Synthesis method for liraglutide with low racemate impurity
11518794 · 2022-12-06
Assignee
Inventors
- Yuqing Fu (Shenzhen, CN)
- Hongji Ma (Shenzhen, CN)
- Xinyu Li (Shenzhen, CN)
- Lixiang Zhang (Shenzhen, CN)
- Qin Zhi (Shenzhen, CN)
- Lifen Wu (Shenzhen, CN)
- Zicheng Liu (Shenzhen, CN)
Cpc classification
International classification
Abstract
A synthesis method for low-racemization impurity liraglutide comprises the following steps: performing synthesis to obtain a propeptide, coupling 2 to 5 peptides comprising Thr-Phe on the propeptide by using a solid-phase synthesis method; further, performing solid-phase synthesis to obtain a liraglutide resin; the liraglutide resin is cracked after modification, or the liraglutide resin is directly cracked, purified and frozen dry, so as to obtain the liraglutide. The provided liraglutide synthesis method effectively restrains or reduces the generation of racemization impurity D-Thr.sup.5 highly similar to a product property, which facilitates the purification of the coarse liraglutide, and the high yield of the liraglutide is ensured, thereby greatly reducing production costs; during the synthesis of the liraglutide, the syntheses between dipeptide fragments, tripeptide fragments, the tetrapeptide fragments and pentapeptide fragments and the Gly-resin or the syntheses between the combination of the dipeptide fragments, the tripeptide fragments, the tetrapeptide fragments and pentapeptide fragments and the Gly resin can be carried out at the same time, and accordingly the synthesis time is shortened to some extent.
Claims
1. A process for synthesizing liraglutide with a low racemate impurity comprising: synthesizing to obtain a propeptide, and then coupling a 2˜5 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 2˜5 amino acid residue-containing peptide with Thr-Phe is selected from the group consisting of Thr-Phe, Gly-Thr-Phe, Thr-Phe-Thr, Gly-Thr-Phe-Thr (SEQ ID NO:2), Glu-Gly-Thr-Phe (SEQ ID NO:3), Ala-Glu-Gly-Thr-Phe (SEQ ID NO:5), Glu-Gly-Thr-Phe-Thr (SEQ ID NO:6), and Gly-Thr-Phe-Thr-Ser (SEQ ID NO:7).
2. The process according to claim 1, wherein pentapeptide Glu-Phe-Ile-Ala-Trp (SEQ ID NO:9) is used during the process of synthesizing the propeptide.
3. The process according to claim 1, wherein during the 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; wherein: the dipeptide fragment is selected from the group consisting of His-Ala, Ala-Glu, Glu-Gly, Thr-Ser, and Ala-Ala; the tripeptide fragment is selected from the group consisting of Glu-Phe-Ile, Ser-Asp-Val, and Thr-Ser-Asp; the tetrapeptide fragment is selected from the group consisting of Lys-Glu-Phe-Ile (SEQ ID NO:10) and Glu-Phe-Ile-Ala (SEQ ID NO:11); the pentapeptide fragment is selected from the group consisting of Glu-Phe-Ile-Ala-Trp (SEQ ID NO:12), Ala-Lys-Glu-Phe-Ile (SEQ ID NO:13), Lys-Glu-Phe-Ile-Ala (SEQ ID NO:14), and Ala-Trp-Leu-Val-Arg (SEQ ID NO:15); and two peptide fragments are not used in combination when a same amino acid residue is present at respective ends of the two peptide fragments to be coupled with each other.
4. The process according to claim 3, wherein the combination comprises a combination of one of Glu-Phe-Ile, Lys-Glu-Phe-Ile (SEQ ID NO:10), and Ala-Lys-Glu-Phe-Ile (SEQ ID NO:13) with Ala-Trp-Leu-Val-Arg (SEQ ID NO:15).
5. The process according to claim 1, wherein the 2˜5 amino acid residue-containing peptide with Thr-Phe is selected from one of Thr-Phe, Gly-Thr-Phe and Thr-Phe-Thr.
6. The process according to claim 3, wherein the combination comprises Ala-Trp-Leu-Val-Arg (SEQ ID NO:15), Ala-Lys-Glu-Phe-Ile (SEQ ID NO:13), Ser-Asp-Val, and Glu-Gly.
7. The process according to claim 3, wherein the combination comprises Glu-Phe-Ile-Ala-Trp (SEQ ID NO:12), Ala-Ala, Ser-Asp-Val, and Ala-Glu.
8. The process according to claim 3, wherein the combination comprises Ala-Trp-Leu-Val-Arg (SEQ ID NO:15), Glu-Phe-Ile, Thr-Ser-Asp, and Ala-Glu.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
DETAILED DESCRIPTION OF THE EMBODIMENTS
(5) 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.
