Cyclic and Lasso Peptides: Sequence Determination,Topology Analysis,and Rotaxane Formation

A broadly applicable chemical cleavage methodology to facilitate MS/MS sequencing was developed for macrocyclic and lasso peptides, which hold promise as exciting new therapeutics. Existing methods such as Edman degradation, CNBr cleavage, and enzymatic digestion are either limited in scope or completely fail in cleavage of constrained nonribosomal peptides. Importantly, the new method was utilized for synthesizing a unique peptide‐based rotaxane (both cyclic and threaded) from the lasso peptide, benenodin‐1 ΔC5.     

Cyclic and Lasso Peptides: Sequence Determination,Topology Analysis,and Rotaxane Formation

A broadly applicable chemical cleavage methodology to facilitate MS/MS sequencing was developed for macrocyclic and lasso peptides, which hold promise as exciting new therapeutics. Existing methods such as Edman degradation, CNBr cleavage, and enzymatic digestion are either limited in scope or completely fail in cleavage of constrained nonribosomal peptides. Importantly, the new method was utilized for synthesizing a unique peptide‐based rotaxane (both cyclic and threaded) from the lasso peptide, benenodin‐1 ΔC5.     

Cyclization mechanism for the synthesis of macrocyclic antibiotic lankacidin in Streptomyces rochei

The lankacidin biosynthetic gene cluster in Streptomyces rochei strain 7434AN4 was found to span 31 kb of the giant linear plasmid pSLA2-L and contain a polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) hybrid gene (lkcA), type I PKS genes, and pyrroloquinoline quinone (PQQ) biosynthetic genes (lkcK-lkcO). Feeding of PQQ to a pqq mutant restored the lankacidin production, suggesting its crucial role in an oxidation process. However, formation of the 17-membered macrocyclic ring was not catalyzed by PQQ-dependent dehydrogenase (Orf23), but was by flavin-dependent amine oxidase (LkcE). Compound LC-KA05 isolated from an lkcE disruptant was an acyclic intermediate lacking the C2-C18 linkage. These results suggested a cyclization mechanism for the synthesis of the lankacidin macrocyclic skeleton.     

Natural product-like macrocyclic N-methyl-peptide inhibitors against a ubiquitin ligase uncovered from a ribosome-expressed de novo library

Naturally occurring peptides often possess macrocyclic and N-methylated backbone. These features grant them structural rigidity, high affinity to targets, proteolytic resistance, and occasionally membrane permeability. Because such peptides are produced by either nonribosomal peptide synthetases or enzymatic posttranslational modifications, it is yet a formidable challenge in degenerating sequence or length and preparing libraries for screening bioactive molecules. Here, we report a new means of synthesizing a de novo library of “natural product-like” macrocyclic N-methyl-peptides using translation machinery under the reprogrammed genetic code, which is coupled with an in vitro display technique, referred to as RaPID (random nonstandard peptides integrated discovery) system. This system allows for rapid selection of strong binders against an arbitrarily chosen therapeutic target. Here, we have demonstrated the selection of anti-E6AP macrocyclic N-methyl-peptides, one of which strongly inhibits polyubiqutination of proteins such as p53.     

Posttranslationally Acting Arginases Provide a Ribosomal Route to Non-proteinogenic Ornithine Residues in Diverse Peptide Sequences

Ornithine is a component of many bioactive nonribosomal peptides but is challenging to incorporate into ribosomal products. We recently identified OspR, a cyanobacterial arginase-like enzyme that installs ornithines in the antiviral ribosomally synthesised and posttranslationally modified peptide (RiPP) landornamide A. Here we report that OspR belongs to a larger family of peptide arginases from diverse organisms and RiPP types. In E. coli, seven selected enzymes converted arginine into ornithine with little preference for the leader type. A broad range of peptide sequences was modified, including polyarginine repeats. We also generated analogues of ornithine-containing nonribosomal peptides using RiPP technology. Five pseudo-nonribosomal products with ornithines at the correct positions were obtained, including a brevicidine analogue containing ornithine and a d -amino acid installed by the peptide epimerase OspD. These results suggest new opportunities for peptide bioengineering.     

