Patent Description:
With the development of society, global incidence of diabetes, which has a significantly increasing trend, was estimated to be <NUM>% in <NUM> and is expected to be <NUM>% by <NUM>, and the number of diabetics will increase from <NUM> million in <NUM> to <NUM> million in <NUM>. Diabetes is divided into gestational diabetes, specific diabetes, type I diabetes, and type II diabetes. Type II diabetes, also known as non-insulin-dependent diabetes, is characterized by the fact that a human body itself can produce insulin, but cells are unable to respond to it, so that the effect of insulin is greatly compromised. There are many types of hypoglycemic agents for type II diabetes, including metformin, sulfonylureas, glucagon-like peptide-<NUM> (GLP-<NUM>) receptor agonist, and the like. The GLP-<NUM> receptor agonist is a hot topic in recent studies.

Liraglutide, one of human glucagon-like peptide-<NUM> (GLP-<NUM>) analogues, with an English name Liraglutide, is a drug developed by Novo Nordisk in Denmark for the treatment of type II diabetes, and its injection was approved under a trade name Victoza by the FDA on January <NUM>, <NUM>, and approved by the SFDA on March <NUM>, <NUM>. As a GLP-<NUM> receptor agonist, Liraglutide can play a good role in lowering blood glucose level.

At present, liraglutide is synthesized mainly by using a gene recombination technology and a stepwise coupling method. The synthesis of liraglutide, when is performed by the genetic recombination technique, has a relatively high technical difficulty and a relatively high cost, and an intermediate GLP-<NUM>(<NUM>-<NUM>)-OH needs to be repeatedly purified by HPLC and then reacted with Na-alkanoyl-Glu(ONSu)-OtBu under a liquid phase condition. Moreover, since the N-terminus of GLP-<NUM>(<NUM>-<NUM>)-OH is unprotected and the side chain protective groups of GLP-<NUM>(<NUM>-<NUM>)-OH are completely removed, many impurities are produced and Liraglutide is difficult to purify. As well known to those skilled in the art, the synthesis of liraglutide, when is performed by the stepwise coupling method, comprises: performing a condensation reaction of a resin as a solid phase carrier with Fmoc-Gly-OH to obtain Fmoc-Gly-resin; condensing, by solid-phase synthesis, amino acids with N-terminal Fmoc protection and side chain protection in sequence according to a main-chain peptide of liraglutide, wherein lysine is used in a form of Fmoc-Lys(X)-OH (X is a side chain protective group of Lys) or Fmoc-Lys (N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-OH and each condensation reaction is performed for <NUM> to <NUM>; cleaving a side chain of lysine in a liraglutide resin after modification or directly cleaving the side chain of lysine in the liraglutide resin; and purifying and lyophilizing to obtain liraglutide. Due to a long sequence of liraglutide and a high proportion of hydrophobic amino acids, in stepwise coupling, β-sheet is easily formed, resulting in severe shrinkage of the resin and prolongation of the reaction time, and further producing, among the crude peptide, more racemate impurity, namely, NH<NUM>-His-Ala-Glu-Gly-D-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-A la-Lys(N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-G ly-COOH (D-Thr<NUM> liraglutide), which has a property very close to that of the product, and making purification difficult; in addition, the resin shrinks severely and the reaction is incomplete, resulting in a lower yield.

On the one hand, the racemate by-product has a structure very similar to that of liraglutide, making much difficulty in purification and separation of the crude peptide of liraglutide, and no good effect can be achieved from separation although various purification separation systems have been tried. If multiple separations are performed, it can be foreseen that the separations will result in significant loss of the product. On the other hand, the racemate impurities have an adverse effect on the quality of the drug, namely, not only affecting stability and efficacy of the drug, but also being harmful to human health. Therefore, in the preparation process of synthesizing liraglutide, the production of racemate by-product should be minimized.

Different specific fragments are used to synthesize liraglutide. For example, <CIT> discloses a method for synthesizing liraglutide, wherein a peptide resin is obtained by coupling a dipeptide fragment, a tripeptide fragment or a combination thereof with an amino acid and a Gly-resin, which is obtained by side chain modification. <CIT> also discloses a method for synthesizing liraglutide similar to that of <CIT> via fragment condensation in solution.

Liraglutide also can be obtained via fragment condensation in solid phase or in solution of fragments obtained via Solid-Phase Peptide Synthesis, see <NPL>.

