WO2021114787A1 - Method of synthesizing teriparatide impurity f - Google Patents
Method of synthesizing teriparatide impurity f Download PDFInfo
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- WO2021114787A1 WO2021114787A1 PCT/CN2020/115240 CN2020115240W WO2021114787A1 WO 2021114787 A1 WO2021114787 A1 WO 2021114787A1 CN 2020115240 W CN2020115240 W CN 2020115240W WO 2021114787 A1 WO2021114787 A1 WO 2021114787A1
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- fmoc
- impurity
- trt
- leu
- synthesis method
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- 239000012535 impurity Substances 0.000 title claims abstract description 75
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 9
- 229960005460 teriparatide Drugs 0.000 title claims description 31
- 108010049264 Teriparatide Proteins 0.000 title claims description 27
- 238000000034 method Methods 0.000 title claims description 26
- OGBMKVWORPGQRR-UMXFMPSGSA-N teriparatide Chemical compound C([C@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)[C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CNC=N1 OGBMKVWORPGQRR-UMXFMPSGSA-N 0.000 title claims description 26
- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 44
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- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 18
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- 239000002994 raw material Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
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- 238000001291 vacuum drying Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
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Definitions
- the present invention provides a method for synthesizing teriparatide impurity F.
- the discovery and synthesis of this impurity is beneficial to the quality control of teriparatide raw materials; by adopting the synthesis method of the present invention, the purity of the crude peptide can reach 42%, and the purity of the refined peptide can reach 92% after purification.
- Step B Remove the PG protecting group of Asp(PG) and close the ring to obtain a peptide resin
- the substitution degree is 0.1 ⁇ 3.0mmol/g, according to the peptide solid phase synthesis method, according to the peptide sequence, the protected amino acids are coupled in sequence from the C-terminus to the N-terminus.
- Asp uses Fmoc-Asp( PG)-OH
- PG is All or Dmab protecting group
- Ser uses Boc-Ser(tBu)-OH
- other residues use conventional amino acids.
- one or more of the coupling agents HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA and HOAt/PyAop/DIPEA are used for coupling in sequence Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(ODmab)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc- Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH
- the impurity F obtained in Example 2 was purified by HPLC. The sample amount was 30g/time. The fractions were collected according to the chromatographic peak, starting from about 10% at the beginning of the main peak, and the fractions were collected according to the chromatographic peak. Stop collecting at about 10%. The fraction was concentrated by rotary evaporation and lyophilized to obtain 15.5 g of impurity F refined peptide with a purity of 92.13% ( Figure 3, Table 2).
- Mobile phase A1 0.1% TFA solution
- mobile phase B1 pure acetonitrile
- ND YAG 355nm, 1000Hz;
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Abstract
Disclosed is a teriparatide-aspartimide impurity, named impurity F. In addition, provided is a solid-phase method of synthesizing impurity F.
Description
本申请要求于2019年12月10日提交中国专利局、申请号为201911259985.9、发明名称为“一种特立帕肽杂质F的合成方法”的中国专利申请的优先权,其全部内容通过引用结合在本申请中。This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on December 10, 2019, the application number is 201911259985.9, and the invention title is "a method for synthesizing teriparatide impurity F", the entire content of which is incorporated by reference In this application.
本发明涉及杂质合成技术领域,特别涉及一种特立帕肽杂质F的合成方法。The present invention relates to the technical field of impurity synthesis, in particular to a method for synthesizing teriparatide impurity F.
特立帕肽(Teriparatide)是人甲状旁腺激素中的1-34位片段,该片段与人甲状旁腺素具有相同的生物活性,由美国Eli Lilly公司开发用于原发性骨质疏松、性腺功能减退性骨质疏松和绝经妇女的骨质疏松。肽序为: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
30-Val
31-His-Asn-Phe-OH。
Teriparatide is a fragment 1-34 in human parathyroid hormone. This fragment has the same biological activity as human parathyroid hormone. It was developed by Eli Lilly Company of the United States for primary osteoporosis, Hypogonadal osteoporosis and osteoporosis in menopausal women. The 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 30 -Val 31 -His-Asn-Phe-OH.
特立帕肽在其生产和储存过程中会产生多种杂质,目前报道发现的杂质有Trp23(C=O)-特立帕肽氧化杂质、Trp23(OH)-特立帕肽氧化杂质、Met8(O)-特立帕肽氧化杂质、Met18(O)-特立帕肽氧化杂质,Met8,18(O)-特立帕肽氧化杂质等。为了保证特立帕肽药品的安全性和有效性,必须控制产品中杂质的含量。新杂质的发现与合成对于特立帕肽原料药的质量控制具有重要的现实意义。Teriparatide produces a variety of impurities during its production and storage. The impurities currently reported include Trp23(C=O)-teriparatide oxidation impurities, Trp23(OH)-teriparatide oxidation impurities, and Met8 (O)-teriparatide oxidation impurity, Met18(O)-teriparatide oxidation impurity, Met8,18(O)-teriparatide oxidation impurity, etc. In order to ensure the safety and effectiveness of teriparatide drugs, the content of impurities in the product must be controlled. The discovery and synthesis of new impurities have important practical significance for the quality control of teriparatide bulk drugs.
发明内容Summary of the invention
有鉴于此,本发明提供了一种特立帕肽杂质F的合成方法。该杂质的发现与合成有利于特立帕肽原料药的质量控制;采用本发明合成方法,粗肽纯度可达到42%,经过纯化后精肽纯度可达到92%。In view of this, the present invention provides a method for synthesizing teriparatide impurity F. The discovery and synthesis of this impurity is beneficial to the quality control of teriparatide raw materials; by adopting the synthesis method of the present invention, the purity of the crude peptide can reach 42%, and the purity of the refined peptide can reach 92% after purification.