Biosynthetic Strategies for Macrocyclic Peptides

Macrocyclic peptides are predominantly peptide structures bearing one or more rings and spanning multiple amino acid residues. Macrocyclization has become a common approach for improving the pharmacological properties and bioactivity of peptides. A variety of ribosomal-derived and non-ribosomal synthesized cyclization approaches have been established. The biosynthesis of backbone macrocyclic peptides using seven new emerging methodologies will be discussed with regard to the features and strengths of each platform rather than medicinal chemistry tools. The mRNA display variant, known as the random nonstandard peptide integrated discovery (RaPID) platform, utilizes flexible in vitro translation (FIT) to access macrocyclic peptides containing nonproteinogenic amino acids (NAAs). As a new discovery approach, the ribosomally synthesized and post-translationally modified peptides (RiPPs) method involves the combination of ribosomal synthesis and the phage screening platform together with macrocyclization chemistries to generate libraries of macrocyclic peptides. Meanwhile, the split-intein circular ligation of peptides and proteins (SICLOPPS) approach relies on the in vivo production of macrocyclic peptides. In vitro and in vivo peptide library screening is discussed as an advanced strategy for cyclic peptide selection. Specifically, biosynthetic bicyclic peptides are highlighted as versatile and attractive modalities. Bicyclic peptides represent another type of promising therapeutics that allow for building blocks with a heterotrimeric conjugate to address intractable challenges and enable multimer complexes via linkers. Additionally, we discuss the cell-free chemoenzymatic synthesis of macrocyclic peptides with a non-ribosomal catalase known as the non-ribosomal synthetase (NRPS) and chemo-enzymatic approach, with recombinant thioesterase (TE) domains. Novel insights into the use of peptide library tools, activity-based two-hybrid screening, structure diversification, inclusion of NAAs, combinatorial libraries, expanding the toolbox for macrocyclic peptides, bicyclic peptides, chemoenzymatic strategies, and future perspectives are presented. This review highlights the broad spectrum of strategy classes, novel platforms, structure diversity, chemical space, and functionalities of macrocyclic peptides enabled by emerging biosynthetic platforms to achieve bioactivity and for therapeutic purposes.     

Characterization of the cereulide NRPS alpha-hydroxy acid specifying modules: activation of alpha-keto acids and chiral reduction on the assembly line

Several nonribosomal peptide natural products are composites of alpha-hydroxy acid and alpha-amino acid monomers. Cereulide, the emetic toxin from the human pathogen Bacillus cereus, and valinomycin, from Streptomyces spp., are closely related macrocyclic K+ ionophores. The macrocyclic core of each natural product contains alternating peptide (six) and ester (six) bonds, and their cyclododecadepsipeptide structures consist of a tetradepsipeptide unit repeated three times. Here we overexpress the cereulide NRPS alpha-hydroxy acid specifying modules from CesA and CesB and demonstrate that each contains an alpha-keto acid activating adenylation domain and a chiral alpha-ketoacyl-S-carrier protein reductase (alpha-KR). The logic used by the cereulide NRPS is likely at work in the valinomycin NRPS and may be the general strategy used in bacterial NRPSs to form alpha-hydroxy acid containing natural products.     

Disulfide Click Reaction for Stapling of S-terminal Peptides

A disulfide click strategy is disclosed for stapling to enhance the metabolic stability and cellular permeability of therapeutic peptides. A 17-membered library of stapling reagents with adjustable lengths and angles was established for rapid double/triple click reactions, bridging S-terminal peptides from 3 to 18 amino acid residues to provide 18- to 48-membered macrocyclic peptides under biocompatible conditions. The constrained peptides exhibited enhanced anti-HCT-116 activity with a locked α-helical conformation (IC₅₀=6.81 μM vs. biological incompetence for acyclic linear peptides), which could be unstapled for rehabilitation of the native peptides under the assistance of tris(2-carboxyethyl)phosphine (TCEP). This protocol assembles linear peptides into cyclic peptides controllably to retain the diverse three-dimensional conformations, enabling their cellular uptake followed by release of the disulfides for peptide delivery.     

Identification and characterization of the lysobactin biosynthetic gene cluster reveals mechanistic insights into an unusual termination module architecture

Lysobactin (katanosin B) is a macrocyclic depsipeptide, displaying high antibacterial activity against human pathogens. In this work, we have identified and characterized the entire biosynthetic gene cluster responsible for lysobactin assembly. Sequential analysis of the Lysobacter sp. ATCC 53042 genome revealed the lysobactin gene cluster to encode two multimodular nonribosomal peptide synthetases. As the number of modules found within the synthetases LybA and LybB directly correlates with the primary sequence of lysobactin, a linear logic of lysobactin biosynthesis is proposed. Investigation of adenylation domain specificities in?vitro confirmed the direct association between the synthetases and lysobactin biosynthesis. Furthermore, an unusual tandem thioesterase architecture of the LybB termination module was identified. Biochemical characterization of the individual thioesterases in?vitro provides evidence that solely penultimate thioesterase domain mediates the cyclization and simultaneous release of lysobactin.     

