Peptide cyclization to form cyclic peptides or cyclized peptides is a frequently used strategy for the development of peptides with enhanced conformational stability (compared to their linear analogs). Cyclic peptides have proven to be useful for several applications:
- The mimicry of (protein) secondary structures (e.g. protein loops)
- The optimization of peptides (e.g. peptide ligands with increased binding potency/selectivity and enhanced protease stability)
Depending on the cyclization site, there are nine general methods to synthesize cyclic peptides, e.g. head-to-tail cyclization, side-chain-to-side-chain, head-to-side-chain, and side-chain-to-tail cyclization (see figure below). Among the mentioned ones, the two latter methods are the most frequently utilized. While head-to-tail cycles are usually formed by amide bond formation, side-chain-to-side-chain cycles are most often synthesized via Cys-Cys or amide bond formation within a cyclic peptide.
Cys-Cys cyclization results from the formation of disulfide bridges between cysteine residues of the peptide. Synthetically, this is the most straightforward and frequently used peptide cyclization method. However, potential problems may still have to be overcome. The main problem is dimerization, which is a frequent side reaction during cyclization. This can be minimized by cyclization under high dilution conditions. When a peptide contains multiple cysteine residues, challenges arise due to the large number of possible regioisomers. To cope with this, disulfide bridges can be formed regiospecifically between specific cysteine positions. JPT can synthesize peptides with up to 4 disulfide bonds in one peptide, employing site-specific orthogonal chemistry or thermodynamic stability methods.
One distinct disadvantage of Cys-Cys cyclized peptides is limited stability, especially against reductive conditions. Therefore, drug discovery projects commonly employ a two-step approach:
1) Screening and initial optimization of Cys-Cys cyclized peptides
2) Replacement of the Cys-Cys disulfide by more stable bonds
Amide bonds – which have the advantage over disulfides that they are chemically more stable – are frequently used for cyclic peptides. Most often the following peptides are prepared:
- Head-to-tail cyclic peptides
- Side-chain-to-side-chain cyclic peptides
As with Cys-Cys cyclization, there are some synthetic challenges with amide cyclizations. Dimerization has to be limited, which can be accomplished by working under high dilution conditions. The most important challenge with head-to-tail cyclizations is, however, the fact that the peptide cyclization is often slow due to entropic reasons. This can lead to undesired side reactions like racemization or peptide capping by coupling reagents. For effective head-to-tail cyclization it is therefore critical to choose beforehand
- The optimal cyclization site
- The optimal cyclization reagent
- The optimal cyclization conditions
Over that last year alone, JPT has used its experience for the successful synthesis of more than a thousand amide cyclic peptides with very high success rates even for challenging peptides.
In addition to disulfide and amide cyclizations, there are many other cyclization methods available. Examples are cyclizations via click chemistry (copper-catalyzed azide-alkyne cycloaddition, CuAAC) or cyclizations leading to thioethers (e.g. by cyclization of cysteine side chains with bromoacetate).
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