Biosynthetic access to the rare antiarose sugar via an unusual reductase-epimerase

Rubrolones, isatropolones, and rubterolones are recently isolated glycosylated tropolonids with notable biological activity. They share similar aglycone skeletons but differ in their sugar moieties, and rubterolones in particular have a rare deoxysugar antiarose of unknown biosynthetic provenance. During our previously reported biosynthetic elucidation of the tropolone ring and pyridine moiety, gene inactivation experiments revealed that RubS3 is involved in sugar moiety biosynthesis. Here we report the in vitro characterization of RubS3 as a bifunctional reductase/epimerase catalyzing the formation of TDP-d-antiarose by epimerization at C3 and reduction at C4 of the key intermediate TDP-4-keto-6-deoxy-d-glucose. These new findings not only explain the biosynthetic pathway of deoxysugars in rubrolone-like natural products, but also introduce RubS3 as a new family of reductase/epimerase enzymes with potential to supply the rare antiarose unit for expanding the chemical space of glycosylated natural products.

Rubrolones, isarubrolones, and rubterolones are recently isolated glycosylated tropolonids with notable biological activity.     

Discovery and biosynthesis of guanipiperazine from a NRPS-like pathway

Nonribosomal peptide synthetases (NRPSs) are modular enzymes that use a thiotemplate mechanism to assemble the peptide backbones of structurally diverse and biologically active natural products in bacteria and fungi. Unlike these canonical multi-modular NRPSs, single-module NRPS-like enzymes, which lack the key condensation (C) domain, are rare in bacteria, and have been largely unexplored to date. Here, we report the discovery of a gene cluster (gup) encoding a NRPS-like megasynthetase through genome mining. Heterologous expression of the gup cluster led to the production of two unprecedented alkaloids, guanipiperazines A and B. The NRPS-like enzyme activates two l-tyrosine molecules, reduces them to the corresponding amino aldehydes, and forms an unstable imine product. The subsequent enzymatic reduction affords piperazine, which can be morphed by a P450 monooxygenase into a highly strained compound through C–O bond formation. Further intermolecular oxidative coupling forming the C–C or C–O bond is catalyzed by another P450 enzyme. This work reveals the huge potential of NRPS-like biosynthetic gene clusters in the discovery of novel natural products.

Genome mining of a NRPS-like gene cluster led to the identification of two novel alkaloids with antimicrobial activity. This work reveals the huge potential of NRPS-like biosynthetic gene clusters in the discovery of novel natural products.     

New insights into the disulfide bond formation enzymes in epidithiodiketopiperazine alkaloids

Epidithiodiketopiperazines (ETPs) are a group of bioactive fungal natural products and structurally feature unique transannular disulfide bridges between α, α or α, β carbons. However, no enzyme has yet been demonstrated to catalyse α, β-disulfide bond formation in these molecules. Through genome mining and gene deletion approaches in Trichoderma hypoxylon, we identified a putative biosynthetic gene cluster of pretrichodermamide A (1), which requires a FAD-dependent oxidoreductase, TdaR, for the irregular α, β-disulfide formation in 1 biosynthesis. In vitro assays of TdaR, together with AclT involved in aspirochlorine and GliT involved in gliotoxin biosynthesis, proved that all three enzymes catalyse not only the conversion of red-pretrichodermamide A (4) to α, β-disulfide-containing 1 but also that of red-gliotoxin (5) to α, α-disulfide-containing gliotoxin (6). These results provide new insights into the thiol-disulfide oxidases responsible for the disulfide bond formation in natural products with significant substrate and catalytic promiscuities.

A FAD-dependent oxidoreductase TdaR was responsible for α, β-disulfide formation in the biosynthesis of pretrichodermamide A. TdaR, together with its homologs AclT and GliT, catalysed not only α, α- but also α, β-disulfide formation in fungi.     

