Introduction

Short peptides (typically 2–20 amino acids) are widely used in bioconjugation, molecular probes, and peptide-based therapeutics. Their synthesis involves forming amide bonds step by step while using protecting groups to prevent unwanted side reactions.

Today, solid-phase peptide synthesis (SPPS) is the most common approach in research laboratories because it is fast, reproducible, and compatible with automation. This guide focuses on the practical basics of short peptide synthesis, with an emphasis on Fmoc-based SPPS for quick and reliable results.

For most short peptide projects, SPPS is generally preferred, as it requires fewer purification steps and is easier to scale and standardize.

Fmoc-SPPS Step-by-Step

What You’ll Need

Resin: Rink Amide MBHA (loading 0.5–1.0 mmol/g)

Amino acids: Fmoc-protected amino acids with appropriate side-chain protection

Coupling reagents: HBTU + DIPEA or DIC + HOBt

Deprotection: 20% piperidine in DMF

Cleavage cocktail: 95% TFA / 2.5% water / 2.5% triisopropylsilane (TIPS)

Equipment: Syringe reactor or manual vessel, HPLC system, lyophilizer

Steps

Prepare the Resin
Swell 100 mg of resin in DMF for 30 minutes, then wash three times with DMF.

Couple the First Amino Acid
Activate the C-terminal Fmoc-amino acid (≈3 equivalents relative to resin) with HBTU/DIPEA and couple for 60 minutes. Wash thoroughly.

Fmoc Deprotection
Treat the resin with 20% piperidine in DMF for 20 minutes, followed by five DMF washes.

Sequential Amino Acid Addition
Repeat the coupling and deprotection steps for each remaining amino acid. Typical coupling times are 20–40 minutes per residue.

Cleavage and Global Deprotection
Treat the resin with the TFA cleavage cocktail for 2–3 hours at room temperature, then filter to remove the resin.

Peptide Isolation and Purification
Precipitate the crude peptide with cold ether, centrifuge, and purify by reverse-phase HPLC to obtain ≥95% purity. Lyophilize the final product.

Troubleshooting

Low coupling efficiency: Use fresh amino acids, extend coupling time to 60 minutes, and ensure the resin is fully swollen.

Peptide aggregation: Add 5% DMSO to the coupling solvent or switch to DIC/HOBt activation.

Impurities after cleavage: Extend cleavage time to 3 hours or increase the amount of scavenger in the TFA cocktail.

Common Questions

What is the best activation reagent for SPPS?

HBTU/DIPEA works well for most sequences, while DIC/HOBt is often better for sensitive amino acids such as cysteine or histidine.

How should synthesized peptides be stored?

Lyophilized peptides should be stored at −20 °C or −80 °C in aliquots to avoid repeated freeze–thaw cycles.

Can modified peptides (e.g., phosphorylated peptides) be synthesized?

Yes. Pre-modified Fmoc amino acids can be incorporated during synthesis, or modifications can be introduced after synthesis.

Do I need an automated peptide synthesizer?

No. Manual SPPS is sufficient for small-scale work. Automated systems (such as CEM Liberty Blue) are useful for higher throughput or longer sequences.

Conclusion

Fmoc-based SPPS is a practical and reliable method for short peptide synthesis. By choosing appropriate reagents, maintaining clean reaction conditions, and addressing common issues early, researchers can consistently obtain high-purity peptides for bioconjugation, biochemical assays, and early-stage drug research.