Total Synthesis of Crocagin A

Crocagin A ( 1 ) combines an attractive molecular structure with an unusual biosynthesis and bioactivity. An efficient synthesis of crocagin A is presented that hinges on an early formation of the heterotricyclic core, an electrophilic amination, and the stereoselective hydrogenation of a tetrasubstituted double bond. This synthesis confirms the absolute configuration of crocagin A and provides access to the natural product and derivatives thereof for further biological testing.     

Discovery and characterisation of an amidine-containing ribosomally-synthesised peptide that is widely distributed in nature

Ribosomally synthesised and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural product with a wide range of bioactivities. Genome mining for RiPP biosynthetic gene clusters (BGCs) is often hampered by poor annotation of the short precursor peptides that are ultimately modified into the final molecule. Here, we utilise a previously described genome mining tool, RiPPER, to identify novel RiPP precursor peptides near YcaO-domain proteins, enzymes that catalyse various RiPP post-translational modifications including heterocyclisation and thioamidation. Using this dataset, we identified a novel and diverse family of RiPP BGCs spanning over 230 species of Actinobacteria and Firmicutes. A representative BGC from Streptomyces albidoflavus J1074 (formerly known as Streptomyces albus) was characterised, leading to the discovery of streptamidine, a novel amidine-containing RiPP. This new BGC family highlights the breadth of unexplored natural products with structurally rare features, even in model organisms.

Genome mining for pathways containing YcaO proteins revealed a widespread novel family of RiPP gene clusters. A model gene cluster was characterised through genetic and chemical analyses, which yielded streptamidine, a novel amidine-containing RiPP.     

Discovery of a Cryptic Depsipeptide from Streptomyces ghanaensis via MALDI-MS-Guided High-Throughput Elicitor Screening

Microbial genomes harbor an abundance of biosynthetic gene clusters, but most are expressed at low levels and need to be activated for characterization of their cognate natural products. In this work, we report the combination of high-throughput elicitor screening (HiTES) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the rapid identification of cryptic peptide natural products. Application to Streptomyces ghanaensis identified amygdalin as an elicitor of a novel non-ribosomal peptide, which we term cinnapeptin. Complete structural elucidation revealed cinnapeptin as a cyclic depsipeptide with an unusual 2-methyl-cinnamoyl group. Insights into its biosynthesis were provided by whole genome sequencing and gene deletion studies, while bioactivity assays showed antimicrobial activity against Gram-positive bacteria and fission yeast. MALDI-HiTES is a broadly applicable tool for the discovery of cryptic peptides encoded in microbial genomes.     

Recently Discovered Secondary Metabolites from Streptomyces Species

The Streptomyces genus has been a rich source of bioactive natural products, medicinal chemicals, and novel drug leads for three-quarters of a century. Yet studies suggest that the genus is capable of making some 150,000 more bioactive compounds than all Streptomyces secondary metabolites reported to date. Researchers around the world continue to explore this enormous potential using a range of strategies including modification of culture conditions, bioinformatics and genome mining, heterologous expression, and other approaches to cryptic biosynthetic gene cluster activation. Our survey of the recent literature, with a particular focus on the year 2020, brings together more than 70 novel secondary metabolites from Streptomyces species, which are discussed in this review. This diverse array includes cyclic and linear peptides, peptide derivatives, polyketides, terpenoids, polyaromatics, macrocycles, and furans, the isolation, chemical structures, and bioactivity of which are appraised. The discovery of these many different compounds demonstrates the continued potential of Streptomyces as a source of new and interesting natural products and contributes further important pieces to the mostly unfinished puzzle of Earth’s myriad microbes and their multifaceted chemical output.     

Type S Non-Ribosomal Peptide Synthetases for the Rapid Generation of Tailormade Peptide Libraries**