(6) Specific meanings of English abbreviations used are shown in Table 1.
(7) TABLE-US-00001 TABLE 1 Specific meanings of the English abbreviations used in the specification and claims English English abbreviation Meaning abbreviation Meaning DIC N,N′-diisopropylcarbodiimide DIPEA N,N′-diisopropylethylamine HONb N-hydroxy-5-norbornene-2,3-dicarboximide HOBT 1-hydroxybenzotriazole DCC N,N′-dicyclohexylcarbodiimide TFA trifluoroacetic acid DCM dichloromethane EDT ethanedithiol Et.sub.2O diethyl ether DMF N,N′-dimethylformamide 20% DBLK 20% hexahydropyridine (v)/N,N′- TFE trifluoroethanol dimethylformamide (v) Anisole anisole EA ethyl acetate TIS triisopropylsilane H.sub.2O water THF tetrahydrofuran NaHCO.sub.3 sodium bicarbonate PIP hexahydropyridine HBTU benzotriazole-N,N,N′,N′- DMAP 4-dimethylaminopyridine tetramethyluronium hexafluorophosphate PyBOP benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate
(8) 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.
(9) 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.
(10) 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 2˜5 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.
(11) The structure of the racemate impurity among the crude peptide of liraglutide herein is NH.sub.2—His-Ala-Glu-Gly-D-Thr-Phe-Thr-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-COOH (SEQ ID NO:1), and is represented by D-Thr.sup.5 liraglutide.
Example 1
(12) 1) 4.25 g of Fmoc-Gly-Wang resin (0.279 mmol/g) was added to a solid-phase reaction column, and washed twice with DMF. The resin was swelled with DMF for 30 min, deprotected with DBLK (5 min plus 7 min), and washed with DMF for 6 times. The ninhydrin test was positive.
(13) 2. Fmoc-Arg(pbf)-OH (3.245 g, 5 mmol) and HOBt (0.426 g, 3.15 mmol) were dissolved in 15 mL of DMF, and DIC (0.49 mL, 3.15 mmol) was added in an ice bath for activation for 5 minutes. The activated solution was added into the above solid-phase reaction column, and reacted under nitrogen for 2 h with stirring. The ninhydrin test was negative, and the Fmoc-Arg(pbf)-Gly-Wang resin was obtained. The reaction solution was drained. The resin was washed with DMF for 3 times, deprotected with DBLK (5 min plus 7 min), and washed with DMF for 6 times. The ninhydrin test was negative, and H-Arg(pbf)-Gly-Wang resin was obtained.
(14) 3. Fmoc-Gly-OH, Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH (SEQ ID NO:16), Fmoc-Ala-Lys(Alloc)-Glu(OtBu)-Phe-Ile-OH (SEQ ID NO:17), 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)-OH, Fmoc-Thr(tBu)-Phe-OH, and Fmoc-Glu(OtBu)-Gly-OH were sequentially coupled to the H-Arg(pbf)-Gly-Wang resin.
(15) 4. Boc-His(Trt)-Ala-OH (2.89 g, 5 mmol) and HOBt (0.426 g, 3.15 mmol) were dissolved in 15 mL of DMF, and DIC (0.49 mL, 3.15 mmol) was added in an ice bath for activation for 5 minutes. The activated solution was added into the above solid-phase reaction column, and reacted under nitrogen for 2 h with stirring. The ninhydrin test was negative. The resin was washed with DMF for 4 times and washed with DCM twice.
(16) 5. To the above solid-phase reaction column, 15 mL of DCM and 1.08 g of phenylsilane were added, followed by 0.289 g of tetrakis(triphenylphosphine)palladium after stirring under nitrogen for 1 minute. The solution was reacted for 0.5 h, and then drained, and the resin was washed with DCM for 6 times. The ninhydrin test was positive.
(17) 6. Fmoc-Glu-OtBu (2.128 g, 5 mmol), HOBt (0.709 g, 5.25 mmol), and PyBOP (2.602 g, 5 mmol) were dissolved in 25 mL of DMF. DIPEA (1.75 mL, 10 mmol) was added in an ice bath for activation for 3 minutes. The activated solution was added into the above solid-phase reaction column, and reacted under nitrogen for 2 h with stirring. The ninhydrin test was negative. The solution was drained, and the resin was washed with DMF for 4 times and washed with DCM twice.
(18) 7) 20 mL of DCM and DIPEA (1.75 mL, 10 mmol) were added into the above solid-phase reaction column. After thoroughly stirring under nitrogen, palmitoyl chloride (1.374 g, 5 mmol) was slowly added dropwise. After adding, the reaction was continued for 2 h. The ninhydrin test was negative. The solution was drained. The resin was washed with DCM for 6 times, shrunk with MeOH, and dried in vacuo to obtain 9.43 g of peptide resin.
