[reaction: see text] Herein we report on the use of siletanes as substrates for the oxidation of carbon-silicon bonds. These tetraalkylsilanes are easy to handle yet susceptible to rapid ring opening and oxidation upon exposure to aqueous fluoride and peroxide. This combination of stability and reactivity presents many practical benefits, including compatibility with silicon protecting groups and electron-rich aromatic rings.

Synthesis and bioconversions of notoamide T: a biosynthetic precursor to stephacidin A and notoamide B.

In an effort to further elucidate the biogenesis of the stephacidin and notoamide families of natural products, notoamide T has been identified as the likely precursor to stephacidin A. The total synthesis of notoamide T is described along with it is C-6-epimer, 6-epi-notoamide T. The chemical conversion of stephacidin A to notoamide T by reductive ring opening is described as well as the oxidative conversion of notoamide T to stephacidin A. Furthermore, [(13)C](2)-notoamide T was synthesized and provided to Aspergillus versicolor and Aspergillus sp. MF297-2, in which significant incorporation was observed in the advanced metabolite, notoamide B.

Comparative analysis of the biosynthetic systems for fungal bicyclo[2.2.2]diazaoctane indole alkaloids: the (+)/(-)-notoamide, paraherquamide and malbrancheamide pathways.

The biosynthesis of fungal bicyclo[2.2.2]diazaoctane indole alkaloids with a wide spectrum of biological activities have attracted increasing interest. Their intriguing mode of assembly has long been proposed to feature a non-ribosomal peptide synthetase, a presumed intramolecular Diels-Alderase, a variant number of prenyltransferases, and a series of oxidases responsible for the diverse tailoring modifications of their cyclodipeptide-based structural core. Until recently, the details of these biosynthetic pathways have remained largely unknown due to lack of information on the fungal derived biosynthetic gene clusters. Herein, we report a comparative analysis of four natural product metabolic systems of a select group of bicyclo[2.2.2]diazaoctane indole alkaloids including (+)/(-)-notoamide, paraherquamide and malbrancheamide, in which we propose an enzyme for each step in the biosynthetic pathway based on deep annotation and on-going biochemical studies.

Concise total synthesis of (+/-)-pseudotabersonine via double ring-closing metathesis strategy.

A concise synthesis of (+/-)-pseudotabersonine from commercially available 1-(phenylsulfonyl)-3-indolecarboxaldehyde has been accomplished. This synthesis features the convergent assembly of a key intermediate via a stepwise variant of a Mannich-type multicomponent coupling process, a double ring-closing metathesis, and a one-pot deprotection/cyclization reaction.