Synthesis of Cyclic Peptides
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At present, a number of cyclic peptide drugs have been used in clinical research, such as romidepsin extracted from Chromobacterium violaceum, which can inhibit histone deacetylase and is used as an anti-tumor drug for the treatment of T cell lymphoma. Cyclosporine, a macro cyclic peptide isolated from cyclosporine, was approved by the FDA in 2021 for the treatment of lupus nephritis (LN). Pasireotide, a second-generation somatostatin analogue, is used clinically for the treatment of surgically refractory Cushing's disease. Therefore, the development of an efficient cyclic peptide synthesis strategy is particularly important for the research of this class of drugs. Based on this, a variety of efficient methods for the synthesis of cyclic peptides have been developed and applied to the research and development of new drugs in recent years.
Synthesis of Lactam-Linked Cyclic Peptides
When synthesizing molecular lactam-linked cyclic peptides, conventional methods rely on the use of natural amino acids such as lysine, glutamic acid, or aspartic acid using the Fmoc/ tert-butyl solid phase synthesis method. In this process, the step of removing the protective group using heavy metals such as Pd usually requires harsh conditions. In order to overcome this problem, Chen et al., this paper propose a new crosslinking strategy. They used tert-butyl disulfide as a protective group, and quickly removed the protective group in mild 2-mercaptoethanol /DIPEA solvent to achieve an efficient solid-phase synthesis of lactam cyclic peptides. This method not only showed comparable biological activity and structure to natural products in the synthesis of the antimicrobial peptide brevibacilin and Bowman-Birk protease inhibitors but also successfully overcame the yield problem caused by the formation of oligomers. This study provides an important basis and reference for the efficient preparation of lactam cyclic peptides under mild and metal-free conditions.
Stapled peptide is a special peptide structure, which is formed by cross-linking the side chains of amino acids in peptides to each other or to the end of a peptide, so as to form a stable α-helix or β-fold conformation, mainly covering α-helical peptides supported by olefin/alkyne. Because α-helices account for about 30% to 40% of ordered proteins and play a key role in biological receptor protein binding, stapled peptides containing α-helices have higher targeting affinity, stronger cell penetration, and less resistance to protease hydrolysis. The synthesis strategy of stapled peptides is usually to introduce unnatural amino acids containing α-methyl and α-alene groups into the solid-phase synthetic peptide chain, and then form them through a cyclization reaction. In this process, the selection of unnatural amino acid insertion sites has a significant impact on the activity of the stapled peptide. In 2018, Hui et al. proposed a new stapled peptide binding method, which does not require the introduction of unnatural amino acids, but through the cyclization of tryptophan inside the peptide molecule. This method links tryptophan within the peptide molecule by acid-mediated condensation with aldehyde. It was found that the yield of tryptophan residues at positions i, i+n (n = 1 to 7) was higher. Although it cannot form α-helix cyclic peptide synthesis, it can greatly enhance protein hydrolysis stability, its half-life is linear peptide high 69 minutes. This method avoids the use of heavy metals, simplifies the purification process, and does not need to use expensive unnatural amino acids. It has the advantages of environmental protection and low cost. If the yield can be improved, it will have a good prospect in industrial production.
Example of peptide stapling. (Moiola M et al.)
Example of i, i + 4 (A), i, i + 7 (B) and double i, i + 4 (C) stapling. (Moiola M et al.)
Peptide Synthesis Services at Creative Peptides
| Services | Description |
|---|
| Peptide Synthesis Services | Peptide synthesis services provide expertise in custom peptide production, offering tailored solutions for research, drug development, and industrial applications. These services utilize state-of-the-art synthesis techniques to efficiently generate peptides of varying lengths, purities, and modifications, supporting diverse scientific endeavors. |
| Peptide Modification Services | Peptide modification services specialize in customizing peptides to meet specific research or therapeutic needs, offering expertise in chemical, structural, or functional alterations. These services employ advanced techniques to introduce modifications such as post-translational modifications, labeling, conjugation, or sequence optimization, enhancing the peptide's stability, activity, or targeting capabilities for various applications. |
| Bicyclic Peptides Synthesis | Bicyclic peptides synthesis involves the creation of peptides with two interconnected loops, offering enhanced stability and target binding properties compared to linear peptides. This process typically employs solid-phase peptide synthesis (SPPS) or solution-phase methods to assemble the peptide backbone, followed by cyclization strategies to form the bicyclic structure. The resulting peptides hold promise for drug development, as their constrained structures often improve bioactivity, specificity, and pharmacokinetic properties, making them valuable candidates for therapeutic applications. |
| Cyclic Peptides Synthesis | Cyclic peptides synthesis refers to the process of creating peptides with closed-loop structures, offering enhanced stability and target binding properties compared to linear peptides. This synthesis typically involves solid-phase peptide synthesis (SPPS) or solution-phase methods to assemble the peptide backbone, followed by cyclization strategies to close the loop. These cyclic peptides are valuable in drug discovery due to their structural constraints, which often improve bioactivity, specificity, and pharmacokinetic properties, making them promising candidates for therapeutic development. |
Construct Cyclic Peptides with Cysteine Residues
Build big cyclic peptides a common strategy is to use cysteine residues to form intra-molecular disulfide bonds. Such methods usually include air oxidation, iodine oxidation, potassium ferricyanide oxidation, dimethyl sulfoxide oxidation, and thallium trifluoroacetate oxidation. If there are two or more pairs of disulfide bonds, the protection group such as triphenylmethyl (Trt), monomethoxytriphenyl (Mmt), acetamide methyl (Acm), tert-butyl (t-Bu), benzyl (Bzl), p-methoxybenzyl (Tmob), etc., is commonly used to protect the sulfhydryl group.