为了实现上述发明目的,本发明提供以下技术方案:In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:
本发明提供了一种特立帕肽杂质F,其结构式如式I所示:The present invention provides a teriparatide impurity F, the structural formula of which is shown in formula I:
本申请在研发过程中发现,在其生产和储存过程中会产生天冬酰亚胺类杂质,命名为杂质F,即特立帕肽残基中的30
#Asp和31
#Val之间发生亲核取代反应形成五元环。肽序为:
In the research and development process of this application, it was found that aspartimide impurities will be produced during the production and storage process, named as impurity F, that is, the affinity between 30 # Asp and 31 # Val in teriparatide residues The nuclear substitution reaction forms a five-membered ring. The 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-Asu-Val-His-Asn-Phe-OH。其氨基酸序列如SEQ ID NO:1所示。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-Asu-Val-His-Asn-Phe-OH. Its amino acid sequence is shown in SEQ ID NO:1.
本发明还提供了该特立帕肽杂质F的合成方法,包括如下步骤:The invention also provides a method for synthesizing teriparatide impurity F, which includes the following steps:
步骤A:采用固相合成法,以树脂为载体,依次偶联Fmoc-Asn(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-Val-OH、Fmoc-Asp(ODmab)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Val-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Ser(tBu)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-Leu-OH、Fmoc-Asn(Trt)-OH、 Fmoc-His(Trt)-OH、Fmoc-Met-OH、Fmoc-Leu-OH、Fmoc-Gln(Trt)-OH、Fmoc-Ile-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH和Boc-Ser(tBu)-OH,得到带有保护基的肽树脂;Asp(PG)中的PG保护基为All或Dmab保护基;Step A: Using solid phase synthesis method, using resin as carrier, sequentially coupling Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(ODmab)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc- Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Ser( tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu )-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH and Boc-Ser(tBu)-OH to obtain peptide resins with protecting groups; the PG protecting group in Asp(PG) is All or Dmab Protecting base
步骤B:脱除Asp(PG)的PG保护基,关环,得到肽树脂;Step B: Remove the PG protecting group of Asp(PG) and close the ring to obtain a peptide resin;
步骤C:将肽树脂进行裂解,得到特立帕肽杂质F。Step C: The peptide resin is cleaved to obtain teriparatide impurity F.
本发明人采用文献报道的方法(DBU催化或酸降解),得到的目标产品纯度<1%,按照常规的合成方法(如HOAt/PyAop/DIPEA关环),得到的目标产品纯度只有约1%,均无法得到纯度较高的产品。本发明提供了一种制备特立帕肽杂质F的方法,其机理是30
#Asp侧链裸露的羧基与叠氮磷酸二苯酯生成活性较高的酰基叠氮化物,再与31
#Val的氨基发生亲核取代,从而得到目标产品。该方法得到的产品纯度高,易于纯化,收率也得到了相应地提高。
The inventors used the methods reported in the literature (DBU catalysis or acid degradation), and the purity of the target product obtained was less than 1%. According to the conventional synthesis method (such as HOAt/PyAop/DIPEA ring closure), the purity of the target product obtained was only about 1%. , The products with higher purity cannot be obtained. The present invention provides a method for preparing teriparatide impurity F. The mechanism is that the exposed carboxyl group of the side chain of 30 # Asp and diphenyl azide phosphate form a more active acyl azide, which is combined with 31 # Val. The amino group undergoes nucleophilic substitution to obtain the target product. The product obtained by the method has high purity, is easy to purify, and the yield is correspondingly improved.
作为优选,步骤A中,树脂为Wang Resinn,替代度为0.1~3.0mmol/g。Preferably, in step A, the resin is Wang Resinn, and the degree of substitution is 0.1-3.0 mmol/g.
在本发明提供的具体实施例中,Wang Resinn的替代度为0.5~0.8mmol/g。In the specific embodiment provided by the present invention, the substitution degree of Wang Resinn is 0.5 to 0.8 mmol/g.
作为优选,步骤A中,偶联采用的偶联剂为HOBt/DIPCDI、HOBt/PyBop/DIPEA、HBTU/HOBt/DIPEA、HOAt/DIPCDI、HATU/HOAt/DIPEA或HOAt/PyAop/DIPEA中的一种或几种。Preferably, in step A, the coupling agent used for coupling is one of HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA or HOAt/PyAop/DIPEA Or several.
作为优选,步骤A中,偶联采用的脱除Fmoc保护基团的试剂为10%~30%哌啶溶液,溶解氨基酸和哌啶溶液所用的溶剂为NMP、THF、DCM、DMF或DMSO中的一种或几种。Preferably, in step A, the reagent used for coupling to remove the Fmoc protective group is a 10%-30% piperidine solution, and the solvent used to dissolve the amino acid and the piperidine solution is NMP, THF, DCM, DMF or DMSO. One or more.
优选地,步骤A中,偶联采用的脱除Fmoc保护基团的试剂为20%哌啶溶液。Preferably, in step A, the reagent for removing the Fmoc protective group used for coupling is a 20% piperidine solution.
作为优选,步骤B中,Asp(PG)中的PG保护基为All保护基,脱除采用的催化剂为四三苯基膦钯,清除剂为苯硅烷或吗啉。Preferably, in step B, the PG protecting group in Asp(PG) is an All protecting group, the catalyst used for removal is tetrakistriphenylphosphine palladium, and the scavenger is phenylsilane or morpholine.
作为优选,步骤B中,Asp(PG)中的PG保护基为Dmab保护基,脱除采用的清除剂为1%~5%水合肼-DMF溶液。Preferably, in step B, the PG protecting group in Asp(PG) is a Dmab protecting group, and the scavenger used for removal is a 1% to 5% hydrazine hydrate-DMF solution.
