Discovery and biosynthesis of bosamycins from Streptomyces sp. 120454

Nonribosomal peptides (NRPs) that are synthesized by modular megaenzymes known as nonribosomal peptide synthetases (NRPSs) are a rich source for drug discovery. By targeting an unusual NRPS architecture, we discovered an unusual biosynthetic gene cluster (bsm) from Streptomyces sp. 120454 and identified that it was responsible for the biosynthesis of a series of novel linear peptides, bosamycins. The bsm gene cluster contains a unique monomodular NRPS, BsmF, that contains a cytochrome P450 domain at the N-terminal. BsmF (P450 + A + T) can selectively activate tyrosine with its adenylation (A) domain, load it onto the thiolation (T) domain, and then hydroxylate tyrosine to form 5-OH tyrosine with the P450 domain. We demonstrated a NRPS assembly line for the formation of bosamycins by genetic and biochemical analysis and heterologous expression. Our work reveals a genome mining strategy targeting a unique NRPS domain for the discovery of novel NRPs.

Genome mining targeting a unique NRPS domain led to the identification of a novel class of peptides named bosamycins.     

Metal-free tandem carbene N–H insertions and C–C bond cleavages

A metal-free C–H [5 + 1] annulation reaction of 2-arylanilines with diazo compounds has been achieved, giving rise to two types of prevalent phenanthridines via highly selective C–C cleavage. Compared to the simple N–H insertion manipulation of diazo, this method elegantly accomplishes a tandem N–H insertion/S_EAr/C–C cleavage/aromatization reaction, and the synthetic utility of this new transformation is exemplified by the succinct syntheses of trisphaeridine and bicolorine alkaloids.

A metal-free C–H [5 + 1] annulation reaction of 2-arylanilines with diazo compounds has been achieved, giving rise to two types of prevalent phenanthridines via highly selective C–C cleavage.     

Forwards and backwards – synthesis of Laurencia natural products using a biomimetic and retrobiomimetic strategy incorporating structural reassignment of laurefurenynes C–F

Laurefurenynes C–F are four natural products isolated from Laurencia species whose structures were originally determined on the basis of extensive nuclear magnetic resonance experiments. On the basis of a proposed biogenesis, involving a tricyclic oxonium ion as a key intermediate, we have reassigned the structures of these four natural products and synthesized the four reassigned structures using a biomimetic approach demonstrating that they are the actual structures of the natural products. In addition, we have developed a synthesis of the enantiomers of the natural products laurencin and deacetyllaurencin from the enantiomer of (E)-laurefucin using an unusual retrobiomimetic strategy. All of these syntheses have been enabled by the use of tricyclic oxonium ions as pivotal synthetic intermediates.

The synthesis and structural reassignment of laurefurenynes C–F has been achieved, with the new structures fitting with a proposed biosynthesis. Also reported is the synthesis of ent-laurencin and ent-deacetyllaurencin via a retrobiomimetic approach.     

Cobalt-catalyzed intramolecular decarbonylative coupling of acylindoles and diarylketones through the cleavage of C–C bonds

We report here cobalt–N-heterocyclic carbene catalytic systems for the intramolecular decarbonylative coupling through the chelation-assisted C–C bond cleavage of acylindoles and diarylketones. The reaction tolerates a wide range of functional groups such as alkyl, aryl, and heteroaryl groups, giving the decarbonylative products in moderate to excellent yields. This transformation involves the cleavage of two C–C bonds and formation of a new C–C bond without the use of noble metals, thus reinforcing the potential application of decarbonylation as an effective tool for C–C bond formation.

A method for cobalt–N-heterocyclic carbene catalytic systems for the intramolecular decarbonylative coupling of ketones was achieved.     

Fungal siderophore biosynthesis catalysed by an iterative nonribosomal peptide synthetase

Siderophores play a vital role in the viability of fungi and are essential for the virulence of many pathogenic fungal species. Despite their importance in fungal physiology and pathogenesis, the programming rule of siderophore assembly by fungal nonribosomal peptide synthetases (NRPSs) remains unresolved. Here, we report the characterization of the bimodular fungal NRPS, SidD, responsible for construction of the extracellular siderophore fusarinine C. The use of intact protein mass spectrometry, together with in vitro biochemical assays of native and dissected enzymes, provided snapshots of individual biosynthetic steps during NPRS catalysis. The adenylation and condensation domain of SidD can iteratively load and condense the amino acid building block cis-AMHO, respectively, to synthesize fusarinine C. Our study showcases the iterative programming features of fungal siderophore-producing NRPSs.

Snapshots of fungal siderophore biosynthesis on the biosynthetic assembly-line captured by intact protein mass-spectrometry.     

