Head-to-tail cyclization remains by far the most intuitive and commonly used strategy to produce vaccine-grade macrocycles. In this methodology the N-terminal amino group of an assembly-intact linear peptide is covalently coupled to the C-terminal carboxylate via an intramolecular amide bond, forming a ring system with a net charge of zero and significantly enhanced proteolytic stability. The reaction is typically conducted off-resin after global side-chain deprotection using high dilution conditions (≤1 mM) in anhydrous DMF with HATU/HOAt and a non-nucleophilic base (e.g. DIPEA) to quench competing oligomerisation. The key parameters of ring strain, propensity for epimerization at the C-terminus, and solubility are initially assessed in silico; peptides >15 residues often require conformational pre-organisation through the use of D-Pro or N-methylated residues to alleviate entropic penalties. Vaccine applications of head-to-tail lactams include the locking of the Group A Streptococcus J8 epitope into an α-helical conformation capable of inducing high titre opsonising antibodies after single immunisation. Automated microwave-assisted SPPS followed by microfluidic cyclisation now allows for gram-scale production with >70 % isolated yield and<1 % racemization, making this technique the work-horse for rapid vaccine candidate generation.
Fig. 1 Strategies of peptide cyclization and stabilization of α-helices, β-sheets, and β-strands.1,2
Side-chain to side-chain cyclization relies on the use of the (synthetically installed) nucleophilic and electrophilic handles present on amino-acid side chains, most commonly Lys/Asp or Lys/Glu lactam bridges, Cys-Cys disulfides or designed thioether and triazole linkages. This can be particularly useful when the antigenic epitope needs to retain a free N- or C-terminus to engage the receptor, or to include multiple disulfide bonds in order to mimic native viral folds. Orthogonal on-resin protection (Alloc-lysine / OAll-glutamate) followed by selective deprotection with Pd(PPh3)4 and ring-closure using BOP/DIPEA in DMSO/NMP allows for >90 % cyclisation efficiency in the fully assembled peptide still covalently tethered to the resin. Even reversible disulfide formation can be an advantage for extracellular vaccine targets, and a two-step oxidative approach (reduced glutathione buffer followed by air oxidation) has been used to properly pair three disulfides in cyclic scaffolds based on the influenza HA stem, to maintain the native β-hairpin fold and elicit broadly neutralising antibodies. An emerging CLIPS (Chemical Linkage of Peptides onto Scaffolds) approach further expands this approach by tethering the peptide to small organic scaffolds to produce bicyclic or tricyclic antigen mimetics with picomolar affinities for B-cell receptors.
The technique of intramolecular sidechain-to-sidechain crosslinking stands out as the strongest approach to interfere with PPIs and remains the preferred strategy. This was originally based on tethering of sidechains at i and i + 3, i and i + 4, or i and i + 7 residues, via a chemical linker to lock the conformation and reduce the flexibility of peptides, thus maintaining the α-helix structures of the peptides. This normally forms i, i + 4 or i, i + 7 hydrocarbon staples, which contain 8 or 11 carbon atoms respectively. Stapled peptides are the most common peptide inhibitors of PPIs due to their restoration and enhancement of peptides' natural α-helical structures. This restoration of structure allows the peptides to bind large and flat surfaces efficiently and specifically, as is required in most PPIs. However, because these peptides often adopt a much wider range of 3D structures than α-helices, there is no optimal staple for cyclic peptides in general, with a systematic exploration of crosslink positions and geometries required in order to design peptides with enhanced functional properties. The most common linkages incorporated into sidechains include disulfide and lactam bridges, ring-closing metathesis, cysteine crosslinking, C-H activation and thioether linkages, though others have been reported. More recently, the incorporation of α-Amino Acid-derived peptidyl staples has been shown to also improve enzymatic stability and binding affinity. In general, stapling has been shown to enhance binding through hydrophobic interactions with the target molecule, and well-designed stapled peptides often have additional benefits such as enhanced proteolytic stability and binding, stabilised structure and, in some cases, cell permeability.
Peptides have been cyclized using: amide, disulfide, thioacetal, thioether, ether, C-C, Cdouble bondC (i.e., alkene metathesis), Ctriple bondC triple bond (alkyne metathesis), triazole formation and multicomponent reactions (e.g. Ugi reaction). With unprotected peptides, the classical amide bond formation between amino and carboxyl groups is poor in chemoselectivity and can lead to epimerization of the C-terminal amino acid, thus not being suitable for cyclization in solution. Therefore, only when a partially protected linear peptide and a mild activation C-terminus are used, can aminolysis-based reactions be used to obtain cyclic peptides in solution. However, epimerization of the C-terminus is still an issue. Cyclization by aminolysis can also be done on resin, for instance by using a diaminobenzyoyl linker to link the peptide to the solid support. The drawbacks of the cyclization through amide bond formation are limited using chemoselective ligations. The main advantages of chemoselective ligations are the possibility to use unprotected peptides, thus solving the solubility issues of partially protected sequences, and minimal or no C-terminus activation.
