Native Chemical Ligation (NCL) is a mild, site-selective reaction allowing the chemoselective joining of two unprotected peptide fragments. When applied to a single peptide chain containing both required functional groups, NCL results in peptide cyclization. NCL allows the formation of a natural peptide bond at the ligation site under physiological conditions.
What are the requirements for NCL?
To achieve NCL-mediated cyclization, the precursor peptide must be bifunctional, containing:
- An N-terminal Cysteine: Featuring a 1,2-aminothiol group.
- A C-terminal Thioester: Represented as -COSR.
NCL follows a two-step "capture and rearrange" reaction sequence:
Transthioesterification (Reversible step) followed by an S-to-N Acyl Shift (Irreversible step).
The thiol group of the N-terminal Cysteine performs a nucleophilic attack on the C-terminal thioester, creating a thioester-linked cyclic intermediate. This step is reversible; however, the high local concentration of the two reacting ends favors the formation of the ring. At pH = 7, besides cysteine, all the other amino acid side chains are less nucleophilic. Only the thiol sidechain of cysteine can dominantly trans-esterify with the C-terminal thioester.
Figure 1: The principle of native chemical ligation (Dawson et al. 1994). In this peptide ligation reaction, the first step is the chemoselective reaction of an unprotected synthetic peptide-α-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. This intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.
The free α-amino group of the same Cysteine residue attacks the internal thioester bond. This leads to an irreversible S -> N acyl shift, forming a stable, native amide bond. This thermodynamic sink reaction drives the reaction to completion. The NCL reaction relies on the entropy-driven S-N acyl shift between the C-terminal thioester and N-terminal Cys.
Enthalpy-driven reactions are generally faster than entropy-driven reactions because they often involve the formation of strong bond interactions, resulting in a lower activation energy.
Enthalpy-driven reactions are fast exothermic reactions (∆H < 0), favored at lower temperatures, characterized by negative enthalpy.
Entropy-driven reactions (Δ𝑆 > 0) are faster at higher temperatures, driven by an increase in disorder (
Δ𝑆 > 0), usually requiring higher temperatures to overcome activation barriers.
A brief timeline
1994: Dawson et al. used the NCL reaction for the chemical synthesis of interleukin 8 (IL-8).
1995: Tam et al. reported the use of orthogonal coupling methods for the synthesis of peptides starting from unprotected smaller peptides.
A thiol-thioester exchange mediated by a trialkylphosphine and an alkylthiol or a thioesterification by C alpha-thiocarboxylic acid reacting with a beta-bromo amino acid allowed the synthesis of peptides up to 54 residues in length.
1996: Botti et al. studied thiazolidine formation reactions for the synthesis of cyclic peptides and reported a general method enabling the synthesis of cyclic peptides from linear, unprotected peptide precursors via intramolecular thiazolidine formation.
Figure 2: Preparation of a cyclic peptide from a linear precursor with a aldehyde group on the C-terminal end to enable end-to-end all amide backbone cyclization (Botti et al.).
Figure 3: NCL cyclization reaction. This entropic chemical ligation reaction starts with a chemoselective capture step merging the N- and C- terminal ends of a peptide as a covalently linked O/S-ester intermediate to permit the subsequent step of an intramolecular O/S-N acyl shift to form an amide (Tam et al. 1997, 2012).
Figure 4: NCL cyclization reaction under control of rig-chain tautomeric equilibrium (1. Zhang & Tam 1997).
Figure 5: NCL cyclization reaction under control of rig-chain tautomeric equilibrium (1. Zhang & Tam 1997).
1997: Zhang & Tam used solid phase peptide synthesis to prepare the peptide thioesters. Next, the researchers used the NCL reaction to synthesize a bicyclic peptide and cyclic peptide dendrimers.
1997: Tam & Lu described the synthesis of a large cyclic knot peptide.
1998: Tam et al. developed a thia zip cyclization reaction allowing a series of intramolecular rearrangements in a cysteine-rich peptide for the synthesis of large end-to-end cyclic peptides. This thia zip reaction allowed the synthesis of a 31-amino acid cyclic peptide, a naturally occurring cyclopsychotride with microbial activity, and the synthesis of a cyclic 33-amino acid animal defensin by replacing the end-to-end disulfide with a lactam, which also showed antimicrobial activities.
1998: Camarero et al. reported on-resin NCL for Boc-SPPS on a buffer-compatible aminomethylated PEGA resin functionalized with thiol groups.
1999: Hackeng et al. described a methodology determining the compatibility of the native chemical ligation strategy for X–Cys ligation sites, where X is any of the 20 naturally occurring amino acids to avoid the necessity of specific amino acid thioester linkers or alkylation of C-terminal thioacid peptides. Using these methods, the research group could manually synthesize two 124-amino acid long proteins by using a three-step, four-piece ligation to yield a fully active human secretory phospholipase A2 and a catalytically inactive analog.
1999: Tam et al. described orthogonal ligation strategies for peptide and protein and described a thia zip reaction for the synthesis of large cyclic peptides.
2000: Coltart introduced a peptide segment coupling strategy via prior ligation and proximity-induced intramolecular acyl transfer.
2001: Tam et al. reviewed ligation strategies for multiple segments including sequential and tandem ligations.
Alberico, (2004) reviewed the current state of peptide synthesis methodologies with emphasis placed on recent developments.