(19) 8) 9.43 g of the liraglutide resin obtained above was added into the mixed acid hydrolysate (containing 10 mL/g liraglutide resin) with a volume ratio of TFA:water:EDT=90:5:5. The reaction solution was stirred thoroughly, and reacted at room temperature for 3 h with stirring. The reaction mixture was filtered through a sand core funnel, and the filtrate was collected. The resin was washed with a small amount of TFA for three times. The filtrate was combined, and then concentrated in vacuo, and anhydrous ethyl ether was added for precipitation. Then, the precipitate was washed with anhydrous ethyl ether for three times, and the solution was drained to obtain an off-white powder. The off-white powder was dried in vacuo to a constant weight.
(20) 4.25 g of the crude peptide of liraglutide was obtained with a yield of 91.2% and a purity of 70.61%. The racemate impurity with a structure similar to that of liraglutide, i.e. D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.74%. The HPLC spectrum was shown in
(21) TABLE-US-00002 TABLE 2 Results of retention time and peak area of characteristic peaks of the crude peptide of liraglutide of example 1 Ser. Retention time Peak area Peak area No. (min) (AU*s) ratio % 1 32.617 9624 0.14 2 37.329 16327 0.24 3 38.844 4797 0.07 4 41.144 9029 0.13 5 42.127 42365 0.62 6 43.029 24319 0.36 7 43.707 26910 0.40 8 44.428 22402 0.33 9 45.251 49349 0.72 10 45.979 38278 0.56 11 46.375 21074 0.31 12 47.375 18961 0.28 13 47.713 27321 0.40 14 49.288 4809989 70.61 15 49.875 50079 0.74 16 50.942 38263 0.56 17 51.307 99047 1.45 18 52.647 292573 4.29 19 53.233 8892 0.13 20 54.267 39871 0.59 21 54.748 49558 0.73 22 55.367 8979 0.13 23 56.982 7451 0.11 24 57.190 6251 0.09 25 58.640 14643 0.21 26 59.986 30740 0.45 27 61.146 7473 0.11 28 62.893 185727 2.73 29 63.405 247896 3.64 30 64.588 29880 0.44 65.895 8106 0.12
Example 2
(22) 1) H-Arg(pbf)-Gly-Wang resin was synthesized according to the method of Example 1, and Fmoc-Gly-OH, Fmoc-Arg(pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH (SEQ ID NO:18), Fmoc-Lys(Alloc)-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-Ser(tBu)-Asp(OtBu)-Val-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, Fmoc-Gly-OH, and Fmoc-Ala-Glu(OtBu)-OH were sequentially coupled to the H-Arg(pbf)-Gly-Wang resin.
(23) 2. Boc-His(Trt)-OH (2.49 g, 5 mmol) and HOBt (0.426 g, 3.15 mmol) were dissolved in 15 mL of DMF, and DIC (0.49 mL, 3.15 mmol) was added in an ice bath for activation for 5 minutes. The activated solution was added into the solid-phase reaction column, and stirred under nitrogen for 2 h. The ninhydrin test was negative. The resin was washed with DMF for 4 times and washed with DCM twice. The remaining steps were referred to the steps 5-8 in example 1, and the product was dried in vacuo to a constant weight.
(24) 4.34 g of the crude peptide of liraglutide was obtained with a yield of 90.2% and a purity of 71.68%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.71%. The HPLC spectrum was similar to that shown in
Example 3
(25) 1) 3.58 g of Fmoc-Gly-2-CTC resin (0.279 mmol/g) was added to a solid-phase reaction column, and washed twice with DMF. The resin was swelled with DMF for 30 min, deprotected with DBLK (5 min plus 7 min), and washed with DMF for 6 times. The ninhydrin test was positive.
(26) 2. Fmoc-Arg(pbf)-OH (3.245 g, 5 mmol) and HOBt (0.426 g, 3.15 mmol) were dissolved in 15 mL of DMF, and DIC (0.49 mL, 3.15 mmol) was added in an ice bath for activation for 5 minutes. The activated solution was added into the above solid-phase reaction column, and reacted under nitrogen for 2 h with stirring. The ninhydrin test was negative, and Fmoc-Arg(pbf)-Gly-2-CTC resin was obtained. The reaction solution was drained. The resin was washed with DMF for 3 times, deprotected with DBLK (5 min plus 7 min), and washed with DMF for 6 times. The ninhydrin test was negative, and H-Arg(pbf)-Gly-2-CTC resin was obtained.
