The following points are guidelines in designing de novo or native-based peptides that have a composition that favors synthesis, purification, storage and solubility.

Cysteine and methionine are susceptible to rapid oxidation, which can negatively influence the cleavage of protecting groups during synthesis and the subsequent peptide purification. To avoid this, cysteine can be replaced with serine and methionine replaced with norleucine (Nle). Multiple cysteines on a peptide are susceptible to forming disulfide linkages unless a reducing agent such as dithiothreitol (DTT) is added to the buffer or the cysteines are replaced with serine residues. Cysteine residues in peptides used for antibody production can affect the avidity of the antibody, because free cysteines are uncommon in vivo and therefore may not be recognized by the native peptide structure.

N-terminal glutamine is unstable, because it forms cyclic pyroglutamate under acidic conditions during protecting group cleavage. This can be prevented by acetylating the N-terminal glutamine or by substituting glutamine with pre-formed pyroglutamic acid or a conservative amino acid.

N-terminal asparagine should be avoided, because the asparagine N-terminal protecting group can be difficult to remove during cleavage. Therefore, either remove or substitute the N-terminal amino acid.

Aspartic acid can undergo hydrolysis and cause peptide cleavage under acidic conditions whenpairedwith glycine, proline or serine. Avoid these combinations if possible by substitution or breaking them up by shifting the sequence.

Multiple serine or proline residues in a sequence can cause significant deletions during synthesis, especially proline residues, which can undergo cis/trans isomerization and reduce peptide purity.

A series of glutamine, isoleucine, leucine, phenylalanine, threonine, tyrosine or valine can cause β sheets, which cause incomplete solvation during peptide synthesis, resulting in deletions. Conservative substitution of asparagine for glutamine or serine for threonine, adding a proline or glycine every third amino acid or shifting the sequence can break up β sheets.