Peptide synthesis, also known as peptide chain synthesis, is a solid phase synthesis sequence generally synthesized from the N-terminus (amino terminus) to the C-terminus (carboxy terminus). Past peptide synthesis is carried out in solution called liquid phase synthesis. The synthesis of polypeptides is mainly divided into two pathways: chemical synthesis of polypeptides and biosynthetic peptides.
Principle of peptide synthesis
Polypeptide synthesis is how to connect various amino acid units according to the amino acid arrangement order and connection mode of natural products. Since amino acids are present in the intramolecular zwitterionic form (H3+NCH(R)COO-) under neutral conditions, the direct condensation of amino acids to form an amide bond is difficult under normal conditions.
The amino acid ester has a high reactivity. The peptide ester can be polymerized by heating at 100 ° C or at room temperature for a long time, but the reaction is not oriented. The esters of the two amino acids a1 and a2 will form a1a2..., a1a1..., a2a1, etc. during polymerization. a mixture of sequences.
In order to obtain a synthetic polypeptide having a specific sequence, it is not feasible to employ any polymerization method, and only a stepwise condensation-directed polypeptide synthesis method can be employed. Generally, it is represented by the following formula, in which an amino group or a carboxyl group which does not require a reaction is temporarily protected with a suitable group, and then a ligation reaction is carried out to ensure the orientation of the synthesis of the polypeptide.
X and Q in the formula are a protecting group for an amino group and a carboxyl group, respectively, which not only prevent the occurrence of a side-by-side reaction, but also have the effect of eliminating the zwitterionic form of the amino acid and making it soluble in an organic solvent.
In some cases, Q may not be a covalently bonded group, but an organic cation composed of an organic strong base such as triethylamine and a carboxyl hydrogen ion of an amino acid. Y is a strong electron withdrawing group which activates the carboxyl group and favors the free amino group of another amino acid, and nucleophilic attack on the carboxyl carbon atom of the activated carboxyl group to form an amide bond.
The ligation product thus obtained is a protective peptide having a protective group at both the N-terminus and the C-terminus, and a free peptide can be obtained after the protective group is removed. If the peptide chain does not end here, but it is also necessary to extend the peptide chain from the N-terminus or the C-terminus, then X or Q can be selectively removed first, followed by a new N-protected amino acid (or peptide) or C-protection. The amino acid (or peptide) is ligated a second time and is repeated in sequence until the desired length of the peptide chain.
For peptide synthesis of long peptides, there are generally two ways of gradual growth and fragment condensation of two elongated peptide chains, the former starting with the starting amino acid (or peptide). Each time it is ligated, one amino acid is lengthened, and the latter is a new long peptide chain obtained by condensing the N-protected peptide with the C-protected peptide to obtain the length of both.
For peptides containing amino acids with side chain functional groups such as glutamic acid, aspartic acid, lysine, arginine, histidine, cysteine, etc., in order to avoid side chain function Side reactions caused by the group generally require temporary protection of the side chain groups with appropriate protecting groups.
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.
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.
Solid phase synthesis of peptides
The synthesis of a polypeptide is a process in which amino acids are repeatedly added, usually from the C-terminus to the N-terminus (amino terminus). The principle of solid phase synthesis of peptide is to connect the first amino acid C-terminus of the peptide of interest to the solid phase carrier through a covalent bond, and then use the N-terminus of the amino acid as a starting point for synthesis, after deprotection of the amino group and excess of activated The two amino acids are reacted, the peptide chain is terminated, and the operation is repeated to achieve the desired length of the synthetic peptide chain. Finally, the peptide chain is cleaved from the resin, and purified to obtain the target polypeptide.
1. Boc peptide synthesis
The Boc method is a classical peptide solid phase synthesis method in which Boc is used as a protecting group for the amino acid α-amino group, benzyl alcohol is used as a side chain protecting group, and Boc removal is usually carried out using trifluoroacetic acid (TFA). When the polypeptide is synthesized, the Boc-protected N-α-amino acid has been covalently cross-linked to the resin, the TFA excises the Boc protecting group, and the N-terminus is neutralized with a weak base.
