Synthesis Methods

Solid-Phase Peptide Synthesis

Solid-phase peptide synthesis (SPPS), developed by Bruce Merrifield in 1963, involves the stepwise addition of amino acids to a growing peptide chain anchored to an insoluble resin support. This method enables efficient synthesis through simplified purification between coupling steps.

The C-terminal amino acid is first attached to the resin through a cleavable linker. Subsequent amino acids are added sequentially from C-terminus to N-terminus, with each addition involving deprotection, coupling, and washing steps.

Protection Strategies

Amino acid side chains require temporary protection during synthesis to prevent unwanted reactions. Two primary protection schemes dominate modern SPPS: Boc (tert-butyloxycarbonyl) and Fmoc (9-fluorenylmethyloxycarbonyl) chemistry.

Coupling Reagents

Coupling reagents activate the carboxyl group of incoming amino acids to enable peptide bond formation. Common coupling reagents include carbodiimides (DIC, EDC), phosphonium salts (PyBOP, PyAOP), and uronium salts (HBTU, HATU).

Reagent selection affects coupling efficiency, racemization potential, and cost. HATU provides high coupling yields but at greater expense, while DIC offers economical coupling for routine syntheses.

Cleavage and Deprotection

Upon completion of chain assembly, the peptide is cleaved from the resin and side chain protecting groups are removed. For Fmoc chemistry, this typically involves treatment with trifluoroacetic acid (TFA) containing scavengers to trap reactive species.

The crude cleavage product contains the target peptide along with protecting group remnants, deletion sequences, and other impurities requiring removal through purification.

Purification Methods

Reversed-phase high-performance liquid chromatography (RP-HPLC) serves as the primary purification method for synthetic peptides. Separation occurs based on hydrophobic interactions between peptides and the column stationary phase.

Gradient elution with increasing organic solvent (typically acetonitrile) in aqueous buffer resolves peptide mixtures. Fractions containing the target peptide are collected, pooled, and lyophilized to obtain the purified product.

Quality Control

Analytical HPLC assesses peptide purity by comparing the target peak area to total peak area. Mass spectrometry confirms molecular identity by comparing observed and theoretical molecular masses.

Additional characterization may include amino acid analysis to verify composition, nuclear magnetic resonance (NMR) for structural confirmation, and endotoxin testing for applications requiring low endotoxin levels.