Synergizing the potential of bacterial genomics and metabolomics to find novel antibiotics

Antibiotic development based on natural products has faced a long lasting decline since the 1970s, while both the speed and the extent of antimicrobial resistance (AMR) development have been severely underestimated. The discovery of antimicrobial natural products of bacterial and fungal origin featuring new chemistry and previously unknown mode of actions is increasingly challenged by rediscovery issues. Natural products that are abundantly produced by the corresponding wild type organisms often featuring strong UV signals have been extensively characterized, especially the ones produced by extensively screened microbial genera such as streptomycetes. Purely synthetic chemistry approaches aiming to replace the declining supply from natural products as starting materials to develop novel antibiotics largely failed to provide significant numbers of antibiotic drug leads. To cope with this fundamental issue, microbial natural products science is being transformed from a ‘grind-and-find’ study to an integrated approach based on bacterial genomics and metabolomics. Novel technologies in instrumental analytics are increasingly employed to lower detection limits and expand the space of detectable substance classes, while broadening the scope of accessible and potentially bioactive natural products. Furthermore, the almost exponential increase in publicly available bacterial genome data has shown that the biosynthetic potential of the investigated strains by far exceeds the amount of detected metabolites. This can be judged by the discrepancy between the number of biosynthetic gene clusters (BGC) encoded in the genome of each microbial strain and the number of secondary metabolites actually detected, even when considering the increased sensitivity provided by novel analytical instrumentation. In silico annotation tools for biosynthetic gene cluster classification and analysis allow fast prioritization in BGC-to-compound workflows, which is highly important to be able to process the enormous underlying data volumes. BGC prioritization is currently accompanied by novel molecular biology-based approaches to access the so-called orphan BGCs not yet correlated with a secondary metabolite. Integration of metabolomics, in silico genomics and molecular biology approaches into the mainstream of natural product research will critically influence future success and impact the natural product field in pharmaceutical, nutritional and agrochemical applications and especially in anti-infective research.

Antimicrobial resistance is a major public concern and novel antibiotics are largely based on natural products. We summarize recent analytical and genome based technological developments that gain increasing importance in the natural products field.     

Catalytic three-component C–C bond forming dearomatization of bromoarenes with malonates and diazo compounds

A Pd-catalyzed dearomative three-component C–C bond formation of bromoarenes with diazo compounds and malonates was developed. Various bromoarenes ranging from benzenoids to azines and heteroles were transformed to the corresponding substituted alicyclic molecules. The key to this reaction is the generation of a benzyl–palladium intermediate, which reacts with malonates to form a Pd–O-enolate species. Strikingly, the present method enabled rapid access to multi-substituted alicycles through subsequent elaboration of dearomatized products.

A catalytic three-component C–C bond forming dearomatization of bromoarenes was developed, enabling rapid access to multi-substituted alicycles.     

Deaminative meta-C–H alkylation by ruthenium(ii) catalysis

Precise structural modifications of amino acids are of importance to tune biological properties or modify therapeutical capabilities relevant to drug discovery. Herein, we report a ruthenium-catalyzed meta-C–H deaminative alkylation with easily accessible amino acid-derived Katritzky pyridinium salts. Likewise, remote C–H benzylations were accomplished with high levels of chemoselectivity and remarkable functional group tolerance. The meta-C–H activation approach combined with our deaminative strategy represents a rare example of selectively converting C(sp³)–N bonds into C(sp³)–C(sp²) bonds.

Precise structural modifications of amino acids are of importance to tune biological properties or modify therapeutical capabilities relevant to drug discovery.     

Nickel-catalyzed C–O/N–H,C–S/N–H,and C–CN/N–H annulation of aromatic amides with alkynes: C–O,C–S,and C–CN activation

The Ni-catalyzed reaction of ortho-phenoxy-substituted aromatic amides with alkynes in the presence of LiO^tBu as a base results in C–O/N–H annulation with the formation of 1(2H)-isoquinolinones. The use of a base is essential for the reaction to proceed. The reaction proceeds, even in the absence of a ligand, and under mild reaction conditions (40 °C). An electron-donating group on the aromatic ring facilitates the reaction. The reaction was also applicable to carbamate (C–O bond activation), methylthio (C–S bond activation), and cyano (C–CN bond activation) groups as leaving groups.

The Ni-catalyzed reaction of ortho-phenoxy-substituted aromatic amides with alkynes in the presence of LiO^tBu as a base results in C–O/N–H annulation with the formation of 1(2H)-isoquinolinones.     

Palladium-catalyzed direct asymmetric C–H bond functionalization enabled by the directing group strategy

In the past decade, selective C–C and C-heteroatom bond construction through palladium-catalyzed direct C–H bond functionalization has been extensively studied by employing a variety of directing groups. Within this category, direct asymmetric C(sp²)–H and C(sp³)–H activation for the construction of highly enantiomerically enriched skeletons still progressed at a slow pace. This minireview briefly introduces the major advances in the field for palladium-catalyzed direct asymmetric C–H bond functionalization via the directing group strategy.

