Cyclic Peptide Drugs

Importance of Cyclic Peptides and Role of Impurities

  In a world of changing lifestyles and new disease discoveries, peptide drugs provide a ray of hope for people. Peptide drugs are biologically active and less toxic than other drugs. They have wide therapeutic use, for instance, in oncology, cardiovascular, diabetic, bone diseases, etc. However, due to their high molecular weight and physiochemical characteristics, the synthesis of peptide drugs is complex. It leads to the formation of by-products and other impurities that may affect the drug's safety. Here, we will discuss cyclic peptide drugs and their impurity profiling. This blog is the second in the Peptide series. Let’s first discuss-

Cyclic Peptide Drugs and Their Importance

Cyclic peptides are a class of molecules that have garnered significant attention in drug development due to their unique properties, including high stability, target specificity, enhanced potency, and cell permeability. Cyclic peptides are polypeptide chains taking cyclic ring structure consisting of 5-14 amino acids with a molecular weight of about 500 to 2000 Da. The ring structure can be formed by linking one end of the peptide and the other with an amide bond, or other chemically stable bonds such as lactone, ether, thioether, disulfide, and so on.

Peptide sequence cyclization helps to bind more efficiently with their respective receptors. The cyclic structure of peptides provides a large surface area for interaction with the targeted site. Cyclic peptide drugs are impervious to enzymatic hydrolysis as they don't have free amino and carboxyl ends, thus improving their stability. Usually, cyclic peptides show better biological activity compared to their linear counterparts due to the conformational structure, therefore allowing the enhanced binding toward target molecules or the selectivity by the receptor.

Some cyclic peptide drugs include Daptomycin, Telavancin, Dalbavancin, Lanreotide, Octreotide, Linaclotide, Plecanatide, Romidepsin, Vasopressin, Oxytocin, and Calcitonin. Many cyclic peptide drugs are from natural sources like cyclosporin A, Bactenecin, Lactocyclicin, and more. However, bio-engineered, synthetic cyclic peptide drugs are becoming common now.

How Do You Synthesize Cyclic Peptides?

The synthesis of Cyclic peptides involves Solid-Phase Peptide Synthesis (SPPS). In this method, peptide synthesis occurs linearly, immobilized on a resin bead with a disulfide linker or other chemically stable bonds such as lactone, ether, and thioether. After the linear peptide formation, possible protecting groups are cleaved, and the peptides are released by adding a base. Further, deprotonation of the N-terminal thiol group strikes the disulfide linker between the peptide and resin in intramolecular cyclization, giving the desired cyclic peptide.

Overall, synthesizing cyclic peptides requires careful control of protecting groups, coupling reagents, and reaction conditions to ensure high yields and purity.

Techniques for Synthesis of Cyclic Peptides

The preparation of cyclic peptide compounds uses various techniques: • combinatorial chemistry • de novo synthesis. • chemoselective ligation-mediated cyclization

Depending on the cyclization, the methods to synthesize cyclic peptides are head-to-tail, side-chain-to-side-chain, head-to-side-chain, and side-chain-to-tail. Cyclization of side chain amino acids occurs with disulfide bridge formation between cysteine. Further, backbone-to-backbone cyclization is by amide bond formation between N-terminal and C-terminal amino acid residues.

Moreover, there are other cyclic peptides like bicyclic/ tricyclic peptides and stapled cyclic peptides. Bicyclic peptides are effective enzyme inhibitors and stapled peptides facilitate peptide cell penetration and use cross-linkers to improve physiochemical properties.

Impurities in Cyclic Peptide Drugs

Impurities may arise from the synthesis of cyclic peptides. The API-related impurities are truncations, functional group modifications, insertion or deletion of amino acids, oxidations or reduction of functional groups, aggregates, and incomplete deprotection. Chromatographic media used in purification, and solvents also may contribute to impurity generation in cyclic peptide drugs. Degradation products occur due to changes in the drug product stored for a long time or exposure to light, temperature, water, or reaction to excipients. To ensure the safety and efficacy of cyclic peptide drugs, it is important to control and minimize impurities during the synthesis and purification processes.