2004: Albericio reviewed developments in peptide and amide synthesis.
2008: Hackenberger & Schwarzer summarizes recent developments in the field of chemoselective ligation and modification strategies and illustrated their application, with examples ranging from the total synthesis of proteins to the semisynthesis of naturally modified proteins.
2011: Sohma et al. reported the synthesis of an O-acyl isopeptide using NCL.
2012: Tam & Wong reviewed methods for the chemical synthesis of circular proteins and showed that key elements entropic chemical ligation consist of a chemoselective capture step merging the N- and C- termini covalently linked O/S-ester intermediates to permit the subsequent step of an intramolecular O/S-N acyl shift to form an amide. This review describes advances of entropy-driven ligation to prepare circular proteins with or without a cysteinyl side chain.
2012: Dittman et al. showed that ligation in organic solvents can be conducted chemoselectively without side reactions with other nucleophilic groups and without racemization of the C-terminal amino acid. The research group found that native chemical ligation can be performed either in aqueous buffer systems or in organic solvents.
2012: Monbaliu & Katrizky reviewed advanced, chemoselective techniques for synthesizing complex peptides using cysteine (Cys) and serine/threonine (Ser/Thr) residues, with focus on NCL for joining unprotected segments and O,S to N acyl transfer strategies.
2012: Hermantha et al. reviewed the concept, chemistry, methods and applications of ligation techniques for the assembly of polypeptides and proteins. This review covers the chemoselective capture step followed by an intramolecular acyl transfer of native chemical ligation and selected case studies. Expressed protein ligation, traceless Staudinger ligation and the O→N acyl migration protocol, such as click or switch peptide chemistry, are also reviewed here.
2012: Raibaut et al. reviewed sequential native peptide ligation strategies for the synthesis of proteins.
2014: Thapa et al. reviewed NCL for the creation of large, complex peptides and proteins of ~250 amino acids.
2015: Tailhades et al. described peptide synthesis methods using intramolecular acyl transfer reactions.
2016: Nguyen et al. reported a protocol for using the recently discovered peptide ligase, butelase 1, for high-efficiency cyclization and ligation of peptides and proteins ranging in size from 10 to ~200 amino acid residues. Butelase 1 has a C-terminal-specific recognition motif that requires Asn/Asp (Asx) at the P1 position and a dipeptide His–Val at the P1′ and P2′ positions. Standard Fmoc (9-fluorenylmethyloxycarbonyl) chemistry or recombinant expression with the minimal addition of this tripeptide Asn–His–Val motif at the C terminus allows the preparation of substrates for butelase-mediated ligation.
2016: de Figueiredo published a review of the amid bond-forming reaction, covering nonclassical routes.
2019: Hemu et al. also described protocols for using butelase 1 for efficient and site-specific peptide and protein ligation, N-terminal labeling, preparation of thioesters, and bioconjugation of dendrimers. Additionally, an example using butelase 1 for protein cyclization in combination with genetic code expansion in order to incorporate unnatural building blocks is describes here as well.
2021: Kerdraon et al. studied different selenols derived from the seleno-cysteamine scaffold for their capacity to promote thiol–thioester exchanges in aqueous conditions at mildly acidic pH to produce peptide thioesters from bis(2-sulfanylethyl)amido (SEA) peptides. The study showed that Bis(N, N-dimethylaminoethyl) diselenide can be produced on a multigram scale. The chemical synthesis of a biologically active 9 kDa granulysin demonstrated the method's usefulness.
2021: Bechtler & Lamers reviewed macrocyclization strategies for cyclic peptides and peptidomimetics, summarizing available macrocyclization chemistries, including traditional lactam formation, azide–alkyne cycloadditions, ring-closing metathesis, and unconventional cyclization reactions, structured according to the resulting functional groups.
Figure 6: NCL strategies: Macrocyclic peptides have enhanced proteolytic stability. By connecting the N-terminus to the C-terminus of a peptide, the peptide becomes much harder to digest by cellular enzymes. Also, a locked, cyclic shape is more selective, fitting into a specific receptor like a key in a lock. Cyclization enables hiding polar groups, allowing the resulting peptide to slip through cell membranes more easily than its linear cousins (Bechtler & Lamers).
2025: More recently, Sanchez-Campillo et al. designed sodium 2-selenoethanesulfonate (SeESNa) as a new selenol catalyst for a faster NCL. SeESNa reacts with alkyl α-thioester, N-acyl benzotriazole, and N-acylurea peptides, generating α-SeESNa species. The research group also determined the rate constants for the ligation with preformed α-SeESNa peptides and showed that it is a superior catalyst compared to the known 4-mercapto-phenylacetic and 4-mercaptobenzoic acids. Using SeESNA, the researchers synthesize cardiotoxin A5, a snake venom peptide containing eight cysteines, without orthogonal cysteine protection.
Advantages of NCL Cyclization
The NCL cyclization reaction is highly specific to cysteine and the thioester. Typically, the NCL cyclization reaction is performed in phosphate buffer at ~pH 7. The resulting cyclic peptides have a natural protein backbone.
Some Considerations when using the NCL cyclization reaction
Small peptides, less than 10 amino acids, may make ring strain difficult, while very long peptides might favor intermolecular ligation unless the reaction is performed under high dilution. Adding a catalyst, such as MPAA (4-mercaptophenylacetic acid), can accelerate the initial transthioesterification step.
References
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