In modern medicine, researchers use peptide applications to diagnose and treat cancer, map epitopes, develop antibiotic drugs, design vaccines, and offer custom antibody sequencing services. Moreover, the processes necessary to create vaccines have also aided the progress of peptide development.
Since peptide synthesis was first discovered, it has been used in many different ways. For example, synthetic peptides are now used to create specific epitope antibodies against pathogenic proteins, study protein functions, and identify or characterize proteins.
Synthetic peptides make it possible to study essential enzymes for cell signaling, such as proteases and kinases, and specifically to figure out how enzymes interact with their substrates.
What is Peptide Synthesis?
Peptide synthesis occurs when a peptide bond is formed between two amino acids. Although there is no definitive definition of a peptide, the term usually refers to flexible chains of approximately 30–50 amino acids that exhibit little secondary structure.
Linking amino acids together with peptide bonds has been possible for more than 100 years, but the first synthetic peptides, such as oxytocin and insulin, were not successfully synthesized for another 50–60 years. This timeline highlights the difficulty of chemically synthesizing chains of amino acids. In the last 50 years, the chemistry and methods for producing proteins have improved to the point where peptide synthesis is now a common tool for biological research, product and drug development, and high-throughput scientific analysis.
The advantage of modern peptide synthesis strategies is that they can replicate peptides found in biological samples. Furthermore, they allow for the creativity and imagination required to create novel peptides designed to elicit specific biological responses or other desired results. This page discusses the most crucial aspects of peptide synthesis, the most common ways to synthesize and purify peptides, and the pros and cons of each method.
Solid Phase Peptide Synthesis
During solid-phase peptide synthesis (SPSS), the polypeptide chain is attached to a solid polymer that acts as a support and anchor. The polymer is specifically functionalized to bind the first amino acid in the sequence and remain unresponsive to subsequent amino acids. Final treatment with a strong acid cleaves the bond between the polymer and the completed peptide. As a result, the peptide chain maintains a temporary bond with the solid polymer throughout the synthesis process.
The free amino acids in solution and the expanding chain attached to the polymer are prevented from reacting incorrectly. This is because amino acids use protective groups, such as Fmoc or Boc, to block their reactive sites. These shields prevent spontaneous reactions that would disrupt the synthesis of a prescribed sequence of amino acids. Researchers can selectively activate individual amino acids by using specific chemical processes to remove the protecting groups at each step.
Once the peptide chain is fully formed, it must be released from the polymer through cleavage. However, the system must be washed to eliminate unwanted soluble compounds and reagents before this final step can begin. Once the polypeptide chain has been isolated from the polymer resin, the resulting solution is much more concentrated and purified.
Peptide Synthesis Method
In laboratory peptide synthesis, the carboxyl group of an incoming amino acid is usually coupled to the N-terminus of the growing peptide chain. This creates a C-to-N synthesis direction, which contrasts with natural protein biosynthesis, in which amino acids are incorporated into a protein chain from the N-terminus to the C-terminus (N-to-C).
The precise, step-by-step, and cyclic addition of amino acids to the burgeoning peptide chain is necessary for the successful execution of in vitro protein synthesis. Although the most popular approaches to peptide synthesis differ in several respects, they all involve the sequential addition of amino acids to an existing peptide chain.
The Role of Peptide Synthesis
The advantages of peptide synthesis include the following:
1. The Convenience of Removing Unwanted Compounds
The initial amino acids in the peptide chain are bonded to a polymer substrate to create a polypeptide using the solid phase method. All molecules bound to the polymer are insoluble, allowing for easy separation from the washable, free-floating impurities. Anchoring a peptide chain to a polymer facilitates the desired chemical reactions between free-floating amino acids and the existing chain. The stability of the anchored chain minimizes the loss of the product during washing steps.
