Peptides are widely present 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 product of incomplete hydrolysis of proteins. So far, a wide variety of peptides have been found in organisms, and they play an important role in participating in and regulating life activities in the body. According to the number of amino acids, they can be divided into oligopeptides (2-10 amino acids) and polypeptides (10-100 amino acids).
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 biosynthesis and chemical synthesis.
1. Biosynthesis
Biosynthesis methods include extraction, enzymatic hydrolysis, genetic recombination, fermentation, etc.
Natural extraction is a method of extracting polypeptide substances from animals and plants. However, the content of polypeptide substances in organisms is rare, the purity of polypeptides obtained by this method is low, and pathogens are easily introduced during the extraction process. Therefore, the extraction method has been gradually replaced by other methods.
Enzymatic hydrolysis is to degrade proteins into polypeptides by biological enzymes. This method has the advantages of mild reaction conditions, but most of the polypeptides obtained by enzymatic hydrolysis are mixtures, and subsequent separation and purification are difficult. In comparison, enzymatic hydrolysis is more common in food, cosmetics, and other industries.
Genetic recombination is to transfer of polypeptides of certain sequences to recombinant DNA in prokaryotic or eukaryotic cells through genetic technology and expression. This method has the advantages of strong directional expression, no need for polypeptide carriers and protective groups, etc. It is the most widely used biosynthesis method at present, but genetic recombination has problems such as purification difficulties.
The fermentation method is a method of producing active peptides through the metabolism of microorganisms. This method is relatively low-cost, but because it relies on microbial metabolism, it can only be used to produce known beneficial active peptides. It has a limited scope of application and is not a mainstream peptide production method.
2. Chemical synthesis
Chemical synthesis includes liquid-phase synthesis and solid-phase synthesis. Currently, chemical synthesis is still the main method for synthesizing polypeptides. In polypeptide synthesis, the formation of peptide bonds (i.e., amide bonds) is a key step. Peptide synthesis reagents are special reagents for building amide bonds, which play an important role in improving the bonding efficiency of amide bonds, product purity, and yield.
In synthesis, the performance of the reagent 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 performance. Condensation agents are mainly used to promote the formation of amide bonds between carboxylic acids and amines, which can effectively reduce the reaction energy barrier and improve the bonding efficiency. These reagents are widely used in the synthesis of peptide drugs and small molecule chemical drugs, including carbodiimide type (such as DCC, DIC, etc.) and onium salt type, the latter of which can be divided into urea cation type (such as HBTU, HATU, etc.) and phosphonium cation type (such as PyBOP); Protective agent is a kind of reagent that protects active groups to temporarily inactivate them, which can effectively avoid the occurrence of non-target reactions, which is very common in organic and drug synthesis. In peptide synthesis, it is often used to protect amino groups, and the commonly used ones 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. Commonly used 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. The commonly used protecting groups in this method are Boc and Cbz (Z), and two main strategies are adopted: the stepwise synthesis method is to connect the amino acids step by step through peptide bonds in the order of amino acids; the fragment synthesis method 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. The amount of reagents and solvents used is small, the cost is lower, and it conforms to the principle of green chemistry. At the same time, the protective groups are more selective, which is conducive to subsequent purification and large-scale production.
The solid phase synthesis method was first proposed in 1963 and has become a mainstream method for polypeptide synthesis. This method uses an insoluble resin as a carrier, fixes the C-terminus of the first amino acid on the resin, connects the pre-deprotected N-terminus with the activated second amino acid through a condensation reaction, and then washes and repeats the above 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. At present, the mainstream methods of solid phase synthesis are Boc solid phase synthesis and Fmoc solid phase synthesis: In Boc solid phase synthesis, α-amino is protected by the Boc group, and the strong acid trifluoroacetic acid is used for deprotection, while harmful hydrofluoric acid is required 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 dangerous and its harm to the body is self-evident. In contrast, the Fmoc protecting group in Fmoc solid phase synthesis is more stable to acid and can be removed under alkaline conditions without the use of dangerous reagents such as hydrofluoric acid. In addition, the Fmoc protecting group can be used to monitor the reaction process, to more accurately control the production process. It is currently the most popular solid-phase peptide synthesis method.
Compared with liquid phase synthesis, solid phase synthesis has shown obvious advantages in the synthesis of long peptides. It is easy to operate, intermediates do not need to be purified and can achieve automated and high-throughput synthesis of peptides. It is currently the mainstream peptide synthesis method.
Against the backdrop of the rapid development of peptide therapeutics, peptide synthesis should be considered from an economic and sustainable perspective. Green peptide synthesis is a major challenge facing scientists around the world. Whether using solid phase synthesis SPPS or liquid phase synthesis LPPS, the ultimate goal is to minimize impurities to reduce purification steps, which again brings economic and sustainable penalties. At the same time, the hot market for peptide drugs means that the peptide pharmaceutical industry will face increasing pressure to shorten the time to market for new drugs. I believe that shortly, there will be more suitable peptide synthesis methods.
With the rapid development of peptide therapeutics, peptide synthesis technology must take into account both economic benefits and sustainability. Whether it is solid phase synthesis (SPPS) or liquid phase synthesis (LPPS), the goal is to minimize the impurity content to reduce cumbersome purification steps, improve synthesis efficiency, and thus reduce the burden on the economy and the environment. At the same time, given the continued growth in market demand for peptide drugs, the pharmaceutical industry is facing increasing pressure to shorten the new drug development cycle. I believe that shortly, there will be more efficient and environmentally friendly peptide synthesis methods.
Suzhou Haofan is committed to the research development and production of peptide synthesis reagents and protection reagents. After 20 years of development and accumulation, the company has become the world's largest and most comprehensive supplier of peptide synthesis reagents, able to provide comprehensive and diverse product selections, covering various condensation reagents from the first to the fourth generation, as well as Fmoc, Boc, Cbz and Troc and other series of protection groups to meet the needs of different customers. We sincerely welcome interested friends to contact us for details, and we look forward to providing you with professional consulting services.
References:
[1] Andersson, L.; Blomberg, L.; Verlander, M.; et al. Large-scale synthesis of peptides[J]. Peptide Sci. 2000, 55, 227-250.
[2] Lau, J.L.; Dunn, M.K. Therapeutic peptides: Historical perspectives, current development trends, and future directions[J]. Bioorg. Med. Chem. 2018, 26, 2700-2707.
[3] Zheng Long, Tian Jiaxin, Hong Wenjing, et al. Research progress in the preparation process of peptide drugs[J]. Journal of Chemical Industry and Engineering. 2021, 72, 3538-3550.
[4] Lin Long, Jiang Suyun, Tang Xinqiang. Research progress in in vitro synthesis methods of peptide drugs[J]. Journal of Dalian Medical University. 2014, 36, 177-181.
[5] Craik, D.J.; Fairlie, D.P.; Price, D.; et al. The Future of Peptide-based Drugs[J]. Chem. Biol. Drug Des. 2013, 81, 136-147.
[6] Sharma, A.; Kumar, A.; Albericio, F.; et al. Liquid-Phase Peptide Synthesis (LPPS): A Third Wave for the Preparation of Peptides[J]. Chem. Rev. 2022, 122, 13516–13546.
[7] Merrifield, R.B. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide[J]. J. Am. Chem. Soc. 1963, 85, 2149–2154.
