How Peptides Are Synthesized: SPPS Explained
Solid-Phase Peptide Synthesis revolutionized peptide chemistry. Understanding this process helps you appreciate what goes into producing research-grade peptides.
Before Bruce Merrifield developed Solid-Phase Peptide Synthesis (SPPS) in 1963, synthesizing peptides was a laborious process requiring isolation and purification after each amino acid addition. SPPS changed everything—and earned Merrifield the 1984 Nobel Prize in Chemistry.
The Basic Principle
In SPPS, the peptide chain is assembled while attached to an insoluble solid support (resin). This allows excess reagents and byproducts to be washed away between steps, eliminating the need for purification at each stage.
Key Advantages of SPPS
- • Simple purification—just wash the resin
- • High yields through use of excess reagents
- • Easily automated for reproducibility
- • Can produce peptides up to ~50 amino acids
- • Enables synthesis of modified and unnatural peptides
The SPPS Cycle
Peptide synthesis proceeds from C-terminus to N-terminus (opposite to biological protein synthesis). Each cycle adds one amino acid:
Step 1: Deprotection
Remove the protecting group from the N-terminus of the growing chain, exposing the amine for the next coupling.
Step 2: Washing
Wash away deprotection reagents and byproducts from the resin.
Step 3: Coupling
React the next protected amino acid with the free amine, forming a peptide bond. Excess amino acid drives the reaction.
Step 4: Washing
Wash away excess amino acid and coupling reagents. Repeat the cycle.
Fmoc vs. Boc Chemistry
Two main protecting group strategies are used in SPPS:
Fmoc (Most Common)
- • Removed with mild base (piperidine)
- • Milder conditions overall
- • Better for sensitive modifications
- • Standard in modern synthesis
Boc (Classic)
- • Removed with acid (TFA)
- • Requires final HF cleavage
- • More robust chemistry
- • Used for difficult sequences
Cleavage and Purification
After synthesis is complete:
- 1Cleavage: The peptide is released from the resin using TFA cocktails that also remove side-chain protecting groups.
- 2Precipitation: Crude peptide is precipitated with cold ether and collected.
- 3Purification: HPLC purification removes deletion sequences, truncated peptides, and other impurities.
- 4Lyophilization: Purified peptide is freeze-dried for stability and storage.
Common Synthesis Impurities
Understanding where impurities come from helps interpret COA data:
- Deletion peptides: Missing one amino acid due to incomplete coupling
- Truncated peptides: Synthesis stopped early (incomplete chains)
- Modified peptides: Oxidation, deamidation, or side reactions
- Residual TFA: Counterion from cleavage (can be exchanged to acetate)
Why This Matters for Quality
Good manufacturing practices throughout synthesis result in:
- Higher purity peptides (≥99% at BioInfinity Lab)
- Consistent batch-to-batch quality
- Well-characterized impurity profiles
- Reliable research outcomes