Bacterial natural products in general, and non-ribosomally synthesized peptides in particular, are structurally diverse and provide us with a broad range of pharmaceutically relevant bioactivities. Yet, traditional natural product research suffers from rediscovering the same scaffolds and has been stigmatized as inefficient, time-, labour- and cost-intensive. Combinatorial chemistry, on the other hand, can produce new molecules in greater numbers, cheaper and in less time than traditional natural product discovery, but also fails to meet current medical needs due to the limited biologically relevant chemical space that can be addressed. Consequently, methods for the high throughput generation of new natural products would offer a new approach to identifying novel bioactive chemical entities for the hit to lead phase of drug discovery programs. As a follow-up to our previously published proof-of-principle study on generating bipartite type S non-ribosomal peptide synthetases (NRPSs), we now envisaged the de novo generation of non-ribosomal peptides (NRPs) on an unreached scale. Using synthetic zippers, we split NRPSs in up to three subunits and rapidly generated different bi- and tripartite NRPS libraries to produce 49 peptides, peptide derivatives, and de novo peptides at good titres up to 145 mg L⁻¹. A further advantage of type S NRPSs not only is the possibility to easily expand the created libraries by re-using previously created type S NRPS, but that functions of individual domains as well as domain-domain interactions can be studied and assigned rapidly.     

Linking chemistry and genetics in the growing cyanobactin natural products family

Ribosomal peptide natural products are ubiquitous, yet relatively few tools exist to predict structures and clone new pathways. Cyanobactin ribosomal peptides are found in ~30% of all cyanobacteria, but the connection between gene sequence and structure was not defined, limiting the rapid identification of new compounds and pathways. Here, we report discovery of four orphan cyanobactin gene clusters by genome mining and an additional pathway by targeted cloning, which represented a tyrosine O-prenylating biosynthetic pathway. Genome mining enabled discovery of five cyanobactins, including peptide natural products from Spirulina supplements. A phylogenetic model defined four cyanobactin genotypes, which explain the synthesis of multiple cyanobactin structural classes and help direct pathway cloning and structure prediction efforts. These strategies were applied to DNA isolated from a mixed cyanobacterial bloom containing cyanobactins.     

Conversion of L-proline to pyrrolyl-2-carboxyl-S-PCP during undecylprodigiosin and pyoluteorin biosynthesis

Several medically and agriculturally important natural products contain pyrrole moieties. Precursor labeling studies of some of these natural products have shown that L-proline can serve as the biosynthetic precursor for these moieties, including those found in coumermycin A(1), pyoluteorin, and one of the pyrroles of undecylprodigiosin. This suggests a novel mechanism for pyrrole biosynthesis. The biosynthetic gene clusters for these three natural products each encode proteins homologous to adenylation (A) and peptidyl carrier protein (PCP) domains of nonribosomal peptide synthetases in addition to novel acyl-CoA dehydrogenases. Here we show that the three proteins from the undecylprodigiosin and pyoluteorin biosynthetic pathways are sufficient for the conversion of L-proline to pyrrolyl-2-carboxyl-S-PCP. This establishes a novel mechanism for pyrrole biosynthesis and extends the hypothesis that organisms use A/PCP pairs to partition an amino acid into secondary metabolism.     

Highly efficient biomimetic total synthesis and structural verification of bistratamides E and J from Lissoclinum bistratum

The interesting biological activities of heterocycle-containing cyclic peptide-derived natural products, isolated from marine organisms over the past twenty years, have attracted the interest of many synthetic and natural products chemists. Bistratamides E-J, members of this class of natural products that were isolated very recently from Lissoclinum bistratum, exhibited cytotoxic activity against a human colon tumor (HCT-116) cell line. Here we report the first total syntheses of bistratamides E (1) and J (2) in overall yields of 19 and 34 %, respectively. The thiazole substructures have been synthesized by oxidation of their corresponding thiazoline substructures, which were obtained from cysteine containing peptides using a novel biomimetic approach wherein Val-Cys dipeptide units were converted to thiazolines by a bisphosphonium salt. The final macrocyclization was promoted efficiently using the combination of PyBOP and DMAP. This approach allows the use of readily available Fmoc-protected amino acids to make complex thiazole and oxazoline-containing natural products.     

Enzymology of acyl chain macrocyclization in natural product biosynthesis

Polyketides and nonribosomal peptides constitute a large and diverse set of natural products with biological activity in microbial survival and pathogenesis, as well as broad pharmacological utility as antineoplastics, antibiotics or immunosupressants. These molecules are biosynthesized by the ordered condensation of monomer building blocks, acyl-CoAs or amino acids, leading to construction of linear acyl chains. Many of these natural products are constrained to their bioactive conformations by macrocyclization whereby, in one of the terminal steps of biosynthesis, parts of the molecule distant in the constructed linear acyl chain are covalently linked to one another. Typically, macrocyclization is catalyzed by a thioesterase domain at the C-terminal end of the biosynthetic assembly line, although alternative strategies are known. The enzymology of these macrocyclization catalysts, their structure, mechanism, and catalytic versatility, is the subject of this review. The diversity of macrocyclic structures accessed by enzyme catalyzed cyclization of linear acyl chains as well as their inherent substrate tolerance suggests their potential utility in reprogramming natural product biosynthesis pathways or accessing novel macrocyclic structures.     

