Best 5 Conditions for Disulfide Cyclization in Peptides

1️⃣ Air Oxidation (Oxygen-Mediated)

⭐ Most commonly used, simplest approach

Typical conditions

• Peptide concentration: 0.05–0.2 mg/mL

• Buffer: 0.1 M ammonium bicarbonate or phosphate

• pH: 7.5–8.5

• Temperature: RT

• Time: 12–48 h

Best for

• Small to medium peptides

• Single disulfide bond

• Clean sequences without aggregation issues

Pros

• No reagents

• Very clean reaction

• Easy scale-up

Cons

• Slow

• Inefficient for sterically hindered peptides

Expert tip:
Dilution is critical to avoid intermolecular dimerization.

2️⃣ DMSO-Mediated Oxidation

⭐ Fast and very popular in SPPS workflows

Typical conditions

• Solvent: 10–20% DMSO in water or buffer

• pH: 6.5–7.5

• Temperature: RT

• Time: 1–6 h

Best for

• Cyclization immediately after cleavage

• Hydrophobic peptides

• Lab-scale synthesis

Pros

• Faster than air oxidation

• Mild and controllable

• Minimal side reactions

Cons

• DMSO removal needed before purification

Expert tip:
Avoid >20% DMSO to prevent peptide oxidation at Met/Trp.

3️⃣ Iodine (I₂) Oxidation

⭐ Fastest and most decisive method

Typical conditions

• Solvent: Acetic acid / MeOH / water

• I₂: 0.1–1 eq

• Temperature: RT

• Time: 5–30 min

Best for

• Cys-protected peptides (Acm, Trt removal)

• Time-sensitive workflows

Pros

• Extremely fast

• High conversion

Cons

• Can oxidize Met, Trp, Tyr

• Requires quenching (ascorbic acid or thiosulfate)

Expert tip:
Monitor by LC–MS closely; overoxidation happens quickly.

4️⃣ Oxidative Folding (Redox Buffer System)

⭐ Best for complex or bioactive peptides

Typical conditions

• Redox pair: GSH / GSSG (5:1 to 10:1)

• Buffer: Tris or phosphate

• pH: 7.5–8.5

• Temperature: RT–25°C

• Time: 6–48 h

Best for

• Multiple cysteines

• Correct native disulfide pairing

• Therapeutic peptides

Pros

• Thermodynamically controlled

• Minimizes mispaired disulfides

Cons

• Longer reaction time

• Optimization required

Expert tip:
Add 0.5–2 M arginine if aggregation is observed.

5️⃣ On-Resin Disulfide Cyclization

⭐ Excellent for improving purity

Typical conditions

• Oxidant: I₂, DMSO, or air

• Solvent: DMF or DCM

• Resin: Wang / Rink amide

• Time: 30 min–4 h

Best for

• Head-to-tail or constrained cyclic peptides

• Preventing dimer formation

Pros

• Cleaner crude peptide

• Reduced intermolecular reactions

Cons

• Resin accessibility matters

• Not suitable for all sequences

Expert tip:
Use low-loading resin to improve cyclization efficiency.

#Cyclization #Peptidechemistry #SPPS #Peptide #Disulphide