Peptide Synthesis: How Peptides Are Made

Peptide synthesis is the chemical assembly of a peptide chain from individual amino acids, and almost all research peptides today are made by solid-phase peptide synthesis (SPPS). In SPPS the first amino acid is anchored to an insoluble resin bead, and the chain is built one residue at a time by repeating two steps: remove a temporary protecting group, then couple the next protected amino acid. The finished chain is cut from the resin with acid, purified by preparative HPLC and freeze-dried to a powder.

This article walks through each stage, explains the two main chemistries (Fmoc/tBu and Boc/Bzl), and shows where the common impurities come from. Those impurities are the reason a certificate of analysis exists.

Peptide synthesis at a glance

Source: summarised from reviews of Fmoc SPPS and protecting-group chemistry [2-4].

Why peptides are built on a solid support

Joining two amino acids in solution is straightforward. Joining forty is not, because the product of every step has to be isolated and purified before the next. In the early 1960s Bruce Merrifield solved this by attaching the growing chain to an insoluble polymer bead [1]. Reagents are added in large excess to drive each reaction close to completion, and everything that is not bonded to the bead is simply washed away by filtration. The chain stays on the resin until the end.

Building on a support also made peptide synthesis repetitive enough to automate. Modern synthesisers run the same deprotect, wash, couple and wash sequence for each residue, often with heating to shorten reaction times.

Protecting groups: Fmoc/tBu vs Boc/Bzl

Amino acids have reactive groups at both ends and, for many residues, on the side chain. If they were left free, they would react in the wrong places. SPPS therefore uses two layers of protecting groups [3]:

• A temporary group on the alpha-amino group, removed before every coupling.
• Permanent groups on reactive side chains (the amines of lysine, the acids of aspartate and glutamate, the thiol of cysteine, the guanidine of arginine and others), removed only at the end.

The two layers must come off under different conditions. That property is called orthogonality, and it defines the two main chemistries:

Source: Fields and Noble [2], Isidro-Llobet et al. [3], Behrendt et al. [4].

Merrifield’s original method used Boc chemistry [1]. Fmoc amino acids entered SPPS in the late 1970s, and by 1990 Fmoc methodology had been extended with a range of supports, linkers and side-chain protecting groups into a fully orthogonal scheme [2]. Today Fmoc SPPS is the method of choice, and high-quality Fmoc building blocks are produced at multi-ton scale [4].

Coupling: forming each peptide bond

A carboxylic acid and an amine do not form an amide on their own at room temperature. The incoming amino acid’s carboxyl group has to be activated first. Coupling reagents do this, and there are dozens of them, a variety one review called “more than a letter soup” [5]. The main families are:

• Carbodiimides such as DIC, used with an additive such as HOBt or Oxyma to form a reactive ester and limit racemisation.
• Uronium/aminium salts such as HBTU and HATU.
• Phosphonium salts such as PyBOP.

Coupling efficiency matters because errors compound. A 20-residue peptide needs 19 couplings. If each coupling reached 99% completion, about 83% of chains would be full length (0.99¹⁹). At 98% per coupling, that figure falls to about 68%. This is why chemists use excess reagents, repeat difficult couplings, and often cap unreacted chains with acetic anhydride. Capping acetylates any free amine that missed a coupling, so the short chain stops growing and becomes easier to separate later.

Some sequences are harder than others. Bulky residues, repeated hydrophobic stretches and chains that aggregate on the resin all couple more slowly, and these are the sequences where impurity levels in the crude product rise.

Cleavage: releasing the peptide from the resin

Once the last residue is coupled and its N-terminal group removed (or acetylated, if the sequence calls for an Ac- end), the chain is released. In Fmoc/tBu chemistry, a concentrated TFA mixture does two jobs at once: it cuts the bond to the resin linker and strips the acid-labile side-chain protecting groups. The cleaved protecting groups form reactive carbocations, so the mixture contains scavengers such as water and triisopropylsilane (and thiols for some sequences) to trap them before they attach to tryptophan, methionine, cysteine or tyrosine side chains.

The resin linker sets the C-terminal group. A Wang-type linker releases a free C-terminal acid, and a Rink amide linker releases a C-terminal amide. The crude peptide is then usually precipitated in cold ether, collected and dried. At this stage it typically contains the target peptide plus a family of closely related by-products.

