Synthesis of peptides is a complex process that usually involves different synthesis strategies and techniques. The following are some common approaches to peptide synthesis processes and techniques:
Solid-Phase Synthesis
This is one of the most commonly used methods for peptide synthesis. It is based on the stepwise addition of amino acid units on a solid-phase support to build peptide chains. The solid phase support materials typically used are highly reactive resins such as Rink-Amide or Fmoc-AM resin. During the synthesis process, the amino acid unit is immobilized on the solid phase by a reactive protecting group, followed by coupling, deprotection and elution to finally obtain the target
Solution-Phase Synthesis
This method synthesizes peptides in solution. It is usually applied to shorter peptide chains. In solution-phase synthesis, the amino acid unit is coupled to other amino acids in a coupling reaction via an activator (e.g., an activated acid or an activated carboxylic acid). After the reaction, steps such as removal of protective groups and purification are required to obtain a pure peptide product.
Chemical Synthesis
Chemical synthesis is a technique for synthesizing peptide by methods of organic synthesis. It usually involves a reaction using chemical reagents such as protecting groups, activators and solvents. Chemical synthesis methods can be used to synthesize complex peptide structures, but are more challenging for long chain peptides due to the number of steps and the potential for side reactions.
Biosynthesis
Biosynthesis is the synthesis of peptides using cellular mechanisms and enzymes in the organism. This method is commonly used in the synthesis of natural peptides, such as in the production of hormones and peptide drugs. Through genetic engineering techniques, genes encoding peptide are introduced into appropriate expression systems to synthesize the target peptide within the cell.
These synthetic methods are widely used in the field of peptide drugs. Each method has its advantages and limitations, and the choice of synthesis depends on factors such as peptide structural complexity, target purity requirements, scale and economics. Researchers choose the most suitable synthesis strategy and technique based on the specific synthesis needs.

Compared to linear peptides, cyclic peptides exhibit excellent biocompatibility and chemical diversity, making them effective in overcoming limitations and playing a crucial role in new drug development. In recent years, there have been significant advances in cyclic peptide synthesis strategies and their applications in drug development.
Protein-protein interactions (PPI) play a crucial role in various biological processes, and the misregulation of PPI is often associated with the onset of diseases. Peptides, especially linear peptides, face limitations such as susceptibility to proteolytic degradation and poor membrane permeability in practical applications. Therefore, modifications of peptides are often necessary. Peptide modifications can be categorized into N-terminal modification, C-terminal modification, side-chain modification, backbone modification, and cyclization. Among them, peptide cyclization is a common and effective way to enhance peptide stability and binding affinity.
Many natural cyclic peptides or their derivatives are used in clinic therapeutics. For instance, romidepsin, a bicyclic peptide extracted from Chromobacterium violaceum, inhibits histone deacetylase and is clinically used as an anti-tumor drug for treating T-cell lymphoma. Sunflower trypsin inhibitor (SFTI) is the bicyclic peptide that found in seeds of sunflower. Voclosporin, a large cyclic peptide derived from cyclosporin, was approved in 2021 for the treatment of lupus nephritis (LN). Pasireotide, an analog of somatostatin, is used clinically to treat Cushing’s disease when surgery is ineffective.
Cyclic Peptides with Amide Bonds
Initially, researchers used natural amino acids like lysine (Lys), glutamic acid (Glu), or aspartic acid (Asp) residues to form intramolecular amide bonds for cyclic peptide synthesis. The Fmoc/t-butyl solid-phase synthesis method is commonly used today. In this method, the lysine side-chain amino group is protected by allyloxycarbonyl (Alloc), and the carboxyl group of Glu or Asp side chains is protected. After synthesizing the linear peptide chain through solid-phase synthesis, the protecting groups are removed using metal Pd, and the cyclization is performed using coupling agents such as PyBOP. A drawback of this method is the use of heavy metals like Pd during deprotection, which can be harsh.