(27) 3. Fmoc-Gly-OH, Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH (SEQ ID NO:16), 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-Thr(tBu)-Ser(tBu)-Asp(OtBu)-OH, Fmoc-Gly-Thr(tBu)-Phe-OH, Fmoc-Ala-Glu(OtBu)-OH, and Boc-His(Trt)-OH were sequentially coupled to the H-Arg(pbf)-Gly-2-CTC resin.
(28) 4. Boc-protective group was removed in 10 mL of 50% TFA/DCM solution per gram of the resin for 20 min. Then the resin was washed with DCM for 6 times, and dried in vacuo to obtain 9.21 g of peptide resin.
(29) 5. The peptide resin was added into a mixed acid hydrolysate (containing 10 mL/g liraglutide resin) with a volume ratio of TFA:Anisole:TIS:H.sub.2O:EDT=92:2:2:2:2. The solution was stirred thoroughly, and reacted at room temperature for 3 h with stirring. The reaction mixture was filtered through a sand core funnel, and the filtrate was collected. The resin was washed with a small amount of TFA for three times. The filtrate was combined, and then concentrated in vacuo, and anhydrous ether was added for precipitation. Then, the precipitate was washed with anhydrous ethyl ether for three times, and the solution was drained to obtain an off-white powder. The off-white powder was dried in vacuo to a constant weight.
(30) 4.31 g of the crude peptide of liraglutide was obtained with a yield of 90.5% and a purity of 72.02%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.70%. The HPLC spectrum was similar to that shown in
Example 4
(31) H-Arg(pbf)-Gly-2-CTC resin was synthesized according to the method of Example 3, and Fmoc-Gly-OH, Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH (SEQ ID NO:16), 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-2-CTC resin. The remaining steps were referred to the steps 4-5 in example 3, and the product was dried in vacuo to a constant weight.
(32) 4.41 g of the crude peptide of liraglutide was obtained with a yield of 91.5% and a purity of 72.72%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.69%. The HPLC spectrum was similar to that shown in
Example 5
(33) H-Arg(pbf)-Gly-2-CTC resin was synthesized according to the method of example 3, 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 (SEQ ID NO:19), 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, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-OH (SEQ ID NO:20), Fmoc-Glu(OtBu)-Gly-OH, and Boc-His(Trt)-Ala-OH were sequentially coupled to the H-Arg(pbf)-Gly-2-CTC resin. The remaining steps were referred to the steps 4-5 in example 3, and the product was dried in vacuo to a constant weight.
(34) 4.50 g of the crude peptide of liraglutide was obtained with a yield of 91.8% and a purity of 70.72%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.70%. The HPLC spectrum was similar to that shown in
Example 6
(35) H-Arg(pbf)-Gly-2-CTC resin was synthesized according to the method of example 3, and Fmoc-Gly-OH, Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH (SEQ ID NO:16), Fmoc-Lys(Alloc)-Glu(OtBu)-Phe-Ile-OH (SEQ ID NO:21), 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 (SEQ ID NO:22), Fmoc-Glu(OtBu)-OH, and Boc-His(Trt)-Ala-OH were sequentially coupled to the H-Arg(pbf)-Gly-2-CTC resin. The remaining steps were referred to the steps 5-8 in example 1, and the product was dried in vacuo to a constant weight.
(36) 4.50 g of the crude peptide of liraglutide was obtained with a yield of 91.5% and a purity of 71.53%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.71%. The HPLC spectrum was similar to that shown in
Example 7
(37) H-Arg(pbf)-Gly-Wang resin was synthesized according to the method of example 1, and Fmoc-Gly-OH, Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH (SEQ ID NO:16), 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 (SEQ ID NO:23), 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 4-5 in example 3, and the product was dried in vacuo to a constant weight.
(38) 4.40 g of the crude peptide of liraglutide was obtained with a yield of 90.6% and a purity of 72.03%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.70%. The HPLC spectrum was similar to that shown in
Example 8
(39) H-Arg(pbf)-Gly-2-CTC resin was synthesized according to the method of example 3, and Fmoc-Gly-OH, Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH (SEQ ID NO:16), 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 (SEQ ID NO:24), Fmoc-Glu(OtBu)-OH, and Boc-His(Trt)-Ala-OH were sequentially coupled to the H-Arg(pbf)-Gly-2-CTC resin. The remaining steps were referred to the steps 4-5 in Example 3, and the product was dried in vacuo to a constant weight.
(40) 4.52 g of the crude peptide of liraglutide was obtained with a yield of 90.8% and a purity of 70.36%. The racemate impurity having a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.71%. The HPLC spectrum was similar to that shown in
Example 9. Preparation of Refined Peptide of Liraglutide
(41) 1) Water was added to the crude liraglutide obtained in Example 1, stirred, and adjusted to pH 8.5 with ammonia water until the crude liraglutide was completely dissolved. The solution was filtered through a 0.45 m microporous membrane and purified for use.