General peptide impurities like N-Ac-impurity, de-amidation at Gln, Asn residues, deamidation at C-terminus, trisulfide impurity, Cyanoalanice @ Asn residue, des-Gly and endo-Gly of the terminus amino acids are common in cyclic peptides too. Degradation and other truncated peptides are also a major class of impurities in these cyclic peptides.

Apart from the above-described type of impurities, other impurities like Parallel and Anti-parallel dimers are often formed in cyclic peptides.

A) In the case of cyclic peptides containing a single disulfide bridge, the formation of parallel and antiparallel dimers are as below considering Vasopressin as an example:
  • Seq: Cys(1)-Tyr-Phe-Gln-Asn-Cys(6)-Pro-Arg-Gly-CONH2 (sulfur linkage between cysteines)
  • Parallel dimers: 1,1’, 6,6’
  • Anti-parallel dimers: 1,6’,1’,6
B) In the case of cyclic peptides containing two disulfide bridges, there are 4 sulfur-containing amino acids (cysteines) and the possible formation of parallel and anti-parallel dimers are as below considering Plecanatide as an example:
  • Seq: H-Asn-Asp-Glu-Cys(4)-Glu-Leu-Cys(7)-Val-Asn-Val-Ala-Cys(12)-Thr-Gly-Cys(15)-Leu-OH(4-12), (7-15)-bis(disulfide)
  • Parallel dimers: 4,4’, 7,7’, 12,12’, 15,15’;
  • Anti-parallel dimers:  4,7’,4’,7, 12,15’, 12’,15; or 4,12’,4’,12,7,15’,7’,15 or 4,15’,4’,15, 7,12’,7’,12
C) In the case of cyclic peptides containing three disulfide bridges, there are 6 sulfur-containing amino acids (cysteines) and the formation of dimer in parallel is one, but for anti-parallel dimers, there may be chances of permutations and combination of the cysteine positions one with another is very complex and isolation also a challenging. Linaclotide is such an example:
  • Seq: H-Cys(1)-Cys(2)-Glu-Tyr-Cys(5)-Cys(6)-Asn-Pro-Ala-Cys(10)-Thr-Gly-Cys(13)-Tyr-OH (3 disulfide bridges between 1-6, 2-10 and 5-13 of cysteines)


Daicel offers, a wide range of impurities of various cyclic peptide drugs containing single disulfide bridge such as Vasopressin, Oxytocin, Desmopressin, Calcitonin, Lanreotide, Octreotide, Somatostatin; containing two disulfide bridges like Plecanatide and three disulfide bridges like Linaclotide and other lipopeptide like Daptomycin, and more.
Please read our other blogs in the Peptide Synthesis series to learn more about peptide impurities.
Looking for peptide synthesis services or Pharma Impurities? Reach out to our Daicel specialists today! Just drop in your contact details and we will get back to you.
Peptide Synthesis: Importance of Impurity Profiling in Therapeutic Peptides

Peptide Synthesis: Importance of Impurity Profiling in Therapeutic Peptides


Therapeutic Peptides are a unique class of pharmaceutical drugs positioned in-between classical small-molecule drugs and large-molecule drugs. The peptide drug market is growing faster because of several inherent advantages concerning specificity, efficacy, safety, and the possibility of synthesizing by chemical and or biological methods. It is essential to study the impurity profile of a therapeutic peptide as impurities arise during any stage of peptide synthesis, either from the starting materials, manufacturing process, or during storage.

This blog is the first in its series and takes you through the importance of impurity profiling during the chemical synthesis of peptides.

Peptide Synthesis

Peptides consist of 2-50 amino acids linked by amide groups. Chemical synthesis allows the preparation of peptides outside a living cell. Synthetic peptide drug examples include hormones such as Oxytocin, Calcitonin, Liraglutide, Octreotide, etc.

Chemical peptide synthesis is by two methods

  • Solid-Phase Peptide Synthesis
  • Liquid-Phase Peptide Synthesis

Solid-Phase Peptide Synthesis

It is the most frequently used, efficient, and quick method of synthesis of peptides. Solid phase peptide synthesis (SPPS) involves a coupling reaction of amino acids consisting of protected side chain amino acid residues attached to insoluble polymeric support (resin).

