1. Introduction to polypeptides
Peptides are widely found in nature and living organisms. They are a type of natural substance composed of two or more amino acids connected by peptide bonds. They are also the products of incomplete hydrolysis of proteins. A wide variety of polypeptides have been discovered in living organisms so far, and they play an important role in participating in and regulating life activities in the body. According to the number of amino acids, it can be divided into oligopeptides (2-10 amino acids) and polypeptides (10-100 amino acids).
2. Synthesis Methods of Polypeptides
Since the successful synthesis of the first peptide, oxytocin, scientists have developed a variety of peptide synthesis methods after decades of unremitting efforts. These synthesis methods are mainly divided into biological synthesis methods and chemical synthesis methods.
Biosynthesis methods include extraction methods, enzymatic hydrolysis methods, gene recombination methods, fermentation methods, etc.
Natural extraction methods are a method of extracting polypeptide substances from animals and plants. However, the content of polypeptide substances in organisms is rare. The polypeptides obtained by this method The purity is low, and germs are easily introduced during the extraction process. Therefore, the extraction method has been gradually replaced by other methods.
Enzymatic hydrolysis uses biological enzymes to degrade proteins into peptides. This method has the advantages of mild reaction conditions, but most of the polypeptides obtained through enzymatic hydrolysis are mixtures, making subsequent separation and purification difficult. In comparison, enzymatic hydrolysis is more common in food, cosmetics, and other industries.
The gene recombination method uses genetic technology to transfer polypeptides of certain sequences into recombinant DNA in prokaryotic or eukaryotic cells and express them. This method has the advantages of strong directional expression, no need for the participation of polypeptide carriers and protective groups, and low production cost. It is the most widely used biosynthetic method at present, but the genetic recombination method has problems such as a long development cycle and difficult purification.
Fermentation is a method that produces active peptides through the metabolism of microorganisms. This method is relatively low-cost. However, since this method relies on microbial metabolism, it can only be used to produce active peptides that are known to be beneficial, and its application scope is limited. Not a mainstream peptide production method.
Chemical synthesis methods include liquid-phase synthesis and solid-phase synthesis. At present, chemical synthesis is still the most important method for synthesizing peptides. In peptide synthesis, the formation of peptide bonds (i.e. amide bonds) is a key step. Peptide synthesis reagents are special reagents for constructing amide bonds, which play an important role in improving the synthesis efficiency of amide bonds, product purity, and yield.
In synthesis, the performance of reagents determines the effectiveness of the synthesis strategy. Peptide synthesis reagents can be divided into condensation agents, protective agents, and chiral racemization inhibitors according to their properties. Condensation agents are mainly used to promote the formation of amide bonds between carboxylic acids and amines, which can effectively reduce condensation reaction barriers and improve reaction efficiency. These reagents are widely used in the R&D and production of peptide drugs and small molecule chemical drugs, specifically including carbodiimide type and onium salt type. The latter can be divided into urea cationic type and phosphorus cationic type; the protective agent is a reagent that protects active groups to temporarily inactivate them and can effectively avoid non-target reactions, which are very common in organic and pharmaceutical synthesis. In polypeptide synthesis, it is often used to protect amino groups. Commonly used are Fmoc series protective agents and Boc series protective agents; chiral racemization inhibitors are reagents that can effectively inhibit product racemization and improve the optical purity of products during synthesis. They are commonly used The racemization inhibitors include HOBt, HOPO, etc.
The application of condensation reagents has greatly promoted the process of peptide synthesis. The basic process of chemical synthesis includes: protecting active groups that do not participate in the reaction: activating carboxyl groups to prepare active intermediates to form peptide bonds; and deprotecting protecting groups.
Liquid phase synthesis is a method of forming peptide bonds through chemical reactions in solution. This method mainly uses two strategies: the stepwise synthesis method, which is to gradually connect the amino acids in sequence through peptide bonds; and the fragment synthesis method, which is to connect two or more synthesized polypeptide fragments into the target polypeptide. Compared with the solid phase synthesis method, the liquid phase synthesis method is more suitable for the synthesis of short peptides. It uses small amounts of reagents, solvents, etc., and has lower costs. At the same time, there are many options for protecting groups, which is beneficial to subsequent purification and large-scale production.
