Native Chemical Ligation (NCL) has had a transformative impact on the synthesis of cyclic peptides, enabling an otherwise inaccessible chemoselective, traceless and biocompatible means for head-to-tail macrocyclization that cannot readily be performed by classical amide-coupling chemistry. Developed as a general strategy for coupling peptide fragments, NCL is based on the unique reactivity of a N-terminal cysteine and a C-terminal thioester, which are joined to give a native peptide bond under mild aqueous conditions. The strategy is thus ideally suited to the preparation of large and densely functionalized macrocycles, and in contrast to earlier cyclization methods which required high dilution, suffer from epimerization, or require cleavable linkers, NCL proceeds through a reversible thiol–thioester exchange reaction, followed by an irreversible S-to-N acyl shift, affording a backbone with an unmodified and seamless peptide bond that is chemically and immunologically indistinguishable from that of ribosomal products. The method has become a key strategy to prepare cyclic peptides containing non-canonical residues, post-translational modifications, or isotopic labels, and has also enabled a number of hybrid synthetic–biosynthetic platforms which exploit recombinant thioesters ligated to synthetic cysteinyl segments. The lack of requirement for side chain protection relieves the need for complex orthogonal protection strategies, and the aqueous reaction conditions avoid denaturing sensitive scaffolds. NCL has thus shifted from being a somewhat esoteric chemical curiosity to a powerful, general and robust methodology that has enabled both academic exploration and industrial production of cyclic peptide drugs.
Fig. 1 Native chemical ligation reaction.1,5
NCL was discovered while attempting to overcome the size and sequence limitations inherent in stepwise solid-phase synthesis. As a convergent synthesis, NCL links unprotected peptide fragments to form a native amide bond. The methodology is important because it has turned the macrocyclization reaction, often viewed as a low-yielding high dilution incompatibility with solid-phase synthesis, into a near-physiological transformation, which can be performed on a milligram-to-gram scale without specialized equipment. NCL is well-suited to macrocyclization because it provides a marriage of selectivity and simplicity: the reaction can only take place between a cysteinyl N-terminus and a thioester C-terminus and so a single cyclic product is obtained even when a complex mixture of precursors are present, and the aqueous buffer system ensures that peptides are soluble, rather than aggregating as so often occurs when cyclizations are performed in the organic phase. The atom efficiency of the ligation, where no atoms from the coupling chemistry are present in the product macrocycle, gives a product which is chemically identical to a ribosomally made product, aiding regulatory acceptance and biological evaluation. Furthermore, the mild, redox-neutral conditions allow preservation of disulfide bonds, phosphorylation states and other labile modifications, allowing late-stage diversification of cyclic peptides with minimal protecting-group manipulations. These properties have made NCL the de facto standard methodology for the preparation of cyclic peptides of the highest fidelity, structural authenticity and synthetic scalability.
The mechanism of Native Chemical Ligation can be described in two steps. The first step is reversible and the C-terminal thioester reacts with the cysteine thiolate to form a transient thioester intermediate that covalently connects the two fragments. This thioester intermediate then undergoes an irreversible S-to-N acyl shift reaction that transfers the acyl group from the sulfur to the cysteine α-amine, completing the peptide backbone with a native amide bond. The reaction conditions for NCL are mild and only require aqueous buffer at close to neutral pH.