This minireview introduces Pd-catalyzed direct asymmetric C–H functionalization reactions using a directing group strategy.     

Sequential C–O decarboxylative vinylation/C–H arylation of cyclic oxalates via a nickel-catalyzed multicomponent radical cascade

A selective, sequential C–O decarboxylative vinylation/C–H arylation of cyclic alcohol derivatives enabled by visible-light photoredox/nickel dual catalysis is described. This protocol utilizes a multicomponent radical cascade process, i.e. decarboxylative vinylation/1,5-HAT/aryl cross-coupling, to achieve efficient, site-selective dual-functionalization of saturated cyclic hydrocarbons in one single operation. This synergistic protocol provides straightforward access to sp³-enriched scaffolds and an alternative retrosynthetic disconnection to diversely functionalized saturated ring systems from the simple starting materials.

A selective, sequential C–O decarboxylative vinylation/C–H arylation of cyclic alcohol derivatives enabled by visible-light photoredox/nickel dual catalysis has been described.     

Ortho C–H arylation of arenes at room temperature using visible light ruthenium C–H activation

A ruthenium-catalyzed ortho C–H arylation process is described using visible light. Using the readily available catalyst [RuCl₂(p-cymene)]₂, visible light irradiation was found to enable arylation of 2-aryl-pyridines at room temperature for a range of aryl bromides and iodides.

A ruthenium-catalyzed ortho C–H arylation process is described using visible light.     

Discovery of branching meroterpenoid biosynthetic pathways in Aspergillus insuetus: involvement of two terpene cyclases with distinct cyclization modes

The aromatic polyketide 3,5-dimethylorsellinic acid (DMOA) serves as a precursor for many fungal meroterpenoids. A large portion of DMOA-derived meroterpenoids are biosynthesized via the cyclization of (6R,10′R)-epoxyfarnesyl-DMOA methyl ester (1). Theoretically, although 1 can be cyclized into many products, only three cyclization modes have been reported. Here, we discovered a meroterpenoid biosynthetic gene cluster in Aspergillus insuetus CBS 107.25, which encodes the biosynthetic enzymes for 1 along with a terpene cyclase that is phylogenetically distantly related to the other characterized cyclases of 1. Intriguingly, InsA7, the terpene cyclase, folds 1 in a pre-boat-chair conformation, generating a new meroterpenoid species with an axially oriented hydroxy group at C3. The A. insuetus strain also harbors an additional gene cluster encoding another cyclase of 1. The second terpene cyclase–InsB2–also synthesizes a new cyclized product of 1, thereby leading to diverging of the biosynthetic pathway in the fungus. Finally, we characterized the tailoring enzymes encoded by the two clusters, collectively obtained 17 new meroterpenoids, and successfully proposed biosynthetic routes leading to apparent end products of both pathways.

Branching meroterpenoid biosynthetic pathways were discovered in the fungus Aspergillus insuetus CBS 107.25, in which two terpene cyclases, InsA7 and InsB2, accept the same substrate but generate distinctly cyclized products.     

Magnesium-mediated sp3 C–H activation in cascade cyclization of 1-arylethynyl-2-alkyl-o-carboranes: efficient synthesis of carborane-fused cyclopentanes

This work reports an unprecedented cascade cyclization of 1-arylethynyl-2-alkyl-o-carboranes promoted by magnesium-mediated sp³ C–H activation. Treatment of 1-arylethynyl-2-alkyl-o-carboranes with MeMgBr gives a series of carborane-fused cyclopentanes in very good yields. Deuterium labelling and control experiments suggest that HMgBr, resulting in situ from the nucleophilic substitution of cage B–H bonds with Grignard reagent, initiates the reaction, in which magnesium-promoted intramolecular sp³ C–H activation serves as a key step. This work not only offers a new route for the synthesis of carborane-fused cyclopentanes, but also sheds some light on Mg-mediated C–H activation and functionalization.

An unprecedented cascade cyclization of 1-arylethynyl-2-alkyl-o-carboranes with Grignard reagent for synthesizing carborane-fused cyclopentanes has been disclosed, in which magnesium-mediated intramolecular sp³ C–H activation serves as a key step.     

Direct amidation of metallaaromatics: access to N-functionalized osmapentalynes via a 1,5-bromoamidated intermediate

The direct C–H amidation or imidation of metallaaromatics with N-bromoamides or imides has been achieved under mild conditions and leads to the formation of a family of N-functionalized metallapentalyne derivatives. A unique 1,5-bromoamidated species has been identified, and can be viewed as a σ^H-adduct intermediate in a nucleophilic aromatic substitution. The 1,5-addition of both electrophilic and nucleophilic moieties into the metallaaromatic framework demonstrates a novel pathway in contrast to the typical radical process of arene C–H amidation involving N-haloamide reagents.

