What is Solid-phase peptide synthesis?

Solid-phase peptide synthesis (SPPS) builds a peptide on a solid support. The C-terminal amino acid is anchored to a polymer resin bead and the chain grows one residue at a time towards the N-terminus; because the growing peptide stays attached, excess reagents and by-products are simply washed away after each step. R. Bruce Merrifield introduced the approach in 1963 and received the 1984 Nobel Prize in Chemistry for it. Today Fmoc-based SPPS is the method of choice, helped by low-cost, high-quality building blocks produced at multi-tonne scale for therapeutic peptides ^[1].

One cycle of Fmoc chemistry

1. Deprotect. A base, usually piperidine in DMF, removes the Fmoc group from the N-terminal amine of the resin-bound chain.
2. Wash.
3. Couple. The next Fmoc-protected amino acid, activated by a coupling reagent, forms a peptide bond with the free amine. Reactive side chains carry acid-labile protecting groups such as tert-butyl or trityl.
4. Wash, then repeat from step 1 for every residue.
5. Cleave. A cocktail based on trifluoroacetic acid releases the peptide from the resin and strips the side-chain protection in the same step.

The crude product is purified by preparative HPLC and lyophilised, which is why most synthetic peptides arrive as trifluoroacetate salts unless they have been exchanged.

Why step efficiency matters: a worked example

If every coupling succeeds with the same efficiency, the fraction of chains that reach full length after n − 1 couplings is efficiency^(n−1):

Small losses compound. At 99% per step, about a quarter of 30-mer chains are incomplete before cleavage, and the failures are not random debris: they are near-copies of the target, missing one residue here or there, which is what makes them hard to separate. The model is deliberately simple: it ignores incomplete deprotection and side reactions, which add further impurities of their own.

Characteristic by-products

A review of peptide impurities links deletion sequences to inefficient Fmoc removal, insertions to excess amino acid reagent, diastereomers to racemisation during Fmoc removal, and protection adducts to incomplete side-chain deprotection ^[2]. Aspartic acid adds a specific hazard: under the basic conditions used to remove Fmoc, Asp residues can cyclise to an aspartimide, a persistent problem that has driven new side-chain and backbone protecting strategies ^[3]. These by-products are what peptide purity testing measures, and most of them shift the mass seen by mass spectrometry.

Common misunderstandings

• Synthetic does not mean different. A correctly made synthetic copy of a natural sequence is the same molecule; what differs is the impurity profile and the counter-ion.
• Direction is reversed. SPPS builds C→N, the opposite of ribosomal synthesis and of the way sequences are written.
• Unusual residues are routine. D-amino acids, Aib and modified termini are introduced as building blocks or on-resin steps, and many modified derivatives are sold ready-made as Fmoc building blocks ^[1].

The how peptides are made guide follows a peptide from resin to sealed vial, and peptide purity explained covers what the synthesis leaves behind.