Elucidation of the Concise Biosynthetic Pathway of the Communesin Indole Alkaloids

The communesins are a prominent class of indole alkaloids isolated from Penicillium species. Owing to their daunting structural framework and potential as pharmaceuticals, communesins have inspired numerous synthetic studies. However, the genetic and biochemical basis of communesin biosynthesis has remained unexplored. Herein, we report the identification and characterization of the communesin (cns) biosynthetic gene cluster from Penicillium expansum. We confirmed that communesin is biosynthesized by the coupling of tryptamine and aurantioclavine, two building blocks derived from L ‐tryptophan. The postmodification steps were mapped by targeted‐gene‐deletion experiments and the structural elucidation of intermediates and new analogues. Our studies set the stage for the biochemical characterization of communesin biosynthesis. This knowledge will aid our understanding of how nature generates remarkable structural complexity from simple precursors.     

Structure,Biosynthesis, and Occurrence of Bacterial Pyrrolizidine Alkaloids

Pyrrolizidine alkaloids (PAs) are widespread plant natural products with potent toxicity and bioactivity. Herein, the identification of bacterial PAs from entomopathogenic bacteria using differential analysis by 2D NMR spectroscopy (DANS) and mass spectrometry is described. Their biosynthesis was elucidated to involve a non‐ribosomal peptide synthetase. The occurrence of these biosynthesis gene clusters in Gram‐negative and Gram‐positive bacteria indicates an important biological function in bacteria.     

Azetidine‐Containing Alkaloids Produced by a Quorum‐Sensing Regulated Nonribosomal Peptide Synthetase Pathway in Pseudomonas aeruginosa

Pseudomonas aeruginosa displays an impressive metabolic versatility, which ensures its survival in diverse environments. Reported herein is the identification of rare azetidine‐containing alkaloids from P. aeruginosa PAO1, termed azetidomonamides, which are derived from a conserved, quorum‐sensing regulated nonribosomal peptide synthetase (NRPS) pathway. Biosynthesis of the azetidine motif has been elucidated by gene inactivation, feeding experiments, and biochemical characterization in vitro, which involves a new S‐adenosylmethionine‐dependent enzyme to produce azetidine 2‐carboxylic acid as an unusual building block of NRPS. The mutants of P. aeruginosa unable to produce azetidomonamides had an advantage in growth at high cell density in vitro and displayed rapid virulence in Galleria mellonella model, inferring functional roles of azetidomonamides in the host adaptation. This work opens the avenue to study the biological functions of azetidomonamides and related compounds in pathogenic and environmental bacteria.     

Asymmetric Total Syntheses of Kopsia Indole Alkaloids

The asymmetric total syntheses of a group of structurally complex Kopsia alkaloids, (−)‐kopsine, (−)‐isokopsine, (+)‐methyl chanofruticosinate, (−)‐fruticosine, and (−)‐kopsanone, has been achieved. The key strategies for the construction of the molecular complexity in the targets included an asymmetric Tsuji–Trost rearrangement to set the first quaternary carbon center at C20, an intramolecular cyclopropanation by diazo decomposition to install the second and third quaternary carbon centers at C2 and C7, respectively, and a SmI₂‐promoted acyloin condensation to assemble the isokopsine core. A radical decarboxylation of an isokopsine‐type intermediate results in a thermodynamic partial rearrangement to give N‐decarbomethoxyisokopsine and N‐decarbomethoxykopsine, two key intermediates for the syntheses of Kopsia alkaloids with different subtype core structures.     

Discovery of a Calcium‐Dependent Enzymatic Cascade for the Selective Assembly of Hapalindole‐Type Alkaloids: On the Biosynthetic Origin of Hapalindole U

Hapalindole U ( 4 ) is a validated biosynthetic precursor to ambiguine alkaloids (Angew. Chem. Int. Ed . 2016 , 55 , 5780), of which biogenetic origin remains unknown. The recent discovery of AmbU4 (or FamC1) protein encoded in the ambiguine biosynthetic pathway (J. Am. Chem. Soc . 2015 , 137 , 15366), an isomerocyclase that can rearrange and cyclize geranylated indolenine ( 2 ) to a previously unknown 12‐epi ‐hapalindole U ( 3 ), raised the question whether 3 is a direct precursor to 4 or an artifact arising from the limited in vitro experiments. Here we report a systematic approach that led to the discovery of an unprecedented calcium‐dependent AmbU1‐AmbU4 enzymatic complex for the selective formation of 4 . This discovery refuted the intermediacy of 3 and bridged the missing links in the early‐stage biosynthesis of ambiguines. This work further established the isomerocyclases involved in the biogenesis of hapalindole‐type alkaloids as a new family of calcium‐dependent enzymes, where the metal ions are shown critical for their enzymatic activities and selectivities.     