Fig. 2 The mechanism of Native Chemical Ligation. R1 can be an Alkyl or an Aryl group.2,5
The thiol–thioester exchange is the first step in NCL and is reversible. This step sets the rate and chemoselectivity of the ligation. It involves an attack of the thiolate anion of the N-terminal cysteine on the carbonyl carbon of the C-terminal thioester to displace the alkylthio leaving group, resulting in formation of a new thioester which covalently links the two fragments with an S-acyl linkage. The reaction must be initiated by a cysteine thiol group; for example, a serine or any other nucleophilic amino acid will not provide the necessary thiol nucleophile, and this requirement imparts NCL with its characteristic cysteine dependency. The C-terminal thioester needs to be sufficiently electrophilic to react with the incoming thiolate, yet also needs to be stable in aqueous buffer for the duration of the ligation. Thioesters can be generated either by solid-phase synthesis using safety-catch linkers or by recombinant expression of an intein-fusion protein, which is then cleaved thiol-mediated to generate the activated ester. The reversibility of this exchange step allows the system to sample a range of thioester conformations and arrangements, and only after a productive thioester conformation has been sampled, does the S-to-N acyl transfer step occur, to form the irreversible structure. Thiols from outside the system, such as thiophenol or benzyl mercaptan, can serve as catalysts, accelerating the rate of equilibration and driving higher effective concentrations of thioester intermediates, without incorporation into the final product. Adjacent large groups can sterically slow down the exchange step, and residues need to be placed carefully away from the ligation junction. The pKa of the cysteine thiol (~8.3) is such that the reaction is driven by a sufficiently large concentration of thiolate at near-neutral pH, where the peptides are also soluble and where the thioester is not subject to base-catalyzed hydrolysis.
Reaction conditions are also an important consideration for transforming the inherent conceptual simplicity of NCL to robust macrocyclisations with high yields. Conditions are usually buffered aqueous solutions of near neutral pH (usually 7.0–7.5) to allow for deprotonation of cysteine while minimizing any effects on thioester stability (phosphate or Tris-HCl buffers are common). The buffer is at low ionic strength to help keep peptides soluble but should not be so concentrated as to cause an increased rate of hydrolysis of the thioester. Temperature is often kept slightly above room temperature to speed up the S-to-N acyl shift but not so high as to risk possible side reactions (for example β-elimination) and still needs to be low enough not to denature peptides containing any heat-labile post-translational modifications. A reducing agent (most commonly tris(2-carboxyethyl)phosphine or TCEP) is used to ensure that cysteine thiols remain reduced, and also to suppress non-specific formation of disulfides that would consume the N-terminal nucleophile. Chaotropic additives (for example, guanidine hydrochloride or urea) are often added at low to moderate concentrations to ensure that both the thioester and the cysteinyl N-terminus are not engaged in transient aggregations and are therefore solvent accessible. Denaturants also decrease the pKa of the cysteine thiol, which can slightly increase the concentration of the thiolate anion without having to use conditions of higher pH that would shorten thioester half-life. Addition of an external thiol catalyst (usually an aryl thiol) can speed up the reversible thiol–thioester exchange, and thus shuttle the acyl group (driving the reversible thiol-ester exchange toward the thioester). The aromaticity of most common catalysts also makes the thiol a better leaving group, which should also increase the rate of thioester formation. If the reaction is conducted in the presence of air, care must be taken that the reaction does not take too long to complete, as air can oxidise TCEP and generate disulfide by-products, particularly if the peptide of interest contains more than one cysteine. Reaction progress can usually be followed by analytical HPLC or mass spectrometry. As the cyclic product is formed, the linear thioester should disappear.
Since its inception as a targeted fragment-coupling tool, NCL has evolved into the workhorse for the assembly of complex cyclic peptides. It now enables a traceless, aqueous, and stereos-preserving approach that eludes classical coupling chemistries. NCL's unique value proposition resides in the seamless conversion of linear precursors (synthetic or recombinant) into head-to-tail macrocycles with no residual atoms, thus allowing to furnish peptide architectures that are truly indistinguishable from ribosomal counterparts. This level of authenticity is crucial in securing regulatory acceptance and in maintaining the unaltered binding signature of bioactive scaffolds, such as cyclotides, defensins, or receptor-directed epitopes. Since NCL is fully tolerant of unprotected side chains, phosphorylated, glycosylated, or isotopically labelled residues are readily accommodated, with no further chemical manipulations needed for structure–activity studies or biophysical interrogations.