The main chain of liraglutide comprises <NUM> amino acids, and there are many forms of synthesis by fragment method, but only an appropriate fragment synthesizing method can ensure the production of less racemate by-product, and reduce the complexity of synthesis process, while guaranteeing the yield and purity of liraglutide. Through long-term experiments, inventors have surprisingly found that the synthesis of liraglutide using a method of the present disclosure allows a greatly reduced production of racemate impurity of liraglutide, namely, NH<NUM>-His-Ala-Glu-Gly-D-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-A la-Lys(N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-G ly-COOH (D-Thr<NUM> liraglutide), the method is simple and the yield will not be lowered.

It is an object of the present invention to provide a method for synthesizing liraglutide with a low racemate impurity.

A technical solution adopted by the present invention is:.

As a further improvement of the aforementioned synthesis method, during a process of synthesizing liraglutide, a dipeptide fragment, a tripeptide fragment, a tetrapeptide fragment, a pentapeptide fragment or a combination thereof is coupled to an amino acid and a Fmoc-Gly-resin to obtain the liraglutide resin.

As a further improvement of the aforementioned synthesis method, the combination includes Ala-Trp-Leu-Val-Arg, Glu-Phe-Ile, Ser-Asp-Val, Glu-Gly, and His-Ala.

The beneficial effects of the present invention are as follows:
Through long-term experiments, inventors have surprisingly found that when a <NUM>~<NUM> amino acid residue-containing peptide with Thr-Phe, i.e. Thr-Phe-Thr, is used in the process of synthesizing liraglutide, the production of the racemate impurity of liraglutide i.e., D-Thr<NUM> liraglutide, can be greatly reduced, while ensuring that the yield will not be lowered.

The method for synthesizing liraglutide provided by the present invention effectively inhibits or reduces the production of the racemate impurity with very similar properties to the product, namely, D-Thr<NUM> liraglutide. In the crude peptide of liraglutide prepared, the racemate impurity D-Thr<NUM> liraglutide has an amount of less than <NUM>% (w/w), which is advantageous for the purification of the crude peptide of liraglutide. In addition, The method of the present invention ensure a high yield and a greatly reduced production cost. In the process of synthesizing liraglutide, the synthesis of the dipeptide fragments, the tripeptide fragments, the tetrapeptide fragments, the pentapeptide fragments or combinations thereof with the Gly-resin can be carried out simultaneously, so that synthesis time is also shortened.

The method for synthesizing liraglutide of the present disclosure will be further described in detail below in conjunction with the specific examples so that those skilled in the art can further understand the present disclosure. The examples should not be construed as limiting the scope of protection.

Specific meanings of English abbreviations used are shown in Table <NUM>.

Protective group is those commonly used in the field of amino acid synthesis for protecting a group, such as an amino group, a carboxyl group, and the like, in a main chain and a side chain of an amino acid from interfering with synthesis, it prevents the group, such as the amino group, the carboxyl group and the like, from reacting and forming an impurity during preparation of a target product. For amino acids in the present disclosure that need to protect side chains, those skilled in the art are well aware of their side chain structures and the use of common protective groups to protect groups, such as an amino group, a carboxyl group and the like, on the side chain of the amino acid. Preferably, in the present disclosure, the side chains of histidine and glutamine are protected by Trt-protective group, the side chains of glutamic acid and aspartic acid are protected by OtBu-protective group, the side chain of tryptophan is protected by Boc-protective group, the side chains of threonine, serine, and tyrosine are protected by tBu-protective group, the side chain of lysine is protected by Alloc-protective group, and the side chain of arginine is protected by Pbf-protective group. In addition, for amino acids involved in the method of the present disclosure, the N-terminus of the amino acids is preferably protected by a Fmoc-protective group, and histidine can also be protected by a Boc-protective group.

The amino acids or peptides used in the present disclosure, particularly the dipeptide, tripeptide, tetrapeptide, pentapeptide, and the like, can be protected by using a protective group according to the requirement of synthesis.

The propeptide herein refers to a polypeptide fragment synthesized from the C-terminus to the N-terminus of the liraglutide peptide sequence in the synthesis of liraglutide, and doesn't contain the <NUM>~<NUM> amino acid residue containing peptide having Thr-Phe. The protective group can be coupled to the side chain of the propeptide. The propeptide can be obtained by custom synthesis (purchase) or synthesized by a known method. In particular, the propeptide is obtained by solid phase peptide synthesis.

