Solid-Phase Peptide Synthesis: An Overview of the Chemistry

The idea

In solid-phase peptide synthesis the first amino acid is attached by its carboxyl end to an insoluble polymer bead, the resin, and the chain is lengthened one residue at a time. Because the growing peptide is held on a solid, excess reagents and by-products can be washed away by filtration after every step instead of purifying after every step.^[1] Chains therefore grow from the C-terminus towards the N-terminus, the opposite direction to biological synthesis.

Today the dominant approach is the Fmoc strategy. A recent review calls Fmoc synthesis the method of choice, supported by the large-scale availability of the protected building blocks it needs.^[2]

One cycle, step by step

1. Deprotection. The incoming residue has its amino group blocked by the Fmoc group. A base, commonly piperidine in a polar solvent, removes the block from the residue already on the resin so the amine is free to react.^[3]
2. Washing. Solvent washes remove the base and the cleaved by-products.
3. Coupling. The next Fmoc-protected amino acid is activated with a coupling reagent, often an aminium-type reagent, and added in excess so the bond-forming reaction goes as close to completion as possible.^{[3, 4]} The coupling step makes the new amide bond.
4. Washing and, optionally, capping. Unreacted amines can be blocked (capped) so that they do not react in later cycles.

Reactive side chains such as the amino group of lysine, the carboxyl of aspartic and glutamic acid, and the guanidine of arginine carry their own protecting groups, chosen to be stable to the base used in each cycle but removable by strong acid at the end.

Cleavage and the crude peptide

After the last residue is added, the finished chain is detached from the resin and the side-chain protecting groups are removed in one step with a strong acid, usually TFA mixed with scavengers that trap reactive fragments before they can damage the peptide. This is cleavage. The peptide is then recovered, for example by precipitation, and the result is the crude peptide: the target sequence together with everything else that formed. Because TFA is used, the crude peptide is a TFA salt; see counter-ions in synthetic peptides.

Special features of the target are built in at the start or end. A C-terminal amide comes from using an amide-forming resin rather than one giving a free acid; an N-terminal acetyl group is added with a final acetylation step. Disulfide bonds are formed after cleavage by oxidizing a pair of cysteines. These details change the mass, which is why the molecular weight estimator has options for them.

Why longer chains lose yield

Every cycle has a yield below 100 %, and the losses multiply. A made-up example shows how fast: with 30 coupling cycles, a yield of 0.995 per cycle leaves 0.995 raised to the 30th power = 0.860, so 86.0 % of chains are complete; a yield of 0.99 per cycle leaves 0.740, or 74.0 %; and a yield of 0.98 per cycle leaves 0.545, or 54.5 %. A fraction of a percent per step becomes a big effect over a long sequence.

Chains that fall behind do not disappear. They remain on the resin and, unless capped, can continue growing from the missing position, which creates the most common impurity class.

Typical impurities and how they look in data

• Deletion sequences. A residue was never added, so the chain is shorter by one. In mass spectrometry the mass is lower than the target by exactly that residue’s mass.
• Truncated chains. Capped chains that stopped early. Much lower mass; usually well separated by chromatography.
• Incomplete deprotection. A protecting group remains on a side chain, giving a mass higher than the target by the mass of that group.
• Racemization. A residue inverted from the L- to the D-form during activation or coupling. The mass is identical, so it is distinguished by chromatography rather than by mass.
• Aspartimide and related side reactions. Aspartic acid residues, especially followed by small residues such as glycine, can form a ring during base exposure, leading to mixtures of products with the same or slightly changed mass.
• Oxidation during or after cleavage. Methionine and tryptophan are susceptible. Added oxygen shows as +15.995 for methionine sulfoxide.

Difficult sequences

The main cause of failure in peptide chemical synthesis is aggregation of the growing chain on the resin, which makes the reactive end hard to reach.^[3] Chemists respond with special building blocks that disrupt structure, such as pseudoproline units and depsipeptide units, and with heating. Microwave heating during coupling and deprotection has become popular, often shortening synthesis time and improving crude purity, particularly for sequences that tend to form beta-sheet-like structures.^[5]

Purification and drying

The crude product is usually purified by preparative reversed-phase HPLC. Fractions are checked by analytical HPLC and mass spectrometry, and only suitable fractions are pooled, so the purity of the final material depends on which fractions were kept. The pooled solution is then freeze-dried; see lyophilization. Peptide synthesis uses large volumes of hazardous solvents, and the current effort to make it greener is a recognized topic in the field.^[6]

What this page does and does not say

This is a general description of a well-known method. It makes no statement about how any particular material listed on this site was made, and it does not describe any use of a peptide. For identity and purity, analytical data on the specific material is what counts; see reading an HPLC chromatogram and mass spectrometry for peptide identity.