How Research
Peptides Are Made

How are peptides made?

Most research peptides are made by chemical synthesis, and most peptides today are made by solid-phase peptide synthesis, or SPPS (D'Hondt et al., 2014). The chain is built one amino acid at a time on resin beads, cut free with acid, purified by preparative HPLC, checked by mass spectrometry and freeze-dried.

Fmoc/tBu chemistry is the most commonly used version (Amblard et al., 2006). This page describes the chemistry in general; for any vial, the lot's certificate is the record of its contents. Disguised Alpha supplies research peptides, with certificates published by lot, for laboratory research use only. Nothing on this page is guidance for use in people.

From Merrifield to Fmoc: a short history

R. Bruce Merrifield described the method in 1963, in "Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide." Anchoring the growing chain to an insoluble polymer lets excess reagents be washed away instead of separated after every step. Merrifield credited much of its success to an easy, fast procedure with good yields of purified product (Merrifield, 1997). The work earned him the 1984 Nobel Prize in Chemistry.

Classical Boc SPPS removes the Boc group with TFA every cycle and releases the peptide with anhydrous hydrogen fluoride (HF). The repeated acid can strip side-chain protection and cleave peptide from the support (Behrendt et al., 2016). In 1978, Chang and Meienhofer reported SPPS with Fmoc amino acids instead: piperidine, a mild base, removes Fmoc, which permits tert-butyl side-chain protection and a resin link cleaved by mild acid, with no final HF step (Chang and Meienhofer, 1978). Most synthetic peptides are now made by Fmoc SPPS, with Boc kept for specialist work (Behrendt et al., 2016).

Peptide synthesis steps in Fmoc SPPS

Each cycle adds one residue, building from the C-terminus toward the N-terminus (Amblard et al., 2006).

Coupling needs an activator: a carbodiimide such as DIC with an additive such as HOBt, or a phosphonium or uronium salt with a base (Behrendt et al., 2016).

Side-chain protecting groups and final cleavage

Side chains stay masked until the end. The standard set is tBu for serine, threonine and tyrosine, OtBu for aspartic and glutamic acid, Boc for lysine and tryptophan, Trt for asparagine, glutamine, cysteine and histidine, and Pbf for arginine, all removed by TFA (Behrendt et al., 2016).

As TFA strips these groups and the linker, the released fragments can attach to tryptophan, tyrosine, methionine and cysteine, so cleavage cocktails add scavengers. Reagent K (82.5% TFA, 5% phenol, 5% water, 5% thioanisole, 2.5% ethanedithiol) worked best of the mixtures tested against a wide range of these side reactions, and was then used to cleave 10 peptides of 20 to 50 residues (King et al., 1990). The crude peptide is then precipitated, usually in cold ether.

Common impurities, and why length matters

Each incomplete step leaves a by-product that closely resembles the target.

• Deletion sequences: an incomplete coupling or Fmoc removal leaves chains missing a residue (D'Hondt et al., 2014; Merrifield et al., 1988).
• Insertion sequences: an extra residue, linked to excess amino acid reagent or to free amino acid in a building block (D'Hondt et al., 2014; Behrendt et al., 2016).
• Truncation sequences: chains that stopped growing, for example when capped; even trace acetic acid in a building block can cap chains (Behrendt et al., 2016).
• Racemization: a residue flips from the L to the D form, giving a diastereomer of identical mass (D'Hondt et al., 2014). Histidine and cysteine are especially prone; with one base-mediated activation, histidine racemization was 1% without preactivation and 7.8% after 5 minutes of preactivation (Behrendt et al., 2016).
• Aspartimide formation: base from deprotection can close an aspartic acid side chain into a ring. Behrendt and colleagues rank it the most serious side reaction in Fmoc chemistry, with up to nine by-products, some co-eluting with the target, and Asp-Gly the worst sequence.
• Oxidation: in a model 15-residue peptide, crude product from three widely used cleavage cocktails was 15 to 55% methionine sulfoxide, while a cocktail designed against it left none detectable (Huang and Rabenstein, 1999).

Errors compound with length. At 99 percent per step, about 82 percent of 20-residue chains come out full length, 61 percent of 50-residue chains and 37 percent of 100-residue chains. Careful chemistry does better: on a model peptide of 20 alanine residues, the best protocol averaged 0.036% deletion and 0.029% insertion per step, a 99.93% stepwise yield (Merrifield et al., 1988). Growing chains can also aggregate on the resin, which Behrendt and colleagues call the major obstacle to peptide synthesis, and many sequences far shorter than the often quoted 50 residues are extremely difficult (Behrendt et al., 2016).

Peptide purification by preparative HPLC

Reversed-phase HPLC is the favored mode for preparative peptide separation, for its resolving power, speed and volatile mobile phases (Mant et al., 2007). The crude peptide is loaded onto a C8 or C18 column and eluted with a rising gradient of acetonitrile in water, both acidified with TFA, which ion-pairs with charged groups.

Fractions are checked by analytical HPLC, and those that meet the purity target are pooled and freeze-dried. In one example, a crude 26-residue peptide loaded at up to 200 mg was split into hydrophilic impurities, pure product and hydrophobic impurities, with very little product in either impurity pool (Mant et al., 2007).

