Cyclization is a key structural modification used to enhance the stability, activity, and bioavailability of peptides. By constraining the linear peptide backbone into a closed-ring structure, cyclization can dramatically improve resistance to proteolysis, reduce conformational flexibility, and increase binding affinity to targets. In nature, many bioactive peptides—including toxins, hormones, and antibiotics—are cyclic, and synthetic efforts to recreate or mimic these structures have long been a central focus in peptide chemistry. While chemical methods of cyclization have been extensively explored, enzymatic strategies are gaining prominence due to their specificity, biocompatibility, and efficiency under mild conditions.
Enzymatic peptide cyclization harnesses naturally evolved catalysts such as sortases, butelases, and inteins to selectively form covalent bonds between peptide termini or side chains. These enzymes operate with remarkable regio- and chemoselectivity, enabling site-specific cyclization with minimal side products. Their ability to function in aqueous environments and their compatibility with biological systems make them attractive for in vitro synthesis and in vivo applications alike. This article reviews the major enzymatic cyclization strategies, focusing on sortase, butelase, and intein-based approaches, comparing their mechanisms, reaction conditions, and potential uses in peptide engineering and therapeutic design.
Cyclic peptides have emerged as crucial molecular scaffolds in drug development and molecular engineering due to their enhanced stability, improved receptor binding, and resistance to enzymatic degradation. While traditional chemical cyclization techniques can generate cyclic structures, they often lack the precision, selectivity, and mild reaction conditions needed for delicate biomolecules. Enzyme-based cyclization offers a biologically inspired alternative, utilizing naturally evolved catalysts—such as sortase A, butelase 1, and inteins—that enable site-specific cyclization under aqueous, near-physiological conditions. These enzymatic systems offer high efficiency, sequence specificity, and compatibility with both synthetic and biologically expressed peptides, making them powerful tools for the development of stable, functional biomolecules.
The most commonly employed enzymes for peptide ring formation are sortase A, butelase 1, and inteins, each offering distinct ligation strategies.
Sortase A, originally derived from Staphylococcus aureus, is a transpeptidase that recognizes a specific LPXTG motif within a peptide substrate. Upon recognition, sortase cleaves the peptide bond between the threonine and glycine residues in the motif, forming an acyl-enzyme intermediate. This intermediate can then be resolved by a nucleophilic N-terminal glycine (or a poly-glycine sequence), leading to the formation of a new peptide bond. By engineering peptides with both the LPXTG motif and an N-terminal glycine, researchers can drive intramolecular reactions that yield macrocyclic products. Sortase-mediated ligation is widely used for its predictable behavior and compatibility with various peptide sequences.
Butelase 1, an asparaginyl endopeptidase derived from the plant Clitoria ternatea, is among the fastest peptide ligases discovered. It recognizes a minimal tripeptide motif, typically Asn-His-Val, at the C-terminus of a peptide. Butelase cleaves after the asparagine residue and catalyzes ligation with an N-terminal amino group of another peptide segment, effectively forming a new amide bond. Notably, butelase demonstrates high turnover rates, broad sequence tolerance, and excellent efficiency, even at low enzyme concentrations. These properties make it exceptionally well-suited for cyclizing small peptides with high precision.
Inteins are autocatalytic protein elements that excise themselves from a host protein and ligate the flanking exteins via a process called protein splicing. This natural mechanism has been harnessed to generate protein macrocycles or even cyclic peptides. The split intein system, in particular, allows cyclization of target proteins by flanking them with N- and C-terminal intein fragments, which, upon assembly, induce backbone ligation. Inteins are especially advantageous for cyclizing larger protein domains or for applications requiring post-translational control in cellular environments.
Together, these enzymes offer a versatile toolbox for researchers aiming to synthesize cyclic peptides or proteins with tailored properties. Their orthogonality, sequence selectivity, and mild operational conditions make them preferable to many traditional cyclization methods.
Chemical cyclization strategies, such as head-to-tail amide bond formation, disulfide bridge formation, or ring-closing metathesis, have long been employed in peptide synthesis. While these methods provide structural diversity and can be adapted to a wide range of chemistries, they are often limited by low yields, side reactions, and the need for extensive purification steps. Moreover, chemical cyclization can lack selectivity, especially in peptides containing multiple reactive sites, necessitating protecting group strategies that increase synthetic complexity.
Enzymatic cyclization, on the other hand, offers site-specificity based on enzyme recognition motifs. This feature dramatically reduces side reactions and simplifies reaction design. Enzymes operate under physiological or near-physiological conditions, preserving sensitive peptide side chains and functional groups. They also offer catalytic efficiency, allowing reactions to proceed with small amounts of enzyme and minimal organic solvents or additives. Another key advantage of enzymatic methods is compatibility with biological systems. For instance, sortase and inteins can be used in living cells to produce cyclic peptides or labeled proteins in situ, which is not possible with most chemical methods. Furthermore, the possibility to use these enzymes in tandem with genetically encoded sequences makes them ideal for high-throughput applications and bio-orthogonal engineering.
