Natural cyclic peptides are a broad class of natural products, found across prokaryotic and eukaryotic domains, that have been evolutionarily conserved to serve important biological functions. They can be biosynthesized by two antagonistic classes of pathways: those that are synthesized by ribosomal synthesis of a linear precursor, followed by post‑translational modifications; and those that are synthesized non‑ribosomally by the polymerization of amino acids. Naturally occurring cyclic peptides are frequently structurally complex, containing non‑canonical amino acids, multiple types of cyclizations and unusual stability. Synthetic cyclic peptides can be designed and synthesized to mimic, alter or improve upon natural products. This may be accomplished by the introduction of non‑natural amino acids, the optimization of the molecule to improve pharmacokinetic properties, or the creation of new unnatural structures. Natural products can provide a useful source of drug leads and privileged structures. Synthetic analogues can also be designed to more precisely control the structure and can be optimized for select targets.
Natural cyclic peptides are a large class of structurally diverse natural products that have been evolved to perform a variety of important biological functions, including as antibiotics and antifungals, pheromones and hormones. Natural cyclic peptides may be products of either of two large biosynthetic classes, which differ by whether or not the cyclic peptide product is originally encoded by mRNA, and further enzymatic steps are then used to cyclize the molecule. The designation for natural cyclic peptides initially synthesized as linear peptides on the ribosome and later cyclized through post‑translational modifications is ribosomally synthesized and post‑translationally modified peptides (RiPPs). In these cases, a dedicated enzyme or enzymes catalyze the cyclization, crosslinking and other modifications to transform the linear peptide precursor into the final bioactive product. In contrast, the non‑ribosomal peptides (NRPs) are instead directly synthesized by large multimodular enzymes called NRP synthetases (NRPSs), which select, activate, and ligate the amino acid monomers. . Unlike those assembled on ribosomes, these monomers may contain non‑proteinogenic D‑amino acids, N‑methylated amides, hydroxy acids and much more. Stable, proteolytically resistant, membrane permeable and often highly target-specific macrocycles can be produced by both NRPS and RiPP biosynthetic machinery. As a result, these two groups represent an abundant source of chemical probes and potential drug leads. Natural cyclic peptides are found in both bacteria and eukaryotes, and many have roles as antimicrobials, antibiotics, quorum sensing molecules, signaling peptides, enzyme inhibitors and more.
The class of cyclic peptides known as RiPPs expands through ribosomal synthesis of precursor proteins followed by enzymatic cleavage and post-translational modification to produce constrained macrocycles. The precursors are typically encoded by a module containing an N‑terminal leader sequence, which mediates recognition by modifying enzymes, and a C‑terminal core region, which is the sequence of the eventual cyclic product. After translation, the core region is modified by a series of modifying enzymes (cyclases, dehydratases, oxidases, etc.), which install crosslinks and other modifications. For example, cyclization is frequently mediated by protease‑like enzymes that cleave the leader sequence, and simultaneously catalyze the formation of a peptide bond between the newly exposed N‑terminus and a C‑terminal carboxylate (head‑to‑tail cyclization). This strategy is exemplified by cyclotides, plant‑derived macrocyclic peptides that contain a cyclic backbone stabilized by a cystine‑knot motif of three disulfide bonds, and which are extremely stable to thermal and proteolytic degradation. The cyclotide structure is a case study in how ribosomal origin combined with post‑translational modification can produce hyperstable scaffolds that may be used in agricultural and therapeutic applications. A second well‑studied RiPP is the Sunflower Trypsin Inhibitor‑1 (SFTI‑1), a 14‑residue macrocycle found in sunflower seeds, which is a serine protease inhibitor that adopts a rigid, cyclic β‑hairpin structure. SFTI-1 represents another instance in which natural product chemistry elegantly combines ribosomal synthesis with proteolytic maturation to create a short, selective inhibitor whose cyclic structure not only imparts proteolytic stability but also high affinity. Bacteriocins represent another class of ribosomally synthesized antimicrobial peptides produced by bacteria. The bacteriocins nisin and subtilosin undergo extensive modifications through dehydration and cyclization reactions, as well as occasional addition of thioether bridges known as lanthionines. These modifications frequently lead to pore‑forming toxins that are specifically targeted against competing microbes. The modifications are installed by synthetase complexes, which recognize the leader peptide and act processively to introduce multiple crosslinks to the target peptide to form a rigid, membrane‑active structure.
