Peptide Cyclisation Methods
Peptide cyclisation is a common peptide modification technique employed to enhance the properties of linear peptides and address their inherent limitations. The resulting cyclic peptides offer a range of advantages, making them valuable in various applications across life sciences and drug discovery. Notably, three applications worth mentioning are their use as cell-penetrating peptides, peptide therapeutics and tools to mimic structural protein motifs. In this article, we outline the most prevalent strategies for peptide macrocyclisation, including head-to-tail, terminus-to-side-chain and side-chain-to-side-chain cyclisations. We delve into their associated advantages and challenges, providing an overview of this important area of research.
Peptide cyclisation via head-to-tail cyclisation
Amide bond formation – backbone cyclisation
Linear polypeptides are chains of amino acids that are linked together by amide bonds. So, one of the most intuitive cyclisation strategies involves joining both ends of the peptide together. Therefore, head-to-tail cyclisation, as the name suggests, involves forming a lactam bond (a cyclic amide bond) between the N and C-termini of the peptide. While the coupling reagents used are typically the same as those for forming linear peptides, the cyclisation process is more complex in practice.
Challenges – Our scientist’s point of view
Factors such as sterics, reaction conditions, and peptide sequence can significantly influence the efficiency and yield of cyclisation. Satwant Sandhu, Senior Peptide Chemist at AltaBioscience explains, “In general, the macrocyclisation step can be challenging because peptides must adopt an entropically disfavoured pre-cyclisation conformation to close the ring. The chain must also be protected to avoid side reactions and we must choose adequate reaction conditions to prevent oligomerisation and epimerisation of the C-terminal amino acid residue during activation.”
Head-to-tail cyclisation strategies
That’s why retrosynthetic planning, identifying potential challenges and knowing how to optimise the synthesis are essential. In particular, choosing the most suitable cleavage site is crucial to ensure the cyclisation reaction is favourable and does not occur between sterically hindered residues. In addition, introducing a turn-inducing element such as a proline residue, D-amino acids or N-methylation can also facilitate the cyclisation.[1] However, there are other approaches available. For instance, another method is to use chemoselective native ligation-mediated cyclisation strategies, such as sulfur-mediated reactions. Originally developed to condense fragments to generate long peptides, this method was later adapted for intramolecular applications to effectively form macrocycles.
Head-to-side-chain and side-chain-to-tail cyclisations
In terminus to side-chain cyclisation, reactions can occur either between the C- or the N terminus and the corresponding side chain group. Similar to backbone cyclisation, closing the loop typically results in the synthesis of lactams, lactones or thiolactones depending on the functionalities involved. Also, un-natural amino acids can also be employed to vary the size of the ring.
Side-chain-to-side-chain cyclisation
Finally, side-chain-to-side chain peptide cyclisation occurs via two side chain functional groups. The reactions are not limited to lactone and lactam formation; they can also include a wide range of bond types.
Cyclic disulfide-bridged peptides
One important reaction is the formation of intramolecular disulfide bonds. Indeed, disulfide-bridged peptides are widely found in nature; for example, insulin is a well-known peptide hormone that contains one cystine bridge. Plant cyclotides are another example of polycyclic peptides. They usually have a cyclic backbone and a cyclic cystine knot motif which makes them highly stable and resistant to thermal, chemical and enzyme degradation.[2]
The formation of disulfide bridge between two cysteine residues often forms spontaneously upon exposure to air, but can be achieved on solid-phase support or in solution for maximum control. In addition, in peptide containing only two residues in close proximity, the reaction occurs readily. When multiple cysteine residues are present and if the peptide is capable of folding naturally, a “one-pot” multiple bridge formation reaction can be attempted. However, this method can lead to the formation of isomers, so orthogonal protection of the cysteine residues is preferably employed to enable regioselective bond formation.
Stapled peptide synthesis
Ring closure metathesis
One of the drawbacks of disulfide-bridged peptides is their instability under reducing conditions and particularly in the cytosol. Therefore, replacing the cystine bridge with non-natural linkages can ensure increased stability whilst retaining the biological activity of the peptides. For example, olefin staples can be introduced via ring-closing metathesis (RCM) using two terminal alkenes.[3] In practice, the introduction of the modified amino acids and the RCM reaction can be performed on resin with the use of Ru-based Grubbs’ catalyst.
Other stapled peptide methodologies
However, there are other peptide cyclisation methods for synthesising stapled peptides, including:
- Thioether formation: This method is useful when designing peptide libraries where the peptide needs to be presented in a constrained shape. The thioether bond is stable under physiological conditions.