2. Reduced Experimental Labor
Sometimes, the potential benefit of automation outweighs the tremendous cost of correcting human errors. In addition to the financial and time costs of error correction, manual projects often face significant delays. By automating the SPPS process, labs can reduce their likelihood of error and free up funds previously allocated for reworking failed syntheses.
3. Accelerated Rate of Discovery
Using automated workstations for custom peptide synthesis has several benefits, the most important being the simplification and improvement of research throughput. With the help of such automated workstations, researchers are able to analyze a larger number of specimens on target surfaces. The overall precision of the experiment is significantly improved with this method.
4. Boosted Efficiency
The increased throughput and decreased error rates in peptide synthesis lead to greater productivity. When fewer problems arise during experiments, more time, effort, and other resources can be devoted to analysis. An added benefit of SPPS automation is that analysts can modify established procedures with greater precision and reproducibility.
5. Control Over Reaction Sequences
Protective groups are added to the free amino acids to prevent them from reacting prematurely with other molecules. Various chemical classes are utilized, each responding differently to deprotection efforts. This allows scientists to control which amino acids are converted into reactive forms. By taking this measure, researchers can be sure that only the intended next-in-line amino acid will link to the anchored chain.
Peptide Deprotection
Peptide synthesis is a process that must be carried out with extreme caution to prevent unwanted side reactions. If these reactions occur, they can shorten the peptide chain or lead to unintended branching. Because amino acids include many reactive side chains, chemical compounds that attach to these reactive groups, acting as a shield to obstruct the functional group, have been devised to ensure peptide production with minimal side reactions.
Even before synthesis begins, certain protecting groups are added to the pure, independent amino acids. These protecting groups are then eliminated from the recently introduced amino acid, a process known as deprotection, shortly after coupling. This enables the subsequent amino acid to attach to the growing peptide chain correctly. Upon completion of peptide synthesis, all leftover protecting groups are cleared from the resulting peptides. Three common protecting groups are discussed below, depending on the peptide synthesis process used.
1. N-terminal Protecting Groups
The N-termini of amino acids are protected by groups referred to as "temporary" protecting groups, as they can be removed relatively easily to permit peptide bond formation. The usage of tert-butoxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) is determined by the specific requirements of the synthesis protocol.
2. C-terminal Protecting Group
The employment of a C-terminal protecting group is dependent on the method of peptide synthesis applied; for instance, liquid-phase peptide synthesis necessitates the protection of the C-terminus of the first amino acid to prevent unwanted polymerization.
3. Side Chain Protecting Groups
These are referred to as "permanent" protecting groups because they can withstand numerous cycles of chemical modification during the synthesis process. They are only removed by a reaction with strong acids after the final peptide chain is complete.
Amino Acid Coupling
Carbodiimides, such as dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC), are required to activate the C-terminal carboxylic acid of the incoming amino acid during synthetic peptide coupling. These coupling agents interact with the carboxyl group to generate an extremely reactive O-acylisourea intermediate. This intermediate is rapidly displaced by a nucleophilic attack from the unprotected primary amino group at the N-terminus of the growing peptide chain to create the nascent peptide bond.
Racemization of the amino acid is possible because carbodiimides create such a reactive intermediate. Therefore, it is common to add reagents that interact with the O-acylisourea intermediate, such as 1-hydroxybenzotriazole (HOBt), which generates a less reactive intermediate and minimizes the likelihood of racemization. In addition, side reactions induced by carbodiimides have led to the development of other coupling agents, such as benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), both of which necessitate the use of activating bases.
Strategies of Peptide Synthesis
The original approach researchers used to synthesize peptides in vitro is known as liquid-phase peptide synthesis (LPPS), and it is still frequently employed for large-scale production. However, this approach is time-consuming and labor-intensive, as the product must be physically purified from the reaction system after every step. Furthermore, this method necessitates an extra chemical group to protect the C-terminus of the initial amino acid. An advantage of liquid-phase synthesis is that side reactions are quickly discovered because the intermediate is purified after each step. Also, convergent synthesis is possible, in which distinct peptide fragments are created and then joined to produce larger peptides.
