Solid-phase peptide synthesis: how a peptide is built, one residue at a time

Merrifield’s idea: keep the chain on a solid support

A peptide bond forms when the carboxyl group of one amino acid reacts with the amino group of another.² To make the bonds in a chosen order, the groups that should not react are blocked with protecting groups, and one is unblocked each time an amino acid is added.² In the conventional method the product had to be separated from by-products and unreacted material after every step, and some was lost each time.² The Royal Swedish Academy of Sciences put a number on the problem: a hundred steps at 90 per cent each would leave 0.003 per cent of the material.²

Bruce Merrifield’s answer was to attach the first amino acid, through its carboxyl group, to a solid polymer.² After each reaction the by-products and leftover starting materials are removed by filtering and washing the polymer, and the peptide is taken off only when the whole chain is finished.² The Academy credited the method with step yields of 99.5 per cent or better and noted that it suits automation; Merrifield received the 1984 Nobel Prize in Chemistry for it.²

Two consequences follow.

• The chain grows backwards. The first residue is held through its carboxyl group,² so it becomes the C-terminal end and the chain is extended toward the N-terminus. Sequences are written the other way round, as the article on peptide nomenclature and modifications explains.
• Nothing is purified until the end. A peptide bound to the resin cannot be tested without chemically changing it, and the European Medicines Agency (EMA) describes the crude material that comes off as relatively complex.⁵ Chains that went wrong along the way come off with the product.

The Fmoc strategy: two kinds of protecting group

Each amino acid going into the chain is protected at its amino group and, where relevant, on its side chain.⁵ The first protection comes off at every cycle and the second has to stay until the end, so one must be removable without touching the other.

Chemistry study Chang and Meienhofer showed in 1978 how to do that with the Fmoc group (9-fluorenylmethyloxycarbonyl), which a mild base, piperidine, removed completely.³ That allowed the side chains to carry tert-butyl-type groups and the peptide to be joined to the resin by a link that mild acid breaks.³ The authors pointed out that this avoided side reactions of the method then in use, which removed its temporary group with acid at every cycle and freed the peptide with hydrogen fluoride.³

Review A 1990 review by Fields and Noble called Fmoc synthesis a truly “orthogonal” scheme.⁷ The word describes protecting groups that come off under unrelated conditions, here base for Fmoc and acid for the side chains.³ A 2016 review, two of whose three authors were at Merck’s Novabiochem unit, describes Fmoc SPPS as the method of choice and says that high-quality building blocks are inexpensive because therapeutic peptides are made on a multi-tonne scale.⁸

The cycle, step by step

The EMA’s guideline on synthetic peptides, in effect since 1 June 2026, summarizes the assembly as repeated cycles of deprotection, washing, coupling and capping on a solid support, followed by a final cleavage step.⁵

1. Resin and linker

The resin is the solid support, and the first amino acid is joined to it through a linker.⁵ The EMA lists swelling volume, mesh size and loading among the typical quality attributes of a resin.⁵

Review A methods chapter on Fmoc synthesis calls the choice of resin and linker a key parameter for success.⁹ It surveys the resins and linkers used to make peptides that end in a C-terminal amide or a carboxylic acid.⁹

2. Deprotection

Chemistry study Piperidine removes the Fmoc group, which frees the amino group of the last residue added for the next coupling.³ The resin is then washed.⁵

3. Coupling

The next protected amino acid reacts through its carboxyl group with the free amino group on the resin, and a new peptide bond forms.² The EMA guideline notes that additives used in the process suppress epimerization, the flipping of a residue into its mirror-image form.⁵

Chemistry study A 2009 study tested one such additive, Oxyma, in peptide-bond formation by the carbodiimide method and reported a strong capacity to inhibit racemization, with coupling efficiency above that of the older additive HOBt.¹⁰

4. Checking, recoupling and capping

The most common in-process check is the Kaiser test, a colour test based on the reaction of ninhydrin with primary amines.⁵ An incomplete coupling is often repeated, and sites that still have not reacted are capped by acetylation so that they cannot grow further.⁵

5. Cleavage

Chemistry study At the end, trifluoroacetic acid (TFA) releases the peptide from the resin and strips the side-chain groups.⁴ King and colleagues noted that the protecting groups and linkers set free can modify tryptophan, tyrosine, methionine and cysteine.⁴ They compared scavengers, chemicals added to suppress those side reactions, found one mixture the most efficient against a wide range of them, and used it in making ten peptides of 20 to 50 residues.⁴

6. Purification

The EMA guideline states that synthetic peptides are generally purified by chromatography, and that the target is not always fully separated from closely related impurities that elute near it.⁵ Manufacturers therefore collect fractions, pool the purest, and may purify the side fractions again.⁵

Review Mant and colleagues review the main modes of high-performance liquid chromatography (HPLC) used for peptides, which are size-exclusion, ion-exchange and reversed-phase, and give preparative reversed-phase protocols.¹¹

The guide to analytical methods for peptides covers the same technique at analytical scale.

