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Synthetic Peptides Part 2: Tips and Tricks for Peptide Synthesis

Two hands reach toward each other to connect chain links to represent various tricks for peptide synthesis

In Part 1 of Synthetic Peptides, we discussed some applications for synthetic peptides, as well as the basics of peptide synthesis, with a brief explanation of blocking reactive side chains so that only the carboxy and amino termini react in the presence of the catalyst.

In Part 2, we’ll explore various design tricks for peptide synthesis, such as improving solubility, designing long peptides, and incorporating post-translational modifications.

Designing Synthetic Peptides

When planning peptide synthesis, you should take certain physical properties into consideration.

Peptide Length

The first is peptide length. You can easily synthesize shorter peptides, from 5 to 10 amino acids long, and it is typically possible to synthesize a peptide of up to 50 amino acids. The exact size you need depends on your specific application, but when you start getting longer than 50 amino acids, you will have issues with purity and yield.

Peptide Solubility

The next consideration is solubility. The more hydrophobic residues you incorporate (e.g. isoleucine and phenylalanine), the less soluble your peptide will be in an aqueous buffer. A good rule of thumb is that every fifth amino acid should be charged. [1]

If you are mimicking a naturally occurring peptide, you might have to modify the sequence by substituting in a charged amino acid to increase solubility. Some trial and error will be required to get a mostly hydrophobic peptide into solution.

Undesirable Secondary Structure

Another interesting consideration is whether significant hydrogen bonding will occur within the peptide. Polar amino acids such as asparagine, glutamine, serine, and threonine can participate in hydrogen bonding with each other and with water. In naturally occurring proteins, these hydrogen bonds help to stabilize secondary structures such as turns, alpha-helices, and beta-sheets.

Even though your peptide may not be long enough for these secondary structures to form, a peptide composed mostly of hydrophilic amino acids could gel in an aqueous solvent. [1]

If you cannot alter the amino acid composition of your peptide, consider modifying the solvent’s pH. Changing the pH can change the charge of polar side groups, thereby affecting their ability to form hydrogen bonds and secondary structures.

Oxidation of Sulfur-containing Amino Acids

Sulfur-containing amino acids, such as cysteine and methionine, can oxidize easily, creating problems during synthesis, and lead to unwanted disulfide bridges (in the case of cysteine). You may be able to substitute these residues for non-sulfur-containing residues such as serine or norleucine.

You could also consider adding reducing agents to your solvent to prevent the formation of disulfide bonds.

Table 1 sums up these various considerations for peptide synthesis.

Table 1: Considerations for Peptide Synthesis



Anything larger than 50 amino acids will decrease synthesis efficiency and purity of the final preparation.


Numerous hydrophobic residues will make solubility in an aqueous solvent difficult. Incorporate charged residues if the peptide sequence contains numerous hydrophobic residues.

Secondary structure

Sequences that promote hydrogen bonding or runs of Gln, Ile, Leu, Phe, Thr, Tyr or Val can promote secondary structures. Modify the solvent pH or introduce conservative substitutions to avoid charged residues and the formation of ionic bonds that may stabilize unwanted secondary structure.


Met and Cys are easily oxidized and can lead to issues during synthesis. Substitute these with Ser and norleucine when possible.

How to Synthesize Long Peptides or Proteins

If you need to make a long peptide or even a whole protein, how do you go about it knowing that synthesis beyond 50 amino acids is impractical? One option is to synthesize smaller peptides and ligate them together via native chemical ligation (NCL).

NCL relies on chemoselective bond formation between a C-terminal thioester and an N-terminal cysteine. Basically, the first peptide ends with a thioester, and the second peptide beings with a cysteine. The two react in an aqueous solution, ligating the two peptides together to create a larger peptide. [2]

What if your peptide sequence lacks a cysteine? One thing you can do is desulfonate the ligated peptide yielding an alanine.

