Keywords :Peptide synthesis, Solid phase synthesis, Peptide impurity, Peptide purification, peptide sequence

1. The Basic Principle of Peptide Synthesis

Solid phase synthesis of peptide is an important breakthrough in the synthesis of peptide chemistry. Its unique feature is that the intermediate product does not need to be purified, and the synthesis process can be carried out continuously, which lays the foundation for the automation of peptide synthesis. At present, the synthesis of fully automatic peptides is basically solid-phase synthesis. The basic process is as follows:

Based on Fmoc chemical synthesis, the C-terminal amino acid carboxyl group of the target peptide to be synthesized is first connected to an insoluble polymer resin in the form of covalent bonds, and then the amino group of this amino acid is used as the starting point of peptide synthesis, and the carboxyl group of other amino acids has been activated to form peptide bonds, and the process is repeated repeatedly to obtain peptides. According to the different amino acid composition of the peptide, the post-treatment of the peptide is different, and the purification method is also different.

2. Does the Purity of Immune Peptides Need to be Very High?

Generally speaking, peptide is sufficient for immunization at 70-85%.

3. How Long and How Much Purity is Suitable for Immunological Peptide?

Generally about 10-15 amino acids, of course, longer immune effect is better, but the cost of synthesis will also increase. MAP peptide is expected to be longer than 15aa, the effect is better. In addition, the immune effect of peptides below 10aa was poor.

4. Peptides don't Dissolve Well. Is That a Problem with Peptides?

It is difficult to accurately predict the solubility of a peptide and what the appropriate solvent is. The notion that there is a problem with peptide synthesis if it is difficult to dissolve is incorrect.

5. What is the Peptide Form? How to Preserve and Dissolve？

The peptides we provide are in powder form, generally gray and white, the composition is different, the color of the peptide powder is different, the peptides generally need to be kept away from light for a long time, and should be stored at -20 degrees, and can be stored at 4 degrees in the short term. Transport at room temperature if possible for a short time.

6. How to Dissolve Peptides?

Dissolving peptides is a very complex matter, and it is generally difficult to determine the right solvent all at once. Usually a little test is taken first, do not dissolve the joint before determining the appropriate solvent. Omizzur Biotech laboratory generally tests several dissolution conditions after peptide synthesis and marks them on the HPLC.

The Following Methods Can Help You Choose the Right Solvent

1: To determine the specific charge of the peptide, acidic amino acids Asp(D),Glu(E) and C-terminal COOH were set as -1; The basic amino acids Lys(K),Arg(R),His(H) and N-terminal NH2 are +1, and the other amino acids have a charge of 0. Calculate the number of charges.

2: If the net charge number is >0, the peptide is alkaline and dissolved with water: if it is insoluble or less soluble, add acetic acid (more than 10%); If the peptide does not dissolve, add a small amount of TFA(25ul) to dissolve, and then add 500ul of water to dilute.

3: If the net charge number is <0, the peptide is acidic and dissolved with water; If it is insoluble or less soluble, add ammonia water (25ul) to dissolve, and then add 500ul water to dilute.

4: If the net charge number is 0, the peptide is neutral and generally needs to be dissolved with organic solvents such as acetonitrile, methanol or isopropyl alcohol, DMSO, etc. It has also been suggested that urea is needed to dissolve very hydrophobic peptides.

7. What Impurities are Present in Non-HPLC Purified Peptides?

Peptide and non-peptide impurities in crude and desalinated peptides: such as non-full-length peptides and some raw materials such as DTT, TFA, etc

Click to learn more about peptide impurity & synthesis: https://www.omizzur.com/peptide-impurities-synthesis/

8. What are the Impurities of HPLC Purified Peptides?

After HPLC purification of peptides, there will still be some impurities, which are mainly short peptides and trace TFA.

9. What Length of Peptide is Appropriate?

Peptide synthesis needs to consider the length of the peptide, electric charge, hydrophilicity and other factors. The longer the length, the purity and yield of synthetic crude products are reduced, and the difficulty of purification and the probability of not being synthesized will be greater. Of course, the sequence of the functional region of the peptide cannot be changed, but in order to successfully synthesize the peptide, sometimes it is necessary to add some auxiliary amino acids in the upstream and downstream of the functional extraction to improve the solubility and hydrophilicity of the peptide.

If the peptide is too short, the synthesis may also have problems, the main problem is that the synthetic peptide has a certain difficulty in the post-treatment process, the peptide below 5 peptides, generally have hydrophobic amino acids, otherwise the post-treatment difficulty is increased. Peptides with less than 15 amino acid residues can generally obtain satisfactory yield and yield.

10. How to Determine the Solubility of A Peptide From a Peptide Sequence

1: Peptides containing a high proportion of highly hydrophobic amino acids such as Leu,Val,IIe,Met,Phe, and Trp are difficult or impossible to dissolve in aqueous solutions. These amino acids, whether purified or synthesized, can be problematic.

2: Under normal circumstances, the proportion of hydrophobic amino acids is less than 50%, and the proportion of charged amino acids (positive charge K,R,H,N-terminus, negative charge D,E, C-terminus) can not be consecutive 5 consecutive aa hydrophobic, and the proportion of charged amino acids (positive charge K,R,H,N-terminus, negative charge D,E, C-terminus) can reach 20%. If the N or C of the peptide is short, the solubility can also be improved.

11. Why are Peptides Containing Cys,Met, or Trp Difficult to Synthesize?

Peptides containing Cys, Met, or Trp are difficult to synthesize, and high purity products are difficult to obtain. Mainly because these groups are unstable and easily oxidized. The use and storage of these peptides require special care to avoid repeatedly opening the lid.

12. Why is the Synthetic Yield or Purity of Some Peptides Low?

In the actual synthesis process, there are often peptides that cannot be synthesized. Such as Val, Ile, Tyr and Trp, Leu, Phe, Gln, and Thr near or repeat these amino acids, peptide chain in the process of synthesis can not completely stretch dissolved, synthetic efficiency decline. In the following cases, the synthesis efficiency and purity of the product are relatively low, such as: repeated Pro,Ser-Ser, repeated Asp, 4 continuous Gly, etc.

13. How are Peptides Purified?

Peptide purification is generally done using a reversed phase column (such as C8, C18, etc.), 214nm. The buffer system is usually a TFA containing solvent, pH 2.0. Buffer A contains 0.1%TFA in ddH2O, and Buffer B contains 1%TFA/ACN/ pH 2.0. Dissolve with Buffer A before purification; If it does not dissolve well, dissolve it with Buffer B, and then dilute it with Buffer A; For hydrophobic peptides, sometimes a small amount of Formic Acid or acetic acid is added.

HPLC analysis of crude peptide products, if the peptide is not long (less than 15aa), there will be a main peak, the main peak is usually the full-length product; For long peptides above 20aa, if there is no main peak, HPLC should be combined with Mass to determine the molecular weight, and then determine which peak is the peptide to be synthesized

The above are some common problems and solutions in the process of peptide synthesis from the design of peptide sequence to the dissolution of peptide. Hope to help you.

About Omizzur Biotech

Omizzur is a company engaged in custom peptide synthesis, with advantages in various research-grade peptides such as antibacterial peptides, RGD peptides, literature peptide synthesis, API peptide & impurity synthesis. Omizzur providing various fmoc/boc-amino acids, unnatural amino acids, and reagents also. 

Inquiry: [email protected]