The direct C–H amidation of metallapentalyne has been achieved under mild conditions in which key 1,5-bromoamidated intermediates was determined.     

Construction of polycyclic structures with vicinal all-carbon quaternary stereocenters via an enantioselective photoenolization/Diels–Alder reaction

All-carbon quaternary stereocenters are ubiquitous in natural products and significant in drug molecules. However, construction of all-carbon stereocenters is a challenging project due to their congested chemical environment. And, when vicinal all-carbon quaternary stereocenters are present in one molecule, they will dramatically increase its synthetic challenge. A chiral titanium promoted enantioselective photoenolization/Diels–Alder (PEDA) reaction allows largely stereohindered tetra-substituted dienophiles to interact with highly active photoenolized hydroxy-o-quinodimethanes, delivering fused or spiro polycyclic rings bearing vicinal all-carbon quaternary centers in excellent enantiomeric excess through one-step operation. This newly developed enantioselective PEDA reaction will inspire other advances in asymmetric excited-state reactions, and could be used in the total synthesis of structurally related complex natural products or drug-like molecules for drug discovery.

An enantioselective PEDA reaction was developed to enable stereohindered dienophiles to interact with transient photoenolized hydroxy-o-quinodimethanes, delivering fused or spiro polycyclic rings bearing 2–3 vicinal all-carbon quaternary centers in good yield and excellent ee.     

Chemical investigations into the biosynthesis of the gymnastatin and dankastatin alkaloids

Electrophilic natural products have provided fertile ground for understanding how nature inhibits protein function using covalent bond formation. The fungal strain Gymnascella dankaliensis has provided an especially interesting collection of halogenated cytotoxic agents derived from tyrosine which feature an array of reactive functional groups. Herein we explore chemical and potentially biosynthetic relationships between architecturally complex gymnastatin and dankastatin members, finding conditions that favor formation of a given scaffold from a common intermediate. Additionally, we find that multiple natural products can also be formed from aranorosin, a non-halogenated natural product also produced by Gymnascella sp. fungi, using simple chloride salts thus offering an alternative hypothesis for the origins of these compounds in nature. Finally, growth inhibitory activity of multiple members against human triple negative breast cancer cells is reported.

Total synthesis sheds light on biosynthetic relationships among the chlorinated gymnastatin and dankastatin alkaloids.     

Electrostatically-directed Pd-catalysis in combination with C–H activation: site-selective coupling of remote chlorides with fluoroarenes and fluoroheteroarenes

Systems incorporating catalyst–substrate non-covalent interactions are emerging as a versatile approach to address site-selectivity challenges in remote functionalization reactions. Given the achievements that have been made in this regard using metals such as iridium, manganese and rhodium, it is surprising that non-covalent catalyst direction has not been utilized in reactions incorporating palladium-catalyzed C–H activation steps, despite palladium being arguably the most versatile metal for C–H activation. Herein, we demonstrate that electrostatically directed, site-selective C–Cl oxidative addition is compatible with a subsequent C–H activation step, proceeding via a concerted metalation deprotonation-type mechanism. This results in site-selective cross-coupling of dichloroarenes with fluoroarenes and fluoroheteroarenes, with selectivity controlled by catalyst structure. This study demonstrates that Pd-catalyzed C–H activation can be used productively in combination with a non-covalently-directed mode of catalysis, with important implications in both fields.

Electrostatically-directed oxidative addition is compatible with a subsequent C–H activation step, enabling site-selective coupling of remote chlorides with fluoroarenes and fluoroheteroarenes.     

Total synthesis of biseokeaniamides A–C and late-stage electrochemically-enabled peptide analogue synthesis

The first total synthesis of cytotoxic cyanobacterial peptide natural products biseokeaniamides A–C is reported employing a robust solid-phase approach to peptide backbone construction followed by coupling of a key thiazole building block. To rapidly access natural product analogues, we have optimized an operationally simple electrochemical oxidative decarboxylation–nucleophilic addition pathway which exploits the reactivity of native C-terminal peptide carboxylates and abrogates the need for building block syntheses. Electrochemically-generated N,O-acetal intermediates are engaged with electron-rich aromatics and organometallic reagents to forge modified amino acids and peptides. The value of this late-stage modification method is highlighted by the expedient and divergent production of bioactive peptide analogues, including compounds which exhibit enhanced cytotoxicity relative to the biseokeaniamide natural products.

A late-stage electrochemical decarboxylation enables rapid access to structural analogues of biseokeaniamides A–C, cytotoxic lipopeptide natural products.