Solid-phase synthesis makes many peptides routine, but some sequences are notoriously troublesome. Chemists call them difficult sequences. Knowing why helps explain differences in purity, price and lead times between peptides of similar length.
On-resin aggregation
As a chain grows on the resin, neighbouring chains can hydrogen-bond to each other and form β-sheet-like aggregates. When that happens, reagents can't reach the reactive end of the chain, couplings and deprotections fail, and deletion sequences build up. Stretches rich in hydrophobic residues such as valine, isoleucine, leucine, alanine and phenylalanine are the usual culprits.
Ways around it
- Pseudoproline dipeptides: temporary ring structures at serine or threonine that put a kink in the chain and break up β-sheets.
- Backbone protection (Hmb or Dmb groups): temporarily blocks the backbone N–H hydrogen-bond donors.
- Heat and microwave synthesis: extra energy disrupts aggregates and speeds up couplings.
- Low-loading or PEG-based resins: more space between chains means less interaction.
Aspartimide formation
Aspartate residues, especially in Asp–Gly, Asp–Ser and Asp–Asn sequences, can cyclise under the basic conditions used to remove Fmoc. The resulting aspartimide can reopen to give the wrong backbone linkage or a racemised product. These by-products often have exactly the same mass as the target, so they're hard to detect. Chemists reduce the problem with bulkier side-chain protection, additives in the deprotection solution, or backbone protection.
Side-chain protection and cleavage
Each reactive side chain needs its own protecting group that survives synthesis and comes off cleanly at the end. Some common examples:
| Residue | Typical protecting group | Watch-out |
|---|---|---|
| Arginine | Pbf | Can be slow to remove, especially with several arginines |
| Cysteine | Trt | Prone to racemisation during coupling |
| Tryptophan | Boc | Its ring can be attacked by reactive fragments during cleavage |
| Aspartate / glutamate | OtBu | Aspartimide risk for Asp |
| Lysine | Boc | Generally well behaved |
During acid cleavage, the protecting groups come off as reactive carbocations. Scavengers such as water, triisopropylsilane and thiols are added to trap them before they damage sensitive residues.
Disulfide-rich peptides
Peptides with several disulfide bonds must be folded so the right cysteines pair up. With three disulfides there are 15 possible pairings. Chemists use orthogonal cysteine protection to form bonds one at a time, or carefully controlled folding conditions.
Very long peptides
Beyond roughly 50 residues, stepwise synthesis yields drop sharply. Native chemical ligation, introduced by Dawson, Muir, Clark-Lewis and Kent in 1994, joins two unprotected peptide fragments: one ending in a thioester and one starting with cysteine. It forms a natural peptide bond at the join, which has made chemical synthesis of whole small proteins possible.
Related articles
- How laboratories validate analytical methods
- D-amino acids and peptidomimetics
- Mass spectrometry explained for peptides
Sources and further reading
- Paradís-Bas M, Tulla-Puche J, Albericio F. The road to the synthesis of "difficult peptides". Chem Soc Rev 2016;45:631–654. doi:10.1039/C5CS00680E · PMID: 26612670
- Isidro-Llobet A, Álvarez M, Albericio F. Amino acid-protecting groups. Chem Rev 2009;109:2455–2504. doi:10.1021/cr800323s · PMID: 19364121
- Dawson PE, Muir TW, Clark-Lewis I, Kent SBH. Synthesis of proteins by native chemical ligation. Science 1994;266:776–779. doi:10.1126/science.7973629 · PMID: 7973629