GLP-1(link:https://www.bloomtechz.com/synthetic-chemical/peptide/glp-1-peptide-cas-87805-34-3.html) is a polypeptide hormone consisting of 30 amino acids. With the in-depth research on GLP-1, more and more synthetic methods have been developed. This article will systematically introduce the currently known synthesis methods of GLP-1.
Method 1, solid phase synthesis:
Solid-phase synthesis is a widely used method for peptide and protein synthesis, and is also commonly used for the synthesis of GLP-1. In solid-phase synthesis, the core structure is formed by linking the first amino acid to the resin. Next, the next amino acid is added in sequence and chemically reacted with an appropriate condensing agent. Finally, the target product can be obtained by cleaving the polypeptide from the resin.
The importance of solid-phase synthesis is that it enables the automation and large-scale production of peptide synthesis. The current mainstream solid-phase synthesis methods include Fmoc and Boc. Among them, the Fmoc method uses the N-Fmoc protecting group to protect the peptide, while the Boc method uses tert-butyloxycarbonyl to protect the carboxyl group.
Method two, liquid phase synthesis:
Liquid-phase synthesis is a traditional method of peptide synthesis in which the reactants are placed in the liquid phase for the reaction. The advantage of liquid-phase synthesis is that the reaction conditions are mild and suitable for the modification of sensitive chemical structures. However, due to too many reactants, the purification process is relatively cumbersome. Chemical reactions in liquid phase synthesis include:
1. Condensation reaction:
Condensation reaction is one of the most basic reactions in peptide synthesis, that is, the carboxyl group initiated by condensing agents such as DCC and HOBt is connected to the amino group of amino acid through acylation reaction. The reaction conditions are mild and the yield is high.
2. Elimination reactions:
The elimination reaction is to reduce the methionine to the dithiol by NaBH4 and other reducing agents, making it inactive. The reaction needs to be carried out under basic conditions.
3. Removal of protecting groups:
Due to the different functions of amino acids in the peptide chain, different protecting groups will be used for protection. After the synthesis is complete, the protecting group needs to be removed. For the Fmoc method, piperidine is usually used to remove Fmoc; while for the Boc method, TFA is used to remove Boc.
Method three, chemical synthesis:
GLP-1 is a polypeptide hormone with important biological activities. Its synthesis can be realized by various methods, among which chemical synthesis is one of the most commonly used methods. The advantage of chemical synthesis is that it can produce highly pure target products, which are suitable for large-scale production. The chemical synthesis method and detailed steps of GLP-1 will be introduced below.
1. Synthetic route and protecting group selection:
The GLP-1 molecule consists of 36 amino acids, including 21 L-type and 15 D-type amino acids. Before carrying out the synthesis, it is necessary to select a suitable synthetic route and select the corresponding protecting group according to the synthetic conditions. Fmoc solid-phase synthesis is usually used for automated large-scale synthesis. This method uses N-9-fluoroimido carboxyl protection (N-Fmoc) as a protecting group, and also needs to select an appropriate secondary protecting group (such as tert-butyl or methyl) to ensure the protection of specific sites. Each time a new amino acid is added, the Fmoc protecting group needs to be removed first, and then the protected coupling substance of the next amino acid is added.
2. Synthesis of the core amino acid sequence:
The core sequence of GLP-1 consists of 21 amino acids, including a key serine and four prolyl-glutamic acid dipeptide sequences. In solid-phase synthesis, the synthesis of the core sequence can be divided into the following steps:
2.1. Add acetic acid carbamate (Fmoc-NH-CH2CO2Et) and 2-Cl-Trt-Cl to solid-phase synthetic resin, and perform condensation reaction with DIC/NMM coupling agent.
2.2. Remove Fmoc protecting group by deprotecting group reaction.
2.3. Add the next amino acid, repeat step 1 and step 2 in sequence until the core sequence is synthesized.
2.4. Formation of pentapeptide structures on solid phase resin. Add the acetalization reagent to the solid-phase resin, react with the N-terminal recognition agent (such as HBTU), add the side chain protection group of serine as an auxiliary reducing agent, and then remove the Fmoc protection group.
2.5. Under the catalysis of Bacillus subtilis transferase (ProTide), the pentapeptide structure undergoes an exchange reaction with the precursor of serine iodoacetate.