优选地,步骤B中,Asp(PG)中的PG保护基为Dmab保护基,脱除 采用的清除剂为2%水合肼-DMF溶液。Preferably, in step B, the PG protecting group in Asp(PG) is a Dmab protecting group, and the scavenger used for removal is a 2% hydrazine hydrate-DMF solution.
作为优选,步骤B中,关环采用叠氮磷酸二苯酯关环。Preferably, in step B, the ring is closed by diphenyl azide phosphate.
作为优选,步骤C中,裂解采用的裂解液为含有捕捉剂的三氟乙酸溶液,捕捉剂为PhSMe、PhOH、EDT、H
2O、TIS、PhOMe中的一种或几种。
Preferably, in step C, the lysis solution used for lysis is a trifluoroacetic acid solution containing a capture agent, and the capture agent is one or more of PhSMe, PhOH, EDT, H 2 O, TIS, and PhOMe.
优选地,捕捉剂为TIS。Preferably, the capture agent is TIS.
作为优选,裂解液中三氟乙酸与TIS的体积比为(90~99):(1~10)。Preferably, the volume ratio of trifluoroacetic acid to TIS in the lysis solution is (90-99): (1-10).
优选地,裂解液中三氟乙酸与TIS的体积比为95:5。Preferably, the volume ratio of trifluoroacetic acid to TIS in the lysis solution is 95:5.
本发明提供了一种特立帕肽杂质F的合成方法。该特立帕肽杂质F的结构式如式I所示。本发明的优点如下:The present invention provides a method for synthesizing teriparatide impurity F. The structural formula of teriparatide impurity F is shown in formula I. The advantages of the present invention are as follows:
本发明发现特立帕肽在其生产和储存过程中会产生天冬酰亚胺类杂质,命名为杂质F,即特立帕肽残基中的30
#Asp和31
#Val之间发生亲核取代反应形成五元环。该杂质的发现与合成有利于特立帕肽原料药的质量控制;
The present invention found that teriparatide will produce aspartimide impurities during its production and storage process, named as impurity F, that is, nucleophilicity occurs between 30 # Asp and 31 # Val in teriparatide residues. The substitution reaction forms a five-membered ring. The discovery and synthesis of this impurity is conducive to the quality control of teriparatide API;
采用文献报道的方法(DBU催化或酸降解),得到的目标产品纯度<1%,按照常规的合成方法(HOAt/PyAop/DIPEA关环),得到的目标产品纯度只有约1%,均无法得到纯度较高的产品;而采用本发明方法,粗肽纯度可达到42%,经过纯化后精肽纯度可达到92%。与现有技术相比,本发明得到的产品纯度高,易于纯化,收率也得到了相应地提高。Using the method reported in the literature (DBU catalysis or acid degradation), the purity of the target product obtained is less than 1%. According to the conventional synthesis method (HOAt/PyAop/DIPEA ring closure), the purity of the target product obtained is only about 1%, and neither can be obtained. A product with higher purity; while using the method of the present invention, the purity of the crude peptide can reach 42%, and the purity of the refined peptide can reach 92% after purification. Compared with the prior art, the product obtained by the present invention has high purity, is easy to purify, and the yield is correspondingly improved.
图1:杂质F的合成路线图;Figure 1: Synthetic route of impurity F;
图2:杂质F粗肽的HPLC图谱(实施例3);Figure 2: HPLC profile of impurity F crude peptide (Example 3);
图3:杂质F精肽的HPLC图谱(实施例8);Figure 3: HPLC profile of impurity F refined peptide (Example 8);
图4:杂质F的一级质谱图;Figure 4: The first-order mass spectrum of impurity F;
图5:杂质F酶切后的一级质谱图;Figure 5: The first-level mass spectrum of impurity F after digestion;
图6:杂质F酶切后母离子M/Z为1455的片段的二级质谱图;Figure 6: The secondary mass spectrum of the fragment with the parent ion M/Z of 1455 after impurity F digestion;
图7:杂质F酶切后母离子M/Z为886的片段的二级质谱图;Figure 7: The secondary mass spectrum of the fragment with the parent ion M/Z of 886 after impurity F digestion;
图8:杂质F酶切后母离子M/Z为702的片段的二级质谱图;Figure 8: The secondary mass spectrum of the fragment with the parent ion M/Z of 702 after impurity F digestion;
图9:杂质F酶切后母离子M/Z为872的片段的二级质谱图;Figure 9: The secondary mass spectrum of the fragment with the parent ion M/Z of 872 after impurity F digestion;
图10:杂质F酶切后母离子M/Z为1455段实测二级碎片峰和目标肽序的理论二级碎片比对情况;Figure 10: The parent ion M/Z after impurity F digestion is the comparison of the theoretical secondary fragments of the 1455 segment of the measured secondary fragment peak and the target peptide sequence;
图11:杂质F酶切后母离子M/Z为886实测二级碎片峰和目标肽序的理论二级碎片比对情况;Figure 11: The parent ion M/Z is 886 after impurity F is digested with the measured secondary fragment peak and the theoretical secondary fragment comparison of the target peptide sequence;
图12:杂质F酶切后母离子M/Z为702的片段实测二级碎片峰和目标肽序的理论二级碎片比对情况;Figure 12: Comparison of the measured secondary fragment peak of the fragment with the parent ion M/Z of 702 after impurity F digestion with the theoretical secondary fragment of the target peptide sequence;
图13:杂质F酶切后母离子M/Z为872的片段实测二级碎片峰和目标肽序的理论二级碎片比对情况。Figure 13: Comparison of the measured secondary fragment peak of the fragment with the parent ion M/Z of 872 after impurity F digestion with the theoretical secondary fragment of the target peptide sequence.