NRPs are a separate class of cyclic peptides that are synthesized non‑ribosomally. NRPSs are large multi‑modular enzyme complexes that operate in a manner analogous to an assembly line. Modules are typically monomer specific, each module incorporating a single amino acid. A module has three major domains: The adenylation domain activates a specific amino acid to form an aminoacyl‑AMP intermediate while the thiolation domain containing a phosphopantetheinyl arm captures this activated monomer as a thioester before the condensation domain catalyzes peptide bond formation between two linked thioester monomers. As modules incorporate a single amino acid at a time, the substrates used are not limited to proteinogenic amino acids and their α‑L‑enantiomers. As a result, many NRPs contain non‑proteinogenic amino acids, D‑enantiomers, N‑methylated residues and hydroxy acids. The typical NRPS cyclisation step is performed by a thioesterase (TE) domain at the C‑terminus of the NRPS. The TE catalyzes nucleophilic attack of the N‑terminus onto the terminal thioester, liberating the mature macrocyclic product. Cyclosporin is a well‑studied non‑ribosomal immunosuppressant synthesized by the fungus Tolypocladium inflatum, which features eleven residues, seven of which are N‑methylated, as well as a D‑amino acid, generating a conformation that protects polar groups in the backbone, allowing it to be orally bioavailable. Its cyclic structure is required for binding cyclophilin and inhibition of calcineurin, thereby inhibiting T‑cell activation. Gramicidin S is a cyclic decapeptide biosynthesized by Bacillus brevis and has broad‑spectrum antimicrobial activity, as it can disrupt bacterial membranes. Its head‑to‑tail cyclisation, which enforces an amphipathic β‑sheet conformation, and D‑phenylalanine residues are responsible for selectively disrupting microbial membranes while leaving eukaryotic cells unreacted. The NRPS responsible for Gramicidin S production is a large assembly line consisting of repeating modules which work in concert to produce the symmetric, cyclo‑decapeptide. This highlights how modular NRPS architectures are capable of generating symmetric macrocycles with biological activity. Despite the many success stories, NRPs are generally difficult to generate through synthetic biology techniques.
Natural cyclic peptides have a variety of biological functions. The most common ones include defense, signaling, and inhibition of enzymes. For instance, natural cyclic peptides can be defensive toxins or antibiotics which enable an organism to compete with other microorganisms. Bacteriocins are a large group of ribosomally synthesized cyclic peptides produced by bacteria, as natural antibiotics to kill other susceptible bacteria or archaea. Their mechanisms of action include pore formation, inhibition of cell‑wall biosynthesis, or membrane potential disruption. In the producing strain, bacteriocins provide competitive advantage, allowing it to outcompete other microbes for scarce resources in the environment. Cyclotides are thought to be plant host‑defense peptides against predators and pathogens. Their membranolytic activity may contribute to this function. Cyclotides are highly stable in the harsh environment of the plant extracellular matrix, which is believed to be a necessary feature of a plant defensive peptide. Microorganisms use cyclic peptides as signaling molecules for intercellular communication and collective behavior coordination. Quorum sensing demonstrates how bacterial cells use secreted cyclic autoinducers to determine cellular population density and modify gene expression patterns. Other examples include hormones or signaling ligands in higher organisms. Cyclic peptides have found use as drug targets because many of them act as enzyme inhibitors. Cyclic peptides can inhibit a range of enzymes including proteases, kinases, and others, often with high selectivity. For example, many natural product macrocycles are potent inhibitors of enzymes and work by shape complementarity and multiple points of interaction. An example of a cyclic peptide with an enzyme target is the immunosuppressant Cyclosporin, which inhibits the enzyme calcineurin. A plant cyclic peptide SFTI‑1 is a competitive inhibitor of trypsin. The rigidity of the macrocyclic structure allows for high entropic selectivity and often presents multiple functional groups in the correct stereochemical orientation to interact with the active site or allosteric pockets of the target.
