Designing Peptides

• Sequence Length
• Peptide Solubility
• Peptide Sequence Stability

When selecting peptides for custom synthesis, several important factors should be considered during the design process. These considerations include sequence length, solubility and sequence stability.

Sequence Length

Peptide purity typically decreases as the sequence length increases.  Give special attention to sequences greater than 30 amino acids in length. Increased length equates to increased number of amino acid couplings, which may result in solubility issues during purification when attempting to remove synthesis impurities.

Peptide Solubility

Amino acids are classified according to a hydropathy index, based on the hydrophobic or hydrophilic properties of their side chains. Inclusion or exclusion of hydrophobic or hydrophilic amino acids in a peptide sequence will impact the ability to synthesize, purify and solubilize the final peptide material in aqueous solutions.

Amino Acid Classifications

Hydrophobic (non-polar): Ala, Ile, Leu, Met, Phe, Trp, Val

Uncharged (polar): Asn, Cys, Gly, Gln, Pro, Ser, Thr, Tyr

Acidic (polar): Asp, Glu

Basic (polar): His, Lys, Arg

Note: Keep hydrophobic amino acid content below 50% of the total sequence length and to include at least one charged amino acid for every five amino acids. At a physiological pH, Asp, Glu, Lys and Arg will contain charged side chains. A single conservative replacement, such as replacing Ala with Gly or adding polar amino acids to the N- or C-terminus may improve solubility.

Peptide Sequence Stability

There are several strategies for improving peptide stability, which will lead to higher purity and optimal solubility. Amino acid composition of the peptide sequence impacts the overall stability and considerations should be made for the following scenarios:

1. Multiple Cys, Met or Trp amino acids may be difficult to obtain in high purity partly due to the susceptibility of oxidation and
    /or side reactions.

Note: Choose sequences which minimize these residues or choose conservative replacements for these amino acids. Norleucine can substitute for Met and Ser can be a less reactive replacement for Cys. If overlapping peptides from a protein sequence are being designed, shifting the starting point of each peptide may also create a better balance between hydrophobic and hydrophilic amino acid residues.

2. N-terminal Gln (Q) is unstable and may cyclize to pyroglutamate when exposed to the acidic conditions of cleavage.

Note: Amidate the N-terminus of the sequence or substitute this amino acid.

3. Asparagine (N) has a protecting group that is difficult to remove when placed at the N-terminus of a peptide sequence.

Note: Remove the Asn at this location, substitute with another amino acid or lengthen the peptide by one amino acid residue.

4. Multiple prolines (P) or adjacent serines (S)  in a sequence can result in a product that is lower in purity or contains many
    deletion products. Multiple prolines can also undergo a cis/trans isomerization, resulting in an apparent lower purity product.

5. Beta sheet formation is a concern as it causes incomplete solvation of the growing peptide chain and will result in a higher
    incidence of deletion sequences in the final product. 

Note: Avoid sequences that contain multiple or adjacent Val, Ile, Tyr, Phe, Trp, Leu, Gln and Thr.  Break the pattern by making conservative replacements, for example, inserting a Gly or Pro at every third residue, replacing Gln with Asn, or replacing Thr with Ser.