Peptide synthesis (revision 15)
Old revision·06:21, 27 Dec 2024·CostPerMgCory
| 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.
See also
- Solid-phase peptide synthesis
- Fmoc chemistry
- Crude peptide
- Preparative HPLC purification
- Peptide coupling reagent
References
- ^ a b 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.