The structure of the racemate impurity among the crude peptide of liraglutide herein is NH<NUM>-His-Ala-Glu-Gly-D-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-A la-Lys(N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-G ly-COOH, and is represented by D-Thr<NUM> liraglutide.

In the following examples, only example <NUM> is covered by the present invention, and the other examples are for illustrative purposes.

<NUM> of the crude peptide of liraglutide was obtained with a yield of <NUM>% and a purity of <NUM>%. The racemate impurity with a structure similar to that of liraglutide, i.e. D-Thr<NUM> liraglutide, was closely adjacent to the main peak, with a relative retention time of about <NUM> and an amount of <NUM>%. The HPLC spectrum was shown in <FIG>. The results of the retention time and peak area of characteristic peaks were shown in Table <NUM>.

<NUM> of the crude peptide of liraglutide was obtained with a yield of <NUM>% and a purity of <NUM>%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr<NUM> liraglutide, was closely adjacent to the main peak, with a relative retention time of about <NUM> and an amount of <NUM>%. The HPLC spectrum was similar to that shown in <FIG>.

H-Arg(pbf)-Gly-<NUM>-CTC resin was synthesized according to the method of Example <NUM>, and Fmoc-Gly-OH, Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-Phe-Ile-OH, Fmoc-Lys(N-ε-(Nα-Palmitoyl-L-γ-glutamyl-OtBu)), Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-Asp(OtBu)-Val-OH, Fmoc-Thr(tBu)-Phe-Thr(tBu)-OH, Fmoc-Glu(OtBu)-Gly-OH, and Boc-His(Trt)- Ala-OH were sequentially coupled to the H-Arg(pbf)-Gly-<NUM>-CTC resin. The remaining steps were referred to the steps <NUM>-<NUM> in example <NUM>, and the product was dried in vacuo to a constant weight.

H-Arg(pbf)-Gly-<NUM>-CTC resin was synthesized according to the method of example <NUM>, and Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Alloc)-Glu(OtBu)-Phe-Ile-Ala-OH, Fmoc-Ala-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, moc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-OH, Fmoc-Glu(OtBu)-Gly-OH, and Boc-His(Trt)-Ala-OH were sequentially coupled to the H-Arg(pbf)-Gly-<NUM>-CTC resin. The remaining steps were referred to the steps <NUM>-<NUM> in example <NUM>, and the product was dried in vacuo to a constant weight.

H-Arg(pbf)-Gly-<NUM>-CTC resin was synthesized according to the method of example <NUM>, and Fmoc-Gly-OH, Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH, Fmoc-Lys(Alloc)-Glu(OtBu)-Phe-Ile-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-Thr(tBu)-Phe-Thr(tBu)-OH, Fmoc-Glu(OtBu)-OH, and Boc-His(Trt)-Ala-OH were sequentially coupled to the H-Arg(pbf)-Gly-<NUM>-CTC resin. The remaining steps were referred to the steps <NUM>-<NUM> in example <NUM>, and the product was dried in vacuo to a constant weight.

H-Arg(pbf)-Gly-Wang resin was synthesized according to the method of example <NUM>, and Fmoc-Gly-OH, Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-Phe-Ile-OH, Fmoc-Lys(N-ε-(Nα-Palmitoyl-L-γ-glutamyl-OtBu)), Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)Gly-Thr(tBu)-Phe-Thr(tBu)-OH, and Boc-His(Trt)-Ala-OH were sequentially coupled to the H-Arg(pbf)-Gly-Wang resin. The remaining steps were referred to the steps <NUM>-<NUM> in example <NUM>, and the product was dried in vacuo to a constant weight.

H-Arg(pbf)-Gly-<NUM>-CTC resin was synthesized according to the method of example <NUM>, and Fmoc-Gly-OH, Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-Phe-Ile-OH, Fmoc-Lys(N-ε-(Nα-Palmitoyl-L-γ-glutamyl-OtBu)), Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, and Boc-His(Trt)-Ala-OH were sequentially coupled to the H-Arg(pbf)-Gly-<NUM>-CTC resin. The remaining steps were referred to the steps <NUM>-<NUM> in Example <NUM>, and the product was dried in vacuo to a constant weight.

