Key points
- In solid-phase peptide synthesis (SPPS) the growing chain is anchored to insoluble resin beads, so excess reagents can be washed away after every step instead of purifying each intermediate.
- Chains are built from the C-terminus to the N-terminus, the opposite direction to ribosomal synthesis.
- Two protecting-group strategies dominate. Fmoc/tBu uses a base to deprotect and TFA for the final cleavage, and is now the default. Boc/Bzl uses TFA to deprotect and HF for cleavage, and survives for difficult sequences.
- Small losses at each step compound. At 99% efficiency per coupling, only about 61% of chains in a 50-residue synthesis are full length.
The idea behind solid-phase synthesis
Making a peptide bond in solution is straightforward; making thirty of them in a row is not. Each coupling in classical solution-phase synthesis produces a mixture that has to be purified before the next step, and the growing peptide becomes harder to dissolve and handle as it lengthens.
In 1963 R. Bruce Merrifield published a different approach. He attached the first amino acid by its carboxyl group to small, cross-linked polystyrene beads and built the chain on the beads. Because the peptide stays attached to an insoluble support, reagents can be used in large excess to push every reaction close to completion, and the excess is removed simply by filtration and washing. The whole process can be run in a single vessel and automated. Merrifield received the 1984 Nobel Prize in Chemistry for the method, which is now how almost all research and many therapeutic peptides are made.
The synthesis cycle
Every amino acid used in SPPS carries a temporary protecting group on its α-amine, so that it cannot react with itself. Reactive side chains carry permanent protecting groups that stay on until the very end. Each residue is then added in a four-step cycle:
- Deprotection. The temporary group on the N-terminal amine of the resin-bound chain is removed, exposing a free amine.
- Washing. The deprotection reagent and by-products are rinsed out.
- Coupling. The next protected amino acid is activated at its carboxyl group and added in excess, typically three to five equivalents. It acylates the free amine and forms the new peptide bond.
- Washing. Excess amino acid and coupling reagents are rinsed out, and the cycle begins again.
After the last residue, the peptide is released from the resin and its side-chain protecting groups are removed, usually in a single acid treatment. The crude peptide is then precipitated, typically in cold diethyl ether, and purified by reversed-phase HPLC.
Fmoc and Boc: two protecting-group strategies
The choice of temporary α-amine protection defines the whole chemistry. The requirement is that the temporary group comes off under conditions that leave the permanent side-chain groups and the resin linkage intact.
Boc/Bzl chemistry
Merrifield's original strategy used the tert-butyloxycarbonyl (Boc) group, removed at every cycle with trifluoroacetic acid (TFA). Side chains were protected with benzyl-based groups that resist TFA but are cleaved by a much stronger acid, anhydrous hydrogen fluoride (HF), at the end. Both protections are acid-labile, and the scheme works because their sensitivity to acid differs by orders of magnitude.
The drawback is HF itself: it is extremely toxic and corrosive, attacks glass, and requires a dedicated apparatus. Boc chemistry is still preferred for some purposes. Repeated TFA treatment keeps the growing chain protonated, which helps break up aggregation in difficult sequences, and the chemistry is well suited to preparing peptide thioesters for native chemical ligation.
Fmoc/tBu chemistry
The 9-fluorenylmethoxycarbonyl (Fmoc) group, introduced by Louis Carpino and Grace Han in 1970, is removed by a mild base, usually 20% piperidine in DMF. Side chains are protected with acid-labile groups based on tert-butyl or trityl, and the final cleavage uses TFA. Because the temporary group is removed by base and the permanent groups by acid, the two are fully independent. Chemists call such a pair of protecting groups orthogonal.
