Methods and reagents of Peptide synthesis


    In the field of organic chemistry, peptide synthesis is the production of peptides which implies that several amino acids are linked with the peptide bonds. On the other hand, the biological process of manufacturing long peptides that is proteins is usually referred to as protein biosynthesis. 

    The idea of linking the amino acids bond to the peptide bonds is 100 years old but it took around 50 years to find the best solutions for the occurring problems when it is linked. Robert Bruce Merrifield is a scientist who pioneered the solid-phase peptide synthesis. It is possible to link peptides with about 50 amino acids with the help of solid-phase peptide synthesis. This technology allows the synthesis of natural peptides that are difficult to express in bacteria to incorporate into the unnatural amino acids. They are also helpful in generating the unique peptides to optimize a desired biological output or any other result. 

    Importance of peptide synthesis

    The invention of peptide synthesis has brought several developments of different application areas in which synthetic peptides are used. The different applications include the following:

    • The development of epitope-specific antibodies against pathogenic proteins
    • The study of protein functions
    • The characterization and identification of proteins

    The synthesis of peptides allows you to research on important cell signaling enzymes like proteases and kinases, mainly to understand the enzyme-substrate interactions. In Mass Spectrometry (MS) based applications, the synthesis of the peptides is very useful as standards and reagents. 

    Solid-phase peptide synthesis (SPPS)

    To start with the solid-phase peptide synthesis, the C-terminus of the first amino acid is linked with activated solid support, commonly chemically unreactive polystyrol. The resin will act as the C-terminal protecting group, where the immobilized protein can be filtered during the filtration process. On the other hand, the liquid-phase reagents and the by-products of the peptide synthesis get washed away. 

    The basic principle of solid-phase peptide synthesis consists of the repeated cycles of deprotection-washing-coupling-washing. The single N-protected terminal of the amino acid unit and the free N-protected amine of a solid-phase are coupled together to the peptide groups. This coupled unit is then deprotected, by giving out a new N-terminal amine to which a further amino acid group may be attached whenever needed. 

    The repeated cycles of Solid-phase peptide synthesis involves the following:

    • Cleavage of the alpha-amino protecting group
    • Washing to remove the cleavage reagent
    • Coupling of the protected amino acid
    • Washing to remove excess material

    Quality control of peptides

    The quality control parameters of the peptides include the following:

    1. Peptide purity: The purity of the target peptide is analyzed by analytical RP-HPLC followed by UV detection at 220nm.
    2. Net peptide content: As the name suggests, the net peptide content is the number of peptide molecules present in the resulting product.
    3. Impurities: Impurities involves unwanted by-products also. Thus, they are allowed to flush off. 
    4. Batch-to-batch variability: The peptide purity may vary from batch to batch. Thus, when the peptide is at 90% purity, the end product would be of 90-99% purity. 

    Overall insight:

    Thus, the importance of peptide synthesis is discussed in brief in the above-mentioned process.