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 asymmetric multiple dearomatizations of phenols enabled by a cascade 1,8-addition and Diels–Alder reaction

A direct catalytic asymmetric multiple dearomatization reaction of phenols was disclosed, which provides expedient access to a series of architecturally complex polycyclic compounds bearing four stereogenic centers in high enantiopurity. The key to achieve such a transformation is the combination of a dearomative 1,8-addition of β-naphthols to para-quinone methides generated in situ from propargylic alcohols and a subsequent intramolecular dearomative Diels–Alder reaction. Noteworthily, this protocol enrichs not only the diversity of dearomatized products but also the toolbox of dearomatization strategies.

The first chiral phosphoric acid catalyzed asymmetric multiple dearomatizations of phenols for the synthesis of bridged polycyclic compounds are reported.     

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.     

Uncovering biosynthetic relationships between antifungal nonadrides and octadrides

Maleidrides are a class of bioactive secondary metabolites unique to filamentous fungi, which contain one or more maleic anhydrides fused to a 7-, 8- or 9- membered carbocycle (named heptadrides, octadrides and nonadrides respectively). Herein structural and biosynthetic studies on the antifungal octadride, zopfiellin, and nonadrides scytalidin, deoxyscytalidin and castaneiolide are described. A combination of genome sequencing, bioinformatic analyses, gene disruptions, biotransformations, isotopic feeding studies, NMR and X-ray crystallography revealed that they share a common biosynthetic pathway, diverging only after the nonadride deoxyscytalidin. 5-Hydroxylation of deoxyscytalidin occurs prior to ring contraction in the zopfiellin pathway of Diffractella curvata. In Scytalidium album, 6-hydroxylation – confirmed as being catalysed by the α-ketoglutarate dependent oxidoreductase ScyL2 – converts deoxyscytalidin to scytalidin, in the final step in the scytalidin pathway. Feeding scytalidin to a zopfiellin PKS knockout strain led to the production of the nonadride castaneiolide and two novel ring-open maleidrides.

Deoxyscytalidin is a common biosynthetic intermediate to the nonadride scytalidin in the fungus Scytalidium album and in Diffractella curvata gives the octadride zopfiellin.     

Biosynthesis of the fungal glyceraldehyde-3-phosphate dehydrogenase inhibitor heptelidic acid and mechanism of self-resistance

Overcoming resistance to bioactive small molecules is a significant challenge for health care and agriculture. As a result, efforts to uncover the mechanisms of resistance are essential to the development of new antibiotics, anticancer drugs and pesticides. To study how nature evolves resistance to highly potent natural products, we examined the biosynthesis and mechanism of self-resistance of the fungal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibitor heptelidic acid (HA). HA is a nanomolar inhibitor of GADPH through the covalent modification of the active site cysteine thiol. The biosynthetic pathway of HA was elucidated, which uncovered the enzymatic basis of formation of the epoxide warhead. Structure–activity relationship study using biosynthetic intermediates established the importance of the fused lactone ring system in HA. The molecular basis of HA inhibiting human GAPDH was illustrated through the crystal structure of Hs-GAPDH covalently bound with HA. A GAPDH isozyme HepG encoded in the HA cluster was characterized to be less sensitive to HA, and therefore contribute to self-resistance for the producing host. Comparison of the crystal structures of human GAPDH and HepG showed mutations both within and remote to the active site can contribute to resistance of inactivation, which was confirmed through mutagenesis. Due to the critical role GAPDH plays in aerobic glycolysis and other cellular functions, knowledge of HA mode of action and self-resistance mechanism could accelerate the development of improved inhibitors.

The structural basis and self-resistance mechanism of fungal glyceraldehyde-3-phosphate dehydrogenase inhibitor heptelidic acid are uncovered.     

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.     

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.     

Unequivocal structure confirmation of a breitfussin analog by anisotropic NMR measurements

Structural features of proton-deficient heteroaromatic natural products, such as the breitfussins, can severely complicate their characterization by NMR spectroscopy. For the breitfussins in particular, the constitution of the five-membered oxazole central ring cannot be unequivocally established via conventional NMR methods when the 4′-position is halogenated. The level of difficulty is exacerbated by 4′-iodination, as the accuracy with which theoretical NMR parameters are determined relies extensively on computational treatment of the relativistic effects of the iodine atom. It is demonstrated in the present study, that the structure of a 4′-iodo breitfussin analog can be unequivocally established by anisotropic NMR methods, by adopting a reduced singular value decomposition (SVD) protocol that leverages the planar structures exhibited by its conformers.