The successful application of enzyme-based cyclization depends heavily on the quality and activity of the enzyme used. Sortase A, for instance, is typically expressed with an N-terminal or C-terminal His-tag, allowing for rapid purification via nickel-affinity chromatography. Butelase purification presents more challenges due to its plant origin and the requirement for post-translational modifications. Historically, it was isolated from Clitoria ternatea, but recombinant systems using insect cells or yeast have since been developed to improve accessibility. Inteins are generally easier to express, often fused to a target protein as part of a larger construct. The splicing activity of inteins is influenced by buffer composition, redox conditions, and temperature, so proper optimization of these parameters is essential. For all enzymatic systems, enzyme storage and handling are critical. Enzymes are typically stored at -80°C in small aliquots to prevent repeated freeze-thaw cycles, and glycerol is often added to improve stability. Buffer conditions must be carefully adjusted based on the specific enzyme, with attention to pH, salt concentration, and the presence of cofactors (e.g., calcium ions for sortase A).
In summary, efficient purification and preparation of enzymes are essential steps in realizing the full potential of enzymatic cyclization strategies. With advances in expression technologies and enzyme engineering, these challenges are increasingly surmountable, enabling broader adoption of enzyme-based peptide and protein cyclization in both research and industrial settings.
Sortase-mediated ligation (SML) has become a widely used tool for site-specific peptide and protein modification. This method exploits the catalytic activity of sortase A, a transpeptidase from Staphylococcus aureus, which performs a transpeptidation reaction by recognizing and cleaving a specific pentapeptide motif. Among enzymatic cyclization techniques, sortase-mediated peptide cyclization offers a high degree of control over the site and nature of the bond formation, making it highly attractive for applications in protein engineering, drug conjugation, and therapeutic design.
The hallmark of sortase A activity is its strict recognition of the LPXTG motif, where X can be any amino acid. This sequence must be engineered near the C-terminus of the peptide or protein intended for cyclization. Upon recognition, sortase cleaves the peptide bond between the threonine (T) and glycine (G), forming an acyl-enzyme intermediate. This intermediate is then resolved through a nucleophilic attack by an N-terminal oligoglycine motif (typically GGG) on the same or a different peptide chain, resulting in ligation.
To induce intramolecular cyclization, researchers typically design peptides with the LPXTG motif at the C-terminus and a glycine-rich sequence at the N-terminus. The proximity and flexibility of these two motifs are critical for favoring intramolecular over intermolecular ligation. Linker length, amino acid composition, and spatial arrangement influence the efficiency of cyclization. Incorporating flexible residues such as glycine, serine, or proline between the two motifs can increase the likelihood of successful ring closure. In some cases, sortase variants (e.g., engineered sortase A mutants) are employed to alter substrate specificity or improve reaction kinetics. Such mutants allow the use of non-canonical motifs or broaden the compatibility with different peptide substrates, thus expanding the scope of peptide cyclization beyond the canonical LPXTG–GGG pairing.
Sortase A functions under mild and aqueous conditions, typically within the pH range of 7.0 to 8.0, which makes it highly compatible with peptides and proteins containing labile or sensitive residues. The enzyme requires calcium ions (Ca²⁺) for activity, usually supplied at concentrations of 5–10 mM. Calcium stabilizes the enzyme's conformation, promoting substrate binding and catalysis. Some engineered sortase variants (e.g., calcium-independent sortase A) have been developed to eliminate this requirement, which is beneficial for certain biological applications where free calcium may interfere.
The standard reaction temperature is room temperature to 37°C, and reactions are often completed within a few hours, depending on the concentration of the enzyme and substrates. High enzyme-to-substrate ratios are sometimes necessary to drive reactions to completion, although this can be mitigated with optimized reaction conditions or more active enzyme mutants. To enhance intramolecular over intermolecular cyclization, reaction concentrations are typically kept low, minimizing the chances of intermolecular ligation (i.e., polymerization or dimerization). In addition, the cyclization reaction is often performed using a dilute solution of the peptide substrate (e.g., 10–50 µM), with enzyme concentrations adjusted based on catalytic turnover and substrate affinity.
Beyond cyclization, sortase-mediated ligation is a highly versatile tool for site-specific labeling of proteins and peptides. Because the enzyme attaches molecules precisely at the LPXTG motif, it enables the attachment of diverse functional moieties—such as fluorescent dyes, biotin, PEG chains, or even drug molecules—to defined locations on a protein or peptide. In the context of drug conjugation, sortase is especially powerful for generating antibody-drug conjugates (ADCs) or protein-drug conjugates, where site-specificity is crucial to maintain uniformity and biological activity. For example, monoclonal antibodies can be engineered to contain an LPXTG tag, and then selectively ligated to cytotoxic drugs bearing an N-terminal glycine, resulting in precise and reproducible conjugates.
