Peptide Synthesis for Cyclic Peptides: Methods and Applications

Definition and Structural Characteristics of Cyclic Peptides

Cyclic peptides represent a unique category within peptide compounds where their peptide chains form a continuous loop by covalent bonding. The closed ring structure of cyclic peptides differentiates them from linear peptides which possess separate N- and C-terminal ends. The chain in cyclic peptides forms a closed loop which results in the absence of free termini. Cyclic peptides demonstrate a broad range in size because small cyclic peptides are formed from just several amino acid residues and larger cyclic peptides can contain multiple dozen residues. The most common cyclic peptides range from 4 to 30 amino acid residues. The ring-like structure gives cyclic peptides special physicochemical characteristics and biological functions. Cyclic peptides show enhanced conformational stability and protection against enzyme breakdown which leads to their special biological functions when compared to linear peptides.

Classification of Cyclic Peptides

By Amino Acid Composition: Cyclic peptides can be categorized into those composed of natural amino acids and those containing non-natural amino acids. Cyclic peptides made of natural amino acids are solely composed of the 20 common amino acids found in nature and exhibit various biological functions. In contrast, cyclic peptides incorporating non-natural amino acids introduce specially designed residues during synthesis, enhancing stability or modifying biological activity.

By Cyclization Method: Cyclic peptides can be classified as directly cyclized peptides or linker-assisted cyclized peptides. Directly cyclized peptides form a ring by directly linking the N- and C-termini of the peptide chain, a relatively straightforward approach. Linker-assisted cyclized peptides involve the introduction of a linker molecule that connects the ends of the peptide chain, increasing structural diversity and flexibility.

By Source: Cyclic peptides can be either naturally occurring or synthetically produced. Natural cyclic peptides are isolated from biological sources such as plants, microbes, and animals. Synthetic cyclic peptides are artificially prepared using chemical synthesis or biotechnological methods, allowing for the design of specific amino acid sequences and structures tailored to particular needs.

Natural Occurrence of Cyclic Peptides

Cyclic peptides are widely distributed in nature across various organisms. In the plant kingdom, many plants produce bioactive cyclic peptides as a defense mechanism. For example, certain legumes synthesize cyclic peptides with antibacterial and antiviral properties to protect against pathogens. In the microbial world, bacteria and fungi are key sources of cyclic peptides. Some bacterial-derived cyclic peptides exhibit antibiotic activity, inhibiting microbial growth and playing a crucial role in microbial competition. For instance, subtilin, produced by Bacillus subtilis, is effective against Gram-positive bacteria. Fungi also synthesize various cyclic peptides, some of which possess immunomodulatory properties. Additionally, certain marine organisms secrete cyclic peptide toxins for predation or defense. These naturally occurring cyclic peptides not only contribute to the survival and reproduction of the organisms that produce them but also serve as valuable resources for developing new drugs and bioactive molecules.

Synthesis Methods of Cyclic Peptides

1. Solid-Phase Synthesis

Principles of Solid-Phase Synthesis

Solid-phase peptide synthesis (SPPS) is based on anchoring the growing peptide chain to an insoluble resin support. The resin plays a crucial role by providing a stable platform for peptide synthesis while facilitating reaction separation and purification. Commonly used resins include Wang resin and Rink amide resin, selected based on the target cyclic peptide's structure and properties.

The synthesis process involves sequentially coupling amino acids to the anchored peptide chain. The first amino acid is covalently attached to the resin, followed by the stepwise addition of other amino acids in a predefined sequence using coupling reagents such as N,N'-dicyclohexylcarbodiimide (DCC) and its derivatives. Each coupling step is followed by washing to remove unreacted reagents and byproducts, ensuring high efficiency and accuracy. Once the desired linear peptide chain is synthesized, a cyclization reaction is performed to form the cyclic peptide.

Procedure of Solid-Phase Synthesis

Resin Pretreatment: The appropriate resin is chosen based on the target cyclic peptide structure and is swelled in a suitable solvent to enhance reactivity. Hydrophobic resins may require swelling in organic solvents like dichloromethane for optimal reaction conditions.

First Amino Acid Coupling: The first amino acid, protected by a suitable protecting group, is covalently attached to the activated resin in an appropriate reaction solvent with a coupling reagent. The resin is then washed to remove unreacted components.

Sequential Amino Acid Coupling: Additional amino acids are coupled sequentially following the designed sequence. Each step involves protecting group removal, amino acid activation, and coupling under controlled conditions to ensure efficient and accurate synthesis.

Cyclization Reaction: Upon completion of the linear peptide sequence, the protecting groups are removed, and cyclization is induced under carefully controlled conditions, including solvent selection, temperature, and pH, to optimize yield.

Cleavage and Purification: The cyclic peptide is cleaved from the resin and purified using high-performance liquid chromatography (HPLC) to remove impurities and unreacted materials, yielding a high-purity cyclic peptide product.

