Preparation of semaglutide & impurities by fragment method
A method for preparing semaglutide The invention adopts the solid-liquid phase combined with segment condensation scheme to synthesize the side chain protection peptide in solid phase, and the segment condensation in liquid phase to obtain the fully protected somaluptin, and then the crude somaluptin peptide is obtained by pyrolysis, and the somaluptin is purified and changed into salt; In order to reduce the by-products caused by racemization, the invention uses a pseudo-proline strategy.
The method of the invention reduces impurities, improves yield, shortens synthesis time, reduces waste liquid discharge, greatly reduces synthesis cost, and is very conducive to industrial production (fmoc-osu)
The method for preparing somaluptin by fragment condensation is characterized in that three side-chain protected peptide fragments are synthesized in solid phase, each peptide fragment is gradually coupled in a solution system to obtain fully protected somaluptin, and then the crude somaluptin peptide is obtained by cleavage and removal of the protective group, and somaluptin is obtained by purification and salt exchange
The sequence of the three peptide fragments is:
The first peptide fragment sequence is the 1-12 amino acid in the semaglutide sequence,
The second peptide fragment sequence is the 13-24 amino acid in the semaglutide sequence,
The third peptide fragment sequence is the 25-31 amino acid in the semaglutide sequence
The method for preparing semaglutide by fragment condensation is characterized in that it comprises the following steps:
(1) The first to third peptide fragments with side chain protection were synthesized in solid phase and cleaved from the resin;
(2) The carboxyl end of the third peptide segment with side chain protection is modified and protected, and its amino protection group is removed and the second peptide segment with side chain protection is coupled to obtain the fourth peptide segment with side chain protection;
(3) The amino protection group was removed from the fourth peptide segment of the side chain protection and coupled with the first peptide segment of the side chain protection to obtain the fully protected semaglutide ;
(4) The fully protected semaglutide was cleaved to remove the protective group to obtain crude semaglutide ;
(5) The crude semaglutide peptide was purified and replaced with salt to obtain semaglutide .
The method for preparing semaglutide by segment condensation according to claim 2, which is characterized in that in step (1), the first to third peptide segments of the side chain protection are obtained by successively coupling amino acids onto the solid carrier; Wherein, the solid phase carrier is acid-sensitive resin
Semaglutide impurity (exenatide impurities):
Fragment liquid phase coupling, its impurities are mainly uncoupled fragments, rather than defective peptides lacking one or several amino acids, and uncoupled fragments can be extracted and removed by appropriate solvent system, which is much easier in the final high performance liquid chromatography purification, thus reducing the number of preparation times and the preparation cost of semaglutide
Compared with the process of solid-phase fragment condensation to synthesize semaglutide , the present invention uses liquid-phase fragment condensation, the molar ratio of fragments is 0.95-1.05 times, which is much lower than the 2-5 times excess of solid-phase fragment condensation to synthesize fragments, saving material costs; The unreacted fragments can be removed by extraction through appropriate reaction system, and the post-treatment is simple and fast.
The sequence purity of the side chain protective peptide fragments of about 10 amino acids synthesized by super acid sensitive resin is very high, and it is not necessary to purify them by chromatography, but only needs to precipitate and grind them; Fragment liquid phase coupling, its impurity is mainly uncoupled fragment, rather than defective peptide lacking one or several amino acids, and uncoupled fragments can be extracted and removed by appropriate solvent system, which is much easier in the final high performance liquid chromatography purification, thus reducing the number of preparation times and the preparation cost of semaglutide .
In order to avoid and solve the problem of racemization during fragment condensation, we introduced Ser-Ser's pseudoproline strategy into the first peptide fragment, which well solved the problem of racemization at this site.
This method has the characteristics of high throughput, low cost, less waste liquid, high efficiency and easy purification, which is conducive to the realization of large-scale and industrialized production
Semaglutide is a new long-acting GLP-1 analog developed by Novo Nordisk. Its sequence is H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (AEEAc-AEEAc- γ- Glu-17-carboxyheptadecanoyl) - Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, molecular formula: C187H291N 45 O59. It can inhibit the hydrolysis of DPP-4 enzyme, prolong the biological half-life, reduce blood sugar for a long time, promote the regeneration of pancreatic islet cells, and is used to treat type 2 diabetes.