Peptide Fragment Ion Analyser (PFIA): a simple and versatile tool for the interpretation of tandem mass spectrometric data and de novo sequencing of peptides

The software Peptide Fragment Ion Analyser (PFIA) aids in the analysis and interpretation of tandem mass spectrometric data of peptides. The software package has been designed to facilitate the analysis of product ions derived from acyclic and cyclic peptide natural products that possess unusual amino acid residues and are heavily post-translationally modified. The software consists of two programmes: (a) PFIA-I lists the amino acid compositions and their corresponding product ion types for 'a queried m/z value' (z = +1) and (b) PFIA-II displays fragmentation pattern diagram(s) and lists all sequence-specific product ion types for the protonated adduct of 'a queried sequence'. The unique feature of PFIA-II is its ability to handle cyclic peptides. The two programmes used in combination can prove helpful for deriving peak assignments in the de novo sequencing of novel peptides.     

Synthesis of Biaryl-Bridged Cyclic Peptides via Catalytic Oxidative Cross-Coupling Reactions

Biaryl-bridged cyclic peptides comprise an intriguing class of structurally diverse natural products with significant biological activity. Especially noteworthy are the antibiotics arylomycin and its synthetic analogue G0775, which exhibits potent activity against Gram-negative bacteria. Herein, we present a simple, flexible, and reliable strategy based on activating-group-assisted catalytic oxidative coupling for assembling biaryl-bridged cyclic peptides from natural amino acids. The synthetic approach was utilized for preparing a number of natural and unnatural biaryl-bridged cyclic peptides, including arylomycin/G0775 and RP 66453 cyclic cores.     

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.     

CLV3/ESR-related (CLE) peptides as intercellular signaling molecules in plants

For many years, the plant hormones auxin, cytokinin, ethylene, gibberellin, abscisic acid, brassinosteroid, jasmonic acid, and salicylic acid have been extensively studied as key regulators of plant growth and development. However, recent biochemical and genetic analyses have revealed that secretory peptides are also responsible for intercellular signaling in plants and regulate various events including wound response, cell division control, and pollen self-incompatibility. We discovered two natural CLAVATA3 (CLV3)/ESR-related (CLE) peptides: tracheary elements differentiation inhibitory factor (TDIF) and CLV3, which are dodecapeptides with two hydroxyproline residues that regulate vascular development and meristem formation, respectively. This discovery enabled us to predict the chemical form of CLE gene products. In the Arabidopsis genome, there are 31 CLE genes that correspond to 26 CLE peptides. The application of all 26 chemically synthesized peptides to plants revealed the existence of distinctive functional groups. From these results, we discuss the functions of CLE peptides in plant development and plant-parasite interactions.     

Characterization of recombinant human serum albumin using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Chromatographically purified recombinant human serum albumin (rHSA), produced in genetically transformed yeast cells, was characterized using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS techniques. The molecular mass of the intact protein was determined to be 66671, in good agreement with that of purified HSA which was used as a standard. The identity of rHSA to its natural counterpart was established with high precision using peptide mass fingerprinting of tryptic peptides. Partial amino acid sequence data for rHSA were obtained using Ettan CAF MALDI Sequencing Kit and post-source decay on the tryptic peptides. The results achieved provide strong evidence that MALDI-TOF-MS is an important analytical technique for characterising gene products and for establishing the identity and bio-compatibility of recombinant proteins relative to their natural counterparts.     