本发明公开了一种特立帕肽杂质F的合成方法,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。The present invention discloses a method for synthesizing teriparatide impurity F. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant persons can make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.
缩写及英文含义如下:The abbreviations and their English meanings are as follows:
本发明提供的具体制备特立帕肽杂质F的方法包括以下步骤:The method for specifically preparing teriparatide impurity F provided by the present invention includes the following steps:
1、以Wang Resin为载体,替代度为0.1~3.0mmol/g,根据多肽固相合成法,按照肽序,从C端至N端依次偶联保护氨基酸,其中30
#Asp采用 Fmoc-Asp(PG)-OH,PG为All或Dmab保护基,1
#Ser采用Boc-Ser(tBu)-OH,其它残基采用常规氨基酸。采用偶联剂HOBt/DIPCDI、HOBt/PyBop/DIPEA、HBTU/HOBt/DIPEA、HOAt/DIPCDI、HATU/HOAt/DIPEA和HOAt/PyAop/DIPEA中的一种或几种,依次偶联Fmoc-Asn(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-Val-OH、Fmoc-Asp(PG)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Val-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Ser(tBu)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-Leu-OH、Fmoc-Asn(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-Met-OH、Fmoc-Leu-OH、Fmoc-Gln(Trt)-OH、Fmoc-Ile-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH和Boc-Ser(tBu)-OH。PG为All或Dmab保护基,脱除Fmoc保护基团的试剂为20%哌啶溶液,所述溶解氨基酸和20%哌啶溶液所用的溶剂为NMP、THF、DCM、DMF和DMSO中的一种或几种。
1. Using Wang Resin as the carrier, the substitution degree is 0.1~3.0mmol/g, according to the peptide solid phase synthesis method, according to the peptide sequence, the protected amino acids are coupled in sequence from the C-terminus to the N-terminus. Among them, 30 # Asp uses Fmoc-Asp( PG)-OH, PG is All or Dmab protecting group, 1 # Ser uses Boc-Ser(tBu)-OH, and other residues use conventional amino acids. One or more of the coupling agents HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA and HOAt/PyAop/DIPEA are used to couple Fmoc-Asn( Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(PG)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc) -OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH , Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc -His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Met -OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH and Boc- Ser(tBu)-OH. PG is the All or Dmab protecting group, the reagent for removing the Fmoc protecting group is 20% piperidine solution, and the solvent used to dissolve the amino acid and 20% piperidine solution is one of NMP, THF, DCM, DMF and DMSO Or several.
2、脱去PG保护基,如PG为All,则采用四三苯基膦钯催化,苯硅烷或吗啉为清除剂除去;如PG为Dmab,则采用2%水合肼-DMF溶液除去。2. Remove the PG protecting group. If PG is All, use tetrakistriphenylphosphine palladium catalysis and phenylsilane or morpholine as scavenger to remove; if PG is Dmab, use 2% hydrazine hydrate-DMF solution to remove.
3、首先采用叠氮磷酸二苯酯关环,然后采用含有捕捉剂的三氟乙酸溶液裂解得到杂质F,所述捕捉剂为PhSMe、PhOH、EDT、H
2O、TIS、PhOMe中的一种或几种,更优选地,裂解液为TFA/TIS的组合物,其中TFA、TIS的体积比为95:5。
3. First, use diphenyl azide phosphate to close the ring, and then use a trifluoroacetic acid solution containing a trapping agent to crack to obtain impurity F. The trapping agent is one of PhSMe, PhOH, EDT, H 2 O, TIS, and PhOMe Or several, more preferably, the lysate is a TFA/TIS composition, wherein the volume ratio of TFA to TIS is 95:5.
具体路线见图1。The specific route is shown in Figure 1.
本发明提供的特立帕肽杂质F及其制备方法中所用试剂或仪器均可由市场购得。The teriparatide impurity F provided by the present invention and the reagents or instruments used in the preparation method thereof can be purchased from the market.
下面结合实施例,进一步阐述本发明:The following examples further illustrate the present invention:
实施例1:氨基酸的偶联Example 1: Coupling of amino acids
称取替代度为0.8mmol/g的Wang Resin 62.5g(50mmol),加入固相反应柱中,用DMF洗涤2次,用DMF溶胀树脂30分钟后,称取19.37g(50mmol)Fmoc-Phe-OH、8.1g(60mmol)HOBt和6.1g(5mmol)DMAP溶于DMF,冰浴下加入8.2g(65mmol)DIPCDI,加入固相反应柱中,室温反应1小时,DMF洗涤6次。再加入79.1g(1000mmol)吡啶和102.1g(1000mmol)乙酸酐混合液封闭树脂6小时,DMF洗涤6次,甲醇收缩抽干,得到71.4g Fmoc-Phe-Wang Resin,检测替代度为0.3mmol/g。Weigh 62.5g (50mmol) of Wang Resin with a degree of substitution of 0.8mmol/g, add it to the solid phase reaction column, wash twice with DMF, swell the resin with DMF for 30 minutes, and weigh 19.37g (50mmol) of Fmoc-Phe- OH, 8.1g (60mmol) HOBt and 6.1g (5mmol) DMAP were dissolved in DMF, added 8.2g (65mmol) DIPCDI under ice bath, added to solid phase reaction column, reacted at room temperature for 1 hour, and washed with DMF 6 times. Then add 79.1g (1000mmol) pyridine and 102.1g (1000mmol) acetic anhydride mixture to seal the resin for 6 hours, wash with DMF 6 times, shrink and drain with methanol to obtain 71.4g Fmoc-Phe-Wang Resin, and the detection substitution degree is 0.3mmol/ g.