The C-terminal of the initial amino acid is linked covalently to an insoluble polymeric support. After removal of the N(α)-protecting group of the last amino acid residue, N(α)-amino protected amino acids are introduced to the anchored amino acid. Subsequently, it involves a process of purification to remove the soluble by-products. The groups used for N(α)-amino acid protection are 9-fluorenylmethoxycarbonyl (Fmoc) or tert-butoxy carbonyl (boc). Repeating the deprotection and coupling cycle gives the desired peptide. The anchored product is cleaved from the polymer support, and the peptide releases into the solution.

SPPS prepares most peptides of 50 amino acid chains. Fully automated machines can prepare small quantities of peptides quickly. Further, microwave-assisted SPPS offers high-quality peptides.

Liquid-Phase Peptide Synthesis

Peptide synthesis occurring in solution is liquid-phase peptide synthesis. It usually utilizes Boc or Z-amino protecting groups. The final purification step in liquid-phase peptide synthesis is simpler than SPPS. Large-scale synthesis of peptides for shorter chains gives good yield by this method.

Impurities Formed During Peptide Synthesis

The possible impurities during the synthetic process of a therapeutic peptide include

  • Truncated amino acid sequences
  • Deletion sequences
  • Incomplete deprotection sequences
  • Modified sequence due to peptide cleavage
  • Amino-acid racemization

Impurities like peptide counter ions and trifluoroacetate arise from purification methods or SPPS. The side chain reactions of amino acids from Deamidation, oxidation, and hydrolysis reactions result in the formation of impurities. Further, peptide impurities can result in storage due to degradation mechanisms such as β-elimination and succinimide formation. Drug substance-excipient interactions also generate peptide-related impurities.

Purification of Peptides

Synthetic peptide purification is critical as impurities can affect a drug's therapeutic efficacy. The purification of synthetic peptides by Reversed-phase HPLC (RP-HPLC) and ion-exchange chromatography are common methodologies. Also, Gel permeation chromatography (GPC) and Supercritical fluid chromatography (SFC) are used as complementary techniques.

Control of Peptide Impurities

There are many techniques involved in the control of peptide impurities. High-Performance Liquid Chromatography (HPLC) separates and quantifies impurities. Liquid chromatography high-resolution mass spectroscopy (LC-HRMS) helps identify and elucidate structurally related peptide impurities. Regulatory agencies recommend an orthogonal approach using sensitive analytical techniques such as UHPLC-HRMS with the standard HPLC / UHPLC methods to establish the impurity profile of a peptide drug. Peptide-related impurities are critical, not only for active pharmaceutical ingredients (APIs) but also for finished drug products. According to the US-FDA guidelines, during ANDA submission for proposed generic synthetic peptides, the level of a peptide-related impurity in generic synthetic peptides should not be greater than the level found in RLD (Reference listed drug). The control of peptide impurities is vital to establish the safety and efficacy of a synthetic peptide drug.

Daicel offers various kinds of high-quality impurity standards of synthetic peptides and partners with peptide drug manufacturers across the globe. Our custom peptide synthesis team provides reliable solutions to complex peptide impurities and stable isotope-labeled peptides.

Daicel uses state-of-the-art automated SPPS, advanced purification, and analytical techniques during peptide synthesis, followed by the generation of a certificate of analysis from a cGMP-compliant quality control facility.

In addition, Daicel offers physicochemical characterization, cell-based bioassay development, process development, and tech transfer of therapeutic peptides.


Contamination of peptide drugs with impurities will impact the quality and efficacy of peptides and hence impurity profile studies are very important. Peptide impurities can introduce immunogenic epitopes within an amino acid sequence of a peptide and may result in undesired immune responses against the peptide drug. Hence, well-characterized impurity standards are necessary to judge the quality of peptide drugs. Daicel provides reliable custom peptide synthesis services to therapeutic peptide drug developers and manufacturers.

Please read our other blogs in the Peptide Synthesis series to learn more about peptide impurities.

If you are looking for peptide synthesis services, reach out to Daicel specialists today! Just drop in your contact details and we will get back to you.

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