Synthetic cyclic peptides are created macrocycles through chemical synthesis to either replicate natural cyclic peptides or modify their structures and functions. Since these molecules are chemically synthesized, there is also the possibility to incorporate unnatural amino acids (e.g. D-amino acids, N-methylated amino acids, or β-amino acids) in an effort to further diversify the chemistry and alter the properties of the molecule. Cyclic peptides can be synthesized in a number of different ways, but two broad strategies include direct chemical synthesis of the cyclic peptide, or chemical synthesis of a linear peptide followed by subsequent cyclization using either chemical or enzymatic means. Chemical synthesis can be performed by solid-phase or solution phase techniques, and typically involves the coupling of amino acids or peptide fragments to assemble the desired sequence, followed by a cyclization step to form the macrocycle. Approaches combining enzymes and chemical catalysts generate cyclic peptides by using ligases to close peptide chains which originate from either chemical or biological synthesis methods.
Fig. 1 The four possible macrocyclization strategies for the synthesis of cyclic peptides.1,5
Synthetic approaches towards cyclic peptides are generally divided into solid-phase peptide synthesis (SPPS) and solution-phase techniques. Each strategy comes with its own tradeoffs in terms of scalability, difficulty of purification, and cyclization yield. SPPS is the preferred approach to synthesize the linear precursor for cyclization. This involves stepwise addition of residues onto a solid polymeric resin starting from the C-terminus to the N-terminus of the peptide chain and using Fmoc or Boc orthogonal protection. The resin itself acts as the C-terminal protecting group for the carboxylate. The side-chains of the residues are often protected by acid-labile or base-labile groups to protect against any side reactions during synthesis. After synthesis of the linear chain, the peptide is cleaved from the resin and deprotected under strongly acidic conditions to produce the fully unmasked precursor which can then be used for cyclization. Cyclization can either be done on-resin or in solution. On-resin cyclization effectively pseudo-dilutes the substrate, preventing intermolecular oligomerization through limited mobility of the peptide chains and encouraging intramolecular cyclization. In-solution cyclization is less restricted by these limitations, but in order to avoid dimerization and polymerization which would severely decrease the reaction rate, extremely low substrate concentrations are required, often leading to large solvent volumes.
Fig.2 Development and optimization of macrocyclization methodology.2,5
Ring closure through amide bond formation is the most prevalent approach. This includes head-to-tail macrolactamization, side-chain-to-tail ligation and side-chain-to-side-chain lactam bridge formation. Head-to-tail cyclization is accomplished by activation of the C-terminal carboxylate with a phosphonium or aminium coupling reagent (HBTU, HATU) and intramolecular nucleophilic attack by the N-terminal amine. This approach is conceptually simple, but is accompanied by epimerization at the C-terminal stereocenter, especially when activating non-glycine residues as the oxazolone intermediates in this process can abstract a proton, causing racemization, before cyclization can occur. Side-chain-to-side-chain cyclization, as mentioned above, often obviates the termini-based epimerization and instead forms lactam bridges between the lysine and aspartate/glutamate side chains. In order to achieve this type of cyclization, a sophisticated orthogonal protection scheme must be used to allow deprotection of side-chain functionalities while still protecting the backbone. An example of this is the synthesis of conotoxin analogs, where cysteine residues form disulfide bridges, with the oxidative folding trapping the peptide in a conformationally rigid scaffold that recapitulates natural cystine-knot topologies. Disulfide bridges may be formed either by air oxidation in slightly basic buffers or by directed thiol-disulfide exchange using redox agents, the latter of which affords greater control over disulfide connectivity in peptides with more than one pair of cysteines. Site selection for ring closure is dependent on the desired ring size, the sequence's ability to adopt a conformation which places the desired reactive termini in proximity and which avoids steric clashes that would prevent ring closure. In many cases, the cyclization junction must be empirically screened as certain sequences are predisposed to form β-turns which will promote macrocyclization, while others will not cyclize, despite extensive optimization of reaction conditions. A major challenge in chemical synthesis is often to balance the competing demands of yield, stereochemical purity and scalability, particularly for large macrocycles (>20 residues) which are subject to ring strain and conformational entropy penalties.