Structural features of proton-deficient heteroaromatic natural products, such as the breitfussins, can severely complicate their characterization by NMR spectroscopy.     

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.     

Cleavable and tunable cysteine-specific arylation modification with aryl thioethers

Cysteine represents an attractive target for peptide/protein modification due to the intrinsic high nucleophilicity of the thiol group and low natural abundance. Herein, a cleavable and tunable covalent modification approach for cysteine containing peptides/proteins with our newly designed aryl thioethers via a S_NAr approach was developed. Highly efficient and selective bioconjugation reactions can be carried out under mild and biocompatible conditions. A series of aryl groups bearing different bioconjugation handles, affinity or fluorescent tags are well tolerated. By adjusting the skeleton and steric hindrance of aryl thioethers slightly, the modified products showed a tunable profile for the regeneration of the native peptides.

A cleavable and tunable covalent modification approach for cysteine by aryl thioethers via a S_NAr approach was developed. The highly efficient and selective bioconjugation reactions can proceed under the mild and biocompatible conditions.     

Pre-regulation of the planar chirality of pillar[5]arenes for preparing discrete chiral nanotubes

Regulating the chirality of macrocyclic host molecules and supramolecular assemblies is crucial because chirality often plays a role in governing the properties of these systems. Herein, we describe pillar[5]arene-based chiral nanotube formation via pre-regulation of the building blocks'' chirality, which is different from frequently used post-regulation strategies. The planar chirality of rim-differentiated pillar[5]arenes is initially regulated by chiral awakening and further induction/inversion through stepwise achiral external stimuli. The pre-regulated chiral information is well stored in discrete nanotubes by interacting with a per-alkylamino-substituted pillar[5]arene. Such pre-regulation is more efficient than post-regulating the chirality of nanotubes.

Pillar[5]arene-based chiral nanotube formation via pre-regulation of the building blocks'' chirality is more efficient than post-regulating the chirality of nanotubes.     

Asymmetric synthesis of dihydro-1,3-dioxepines by Rh(ii)/Sm(iii) relay catalytic three-component tandem [4 + 3]-cycloaddition

Heterocycles have been widely used in organic synthesis, agrochemical, pharmaceutical and materials science industries. Catalytic three-component ylide formation/cycloaddition enables the assembly of complex heterocycles from simple starting materials in a highly efficient manner. However, asymmetric versions remain a yet-unsolved task. Here, we present a new bimetallic catalytic system for tackling this challenge. A combined system of Rh(ii) salt and chiral N,N′-dioxide–Sm(iii) complex was established for promoting the unprecedented tandem carbonyl ylide formation/asymmetric [4 + 3]-cycloaddition of aldehydes and α-diazoacetates with β,γ-unsaturated α-ketoesters smoothly, affording various chiral 4,5-dihydro-1,3-dioxepines in up to 97% yield, with 99% ee. The utility of the current method was demonstrated by conversion of products to optically active multi-substituted tetrahydrofuran derivatives. A possible reaction mechanism was provided to elucidate the origin of chiral induction based on experimental studies and X-ray structures of catalysts and products.

Catalytic asymmetric tandem carbonyl ylide formation/[4 + 3]-cycloaddition of β,γ-unsaturated α-ketoesters, aldehydes and α-diazoacetates was achieved by using a bimetallic rhodium(ii)/chiral N,N′-dioxide–Sm(iii) complex catalyst.     

