Solid-phase peptide synthesis (revision 17)
Old revision·20:54, 30 Dec 2024·CounterfeitCato
| Solid-phase peptide synthesisManufacture | |
|---|---|
Deprotect, wash, couple, wash — repeated once per residue. | |
| Abbreviation | SPPS |
| Introduced | 1963 |
| Support | Polystyrene or polyethylene glycol resin |
| Dominant chemistry | Fmoc |
| Analytical method infobox · conventions | |
Solid-phase peptide synthesis (SPPS) assembles a peptide on an insoluble polymeric support. Because the growing chain is anchored, excess reagents and soluble by-products are removed simply by washing, which is what makes stepwise synthesis of long sequences practical.[1]
The cycle is: remove the temporary N-terminal protecting group, wash, couple the next protected amino acid in excess, wash. Excess reagent drives each coupling towards completion, and the washing that removes it is the operation solution-phase synthesis cannot perform cheaply.[2]
Its principal limitation follows from the same design. Intermediates cannot be purified — everything anchored to the resin travels together — so a failed coupling generates a deletion sequence that persists to the end and must be separated at the purification stage.[2]
Support and linker
[edit]The resin is typically cross-linked polystyrene or a polyethylene glycol–polystyrene composite, functionalised with a linker that attaches the first residue and defines how the finished peptide is released.[2]
The linker determines the C-terminal functionality. Wang and related linkers give a free acid on cleavage; Rink amide linkers give a C-terminal amide. Since several therapeutic peptides are C-terminally amidated, linker choice is a specification-level decision rather than a convenience.
Resin loading — millimoles of site per gram — sets the scale and also affects chain crowding. High loading gives more material per batch but increases the chance of interchain aggregation on the support, which is the main cause of difficult couplings. See Peptide aggregation for the solution-phase analogue of the same phenomenon.[1]
Difficult sequences
[edit]Some sequences couple readily and some do not. Stretches rich in β-branched or hydrophobic residues promote secondary structure on the resin, burying the reactive N-terminus and depressing coupling yield locally.[2]
| Countermeasure | Mechanism |
|---|---|
| Elevated temperature or microwave heating | Disrupts on-resin structure |
| Chaotropic or polar aprotic solvents | Reduces interchain association |
| Double coupling | Second exposure to activated residue |
| Pseudoproline dipeptides | Introduces a kink that prevents β-sheet |
| Lower resin loading | Reduces chain crowding |
These are process choices rather than quality attributes, and they do not appear on a certificate. Their consequences do: a sequence handled poorly gives a crude with a characteristic deletion at the difficult position, which a sufficiently specific method will resolve as a distinct related substance.[3]
Racemisation is a separate risk, concentrated at histidine and cysteine and at any residue activated for too long before coupling. It is invisible to mass spectrometry, since the epimer is isobaric, and requires a chiral method to detect.[2]
See also
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 e Behrendt R, White P, Offer J. "Advances in Fmoc solid-phase peptide synthesis." Journal of Peptide Science 22(1):4–27 (2016). PMID 26785684.
- ^ United States Pharmacopeia, General Chapter <1503>, Quality Attributes of Synthetic Peptide Drug Substances.