Cyclic peptides and disulfide-bridged peptides need extra steps, either on the resin or in solution after cleavage. Proteins too long to build in one run can be assembled from synthetic segments; native chemical ligation, introduced in 1994, joins two unprotected peptide segments and leaves a native peptide bond at the junction [6].

Common peptide synthesis impurities

The by-products of SPPS are well catalogued [7]. Most are peptides that differ from the target by one residue, one stereocentre or one chemical group, which is what makes them hard to remove and why they are tested for.

Source: D’Hondt et al. [7]; aspartimide chemistry from Kong et al. [8].

Aspartimide formation deserves a note because it is specific to Fmoc chemistry. The piperidine used to remove Fmoc can drive the aspartate side chain to close into a five-membered ring, which then reopens into a mixture of normal and rearranged (beta-linked) peptides, some with an inverted stereocentre. Strategies to suppress it include bulkier side-chain esters, backbone protection and Fmoc alternatives that come off under non-basic conditions [8].

Purification by preparative HPLC

Crude peptides are purified by preparative reversed-phase HPLC. The crude material is loaded onto a large column packed with hydrophobic C18 or C8 silica, and a gradient of water and acetonitrile, each containing about 0.1% TFA, elutes the components in order of hydrophobicity. Fractions are collected across the main peak, checked by analytical HPLC and MS, and the cleanest fractions are pooled.

TFA is in the mobile phase for a reason: it pairs with the positively charged groups on the peptide and sharpens the peaks. Work on preparative peptide separations has shown directly that peptide-TFA ion pairs form in acetonitrile-water and that TFA concentration changes both peak shape and selectivity between peptides [9]. Separation is never perfect. Impurities that elute very close to the main peak, such as some diastereomers, are the hardest to remove, and the purity of the pooled fractions is a trade-off against yield. How purity is measured afterwards is covered in our guide to HPLC peptide purity testing.

Freeze-drying and counter-ions

The pooled fractions are a dilute solution of peptide in water, acetonitrile and TFA. Freeze-drying (lyophilisation) turns this into a stable solid: the solution is frozen, ice is removed by sublimation under vacuum (primary drying), and bound water is then driven off at a higher shelf temperature (secondary drying) [10]. The result is the dry cake or powder seen in a sealed vial. Our explainer on lyophilised peptides covers the process and what the cake should look like.

Because TFA is present during cleavage and purification, peptides with basic groups are usually isolated as trifluoroacetate salts [11]. The counter-ion is part of the powder’s mass but not part of the peptide, so it lowers net peptide content without affecting HPLC purity. It also matters for laboratory work. In fetal rat osteoblast cultures, researchers reported lower cell numbers with TFA alone at nanomolar concentrations, and lower measured proliferation with the TFA salts of several peptides than with their hydrochloride salts, enough to mask or invert an assay result [12]. For that reason, peptides intended for cell-based assays are often supplied as acetate or hydrochloride salts. TFA can be exchanged by reverse-phase HPLC with a different acid, by ion-exchange resin, or by repeated freeze-drying from a stronger or weaker acid [11, 13], and counter-ion content can be measured by ion chromatography or capillary electrophoresis [13].

Why the certificate of analysis matters

Every stage above can leave a trace. A synthesis can produce the right mass at low purity, high purity with the wrong counter-ion, or a clean main peak that hides a diastereomer. The certificate of analysis is where those questions are answered for a given sample:

• Identity by mass spectrometry catches deletion and insertion sequences, adducts and oxidation, because each shifts the mass.
• HPLC purity catches impurities that separate on the column, including some of the same-mass ones MS cannot see.
• Net peptide content accounts for counter-ions and water that neither test sees.
• Contaminant tests, where a lab runs them, cover things synthesis chemistry does not address, such as endotoxin from water and handling (see our endotoxin testing explainer).

Our field-by-field guide on how to read a peptide COA works through two real lab reports, and the background chemistry of chains and modifications is in what are peptides.

Frequently asked questions

How are peptides made?

Most research peptides are made by solid-phase peptide synthesis. The first amino acid is attached to a resin bead, and the chain is built one residue at a time by removing a protecting group and coupling the next amino acid. The finished chain is cleaved with acid, purified by preparative HPLC and freeze-dried.