Prolongation of the peptide chain is carried out by activation and coupling of dicyclohexylcarbodiimide (DCC), and finally the synthesized target polypeptide is dissociated from the resin by strong acid hydrofluoric acid (HF) method or trifluoromethanesulfonic acid (TFMSA). . In the Boc polypeptide synthesis method, in order to facilitate the next step of polypeptide synthesis, the acid is deprotected repeatedly, and some side reactions are brought into the experiment, for example, the polypeptide is easily cleaved from the resin, and the amino acid side chain is unstable under acidic conditions.
2. Fmoc peptide synthesis method
Carpino and Han developed a new method for solid phase synthesis of peptides based on Boc peptide synthesis - Fmoc peptide synthesis.
Fmoc polypeptide synthesis uses Fmoc as a protecting group for the amino acid a-amino group. The advantage is that it is stable under acidic conditions, is not affected by reagents such as TFA, and can be deprotected by mild alkali treatment, so the side chain can be protected by a Boc protecting group which is easily removed by acid.
The final excision of the peptides can be quantified from the resin using TFA/dichloromethane (DCM), avoiding the use of strong acids. At the same time, compared with the Boc method, the Fmoc method has mild reaction conditions, few side reactions, high yield, and the Fmoc group itself has characteristic ultraviolet absorption, which is easy to monitor and control the reaction. The Fmoc method is more and more widely used in the field of peptide solid phase synthesis.
Liquid phase segmentation synthesis of peptide
With the development of peptide synthesis, the liquid phase segmentation synthesis of peptides (ie, the synthesis of peptide fragments in solution based on their chemical specificity or chemical selectivity, spontaneously linked to growth peptides) plays an increasingly prominent role in the field of peptide synthesis. . It is characterized by its ability to be used for the synthesis of long peptides with high purity and easy purification.
Liquid phase fractionation of peptides is mainly divided into natural chemical linkages and Staudinger linkages. Natural chemical ligation is the basic method for the segmental synthesis of peptides. The limitation is that the synthesized peptide must contain a semi-leucine (Cys) residue, thus limiting the range of applications of natural chemical ligation methods. Extensions to natural chemical ligation methods include chemical region selective ligation, removable co-ligation linkages, and light sensitive auxiliary ligand linkages.
The Staudinger ligation method is another basic fragment ligation method that opens up broader ideas for the linking of polypeptide fragments. The orthogonal chemical joining method is an extension of the Staudinger joining method, and the condensation rate between the fragments is improved by simplifying the phosphine sulfide auxiliary group.
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.
Principle of peptide synthesis
Polypeptide synthesis is how to connect various amino acid units according to the amino acid arrangement order and connection mode of natural products. Since amino acids are present in the intramolecular zwitterionic form (H3+NCH(R)COO-) under neutral conditions, the direct condensation of amino acids to form an amide bond is difficult under normal conditions.
The amino acid ester has a high reactivity. The peptide ester can be polymerized by heating at 100 ° C or at room temperature for a long time, but the reaction is not oriented. The esters of the two amino acids a1 and a2 will form a1a2..., a1a1..., a2a1, etc. during polymerization. a mixture of sequences.
In order to obtain a synthetic polypeptide having a specific sequence, it is not feasible to employ any polymerization method, and only a stepwise condensation-directed polypeptide synthesis method can be employed. Generally, it is represented by the following formula, in which an amino group or a carboxyl group which does not require a reaction is temporarily protected with a suitable group, and then a ligation reaction is carried out to ensure the orientation of the synthesis of the polypeptide.
X and Q in the formula are a protecting group for an amino group and a carboxyl group, respectively, which not only prevent the occurrence of a side-by-side reaction, but also have the effect of eliminating the zwitterionic form of the amino acid and making it soluble in an organic solvent.
In some cases, Q may not be a covalently bonded group, but an organic cation composed of an organic strong base such as triethylamine and a carboxyl hydrogen ion of an amino acid. Y is a strong electron withdrawing group which activates the carboxyl group and favors the free amino group of another amino acid, and nucleophilic attack on the carboxyl carbon atom of the activated carboxyl group to form an amide bond.
The ligation product thus obtained is a protective peptide having a protective group at both the N-terminus and the C-terminus, and a free peptide can be obtained after the protective group is removed. If the peptide chain does not end here, but it is also necessary to extend the peptide chain from the N-terminus or the C-terminus, then X or Q can be selectively removed first, followed by a new N-protected amino acid (or peptide) or C-protection. The amino acid (or peptide) is ligated a second time and is repeated in sequence until the desired length of the peptide chain.