Advantages and Disadvantages of Solid-Phase Synthesis

2. Liquid-Phase Synthesis Method

Principles of Liquid-Phase Synthesis

Liquid-phase synthesis of cyclic peptides is conducted in a homogeneous solution system. In solution, amino acid molecules are in a free state, and their reactions primarily follow the principles of chemical reaction kinetics. First, amino acids involved in the reaction need to be appropriately protected with protective groups to prevent unwanted side reactions and ensure that the reaction proceeds as expected. For instance, Boc (tert-butoxycarbonyl) and Fmoc (9-fluorenylmethoxycarbonyl) protecting groups are commonly used to protect the amino or carboxyl groups of amino acids.

Controlling reaction conditions is crucial, including reaction temperature, pH, reaction time, and reactant concentration. Suitable reaction temperature can regulate reaction rate and prevent side reactions caused by excessive heat; precise pH control affects amino acid reactivity and reaction selectivity; reaction time directly determines the extent of reaction completion; reactant concentration influences the driving force of the reaction. During the reaction, appropriate coupling agents are added to promote the condensation reaction between amino acids, forming peptide bonds. As the reaction progresses, the peptide chain is gradually extended, and finally, under specific conditions, a cyclization reaction is carried out to form the cyclic peptide.

Operational Process of Liquid-Phase Synthesis

1. Preparation of the Reaction System

Based on the amino acid sequence of the target cyclic peptide, suitable protective groups are selected to protect the amino acids. The protected amino acids, coupling agents, and appropriate reaction solvents are added into the reaction vessel to form a homogeneous reaction system. Common reaction solvents include dimethylformamide (DMF) and dichloromethane, which are selected based on the solubility and reaction characteristics of the reactants.

2. Amino Acid Coupling Reaction

Under controlled temperature and stirring conditions, the amino acids undergo condensation reactions to form peptide bonds. The reaction progress needs to be closely monitored using techniques such as thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to determine the extent of the reaction. Once the reaction reaches the desired level, the next step is carried out.

3. Deprotection and Addition of New Amino Acids

After completing a condensation reaction, the protective group is removed using an appropriate method based on its properties. Then, a new protected amino acid is added, and the condensation reaction steps are repeated to gradually extend the peptide chain. Each reaction step must ensure completeness and product purity.

4. Cyclization Reaction

Once the linear peptide chain is synthesized, the reaction conditions are adjusted—such as changing the solvent, temperature, and pH—to promote the cyclization reaction between the peptide's terminal groups. This step requires precise control of reaction conditions to improve the yield of the cyclization process. The progress of cyclization can be monitored by observing changes in physical or chemical properties of the reaction system, such as UV absorption and NMR signals.

5. Product Separation and Purification

After the reaction is complete, separation techniques such as extraction and column chromatography are used to remove impurities and unreacted substances. Further purification is carried out using methods such as recrystallization and high-performance liquid chromatography (HPLC) to obtain a high-purity cyclic peptide product.

Advantages and Disadvantages of the Liquid-Phase Synthesis Method

Other Synthesis Methods

Enzymatic Synthesis Method

The enzymatic synthesis of cyclic peptides utilizes enzyme catalysis to achieve peptide bond formation. The principle is that certain enzymes can specifically recognize amino acids or peptide chains and catalyze their linkage. Commonly used enzymes include proteases and transaminases. For example, some proteases can catalyze cyclization reactions under specific conditions.

This method has unique advantages. Firstly, enzymatic reactions exhibit high specificity, allowing precise catalysis between specific amino acids, reducing side reactions and improving product purity. Secondly, enzymatic reactions typically occur under mild conditions, such as near physiological temperature and pH, making them particularly beneficial for cyclic peptides sensitive to temperature and chemical environments. Additionally, enzymes function as biological catalysts with good biocompatibility, minimizing the introduction of impurities during synthesis and facilitating subsequent applications.

Chemoselective Cyclization Method

The chemoselective cyclization method employs specific chemical strategies to induce cyclization at predetermined positions within the peptide chain. The core concept is to use selective chemical reagents or reaction conditions that target specific functional groups or regions of the peptide chain, enabling cyclization.

Several strategies exist for achieving chemoselective cyclization, such as utilizing the specific reaction between thiol (-SH) groups and maleimide groups. When a peptide chain contains thiol and maleimide groups, they undergo a reaction under appropriate conditions to form a cyclic structure. In targeted cyclic peptide synthesis, this method allows precise control over the cyclization site and process. It is particularly valuable for synthesizing cyclic peptides with specific structures and functions, as it enables the accurate incorporation of functional groups into the cyclic peptide framework to meet various application needs.

