Peptide Synthesis

Peptide synthesis refers to all methods of producing a peptide with a defined sequence, with fine control of purity, yield, and, when needed, non-natural modifications. Four main routes coexist: solid-phase chemical synthesis (SPPS), solution-phase chemical synthesis, recombinant production by heterologous expression, and chemoenzymatic synthesis (native chemical ligation, enzymatic ligation by sortases or subtiligases).

SPPS (solid-phase peptide synthesis), introduced by Merrifield in 1963 (Nobel Prize 1984), became the reference method for peptides of 5 to 50 residues. The principle: covalent anchoring of the C-terminus to an insoluble resin, then iterative cycles of N-terminal deprotection, washing, coupling of a new protected amino acid, washing, and repetition until full sequence is built. The Fmoc/tBu strategy dominates today (mild conditions, DMF, piperidine for deprotection); the Boc/Bzl strategy (TFA then final HF) remains used for certain difficult cases. Final quality depends on efficiency of each coupling (targeting 99 %+ per cycle) and real-time monitoring by colorimetric tests (Kaiser, chloranil).

Solution-phase synthesis is suited for very short peptides (2-5 residues) or for ligation of large fragments previously produced by SPPS. It offers superior control over each step but requires costly intermediate purifications.

Recombinant production (E. coli, Pichia pastoris yeast, mammalian cells) remains the economical route for long peptides (>50 residues) and therapeutic proteins. It requires cloning, expression, cell lysis, purification (affinity, ion-exchange, gel filtration chromatography), and validation steps. Post-translational modifications (glycosylation, native disulfide bridges) are easier to reproduce than in chemical synthesis.

Chemoenzymatic synthesis combines synthetic fragments and ligases (sortase A, butelase, subtiligases, trypsiligase) to join fragments bearing modifications not accessible through SPPS. Native Chemical Ligation (NCL) exploits an N-terminal cysteine and a C-terminal thioester to form a native amide bond under mild conditions, enabling assembly of synthetic proteins longer than 150 residues.

Critical steps common to all routes are orthogonal protection of reactive functions, prevention of racemization (choice of coupling agents, controlled basicity), management of difficult sequences (β-sheet aggregation, consecutive prolines), and final purification by preparative HPLC followed by purity analysis (analytical HPLC, mass spectrometry, amino acid analysis).