Method for preparing teriparatide by fragment method and solid-liquid combination

Abstract

The invention belongs to the field of polypeptide synthesis, relating to a method for preparing teriparatide by solid-liquid combination. The method adopts a liquid phase mode to synthesize a part of dipeptide and tripeptide fragments, feeding is performed by the synthesized dipeptide and tripetide fragments, 13 steps of solid phase coupled reaction are reduced, the coupling efficiency is greatly improved, not only is the generation of racemization impurities difficult to be purified and removed at the 34th site tail end avoided, but also the generation of missing peptides at multiple sites is avoided, and the purity of the final target peptide reaches 75 percent or more. Compared with the prior art, the method provided by the invention is simple in synthetic route, less in solid phase coupling steps, mainly solves the problems that a peptide sequence is too long and coupling is difficult, and that the missing peptides are easily generated, the fragment synthesis technology is well developed, the materiel cost is lowered, and the industrialized mass production can be carried out.

Description

technical field [0001] The invention relates to the field of polypeptide synthesis, in particular to a method for preparing teriparatide by solid-liquid combination of a fragment method. Background technique [0002] Teriparatide, whose English name is Teriparatide, is composed of 34 natural amino acids. It is a fragment of 1-34 amino acids in the N-terminal region of endogenous parathyroid hormone with biological activity. Its peptide sequence is: H-Ser-Val- Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys- Leu-Gln-Asp-Val-His-Asn-Phe-OH. [0003] Teriparatide was developed by Eli Lilly and Company, and it was first launched in the United States in December 2002. It is the first new bone-forming agent drug approved by the U.S. Food and Drug Administration. It is mainly used for the treatment of primary osteoporosis, women Postmenopausal osteoporosis and postmenopausal osteoporosis in men. The current conventional osteopo...

AI Technical Summary

Problems solved by technology

The original manufacturer Lilly obtained teriparatide through gene expression. In addition, there are many patents on gene expression, such as: US6590081. However, the gene expression method has a high technical threshold, complicated work, and three wastes. Serious and other defects

[0005] Another relatively common method for preparing teriparatide is solid-phase chemical synthesis. For example, in patent 201210213044.3, five peptide resin fragments of teriparatide are prepared first, and then each peptide resin fragment is prepared on the solid-state by fragment coupling. The teriparatide resin is gradually coupled to the teriparatide resin to synthesize the teriparatide resin, and finally the crude teriparatide is obtained by cleavage. This method of fragment coupling requires purification and lyophilization of the fragment peptides, which is complicated to operate. The cost is high; patent 201510005427.5 adopts the traditional solid-phase synthesis method to gradually synthesize teriparatide from the C-terminus of the peptide chain to the N-terminus. Although the operation is simple, it cannot solve the problem of difficult coupling of long peptides in the later stage, and the final target peptide purity Not high, difficult to purify; Patent No. 201410262511.0 adopts pseudo-proline dipeptide feeding in the 16-17 positions of the peptide chain, which avoids the production of special process impurities, but cannot avoid various missing peptides due to too long peptide chains ;Patent 201510295556.2 uses HMPLinker to link the teriparatide peptide chain with the resin, and the subsequent sites adopt a step-by-step coupling method, which also has the problem of too many impurities and difficult purification; patent 201310403743.9 adopts a step-by-step coupling method. The free hydroxyl group of the Ser position is connected to the α-carboxyl group of the 16-position Asn with an ester bond. After the synthesis of the peptide chain is completed, teriparatide is obtained through a one-step ester-amine conversion reaction in the solution, although the subsequent position can be reduced by changing the spatial configuration of the target peptide. Coupling is difficult, but there is still the problem of too many solid-phase coupling steps. At the same time, there is also a serious yield problem in the last step of esteramine conversion, which leads to an increase in the final cost.

[0006] Teriparatide is a polypeptide composed of 34 amino acids. From the perspective of solid-phase synthesis, the peptide chain is longer, and the coupling reaction will be more difficult with the extension of the peptide chain, and it is easy to produce more process impurities, which is not conducive to the later stage. purification

The existing solid-phase synthesis methods of teriparatide are mainly divided into two categories, one is to couple amino acids one by one, the other is to synthesize fully protected fragments through solid phase, and then synthesize the target peptide through fragment coupling. The first type of synthesis method cannot solve the problem of low purity of the target peptide due to the long peptide chain, and the second type of synthesis method increases the cost of materials due to the purification of fully protected fragments