Cyclic Peptides with Cysteine Residues
Utilizing cysteine residues to form intramolecular disulfide bonds is a common strategy for constructing large cyclic peptides. Various methods, such as air oxidation, iodine oxidation, potassium ferricyanide oxidation, dimethyl sulfoxide oxidation, and thallium trifluoroacetate oxidation, are employed to protect the thiol groups of cysteine residues when two or more disulfide bonds are present, using protective groups such as trityl (Trt), monomethoxytrityl (Mmt), acetylamidoethyl (Acm), t-butyl (t-Bu), benzyl (Bzl), or p-methoxybenzyl (Tmob) for thiol protection. When constructing cyclic peptides via disulfide bonds, an appropriate linker is required to enhance the affinity and membrane permeability of the cyclic peptide.
Stapled Peptides
Stapled peptides involve cross-linking the side chains of any two amino acids in a peptide or cross-linking one amino acid side chain to the end of the peptide, forming a stable α-helix or β-fold conformation, primarily referring to α-helical peptides supported by olefinic or alkyne linkers. Since α-helices constitute 30% to 40% of ordered proteins and play a crucial role in the binding of many important biological receptor proteins, stapled peptides with α-helices exhibit higher target affinity, stronger cell penetration, and resistance to proteolytic degradation. The synthesis strategy for stapled peptides involves introducing non-natural amino acids with α-methyl or α-alkene into the solid-phase peptide chain and then cyclizing through ring-closing metathesis (RCM). In RCM reactions, non-natural amino acids are usually inserted at positions i, i+3, i+4, or i+7 in the peptide sequence.
Bicyclic Peptides
Naturally occurring cyclic peptides generally consist of no more than 10 amino acids, resulting in relatively small molecular masses. Such peptides are less susceptible to proteolytic degradation in the body, but they are poorly absorbed orally. On the other hand, cyclic peptides with more than 10 amino acids, although more structurally diverse and stable than linear peptides, still lack sufficient stability in vivo. To address the issue of poor stability of large cyclic peptides in vivo, researchers have turned to synthesizing bicyclic peptides. Compared to monocyclic peptides, bicyclic peptides have a more rigid structure, and with proper design, one ring is responsible for binding to cell surface proteins, providing them with targeting properties. The other ring, after the entire molecule is transported into the cell, binds to the target protein, exhibiting inhibitory activity.
Plecanatide is a peptide with a structure almost identical to human uroguanylin, consisting of 16 amino acids. Plecanatide, through two pairs of disulfide bonds, exhibits an active conformation at physiological pH 5.0 in the proximal duodenum and jejunum, binding to guanylate cyclase C (GC-C) receptors. It acts as a guanylate cyclase receptor agonist, promoting the excretion of urinary sodium, regulating acid-base ions in the gastrointestinal tract, inducing fluid transport into the gastrointestinal tract, and increasing gastrointestinal motility. Plecanatide is suitable for treating chronic idiopathic constipation in adults.
Conclusion and Prospects
The trend in peptide drug development has shifted from naturally isolated cyclic peptides to optimized cyclic peptide analogs to enhance peptide efficacy, stability, and pharmacokinetic properties. The application of cyclic peptides has also expanded to various areas, including disease treatment, drug targeting, imaging, and diagnostics. Current research on cyclic peptides still focuses on stability, and their drug-like properties require further investigation. Rational drug design could be based on specific disease targets, focusing on the binding sites of key amino acids. Subsequently, through biological activity evaluations, cyclic peptides with optimal drug-like properties may be discovered.
References:
Ji, X., et al., Cyclic Peptides for Drug Development, Angew Chem. Int. Ed., 2023.
Walensky, L. D., and Bird, G. H., Hydrocarbon-Stapled Peptides: Principles, Practice, and Progress, J Med Chem., 2014, 57(15): 6275-6288.

The formation of 2,5-diketopiperazine (DKP) with a bicyclic structure is one of the most detrimental side reactions and degradation pathways affecting peptide synthesis. Despite its crucial role as a versatile building block in drug discovery, the formation of DKP reactions is highly undesirable in the production of peptide drugs. DKP formation can occur during the process of peptide drug formulation, as well as in the storage of starting materials and finished products, and may even occur under solid phase synthesis conditions.