Robust platform for de novo production of heterologous polyketides and nonribosomal peptides in Escherichia coli

During the past decade, numerous gene clusters responsible for the biosynthesis of important natural products have been identified from a variety of organisms. Heterologous expression utilizing E. coli has been employed to provide proteins for mechanistic understanding and structural analyses. It was very recently shown that this system is also capable of de novo production of biologically active forms of heterologous nonribosomal peptides, echinomycin and triostin A, through the introduction of genes encoding modules responsible for their assembly into this model bacterial host. The superlative advantage of using E. coli as a heterologous host is the availability of a wealth of well-established molecular biological techniques for its genetic and metabolic manipulation. The platform described above which was developed in our laboratory is ideal for use in the production of metabolites found in marine and symbiotic bacteria that are not amenable to artificial cultivation. Development and tailoring of our system will allow for the design of these natural products and ultimately combinatorial yet rational modification of these compounds. This review focuses on the heterologous expression of biosynthetic gene clusters for the assembly of therapeutically potent compounds.     

New Scabimycins A-C Isolated from Streptomyces acidiscabies (Lu19992)

Peptide natural products displaying a wide range of biological activities have become important drug candidates over the years. Microorganisms have been a powerful source of such bioactive peptides, and Streptomyces have yielded many novel natural products thus far. In an effort to uncover such new, meaningful compounds, the metabolome of Streptomyces acidiscabies was analyzed thoroughly. Three new compounds, scabimycins A–C (1–3), were discovered, and their chemical structures were elucidated by NMR spectroscopy. The relative and absolute configurations were determined using ROESY NMR experiments and advanced Marfey’s method.     

Lasso peptides: structure, function, biosynthesis, and engineering

Covering: up to May 2012Lasso peptides are a class of ribosomally-synthesized and posttranslationally-modified natural products with diverse bioactivities. This review describes the structure and function of all known lasso peptides (as of mid-2012) and covers our current knowledge about the biosynthesis of those molecules. The isolation and characterization of lasso peptides are also covered as are bioinformatics strategies for the discovery of new lasso peptides from genomic sequence data. Several studies on the engineering of new or improved function into lasso peptides are highlighted, and unanswered questions in the field are also described.     

NosA catalyzing carboxyl-terminal amide formation in nosiheptide maturation via an enamine dealkylation on the serine-extended precursor peptide

The carboxyl-terminal amide group has been often found in many bioactive peptide natural products, including nosiheptide belonging to the over 80 entity-containing thiopeptide family. Upon functional characterization of a novel protein NosA in nosiheptide biosynthesis, herein we report an unusual C-terminal amide forming strategy in general for maturating certain amide-terminated thiopeptides by processing their precursor peptides featuring a serine extension. NosA acts on an intermediate bearing a bis-dehydroalanine tail and catalyzes an enamide dealkylation to remove the acrylate unit originating from the extended serine residue.     

Structure and Biosynthesis of Xenoamicins from Entomopathogenic Xenorhabdus

During the search for novel natural products from entomopathogenic Xenorhabdus doucetiae DSM17909 and X. mauleonii DSM17908 novel peptides named xenoamicins were identified in addition to the already known antibiotics xenocoumacin and xenorhabdin. Xenoamicins are acylated tridecadepsipeptides consisting of mainly hydrophobic amino acids. The main derivative xenoamicin A ( 1 ) was isolated from X. mauleonii DSM17908, and its structure elucidated by detailed 1 D and 2 D NMR experiments. Detailed MS experiments, also in combination with labeling experiments, confirmed the determined structure and allowed structure elucidation of additional derivatives. Moreover, the xenoamicin biosynthesis gene cluster was identified and analyzed in X. doucetiae DSM17909, and its participation in xenoamicin biosynthesis was confirmed by mutagenesis. Advanced Marfey’s analysis of 1 showed that the absolute configuration of the amino acids is in agreement with the predicted stereochemistry deduced from the nonribosomal peptide synthetase XabABCD. Biological testing revealed activity of 1 against Plasmodium falciparum and other neglected tropical diseases but no antibacterial activity.     

Synthesis of Biaryl‐Bridged Cyclic Peptides via Catalytic Oxidative Cross‐Coupling Reactions

Biaryl‐bridged cyclic peptides comprise an intriguing class of structurally diverse natural products with significant biological activity. Especially noteworthy are the antibiotics arylomycin and its synthetic analogue G0775, which exhibits potent activity against Gram‐negative bacteria. Herein, we present a simple, flexible, and reliable strategy based on activating‐group‐assisted catalytic oxidative coupling for assembling biaryl‐bridged cyclic peptides from natural amino acids. The synthetic approach was utilized for preparing a number of natural and unnatural biaryl‐bridged cyclic peptides, including arylomycin/G0775 and RP 66453 cyclic cores.