The polypeptide synthesis method is a synthesis method in which different or the same plurality of amino acids are linked in a specified order to form a peptide chain (containing a plurality of peptide bonds -CONH-) compounds. First created by the German chemist E. Fischer.
For peptide synthesis, two problems must be solved first:
1. To block one of the two functional groups of the amino acid (usually an amino group), that is, to protect it with a protecting group, and only let the unprotected group (carboxyl group) react with another amino acid molecule. The protecting group employed is not only easy to react with the protected group but also easily removed after the formation of the peptide bond.
2. Activate the unblocked functional group to allow it to react under milder conditions. As the amino protective reagent, there are benzyl chloroformate and t-butoxycarbonyl chloride. The method for activating the carboxyl group is to convert the carboxyl group into a derivative such as an acid chloride, an ester or a mixed acid anhydride or a condensing agent (water-reducing agent). Cyclohexylcarbodiimide combines an amino group and a carboxyl group.
In 1962, R. Merrifield introduced a new peptide synthesis method, solid phase peptide synthesis. The principle is that the first amino acid is attached to an insoluble polymer molecule and then linked to another amino acid one by one. The growing peptide chain is attached to the insoluble polymer, which greatly reduces the separation operation. Loss, simplifying the purification process and eliminating the hassle of purifying individual intermediates. It can mechanize and automate the feeding process and shorten the peptide synthesis cycle. After completion of the polypeptide ligation, the polypeptide is separated from the polymer by a reductive cleavage method and then purified by chromatography. The establishment and application of solid phase methods have greatly facilitated the study of peptide synthesis.
Classification of peptide synthesis methods
The synthesis of polypeptides is mainly divided into two pathways: chemical synthesis of polypeptides and biosynthetic peptides.
Chemical synthesis is mainly carried out in the form of condensation between amino acids and amino acids. In the synthesis of a polypeptide containing a specific sequence, since the polypeptide synthesis raw material contains an amino acid monomer having a functionality of more than 2, the polypeptide should be temporarily protected by a group which does not require a reaction, and the peptide reaction can be carried out, thereby ensuring the polypeptide. The directionality of the target product is synthesized. The chemical synthesis of polypeptides is further divided into liquid phase synthesis and solid phase synthesis.
The liquid phase synthesis of peptides is mainly divided into two strategies: stepwise synthesis and fragment combination. The step-by-step synthesis is simple and rapid, and can be used for the synthesis of various biologically active polypeptide fragments. Fragment combination methods mainly include natural chemical linkages and Staudinger linkages. In recent years, peptide liquid phase synthesis has developed rapidly, and major breakthroughs have been made in the field of peptide and protein synthesis. In the polypeptide fragment synthesis method, depending on the chemical specificity or chemoselectivity of the polypeptide fragment, the polypeptide fragment can be spontaneously ligated to obtain the target polypeptide. Because polypeptide fragments contain relatively few amino acid residues, they are relatively pure and easy to purify.
In 1963, the famous American biochemist Merrifield proposed a solid phase synthesis method, which carried out the solid phase synthesis of peptides, that is, the C-terminal (carboxy terminal) of the amino acid was attached to the insoluble resin, and then the amino acid was condensed and extended sequentially on the resin. Peptide chain. The solid phase synthesis method can be divided into a tert-butoxycarbonyl (Boc) method and a 9-fluorenylmethyloxycarbonyl (Fmoc) method. Based on the liquid phase and solid phase synthesis methods of peptides, the carboxylic acid anhydride (NCA) method, combinatorial chemistry method, etc. of amino acids have been developed.
Biosynthesis methods of peptides mainly include fermentation and enzymatic hydrolysis. With the development of bioengineering technology, genetic engineering, which is dominated by DNA recombination technology, has also been applied to the synthesis of peptides.
Other polypeptide synthesis methods
1. Carboxylic acid anhydride method (NCA) for amino acids
The amino protecting group of the carboxylic acid anhydride of an amino acid also activates the carboxyl group.
Principle of NCA: Under alkaline conditions, the amino acid anion forms a more stable carbamate ion with NCA, which loses carbon dioxide and forms a dipeptide when acidified. The resulting dipeptide is combined with other NCAs and repeated.