For peptide synthesis of long peptides, there are generally two ways of gradual growth and fragment condensation of two elongated peptide chains, the former starting with the starting amino acid (or peptide). Each time it is ligated, one amino acid is lengthened, and the latter is a new long peptide chain obtained by condensing the N-protected peptide with the C-protected peptide to obtain the length of both.
For peptides containing amino acids with side chain functional groups such as glutamic acid, aspartic acid, lysine, arginine, histidine, cysteine, etc., in order to avoid side chain function Side reactions caused by the group generally require temporary protection of the side chain groups with appropriate protecting groups.
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.
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.
Solid phase synthesis of peptides
The synthesis of a polypeptide is a process in which amino acids are repeatedly added, usually from the C-terminus to the N-terminus (amino terminus). The principle of solid phase synthesis of peptide is to connect the first amino acid C-terminus of the peptide of interest to the solid phase carrier through a covalent bond, and then use the N-terminus of the amino acid as a starting point for synthesis, after deprotection of the amino group and excess of activated The two amino acids are reacted, the peptide chain is terminated, and the operation is repeated to achieve the desired length of the synthetic peptide chain. Finally, the peptide chain is cleaved from the resin, and purified to obtain the target polypeptide.
1. Boc peptide synthesis
The Boc method is a classical peptide solid phase synthesis method in which Boc is used as a protecting group for the amino acid α-amino group, benzyl alcohol is used as a side chain protecting group, and Boc removal is usually carried out using trifluoroacetic acid (TFA). When the polypeptide is synthesized, the Boc-protected N-α-amino acid has been covalently cross-linked to the resin, the TFA excises the Boc protecting group, and the N-terminus is neutralized with a weak base.
Prolongation of the peptide chain is carried out by activation and coupling of dicyclohexylcarbodiimide (DCC), and finally the synthesized target polypeptide is dissociated from the resin by strong acid hydrofluoric acid (HF) method or trifluoromethanesulfonic acid (TFMSA). . In the Boc polypeptide synthesis method, in order to facilitate the next step of polypeptide synthesis, the acid is deprotected repeatedly, and some side reactions are brought into the experiment, for example, the polypeptide is easily cleaved from the resin, and the amino acid side chain is unstable under acidic conditions.
2. Fmoc peptide synthesis method
Carpino and Han developed a new method for solid phase synthesis of peptides based on Boc peptide synthesis - Fmoc peptide synthesis.
Fmoc polypeptide synthesis uses Fmoc as a protecting group for the amino acid a-amino group. The advantage is that it is stable under acidic conditions, is not affected by reagents such as TFA, and can be deprotected by mild alkali treatment, so the side chain can be protected by a Boc protecting group which is easily removed by acid.
The final excision of the peptides can be quantified from the resin using TFA/dichloromethane (DCM), avoiding the use of strong acids. At the same time, compared with the Boc method, the Fmoc method has mild reaction conditions, few side reactions, high yield, and the Fmoc group itself has characteristic ultraviolet absorption, which is easy to monitor and control the reaction. The Fmoc method is more and more widely used in the field of peptide solid phase synthesis.
Liquid phase segmentation synthesis of peptide
With the development of peptide synthesis, the liquid phase segmentation synthesis of peptides (ie, the synthesis of peptide fragments in solution based on their chemical specificity or chemical selectivity, spontaneously linked to growth peptides) plays an increasingly prominent role in the field of peptide synthesis. . It is characterized by its ability to be used for the synthesis of long peptides with high purity and easy purification.
Liquid phase fractionation of peptides is mainly divided into natural chemical linkages and Staudinger linkages. Natural chemical ligation is the basic method for the segmental synthesis of peptides. The limitation is that the synthesized peptide must contain a semi-leucine (Cys) residue, thus limiting the range of applications of natural chemical ligation methods. Extensions to natural chemical ligation methods include chemical region selective ligation, removable co-ligation linkages, and light sensitive auxiliary ligand linkages.
The Staudinger ligation method is another basic fragment ligation method that opens up broader ideas for the linking of polypeptide fragments. The orthogonal chemical joining method is an extension of the Staudinger joining method, and the condensation rate between the fragments is improved by simplifying the phosphine sulfide auxiliary group.
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.
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