Nonribosomal peptides: from genes to products

The ability to synthesize nonribosomally small bioactive peptides that find application in modern medicine is widely spread among microorganisms. As broad as the spectrum of biological activities is the structural diversity of these peptides, which are mostly cyclic or branched cyclic compounds containing non-proteinogenic amino acids, small heterocyclic rings and other unusual modifications in the peptide backbone. They are synthesized by multimodular enzymes, the so-called nonribosomal peptide synthetases (NRPSs), from simple building blocks. Biochemical and genetic studies have unveiled the key principles of nonribosomal peptide syntheses, as well as the realization of many structural features of these peptides. This review focuses on recent results in NRPS research and highlights how this knowledge can be exploited for biotechnological purposes. In addition, possibilities and limitations for prediction of structural features of uncharacterized NRPSs and approaches for their engineering are discussed.     

Preparation of large macrocyclic peptides using the oxime resin

We exploited a peptide cyclization-cleavage reaction on oxime resin (PCOR) to obtain in one key step large macrocyclic peptides. Herein, we report on the different parameters affecting the cyclization-oligomerization reaction, whether to favor cyclic monomer or cyclic oligomers formation.     

Tuning Peptide Structure and Function through Fluorobenzene Stapling

Cyclic peptides are promising next-generation therapeutics with improved biological stability and activity. A catalyst-free stapling method for cysteine-containing peptides has been developed that enables fine-tuning of the macrocycle by using the appropriate regioisomers of fluorobenzene linkers. Stapling was performed on the unprotected linear peptide or, more conveniently, directly on-resin after peptide synthesis. NMR spectroscopy and circular dichroism studies demonstrate that the type of stapling can tune the secondary structures of the peptides. The method was applied to a set of potential agonists for melanocortin receptors, generating a library of macrocyclic potent ligands with ortho, meta or para relationships between the thioethers. Their small but significant differences in potency and efficacy demonstrate how the method allows facile fine-tuning of macrocyclic peptides towards biological targets from the same linear precursor.     

Secondary Amino Alcohols: Traceless Cleavable Linkers for Use in Affinity Capture and Release

Capture and release of peptides is often a critical operation in the pathway to discovering materials with novel functions. However, the best methods for efficient capture impede facile release. To overcome this challenge, we report linkers based on secondary amino alcohols for the release of peptides after capture. These amino alcohols are based on serine (seramox) or isoserine (isoseramox) and can be incorporated into peptides during solid‐phase peptide synthesis through reductive amination. Both linkers are quantitatively cleaved within minutes under NaIO₄ treatment. Cleavage of isoseramox produced a native peptide N‐terminus. This linker also showed broad substrate compatibility; incorporation into a synthetic peptide library resulted in the identification of all sequences by nanoLC‐MS/MS. The linkers are cell compatible; a cell‐penetrating peptide that contained this linker was efficiently captured and identified after uptake into cells. These findings suggest that such secondary amino alcohol based linkers might be suitable tools for peptide‐discovery platforms.     

A Self‐Sacrificing N‐Methyltransferase Is the Precursor of the Fungal Natural Product Omphalotin

Research on ribosomally synthesized and posttranslationally modified peptides (RiPPs) has led to an increasing understanding of biosynthetic mechanisms, mostly drawn from bacterial examples. In contrast, reports on RiPPs from fungal producers, apart from the amanitins and phalloidins, are still scarce. The fungal cyclopeptide omphalotin A carries multiple N‐methylations on the peptide backbone, a modification previously known only from nonribosomal peptides. Mining the genome of the omphalotin‐producing fungus for a precursor peptide led to the identification of two biosynthesis genes, one encoding a methyltransferase OphMA that catalyzes the automethylation of its C‐terminus, which is then released and cyclized by the protease OphP. Our findings suggest a novel biosynthesis mechanism for a RiPP in which a modifying enzyme bears its own precursor peptide.     

Affinity Maturation of Macrocyclic Peptide Modulators of Lys48-Linked Diubiquitin by a Twofold Strategy

Messenger RNA display of peptides containing non-proteinogenic amino acids, referred to as RaPID system, has become one of the leading methods to express libraries consisting of more than trillion-members of macrocyclic peptides, which allows for discovering de novo bioactive ligands. Ideal macrocyclic peptides should have dissociation constants (K_D) as low as single-digit values in the nanomolar range towards a specific target of interest. Here, a twofold strategy to discover optimized macrocyclic peptides within this affinity regime is described. First, benzyl thioether cyclized peptide libraries were explored to identify tight binding hits. To obtain more insights into critical sequence information, sequence alignment was applied to guide rational mutagenesis for the improvement of their binding affinity. Using this twofold strategy, benzyl thioether macrocyclic peptide binders against Lys48-linked ubiquitin dimer (K48-Ub2) were successfully obtained that display K_D values in the range 0.3–1.2 nm , which indicate binding two orders of magnitude stronger than those of macrocyclic peptides recently reported. Most importantly, this macrocyclic peptide also showed an improved cellular inhibition of the K48-Ub2 recognition by deubiquitinating enzymes and the 26S proteasome, resulting in the promotion of apoptosis in cancer cells.     