20%哌啶溶液脱除Fmoc保护基团(反应时间5+7分钟),DMF洗涤6次。按照肽序,采用偶联剂HOBt/DIPCDI、HOBt/PyBop/DIPEA、HBTU/HOBt/DIPEA、HOAt/DIPCDI、HATU/HOAt/DIPEA和HOAt/PyAop/DIPEA中的一种或几种,依次偶联Fmoc-Asn(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-Val-OH、Fmoc-Asp(OAll)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Val-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Ser(tBu)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-Leu-OH、Fmoc-Asn(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-Met-OH、Fmoc-Leu-OH、Fmoc-Gln(Trt)-OH、Fmoc-Ile-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH和Boc-Ser(tBu)-OH。20% piperidine solution removes the Fmoc protective group (reaction time 5+7 minutes), and DMF washes 6 times. According to the peptide sequence, one or more of the coupling agents HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA and HOAt/PyAop/DIPEA are used for coupling in sequence Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(OAll)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc- Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc -Val-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu -OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH , Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val- OH and Boc-Ser(tBu)-OH.
实施例2:氨基酸的偶联Example 2: Coupling of amino acids
称取替代度为0.5mmol/g的Wang Resin 100.0g(50mmol),加入固相反应柱中,用DMF洗涤2次,用DMF溶胀树脂30分钟后,称取38.74g(100mmol)Fmoc-Phe-OH、16.2g(120mmol)HOBt和52.1g(100mmol)PyBOP溶于DMF,冰浴下加入25.9g(200mmol)DIPEA,加入固相反应柱中,室温反应2小时,DMF洗涤6次。再加入79.1g(1000mmol)吡啶和 102.1g(1000mmol)乙酸酐混合液封闭树脂6小时,DMF洗涤6次,甲醇收缩抽干,得到115.4g Fmoc-Phe-Wang Resin,检测替代度为0.26mmol/g。Weigh 100.0 g (50 mmol) of Wang Resin with a substitution degree of 0.5 mmol/g, add it to the solid phase reaction column, wash twice with DMF, swell the resin with DMF for 30 minutes, and weigh 38.74 g (100 mmol) of Fmoc-Phe- OH, 16.2g (120mmol) HOBt and 52.1g (100mmol) PyBOP were dissolved in DMF, 25.9g (200mmol) DIPEA was added under ice bath, added to the solid phase reaction column, reacted at room temperature for 2 hours, and washed with DMF 6 times. Then add 79.1g (1000mmol) pyridine and 102.1g (1000mmol) acetic anhydride mixture to seal the resin for 6 hours, wash with DMF 6 times, shrink and drain with methanol to obtain 115.4g Fmoc-Phe-Wang Resin, and the detection substitution degree is 0.26mmol/ g.
20%哌啶溶液脱除Fmoc保护基团(反应时间5+7分钟),DMF洗涤6次。按照肽序,采用偶联剂HOBt/DIPCDI、HOBt/PyBop/DIPEA、HBTU/HOBt/DIPEA、HOAt/DIPCDI、HATU/HOAt/DIPEA和HOAt/PyAop/DIPEA中的一种或几种,依次偶联Fmoc-Asn(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-Val-OH、Fmoc-Asp(ODmab)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Val-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Ser(tBu)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-Leu-OH、Fmoc-Asn(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-Met-OH、Fmoc-Leu-OH、Fmoc-Gln(Trt)-OH、Fmoc-Ile-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH和Boc-Ser(tBu)-OH。20% piperidine solution removes the Fmoc protective group (reaction time 5+7 minutes), and DMF washes 6 times. According to the peptide sequence, one or more of the coupling agents HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA and HOAt/PyAop/DIPEA are used for coupling in sequence Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(ODmab)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc- Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc -Val-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu -OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH , Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val- OH and Boc-Ser(tBu)-OH.
实施例3:杂质F的合成Example 3: Synthesis of impurity F
向实施例1的肽树脂反应柱中加入四三苯基膦钯12.4g(0.5eq)、苯硅烷23.2g(10eq)和二氯甲烷500mL,室温反应1小时,抽去反应液,二氯甲烷洗涤六次,四氢呋喃洗涤三次,转移至三口瓶中,加入三乙胺43.4g(20eq)、叠氮磷酸二苯酯117.9g(20eq)和四氢呋喃500mL,加热至回流反应8小时,冷却至室温,过滤,四氢呋喃洗涤六次,甲基叔丁基醚收缩三次,真空干燥得到肽树脂140g。Add 12.4 g (0.5 eq) of tetratriphenylphosphine palladium, 23.2 g (10 eq) of phenylsilane and 500 mL of dichloromethane to the peptide resin reaction column of Example 1, and react at room temperature for 1 hour. Remove the reaction solution and dichloromethane. Washed six times, washed with tetrahydrofuran three times, transferred to a three-necked flask, added 43.4g (20eq) of triethylamine, 117.9g (20eq) of diphenyl azide phosphate and 500mL of tetrahydrofuran, heated to reflux and reacted for 8 hours, cooled to room temperature, Filter, wash with tetrahydrofuran six times, shrink three times with methyl tert-butyl ether, and dry under vacuum to obtain 140 g of peptide resin.
加入裂解液(TFA:TIS=95:5,体积比)1.2L室温裂解2小时,过滤,滤液倒入12L***中沉降,离心洗涤,真空干燥得到杂质F粗肽90g,纯度42.96%(图2、表1)。Add lysis buffer (TFA:TIS=95:5, volume ratio) 1.2L to lyse at room temperature for 2 hours, filter, pour the filtrate into 12L ether to settle, centrifuge, wash, vacuum dry to obtain impurity F crude peptide 90g, purity 42.96% (Figure 2 ,Table 1).