An archetypal NCL-based head-to-tail workflow starts from the design of a linear precursor whose sequence encodes the macrocyclic topology of interest: the N-terminus is designed to be cysteine, and the C-terminus activated as an alkyl thioester, most conveniently installed on-resin with a safety-catch linker or produced recombinantly with an intein fusion. Chain assembly and global deprotection is followed by dissolution of the crude linear peptide in aqueous buffer at near-neutral pH, with the addition of catalytic aryl thiol and a mild reductant to preserve cysteine integrity. Warming to physiologic temperature allows the cysteine thiolate to attack the thioester carbonyl, producing a short-lived S-acyl intermediate that rapidly and irreversibly rearranges as an S-to-N acyl shift, to close the backbone into a continuous ring. The intramolecular nature of the reaction allows this to happen efficiently at low precursor concentrations, eliminating the need for the severe dilution needed in many classical macrolactamisations and allowing gram-scale cyclisations in standard glassware. The synthesis of cyclotides is a prime example. Cyclotides are ultra-stable plant macrocycles that also contain three conserved disulfides arranged in a cystine-knot topology. Applying NCL, a linear precursor spanning the cyclotide core can be assembled with designed cysteine pairing, cyclized, and then subjected to oxidative folding, either in one-pot or tandem protocol. The ligation step locks the backbone, and subsequent air oxidation installs the knotted disulfide network to give the native fold without any need for protecting groups on thiols. The entire sequence, from linear chain to correctly folded cyclotide can be achieved within days, and recent reports describe multi-gram batches in a single run, highlighting the scalability of the NCL route. The mild redox-neutral conditions additionally preserve any pre-existing post-translational modifications, opening the possibility of constructing glycosylated or phosphorylated cyclotide analogues that would not be accessible through purely biological expression systems. The traceless nature of the ligation also means that structure–activity studies can be done with confidence that any biological read-out arises from the intended topology rather than linker artefacts.
In cases where the desired macrocycle is beyond the practical reach of stepwise solid-phase synthesis, NCL can be applied in a convergent manner using segmental ligation: the target sequence is split into two or more shorter fragments, each of which carries an internal cysteine and a thioester, and which are ligated in a stepwise fashion before the final cyclisation reaction. In a common strategy, a central fragment with N-terminal cysteine and C-terminal thioester (the "middle" fragment) is first ligated to an N-terminal fragment with its own C-terminal thioester, lengthening the chain and providing an extra cysteine that will ultimately be used for cyclisation. This process is repeated, adding new fragments in a stepwise, directional manner until the desired length is reached; finally, the last fragment is prepared such that the N-terminal cysteine will attack the most remote thioester, thus cyclizing the entire construct into a head-to-tail macrocycle intramolecularly, in one step. This modular approach bypasses the cumulative deletion, racemization and aggregation that limits very long linear syntheses, and each fragment can be purified separately before being joined in essentially quantitative yield. Segmental ligation is also particularly useful for preparing bicyclic or heavily modified peptides. In one recent example, a kinase-inhibitory macrocycle with non-natural γ-aminobutyric acid linkers, isopeptide side chains and a PEGylated lysine was prepared by ligating three fragments: an N-terminal thioester fragment with the PEG unit, a central cysteinyl fragment with the isopeptide and a C-terminal cysteinyl fragment that ultimately provided the nucleophile for cyclisation. The convergent approach allowed each non-natural feature to be incorporated at the fragment level (where purification is not an issue), rather than trying to incorporate all of it in a single, massive linear synthesis. As each ligation junction restores a native amide bond, the final macrocycle is fully biochemically authentic, which is of course a prerequisite for any downstream structural studies or in vivo assessments. In the future, the use of recombinant thioester fragments with synthetic cysteinyl segments (expressed protein ligation) will open the door to even larger cyclic constructs in the segmental NCL format, including protein-sized macrocycles with the stability of a closed backbone and the functional complexity of the full-length protein.