Mixing and matching genes of marine and terrestrial origin in the biosynthesis of the mupirocin antibiotics

With growing understanding of the underlying pathways of polyketide biosynthesis, along with the continual expansion of the synthetic biology toolkit, it is becoming possible to rationally engineer and fine-tune the polyketide biosynthetic machinery for production of new compounds with improved properties such as stability and/or bioactivity. However, engineering the pathway to the thiomarinol antibiotics has proved challenging. Here we report that genes from a marine Pseudoalternomonas sp. producing thiomarinol can be expressed in functional form in the biosynthesis of the clinically important antibiotic mupirocin from the soil bacterium Pseudomonas fluorescens. It is revealed that both pathways employ the same unusual mechanism of tetrahydropyran (THP) ring formation and the enzymes are cross compatible. Furthermore, the efficiency of downstream processing of 10,11-epoxy versus 10,11-alkenic metabolites are comparable. Optimisation of the fermentation conditions in an engineered strain in which production of pseudomonic acid A (with the 10,11-epoxide) is replaced by substantial titres of the more stable pseudomonic acid C (with a 10,11-alkene) pave the way for its development as a more stable antibiotic with wider applications than mupirocin.

Where the sea meets the land: the mupirocin biosynthetic gene cluster (BGC) from the terrestrial bacterium Pseudomonas fluorescens was repurposed via a plug-and-play approach with heterologous genes from the marine strain that produces thiomarinol.     

Salient features of the aza-Wacker cyclization reaction

The intramolecular aza-Wacker reaction has unparalleled potential for the site-selective amination of olefins, but it is perhaps underappreciated relative to other alkene oxidations. The first part of this review makes the distinction between classical and tethered aza-Wacker cyclization reactions and summarizes examples of the latter. The second portion focuses on developments in asymmetric aza-Wacker cyclization technology. The final part of the review summarizes applications of all classes of aza-Wacker cyclization reactions to natural product assembly.

The aza-Wacker cyclization reaction is a powerful strategy for alkene amination.     

A chemical circular communication network at the nanoscale

In nature, coordinated communication between different entities enables a group to accomplish sophisticated functionalities that go beyond those carried out by individual agents. The possibility of programming and developing coordinated communication networks at the nanoscale—based on the exchange of chemical messengers—may open new approaches in biomedical and communication areas. Here, a stimulus-responsive circular model of communication between three nanodevices based on enzyme-functionalized Janus Au–mesoporous silica capped nanoparticles is presented. The output in the community of nanoparticles is only observed after a hierarchically programmed flow of chemical information between the members.

A community of three nanodevices communicates through a hierarchically programmed circular flow of chemical information between members.     

Expedient synthesis of conjugated triynes via alkyne metathesis

The first synthesis of conjugated triynes by molybdenum-catalysed alkyne metathesis is reported. Strategic to the success of this approach is the utilization of sterically-hindered diynes that allowed for the site-selective alkyne metathesis to produce the desired conjugated triyne products. The steric hindrance of the alkyne moiety was found to be crucial in preventing the formation of diyne byproducts. This novel synthetic strategy was amenable to self- and cross-metathesis providing straightforward access to the corresponding symmetrical and dissymmetrical triynes with high selectivity.

The first synthesis of symmetrical and dissymmetrical conjugated triynes by self- and cross-metathesis was successfully achieved thanks to the use of hindered diynes.     

Kinetic resolution of racemic allylic alcohols via iridium-catalyzed asymmetric hydrogenation: scope,synthetic applications and insight into the origin of selectivity

Asymmetric hydrogenation is one of the most commonly used tools in organic synthesis, whereas, kinetic resolution via asymmetric hydrogenation is less developed. Herein, we describe the first iridium catalyzed kinetic resolution of a wide range of trisubstituted secondary and tertiary allylic alcohols. Large selectivity factors were observed in most cases (s up to 211), providing the unreacted starting materials in good yield with high levels of enantiopurity (ee up to >99%). The utility of this method is highlighted in the enantioselective formal synthesis of some bioactive natural products including pumiliotoxin A, inthomycin A and B. DFT studies and a selectivity model concerning the origin of selectivity are presented.

Asymmetric hydrogenation is one of the most commonly used tools in organic synthesis, whereas, kinetic resolution via asymmetric hydrogenation was less developed.