What is the difference between Fmoc and Boc synthesis?

They use different temporary protecting groups on the amino end. Fmoc is removed with a base and the final cleavage uses TFA. Boc is removed with TFA and the final cleavage needs a much stronger acid such as hydrogen fluoride. Fmoc chemistry is the standard method today.

What impurities come from peptide synthesis?

The main ones are deletion and insertion sequences, truncated chains, diastereomers from racemisation, aspartimide-derived products, leftover protecting groups and oxidised forms. Most differ from the target by one residue or one small group, which is why they are measured by HPLC and mass spectrometry.

Why do synthetic peptides contain TFA?

Trifluoroacetic acid is used to cleave the peptide from the resin and in the HPLC mobile phase, so peptides with basic groups are usually isolated as trifluoroacetate salts. TFA can be exchanged for acetate or chloride, and its level can be measured by ion chromatography.

Research materials: Vortex supplies peptides as sealed lyophilised research reagents. Browse the catalogue at /shop/. Published lab reports are at /coa/.

References

1. Merrifield B. Solid phase synthesis. Science. 1986;232(4748):341-7. PMID 3961484. doi:10.1126/science.3961484
2. Fields GB, Noble RL. Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int J Pept Protein Res. 1990;35(3):161-214. PMID 2191922. doi:10.1111/j.1399-3011.1990.tb00939.x
3. Isidro-Llobet A, Alvarez M, Albericio F. Amino acid-protecting groups. Chem Rev. 2009;109(6):2455-504. PMID 19364121. doi:10.1021/cr800323s
4. Behrendt R, White P, Offer J. Advances in Fmoc solid-phase peptide synthesis. J Pept Sci. 2016;22(1):4-27. PMID 26785684. doi:10.1002/psc.2836
5. El-Faham A, Albericio F. Peptide coupling reagents, more than a letter soup. Chem Rev. 2011;111(11):6557-602. PMID 21866984. doi:10.1021/cr100048w
6. Dawson PE, Muir TW, Clark-Lewis I, Kent SB. Synthesis of proteins by native chemical ligation. Science. 1994;266(5186):776-9. PMID 7973629. doi:10.1126/science.7973629
7. D’Hondt M, Bracke N, Taevernier L, et al. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014;101:2-30. PMID 25044089. doi:10.1016/j.jpba.2014.06.012
8. Kong MJW, van den Braak TJHP, Neumann K. Aspartimide formation and its prevention in Fmoc chemistry solid phase peptide synthesis. Chembiochem. 2025;26(18):e202500490. PMID 40857621. doi:10.1002/cbic.202500490
9. Åsberg D, Langborg Weinmann A, Leek T, et al. The importance of ion-pairing in peptide purification by reversed-phase liquid chromatography. J Chromatogr A. 2017;1496:80-91. PMID 28363419. doi:10.1016/j.chroma.2017.03.041
10. Tang X, Pikal MJ. Design of freeze-drying processes for pharmaceuticals: practical advice. Pharm Res. 2004;21(2):191-200. PMID 15032301. doi:10.1023/b:pham.0000016234.73023.75
11. Roux S, Zékri E, Rousseau B, et al. Elimination and exchange of trifluoroacetate counter-ion from cationic peptides: a critical evaluation of different approaches. J Pept Sci. 2008;14(3):354-9. PMID 18035848. doi:10.1002/psc.951
12. Cornish J, Callon KE, Lin CQ, et al. Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes. Am J Physiol. 1999;277(5):E779-83. PMID 10567002. doi:10.1152/ajpendo.1999.277.5.E779
13. Mrozik W, Markowska A, Guzik L, Kraska B, Kamysz W. Determination of counter-ions in synthetic peptides by ion chromatography, capillary isotachophoresis and capillary electrophoresis. J Pept Sci. 2012;18(3):192-8. PMID 22252914. doi:10.1002/psc.1436

Methods note: written by the Vortex Research Editorial Team. Literature was selected from PubMed for synthetic and analytical chemistry topics, and every PMID was checked against its PubMed record for title, journal and year. Coupling-yield figures are simple arithmetic examples, not measured values.

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