(42) 2. Purification was performed by high performance liquid chromatography, by a column (50 mm*250 mm) with 10 m reverse phase C18 chromatographic packing, a mobile phase system of 0.1% TFA/aqueous solution −0.1% TFA/acetonitrile solution, and a flow rate of 90 mL/min for purification. Purification was performed by gradient system elution and cyclic sampling. A solution of the crude product was added to the column, and elution was run with a mobile phase. The main peak was collected, and acetonitrile was evaporated to obtain a concentrated solution of a purified intermediate of liraglutide. The concentrated solution of the purified intermediate of liraglutide was filtered through 0.45 μm microporous membrane for use.
(43) 3. Salt exchange was performed by high performance liquid chromatography, by a mobile phase system of 1% acetic acid/aqueous solution acetonitrile, a column (77 mm*250 mm) with 10 m reverse phase C18 chromatographic packing, and a flow rate of 90 mL/min for purification. The process was performed by gradient system elution and cyclic sampling. The sample was added to the column, and elution was run with a mobile phase. Spectrum was acquisited and changes in absorbance was observed. The salt-exchanged main peak was collected and detected for purity by analytical liquid phase. The solution of salt-exchanged main peak was combined and concentrated in vacuo to obtain an aqueous acetic acid solution of liraglutide for use.
(44) 4. Purification and then desalting was performed by HPLC. The chromatographic column was set as follows: stationary phase: octadecylsilane-bonded silica gel; diameter and length of the column: 150 mm×250 mm; phase A: aqueous solution of 0.06% ammonia water; phase B: chromatographic grade acetonitrile; flow rate: 480 ml/min; gradient: 32% B-65% B; detection wavelength: 275 nm. The purified solution was concentrated by rotary evaporation, and lyophilized.
(45) 1.17 g of the pure product of liraglutide was obtained with a purity of 99.27% and a total yield of 25.12%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.27%. The HPLC spectrum was shown in
(46) TABLE-US-00003 TABLE 3 Results of retention time and peak area of characteristic peaks of liraglutide of Example 9 Ser. Retention time Peak area Peak area No. (min) (AU*s) ratio % 1 48.626 7474 0.15 2 50.387 5525 0.11 3 52.099 4908123 99.27 4 52.775 13237 0.27 5 53.709 7596 0.15 6 58.002 2237 0.05
Example 10. Preparation of Refined Peptide of Liraglutide
(47) The crude peptide of liraglutide prepared in Example 2 was purified by the same purification method as in Example 9.
(48) 1.15 g of the pure product of liraglutide was obtained with a purity of 99.31% and a total yield of 23.14%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.25%. The HPLC spectrum was similar to that shown in
Example 11. Preparation of Refined Peptide of Liraglutide
(49) The crude peptide of liraglutide prepared in Example 3 was purified by the same purification method as that in Example 9.
(50) 1.16 g of the pure product of liraglutide was obtained with a purity of 99.23% and a total yield of 24.14%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.26%. The HPLC spectrum was similar to that shown in
Example 12. Preparation of Refined Peptide of Liraglutide
(51) The crude peptide of liraglutide prepared in Example 4 was purified by the same purification method as that in Example 9.
(52) 1.19 g of the pure product of liraglutide was obtained with a purity of 99.36% and a total yield of 24.36%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.27%. The HPLC spectrum was similar to that shown in
Example 13. Preparation of Refined Peptide of Liraglutide
(53) The crude peptide of liraglutide prepared in Example 5 was purified by the same purification method as that in Example 9.
(54) 1.23 g of the pure product of liraglutide was obtained with a purity of 99.48% and a total yield of 25.14%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.26%. The HPLC spectrum was similar to that shown in
Example 14. Preparation of Refined Peptide of Liraglutide
(55) The crude peptide of liraglutide prepared in Example 6 was purified by the same purification method as that in Example 9.
(56) 1.21 g of the pure product of liraglutide was obtained with a purity of 99.50% and a total yield of 24.65%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.27%. The HPLC spectrum was similar to that shown in
Example 15. Preparation of Refined Peptide of Liraglutide
(57) The crude peptide of liraglutide prepared in Example 7 was purified by the same purification method as that in Example 9.
(58) 1.14 g of the pure product of liraglutide was obtained with a purity of 99.51% and a total yield of 23.46%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.26%. The HPLC spectrum was similar to that shown in
Example 16. Preparation of Refined Peptide of Liraglutide
(59) The crude peptide of liraglutide prepared in Example 8 was purified by the same purification method as that in Example 9.