Mechanism of DKP Formation
DKP can be formed through acid- or base-catalyzed reactions. The nucleophilic attack by the Nα group of the peptide on the carbonyl group between the second and third amino acid residues, either as an amide or ester carbonyl, leads to the cleavage of the amide or ester bond. The N-terminal dipeptide, as a derivative of the six-membered ring DKP, is separated from the peptide molecule. Consequently, the peptide molecule is truncated at its N-terminus by two amino acid residues, forming a byproduct characteristic of DKP. When the involved peptide is a depsipeptide (X=O in Figure 1), the formation of DKP is accelerated, as hydroxy derivatives act as better leaving groups than their amino counterparts.
FDA-approved Peptide Drugs
Considering the common occurrence of degradation in peptides with Nα groups, whether the N-terminus should be blocked to prevent DKP formation becomes a crucial consideration in peptide drug design. In fact, there are peptide drugs on the market that have a free Nα group. It is noteworthy that several peptide entities at their N-terminus are blocked through acylation, cyclization, or pyroglutamation. In peptide drugs where Nα is not blocked, they are often cyclic peptides with disulfide bonds or amide cyclization, or they may be prodrugs. Cyclization is known to effectively limit Nα activity, thereby reducing the formation of DKP. There are also three peptide drugs with free Nα, namely Golotimod, Thymodepressin, and NOV-002, which are actually derivatives modified from (L/D)-Glu side chain carboxylate salts. In these cases, the formation of DKP is also hindered.
Characteristics of Peptides Prone to DKP Formation
N-terminal Secondary Amino Acid (Proline, Sarcosine)
When Pro or Sar is positioned as the second residue at the N-terminus of a peptide, the cis configuration of the peptide bond between the second and first amino acid residues is strengthened due to the properties of these secondary amino acids. This stimulation leads to DKP formation in peptides with sequences such as Phe-Pro at the N-terminus, as seen in recombinant human growth hormone (rhGH). Substituting Pro with other amino acids significantly inhibits DKP formation. N-alkyl amino acids appear at the first amino acid position in some marketed peptide drugs, such as oritavancin (N-Me-D-Val) and saralasin (Sar-Arg). It is noteworthy that despite the potential high risk of DKP with Xaa-Pro as the peptide N-terminus, approved peptide drugs like nesiritide (32-amino acid disulfide-bonded peptide) and bivalirudin (20-amino acid linear peptide) have N-terminal sequences containing H-Ser-Pro and H-D-Phe-Pro, respectively.
Cα,α-Dialkylated Amino Acids
Cα,α-dialkylated amino acids synergistically contribute to peptide DKP formation. Although they do not promote cis amide bonds, they impose enhanced conformational restrictions on Cα-C' and N-Cα bonds, favoring the helical secondary conformation. This conformational preference brings Nα closer spatially to the unstable amide carbonyl, promoting DKP formation. While introducing Cα,α-dialkylated amino acids may enhance susceptibility to DKP formation, some marketed peptide drugs like semaglutide (H-His-Aib, GLP-1 analog, 31 amino acids) and macimorelin (H-Aib-D-Trp) contain Aib (2-aminoisobutyric acid, Cα,α-dialkylated amino acid) residues near the N-terminus structural domain.
N-terminal Glycine (Gly):
The correlation between DKP formation and N-terminal residues can be influenced by pKa, volume, amino acid configuration, and the conformational stability of DKP molecules. H-Gly-Pro-peptide is a highly sensitive N-terminal sequence that readily forms DKP due to the spatial promotion effect of Gly as the N-terminal residue and the cis configuration driven by Pro. H-Gly-Gly is another sequence highly sensitive to DKP formation. Marketed peptide drug peginesatide has a branched double N-terminal sequence Gly-Gly, but its N-terminal Gly is acetylated. Another marketed peptide drug terlipressin (cyclic peptide with disulfide bonds) has the N-terminal sequence H-Gly-Gly-Gly, and it is found to be susceptible to DKP formation during the formulation process. Peptide sequences with Gly as the N-terminal third residue also undergo DKP formation. Other marketed peptide drugs with free N-terminal Gly residues include desmopressin (H-Gly-Arg) and PepGen P-15 (H-Gly-Thr).
