Automated peptide synthesis revolutionized the creation of complex protein chains. By attaching amino acids to polymer beads, scientists can efficiently build peptides step-by-step, using protecting groups and coupling agents to control the process.

This method offers huge advantages over traditional techniques. It allows for longer peptides, higher yields, and easier purification. With automated synthesizers, researchers can now produce custom peptides quickly and reliably for various applications in biochemistry and medicine.

Automated Peptide Synthesis

Steps in Merrifield solid-phase synthesis

1. Attach first amino acid to insoluble polymer support (resin bead) via linker

   • Linker chosen based on desired C-terminal functionality of peptide
   • Amino acid protected at N-terminus and side chain (if necessary) prevents unwanted reactions

2. Remove N-terminal protecting group (deprotection) allows coupling of next amino acid

3. Activate carboxyl group of next amino acid and couple to free N-terminus of growing peptide chain

   • Coupling reagents (DCC, DIC, PyBOP) facilitate formation of peptide bond
   • Peptide bond formation is a crucial step in building the amino acid sequence

4. Repeat deprotection and coupling steps until desired peptide sequence obtained

5. Cleave completed peptide from solid support and remove remaining side chain protecting groups

   • Trifluoroacetic acid (TFA) commonly used for cleavage and final deprotection

Advantages of polymer resin beads

• Allows use of excess reagents to drive reactions to completion
  • Excess reagents and byproducts easily removed by washing resin beads, simplifies purification
• Growing peptide chain remains attached to insoluble resin throughout synthesis
  • Eliminates need for intermittent purification steps required in solution-based methods
• Automated peptide synthesizers streamline process
  • Synthesizer performs repetitive deprotection, coupling, and washing steps, reduces manual labor and increases efficiency
• Enables synthesis of longer peptide sequences with higher yields compared to solution-based methods
• Solid support provides a stable platform for the growing peptide chain

Resins and protecting groups

• Wang resin with Fmoc (9-fluorenylmethoxycarbonyl) protecting group
  • Polystyrene-based resin yields peptides with free carboxylic acid at C-terminus upon cleavage
  • Fmoc is base-labile protecting group for N-terminus, removed using piperidine (20% in DMF)
  • Side chain protecting groups typically acid-labile (tBu, Boc, Trt), removed during final TFA cleavage step
  • Fmoc/tBu strategy is orthogonal, allows selective deprotection of N-terminus while leaving side chain protecting groups intact
• PAM (4-hydroxymethylphenylacetamidomethyl) resin with Boc (tert-butyloxycarbonyl) protecting group
  • Polystyrene-based resin yields peptides with C-terminal amide upon cleavage with anhydrous HF
  • Boc is acid-labile protecting group for N-terminus, removed using TFA
  • Side chain protecting groups also acid-labile (Bzl, Cbo), removed during final HF cleavage step
  • Boc/Bzl strategy requires graduated acidolysis, stronger acids (HF) needed for final cleavage and deprotection
• Fmoc/tBu strategy more widely used in automated peptide synthesis due to milder cleavage conditions and avoidance of hazardous HF

Considerations in Automated Peptide Synthesis

• Coupling efficiency is crucial for obtaining high-quality peptides
• Orthogonal protection strategies prevent unwanted side reactions
• Racemization must be minimized to maintain the correct stereochemistry of amino acids
• Automated synthesis improves reproducibility and reduces human error in the peptide synthesis process