An Unprecedented Cyclization Mechanism in the Biosynthesis of Carbazole Alkaloids in Streptomyces

Carquinostatin A (CQS), a potent neuroprotective substance, is a unique carbazole alkaloid with both an ortho‐quinone function and an isoprenoid moiety. We identified the entire gene cluster responsible for CQS biosynthesis in Streptomyces exfoliatus through heterologous production of CQS and gene deletion. Biochemical characterization of seven CQS biosynthetic gene products (CqsB1–7) established the total biosynthetic pathway of CQS. Reconstitution of CqsB1 and CqsB2 showed that the synthesis of the carbazole skeleton involves CqsB1‐catalyzed decarboxylative condensation of an α‐hydroxyl‐β‐keto acid intermediate with 3‐hydroxybutyryl‐ACP followed by CqsB2‐catalyzed oxidative cyclization. Based on crystal structures and mutagenesis‐based biochemical assays, a detailed mechanism for the unique deprotonation‐initiated cyclization catalyzed by CqsB2 is proposed. Finally, analysis of the substrate specificity of the biosynthetic enzymes led to the production of novel carbazoles.     

Design and Use of de novo Cascades for the Biosynthesis of New Benzylisoquinoline Alkaloids

The benzylisoquinoline alkaloids (BIAs) are an important group of secondary metabolites from higher plants and have been reported to show significant biological activities. The production of BIAs through synthetic biology approaches provides a higher‐yielding strategy than traditional synthetic methods or isolation from plant material. However, the reconstruction of BIA pathways in microorganisms by combining heterologous enzymes can also give access to BIAs through cascade reactions. Most importantly, non‐natural BIAs can be generated through such artificial pathways. In the current study, we describe the use of tyrosinases and decarboxylases and combine these with a transaminase enzyme and norcoclaurine synthase for the efficient synthesis of several BIAs, including six non‐natural alkaloids, in cascades from l ‐tyrosine and analogues.     

Biosynthesis of the Indole Alkaloids. Cell-free Systems from Catharanthus roseus Plants

Cell-free systems from Catharanthus roseus plants are utilized for various studies relating to the biosynthesis of indole alkaloids. Tryptamine ( 5 ) and secologanin ( 6 ), two fundamental building units, are shown to be incorporated into the alkaloid vindoline ( 7 ). In another study, catharanthine ( 18 ) and vindoline ( 7 ) are utilized by this enzyme system and coupled to the important bisindole biointermediate 3′,4′-anhydrovinblastine

1 The previously [20] used name for 17 , 3′, 4′-dehydrovinblastine, is incorrect.

( 17 ). The latter substance is, in turn, incorporated and converted to the natural alkaloids leurosine ( 8 ), Catharine ( 9 ) and vinblastine ( 10 ), thereby providing information about the biosynthesis of these complex molecules. High pressure liquid chromatography assay of the enzymic mixture sheds light on the enzymes involved in the coupling of 18 and 7 .     

Astellifadiene: Structure Determination by NMR Spectroscopy and Crystalline Sponge Method,and Elucidation of its Biosynthesis

Genome mining of a terpene synthase gene from Emericella variecolor NBRC 32302 and its functional expression in Aspergillus oryzae led to the production of the new sesterterpene hydrocarbon, astellifadiene ( 1 ), having a 6‐8‐6‐5‐fused ring system. The structure of 1 was initially investigated by extensive NMR analyses, and was further confirmed by the crystalline sponge method, which established the absolute structure of 1 and demonstrated the usefulness of the method in the structure determination of complex hydrocarbon natural products. Furthermore, the biosynthesis of 1 was proposed on the basis of isotope‐incorporation experiments performed both in vivo and in vitro. The cyclization of GFPP involves a protonation‐initiated second cyclization sequence, 1,2‐alkyl migration, and 1,5‐hydride shift to generate the novel scaffold of 1 .     

氧化偶联策略在复杂吲哚生物碱全合成中的应用

氧化偶联反应作为一种高效、经济的C—C键构建策略,在天然产物的全合成中得到了重要的应用.近年来,马大为课题组通过发展高效的LiHMDS/I2偶联条件,成功实现了四种复杂吲哚生物碱的全合成,在该领域取得了重要的进展.本文就马大为课题组近年在该领域的工作作一亮点评述.     

Insights into the Biosynthesis of Cyclic Guanidine Alkaloids from Crambeidae Marine Sponges

Among the outstanding chemical diversity found in marine sponges, cyclic guanidine alkaloids, present in species of the family Crambeidae, are particularly attractive, not only because of their unique chemical features, but also due to a broad range of biological activities. Despite a growing interest in these natural products as therapeutic agents, their metabolic pathway has not been experimentally investigated. Ex situ feeding experiments using radiolabeled precursors performed on the Mediterranean sponge Crambe crambe suggest arginine and fatty acids as precursors in the metabolic pathway of crambescins. A subsequent bio‐inspired approach supported the change of paradigm in the metabolic pathway of cyclic guanidine alkaloids. A large part of the chemical diversity of this family would therefore originate from a tethered Biginelli‐like reaction between C‐2/C‐3 activated fatty acids and a central guanidinylated pyrrolinium.