Removal of Fmoc releases dibenzofulvene, which forms an adduct with piperidine that absorbs strongly in the UV around 301 nm. Automated synthesisers use this signal to monitor each deprotection in real time.
| Fmoc/tBu | Boc/Bzl | |
|---|---|---|
| Temporary α-amine group | Fmoc | Boc |
| Removed by | Base (20% piperidine in DMF) | Acid (TFA, typically 25–50% in DCM) |
| Side-chain protection | tBu, Trt, Boc, Pbf | Benzyl-based |
| Final cleavage | TFA with scavengers | Anhydrous HF or TFMSA |
| Orthogonal | Yes | No (graded acid lability) |
| Main advantages | Mild conditions, no HF, easy automation, compatible with many modifications | Better for some aggregation-prone sequences; thioester chemistry |
| Main risks | Base-driven side reactions such as aspartimide formation | HF handling; repeated acid exposure |
Common side-chain protecting groups in Fmoc chemistry are tBu for Ser, Thr, Tyr, Asp and Glu; Trt for Cys, His, Asn and Gln; Boc for Lys and Trp; and Pbf for Arg.
Resins and the C-terminus
The linker that holds the peptide to the resin decides what the C-terminus will look like after cleavage. This is why the choice of resin comes first when planning a synthesis:
| Resin | Chemistry | C-terminus after cleavage |
|---|---|---|
| Wang | Fmoc | Free acid (COOH) |
| 2-Chlorotrityl chloride | Fmoc | Free acid; very mild acid releases fully protected fragments |
| Rink amide | Fmoc | Amide (CONH₂) |
| Merrifield / PAM | Boc | Free acid |
| MBHA | Boc | Amide |
Since many natural peptide hormones are C-terminally amidated, Rink amide resin is among the most used. The difference matters for analysis: an amide is 0.984 Da lighter than the corresponding acid and has one fewer acidic group, which changes both the molecular weight and the isoelectric point.
Polystyrene beads cross-linked with about 1% divinylbenzene remain the standard support. PEG-based and PEG-grafted resins swell better in a wider range of solvents and are often used for long or aggregation-prone sequences.
Coupling reagents
A carboxylic acid does not react with an amine at room temperature on its own. Coupling reagents convert it into a reactive intermediate, an active ester or similar species, that the amine attacks readily. The main families are:
- Carbodiimides, above all DIC (diisopropylcarbodiimide), used together with an additive such as Oxyma Pure or HOBt that suppresses racemisation. DIC/Oxyma is a common default for automated and heated synthesis.
- Aminium and uronium salts such as HBTU and HATU, used with a tertiary base such as DIPEA. HATU is among the most reactive reagents and is often reserved for difficult couplings.
- Phosphonium salts such as PyBOP, with similar uses.
Oxyma-based additives have largely replaced HOBt in many laboratories, partly because anhydrous HOBt and related benzotriazoles are classified as explosive and are difficult to ship.
Why step efficiency matters
Every coupling that fails to go to completion leaves some chains one residue short. Those chains keep growing in later cycles, producing deletion sequences: peptides that lack a single internal residue and are chemically very similar to the target. The fraction of full-length product falls exponentially with the number of steps:
| Peptide length | 97% per step | 99% per step | 99.5% per step | 99.9% per step |
|---|---|---|---|---|
| 10 residues | 76% | 91% | 96% | 99% |
| 20 residues | 56% | 83% | 91% | 98% |
| 30 residues | 41% | 75% | 87% | 97% |
| 50 residues | 22% | 61% | 78% | 95% |
| 70 residues | 12% | 50% | 71% | 93% |
The table assumes the same efficiency for all n − 1 couplings and ignores other side reactions, so real crude purities are usually lower. It explains two standard practices. Difficult couplings are often repeated ("double coupling"). And after a coupling, any unreacted amines can be capped with acetic anhydride, which stops the failed chains permanently. Capped truncations are shorter and easier to separate from the product than deletion sequences.
The table also shows why linear SPPS becomes impractical beyond roughly 50–70 residues. Longer peptides and small proteins are usually assembled from two or more synthetic fragments by native chemical ligation, a method introduced by Stephen Kent and colleagues in 1994. It joins a peptide with a C-terminal thioester to a peptide with an N-terminal cysteine, forming a normal peptide bond at the junction.
