Peptide Synthesis
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What is Peptide Synthesis?
Peptide synthesis, marked by the bonding of two amino acids through a peptide bond, is the fundamental process of producing peptides. Initially constrained by inefficient methods, advancements in chemistry and technology have significantly enhanced synthesis techniques. Synthetic peptides are poised to remain crucial in advancing scientific and medical frontiers in the contemporary era.
How Peptides are Synthesized?
Peptides are formed through the linkage of two amino acids, typically by attaching the carboxyl group (C-terminus) of one amino acid to the amino group (N-terminus) of another. Unlike protein biosynthesis, which proceeds from N- to C-terminus, peptide synthesis occurs in the opposite direction, known as C-to-N fashion.
While the natural world offers twenty common amino acids, the synthesis of additional amino acids expands the possibilities for peptide creation. However, the synthesis process is intricate due to the reactive groups present in amino acids, which can lead to unwanted interactions, such as truncation or branching of the peptide chain, resulting in suboptimal purity or yield. Expert execution is essential in peptide synthesis to mitigate such complexities.
To ensure the success of peptide synthesis and prevent unintended reactions, scientists employ protecting groups designed to shield reactive amino acid groups. These groups fall into three categories:
- N-terminal protecting groups: These safeguard the N-termini of amino acids and are easily removable to facilitate peptide bond formation. Common examples include tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc).
- C-terminal protecting groups: These are employed in liquid-phase peptide synthesis to protect the C-terminus of amino acids. However, they are not used in solid-phase peptide synthesis.
- Side chain protecting groups: These shield the reactive side chains of amino acids, remaining intact throughout the synthesis process. Referred to as permanent protecting groups, they are removed with strong acids after peptide synthesis is complete.
Peptide Synthesis Processes
Originally a popular method, solution phase synthesis (SPS) for peptide synthesis has been largely supplanted by solid-phase peptide synthesis (SPPS) due to its superior attributes including heightened yield, purity, and production velocity. SPPS entails a cyclical process consisting of five key steps: amino acid attachment to the polymer, protection, coupling, deprotection, and polymer removal to yield the free peptide. A variant known as microwave-assisted SPPS further augments yield and pace, particularly advantageous for synthesizing extensive peptide sequences, albeit potentially at an elevated cost.
Despite the advancements in SPPS, the occurrence of impurities and imperfections remains, particularly pronounced with longer peptide sequences, necessitating the employment of purification techniques such as reverse-phase chromatography (RPC) and high-performance liquid chromatography (HPLC). These methodologies leverage peptides’ physicochemical properties to segregate impurities from the desired peptide, with RPC emerging as the predominant purification method in contemporary peptide synthesis practices.