How are Peptides Made? (Synthesis)

While biological organisms synthesize peptides using ribosomes, laboratory production relies on a highly sophisticated chemical process known as Solid-Phase Peptide Synthesis (SPPS). Developed by Bruce Merrifield in 1963—a feat that later earned him a Nobel Prize—this method allows for the precise, automated creation of specific amino acid sequences.

The Foundation: The Solid Support

The defining feature of SPPS is that the peptide chain is “anchored” to a solid surface rather than floating freely in a liquid solution.

  • The Resin: The process begins with tiny, porous beads made of a polymer (usually polystyrene). These beads are functionalized with “linkers” that serve as the attachment point for the first amino acid.

  • The Advantage: Because the growing peptide is stuck to a physical bead, all the excess chemicals, solvents, and byproducts used in each step can be simply washed away. This allows for extremely high purity without the need for complex purification at every single step.

The Synthesis Cycle: Step-by-Step

Building a peptide is a repetitive, four-step cycle. To ensure the amino acids bond in the correct order, they are “protected” with chemical caps (most commonly Fmoc or Boc) that prevent them from reacting prematurely.

  1. Deprotection: The “cap” on the amino acid currently attached to the resin is removed using a base (like piperidine). This “unmasks” the amino group, making it ready to accept the next link in the chain.

  2. Activation & Coupling: The next amino acid in the sequence is “activated” using a coupling reagent (such as HATU or DIC). This makes the carboxyl end of the new amino acid highly reactive so it can successfully form a peptide bond with the unmasked end of the chain on the resin.

  3. Washing: The resin is flushed with a solvent (usually DMF) to remove any unreacted amino acids or leftover reagents.

  4. Capping (Optional): If any chains failed to bond during the coupling step, a “capping” reagent is added to permanently block them. This prevents “deletion sequences” (missing links) from occurring later in the chain.

This cycle is repeated—Deprotection, Coupling, Washing—until the entire desired sequence is constructed.

Cleavage and Global Deprotection

Once the full sequence is complete, the peptide is still technically a “protected” molecule attached to a plastic bead. It must be “harvested.”

  • The “Cut”: A strong acid (typically Trifluoroacetic Acid, or TFA) is used to cleave the bond between the peptide and the resin.

  • Global Deprotection: In the same step, the TFA removes the “side-chain protectors”—additional caps that were placed on the amino acids’ reactive side groups to prevent branching.

  • Precipitation: The crude peptide is then precipitated out of the acid solution, usually using cold ether, appearing as a raw white solid.

Purification: Ensuring Research Integrity

The raw product from the cleavage step is rarely pure enough for sensitive research. To reach the industry standard of 95% to 99%+ purity, the peptide undergoes High-Performance Liquid Chromatography (HPLC).

  • The Sieve: The crude peptide is pumped through a column at high pressure. Different molecules move through the column at different speeds based on their size and charge.

  • Fractionation: Scientists collect only the “peaks” that represent the pure, correctly sequenced peptide, discarding any fragments or impurities.

  • Final Verification: The final product is verified using Mass Spectrometry (MS) to ensure the molecular weight exactly matches the theoretical weight of the requested sequence.