RESUMO
Halogenation of pyrrole requires strong electrophilic reagents and often leads to undesired polyhalogenated products. Biocatalytic halogenation is a highly attractive approach given its chemoselectivity and benign reaction conditions. While there are several reports of enzymatic phenol and indole halogenation in organic synthesis, corresponding reports on enzymatic pyrrole halogenation have been lacking. Here we describe the in vitro functional and structural characterization of PrnC, a flavin-dependent halogenase that can act on free-standing pyrroles. Computational modeling and site mutagenesis studies identified three key residues in the catalytic pocket. A moderate resolution map using single-particle cryogenic electron microscopy reveals PrnC to be a dimer. This native PrnC can halogenate a library of structurally diverse pyrrolic heterocycles in a site-selective manner and be applied in the chemoenzymatic synthesis of a chlorinated analog of the agrochemical fungicide Fludioxonil.
RESUMO
In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes strain-dependent, and the commonly used Streptococcus pyogenes Cas9 (SpCas9) is notorious for having high levels of off-target toxicity effects. Thus, a variety of Cas proteins is required for greater flexibility of genetic manipulation within a wider range of Streptomyces strains. This study explored the first use of Acidaminococcus sp. Cas12j, a hypercompact Cas12 subfamily, for genome editing in Streptomyces and its potential in activating silent biosynthetic gene clusters (BGCs) to enhance natural product synthesis. While the editing efficiencies of Cas12j were not as high as previously reported efficiencies of Cas12a and Cas9, Cas12j exhibited higher transformation efficiencies compared to SpCas9. Furthermore, Cas12j demonstrated significantly improved editing efficiencies compared to Cas12a in activating BGCs in Streptomyces sp. A34053, a strain wherein both SpCas9 and Cas12a faced limitations in accessing the genome. Overall, this study expanded the repertoire of Cas proteins for genome editing in actinomycetes and highlighted not only the potential of recently characterized Cas12j in Streptomyces but also the importance of having an extensive genetic toolbox for improving the editing success of these beneficial microbes.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Streptomyces , Streptomyces/genética , Streptomyces/metabolismo , Edição de Genes/métodos , Acidaminococcus/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Família Multigênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Genoma BacterianoRESUMO
Milk cap mushrooms in the genus Lactarius are known to produce a wide variety of terpene natural products. However, their repertoire of terpene biosynthetic enzymes has not been fully explored. In this study, several candidate sesquiterpene synthases were identified from the genome of the saffron milk cap mushroom L. deliciosus and expressed in a sesquiterpene-overproducing Escherichia coli strain. In addition to enzymes that produce several known terpenes, we identified an enzyme belonging to a previously unknown clade of sesquiterpene synthases that produces a terpene with a unique spiro-tricyclic scaffold. These findings add to the rich diversity of terpene scaffolds and mushroom terpene synthases and are valuable for biotechnological applications in producing these terpenoids.
Assuntos
Agaricales , Alquil e Aril Transferases , Basidiomycota , Sesquiterpenos , Terpenos , Alquil e Aril Transferases/genéticaRESUMO
RadH is one of the flavin-dependent halogenases that has previously exhibited promising catalytic activity towards hydroxycoumarin, hydroxyisoquinoline, and phenolic derivatives. Here, we evaluated new functional homologs of RadH and expanded its specificities for the halogenation of non-tryptophan-derived, heterocyclic scaffolds. Our investigation revealed that RadH could effectively halogenate hydroxyquinoline and hydroxybenzothiophene. Assay optimization studies revealed the need to balance the various co-factor concentrations and where a GDHi co-factor recycling system most significantly improves the conversion and efficiency of the reaction. A crystal structure of RadH was also obtained with a resolution of 2.4 Å, and docking studies were conducted to pinpoint the binding and catalytic sites for substrates.
Assuntos
Halogenação , Oxirredutases , Oxirredutases/metabolismo , Domínio Catalítico , Flavinas/química , Flavinas/metabolismoRESUMO
As less attention has been focussed on the design of highly efficient palladium precatalysts to ensure the smooth formation of the active catalyst for metal-mediated cross coupling reactions, we herein demonstrate that combining the bulky N-heterocyclic carbene (NHC) 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene (IPr) with cyclopalladated acetanilide as the optimal palladium precatalyst leads to superior catalytic activity compared with the state-of-the-art NHC-Pd catalysts. The complex was discovered through the evaluation of a small, rationally designed library of NHC-palladacycles prepared by a novel, practical and atom-economic method, the direct reaction of IPrHCl with palladacycle acetate dimers.