A combination of LC–MS and NMR provides orthogonal experimental support of NCL: these techniques are the ultimate arbiter of the molecular identity and correct structure of the cyclic product. LC–MS is typically the first diagnostic tool used: the simultaneous loss of the linear thioester precursor and appearance of a single new peak, at a mass that is consistent with the expected value for the target macrocycle, unambiguously demonstrates that ligation has taken place. High-resolution full-scan MS can be used to determine whether the cysteine footprint is still present (standard NCL) or absent (post-ligation desulfurization), and is able to detect trace-level amounts of hydrolyzed thioester or oligomeric side-products which may be undetectable by UV. Tandem MS sequencing of the cyclic peptide product can also be carried out, though assignment of peptide sequence is more difficult for cyclic peptides since the loss of terminal charges reduces the number of sequence-specific fragmentation pathways. Collision-induced dissociation of the intact peptide, after labelling of the free C terminus and then after ring-opening with reducing agent or protease, allows assignment of the novel amide linkage to the correct junction, and is also able to confirm that no epimerization has taken place during ligation. NMR also provides atomic-level proof of connectivity and conformation: the diagnostic down-field shift of the cysteine α-proton, upon conversion from free thiol to amide-linked residue, and the disappearance of the thioester carbonyl signal, together with appearance of a new backbone amide cross-peak in ¹H–¹³C HSQC spectra, is proof that the ligation is regio- and chemoselective. Comparison of the NOE patterns of the linear precursor and the cyclic product provides direct evidence of the expected decrease in conformational entropy, with new medium-range NOEs (across the ligation junction) providing evidence of the enforced proximity of formerly non-adjacent residues. The absence of extra signals, due to linker atoms or protecting groups, provides atomic-level evidence of the traceless nature of NCL. This is in contrast to many amide-coupling based synthesis strategies, which often have associated non-peptide residual mass and/or stereochemical ambiguity. Taken together, these orthogonal analytical signatures are the synthetic chemist's ultimate validation suite, providing confidence in the product to allow subsequent biological evaluation.
Chemoselective, traceless, and operationally aqueous, NCL is by many metrics an ideal method for the construction of cyclic peptides, although several technical drawbacks have limited its widespread implementation. Advantages include the production of a true amide bond (rather than an auxiliary-containing pseudopeptide bond) without additional protecting groups, making the final macrocycle indistinguishable from ribosomally synthesized counterparts, an appealing property for studies of mechanistic and pharmacological nature as well as ease of regulatory approval; reaction conditions are mild and metal-free, compatible with disulfide, phosphorylated, or otherwise labile post-translational modifications to allow late-stage diversification of cyclic scaffolds without resort to orthogonal protection strategies; protection group-free side chains simplify synthetic design and can minimize synthetic steps to otherwise complex targets; and cyclisation is intramolecular, permitting useful yields at synthetically convenient dilution, obviating the extreme dilution required for many classical macrolactamisations. Disadvantages include the requirement for an anchor point Cys residue, the 20th most scarce residue in nature by count, often requiring introduction of a non-native Cys or post-ligation desulfurization to Ala, which often requires optimization and can be incompatible with existing disulfides; the instability of thioester precursors to hydrolysis or side reaction with even weak nucleophbiles, requiring careful control of pH and exclusion of such reagents; the requirement for anhydrous conditions, difficult to achieve for precursors made via Boc-SPPS as the cleavage cocktail is often incompatible with moisture exclusion, or for Fmoc-SPPS often requiring special linkers or in-situ activation methods; a ligation site which is sterically encumbered (such as Pro-Cys or Val-Cys) slows the initial transthioesterification step to a rate-limiting crawl, often requiring a temperature or thiol catalyst which may also increase side reactions, including β-elimination and disulfide scrambling; and the high chemoselectivity of NCL can result in branched or oligomeric byproducts when any other Cys is left unprotected in the sequence, requiring masking by reversible oxidation or strategic desulfurisation. Methodological improvements, including the use of masked thioesters and catalyst accelerated ligation conditions, are steadily pushing the envelope of what is practically possible via NCL.
