Peptide Synthesis
Solid-Phase Peptide Synthesis (SPPS)
The most widely used method for synthetic peptide production. Developed by Robert Bruce Merrifield in 1963 (Nobel Prize, 1984).
Fmoc Strategy
The Fmoc (9-fluorenylmethyloxycarbonyl) protecting group strategy is the most common approach:
// Cycle:
1. Deprotection: 20% piperidine in DMF
2. Coupling: Fmoc-AA-OH + HBTU + DIPEA
3. Wash: DMF, DCM
4. Repeat for next residue
// After last residue:
5. Final deprotection
6. Cleavage: TFA + scavengers
Key Reagents
| Reagent | Full Name | Purpose |
|---|---|---|
| HBTU | O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate | Coupling activator |
| DIPEA | N,N-Diisopropylethylamine | Base |
| DMF | Dimethylformamide | Solvent |
| TFA | Trifluoroacetic acid | Cleavage/deprotection |
Liquid-Phase Synthesis
Traditional solution-phase synthesis. Used for large-scale production where SPPS cost is prohibitive. Requires selective protecting groups and purification at each step.
Recombinant Production
Expression in E. coli, yeast, or mammalian cells. Required for peptides >50 aa or those requiring post-translational modifications. Includes fusion tags for purification (His₆, GST, MBP).
Purification & Characterization
HPLC
Reversed-phase C18 columns with acetonitrile/water gradient. Primary purity assessment method.
Mass Spectrometry
MALDI-TOF or ESI-MS for molecular weight confirmation. Peptide fingerprinting for identity.
Amino Acid Analysis
Acid hydrolysis followed by derivatization and HPLC. Quantitative composition determination.
Edman Degradation
Sequential N-terminal sequencing. Confirms sequence identity for peptides <50 aa.