表1Table 1
实施例4:杂质F的合成Example 4: Synthesis of impurity F
向实施例2的肽树脂反应柱中加入2%水合肼-DMF溶液脱除Dmab保护基(反应时间10+10分钟),DMF洗涤6次,四氢呋喃洗涤三次,转移至三口瓶中,加入三乙胺30.4g(10eq)、叠氮磷酸二苯酯82.6g(10eq)和四氢呋喃1L,加热至回流反应15小时,冷却至室温,过滤,四氢呋喃洗涤六次,甲基叔丁基醚收缩三次,真空干燥得到肽树脂200g。Add 2% hydrazine hydrate-DMF solution to the peptide resin reaction column of Example 2 to remove the Dmab protecting group (reaction time 10+10 minutes), wash with DMF 6 times, wash with tetrahydrofuran three times, transfer to a three-necked flask, and add triethyl Amine 30.4g (10eq), 82.6g (10eq) of diphenyl azide phosphate and 1L of tetrahydrofuran, heated to reflux for 15 hours, cooled to room temperature, filtered, washed with tetrahydrofuran six times, methyl tert-butyl ether shrinked three times, vacuum 200 g of peptide resin was obtained by drying.
加入裂解液(TFA:H
2O=95:5,体积比)1.6L室温裂解2小时,过滤,滤液倒入16L***中沉降,离心洗涤,真空干燥得到杂质F粗肽120g,纯度40.35%。
Add lysis solution (TFA:H 2 O=95:5, volume ratio) 1.6L for room temperature lysis for 2 hours, filter, pour the filtrate into 16L ether to settle, centrifuge, wash, and vacuum dry to obtain 120g of impurity F crude peptide with a purity of 40.35%.
实施例5:杂质F的合成Example 5: Synthesis of impurity F
称取实施例1的肽树脂的1/10,加入四三苯基膦钯1.2g(0.5eq)、苯硅烷2.3g(10eq)和二氯甲烷50mL,室温反应1小时,抽去反应液,二氯甲烷洗涤六次,另称取1.6g(12mmol)HOAt和5.2g(10mmol)PyAOP溶于DMF,冰浴下加入2.6g(20mmol)DIPEA,加入固相反应柱中,室温反应15小时,抽去反应液,DMF洗涤六次,甲基叔丁基醚收缩三次,真空干燥得到肽树脂15g。加入裂解液(TFA:TIS=95:5,体积比)150mL室温裂解2小时,过滤,滤液倒入1.5L***中沉降,离心洗涤,真空干燥得到杂质F粗肽9g,纯度约1%。 Weigh 1/10 of the peptide resin of Example 1, add 1.2 g (0.5 eq) of tetrakistriphenylphosphine palladium, 2.3 g (10 eq) of phenylsilane and 50 mL of dichloromethane, and react at room temperature for 1 hour. The reaction solution is removed. Wash with dichloromethane for six times, weigh 1.6g (12mmol) HOAt and 5.2g (10mmol) PyAOP in DMF, add 2.6g (20mmol) DIPEA under ice bath, add to solid phase reaction column, react at room temperature for 15 hours, The reaction solution was removed, washed with DMF six times, MTBE contracted three times, and dried in vacuum to obtain 15 g of peptide resin. 150 mL of lysate (TFA:TIS=95:5, volume ratio) was added for lysis at room temperature for 2 hours, filtered, and the filtrate was poured into 1.5L of ether to settle, centrifuged, washed, and vacuum dried to obtain 9g of crude impurity F peptide with a purity of about 1%.
实施例6:杂质F的合成Example 6: Synthesis of impurity F
取特立帕肽肽树脂5g,加入50%DBU-DMF溶液,室温反应7d,抽去反应液,DMF洗涤六次,甲基叔丁基醚收缩三次,真空干燥后加入裂解液(TFA:TIS=95:5,体积比)50mL室温裂解2小时,过滤,滤液倒入500mL***中沉降,离心洗涤,真空干燥得到杂质F粗肽2g,纯度0.4%。Take 5 g of teriparatide peptide resin, add 50% DBU-DMF solution, react at room temperature for 7 days, extract the reaction solution, wash with DMF six times, shrink three times with methyl tert-butyl ether, add the lysate (TFA:TIS) after vacuum drying =95:5, volume ratio) 50mL was lysed at room temperature for 2 hours, filtered, and the filtrate was poured into 500mL of ether to settle, centrifuged, washed, and vacuum dried to obtain 2g of crude impurity F peptide with a purity of 0.4%.
实施例7:杂质F的合成Example 7: Synthesis of impurity F
取特立帕肽精肽0.1g溶于5mL纯化水,稀盐酸调节pH=3,加热至90℃反应3d,HPLC检测杂质F纯度0.8%。Dissolve 0.1 g of teriparatide refined peptide in 5 mL purified water, adjust the pH to 3 with dilute hydrochloric acid, heat to 90° C. and react for 3 days. The purity of impurity F is 0.8% by HPLC.
实施例8:杂质F的纯化Example 8: Purification of Impurity F
将实施例2得到的杂质F采用HPLC进行纯化,上样量30g/次,根据色谱峰分段收集馏分,从主峰起始处约10%开始,根据色谱峰分段收集馏分,在主峰下降至约10%处,停止收集。馏分旋转蒸发浓缩,冻干得到杂质F精肽15.5g,纯度92.13%(图3、表2)。The impurity F obtained in Example 2 was purified by HPLC. The sample amount was 30g/time. The fractions were collected according to the chromatographic peak, starting from about 10% at the beginning of the main peak, and the fractions were collected according to the chromatographic peak. Stop collecting at about 10%. The fraction was concentrated by rotary evaporation and lyophilized to obtain 15.5 g of impurity F refined peptide with a purity of 92.13% (Figure 3, Table 2).