As a complementary approach to chemical cyclization, enzymatic and hybrid synthetic methods have also been used to cyclize peptides. In addition to providing a means to carry out ring closure under mild and aqueous conditions, these methods can avoid many of the side reactions that often occur with conventional coupling reactions. The general concept of the hybrid approach involves the chemical synthesis of a linear peptide precursor containing a suitable recognition motif, which is then enzymatically ligated by an appropriate ligase, in a process that combines the flexibility of chemical synthesis with the highly specific substrate recognition of a biocatalyst. The most common use of this approach involves the chemical synthesis of a linear peptide on solid support containing a C-terminal thioester or activated ester, as well as an N-terminal cysteine or polyglycine, which can serve as the nucleophile for enzymatic cyclization. The resin is then cleaved and deprotected, and the crude peptide precursor is incubated with a purified ligase that can recognize the introduced motifs and catalyze intramolecular peptide bond formation to yield the cyclic peptide product. Many different enzymes can be used in a hybrid synthesis, including sortase A, butelase 1, and engineered subtilisin variants. Sortase A is a transpeptidase enzyme that recognizes the LPXTG motif in Gram-positive bacteria and cleaves between threonine and glycine to form an acyl-enzyme thioester intermediate, which is resolved through nucleophilic attack of the N-terminal oligoglycine to form a peptide product, allowing cyclization of peptides with this recognition sequence at the termini. Butelase 1 is a type of ligase asparaginyl endopeptidase, an enzyme class from plants, that cyclizes peptides in a process called reverse-proteolysis. Peptides with a C-terminal Asn-His-Val recognition tag are cyclized by butelase 1 through cleavage of the amide bond after asparagine, followed by capture of the released N-terminal amine to form a head-to-tail peptide macrocycle with high efficiency and low levels of epimerization. Engineered subtiligase variants, based on subtilisin, a serine protease enzyme, have been engineered by rational design to favor aminolysis over hydrolysis, allowing for cyclization of peptide thioesters under neutral conditions with broad substrate scope. The main advantage of the hybrid approach is that the enzyme only reacts with the desired N-terminal nucleophile, but not other nucleophiles such as lysine side chains, to give the desired cyclized product, suppressing many of the side reactions that can occur during a chemical cyclization. This can be especially advantageous in aqueous reaction conditions, which can improve solubility of the peptide over organic solvents, particularly for more hydrophobic sequences.