<NUM> of the crude peptide of liraglutide was obtained with a yield of <NUM>% and a purity of <NUM>%. The racemate impurity having a structure similar to that of liraglutide, i.e., D-Thr<NUM> liraglutide, was closely adjacent to the main peak, with a relative retention time of about <NUM> and an amount of <NUM>%. The HPLC spectrum was similar to that shown in <FIG>.

<NUM> of the pure product of liraglutide was obtained with a purity of <NUM>% and a total yield of <NUM>%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr<NUM> liraglutide, was closely adjacent to the main peak, with a relative retention time of about <NUM> and an amount of <NUM>%. The HPLC spectrum was shown in <FIG>. The results of the retention time and peak area of characteristic peaks were shown in Table <NUM>.

The crude peptide of liraglutide prepared in Example <NUM> was purified by the same purification method as in Example <NUM>.

<NUM> of the pure product of liraglutide was obtained with a purity of <NUM>% and a total yield of <NUM>%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr<NUM> liraglutide, was closely adjacent to the main peak, with a relative retention time of about <NUM> and an amount of <NUM>%. The HPLC spectrum was similar to that shown in <FIG>.

The crude peptide of liraglutide prepared in Example <NUM> was purified by the same purification method as that in Example <NUM>.

The crude peptide of liraglutide has a yield of <NUM>% and a purity of <NUM>%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr<NUM> liraglutide, was closely adjacent to the main peak, with a relative retention time of about <NUM> and an amount of <NUM>%. The HPLC spectrum was shown in <FIG>. The results of the retention time and peak area of characteristic peaks were shown in Table <NUM>.

The crude peptide of liraglutide has a yield of <NUM>% and a purity of <NUM>%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr<NUM> liraglutide, was closely adjacent to the main peak, with a relative retention time of about <NUM> and an amount of <NUM>%. The HPLC spectrum was similar to that of <FIG>.

As can be seen from the HPLC spectra and the corresponding data of examples <NUM>-<NUM> and comparative examples <NUM> and <NUM>, the peak of racemate impurity, i.e., the peak of D-Thr<NUM> liraglutide, was closely adjacent to the main peak of liraglutide. Their retention times were <NUM> and <NUM> in example <NUM>, respectively and were <NUM> and <NUM> in comparative example <NUM>, respectively. The relative retention time of D-Thr<NUM> liraglutide was about <NUM>, which was far from the requirement of separation. If D-Thr<NUM> liraglutide is present in a large amount, it will be very difficult to purify and separate the crude peptide. However, compared with comparative examples <NUM> and <NUM>, the amount of D-Thr<NUM> liraglutide was reduced from respective <NUM>% and <NUM>% to <NUM>% in example <NUM>, relatively reducing by <NUM>% and <NUM>%, respectively. Compared with comparative examples <NUM> and <NUM>, the amount of D-Thr<NUM> liraglutide was reduced from respective <NUM>% and <NUM>% to <NUM>% in example <NUM>, relatively reducing by <NUM>% and <NUM>%, respectively. Compared with comparative examples <NUM> and <NUM>, the amount of D-Thr<NUM> liraglutide was reduced from respective <NUM>% and <NUM>% to <NUM>% in example <NUM>, relatively reducing by <NUM>% and <NUM>%, respectively. Compared with comparative examples <NUM> and <NUM>, the amount of D-Thr<NUM> liraglutide was reduced from respective <NUM>% and <NUM>% to <NUM>% in example <NUM>, relatively reducing by <NUM>% and <NUM>%, respectively. Compared with comparative examples <NUM> and <NUM>, the amount of D-Thr<NUM> liraglutide was reduced from respective <NUM>% and <NUM>% to <NUM>% in example <NUM>, relatively reducing by <NUM>% and <NUM>%, respectively. Compared with comparative examples <NUM> and <NUM>, the amount of D-Thr<NUM> liraglutide was reduced from respective <NUM>% and <NUM>% to <NUM>% in example <NUM>, relatively reducing by <NUM>% and <NUM>%, respectively. Compared with comparative examples <NUM> and <NUM>, the amount of D-Thr<NUM> liraglutide was reduced from respective <NUM>% and <NUM>% to <NUM>% in example <NUM>, relatively reducing by <NUM>% and <NUM>%, respectively. Compared with comparative examples <NUM> and <NUM>, the amount of D-Thr<NUM> liraglutide was reduced from respective <NUM>% and <NUM>% to <NUM>% in example <NUM>, relatively reducing by <NUM>% and <NUM>%, respectively. It can be seen from the above that the method for synthesizing liraglutide provided in the present disclosure can greatly reduce the amount of the racemate impurity, i.e., D-Thr<NUM> liraglutide, which produced in the synthesis of the crude peptide of liraglutide. D-Thr<NUM> liraglutide is in an amount of less than <NUM>%, which is very advantageous for purification.