1、HPLC制备纯化色谱条件如下:1. HPLC preparation and purification chromatographic conditions are as follows:
(1)流动相A1:0.1%TFA溶液,流动相B1:纯乙腈;(1) Mobile phase A1: 0.1% TFA solution, mobile phase B1: pure acetonitrile;
(2)Novasep LC150色谱***,15cm色谱柱内装Welch Ultimate H S-C18 8μm
填料(柱效值的理论塔板数不低于5000);
(2) Novasep LC150 chromatographic system, 15cm column with Welch Ultimate H S-C18 8μm Packing (the theoretical plate number of the column efficiency value is not less than 5000);
(3)监测波长:220nm;(3) Monitoring wavelength: 220nm;
(4)流速:500mL/min(4) Flow rate: 500mL/min
(5)纯化梯度:(5) Purification gradient:
时间(min)Time (min) | 流动相A1(%)Mobile phase A1 (%) | 流动相B1(%)Mobile phase B1 (%) |
00 | 9090 | 1010 |
55 | 7575 | 2525 |
105105 | 6565 | 3535 |
115115 | 3030 | 7070 |
2、HPLC分析检测色谱条件如下:2. HPLC analysis and detection chromatographic conditions are as follows:
(1)流动相:A相:乙腈:硫酸氨缓冲液=10:90(V:V),B相:乙腈:硫酸氨缓冲液=50:50(V:V);(1) Mobile phase: Phase A: Acetonitrile: ammonium sulfate buffer = 10: 90 (V: V), Phase B: Acetonitrile: ammonium sulfate buffer = 50: 50 (V: V);
(2)色谱柱:Waters BEHPeptide 300-C18-1.7μm 2.1*100mm;(2) Chromatographic column: Waters BEHPeptide 300-C18-1.7μm 2.1*100mm;
(3)流速:0.4mL/min;(3) Flow rate: 0.4mL/min;
(4)监测波长:214nm;(4) Monitoring wavelength: 214nm;
(5)柱温:60℃;(5) Column temperature: 60℃;
(6)检测梯度:(6) Detection gradient:
时间(min)Time (min) | 流动相A(%)Mobile phase A (%) | 流动相B(%)Mobile phase B (%) |
00 | 100100 | 00 |
55 | 6565 | 3535 |
2929 | 6161 | 3939 |
3030 | 00 | 100100 |
3535 | 00 | 100100 |
35.135.1 | 100100 | 00 |
4040 | 100100 | 00 |
表2Table 2
实施例9:杂质F的结构确认Example 9: Confirmation of structure of impurity F
为了确定杂质F的结构,对其进行一级质谱分析,并用Trypsin酶进行处理,分析酶切后的一级、二级质谱。In order to determine the structure of impurity F, it was subjected to primary mass spectrometry and treated with Trypsin enzyme to analyze the primary and secondary mass spectra after digestion.
1、实验条件:1. Experimental conditions:
(1)仪器型号:MALDI-TOF-TOF,Autoflex Speed;(1) Instrument model: MALDI-TOF-TOF, Autoflex Speed;
(2)一级质谱(2) Primary mass spectrometry
激光器波长和频率:ND:YAG 355nm,1000Hz;Laser wavelength and frequency: ND: YAG 355nm, 1000Hz;
极性:Positive;Polarity: Positive;
操作模式:RP_700-3500;Operation mode: RP_700-3500;
检测器电压:2000V;Detector voltage: 2000V;
分析软件:FlexAnalysis;Analysis software: FlexAnalysis;
靶型号:MTP 384 ground steel;Target model: MTP 384 ground steel;
基质:DHB用30%的乙腈水溶液溶解成20mg/mL;Substrate: DHB is dissolved into 20mg/mL with 30% acetonitrile aqueous solution;
基质与样品:按1:1体积比混合,自然晾干后放入质谱测试仓。Matrix and sample: mix in a 1:1 volume ratio, dry naturally, and put into the mass spectrometer test chamber.
(3)二级质谱(3) Secondary mass spectrometry
激光器波长和频率:ND:YAG 355nm,1000Hz;Laser wavelength and frequency: ND: YAG 355nm, 1000Hz;
极性:Positive reflector;Polarity: Positive reflector;
操作模式:LIFT;Operation mode: LIFT;
检测器电压:2000V;Detector voltage: 2000V;
分析软件:FlexAnalysis、BioTools、Sequence Editor;Analysis software: FlexAnalysis, BioTools, Sequence Editor;
靶型号:MTP 384 ground steel;Target model: MTP 384 ground steel;
基质:DHB用30%的乙腈水溶液溶解成20mg/mL;Substrate: DHB is dissolved into 20mg/mL with 30% acetonitrile aqueous solution;
基质与样品:按1:1体积比混合,自然晾干后放入质谱测试仓。Matrix and sample: mix in a 1:1 volume ratio, dry naturally, and put into the mass spectrometer test chamber.
(4)酶切前样品的处理:将杂质F精肽用0.1%TFA溶液溶解成浓度约为0.1mg/mL水溶液,对溶液进行一级质谱分析。(4) Sample treatment before digestion: the impurity F refined peptide was dissolved with 0.1% TFA solution into an aqueous solution with a concentration of about 0.1 mg/mL, and the solution was analyzed by mass spectrometry.
(5)酶切样品的处理:将杂质F精肽用50mM碳酸氢铵溶液溶解成浓度约为1mg/mL的水溶液,用Trypsin对其进行酶切,分析酶切后的一、二级质谱。(5) Treatment of digested samples: Dissolve the impurity F refined peptide with 50mM ammonium bicarbonate solution into an aqueous solution with a concentration of about 1mg/mL, digest it with Trypsin, and analyze the primary and secondary mass spectra after digestion.