Although there are several approaches to constructing cyclic peptides through disulfide bonds, finding suitable linkers to improve the affinity and membrane permeability of cyclic peptides remains a challenge. Based on previous disulfide bond construction of macro cyclic peptides, Sun et al. used small rigid isobutene cross-linked to cysteine residues to cyclize the peptides. It has been found that the isobutylene skeleton is more flexible than the synthesis strategy based on brominated toluene, especially in the presence of thiols in the biological environment. This reaction can even be carried out in water with 10% DMF. In the synthesis and biological activity of somatostatin, in this way can easily obtain quantitative somatostatin, and somatostatin derivatives in glutathione in human plasma and, under the condition of 37 °C incubation 48 hours not happen decomposition, showing good stability and biological activity.
Chiral-Induced Coiling
In addition to relying on amide bonds, alene groups, and sulfhydryl groups, researchers have also proposed the concept of "chirality-induced helicity" (CIH) to stabilize the secondary structure of peptides. Wang's group synthesized staked peptides by replacing the side chain with cysteine residues and olefins. When a chiral center was introduced into the side loop of the stable peptide, and the absolute configuration of the chiral center was R type, the short peptide containing at least 5 amino acids could be promoted to form a standard helix structure with a stable structure. By studying two CIH peptides (diastereomers) with completely consistent amino acid sequences but significantly different secondary conformations, we found that increasing the helicity of the peptides could significantly enhance the stability of the peptides. For peptides with the same amino acid sequence, increasing the helicity of peptides can significantly increase the membrane permeability and target protein binding ability of peptides.
Structural Design
Cyclic peptides found in nature are generally no more than 10 amino acids and have a small relative molecular mass. Such peptides are difficult to be degraded by proteases in vivo and have poor oral absorption. For cyclic peptides with more than 10 amino acids, although they have more abundant molecular configurations and are more stable than linear peptides, their in vivo stability is still not satisfactory. Therefore, researchers have solved the problem of poor stability of micro peptides in vivo by synthesizing cyclopeptides. Compared with monocyclic peptides, dicyclopeptides have a more rigid structure, and after rational design, one of the rings is responsible for binding to proteins on the cell surface, making them targeted. The other loop also binds to the target protein to exert inhibitory activity after the entire molecule is transported into the cell.
Timmerman et al. used tribromometh-ylbenzene (TBMB) to construct bicyclic peptides. The three BRS on TBMB form covalent bonds with the three cysteine residues -SH groups on the peptide chain to form the structure of the bicyclic peptides. The only requirement and key to this technique is that cysteines (Cys) must be at positions 2, 9, and 16 of the bicyclic peptides in order to form a covalent bond with TBMB.
Conclusion
The development of cyclic peptide drugs has benefited from the progress of synthetic technology. A variety of cyclic peptide synthesis strategies have been developed to improve peptide stability, cell permeability, and biological activity. Modern synthetic methods allow more gentle and efficient preparation of cyclic peptides and improve the stability and targeting of the molecules. Cyclic peptides are more and more widely used and are expected to surpass the status of small molecules and antibodies in the pharmaceutical industry in the future.
References
- Lambert J N, Mitchell J P, Roberts K D. The synthesis of cyclic peptides[J]. Journal of the Chemical Society, Perkin Transactions 1, 2001 (5): 471-484.
- Moiola M, Memeo M G, Quadrelli P. Stapled peptides—a useful improvement for peptide-based drugs[J]. Molecules, 2019, 24(20): 3654.
