Causes of Peptide Instability
Deamidation reaction: In the deacylation reaction, Asn/Gln residues are hydrolyzed to form Asp/Glu. The non-enzyme-catalyzed deamidation reaction proceeds. The amide group in the Asn-Gly- structure is easier to hydrolyze, and the amide group on the surface of the molecule is also easier to hydrolyze than the amide group inside the molecule.
There are two main reasons for the easy oxidation of oxidized polypeptide solution. One is the pollution of peroxide in the solution, and the other is the spontaneous oxidation of the polypeptide. Among all amino acid residues, Met, Cys and His, Trp, Tyr, etc. are the most easily oxidized. Oxygen partial pressure, temperature and buffer solution also have an effect on oxidation.
Hydrolysis: Peptide bonds in polypeptides are easily broken by hydrolysis. The peptide bond formed by Asp is easier to break than other peptide bonds, especially the Asp-Pro and Asp-Gly peptide bonds. Formation of wrong disulfide bonds: exchanges between disulfide bonds or between disulfide bonds and sulfhydryl groups can form wrong disulfide bonds, resulting in changes in tertiary structure and loss of activity.
Racemization: Except for Gly, the α carbon atoms of all amino acid residues are chiral, and racemization reaction occurs easily under base catalysis. Among them, Asp residues are most prone to racemization. β-elimination: β-elimination refers to the elimination of the group on the β carbon atom in the amino acid residue. Cys, Ser, Thr, Phe, Tyr and other residues can be degraded by β-elimination. Β-elimination is prone to occur at alkaline pH, and temperature and metal ions also affect it. Denaturation, adsorption, aggregation or precipitation denaturation are generally related to the destruction of tertiary structure and secondary structure.
In the denatured state, peptides are often more prone to chemical reactions, and it is difficult to recover their activity. In the process of polypeptide denaturation, intermediates are first formed. Generally, intermediates have low solubility and are easy to aggregate to form aggregates, which in turn form visible precipitates.
Suggestions for Improving Peptide Stability
- Site-directed mutagenesis: Replacement of residues that cause polypeptide instability by genetic engineering methods or introduction of residues that can increase the stability of the polypeptide can improve the stability of the polypeptide.
- Chemical modification: There are many chemical modification methods for peptides, and PEG modification is the most studied. PEG is a water-soluble polymer compound, which is degradable in the body and is non-toxic. The combination of PEG and polypeptide can improve the solubility of the polypeptide, can adjust the biocompatibility, improve thermal stability, resist degradation by proteases, reduce antigenicity, and extend the half-life in vivo. Choosing an appropriate modification method and controlling the degree of modification can improve physical fitness or improve the original biological activity.
- Additives: By adding additives, such as sugars, polyols, gelatin, amino acids and certain salts, the stability of the peptide can be improved. Sugars and polyols force more water molecules to surround the protein at low concentrations, thus improving the stability of the peptide. During the freeze-drying process, the above substances can also replace water to form hydrogen bonds with the polypeptide to stabilize the natural conformation of the polypeptide, and can also increase the glass transition temperature of the freeze-dried product. In addition, surfactants such as SDS, Tween, and Pluronic can prevent peptide surface adsorption, aggregation and precipitation.
Freeze-drying: A series of chemical reactions such as deamidation, β-elimination, and hydrolysis of peptides require water to participate, and water can also be used as a mobile phase for other reactants. In addition, a decrease in water content can increase the denaturation temperature of the polypeptide. Therefore, lyophilization can improve the stability of the polypeptide.
Difficulties in the Synthesis of Long Peptides and their Solutions
In the synthesis of long peptides, we have always faced a contradiction, that is, as the sequence grows, the steric hindrance of the condensation reaction increases. In order to complete the reaction, the reaction time needs to be increased; however, as the reaction time increases, side reactions will occur. The more, some target peptides lacking residues will be produced. These peptide chains lacking residues are also the main impurities produced during the synthesis of long peptides. Therefore, during the synthesis of long peptides, the main difficulty we need to overcome is to explore better reaction conditions and reaction methods, so that the amino acid condensation reaction can proceed more fully and thoroughly. At the same time, shorten the reaction time, because the longer the reaction time, the more uncontrollable side reactions and the more complicated the by-products.
To this end, after years of exploration and accumulation, Peptide Valley Bio has summed up the following experience:
- Accurate and flexible control of reaction time: Our accumulated experience in peptide synthesis for many years allows us to accurately control the reaction time of peptides, and find a balance in complete reaction-reduction of by-products;
Microwave synthesis method: We use microwave method to synthesize some amino acids that are difficult to condense during the synthesis process. This method has significant effects, greatly shortens the reaction time, and reduces the production of two main by-products.
Using fragment synthesis method: When some peptides are difficult to synthesize by conventional synthetic methods and are difficult to purify, we will also use the condensation of certain amino acids in a certain segment of the polypeptide and then condense them as a whole to the peptide chain. This method can also solve many problems in the synthesis.
Adopting hydrazide synthesis method: The method for synthesizing peptides by the hydrazide method is a chemical selective reaction between the N-terminal Cys peptide and the C-terminal peptide hydrazide synthesized in a solid phase to form an amide bond to realize the peptide connection. According to the position of Cys in the peptide chain, this method divides the entire peptide chain into multiple sequences to synthesize separately, and finally obtain the target peptide through the liquid phase condensation reaction. This method not only greatly shortens the long peptide synthesis time, but also significantly improves the final The purity of the product is therefore widely applicable to the synthesis of long-chain polypeptides containing Cys.
Long Peptide Purification
The particularity of long peptides inevitably leads to the complexity of the final crude peptide components. Therefore, the HPLC purification of long peptides is also a challenge; we have learned a lot of experience and successfully used it in the purification process of amyloid series peptides. For purification, we have greatly improved the success rate of our long peptide purification through the use of advanced equipment, multiple purification systems, and repeated separation.