In addition to sortase-mediated ligation, two other enzymatic systems—butelase and intein-based splicing—have gained considerable attention for their ability to cyclize peptides and proteins with high specificity, speed, and versatility. These enzymes originate from distinct biological contexts and exhibit unique catalytic properties, yet both can facilitate backbone cyclization and functional ligation in a site-specific manner. Their growing use reflects the broader push toward bioorthogonal tools that offer precise control over peptide architecture without compromising functionality or biocompatibility.
Butelase 1, a member of the legumain/asparaginyl endopeptidase (AEP) family, is a plant-derived enzyme first identified in Clitoria ternatea. Unlike proteases that primarily cleave peptide bonds, butelase 1 functions primarily as a peptide ligase, catalyzing the formation of new peptide bonds through a transpeptidation reaction. It specifically recognizes Asn/Asp-containing motifs—typically NHV or NHV-like sequences—at the C-terminus of substrates and forms a bond between the cleaved asparagine and the N-terminal amine of a second peptide or the same molecule in the case of cyclization. The general logic of butelase-mediated cyclization involves engineering the target peptide with a C-terminal recognition sequence (e.g., NHV) and an unmodified N-terminal amine. When these termini are brought into proximity, butelase cleaves after the Asn and simultaneously forms a new peptide bond with the N-terminus, resulting in a head-to-tail cyclic product. Unlike sortase, butelase does not require exogenous cofactors like calcium, and the reaction occurs under mild conditions, usually in neutral buffers.
Inteins, by contrast, are autocatalytic protein domains that catalyze protein splicing. Found in a variety of prokaryotic and eukaryotic organisms, inteins excise themselves from a larger protein and ligate the flanking peptide sequences (known as exteins). Engineered versions—especially split inteins—allow researchers to cyclize proteins or peptides by placing the split intein fragments at the termini of a target sequence. Upon intein reconstitution, the intervening peptide becomes covalently cyclized via native peptide bonds. The cyclization logic of inteins typically involves fusing N-terminal and C-terminal split intein segments (such as Npu DnaE or Ssp DnaE) to a target peptide or protein. When expressed or combined in vitro, the intein fragments assemble and catalyze trans-splicing, resulting in peptide cyclization. This method is particularly valuable for larger proteins, where other ligases may be inefficient or incompatible due to steric constraints.
Butelase is renowned for its exceptional efficiency, with reaction rates significantly faster than most peptide ligases. Cyclization can often be achieved in minutes with catalytic amounts of enzyme, even at low micromolar substrate concentrations. Moreover, butelase displays broad substrate tolerance, accommodating a range of peptide sequences flanking the recognition site. However, its strict requirement for a C-terminal Asn residue can be a limitation in some contexts, though ongoing engineering efforts are addressing this issue.
Inteins, while generally slower than butelase, offer sequence independence and are highly modular. Their splicing activity depends more on the protein context and folding than on specific short recognition sequences. This flexibility enables intein-mediated cyclization of full-length proteins, large domains, or even multidomain constructs—applications beyond the reach of sortase or butelase. Advancements in split intein engineering have led to variants with improved kinetics, fidelity, and orthogonality, enabling parallel or multiplexed protein cyclization strategies. While intein systems typically require hours to complete cyclization, they do so with high chemoselectivity and generate native amide bonds, leaving no scar or extra residues at the ligation site.
Both butelase and inteins can be deployed in in vitro and in vivo environments, but their utility differs significantly depending on context.
In vitro, both enzymes allow highly controlled cyclization reactions. Butelase can be used to cyclize synthetic peptides post-synthesis, or to modify recombinant proteins after purification. Its fast kinetics and minimal cofactor requirements (no need for ATP or metal ions) make it suitable for high-throughput applications and manufacturing processes. Its robustness also supports applications in diagnostic assays, drug discovery platforms, and the generation of macrocyclic peptide libraries for screening. Inteins have also proven useful in in vitro applications, especially for generating site-specific modifications, circularly permuted proteins, or stable protein scaffolds. The split-intein system allows orthogonal design—different intein pairs do not cross-react—enabling multiplexed protein engineering. Moreover, intein-based cyclization can be integrated with protein purification systems, such as intein-chitin-binding tag fusions, allowing seamless purification and cyclization in one workflow.
In vivo, inteins demonstrate superior adaptability. Because intein-mediated splicing is genetically encoded and self-sufficient, it is easily incorporated into plasmid constructs and expressed in living cells. This enables protein cyclization to occur co-translationally or post-translationally inside bacteria, yeast, or even mammalian cells. In vivo cyclization enhances protein stability, reduces proteolytic degradation, and can improve intracellular retention or bioavailability. Butelase, though promising, faces challenges for in vivo use. Since butelase is a plant enzyme, it must be co-expressed or delivered exogenously in cellular systems. Its large size and potential immunogenicity complicate expression in animal systems. Moreover, delivery of synthetic peptide substrates into cells remains a limiting step.
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