(60) 1.21 g of the pure product of liraglutide was obtained with a purity of 99.48% and a total yield of 24.12%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 0.25%. The HPLC spectrum was similar to that shown in
Comparative Example 1. Preparation of Crude Peptide of Liraglutide
(61) 1) 4.25 g of Fmoc-Gly-Wang resin (0.279 mmol/g) was added to a solid-phase reaction column, and washed twice with DMF. The resin was swelled with DMF for 30 min, deprotected with DBLK (5 min plus 7 min), and washed with DMF for 6 times. The ninhydrin test was positive.
(62) 2. Fmoc-Arg(pbf)-OH (3.245 g, 5 mmol) and HOBt (0.426 g, 3.15 mmol) were dissolved in 15 mL of DMF, and DIC (0.49 mL, 3.15 mmol) was added in an ice bath for activation for 5 minutes. The activated solution was added into the above solid-phase reaction column, and reacted under nitrogen for 2 h with stirring. The ninhydrin test was negative, and Fmoc-Arg(pbf)-Gly-Wang resin was obtained. The reaction solution was drained. The resin was washed with DMF for 3 times, deprotected with DBLK (5 min plus 7 min), and washed with DMF for 6 times. The ninhydrin test was negative, and H-Arg(pbf)-Gly-Wang resin was obtained.
(63) 3. Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Alloc)-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-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, and Boc-His(Trt)-OH were sequentially coupled to the H-Arg(pbf)-Gly-Wang resin according to the above Fmoc-Arg(pbf)-OH coupling method.
(64) 4) 15 mL of DCM and 1.08 g of phenylsilane were added to the above solid-phase reaction column, followed by 0.289 g of tetrakis(triphenylphosphine)palladium after stirring under nitrogen for 1 minute. The solution was reacted for 0.5 h, drained, and the resin was washed with DCM for 6 times. The ninhydrin test was positive.
(65) 5. Fmoc-Glu-OtBu (2.128 g, 5 mmol), HOBt (0.709 g, 5.25 mmol), and PyBOP (2.602 g, 5 mmol) were dissolved in 25 mL of DMF, and DIPEA (1.75 mL, 10 mmol) was added in an ice bath for activation for 3 minutes. The activated solution was added into the above solid-phase reaction column, and reacted under nitrogen for 2 h with stirring. The ninhydrin test was negative. The solution was drained, and the resin was washed with DMF for 4 times and washed with DCM twice.
(66) 6) 20 mL of DCM and DIPEA (1.75 mL, 10 mmol) were added into the above solid-phase reaction column. After thoroughly stirring under nitrogen, palmitoyl chloride (1.374 g, 5 mmol) was slowly added dropwise. After adding, the reaction was continued for 2 h. The ninhydrin test was negative. The solution was drained. The resin was washed with DCM for 6 times, shrunk with MeOH, and dried in vacuo to obtain 7.46 g of peptide resin.
(67) 7. A mixed acid hydrolysate (containing 10 mL/g liraglutide resin) having a volume ratio of TFA:water:EDT=90:5:5 was added to a round-bottom flask with 7.46 g of the peptide resin of liraglutide. The solution was stirred thoroughly, and reacted at room temperature for 3 h with stirring. The reaction mixture was filtered through a sand core funnel, and the filtrate was collected. The resin was washed with a small amount of TFA for three times. The filtrates were combined, and then concentrated in vacuo, and anhydrous ethyl ether was added for precipitation. Then, the precipitate was washed with anhydrous ethyl ether for three times, and the solution was drained to obtain an off-white powder. The off-white powder was dried in vacuo to a constant weight to obtain 3.68 g of the crude peptide of liraglutide.
(68) The crude peptide of liraglutide has a yield of 80.5% and a purity of 68.63%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 2.39%. The HPLC spectrum was shown in
(69) TABLE-US-00004 TABLE 4 Results of retention time and peak area of characteristic peaks of the crude peptide of liraglutide of Comparative Example 1 Ser. Retention time Peak area Peak area No. (min) (AU*s) ratio % 1 34.447 43379 0.39 2 35.325 13387 0.12 3 36.185 40142 0.36 4 37.324 14414 0.13 5 38.160 12441 0.11 6 39.443 7592 0.07 7 39.981 13136 0.12 8 41.167 45452 0.41 9 41.459 105145 0.94 10 42.465 27602 0.25 11 43.168 51138 0.46 12 44.602 178039 1.59 13 45.370 92060 0.82 14 46.506 182605 1.64 15 48.253 7661983 68.63 16 48.835 266500 2.39 17 49.758 87000 0.78 18 50.127 290537 2.60 19 51.434 166104 1.49 20 53.008 41115 0.37 21 53.573 129362 1.16 22 56.210 38923 0.35 23 59.583 24725 0.22 24 59.741 29094 0.26 25 61.502 269424 2.41 26 62.509 76005 0.68 27 63.372 58826 0.53 28 64.737 63926 0.57
Comparative Example 2. Preparation of Crude Peptide of Liraglutide
(70) 1) 3.58 g of Fmoc-Gly-2-CTC resin (0.279 mmol/g) was added to a solid-phase reaction column, and washed twice with DMF. The resin was swelled with DMF for 30 min, deprotected with DBLK (5 min plus 7 min), and washed with DMF for 6 times. The ninhydrin test was positive.
