The characteristics and main problems of solid-phase synthesis.

The solid phase synthesis method has significant advantages for peptide synthesis: simplify and accelerate multi-step synthesis, because the reaction can be carried out in a simple reactor vessel, avoid losses due to manual operation and repeated transfer of materials, solid phase carrier co-priced peptide chain is in a suitable physical state, through rapid pumping, washing not completed intermediate purification, avoid the liquid phase peptide synthesis in the long recrystration or column, can avoid a large amount of separation in the middle of the loss;
The use of excessive reactants forces individual reactions to be complete in order to achieve a high yield of the final product, increase solventization and reduce intermediate product focus, and the polymeric chains of peptide chains and mild crosslinks on solid-phase vectors are closely mixed to produce a mutual solvent effect, which is detrimental to peptide self-aggregation thermodynamics and suitable for reaction.
main problem of solid phase synthesis is that intermediate hemopeptides on solid phase carriers cannot be separated, so that the purity of the final product is not as good as that of liquid phase synthesis, which must be purified by reliable separation means.
instability of
polypeptides
is one of the main problems in the study of their preparations, and there are many reasons. But there are not many main causes of instability for a peptide.
the effects of external conditions (e.g. PH, temperature, lighting, oxygen concentration, etc.) on peptide stability can be used to help design reasonable formulations. Although the mechanism of additive stabilization of peptides is not very clear, the use of additives is still one of the main means to improve the stability of peptide preparations. The application of CD, DSC and other analytical means can help to quickly screen the right additives.
cause of peptide instability is
. 1. Deamide reaction
in the deacid reaction, Asn/Gln residual hydrolysing forms Asp/Glu. Non-enzyme catalytic deamide reaction. The acrylamide group in the Asn-Gly-structure is more hydrolysed, and the alamide group located on the molecular surface is also more hydrolysed than the alamide group inside the molecule.
2. There are
main reasons for the oxidation of peptide solutions, one is the contamination of oxides in the solution and the other is the spontaneous oxidation of peptides. Met, Cys
His, Trp
, Tyr, etc. are the most easily oxidized of all amino acids and residues. Oxygen pressure, temperature and buffer solution also have an effect on oxidation.
3. The peptide bonds
peptides in hydrolysing are prone to hydrolysaling. The peptide bonds involved in asp are more likely to break than other peptide bonds, especially Asp-Pro and Asp-Gly peptide bonds.
4. The formation of the wrong disulfur bond
the exchange between the disulfur bond or between the disulfur bond and the -base can result in the wrong disulfur bond, resulting in three-stage structural changes and loss of activity.
5.
with the exception of Gly, the α carbon atoms of all amino acid residues are hand-based and prone to decarbonization under alkali catalysis. Among them, Asp residue base is most prone to cyclone reaction.
6. β-elimination
β-elimination refers to the elimination of groups β carbon atoms in amino acid residues. Remnants such as Cys, Ser, Thr, Phe, and Tyr can β-eliminate degradation. Easy to occur under alkaline PH β-elimination, temperature and metal ions also have an impact on it.
7. Denaturation, adsorption, aggregation or precipitation
degeneration are generally associated with the destruction of secondary structures and secondary structures. In denatured states, peptides tend to be more
bio
, and activity is difficult to recover. In the process of peptide denaturation, intermediates are first formed. Usually the intermediates have low solubility and are easy to gather, forming aggregates, which in turn form precipitation visible to the naked eye.
surface adsorption of
proteins is another headache encountered during its storage and use, such as riL-2 which is absorbed on the surface of the pipe during crank filling, resulting in loss of activity.
ways to improve the stability of peptides
. 1. Fixed-point mutations
replace residues that cause peptide instability by
gene
engineering or introduce residues that increase peptide stability, which can improve the stability of peptides.
2. There are
chemical modification methods for chemical modification of peptides, the most studied is PEG modification. PEG is a water-soluble polymer compound that degrades and is non-toxic in the body. PeG combined with polypeptides can improve thermal stability, resist protease degradation, reduce
antigen
, and prolong the half-life of the body. Choosing the right modification method and controlling the degree of modification can improve physical fitness or improve progeny activity.
3.
additives can improve the stability of peptides by adding additives such as sugars, polyols, gelatins, amino acids and certain salts. Sugar and polyols force more water molecules around proteins at low concentrations, improving the stability of peptides.
in the process of freeze-drying, the above-mentioned substances can also replace water and form hydrogen bonds with peptides to stabilize the natural composition of peptides, but also can improve the glass temperature of freeze-dried products. In
surfactants
se
SDS
, Tween, Pluronic) prevent peptide surface adsorption, aggregation and precipitation.
4. A series of chemical reactions such as deamide, β-elimination, hydrolysing, etc. that occur in freeze-dried
peptides require water participation, which can also be used as the flow phase of other reactants. In addition, lower water content can increase the denatured temperature of peptides. Therefore, freeze-dried can improve the stability of peptides.
.