In addition, liraglutide in example <NUM> has a yield of <NUM>% and a purity of <NUM>%, which were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively, and were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively. Liraglutide in example <NUM> has a yield of <NUM>% and a purity of <NUM>%, which were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively, and were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively. Liraglutide in example <NUM> has a yield of <NUM>% and a purity of <NUM>%, which were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively, and were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively. Liraglutide in example <NUM> has a yield of <NUM>% and a purity of <NUM>%, which were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively, and were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively. Liraglutide in example <NUM> has a yield of <NUM>% and a purity of <NUM>%, which were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively, and were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively. Liraglutide in example <NUM> has a yield of <NUM>% and a purity of <NUM>%, which were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively, and were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively. Liraglutide in example <NUM> has a yield of <NUM>% and a purity of <NUM>%, which were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively, and were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively. Liraglutide in example <NUM> has a yield of <NUM>% and a purity of <NUM>%, which were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively, and were <NUM>% and <NUM>% higher than those of comparative example <NUM>, respectively. It can be seen that the method for synthesizing liraglutide provided by the present disclosure can improve the yield and purity of the crude peptide of liraglutide, which is advantageous for purification.

The method of the present disclosure can greatly reduce the racemate impurity, i.e., D-Thr<NUM> liraglutide which produced during the synthesis of liraglutide, while the yield is not lowered. It is advantageous for purifying the crude peptide of liraglutide to obtain the refined peptide.

The crude peptide of liraglutide prepared in comparative example <NUM> was purified according to the above purification method of Example <NUM> to obtain <NUM> of the pure product of liraglutide with a purity of <NUM>% and a total yield of <NUM>%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr<NUM> liraglutide, was closely adjacent to the main peak, with a relative retention time of about <NUM> and an amount of <NUM>%. The HPLC spectrum was shown in <FIG>. The results of the retention time and peak area of characteristic peaks were shown in Table <NUM>.

The crude peptide of liraglutide prepared in comparative example <NUM> was purified according to the above purification method of Example <NUM> to obtain <NUM> of the pure product of liraglutide with a purity of <NUM>% and a total yield of <NUM>%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr<NUM> liraglutide, was closely adjacent to the main peak, with a relative retention time of about <NUM> and an amount of <NUM>%. The HPLC spectrum was similar to that shown in <FIG>.

As can be seen from the HPLC spectra of examples <NUM> to <NUM> and comparative example <NUM>, after simple purification steps, D-Thr<NUM> liraglutide among liraglutide had been substantially removed, and its maximum amount is only <NUM>% in the refined peptide. However, D-Thr<NUM> liraglutide in the comparative examples is more difficult to remove, which has an amount as high as <NUM>% in the refined peptide and is larger than that in the crude peptide prepared by the method of the present disclosure. It can be foreseen that if further purification is carried out in order to reduce the amount of racemate impurity D-Thr<NUM> liraglutide, the lower original yield will be further lowered.

It can be seen that the method of the present disclosure substantially reduces the production of the racemate impurity D-Thr<NUM> liraglutide in the synthesis of liraglutide and ensures the yield of liraglutide. It is advantageous for purifying to obtain the refined peptide of liraglutide with a low content of D-Thr<NUM> liraglutide.

Claim 1:
A process for synthesizing liraglutide with a low racemate impurity comprising:
synthesizing to obtain a propeptide, and then coupling a <NUM>~<NUM> amino acid residue-containing peptide with Thr-Phe to the propeptide by using solid-phase synthesis,
further performing a solid-phase synthesis to obtain a liraglutide resin, cleaving the liraglutide resin after side chain modification, or directly cleaving the liraglutide resin, purifying, and lyophilizing to give liraglutide,
wherein the propeptide is Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly,
wherein the <NUM>~<NUM> amino acid residue-containing peptide with Thr-Phe is Thr-Phe-Thr.