NCA is suitable for peptide synthesis of short-chain peptide fragments. It has a short cycle, simple operation, low cost, high molecular weight of the product, and a large proportion in the current peptide synthesis, and the technology is also relatively versatile.
2, combinatorial chemistry
In the 1980s, based on the synthesis of solid phase peptides, a combinatorial chemistry method was proposed, in which the building blocks of amino acids were linked by a combination, and a chemical library containing a large number of compounds was synthesized, and a certain physical and chemical property or pharmacology was selected. A set of peptide synthesis strategies and screening protocols for active compounds.
The peptide synthesis strategy of combinatorial chemistry mainly includes: mixed-average method, iterative method, light-controlled positioning combined library method, tea bag method and the like. The greatest advantage of combinatorial chemistry is the ability to synthesize multiple compounds simultaneously and maximize the screening of new compounds and their isomers.
3. Enzymatic hydrolysis Enzymatic hydrolysis is the use of biological enzymes to degrade plant proteins and animal proteins to obtain small molecular peptides. The enzymatic hydrolysis method failed to achieve industrial production due to its low polypeptide yield, large investment, long cycle and serious pollution. The polypeptide obtained by enzymatic hydrolysis can retain the original nutritional value of the protein, and can obtain more functions than the original protein, and is greener and healthier.
4. Genetic engineering method The genetic engineering method is mainly based on DNA recombination technology, and the sequence synthesis of the polypeptide is controlled by a suitable DNA template. Some researchers have obtained the quasi-elastin-poly-proline-valine-glycine-valine-glycine peptide (VPGVG) by genetic engineering.
Active peptides produced by genetic engineering techniques include peptide antibiotics, interferons, interleukins, growth factors, tumor necrosis factor, human growth hormone, blood coagulation factors, erythropoietin, tissue non-protein plasminogen Wait.
Genetically engineered peptides have the advantages of strong orientation, safety and hygiene, wide source of raw materials and low cost. However, due to the high expression, separation and low yield, it is difficult to achieve large-scale production.
5. Fermentation Fermentation is a method of obtaining a polypeptide from a microbial metabolite. Although the cost of fermentation is low, its application range is narrow, because the polyamino acids that microorganisms can independently synthesize now are only ε-polylysine (ε-PL), γ-polyglutamic acid (γ-PGA) and cyanobacteria. Peptide.
For peptide synthesis, two problems must be solved first:
1. To block one of the two functional groups of the amino acid (usually an amino group), that is, to protect it with a protecting group, and only let the unprotected group (carboxyl group) react with another amino acid molecule. The protecting group employed is not only easy to react with the protected group but also easily removed after the formation of the peptide bond.
2. Activate the unblocked functional group to allow it to react under milder conditions. As the amino protective reagent, there are benzyl chloroformate and t-butoxycarbonyl chloride. The method for activating the carboxyl group is to convert the carboxyl group into a derivative such as an acid chloride, an ester or a mixed acid anhydride or a condensing agent (water-reducing agent). Cyclohexylcarbodiimide combines an amino group and a carboxyl group.
In 1962, R. Merrifield introduced a new peptide synthesis method, solid phase peptide synthesis. The principle is that the first amino acid is attached to an insoluble polymer molecule and then linked to another amino acid one by one. The growing peptide chain is attached to the insoluble polymer, which greatly reduces the separation operation. Loss, simplifying the purification process and eliminating the hassle of purifying individual intermediates. It can mechanize and automate the feeding process and shorten the peptide synthesis cycle. After completion of the polypeptide ligation, the polypeptide is separated from the polymer by a reductive cleavage method and then purified by chromatography. The establishment and application of solid phase methods have greatly facilitated the study of peptide synthesis.
The synthesis of polypeptides is mainly divided into two pathways: chemical synthesis of polypeptides and biosynthetic peptides.
Chemical synthesis is mainly carried out in the form of condensation between amino acids and amino acids. In the synthesis of a polypeptide containing a specific sequence, since the polypeptide synthesis raw material contains an amino acid monomer having a functionality of more than 2, the polypeptide should be temporarily protected by a group which does not require a reaction, and the peptide reaction can be carried out, thereby ensuring the polypeptide. The directionality of the target product is synthesized. The chemical synthesis of polypeptides is further divided into liquid phase synthesis and solid phase synthesis.