Assuntos
Reagentes de Ligações Cruzadas/química , Compostos Heterocíclicos/química , Imidazóis/química , Metano/análogos & derivados , Paládio/química , Catálise , Cristalografia por Raios X , Dimerização , Metano/química , Modelos MolecularesRESUMO
The wide dissemination of catalytic protocols in academic and industrial laboratories is facilitated by the development of catalysts that are not only highly active but also user-friendly, stable to moisture, air and long term storage and easy to prepare on a large scale. Herein we describe a protocol for the Heck-Mizoroki reaction mediated by cyclopalladated N,N-dimethylbenzylamine (dmba) ligated with a N-heterocyclic carbene, 1,3-bis(mesityl)imidazol-2-ylidene (IMes), that fulfils these criteria. The precatalyst can be synthesized on approximately 100 g scale by a tri-component, sequential, one-pot reaction of N,N-dimethylbenzylamine, PdCl2 and IMes.HCl in refluxing acetonitrile in air in the presence of K2CO3. This single component catalyst is stable to air, moisture and long term storage and can be conveniently dispensed as a stock solution in NMP. It mediates the Heck-Mizoroki reaction of a range of aryl- and heteroaryl bromides in reagent grade NMP at the 0.1-2 mol% range without the need for rigorous anhydrous techniques or a glovebox, and is active even in air. The catalyst is capable of achieving very high levels of catalytic activity (TON of up to 5.22 x 10(5)) for the coupling of a deactivated arylbromide, p-bromoanisole, with tBu acrylate as a benchmark substrate pair. A wide range of aryl bromides, iodides and, for the first time with a NHC-Pd catalyst, a triflate was coupled with diverse acrylate derivatives (nitrile, tert-butyl ester and amides) and styrene derivatives. The use of excess (>2 equiv.) of the aryl bromide and tert-butyl acrylate leads to mixture of tert-butyl beta,beta-diarylacrylate and tert-butyl cinnamate derivatives depending on the substitution pattern of the aryl bromide. Electron rich m- and p-substituted arylbromides give the diarylated products exclusively, whereas electron-poor aryl bromides give predominantly mono-arylated products. For o-substituted aryl bromides, no doubly arylated products could be obtained under any conditions. Overall, the active catalyst (IMes-Pd) shows higher activity with electron-rich aryl halides, a marked difference compared with the more commonly used phosphane-Pd or non-ligated Pd catalysts.
Assuntos
Acrilatos/química , Benzilaminas/química , Catálise , Metano/análogos & derivados , Paládio/química , Brometos/química , Cinamatos/química , Ésteres/química , Imidazóis/química , Cinética , Metano/química , Preparações Farmacêuticas , Fosfinas/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Two one-pot oxidative annulative approaches to spiroacetal synthesis are described. One approach uses a Lewis acid mediated Ferrier reaction in the fragment-coupling stage followed by DDQ-promoted oxidative carbon-hydrogen bond cleavage and cyclization. An alternative approach employs a Heck reaction for fragment coupling followed by DDQ-mediated enone formation and cyclization. These strategies provide convergent routes to common subunits in natural products, medicinal agents, and chemical libraries under mild reaction conditions.
Assuntos
Produtos Biológicos/síntese química , Compostos de Espiro/síntese química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Técnicas de Química Combinatória , Ciclização , Ácidos de Lewis/química , Estrutura Molecular , Oxirredução , Compostos de Espiro/química , Compostos de Espiro/farmacologiaRESUMO
Cyclopropane-substituted allylic ethers react with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to form oxocarbenium ions with no competitive ring cleavage. This reaction can be used for the preparation of cyclopropane-substituted tetrahydropyrans. The protocol was used as a key step in the total synthesis of the sponge-derived macrolide clavosolide A.
Assuntos
Ciclopropanos/química , Macrolídeos/síntese química , Animais , Benzoquinonas/química , Macrolídeos/química , Estrutura Molecular , Poríferos/químicaRESUMO
A highly active, N-heterocyclic carbene-palladacycle precatalyst for the Heck-Mizoroki reaction was rationally designed. The complex can be synthesized on a large scale in excellent yield by a novel, one-pot, three-component reaction and is tolerant to air, moisture, and long-term storage. A wide range of challenging substrates is successfully coupled under a simple and user-friendly reaction protocol.
Assuntos
Compostos Heterocíclicos/química , Metano/análogos & derivados , Compostos Organometálicos/química , Paládio/química , Catálise , Metano/químicaRESUMO
The coupling of electrophiles with activated alkenes by using tertiary amines or phosphines is generally known as the Baylis-Hillman reaction. It is a useful and atom-economical carbon-carbon bond-forming reaction that generates multifunctionalized products. This reaction is notoriously slow; yields are often low and substrate-dependent. The asymmetric reaction is still limited especially for unactivated olefins such as acrylates. Imidazolines have been developed as ligands in metal-catalyzed reactions and have also been used as privileged structures in diversity-oriented synthesis. A series of novel chiral imidazolines were prepared and used to develop asymmetric Baylis-Hillman reactions. These imidazolines promote the reactions of various aromatic aldehydes with unactivated acrylates. Enantiomeric excesses of up to 60% and high yields were obtained by using stoichiometric amounts of the promoter. Furthermore, the imidazolines are also suitable promoters for the reactions between aromatic aldehydes and alkyl vinyl ketones. Enantiomeric excesses of up to 78% and high yields were obtained with 50 mol % of an imidazoline with a chiral methylnaphthyl group. These chiral imidazolines are easily prepared from commercially available amino alcohols and can be easily recovered for reuse without loss of product enantioselectivity.