7. Isolation

Lyophilization (freeze-drying) is common practice for isolating the purified peptide, with precipitation, crystallization and spray drying as alternatives.⁵ The powder often carries a counter-ion: the guideline says acetate is the usual one, that trifluoroacetate or chloride are also possible, and that the type of counter-ion can affect a peptide’s biological and physicochemical properties.⁵ The drying step has its own guide: why peptides are lyophilized.

Where impurities come from

The table lists the main peptide-related impurities and the step each comes from.

Missing residues: deletion and truncation

Both kinds lack residues, in different ways. A capped chain stops where it failed and is left as an acetylated fragment, while a chain that only missed a step keeps growing and can end up a single residue short.⁵

Chemistry study A 2015 quality study from Ghent University evaluated a set of quorum-sensing peptides supplied for research with a requested purity of at least 95 per cent.¹⁴ In the authors’ own testing only 44 per cent met that figure, most of the related impurities lacked one or more amino acids, and the main compound in one sample had a different structure from the peptide wanted.¹⁴

Racemization

Every natural amino acid except glycine has at least one stereocentre, so a residue can flip into its mirror-image form and give a stereoisomer of the target.⁵ A stereoisomer that differs at one centre only is called an epimer. The EMA guideline notes that building blocks with urethane-type protecting groups such as Fmoc are rather resistant to the ring formation that leads to this change.⁵

Chemistry study In a study from CEM Corporation, a model 20-residue peptide containing all twenty natural amino acids was assembled with microwave heating: cysteine, histidine and aspartic acid were susceptible to racemization, and a lower coupling temperature limited it for histidine and cysteine.¹³

Review A 2023 review notes that epimerized products have very similar physical characteristics, which makes them difficult to purify, and that the change can alter a peptide’s bioactivity.¹⁵

Rings that should not form

Chemistry study The same microwave study found that aspartimide formation, and the racemization of aspartic acid that follows it, fell when an additive was put in the deprotection solution or its base was changed.¹³

Chemistry study A 2022 study from Eli Lilly examined diketopiperazine formation during the solid-phase synthesis of tirzepatide.¹² The side reaction gave impurities with two amino acids missing.¹² It occurred mainly during Fmoc removal and while a resin-bound intermediate was held after coupling, and intermediates with proline as the second residue from the growing end were prone to it.¹² Replacing Fmoc with another temporary protecting group eliminated the by-products, and an additive and a lower storage temperature made the intermediates markedly more stable.¹²

Adducts, oxidation and stray peptides

Review A review of impurities in peptide medicines lists three more groups: adducts left by incomplete removal of side-chain groups, oxidized side chains, and dimers or larger oligomers.¹ It also notes that trifluoroacetate can reach the final product as a counter-ion, and records cases of contamination with an unrelated peptide, which the authors attribute to a lack of appropriate manufacturing practice.¹

Why long and difficult sequences are harder

Yields multiply. If every cycle delivers the same share of correct chains, the share that is full length at the end is that figure raised to the number of cycles. The table is arithmetic, not measurement.

What is not full length still resembles the target, and such closely related impurities are the ones chromatography does not always separate completely.⁵

Review A 2016 review defines “difficult peptides”, a term from the 1980s, as sequences whose chains form beta-sheet contacts strong enough to aggregate during synthesis, held together by hydrogen bonds along the backbone.⁶ The EMA guideline names special building blocks used against such problems: pseudoproline dipeptides and a modified glycine, Dmb-glycine, to limit aggregation, and other dipeptides to reduce diketopiperazine formation.⁵

Review A 2019 perspective by chemists from eight pharmaceutical companies describes current peptide synthesis as relying on large amounts of highly hazardous reagents and solvents.¹⁶ The EMA guideline notes that the solid-phase process requires extensive washing of the resin.⁵

Manufacturers may also make fragments on resin and join them in solution, a hybrid approach that the EMA says may become more popular for large-scale production.⁵ What these factors do to a price is the subject of why peptide prices vary.

Reading a purity figure with the synthesis in mind

For peptides used as active substances in medicines, the EMA guideline cites the European Pharmacopoeia: peptide-related impurities are reported above 0.1 per cent, identified above 0.5 per cent and qualified above 1.0 per cent.⁵ Those thresholds are for substances used in medicines, not a description of research materials, on this site or anywhere else.

The synthesis also shows what a purity figure leaves open. The guideline warns of the risk of co-eluting impurities, and it lists counter-ion content and water content as tests separate from purity.⁵ The guide on what 99% purity means takes this further, and the certificate of analysis guide shows where each result appears. Batch documents, when they exist, are published on the Lab Results page.

Human evidence

None applies. This guide is about how peptides are made, and none of its sources is a study of what a peptide does in people or in animals. Nothing here bears on the effects or the safety of any compound.