Solution Phase Synthesis (SPS) was the original method for producing peptides and is still used in certain applications. For most modern research and large-scale production, however, Solid-Phase Peptide Synthesis (SPPS) has become the preferred approach because it offers higher purity, faster production, and improved yield.

SPPS follows a cyclical, five-step process, repeated until the full sequence is assembled:

1. Attachment. The first amino acid is attached to a solid polymer support.
2. Protection. The amino acid is chemically protected to prevent unwanted reactions.
3. Coupling. Additional protected amino acids are added to the growing chain.
4. Deprotection. The protective group is removed so the next amino acid can react.
5. Cleavage. The completed peptide is released from the polymer support.

Microwave-assisted SPPS can further improve yield and speed, particularly for long sequences, though it costs more than traditional SPPS. Even with SPPS, longer peptides remain prone to impurities and incomplete reactions, so purification is essential. Reverse-phase chromatography (RPC) is the most widely used technique, often alongside high-performance liquid chromatography (HPLC), to separate impurities while preserving the peptide’s physicochemical properties.

In synthesis, peptides are built by linking the C-terminus (carboxyl group) of one amino acid to the N-terminus (amino group) of another, creating a C-to-N directional chain. This is the reverse of natural protein biosynthesis, which proceeds from N to C.

While the 20 standard amino acids such as arginine, lysine, and glutamine are commonly used, synthetic amino acids expand the possibilities for creating novel peptides. Because amino acids contain multiple reactive groups, careful control is needed to avoid truncation, branching, and reduced purity or yield.

To prevent unwanted reactions during synthesis, protecting groups temporarily deactivate specific reactive sites on amino acids. They fall into three categories:

1. N-terminal protecting groups temporarily block the amino terminus to enable peptide bond formation. Common examples are tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc).
2. C-terminal protecting groups protect the carboxyl terminus and are used mainly in solution-phase synthesis.
3. Side-chain protecting groups permanently protect reactive side chains across multiple synthesis cycles and are removed only at the end using strong acids.

By using protecting groups strategically, scientists can achieve high-purity peptide production while avoiding unwanted side reactions.