Currently, the most common technique for peptide synthesis is solid-phase synthesis (SPPS). Instead of protecting the C-terminus with a chemical group, the C-terminus of the first amino acid is connected to an insoluble solid support, such as polystyrene or polyacrylate resin. This method serves a dual purpose: the resin functions as a C-terminal protective group and provides a quick method for separating the growing peptide product from the various reaction mixtures. As with numerous other biological manufacturing techniques, automated peptide synthesizers have been created for high-throughput peptide manufacturing.
Purification of Peptides
No method for producing peptides is perfect, even though synthesis procedures have been refined and automated. Truncated or deleted sequences, isomers, or other byproducts might result from factors like inadequate deprotection or reactions with residual protecting groups. These occurrences can happen at any point in the peptide synthesis process. Thus, the lengthier the peptide sequence, the higher the likelihood that impurities will interfere with the production of the desired peptide. Therefore, the relationship between peptide yield and peptide size is inverse.
Typically, purification tactics depend on a mix of separation techniques that leverage the physicochemical properties of peptides, such as their size, charge, and hydrophobicity. Common purification processes involve:
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Size-exclusion chromatography
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Ion-exchange chromatography (IEC)
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Reverse-phase chromatography (RPC)
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High-performance liquid chromatography (HPLC)
The most flexible and extensively applied technique for purifying peptides is reverse-phase high-performance liquid chromatography (RP-HPLC). In conventional normal-phase HPLC techniques, polar, hydrophilic molecules are captured by the stationary phase and then selectively eluted by raising the percentage of polar solvents in the mobile phase. As the name indicates, in RPC, hydrophobic molecules in aqueous systems are bound to the stationary phase employing hydrophobic C4, C8, or C18 n-alkyl hydrocarbon ligands. Their retention time is dependent on the hydrophobicity of the peptide and the composition of the mobile phase.
RPC separates the target peptides from contaminants produced during the synthesis steps. These impurities can include isomers, deletion sequences, and peptide products from side reactions with free coupling and protecting groups. Peptide purity is determined by the ratio of the target peptide to contaminants that absorb at the wavelength where peptide bonds typically absorb light (214–220 nm). Different purity grades are sold commercially depending on how the peptides will be used.
Categories of Treatment-Relevant Custom Peptide Drugs
- Antimicrobial Peptides (AMPs) - AMPs are employed to treat a variety of microbial illnesses and fungal infections. After they have penetrated the cell membrane, they may produce barrel-stave, toroidal, or carpet-like pores, all of which result in the death of the infectious agents.
- Anti-inflammatory Peptides (AIPs) - These are anti-inflammatory agents found in nearly all living organisms. AIPs can suppress, reduce, or modulate the production and activity of inflammatory mediators.
- Opioid peptides: These peptides belong to a family that has the capacity to engage with specific opioid receptors. Both pain management and the treatment of substance addiction are possible applications.
- Anticoagulant peptides - These help prevent blood clotting from occurring within the body. The application of anti-thrombotic peptides has been investigated to prevent and treat potentially fatal blood clots.
- Anorexigenic (Appetite-Suppressing) Peptides - These represent a group of peptides effective in treating obesity. They inhibit appetite, thereby preventing weight gain.
- Anti-angiogenic Peptides (AAPs) - AAPs are effective against various cancer types. They work by suppressing the angiogenic process, thereby preventing the growth of new blood vessels that feed tumors.
Conclusion
It is critical to understand that different peptide synthesis companies use various media, biochemicals, and approaches in their labs. Peptide purification generally necessitates the use of multiple separation techniques that take advantage of the peptide's unique physicochemical properties, such as its size, charge, and hydrophobicity. In the modern era, it is essential to maintain the credibility and complexity of synthetic chemistry, as numerous researchers and laboratories consistently seek new and improved ways to produce state-of-the-art medical treatments.