The liquid phase synthesis of peptides is mainly divided into two strategies: stepwise synthesis and fragment combination. The step-by-step synthesis is simple and rapid, and can be used for the synthesis of various biologically active polypeptide fragments. Fragment combination methods mainly include natural chemical linkages and Staudinger linkages. In recent years, peptide liquid phase synthesis has developed rapidly, and major breakthroughs have been made in the field of peptide and protein synthesis. In the polypeptide fragment synthesis method, depending on the chemical specificity or chemoselectivity of the polypeptide fragment, the polypeptide fragment can be spontaneously ligated to obtain the target polypeptide. Because polypeptide fragments contain relatively few amino acid residues, they are relatively pure and easy to purify.
In 1963, the famous American biochemist Merrifield proposed a solid phase synthesis method, which carried out the solid phase synthesis of peptides, that is, the C-terminal (carboxy terminal) of the amino acid was attached to the insoluble resin, and then the amino acid was condensed and extended sequentially on the resin. Peptide chain. The solid phase synthesis method can be divided into a tert-butoxycarbonyl (Boc) method and a 9-fluorenylmethyloxycarbonyl (Fmoc) method. Based on the liquid phase and solid phase synthesis methods of peptides, the carboxylic acid anhydride (NCA) method, combinatorial chemistry method, etc. of amino acids have been developed.
Biosynthesis methods of peptides mainly include fermentation and enzymatic hydrolysis. With the development of bioengineering technology, genetic engineering, which is dominated by DNA recombination technology, has also been applied to the synthesis of peptides.
1. Carboxylic acid anhydride method (NCA) for amino acids
The amino protecting group of the carboxylic acid anhydride of an amino acid also activates the carboxyl group.
Principle of NCA: Under alkaline conditions, the amino acid anion forms a more stable carbamate ion with NCA, which loses carbon dioxide and forms a dipeptide when acidified. The resulting dipeptide is combined with other NCAs and repeated.
NCA is suitable for peptide synthesis of short-chain peptide fragments. It has a short cycle, simple operation, low cost, high molecular weight of the product, and a large proportion in the current peptide synthesis, and the technology is also relatively versatile.
2, combinatorial chemistry
In the 1980s, based on the synthesis of solid phase peptides, a combinatorial chemistry method was proposed, in which the building blocks of amino acids were linked by a combination, and a chemical library containing a large number of compounds was synthesized, and a certain physical and chemical property or pharmacology was selected. A set of peptide synthesis strategies and screening protocols for active compounds.
The peptide synthesis strategy of combinatorial chemistry mainly includes: mixed-average method, iterative method, light-controlled positioning combined library method, tea bag method and the like. The greatest advantage of combinatorial chemistry is the ability to synthesize multiple compounds simultaneously and maximize the screening of new compounds and their isomers.
3. Enzymatic hydrolysis Enzymatic hydrolysis is the use of biological enzymes to degrade plant proteins and animal proteins to obtain small molecular peptides. The enzymatic hydrolysis method failed to achieve industrial production due to its low polypeptide yield, large investment, long cycle and serious pollution. The polypeptide obtained by enzymatic hydrolysis can retain the original nutritional value of the protein, and can obtain more functions than the original protein, and is greener and healthier.
4. Genetic engineering method The genetic engineering method is mainly based on DNA recombination technology, and the sequence synthesis of the polypeptide is controlled by a suitable DNA template. Some researchers have obtained the quasi-elastin-poly-proline-valine-glycine-valine-glycine peptide (VPGVG) by genetic engineering.
Active peptides produced by genetic engineering techniques include peptide antibiotics, interferons, interleukins, growth factors, tumor necrosis factor, human growth hormone, blood coagulation factors, erythropoietin, tissue non-protein plasminogen Wait.
Genetically engineered peptides have the advantages of strong orientation, safety and hygiene, wide source of raw materials and low cost. However, due to the high expression, separation and low yield, it is difficult to achieve large-scale production.
5. Fermentation Fermentation is a method of obtaining a polypeptide from a microbial metabolite. Although the cost of fermentation is low, its application range is narrow, because the polyamino acids that microorganisms can independently synthesize now are only ε-polylysine (ε-PL), γ-polyglutamic acid (γ-PGA) and cyanobacteria. Peptide.
Wenzhou Celecare Medical Instruments Co.,Ltd , https://www.celecaremed.com