Detailed analysis of peptide sequences
When designing peptides we need to take into account the formation of beta folds. During the synthesis process, as the peptide chain lengthens, the β-fold structure results in a very large number of missing sequences in the final product. Choosing not to contain more consecutive sequences of valine, isoleucine, tyrosine, phenylalanine, tryptophan, leucine, glutamine, and threonine can avoid this problem. We usually analyze and evaluate the peptide sequence before performing custom peptide synthesis. If a sequence containing consecutive residues cannot be avoided, we can insert a glycine or proline into every three residues, replace glutamine with asparagine, or replace threonine with serine.
Positively charged residues: lysine, arginine, histidine, N-terminal.
Negatively charged residues: aspartate, glutamic acid, C-terminal.
Hydrophobic residues without charge: phenylalanine, isoleucine, leucine, methionine, valine, tryptophan and tyrosine.
Uncharged residues: Glycine, alanine, threonine, serine, cysteine, asparagine, glutamine, proline, acetylation and amidation.
N-terminal glutamine is easily cycled into pyroglutamate under acidic conditions of cutting. We recommend replacing the n-terminal glutamine with pyroglutamate, removing the glutamine, replacing the glutamine with another amino acid, or modifying the n-terminal glutamine with acetylation. When asparagine is at the N-terminal, it forms a protective group that is difficult to remove. We recommend removing the n-terminal asparagine or replacing it with another amino acid.
If the non-natural amino acids at the C-terminal include D-type amino acids, the C-terminal should be amidezed. If a modification is required at the C-terminal (fluorescent dyes, biotin, etc.), the modification must be linked through the side chain of the lysine, and the C-terminal of these peptides is also amidezed.
As the length of the peptide increases, the efficiency of the coupling reaction will become less and less, and the purity of the crude product will also become less and less. However, peptides with fewer than 5 residues can also cause problems during the cutting and purification process. Sequences containing 3-5 residues should contain at least one hydrophobic residue (leucine, isoleucine, tryptophan, valine, phenylalanine, tyrosine, methionine) or a hydrophobic modification group.
a) The presence of multiple prolines in the sequence tends to form cis/trans isomers, resulting in reduced purity.
b) Continuous serine sequences lead to fragment deletion and reduced purity.
c) Multiple aspartic acid in the sequence, easy to form aspartic imide resulting in reduced purity.
d) Too many modifications in the sequence usually affect the purity and yield of the peptide as well.
e) Continuous multiple glycine (4 or more), the peptide skeleton is prone to form hydrogen bonds, this hydrogen bond makes the peptide difficult to dissolve and purify.
f) Phosphorylation leads to reduced coupling efficiency. Because synthesis is carried out from the C-terminal to the N-terminal, there should be fewer than 10 residues between the phosphorylated modified amino acid and the N-terminal.
There is usually at least one charged residue for every five amino acid residues (including the unsealed C-terminal and N-terminal). If there is enough charge, ensure that the number of consecutive uncharged residues does not exceed 5.
The above are some precautions for analyzing peptide sequences before custom peptide synthesis, hoping to help you.
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