3. Synthesis of the remaining amino acid sequence:
After completing the synthesis of the core sequence, it is necessary to continue to add the remaining amino acids, including L- and D-type amino acids. The addition of these amino acids needs to start from the core sequence, add the next amino acid in sequence, and use the corresponding condensing agent to carry out chemical reactions until a complete GLP-1 polypeptide molecule is synthesized. During this process, it is also necessary to select an appropriate protecting group as required, and to perform the steps of reaction, removal of protecting group and addition of amino acid in sequence.
4. Sodium hydroxide treatment:
After all the amino acids have been added, an incompletely synthesized peptide chain is formed on the solid-phase resin and needs to be processed to form a fully formed peptide molecule. Firstly, the unformed peptide should be hydrolyzed by sodium hydroxide, so that the C-terminal carboxyl group originally attached to the resin is detached from the resin, and the protective group is detached in water. After hydrolysis reaction, the target product is obtained.
5. Precipitation and washing:
After the treatment, the hydrolyzed solution is treated with acid to precipitate the target product. Next, the pellet was resuspended in water, followed by intensive washing to remove impurities.
6. Purification:
The final step is purification of the desired product, usually using high performance liquid chromatography. During this process, the purity of the product can be determined by detecting the peak of the solution in the mass spectrum. In short, the chemical synthesis of GLP-1 requires multiple rounds of complex reactions and strict purification processes to finally obtain the active target product.
Method four, biosynthesis:
GLP-1 is an important polypeptide hormone with various physiological effects, including promoting insulin secretion, suppressing appetite, reducing body weight and maintaining insulin sensitivity, etc. The biosynthesis method of GLP-1 is mainly synthesized by L cells in the pancreatic gland, and its synthesis rate is regulated with dietary intake. The detailed steps are introduced as follows:
1. Preparatory work before synthesis:
Before the biosynthesis of GLP-1, some preparatory work needs to be done, including determining the cell type used, setting the culture conditions and selecting the appropriate catalytic enzyme. L cells are the main source of GLP-1 synthesis because they contain precursors of two hormones, GIP (glucagon-like peptide 1) and GLP-1. L cells can be isolated from the intestinal epithelium of rabbits or mice. Before biosynthesis, cells need to be cultured to a sufficient number, and sufficient nutrients and suitable culture conditions should be provided. In addition, it is necessary to select the appropriate catalytic enzyme to promote the reaction.
2. Synthesis and processing of precursors:
The biosynthesis of GLP-1 mainly occurs in L cells, and its precursor is composed of two hormones, GIP and GLP-1. After entering endocrine cells, GIP and GLP-1 are processed by proteolytic enzymes and cleaved into individual peptides. A series of enzymes and cofactors are involved in this process, including precursor polypeptide acidase (PC2), isomerase, and late adhesion factors.
3. Mutual conversion between polypeptide segments:
After processing, the GIP and GLP-1 peptides are recombined to form the GLP-1 polypeptide. This process requires the use of glucagon-like peptide 1 (GLP-1) as a template to which other individual peptides are combined to form new composite polypeptides. This process also requires some specific enzymes and factors, including Prohormone Convertase 1/3 (PC1/3) and Carboxypeptidase E (CPE).
4. GLP-1 secretion:
After GLP-1 is synthesized and processed, it is stored in the cytoplasm and internal vesicles of endocrine cells. When stimulated by diet, endocrine cells release GLP-1 and enter blood circulation through microvessels. This process is regulated and controlled through a series of signal transduction pathways, including cAMP-Ca2+ and so on.
In short, the biosynthesis of GLP-1 involves the joint action of multiple links and factors. The combination of biosynthesis and chemical synthesis can provide a better foundation and support for the research and production of GLP-1.
Method five, enzymatic synthesis:
Enzymatic synthesis is the synthesis of peptide chains through the catalysis of biological enzymes. Compared with traditional liquid-phase synthesis methods, enzymatic synthesis can be carried out at room temperature, and a wide range of raw materials can be selected. Enzymes such as theta-liquid synthase, AEP, ACE, etc. are usually used to catalyze the synthesis.
In conclusion, the above-mentioned methods are feasible methods for GLP-1 synthesis. Different methods are suitable for different experimental conditions and pharmaceutical production environments.