Peptides have many applications, such as preparing epitope-specific antibodies, mapping antibody epitopes, and the sites for enzyme binding to design novel drugs, enzymes, and vaccines. Improved production methods and peptide chemistry have increased the availability of peptide synthesis for general research applications. That is unlike in the past when it used to be labor-intensive and low-yielding.
Peptide synthesis refers to forming a peptide bond between two amino acids. There is no universal definition of a peptide, but usually, it relates to flexible chains of between 30 and 50 amino acids. The first peptides to be synthesized included insulin and oxytocin, and after that, it did not occur until another 50 to 60 years. This demonstrates the difficult job of synthesizing amino acid chains chemically.
Until this far, there have been a lot of advancements in protein synthesis chemistry in the recent past. Today, the benefit of peptide synthesis strategies includes the ability to make peptides that are found in biological specimens creatively and imaginatively. That has created the ability to trap biological specimens to generate unique peptides to optimize a desired physical response.
Synthetic Peptides-Its applications
The fifties and sixties spurred the invention of peptide synthesis. That further led to the development of different application areas in which synthetic peptides are currently used. It includes developing epitope-specific antibodies against pathogens, the characterization and application of proteins, and the study of protein functions. Synthetic peptides have been further utilized to study enzyme-substrate interactions within crucial enzyme classes like proteases and kinases (they play a vital role in cell signaling).
Peptide Synthesis- Process
Most frequently, the synthesis of peptides occurs through the coupling of the carboxyl group of the amino acid that is incoming to the growing peptide chain’s N-terminus. C-to-N synthesis is the opposite of protein biosynthesis. In that event, the N-terminus of the incoming amino acid is linked to the protein chain’s C-terminus. The process of peptide synthesis includes;
Peptide deprotection
Since amino acids contain many reactive groups, peptide synthesis should be performed carefully to avoid side reactions which can reduce the length and lead to branching of the peptide chain. There has been the development of chemical groups to facilitate the formation of peptides with minimal side effects or reactions. The chemical groups bind the reactive groups and block amino acids for the protection of the functional group from reactions that are not specific.
The amino acids for protein synthesis of peptides are purified and then reacted with the protecting groups before synthesis. After that, the specific protecting groups are removed from the amino acid that is newly added. This step is known as deprotection, and it allows an incoming amino acid to bind to the growing chain of peptides in a proper or correct orientation.
Coupling of Amino Acid
The synthetic coupling of synthetic peptides needs the activation of the carboxylic acid’s C-terminal on the incoming amino acid. This process uses carbodiimides like dicyclohexylcarbodiimide (DCC) or diisopropyl carbodiimide (DIC). The coupling reagents react with the carboxyl group forming a highly reactive O-acylisourea intermediate which is displaced quickly by a nucleophilic attack.
Peptide Cleavage
After many cycles of deprotection and coupling of amino acids, the remaining protecting groups should be removed from the nascent peptide. Acidolysis cleaves these groups, and the cleavage chemical depends on the scheme of protection used. Strong acids like hydrogen bromide, hydrogen fluoride, or trifluoromethane sulfonic acid are used to cleavage Boc and Bzl groups. On the other hand, a relatively milder acid-like TFA cleaves Fmoc and tBut groups.
Strategies of Peptide Synthesis
Scientists used the liquid-phase synthesis method during the first discovery of how peptides in vitro are generated. It is still common in large-scale synthesis. This method is labor-intensive and slow, even though the product must be removed manually from the reaction solution after every step. In addition to that, this approach needs another chemical group to protect the first amino acid’s C-terminus.
Peptide Purification
Despite the strategies of peptide synthesis having been optimized and could be mass-produced, the process of peptide generation is perfect, by no means. Events like incomplete deprotection or reaction with the free protecting groups could lead to the deletion of truncated sequences or isomers. These events can take place at any step during the synthesis of peptides. Thus, the longer the peptide sequence, the greater the probability of something negatively affecting the synthesis of a target peptide. Therefore, the yield of peptides has an inverse correlation with the length of a peptide.
The purification strategies are generally based on a combination of separation methods, which exploits the physiochemical peptide characteristics, including charge, size, and the techniques of hydrophobicity techniques. These techniques include; partition chromatography, Ion exchange chromatography, high-performance liquid chromatography, and size-exclusion chromatography.
Final Thoughts
Synthetic peptides act as biological functions and structure probes. They also act as essential intermediates for developing enzyme inhibitors and peptidomimetics as therapeutic agents. The solid-phase synthesis automation has prompted many researchers to utilize synthetic peptides effectively. The Association of Biomolecular Resource Facilities contains close to one hundred and thirty member laboratories engaged in protein synthesis and structurally analyzing proteins and peptides as a service in government, academic, and research institutions and private industries.
The ABRF Committee on Mass Spectrometry and Peptide Synthesis was formed to evaluate the quality of the synthetic methods used in member laboratories to synthesize peptides.
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