Peptides are used to prepare epitope-specific antibodies, map antibody epitopes and enzyme binding sites, and develop new enzymes, drugs, and vaccines. Although peptide synthesis was labour-intensive and yielded low yields, improved production and peptide chemistry techniques have enabled it to be utilized in general research.
Introduction To Peptides
The process of peptide synthesis involves the creation of a peptide bond between two amino acids. The term peptide is generally applied to flexible (little secondary structure) chains of between 30 and 50 amino acids.
Besides making peptides found in biological specimens, peptide synthesis strategies today allow creativity and imagination to be tapped to generate unique peptides that optimize biological responses or other outcomes. It is also critical to understand the strengths and limitations of the standard methods used in peptide synthesis.
Process Of Synthesizing Peptides
It is mainly the carboxyl group of amino acids that becomes part of a peptide by joining it to the N-terminus. Meanwhile, ELISA kit manufacturers utilize specialized products made primarily to measure protein concentrations to achieve uniformity. Different antibody layers are introduced during the formulation process to amplify signals.
1. Peptide Deprotection
Amino acids possess multiple reactive groups. This results in further reactions that can reduce the chain’s length and cause it to branch. To manufacture peptides with minimal side reactions, an amino acid reactive group has been analyzed for its ability to bind to a functional group and prevent nonspecific reactions.
Individual amino acids are first reacted with these protective groups before being used to synthesize peptides. The newly added amino acids are deprotected (a step called deprotection) shortly after coupling. This allows the following amino acid to bind properly to the growing peptide chain. When the peptide synthesis is complete, all protecting groups remain until the nascent peptides get removed.
2. Amino Acid Coupling
Carbodiimide activation is necessary for synthetic peptide coupling using dicyclohexylcarbodiimide (DCC) or diisopropyl carbodiimide (DIC). This coupling reagent reacts with the carboxyl group to form a highly reactive O-acylisourea intermediate quickly displaced by nucleophilic attack from the N-terminal primary amino group.
Due to the reactive nature of carbohydrate amino acids, several racemizations can occur. Here, a researcher reduces the risk of racemization and adds reagents that react with the O-acylisourea intermediate, such as 1-hydroxy benzotriazole (HOBt), which forms a less-reactive intermediate, and require activating bases to mediate amino acid coupling.
3. Peptide cleavage
It is necessary to remove all intact protecting groups from a nascent peptide after successive rounds of deprotection and coupling. By acidolysis, the groups are broken up, and the chemical used to do this depends on the protection scheme. Strong acids such as hydrogen bromide (HBr), hydrogen fluoride (HF), or trifluoromethane sulfonic acid (TFMSA) cleaves Boc and Bzl. In contrast, TFA is a relatively mild acid that cleaves Fmoc and tBut groups.
Cleavage, when properly conducted, produces the removal of the N-terminal protection group of the last amino acid. Also, remove the C-terminal protection group (either chemical or resin) from the first amino acid and any side-chain protective groups.
During this step, scavengers are also included to react to free protecting groups. To avoid acid-catalyzed side reactions, researchers tend to optimize peptide synthesis to prevent the cleavage reaction.
4. Peptide synthesis strategies
Until recent years, scientists have been synthesizing peptides using liquid-phase peptide synthesis, and today, it is still one of the most common methods. As a result of the manual removal of the product from the reaction solution after each step, this method is slow and labour-intensive. Moreover, this method involves another chemical group to protect the C-terminus of the first amino acid.
- In liquid-phase synthesis, however, side reactions are easy to detect due to the product’s purification after every step. It is also possible to perform convergent synthesis, which involves synthesizing peptides in sequence and linking them together to make larger molecules.
- The most common peptide synthesis procedure used today, however, is solid-phase synthesis.
- Rather than a chemical group protecting its C-terminus, the first amino acid has its C-terminus coupled to a rigid, activated support, like polystyrene or polyacrylamide. It is a two-fold method: the resin functions as a C-terminal protective group. It provides a quick way to separate the growing peptide product from the various reaction mixtures during synthesis.
Peptide synthesizers are used in several kinds of biological manufacturing processes, allowing for the high-throughput production of peptides.
5. Peptide Purification
It has now become possible to mass-produce peptides. Several reactions with free protecting groups, such as incomplete deprotection, can cause truncated or deletion sequences or isomers. In addition, a negative effect on peptide synthesis can occur at any point in the process, so longer peptide sequences have a greater chance of producing a negative impact on the synthesis of the target peptide. Therefore, peptide yield is inversely related to peptide length.
Generally, peptide purification is accomplished by using a combination of separation strategies that leverage their physicochemical properties, including size, charge, and hydrophobicity. However, reverse-phase chromatography (RPC) is the most widely and versatile method used in peptide purification. This process removes impurities during syntheses, such as isomers, deletion sequences, and peptides that have undergone side reactions with free couplings and protecting groups.
Historically, peptide synthesis was labour-intensive and produced low yields. The use of peptides to develop new enzymes, drugs, and vaccines, including preparing antibodies that target specific epitopes and mapping enzyme binding sites, is widespread. Nevertheless, peptide synthesis has been used in general research studies using improved manufacturing and peptide chemistry techniques.