Overview of Peptide Synthesis
Peptide synthesis is the process of forming peptide bonds between amino acids to create specific sequences. Modern synthesis techniques enable researchers to produce peptides with precise sequences for various research applications. The two primary approaches are solid-phase peptide synthesis (SPPS) and solution-phase synthesis.
Solid-Phase Peptide Synthesis (SPPS)
Developed by Bruce Merrifield in 1963 (for which he received the Nobel Prize in Chemistry in 1984), SPPS revolutionized peptide production. In this method:
- The C-terminal amino acid is attached to an insoluble resin support
- Amino acids are added sequentially from C-terminus to N-terminus
- Protecting groups prevent unwanted reactions during coupling
- The completed peptide is cleaved from the resin
Fmoc Chemistry
Fluorenylmethyloxycarbonyl (Fmoc) protection is the most common SPPS strategy today. The Fmoc group protects the α-amino group and is removed with mild base (typically piperidine), allowing synthesis to proceed under conditions that preserve sensitive amino acid side chains.
Boc Chemistry
tert-Butyloxycarbonyl (Boc) protection is an alternative strategy using acid-labile protecting groups. While more robust, it requires harsher cleavage conditions (hydrogen fluoride) and is less commonly used in standard laboratories.
Solution-Phase Synthesis
In solution-phase synthesis, reactions occur in homogeneous solution rather than on a solid support. This traditional approach offers advantages for:
- Large-scale industrial production
- Synthesis of short peptides
- Fragment condensation strategies
- Applications where solid-phase limitations apply
Key Steps in Peptide Synthesis
1. Protection Strategy
Amino acids have multiple reactive groups that must be selectively protected:
- α-Amino protection: Fmoc or Boc groups
- Side chain protection: Various groups depending on the amino acid
- C-terminal protection: Ester linkage to resin in SPPS
2. Coupling Reaction
The carboxyl group of the incoming amino acid is activated to form a peptide bond with the free amino group. Common coupling reagents include:
- Carbodiimides (DIC, EDC)
- Phosphonium reagents (PyBOP, PyAOP)
- Uronium reagents (HBTU, HATU)
3. Deprotection
After each coupling, the N-terminal protecting group is removed to expose the amino group for the next coupling cycle.
4. Cleavage and Global Deprotection
Once synthesis is complete, the peptide is released from the resin and all side chain protecting groups are removed, typically using trifluoroacetic acid (TFA) cocktails.
Quality Considerations
Several factors affect synthesis quality:
- Coupling efficiency: Each coupling should exceed 99% completion
- Aggregation: Hydrophobic sequences may aggregate during synthesis
- Racemization: Improper conditions can cause chiral inversion
- Deletion sequences: Incomplete couplings create truncated products
Applications
Synthetic peptides serve numerous research applications including antibody production, enzyme substrates, receptor ligands, and drug development. The ability to synthesize peptides with specific modifications enables detailed structure-function studies.