Synthesis of diamino-furostan sapogenins and their use as scaffolds for positioning peptides in a preorganized form

The synthesis of peptide-furostane conjugates from natural steroidal sapogenins is reported. The approach comprises the introduction of α-oriented amino groups into spirostanic sapogenins followed by reductive opening of the spiroketal chain, thus producing diamino-furostanic scaffolds suitable for further functionalization. Solid and solution-phase coupling processes were utilized for the incorporation of various α-amino acids and peptides into the furostanic skeletons. The attachment position depends on the steroidal sapogenin originally used, i.e., diosgenin or hecogenin. The resulting furostanic skeletons feature a trans A/B-ring fusion and hold the peptides in axial disposition. This characteristic ensures a preorganized alignment of the peptidic motifs, an important structural feature for future applications in molecular recognition and catalysis. A macrocyclic tripeptide-furostane conjugate was also produced by a combination of peptide coupling, Staudinger ligation, and a cyclization protocol. This work constitutes the first report on the use of furostanic sapogenins as scaffolds for positioning natural amino acids and (cyclo)peptides.     

Apparent inhibition by arginine of macrocyclic <Emphasis Type="Italic">b</Emphasis> ion formation from singly charged protonated peptides

There is now strong evidence for the existence of macrocyclic isomers of b_n⁺ ions, the formation and subsequent opening of which can lead to loss of sequence information from protonated peptides in multiple-stage tandem mass spectrometry experiments. In this study, the fragmentation patterns of protonated YARFLG and permuted isomers of the model peptide were investigated by collision-induced dissociation. Of interest was the potential influence of the arginine residue, and its position in the peptide sequence, on formation of the presumed macrocyclic b₅ ion isomer and potential loss of sequence information. We find that regardless of the sequence position (either internal or at the N- or C-terminus), only direct sequence ions or ions directly related to fragmentation of the arginine side chain are observed.     

Insights into the biosynthesis of hormaomycin, an exceptionally complex bacterial signaling metabolite

Hormaomycin produced by Streptomyces griseoflavus is a structurally highly modified depsipeptide that contains several unique building blocks with cyclopropyl, nitro, and chlorine moieties. Within the genus Streptomyces, it acts as a bacterial hormone that induces morphological differentiation and the production of bioactive secondary metabolites. In addition, hormaomycin is an extremely potent narrow-spectrum antibiotic. In this study, we shed light on hormaomycin biosynthesis by a combination of feeding studies, isolation of the biosynthetic nonribosomal peptide synthetase (NRPS) gene cluster, and in vivo and in vitro functional analysis of enzymes. In addition, several nonnatural hormaomycin congeners were generated by feeding-induced metabolic rerouting. The NRPS contains numerous highly repetitive regions that suggest an evolutionary scenario for this unusual bacterial hormone, providing new opportunities for evolution-inspired metabolic engineering of novel nonribosomal peptides.     

Introduction of a non-natural amino acid into a nonribosomal peptide antibiotic by modification of adenylation domain specificity

Calcium-dependent antibiotics (CDA) are cyclic lipopeptides assembled by nonribosomal peptide synthetase (NRPS) enzymes. Active site modification of the 3-methyl glutamate activating adenylation (A) domain of the CDA NRPS enables the incorporation of synthetic 3-methyl glutamine into CDA. This provides the first example of how A-domains can be engineered to introduce synthetic "non-natural" amino acids into nonribosomal peptides.     

Tryptophan in Multicomponent Petasis Reactions for Peptide Stapling and Late-Stage Functionalisation

Peptide stapling is a robust strategy for generating enzymatically stable, macrocyclic peptides. The incorporation of biologically relevant tags (such as cell-penetrating motifs or fluorescent dyes) into peptides, while preserving their binding interactions and enhancing their stability, is highly sought after. Despite the unique opportunities offered by tryptophan‘s indole scaffold for targeted functionalisation, its utilisation in peptide stapling has been limited as compared to other amino acids. Herein, we present an approach for peptide stapling using the tryptophan-mediated Petasis reaction. This method enables the synthesis of both stapled and labelled peptides and is applicable to both solution and solid-phase synthesis. Importantly, the use of the Petasis reaction in combination with tryptophan facilitates the formation of stapled peptides in a straightforward, multicomponent fashion, while circumventing the formation of undesired by-products. Furthermore, this approach allows for efficient and diverse late-stage peptide modifications, thereby enabling rapid production of numerous conjugates for biological and medicinal applications.