- Methionine- Methionine linkage through chemoselective bis-alkylation. The additional positive charges on the tether facilitate cellular uptake of the peptide. In cellular environment, the tether is reduced and the native peptide is released.[4]
Peptide cyclisation with “Click Chemistry”
Click chemistry is a powerful synthetic approach that enables the formation of cyclic polypeptides. In particular, the copper-catalysed azide-alkyne cycloaddition (CuAAC) is a mild and high-yielding reaction that forms a 1,2,3-triazole linkage. As a result, this has several advantages:
- Chemo- and regioselectivity: The reaction is compatible with a wide range of functional groups and is usually performed without side chain protection, either on resin or in solution.
- Stabilisation of secondary structures: The 1,2,3 triazole bridge can also stabilise the peptide secondary structure, such as alpha-helix.
- Bioisosterism: Finally, the 1,2,3-triazole ring is a common amide bond bioisostere used in medicinal chemistry. However, unlike the latter, it is resistant to proteases and can be used to increase the peptide’s in vivo stability.
In conclusion, peptide cyclisation has become an indispensable tool for enhancing the properties of linear peptides, offering significant advantages in stability, bioavailability, and specificity. The development of cost-effective Fmoc-protected amino acids and innovative building blocks has greatly expanded the chemistry toolbox available, enabling the design of diverse molecular structures and driving further innovations in the field. In addition to the techniques discussed, several alternative methods exist for peptide cyclisation, each with their own unique advantages. For a more in-depth exploration of these approaches, we encourage you to consult the comprehensive reviews referenced below. [5-10]
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To learn more about our capabilities or to discuss your unique requirements, please contact us or email info@altabioscience.com.
References
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[2] David J Craik, Uru Malik, Cyclotide biosynthesis, Current Opinion in Chemical Biology, Volume 17, Issue 4, 2013, Pages 546-554, ISSN 1367-5931, https://doi.org/10.1016/j.cbpa.2013.05.033.
[3] Ellen C. Gleeson, W. Roy Jackson, Andrea J. Robinson, Ring-closing metathesis in peptides, Tetrahedron Letters, Volume 57, Issue 39, 2016, Pages 4325-4333, ISSN 0040-4039, https://doi.org/10.1016/j.tetlet.2016.08.032.
[4] Xiaodong Shi, Rongtong Zhao, Yixiang Jiang, Hui Zhao, Yuan Tian, Yanhong Jiang, Jingxu Li, Weirong Qin, Feng Yin, Zigang Li, Reversible stapling of unprotected peptides via chemoselective methionine bis-alkylation/dealkylation Chemical Science, Volume 9, Issue 12, 2018, Pages 3227-3232, ISSN 2041-6520, https://doi.org/10.1039/c7sc05109c.
[5] Clément Bechtler, Christina Lamers, Macrocyclization strategies for cyclic peptides and peptidomimetics, RSC Med. Chem., 2021, 12, 1325-1351, DOI: 10.1039/D1MD00083G
[6] Yulei Li, Minghao Wu, Yinxue Fu, Jingwen Xue, Fei Yuan, Tianci Qu, Anastassia N. Rissanou, Yilin Wang, Xiang Li, Honggang Hu, Therapeutic stapled peptides: Efficacy and molecular targets, Pharmacological Research, Volume 203, 2024, 107137, ISSN 1043-6618, https://doi.org/10.1016/j.phrs.2024.107137.
[7] Costa L, Sousa E, Fernandes C. Pharmaceuticals (Basel). 2023 Jul 12;16(7):996. doi: 10.3390/ph16070996. PMID: 37513908; PMCID: PMC10386233.
[8] Yu Heng Lau , Peterson de Andrade , Yuteng Wu, David R. Spring, Peptide stapling techniques based on different macrocyclisation chemistries, (Tutorial Review) Chem. Soc. Rev., 2015, 44, 91-102 DOI: 10.1039/C4CS00246F
[9] Junming He, Pritha Ghosh and Christoph Nitsche, Biocompatible strategies for peptide macrocyclization, (Perspective) Chem. Sci., 2024, 15, 2300-2322, DOI: 10.1039/D3SC05738K
[10] Choi JS, Joo SH. Biomol Ther (Seoul). 2020 Jan 1;28(1):18-24. doi: 10.4062/biomolther.2019.082. PMID: 31597413; PMCID: PMC6939695.
