There are mainly two methods for chemically synthesizing peptides: Fmoc and tBoc. Because Fmoc has more advantages than tBox, Fmoc is more prevalent now. Peptide synthesis is a process of adding amino acid repeatedly and it synthesize from carboxyl terminal to amino terminal. In the past, most peptide synthesis were operated in liquid phases, but now most of them are operated in solid phases, which may greatly reduce the difficulty of purification of each step of product. To prevent the occurrence of side reactions, the synthetic columns and the side chains of added amino acids are protected in advance. Only the carboxyl terminal is free, and it should be activated with chemical reagents before reaction.

Specific synthesis steps are as follows:

1、Deprotection: Same alkaline solvent (piperidine) should be used in the column and monomer Fmoc protects to remove the protecting group of amino.

2、Activation and cross-linking: The carboxyl of the next amino acid is activated and dissolved by the same activator. Under the effect of crosslinking agent, the peptide bond forms between activated monomer and free amino acid.

3、Cycle: Repeat the two steps of reaction until the entire peptide chain is synthesized.

4、Elution and deprotection: According to the different residues in peptide chains, we use different resin solvents to elute it from the column. The protecting group is eluted and deprotected by deprotective agent (TFA).

Polypeptides are complex macromolecules, so the physical and chemical properties are distinct between every sequence. Some peptides are difficult to synthesize, while some peptides are relatively easy to synthesize but difficult to purify. The most common problem is that many peptides are insoluble in aqueous solution, so these hydrophobic peptides must dissolve in non-aqueous solutions or particular buffers during purification. Some of these solutions may not suitable for the biological experiment system, so researchers could not achieve their purpose using these peptides. Thus, here are some advice for researchers to design peptides.

How to reduce the difficulty of peptide chain synthesis?

1. Cut down the length of sequence.

Since the increased length of peptides would lead to the decrease of the original product purity, it will be easy to get high purity of head products in the peptide with less than 15 residues. The quantity of the right product is a main problem when the length of peptides increases to more than 20 residues. Decreasing the number of residues to less than 20 could often get a better result in many experiments.

2. Decrease the number of hydrophobic residues.

It always causes synthetic difficulties in the peptides which hydrophobic residues dominate, especially in the region 7-12 residues distant from the carboxyl terminal. It is usually considered to be due to the formation of β-pleated sheet during synthesis, which may cause incomplete paring. It might help to replace with 1 to several polar residues or add Gly or Pro to open the peptide structure.

3. Decrease the number of “difficult” residues

It’s always difficult to synthesize when several Cys、Met、Arg、Try residues exist. Ser is usually used as a non-oxidative replacement for Cys.

How to strengthen the solubility of peptides?

•  Change the amino terminal or carboxyl terminal

We recommend acetylation (amino terminal acetylation with free carboxyl groups in carboxyl terminal) to increase the number of negative charges for acid peptides (carry negative charges when pH value is 7), while amination (free amino groups in amino terminal with carboxyl terminal amination) to increase the number of positive charges for alkaline peptides (carry positive charges when pH value is 7)

•  Decrease or increase the length of sequence

Some sequences contains a large amount of hydrophobic amino acids, such as Trp、Phe、Val、Ile、Leu、Met、Tyr and Ala, etc. Usually it is difficult to dissolve when the amount of hydrophobic residues is more than 50% of the total. Lengthen the sequence might help to increase the peptides polarity. Another method is decreasing the length of peptide chains to increase the polarity by reducing hydrophobic residues. The higher the polarity of the peptide chains, the more likely it is to dissolve in water.

•  Add soluble residues

For some peptide chains, it may change the solubility by adding some polar amino acids. We recommend adding Glu-Glu to amino terminal or carboxyl terminal of acid peptides and adding Lys-Lys to amino terminal of alkaline peptides. If the charged group couldn’t be added, we can add Ser-Gly-Ser to amino terminal or carboxyl terminal. But it’s not suitable when the two terminals couldn’t be changed.

•  Change the sequence by replacing one or more residues

 The solubility of peptides could be improved by changing some residues in the sequence. Usually the replacement of a single residue could improve the hydrophobicity dramatically. But the replacement is always conserved, for example, use Gly instead of Ala.

•  Change the sequence by choosing different “frame”

The sequence would change by altering the starting point of each peptide if a sequence could be used to produce many polypeptides in series or overlapped with certain length. The principle is: Create a new and better balance between hydrophilic and hydrophobic residues in the same polypeptide, or place the “difficult” residues (such as 2 Cys) in two different polypeptides instead of one molecular.

Some points about the influence of particular amino acid residues on the peptides feature

There are several methods to classify the 20 amino acids and other amino acids common in proteins coded by genetic code according to its feature. The most common methods are three letters code and single letter code of amino acids, and others.