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Applications of Cyclic Peptides

1. Applications in the Pharmaceutical Field

• Cyclic Peptides as Drug Molecules

Several cyclic peptide drugs have been successfully marketed or are in clinical trials. For example, octreotide is a synthetic cyclic peptide drug that targets the somatostatin receptor. It is primarily used to treat acromegaly and other hormone-related disorders. By binding to the somatostatin receptor, octreotide inhibits excessive growth hormone secretion, alleviating symptoms of acromegaly. Compared to traditional drugs, cyclic peptide drugs offer unique advantages. Their cyclic structure enhances stability and affinity, allowing for more precise target binding and improved therapeutic efficacy. Additionally, cyclic peptides exhibit high specificity, resulting in fewer side effects. Another example is exenatide, which acts on the glucagon-like peptide-1 (GLP-1) receptor and is used to treat type 2 diabetes. Exenatide mimics the function of natural GLP-1, promoting insulin secretion and lowering blood glucose levels. Compared to conventional hypoglycemic drugs, exenatide not only effectively controls blood sugar but also aids in weight reduction, demonstrating promising therapeutic effects.

• The Role of Cyclic Peptides in Drug Development

Cyclic peptides play a crucial role in drug development. First, they serve as templates for optimizing lead compounds. Due to their diverse structures and unique biological activities, researchers can modify and refine natural cyclic peptides by altering amino acid sequences or introducing specific chemical groups to enhance activity and pharmacokinetic properties, leading to the development of more effective drug molecules. Second, cyclic peptides are valuable for constructing drug screening libraries. Using combinatorial chemistry, researchers can generate large libraries of cyclic peptides by combining different amino acids and screen for molecules with potential therapeutic activity. This approach enables rapid and efficient discovery of new lead compounds. Furthermore, cyclic peptides help in studying drug-target interactions. By analyzing how cyclic peptides bind to their targets, researchers can design more targeted drugs, increasing the success rate of drug development and accelerating the discovery of new therapeutics.

2. Applications in Biotechnology

• Cyclic Peptides in Protein Interaction Studies

Cyclic peptides play an important role in studying protein-protein interactions. Experimentally, their ability to bind specifically to target proteins is often exploited. First, cyclic peptides can be labeled with fluorescence or radioisotopes. These labeled peptides are then incubated with samples containing the target protein. If the protein interacts with the cyclic peptide, binding occurs, and the interaction can be detected through the labeled signal. For instance, in studying intracellular signal transduction pathways, cyclic peptides that specifically bind to key signaling proteins can be designed. By analyzing their interactions with other proteins, new protein interaction networks have been discovered, providing important insights into signaling mechanisms. Compared to traditional methods, this approach offers higher specificity and sensitivity, allowing for more precise characterization of complex protein interactions.

• Cyclic Peptides in Biosensors

Cyclic peptides demonstrate unique applications in biosensor development. The principle of their application lies in their ability to selectively recognize and bind specific biomolecules. By immobilizing cyclic peptides onto the sensing elements of a biosensor, they can specifically interact with target biomolecules, causing changes in the physical or chemical properties of the sensor, such as optical or electrical signals, thereby enabling target detection. In practical applications, cyclic peptide-based biosensors have been developed to detect specific tumor markers. Researchers design cyclic peptides that selectively recognize tumor markers and integrate them into biosensors. When a sample containing the tumor marker is tested, the sensor generates a detectable signal, allowing for rapid and sensitive tumor marker detection. This technology provides a powerful tool for early cancer diagnosis.

3. Applications in Materials Science

• Preparation of Cyclic Peptide-Based Materials

Cyclic peptides are used to develop novel materials, particularly self-assembling materials. The preparation process involves several steps. First, appropriate cyclic peptide sequences are selected based on their specific amino acid composition and structure to ensure optimal self-assembly properties. Next, the cyclic peptides are dissolved in a suitable solvent to form a uniform solution. By controlling parameters such as concentration, temperature, and pH, the self-assembly of cyclic peptide molecules is induced. During self-assembly, cyclic peptide molecules arrange in an ordered manner through non-covalent interactions such as hydrogen bonding and hydrophobic interactions, forming structures like nanofibers and nanotubes. For example, certain cyclic peptides containing specific amino acid residues can self-assemble into nanofiber networks under appropriate conditions. By adjusting the preparation conditions, the morphology and size of the self-assembled materials can be precisely controlled to meet the requirements of different applications.

• Properties and Applications of Cyclic Peptide-Based Materials

Cyclic peptide-based materials possess several unique properties. In terms of biocompatibility, cyclic peptides, being composed of amino acids, are highly compatible with biological systems, minimizing immune responses and side effects in biomedical applications. Functionally, specific amino acid sequences can be designed to impart various properties, such as targeted recognition or catalytic activity. In materials science, cyclic peptide-based materials have a wide range of applications. In tissue engineering, self-assembling cyclic peptide materials can serve as cell scaffolds, providing a suitable microenvironment for cell growth and differentiation, thus promoting tissue repair and regeneration. In drug delivery, cyclic peptide-based materials can encapsulate drug molecules, enabling controlled release and enhancing therapeutic efficacy. Additionally, in nanotechnology, cyclic peptide-based nanomaterials can be used to develop high-performance sensors and electronic devices, showcasing significant potential for future applications.