The contrast between naturally occurring and synthetic cyclic peptides is not merely one of their origin, but rather it is characterized by distinct philosophies that influence their structural intricacy, functional diversity, and translational pathways. Natural cyclic peptides are the culmination of an evolutionary process shaped by selection pressures that favor structures with specific biological roles, such as antimicrobial activity, signaling, or nutrient capture, but are not necessarily aligned with therapeutic needs. As a result, these peptides embody a series of evolutionary trade-offs, which dictate their ability to be synthesized by the host organism, stability within a competitive environment, and their interaction with targets in a way that avoids detrimental effects on the host itself. This evolutionary sculpting often leads to non-canonical structures that may defy traditional drug-likeness metrics but possess significant bioactivity due to their high evolutionary fitness, as seen in the case of cyclosporin, which is a potent immunosuppressant despite its relatively large size and lipophilicity. In stark contrast, synthetic cyclic peptides are a product of human ingenuity, purposefully crafted to meet specific therapeutic targets with a focus on enhancing binding affinity, pharmacokinetic properties, and ease of synthesis. The synthetic approach to cyclic peptide design allows for the integration of non-natural modifications such as D-amino acids, N-methylations, or β-amino acids, which can extend the chemical diversity beyond what is typically found in nature, but which would be unavailable to natural biosynthetic enzymes. Yet, this process-driven design often lacks the nuanced details that natural selection can introduce, such as fine-tuned conformational preorganization or metabolite-mimetic features that can help the peptide evade host detoxification mechanisms. Furthermore, the practical aspects of production also set these two classes apart: natural cyclic peptides can be obtained from fermentation processes that utilize renewable feedstocks, but their production is often hampered by low yields, cryptic biosynthetic gene clusters, and complex purification from metabolite mixtures.
| Aspect | Natural | Synthetic |
| Diversity | Limited | Unlimited |
| Production | Enzymatic | Chemical |
| Applications | Biological roles | Therapeutic, industrial |
| Scalability | Difficult | Controllable |
Natural and synthetic cyclic peptides are thus often optimized for different purposes and targets: natural peptides are optimized for antimicrobial activity, whereas synthetic peptides are typically optimized for their drug-likeness in humans. On one hand, many natural cyclic peptides, including bacteriocins and cyclotides, were developed by microorganisms or plants for antimicrobial activity as weapons against competing microorganisms or as defenses against herbivores, and their mechanism of action often involves the perturbation of the membrane of these competitors. They are used as low-concentration antibacterial, antiviral, or antifungal agents in their natural environments. On the other hand, when developing a drug, the target is often a specific bacterial protein, or the desired mechanism of action is a targeted inhibition of bacterial protein synthesis or bacterial metabolism, without affecting mammalian cells. To this end, natural cyclic peptide scaffolds can be rationally modified to increase their selectivity, as they are usually not selective for human cell targets and can be hemolytic or immunogenic, such as by mimicking N-methylation patterns or D-amino acids to improve cell membrane permeation, or by charge reversal to abrogate hemolytic activity. Libraries of such derivatives of natural products have been developed, for example, of gramicidin S. Modifications for improved therapeutic index and desired pharmacokinetics in the human body may include factors such as improved stability for systemic administration, oral bioavailability, and target clearance, leading to approaches that take naturally-occurring antimicrobial peptides and adapt them into more drug-like cyclic peptides, for example by dissecting a natural antimicrobial scaffold into its minimal pharmacophoric elements, and then rebuilding it with the same pharmacophoric elements within a synthetic cyclic peptide scaffold optimized for drug-likeness.
Synthetic and natural cyclic peptides have many uses as tools in molecular and cell biology. They are used to study protein function, as imaging probes and as probes of signaling cascades. They can be tagged with fluorophores or biotin handles or chemical crosslinkers. In the former case they can be used as reagents in pull-down assays and in fluorescence microscopy, and in the latter case to study protein-protein interactions. Cyclic peptide antagonists of protein domains can be used as inhibitors of protein function that can be titrated to reversibly, and dose-dependently block signaling. This can be useful in dissecting signaling pathways, and in some cases can reveal compensatory feedback signaling that may be hidden in a genetic knockout study. As imaging agents, near-infrared dye or radiometal labeled cyclic peptides, are used as they can enter tissue quickly and are rapidly cleared from tissues. Because cyclic peptides are smaller and less flexible than most proteins they give off less background signal, which can make it easier to get a good signal. Cyclic peptides can also be used to cross cell membranes and deliver other molecules like antisense oligonucleotides or small interfering RNAs or protein degradation tags to modify gene expression or protein levels in specific cell types. This can be used in combination with tags or labels on the cyclic peptides themselves to target specific cells. Cyclic peptides can be engineered to include non-canonical amino acids that have bioorthogonal chemical handles that can then be used to conjugate the cyclic peptide to a payload of choice using a click chemistry reaction. In this manner, their cargo can be modified after the cyclic peptide has been synthesized. Cyclic peptides have also been used as crystallization chaperones, which bind and stabilize flexible regions in proteins, allowing them to be more easily crystallized for X-ray crystallography or cryo-electron microscopy. Finally, since many types of RiPPs, including cyclic peptides, are genetically tractable, they can be used to select high affinity binders by phage display against any target of interest, which can then be further chemically modified to add stability or reporter tags.