N-terminal Histidine (His)
Peptides with the sequence H-His-Pro at the N-terminus are highly sensitive to DKP formation, attributed to the catalytic role of the imidazole group in the His side chain. In this structure, the His-Pro peptide bond adopts a cis conformation favorable for DKP, promoting nucleophilic attack by His-Nα on -Pro-Xaa3- main chain amide. The enhanced nucleophilicity of His-Nα is facilitated by the deprotonation of His's basic imidazole group. This His-mediated catalytic process results in the cleavage of the Pro-Xaa3 peptide bond, ultimately forming the cyclic derivative His-Pro-DKP and complementary truncated peptide fragments.
In terms of DKP formation, the H-His-Gly sequence appears more stable than H-His-Pro. Many approved GLP-1/2 analog peptides, such as exenatide, dulaglutide, lixisenatide, and teduglutide, feature N-terminal H-His-Gly sequences. Additionally, Alloferon also has an H-His-Gly N-terminal sequence.
Opposite Configuration of N-terminal Amino Acids
Peptide sequences composed of alternating L- and D-amino acids significantly affect DKP formation. In this case, the formed DKP molecule is stabilized thermodynamically due to the positioning of the two side chain groups, R1 and R2, on both sides of the DKP molecule plane, favoring DKP formation.
N-terminal dipeptide sequences with opposite configurations of amino acid residues are found in some marketed peptide drugs, such as icatibant (H-D-Arg-Arg) and bivalirudin (H-D-Phe-Pro).
Disulfide-Bridged Peptides:
Disulfide bonds can effectively suppress DKP formation by limiting the spatial position of the nucleophilic Nα group. This feature may explain the higher proportion of free Nα groups in marketed disulfide-bridged peptides, such as octreotide, oxytocin, and lanreotide. Compared to N-terminal unacetylated short linear peptides, they are less likely to form DKP.
N-terminal H-Xaa-Asp Peptides
If Asp or Asn is located at the second position of the peptide N-terminus, the formation of aspartimide can promote peptide DKP formation. This aspartimide-induced DKP formation has been detected in many studies. Asn/Asp at the second N-terminal position initially forms aspartimide, and the formed aspartimide peptide intermediate can undergo differential isomerization into the corresponding non-enantiomeric isomer aspartimide or hydrolyze into Asp- or β-Asp peptides. Additionally, the aspartimide at the second N-terminal position can undergo nucleophilic attack from Nα and convert into the corresponding cis-DKP compound. Under neutral to alkaline conditions at room temperature or elevated temperatures, cis-DKP can rapidly isomerize into trans-DKP. The sensitivity of aspartimide at the second N-terminal position to DKP is attributed to the increased electrophilicity of the acyl imide part and the easy racemization of the aspartimide intermediate. In this mode, the six-membered ring of DKP is not excluded from the peptide molecules, resulting in the presence of DKP isomers in the peptides.
Plecanatide is a marketed peptide drug with both linear and Asp as the second N-terminal residue (H-Asn-Asp). Thymosin α1 has a Ser-Asp N-terminus, but its Nα group is acetylated.
Summary
In FDA-approved short linear peptide drugs, the majority of Nα groups are acetylated and capped.
Nα acetylation and capping can have a significant impact on the physicochemical properties of the parent peptide molecule, such as secondary structure, isoelectric point (PI), solubility, etc., particularly for short peptides.
Long peptide drugs tend to have a relatively higher proportion of free Nα groups.
The occurrence of unacetylated Nα is more common in disulfide-bridged peptides and amide-cyclized peptides.
Special attention should be given to the possibility of DKP formation for short peptides containing sensitive N-terminal dipeptide sequences.