Selecting the ideal cyclization strategy for a peptide vaccine involves multiple criteria rather than a simple “head-to-tail vs. side-chain” choice. A first key factor is the linker distance between the residues to be connected. If the N- and C-termini are less than 15–20 Å apart in the native epitope, head-to-tail lactamization is generally possible and affords the most native-like global topology. If they are more distant or if the epitope is β-hairpin stabilized, side-chain-to-side-chain cyclization (e.g., Lys-Asp lactam or Cys-Cys disulfide) may better maintain the conformation required for antigenicity without imposing ring strain. Ring size is another crucial parameter: peptides < 8 residues in length are prone to high entropic penalties upon cyclization, while >25-mers often aggregate and need pseudo-dilution microfluidics or on-resin cyclization to suppress oligomerization. Sequence-specific features also matter, such as Pro/Gly residues at the C-terminus minimizing the risk of epimerization, or bulky aromatic residues lowering the cyclization yield. The production approach (chemical vs. recombinant synthesis) impacts the choice of linker types: chemical synthesis is more permissive of unnatural linkers (triazoles, hydrocarbons), while recombinant methods require traceless intein-mediated cyclization. Finally, application-specific requirements, such as the need for redox-stable disulfides for extracellular vaccines, or hydrophobic staples for membrane penetration, may be more or less compatible with large-scale manufacturability and cost. A decision matrix encompassing all these factors is used to guide our clients towards the best cyclization chemistry in a single design iteration.
Through cyclization of antigens a stable thermodynamic shape emerges that influences their ability to provoke an immune response. Common head-to-tail lactams enforce either α-helices or β-turns. These constrained epitopes often display increased exposure, improving MHC binding affinities by up to two orders of magnitude over linear controls. The addition of disulfide or thioether bridges can rigidify β-hairpins in order to maintain critical neutralisation residues in solvent-exposed conformations. This approach has been used in the design of cyclic HA-stem vaccines that elicit broadly neutralising antibodies. Excessive ring strain can lead to distorted epitopes with lower affinity. Therefore, computational modelling (RosettaRemodel) is often used to predict resulting strain energies, and linker length/residue identity is optimized based on these calculations. Stapled peptides also introduce hydrophobic patches that can improve membrane insertion and APC uptake, but may also alter solubility and therefore require lipidation or PEGylation to remain formulation compatible. Importantly, cyclization reduces conformational entropy, and the resulting slower off-rates increase residence time on B-cell receptors, improving germinal-center reactions and memory formation. The current reseacher integrated workflow links structural prediction, in-silico binding assays and rapid in-vitro validation in order to ensure that every cyclization step is optimal and does not compromise the desired immune response.
Peptide Modification Services at Creative Peptides
Each vaccine candidate has its own topological complexity – multiple disulfides, ncAAs, lipidated residues, or large glycans that require special handling to avoid conventional head-to-tail lactamization approaches. Thus our platform utilizes a toolbox of orthogonal cyclization chemistries guided by retrosynthetic analysis (AI-driven when the structure becomes particularly challenging). For cysteine-rich peptides, orthogonal on-resin Acm/Trt protection schemes combined with well-defined glutathione redox buffers yields regioselective disulfide patterns and routinely >80 % correctly folded product with<1 % racemization. If ring strain impinges on efficiency in 18- to 25-mer constructs, we transition to ring-closing metathesis (RCM) via second-generation Hoveyda–Grubbs catalysts in flow reactors; this approach has delivered 15-membered macrocycles in 65–78 % isolated yield with no detectable epimerization (see industrial-scale synthesis of hepatitis-C peptidomimetic above). Cu-free click cyclization (SPAAC) is used when a sequence contains azide or alkyne ncAAs, so triazole-bridged scaffolds can be made that are both redox-stable and membrane-permeable. All routes are conducted in GMP-grade clean rooms with in-line PAT (Raman, UPLC-MS) that can detect incomplete cyclization in minutes, preventing failure modes that would otherwise require expensive re-synthesis.
Scale-up from milligram samples of discovery compounds to kilogram-sized lots of vaccines is accomplished with straightforward process transfer and statistical process control.
Peptide Synthesis Services at Creative Peptides
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