Fmoc solid-phase peptide synthesis: the reagents you need, step by step

Fmoc solid-phase peptide synthesis (SPPS) builds a peptide one residue at a time on an insoluble resin. Every cycle is the same: remove the Fmoc group, wash, couple the next protected amino acid, wash. The reagents you choose at each step decide your yield and purity.

1. Choose the resin for your C-terminus

The resin sets what you get at the C-terminus after cleavage:

• Rink Amide MBHA resin gives a C-terminal amide, which many bioactive peptides need.
• Wang resin, 1% crosslinked gives a C-terminal carboxylic acid.

Loading (mmol/g) matters. Lower loading reduces chain aggregation in long or hydrophobic sequences, at the cost of less peptide per gram of resin.

2. Protected amino acids

Each residue carries a base-labile Fmoc group on the α-amine and, where needed, an acid-labile side-chain protecting group that comes off in the final TFA cleavage. Common choices are Pbf for arginine (Fmoc-Arg(Pbf)-OH) and tert-butyl esters for aspartate and glutamate (Fmoc-Asp(OtBu)-OH).

Non-proteinogenic residues are increasingly common in research sequences. α-Aminoisobutyric acid (Fmoc-Aib-OH) restricts backbone conformation and slows enzymatic degradation, and is a feature of several GLP-1 analogues studied today. It is also sterically hindered, so plan longer or double couplings.

3. Coupling reagents

Two families cover most labs:

• Uronium/aminium salts such as HBTU, used with a tertiary base. Fast and reliable for routine couplings.
• Carbodiimide plus additive. The additive suppresses racemisation and speeds the reaction. HOBt hydrate is the classic choice; Ethyl (hydroxyimino)cyanoacetate (Oxyma), known as Oxyma, is a widely used non-explosive alternative.

4. Orthogonal protection for branching and labelling

To modify a single lysine on resin, for a fluorophore, a lipid or a branch, you need a side-chain group that comes off under conditions that leave Fmoc and the acid-labile groups intact. Fmoc-Lys(Dde)-OH and the more robust Fmoc-Lys(ivDde)-OH are removed with dilute hydrazine. Fmoc-Lys(N3)-OH carries an azide for copper-catalysed or strain-promoted click chemistry after assembly; see our Click Chemistry & Linkers collection.

Pre-assembled side-chain building blocks such as Fmoc-Lys(tBuO-Ara-Glu(AEEA-AEEA)-OtBu)-OH let you install a complete spacer-and-lipid motif in one coupling, instead of building it step by step on the resin.

5. Quality checks

Use a colour test after each coupling for manual work, and check the crude peptide by HPLC and mass spectrometry. Ask for the certificate of analysis for each amino acid lot: enantiomeric purity and free-amine content both affect the final crude profile.

Browse the full range in Peptide Synthesis and Amino Acids.

Products in this guide

Rink Amide MBHA resinCAS 431041-83-7 Wang resin, 1% crosslinkedCAS 201058-08-4 Fmoc-Arg(Pbf)-OHCAS 154445-77-9 Fmoc-Asp(OtBu)-OHCAS 71989-14-5 Fmoc-Aib-OHCAS 94744-50-0 Fmoc-Lys(Dde)-OHCAS 150629-67-7 Fmoc-Lys(ivDde)-OHCAS 204777-78-6 HBTUCAS 94790-37-1 HOBt hydrateCAS 123333-53-9 Ethyl (hydroxyimino)cyanoacetate (Oxyma)CAS 3849-21-6 Fmoc-Lys(N3)-OHCAS 159610-89-6 Fmoc-Lys(tBuO-Ara-Glu(AEEA-AEEA)-OtBu)-OHCAS 2460751-66-8

Related collections

Peptide Synthesis 433 Amino Acids 259 Biochemicals & Peptide Reagents 1660 Click Chemistry & Linkers 17