(71) 2. Fmoc-Arg(pbf)-OH (3.245 g, 5 mmol) and HOBt (0.426 g, 3.15 mmol) were dissolved in 15 mL of DMF, and DIC (0.49 mL, 3.15 mmol) was added in an ice bath for activation for 5 minutes. The activated solution was added into the above solid-phase reaction column, and reacted under nitrogen for 2 h with stirring. The ninhydrin test was negative, and Fmoc-Arg(pbf)-Gly-2-CTC resin was obtained. The reaction solution was drained. The resin was washed with DMF for 3 times, deprotected with DBLK (5 min plus 7 min), and washed with DMF for 6 times. The ninhydrin test was negative, and H-Arg(pbf)-Gly-2-CTC resin was obtained.
(72) 3. Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-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-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, and Boc-His(Trt)-OH were sequentially coupled to the H-Arg(pbf)-Gly-Wang resin.
(73) 4. Boc-protective group was removed in 10 mL of 50% TFA/DCM solution per gram of the resin for 20 min, and then the resin was washed with DCM for 6 times, and dried in vacuo to obtain 8.52 g of peptide resin.
(74) 5. A mixed acid hydrolysate (containing 10 mL/g liraglutide resin) with a volume ratio of TFA:Anisole:TIS:H.sub.2O:EDT=92:2:2:2:2 was added to a round-bottom flask with 8.52 g of the peptide resin of liraglutide. The solution was stirred thoroughly, and reacted at room temperature for 3 h with stirring. The reaction mixture was filtered through a sand core funnel, and the filtrate was collected. The resin was washed with a small amount of TFA for three times. The filtrate was combined, and then concentrated in vacuo, and anhydrous ethyl ether was added for precipitation. Then, the precipitate was washed with anhydrous ethyl ether for three times, and the solution was drained to obtain an off-white powder. The off-white powder was vacuum dried in vacuo to a constant weight and 3.68 g of the crude peptide of liraglutide was obtained.
(75) The crude peptide of liraglutide has a yield of 79.8% and a purity of 68.61%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 2.40%. The HPLC spectrum was similar to that of
(76) As can be seen from the HPLC spectra and the corresponding data of examples 1-8 and comparative examples 1 and 2, the peak of racemate impurity, i.e., the peak of D-Thr.sup.5 liraglutide, was closely adjacent to the main peak of liraglutide. Their retention times were 49.288 min and 49.875 min in example 1, respectively and were 48.253 min and 48.835 min in comparative example 1, respectively. The relative retention time of D-Thr.sup.5 liraglutide was about 1.0, which was far from the requirement of separation. If D-Thr.sup.5 liraglutide is present in a large amount, it will be very difficult to purify and separate the crude peptide. However, compared with comparative examples 1 and 2, the amount of D-Thr.sup.5 liraglutide was reduced from respective 2.39% and 2.40% to 0.74% in example 1, relatively reducing by 69.04% and 69.17%, respectively. Compared with comparative examples 1 and 2, the amount of D-Thr.sup.5 liraglutide was reduced from respective 2.39% and 2.40% to 0.71% in example 2, relatively reducing by 70.29% and 70.42%, respectively. Compared with comparative examples 1 and 2, the amount of D-Thr.sup.5 liraglutide was reduced from respective 2.39% and 2.40% to 0.70% in example 3, relatively reducing by 70.71% and 70.83%, respectively. Compared with comparative examples 1 and 2, the amount of D-Thr.sup.5 liraglutide was reduced from respective 2.39% and 2.40% to 0.69% in example 4, relatively reducing by 71.13% and 71.25%, respectively. Compared with comparative examples 1 and 2, the amount of D-Thr.sup.5 liraglutide was reduced from respective 2.39% and 2.40% to 0.70% in example 5, relatively reducing by 70.71% and 70.93%, respectively. Compared with comparative examples 1 and 2, the amount of D-Thr.sup.5 liraglutide was reduced from respective 2.39% and 2.40% to 0.71% in example 6, relatively reducing by 70.29% and 70.42%, respectively. Compared with comparative examples 1 and 2, the amount of D-Thr.sup.5 liraglutide was reduced from respective 2.39% and 2.40% to 0.70% in example 7, relatively reducing by 70.71% and 70.93%, respectively. Compared with comparative examples 1 and 2, the amount of D-Thr.sup.5 liraglutide was reduced from respective 2.39% and 2.40% to 0.71% in example 8, relatively reducing by 70.29% and 70.42%, 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.sup.5 liraglutide, which produced in the synthesis of the crude peptide of liraglutide. D-Thr.sup.5 liraglutide is in an amount of less than 0.8%, which is very advantageous for purification.
