Solid-Phase Peptide Synthesis (SPPS) Explained

Solid-phase peptide synthesis (SPPS) builds a peptide one amino acid at a time on insoluble resin beads, starting from the C-terminal residue. Each round removes a temporary protecting group from the end of the growing chain and couples the next protected amino acid, and everything not attached to the resin is washed away. When the chain is complete it is cleaved from the resin, purified by preparative reversed-phase HPLC and freeze-dried to a powder.

The majority of synthetic peptides are now made by the Fmoc version of SPPS. This article follows that route from resin to powder. For the basic chemistry of amino acids and peptide bonds, see what peptides are.

Merrifield's idea: synthesis on a solid support

Before SPPS, peptides were made entirely in solution, and the product had to be isolated and purified after every coupling. In 1963 R. Bruce Merrifield anchored the first amino acid to an insoluble polymer and built a tetrapeptide on it. Because the growing chain stays attached to the beads, excess reagents and by-products are removed simply by filtering and washing, so each reagent can be used in large excess to push its step towards completion. The same short sequence of operations repeats for every residue, which made the method easy to automate, and it earned Merrifield the 1984 Nobel Prize in Chemistry.

The first amino acid is attached through its carboxyl group, so the chain can only grow at its amino end. Synthesis therefore runs from the C-terminus to the N-terminus.

Fmoc/tBu chemistry: temporary and permanent protection

Each amino acid building block carries two kinds of protection:

• Temporary protection on the α-amino group: the Fmoc (9-fluorenylmethoxycarbonyl) group, introduced by Carpino and Han in 1970. It is removed at every round by a secondary amine base, usually 20% piperidine in DMF.
• Permanent protection on reactive side chains: acid-labile groups, mostly based on tert-butyl or trityl. They are stable to piperidine and come off only at the end, in trifluoroacetic acid (TFA).

Because one set is removed by base and the other by acid, the two are orthogonal: removing one leaves the other intact. The usual side-chain groups are:

The older Boc/benzyl approach removed the temporary Boc group with TFA at every round, which could slowly strip side-chain protection and lose chains from the support, and its final cleavage needed anhydrous hydrogen fluoride. Fmoc chemistry avoids both, and the fluorene-based by-product released at each deprotection absorbs strongly in the ultraviolet, which gives a convenient way to monitor the synthesis.

Resins and linkers

The resin is a bead of cross-linked polymer, and a linker joins it to the first amino acid. The linker decides what the C-terminus looks like after cleavage:

A sequence such as SS-31, D-Arg-Dmt-Lys-Phe-NH2, ends in an amide, so making it by Fmoc SPPS calls for an amide-forming linker, while a free-acid sequence such as BPC-157 calls for an acid-releasing one.

The synthesis round, step by step

Each residue is added by the same sequence of operations:

1. Deprotection. Piperidine removes the Fmoc group from the chain's N-terminal residue, exposing a free α-amine. The by-product, dibenzofulvene, is trapped by excess piperidine and washed away with it.
2. Washing. Repeated DMF washes clear the base and by-products from the resin.
3. Activation and coupling. The next Fmoc-amino acid is activated at its carboxyl group and added in excess, forming a new peptide bond with the resin-bound amine.
4. Washing. Excess amino acid and reagents are washed out.
5. Checking and capping. A colour test such as the ninhydrin (Kaiser) test shows whether free amines remain. If they do, the coupling can be repeated, or the unreacted chains can be capped with acetic anhydride so they stop growing.

After the last residue, the final Fmoc group is removed. If the sequence starts with an N-terminal acetyl group, written Ac-, the free amine is acetylated on the resin before cleavage.

Coupling reagents

Oxyma, ethyl cyano(hydroxyimino)acetate, was developed to replace the benzotriazole additives HOBt and HOAt with a lower risk of explosion. Non-standard residues are introduced as ready-made Fmoc building blocks: an Fmoc-D-amino acid for a D-residue, or Fmoc-Aib-OH for α-aminoisobutyric acid, a sterically hindered residue that couples more slowly than most. Ipamorelin, Aib-His-D-2-Nal-D-Phe-Lys-NH2, contains three such residues in five.

Why every step counts

Small losses compound. If every coupling went to 99% completion and every other step were perfect, only about 75% of the chains in a 30-residue synthesis, which needs 29 couplings, would come off the resin full length; at 98% per coupling, the figure would be about 56%. The length that can be made routinely depends on the sequence more than on the number of residues: some short sequences are difficult because the growing chains aggregate on the resin, and pseudoproline dipeptides or backbone-protected building blocks are used to break that aggregation up.

Cleavage and global deprotection

A single exposure to concentrated TFA cleaves the linker and removes the side-chain protecting groups together. The cleavage releases reactive species, such as tert-butyl and trityl cations and fragments of the Pbf group, that can alkylate electron-rich side chains such as those of tryptophan, methionine, cysteine and tyrosine. The cleavage mixture therefore contains scavengers: water and triisopropylsilane (TIS), plus a thiol such as 1,2-ethanedithiol (EDT) when cysteine or methionine is present.

The peptide is then precipitated from the TFA solution with cold diethyl ether, collected and washed, giving the crude peptide. Ring-forming bonds are made at different stages: disulfide bridges are usually formed after cleavage by oxidising free cysteine thiols, while side-chain lactam bridges are often closed on the resin, using protecting groups such as allyl esters that can be removed selectively.

Typical impurities in crude peptides

The crude product contains the target alongside related peptides. The common ones, with the mass shift each shows in a mass spectrum:

Aspartimide formation is the most serious side reaction in Fmoc chemistry, because the repeated piperidine steps expose aspartic acid to base, and several of its products elute close to the target. The last rows of the table also show why two analytical methods are used: isomers and diastereomers have the same mass, so mass spectrometry alone cannot distinguish them, while chromatography often can. See HPLC and peptide purity and mass spectrometry and peptide identity.

Purification by preparative RP-HPLC

The crude peptide is dissolved and loaded onto a preparative reversed-phase column, usually packed with C18-bonded silica, and eluted with a gradient of acetonitrile in water, both containing a small amount of TFA as an ion-pairing agent. Analytical separations typically run gradients of 0.5–2% acetonitrile per minute; for preparative work a much shallower gradient, down to about 0.1% per minute, separates impurities that elute just before or after the target. Fractions are collected across the main peak, checked by analytical HPLC and mass spectrometry, and those that meet the specification are pooled.

Because TFA is present during both cleavage and purification, a peptide with basic groups is normally isolated as its trifluoroacetate salt. An extra exchange step, such as running the final purification with acetic acid instead of TFA, gives the acetate salt.

Lyophilisation: from solution to powder

The pooled fractions are mostly water and acetonitrile. They are frozen, then held under vacuum so that the ice sublimes directly to vapour, a stage called primary drying; secondary drying at a higher shelf temperature then removes most of the water still bound to the solid. The volatile components, including excess TFA, leave with the water, and the peptide remains as a dry, white to off-white powder or cake. Freeze-drying avoids heating the peptide in solution and gives a solid that can be weighed and sealed in a vial.

Even after drying, the solid holds counter-ions and some residual water, which is why net peptide content is stated separately from purity. The physical form itself is covered in lyophilised peptides, and each term used here is defined in the glossary.