Emerging roles for cyclic peptides also include applications in cosmetics and materials. Cyclic peptides show promise in cosmetic formulations as ingredients that can potentially improve skin appearance, particularly against aging, hyperpigmentation, and impaired skin barrier function. Cyclic peptides are often more stable than linear peptides when applied topically as they are resistant to enzymatic degradation by proteases present on the skin surface and are more likely to penetrate the stratum corneum if they are sufficiently hydrophobic and small in size. The sustained presence of these peptides on the skin can allow continuous delivery of the bioactive peptide to skin cells such as fibroblasts or melanocytes. The peptides can bind more selectively to the target receptors in the skin, such as integrins or growth factor receptors. By doing so, they can more specifically modulate the production of extracellular matrix components such as collagen or elastin, or sebum production. Cyclic peptides with synthetic modifications that enhance skin permeation and reduce immunogenicity, such as N-methylation and the inclusion of D-amino acids, can be used to develop next-generation cosmeceuticals that deliver drug-like potency in a convenient, over-the-counter format. In materials, cyclic peptides have been evaluated as antimicrobial coatings, wound dressings, and anti-biofilm coatings for medical devices. Cyclic peptides can be used to form ordered nanostructures, such as hydrogels or surface films, that allow for sustained release of antimicrobial agents without the release of toxic chemicals, which is a common limitation of traditional biocides. In material science, cyclic peptide-based supramolecular structures can be used as templates for the synthesis of inorganic nanoparticles or to create porous scaffolds for tissue engineering. The cystine-knot scaffold of cyclotides, for example, has been used to stabilize colloidal silver or gold nanoparticles, producing materials with improved catalytic or plasmonic activity. Cyclic peptides with polymerizable groups can also be used to be embedded in polymer networks to create stimuli-responsive materials that can release payloads in response to pH or enzymatic cues, with potential applications in smart packaging or controlled release drug delivery systems.
Synthetic biology can provide another strategy for cyclic peptide discovery. Cyclic peptides of potentially interesting structures and activities can be designed by natural and artificial means to enhance the number and diversity of cyclic peptides. This will in turn increase the number of potential antimicrobials and therapeutics to be discovered. Semi-synthetic hybrid peptides will also be made. The scale-up necessary to make a cyclic peptide therapeutic feasible has led to the development of semi-synthetic hybrid strategies, which combines the speed and efficiency of biological fermentation with the complexity manipulation of chemical post-translation modification to provide cost-effective routes to complex macrocycles that are impractical or impossible to make entirely by chemical or biological means alone. Such strategies generally begin with microbial fermentation of a biosynthetic precursor (linear peptide or partially cyclized scaffold) by an engineered strain optimized to direct metabolic flux to the target molecule and secrete the product into the fermentation broth for facile downstream capture. The isolated precursor is then elaborated by chemical or chemoenzymatic means: specific residues can be modified through site-selective chemistry, non-canonical amino acids incorporated, or additional crosslinks added to increase stability or provide new functional groups. This approach reduces the synthetic load of having to construct the entire macrocycle through chemical synthesis, both in terms of reagent cost and the difficulty of purification, while avoiding the limited substrate range of biosynthetic enzymes that typically precludes direct integration of designer elements.
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