(77) In addition, liraglutide in example 1 has a yield of 91.2% and a purity of 70.61%, which were 10.7% and 1.98% higher than those of comparative example 1, respectively, and were 11.4% and 2.0% higher than those of comparative example 2, respectively. Liraglutide in example 2 has a yield of 90.2% and a purity of 71.68%, which were 9.7% and 3.05% higher than those of comparative example 1, respectively, and were 10.4% and 3.07% higher than those of comparative example 2, respectively. Liraglutide in example 3 has a yield of 90.5% and a purity of 72.02%, which were 10.0% and 3.39% higher than those of comparative example 1, respectively, and were 10.7% and 3.41% higher than those of comparative example 2, respectively. Liraglutide in example 4 has a yield of 91.5% and a purity of 72.72%, which were 11.0% and 4.09% higher than those of comparative example 1, respectively, and were 11.7% and 4.11% higher than those of comparative example 2, respectively. Liraglutide in example 5 has a yield of 91.8% and a purity of 70.72%, which were 11.3% and 2.09% higher than those of comparative example 1, respectively, and were 12.0% and 2.11% higher than those of comparative example 2, respectively. Liraglutide in example 6 has a yield of 91.5% and a purity of 71.53%, which were 11.0% and 2.9% higher than those of comparative example 1, respectively, and were 11.7% and 2.92% higher than those of comparative example 2, respectively. Liraglutide in example 7 has a yield of 90.6% and a purity of 72.03%, which were 10.1% and 3.40% higher than those of comparative example 1, respectively, and were 10.8% and 3.42% higher than those of comparative example 2, respectively. Liraglutide in example 8 has a yield of 90.8% and a purity of 70.36%, which were 10.3% and 1.73% higher than those of comparative example 1, respectively, and were 11.0% and 1.75% higher than those of comparative example 2, 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.
(78) The method of the present disclosure can greatly reduce the racemate impurity, i.e., D-Thr.sup.5 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.
Comparative Example 3. Preparation of Refined Peptide of Liraglutide
(79) The crude peptide of liraglutide prepared in comparative example 1 was purified according to the above purification method of Example 9 to obtain 0.47 g of the pure product of liraglutide with a purity of 98.36% and a total yield of 11.34%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 1.23%. The HPLC spectrum was shown in
(80) TABLE-US-00005 TABLE 5 Results of retention time and peak area of characteristic peaks of liraglutide of Comparative Example 3 Ser. Retention time Peak area Peak area No. (min) (AU*s) ratio % 1 45.208 1714 0.04 2 47.734 2516 0.06 3 48.841 20819 0.46 4 50.246 12611 0.28 5 52.080 4385374 97.53 6 52.860 55484 1.23 7 53.676 10013 0.22 8 55.455 2571 0.06 9 57.310 2570 0.06 10 57.968 2576 0.06
(81) The crude peptide of liraglutide prepared in comparative example 2 was purified according to the above purification method of Example 9 to obtain 0.48 g of the pure product of liraglutide with a purity of 97.53% and a total yield of 12.58%. The racemate impurity with a structure similar to that of liraglutide, i.e., D-Thr.sup.5 liraglutide, was closely adjacent to the main peak, with a relative retention time of about 1.0 and an amount of 1.23%. The HPLC spectrum was similar to that shown in
(82) As can be seen from the HPLC spectra of examples 9 to 15 and comparative example 3, after simple purification steps, D-Thr.sup.5 liraglutide among liraglutide had been substantially removed, and its maximum amount is only 0.27% in the refined peptide. However, D-Thr.sup.5 liraglutide in the comparative examples is more difficult to remove, which has an amount as high as 1.23% 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.sup.5 liraglutide, the lower original yield will be further lowered.
(83) It can be seen that the method of the present disclosure substantially reduces the production of the racemate impurity D-Thr.sup.5 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.sup.5 liraglutide.
(84) Although the disclosure is illustrated and described herein with reference to specific embodiments, the disclosure is not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the disclosure.