Identity by mass spectrometry

Electrospray ionization produces intact, multiply charged ions of large molecules from solution (Fenn et al., 1989), and the measured mass is compared with the mass calculated from the sequence. A deletion is lighter by one residue, an insertion heavier, and oxidized methionine adds 16 daltons; mass spectrometry can measure deletion and insertion peptides across a whole crude product (Merrifield et al., 1988). It cannot see a racemized residue, which has the same mass, and an epimerized aspartyl peptide can be very difficult or impossible to resolve by reversed-phase HPLC (Behrendt et al., 2016).

Counterions and lyophilization of peptides

Because TFA is used in cleavage and purification, peptides with basic residues usually come out as trifluoroacetate salts. The counterion can be exchanged: repeated freeze-drying with excess hydrochloric acid, the classic route to the chloride, runs below pH 1 and can degrade the peptide, while reversed-phase HPLC, ion-exchange resin or a deprotonation and reprotonation cycle can give acetate or other salts (Roux et al., 2008).

Counterions and water add mass, so net peptide content usually sits below HPLC purity (see What is a COA?). Last, the pooled fractions are freeze-dried to a dry powder, the usual form for research peptides; the lyophilized peptides guide covers the process.

Liquid-phase, ligation and recombinant routes for long peptides

Classical solution synthesis remains in use alongside SPPS (Merrifield, 1997), and liquid-phase peptide synthesis (LPPS) also builds in solution, on a soluble tag, and allows large-scale production with less excess reagent and solvent (Sharma et al., 2022). Long chains can be made as segments and joined: in native chemical ligation, two unprotected segments react through a thioester and rearrange to a native peptide bond at the join (Dawson et al., 1994).

Recombinant expression is the biological route: in 1979, synthetic genes for the human insulin A and B chains were expressed in E. coli as fusion proteins, and the cleaved, purified chains were mixed to give detectable insulin (Goeddel et al., 1979). Expression suits long chains of standard amino acids; synthesis can place D-amino acids and modified residues anywhere, as in the all-D 26-residue peptide above (Mant et al., 2007).

How synthesis shows up on a certificate

Each line of a certificate of analysis traces back to a step above.

• Purity by HPLC: the target peak's share of the total peak area. By-products that separate show as their own peaks; any that co-elute count as target.
• Identity by MS: the measured mass against the mass calculated from the sequence. Identity by HPLC retention time against a reference does not weigh the molecule.
• Net content: the measured amount in the vial beside the labeled amount, or net peptide content, the share of the powder that is peptide after water and counterions.

Disguised Alpha's COA portal lists certificates by product and can be searched by compound name or by the batch number printed on a vial. Its identity testing page names the method behind each identity result, HPLC retention time or LC-MS, and its fill accuracy page lists each measured amount beside the labeled amount. The peptide supplier checklist covers what else to check.

Key sources

1. Behrendt R, et al. "Advances in Fmoc solid-phase peptide synthesis." J Pept Sci. 2016. PMID 26785684. DOI 10.1002/psc.2836.
2. D'Hondt M, et al. "Related impurities in peptide medicines." J Pharm Biomed Anal. 2014. PMID 25044089. DOI 10.1016/j.jpba.2014.06.012.
3. Mant CT, et al. "HPLC analysis and purification of peptides." Methods Mol Biol. 2007. PMID 18604941. DOI 10.1007/978-1-59745-430-8_1.
4. Merrifield RB. "Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide." J Am Chem Soc. 1963. DOI 10.1021/ja00897a025. The original method; not indexed in PubMed.
5. Also named: the Nobel Foundation's 1984 Chemistry prize announcement.

Frequently asked questions

How are peptides made?

Mostly by chemical synthesis: Fmoc SPPS on resin, cleavage with a TFA cocktail, purification by preparative HPLC, then freeze-drying.

How are peptides synthesized, step by step?

In order: resin loading; deprotection, washing and coupling for each residue; then cleavage, purification and lyophilization.

What does SPPS stand for?

Solid-phase peptide synthesis, described by R. Bruce Merrifield in 1963 and now the way most peptides are made (D'Hondt et al., 2014).

What is Fmoc peptide synthesis?

SPPS in which piperidine removes an Fmoc group every cycle while tBu-type side-chain groups wait for the final TFA cleavage, an orthogonal scheme. It replaced HF-based Boc chemistry for most work (Behrendt et al., 2016).

How does peptide purification by HPLC work?

The crude peptide runs through a reversed-phase column in a water and acetonitrile gradient with TFA; fractions that pass analytical HPLC are pooled (Mant et al., 2007).

Why are synthetic peptides lyophilized?

Freeze-drying removes water from the pooled fractions and leaves a dry powder for storage. See What does lyophilized mean?

Related guides

• What is a peptide?
• What is a COA?
• What does lyophilized mean?
• How to choose a peptide supplier
• TFA vs acetate peptide salts
• Peptide modifications explained
• COA portal: published certificates, by lot
• HPLC purity testing
• Identity testing
• Amino acid codes and residue masses
• Research hub

Disguised Alpha products are for research use only. They have not been evaluated for safety or effectiveness in humans. Not for human consumption. All products are intended for laboratory research purposes only.