Difficult sequences and side reactions
Aggregation on the resin
As a chain grows, it can fold back or pair with neighbouring chains on the same bead, forming β-sheet-like aggregates. The N-terminus becomes buried and couplings slow down dramatically, often after 5–15 residues in hydrophobic stretches rich in Val, Ile, Ala and Gln. A strongly positive hydropathy profile is a useful warning sign. Remedies include:
- Pseudoproline dipeptides, derived from Ser, Thr or Cys, which introduce a temporary kink that disrupts β-sheets and is removed during the final TFA cleavage.
- Backbone protection with Hmb or Dmb groups on selected amide nitrogens.
- Elevated temperature, including microwave-assisted synthesis, which speeds up couplings and disrupts aggregates.
- More polar solvents, chaotropic salts, and PEG-based resins.
Common side reactions
| Side reaction | Where it happens | Consequence |
|---|---|---|
| Aspartimide formation | Asp followed by Gly, Asn, Ser; promoted by piperidine | Ring-closed by-product, then mixtures of α- and β-linked Asp and racemised forms |
| Racemisation | Cys and His during activation | D-isomer impurities that are hard to separate |
| Diketopiperazine formation | At the dipeptide stage, especially with Pro or Gly | Loss of the first two residues from the resin |
| Incomplete deprotection or coupling | Aggregated sequences, bulky residues | Deletion sequences |
| Alkylation during cleavage | Trp, Met, Cys, Tyr | Adducts from carbocations released by protecting groups |
The last row is the reason TFA cleavage cocktails contain scavengers. Removing tBu, Trt and Pbf groups generates reactive carbocations, and scavengers such as water, triisopropylsilane and, for peptides with Cys or Met, thiols trap them before they attack the peptide. A common general-purpose cocktail is TFA with a few percent each of water and triisopropylsilane.
After synthesis
Crude peptides are purified by reversed-phase HPLC, with UV detection of the peptide bond at 214–220 nm, and their identity is confirmed by mass spectrometry, described in the guide to peptide mass spectrometry. Because TFA is present in both the cleavage and the HPLC mobile phase, the purified product is normally obtained as a TFA salt, which affects how much peptide a weighed sample actually contains.
Disulfide bonds are usually formed after cleavage, by air oxidation, with oxidising agents, or selectively when different Cys pairs are protected with orthogonal groups.
Frequently asked questions
Why is SPPS done from C to N when ribosomes work from N to C?
Coupling from C to N activates the carboxyl group of the incoming amino acid, whose amine is protected as a carbamate (Fmoc or Boc). Carbamate-protected amino acids resist racemisation during activation. Building in the other direction would require activating the C-terminus of the growing chain at every cycle, which racemises readily.
What purity should I expect?
It depends on length and sequence. Crude purities of 50–80% are typical for well-behaved peptides of 10–20 residues; aggregation-prone or long sequences can be much lower. After HPLC purification, research-grade peptides are commonly supplied at above 95% purity.
Can non-standard amino acids be incorporated?
Yes. This is one of the main strengths of chemical synthesis. D-amino acids, N-methylated residues, unnatural side chains, fluorescent labels, biotin, lipids and phosphorylated residues can all be introduced as suitably protected building blocks.
Is liquid-phase synthesis obsolete?
No. Solution-phase and hybrid approaches, in which fragments made by SPPS are joined in solution, are used for large-scale manufacture of some therapeutic peptides, where the cost of resin and excess reagents matters.
References
- Merrifield RB (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society 85:2149–2154.
- Carpino LA, Han GY (1970) 9-Fluorenylmethoxycarbonyl function, a new base-sensitive amino-protecting group. Journal of the American Chemical Society 92:5748–5749.
- Dawson PE, Muir TW, Clark-Lewis I, Kent SBH (1994) Synthesis of proteins by native chemical ligation. Science 266:776–779.
- Behrendt R, White P, Offer J (2016) Advances in Fmoc solid-phase peptide synthesis. Journal of Peptide Science 22:4–27.