There are also synthetic thiol-derived amino acids that can be incorporated at the N-terminus of the second peptide. After ligation, desulfonation will yield a native polypeptide. For example, beta-thio-leucine can be used as the N-terminal amino acid in the second peptide. After ligation and desulfonation, you will be left with a native peptide joined by a leucine.

Combining synthetic peptides with NCL can be a powerful technique and lends itself to efficient protein production without the need for a recombinant expression system.

How to Introduce Post-Translational Modifications and Unnatural Amino Acids Through Peptide Synthesis

Can you add post-translational modifications to peptides? Yes, you can! A few you might think of right away are disulfide bridges, phosphorylation, and glycosylation. Let’s take a look at each one:

Disulfide Bridges

Creating disulfide bridges can be done in a single step or sequentially. [3] With the former technique, you simply deprotect the side groups of all residues and then expose the peptide to an oxidant such as dimethylsulfoxide (DMSO).

While relatively straightforward, this approach is inefficient and depends on the ability of the peptide to fold into a native state so that the desired disulfide linkages are made. Without this ability, you could end up with multiple and undesired linkages.

In the sequential method, cysteine residues are protected with different protectants that can be differentially removed. You deprotect as desired, expose to an oxidant, and repeat. This limits the number of available cysteine side groups available for linkage and increases the chance that the desired secondary structure is created.


Adding phosphorylated sites is a bit easier. You could simply synthesize the peptide using phosphorylated residues, such as phosphoserine or phosphotyrosine. [4]

Another option is to synthesize an unphosphorylated peptide and treat it with a kinase. The choice would depend on whether the use of a pre-phosphorylated residue would be problematic during synthesis or whether the kinase is able to recognize the intended phosphorylation target within the synthetic peptide.


There are two methods for glycosylating peptides—direct and convergent. [5] In the direct method, a pre-glycosylated serine (O-linked glycosylation) is added into the growing synthetic chain. For the convergent method, which is used for N-linked glycosylation, the peptide chain is made and the glycosylamine moiety is conjugated to a free asparagine residue.

Unnatural Amino Acids

Unnatural amino acids (UAAs) are amino acids not among the 20 amino acids used by living cells in proteins. Some, such as citrulline, occur naturally but are not proteinogenic.

UAAs are typically used in applications such as photo-crosslinking and activation, fluorescent tagging, and protein engineering. If your research can benefit from these applications, UAAs can be incorporated into synthetic peptides in the same ways as the 20 proteinogenic amino acids.

Design Tools

If you are convinced that synthetic peptides are for you, here are some design tools to help you get started!

Table 2: Design Tools for Peptide Synthesis


Thermo Fisher Scientific

•Analyzes physio-chemical properties and estimates ease of synthesis


•Epitope mapping •Alanine scanning among other analyses


•Overlapping peptide scan •Alanine scanning among other analyses

Peptide 2.0 Inc

•Peptide design and epitope mapping

We hope you enjoyed both articles on peptide synthesis; we’d love to hear about your experiences in the comments.


  1. Peptide Design. Thermo Fisher Scientific. Accessed on 03 Oct 2020.
  2. Kulkarni, S., et al. Rapid and efficient protein synthesis through expansion of the native chemical ligation concept. Nat Rev Chem., 2, (2018). doi: 10.1038/s41570-018-0122
  3. Yang, Y., et al. Two-step selective formation of three disulfide bridges in the synthesis of the C-terminal epidermal growth factor-like domain in human blood coagulation factor IX. Protein Sci., 3, 8 (1994). doi: 10.1002/pro.5560030813
  4. Chen, Z., and Cole, P.A., Synthetic approaches to protein phosphorylation. Curr Opin Chem Biol., 28 (2015). doi: 10.1016/j.cbpa.2015.07.001
  5. Moradi, S.V., et al. Glycosylation, an effective synthetic strategy to improve the bioavailability of therapeutic peptides. Chem Sci., 7, 4, (2016). doi: 10.1039/c5sc04392a.
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