After solid-phase synthesis was first proposed in 1963, it has become a mainstream peptide synthesis method. This method uses an insoluble resin as a carrier, fixes the C-terminal of the first amino acid on the resin, connects the pre-deprotected N-terminal to the activated second amino acid through a condensation reaction, and then undergoes a series of condensation and washing. , deprotection, washing, condensation, and other steps until the synthesis of the target polypeptide is completed. As one of the cores of solid-phase synthesis, the selection of protecting groups plays a key role. Currently, the mainstream methods of solid-phase synthesis include Boc solid-phase synthesis and Fmoc solid-phase synthesis: In Boc solid-phase synthesis, α-amino group The Boc group is used for protection. The strong acid trifluoroacetic acid is used for deprotection, while harmful hydrofluoric acid is needed to cut the peptide chain. On the one hand, trifluoroacetic acid is too acidic and may cause the peptide chain to break during the synthesis process. On the other hand, hydrofluoric acid is very dangerous and its harm to the body is self-evident.
In contrast, the Fmoc protecting group in the Fmoc solid-phase synthesis method is relatively stable to acidity and can be removed under alkaline conditions without the use of dangerous reagents such as hydrofluoric acid. In addition, the reaction process can be monitored through the FMOC protecting group, making it more accurate. The controlled production process is currently the most popular solid-phase peptide synthesis method.
Compared with liquid-phase synthesis, solid-phase synthesis has obvious advantages in the synthesis of long peptides. It is easy to operate, intermediates do not need to be purified, and can achieve an automated and high-throughput synthesis of peptides. It is currently the mainstream peptide synthesis method.
3. Polypeptide drugs and other applications of peptides
The unique advantages of peptides have provided support for peptide drugs to gain a foothold in the biomedical field. Since insulin was first used for clinical treatment in 1922, scientists have successfully developed various peptide drugs. In the 21st century, with the clinical application of peptide therapy, With the successful application of peptide drugs, people are paying increasing attention to peptide drugs, which has injected new vitality into the research of such drugs. In the past decade, the outstanding performance of peptide drugs in clinical applications has gradually established their position in the medical field. At present, hundreds of peptide drugs have been approved for marketing and are used for the treatment and diagnosis of different diseases, covering tumors, cardiovascular diseases, diabetes, metabolic dysfunction and endocrine diseases, skin and bone diseases, diagnostic reagents, autoimmunity, etc. Fields, specific manifestations include: in tumor treatment, peptide drugs are mainly used in the treatment of prostate cancer and breast cancer, such as leuprorelin, goserelin, degarelix, etc.; in the field of cardiovascular diseases, they are mainly used as ACE inhibitors, anticoagulants, etc., such as moexipril, eptifibatide, etc.; polypeptide drugs in the field of diabetes are mainly GLP-1 receptor agonists, which are used to treat type 2 diabetes and have good effects in weight management, such as Semaglutide, liraglutide, tilpotide, etc.; voxireprevir, dalbavancin used for anti-infective drugs, as well as lutetium octreotide, Pluvicto, etc. for diagnostic reagents and radioactive therapeutic drugs, are also used in polypeptides. occupies a place in the wide range of drug applications.
Domestic peptide drugs are mainly anti-tumor and immunomodulatory products, among which immunotherapy is mostly used as auxiliary drugs in China, such as thymopentin, etc.
Peptide substances can not only be used directly as drugs, but can also assume the role of drug carriers and participate in drug delivery, especially in the fields of cancer treatment, gene delivery, antibacterial, anti-infection, and antioxidant. For example, a targeted drug delivery system is formed by combining a targeted peptide (targeting peptide) with a drug—polypeptide drug conjugate (PDC). Polypeptide-conjugated drugs are composed of targeting peptides, linkers, and payload drugs. They can specifically recognize the target and deliver the drug to the target cells, thereby increasing the drug concentration in the target area and reducing the toxicity to normal cells, achieving the goal of treatment. Purpose. Melflufen and lutetium oxyoctreotide, launched in recent years, are examples of this technology.
As a natural starting point for drug research and development, peptides have huge therapeutic potential and broad market prospects. Although the development of peptide drugs faces challenges such as oral administration and their utilization, with the continued innovation of synthetic processes and biotechnology, the production cost of peptide substances will be further reduced in the future, and it is expected that more new peptide drugs will be released. In general, peptide drugs show great development potential with their unique advantages. It is believed that peptide drugs will play a more important role in clinical medicine shortly.
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