Peptide synthesis (revision 21)
Old revision·17:26, 21 Apr 2025·BalanceBirdie
| Peptide synthesisManufacture | |
|---|---|
Coupling and deprotection repeated once per residue, from the C-terminus. | |
| Direction | C-terminus to N-terminus |
| Dominant technique | Solid phase, Fmoc chemistry |
| Alternative | Solution phase; recombinant expression |
| Topic infobox · conventions | |
Peptide synthesis is the chemical assembly of a peptide from protected amino acid building blocks. It proceeds from the C-terminus towards the N-terminus, in the opposite direction to biological translation, because that ordering avoids racemisation of the activated residue.[1]
Chemical synthesis is the only practical route for peptides containing non-proteinogenic residues, and almost every engineered therapeutic peptide contains at least one — the α-aminoisobutyric acid of semaglutide and tirzepatide cannot be introduced by a ribosome.[2]
Three approaches exist: solid-phase synthesis, in which the growing chain is anchored to an insoluble support; solution-phase synthesis, in which it is not; and hybrid approaches in which fragments made on solid phase are joined in solution. Solid phase dominates for research quantities and for most therapeutic peptides.[2]
The elementary cycle
[edit]Each residue is added by a two-step cycle. The N-terminal protecting group of the growing chain is removed, then the next amino acid — protected at its own N-terminus and on any reactive side chain — is activated and coupled.[1]
Protecting groups are what make selectivity possible. The temporary N-terminal group is removed once per cycle; side-chain groups are orthogonal to it and survive until final cleavage. The choice of scheme — Fmoc with acid-labile side chains, or Boc with more forcing conditions — defines the whole chemistry that follows. See Fmoc chemistry.
Coupling is driven by an activating reagent that converts the carboxyl group into a reactive species. Reagent choice affects both speed and the degree of racemisation at the activated centre, and is one of the main levers in optimising a difficult sequence. See Peptide coupling reagent.[2]
Why yield falls with length
[edit]Because a peptide of n residues requires n couplings, overall yield is the product of the individual coupling yields. At 99% per coupling a 30-residue peptide is obtained in about 74% yield; at 98% it is about 55%; at 95% it is about 21%.[2]
The material lost is not simply absent — it is present as truncated and deletion sequences, which remain attached to the support and are cleaved along with the target. These are the impurities that dominate a crude peptide and that a purity method must resolve.
Difficult sequences make the arithmetic worse. Aggregation of the growing chain on the support hides the reactive terminus and depresses coupling yields locally, which is why particular regions of a sequence rather than the whole are usually responsible for a poor crude. See Solid-phase peptide synthesis.[1]
See also
- Solid-phase peptide synthesis
- Fmoc chemistry
- Crude peptide
- Preparative HPLC purification
- Peptide coupling reagent
- Resin cleavage
References
- ^ a b c Merrifield RB. "Solid phase peptide synthesis. I. The synthesis of a tetrapeptide." Journal of the American Chemical Society 85(14):2149–2154 (1963). DOI:10.1021/ja00897a025.
- ^ a b c d Behrendt R, White P, Offer J. "Advances in Fmoc solid-phase peptide synthesis." Journal of Peptide Science 22(1):4–27 (2016). DOI:10.1002/psc.2836. PMID 26785684.