| Parameter | NCL | Amide Coupling |
| Selectivity | High | Moderate |
| Conditions | Mild | Harsh |
| Substrate scope | Narrow | Broad |
Expressed Protein Ligation (EPL) takes the concept of NCL into the recombinant space, leveraging inteins to produce native folded protein thioesters of large size for cyclisation or ligation to synthetic peptides, effectively sidestepping the limitations of solid phase synthesis in peptide length. In its most common implementation, the protein of interest is C-terminally fused to an engineered intein that self-cleaves upon addition of a thiol to give rise to a recombinant polypeptide with a reactive thioester terminus. This recombinant thioester fragment can be mixed under NCL conditions with a synthetic peptide containing an N-terminal cysteine, to promote native peptide bond formation and the site-specific introduction of chemical modifications of various types (post-translational modifications, fluorophores, non-canonical amino acids) in the protein context. For cyclic peptides, EPL enables the production of protein-sized macrocycles with native tertiary structure and the metabolic robustness associated with backbone cyclisation; the recombinant thioester can be engineered to contain a cysteine near its own N-terminus to promote efficient intramolecular cyclisation without the high dilution normally required for NCL. EPL has been used in the preparation of cyclic enzymes, ubiquitin conjugates and phosphorylated signalling scaffolds otherwise beyond the reach of synthetic methods, and the method is compatible with cell-free expression, which should speed up screening of ultra-large cyclic peptides for drug and materials development. Traceless Staudinger Ligation provides a cysteine-independent alternative for the formation of a native amide bond between a C-terminal phosphinothioester (Pin ester) and an N-terminal azide, to a native amide bond. The reaction involves iminophosphorane formation, which collapses to expel a phosphine oxide leaving group. Traceless Staudinger ligation is attractive for peptides lacking cysteine, or where the insertion of cysteine would be detrimental to the peptide structure or function, and the reaction conditions are aqueous and slightly acidic, which is compatible with most unprotected side chains. The main drawbacks are the requirement to synthesize an azide-containing peptide, and the air-sensitivity of the phosphinothioester, although recent work on water-soluble phosphines, and on a one-pot method for azide installation should reduce the entry barrier. Although currently less widely used than NCL, traceless Staudinger ligation has found use in the construction of cysteine-free cyclic antimicrobial peptides and in the generation of D-mirror-image macrocycles.
Scaling NCL for discovery will require its integration into fully automated solid-phase workflows on instruments equipped with on-resin thioester generation (using safety-catch linkers), automated cleavage, pH adjustment and cyclisation in a single fluidic path. Instrumental feedback loops based on real-time LC-MS data will allow the system to automatically prolong the reaction time or automatically inject fresh thiol catalysts in case of incomplete ligation. Robotic liquid handlers will allow hundreds of analogues to be cyclized in parallel for screening campaigns, and next-generation microfluidic chips will further miniaturize the reaction into nanoliter droplets, enabling efficient macrocyclization at precursor concentrations that minimize both solvent use and waste generation, and thus helping NCL to meet green-chemistry requirements. Prediction of most favorable ligation sites can be done in silico with cloud-based software and influence the order of fragment acquisition for synthesis. These and other examples of hardware and software integration will further streamline the entire design-make-test-analyze cycle and increase the pace of lead optimization. In addition to classical cyclic peptides, future NCL workflows will target an interface between peptides and proteins to construct new types of hybrid macrocycles that fuse the targeting precision of peptides with the catalytic or structural complexity of proteins. In one emerging semi-synthetic strategy, a cyclic peptide agonist is ligated to a recombinant protein receptor domain to yield a covalently linked, circularized complex that permanently fixes the signalling assembly into an active conformation. Cyclic protein scaffolds can also be used to define synthetic peptide loops that act as modular and programmable recognition surfaces, paving the way to next-generation vaccines or enzymatic catalysts whose active sites are precisely defined by synthetic chemistry and whose overall fold is provided by the protein scaffold. These and other strategies will be facilitated by next-generation cell-free expression systems that tolerate a growing list of non-canonical amino acids and by continued diversification of chemoselective ligation chemistries, bringing us closer to a routine production of peptide-protein hybrid macrocycles on demand, each custom-designed for a given therapeutic or biotechnological purpose, and placing NCL and its derivative variants at the centre of this ongoing transformation.
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