(6)结果如下:(6) The results are as follows:
1)、杂质F的一级质谱结果见图4。1) The first-stage mass spectrum result of impurity F is shown in Figure 4.
2)、杂质F酶切后的一级质谱结果见图5。2) Figure 5 shows the mass spectrum result of impurity F after digestion.
3)、杂质F酶切后二级质谱结果见图6-9。3) The results of the secondary mass spectrometry after impurity F digestion are shown in Figure 6-9.
4)、用BioTools软件处理MALDI-TOF-TOF二级质谱的结果见图10-13。4). The results of MALDI-TOF-TOF secondary mass spectrometry processing with BioTools software are shown in Figure 10-13.
(7)数据分析(7) Data analysis
表3:杂质F实测一级质谱与理论值比较Table 3: Comparison of measured mass spectra and theoretical values of impurity F
样品sample | 一级质谱理论值Theoretical value of primary mass spectrometry | 一级质谱实测值Measured value of primary mass spectrometer |
杂质FImpurity F | 4100.7274100.727 | 4100.8244100.824 |
表4:杂质F酶切后实测一级质谱与理论值比较Table 4: Comparison of actual mass spectra and theoretical values after impurity F digestion
片段肽序Fragment peptide sequence | 一级质谱理论值Theoretical value of primary mass spectrometry | 一级质谱实测值Measured value of primary mass spectrometer |
SVSEIQLMHNLGKSVSEIQLMHNLGK | 1455.7621,455.762 | 1455.7321,455.732 |
HLNSMERHLNSMER | 886.420886.420 | 886.461886.461 |
VEWLRVEWLR | 702.393702.393 | 702.387702.387 |
LQDVHNFLQDVHNF | 872.426872.426 | 872.406872.406 |
A、从表3可见,杂质F的一级质谱与理论值一致。A. It can be seen from Table 3 that the primary mass spectrum of impurity F is consistent with the theoretical value.
B、Trypsin能选择性地水解蛋白质中赖氨酸或精氨酸的羧基端肽键,从表4可见杂质F酶切后肽段的二级碎片峰与理论一致,母离子M/Z为1455、886、702、872的肽段二级碎片峰分别与肽段SVSEIQLMHNLGK、HLNSMER、VEWLR、LQDVHNF的理论碎片峰一致,其中LQDVHNF肽段是在酶切过程中由LQ(Asu)VHNF水解得到的。B. Trypsin can selectively hydrolyze the carboxyl terminal peptide bond of lysine or arginine in protein. From Table 4, it can be seen that the secondary fragment peak of the peptide fragment after impurity F digestion is consistent with the theory, and the parent ion M/Z is 1455 The secondary fragment peaks of the peptides of 886, 702, and 872 are consistent with the theoretical fragment peaks of peptides SVSEIQLMHNLGK, HLNSMER, VEWLR, and LQDVHNF respectively. The LQDVHNF peptide is obtained by hydrolysis of LQ(Asu)VHNF during the digestion process.
因此,可以确定杂质F的肽序为: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-Asu-Val-His-Asn-Phe-OH(SEQ ID NO:1)。Therefore, the peptide sequence of impurity F can be determined as: 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-Asu-Val-His-Asn-Phe-OH (SEQ ID NO:1).
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.
Claims (9)
- 一种特立帕肽杂质F的合成方法,其特征在于,所述特立帕肽杂质F的结构式如式I所示:A method for synthesizing teriparatide impurity F is characterized in that the structural formula of teriparatide impurity F is as shown in formula I:合成方法包括如下步骤:The synthesis method includes the following steps:步骤A:采用固相合成法,以树脂为载体,依次偶联Fmoc-Asn(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-Val-OH、Fmoc-Asp(ODmab)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Val-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Ser(tBu)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-Leu-OH、Fmoc-Asn(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-Met-OH、Fmoc-Leu-OH、Fmoc-Gln(Trt)-OH、Fmoc-Ile-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH 和Boc-Ser(tBu)-OH,得到带有保护基的肽树脂;所述Asp(PG)中的PG保护基为All或Dmab保护基;Step A: Using solid phase synthesis method, using resin as carrier, sequentially coupling Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(ODmab)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc- Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Ser( tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu )-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH and Boc-Ser(tBu)-OH to obtain a peptide resin with protecting groups; the PG protecting group in Asp(PG) is All Or Dmab protecting group;步骤B:脱除Asp(PG)的PG保护基,关环,得到肽树脂;Step B: Remove the PG protecting group of Asp(PG) and close the ring to obtain a peptide resin;步骤C:将肽树脂进行裂解,得到特立帕肽杂质F。Step C: The peptide resin is cleaved to obtain teriparatide impurity F.
- 根据权利要求1所述的合成方法,其特征在于,步骤A中,所述树脂为Wang Resinn,替代度为0.1~3.0mmol/g。The synthesis method according to claim 1, wherein in step A, the resin is Wang Resinn, and the degree of substitution is 0.1-3.0 mmol/g.
- 根据权利要求1所述的合成方法,其特征在于,步骤A中,所述偶联采用的偶联剂为HOBt/DIPCDI、HOBt/PyBop/DIPEA、HBTU/HOBt/DIPEA、HOAt/DIPCDI、HATU/HOAt/DIPEA或HOAt/PyAop/DIPEA中的一种或几种。The method of synthesis according to claim 1, wherein in step A, the coupling agent used in the coupling is HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/ One or more of HOAt/DIPEA or HOAt/PyAop/DIPEA.
- 根据权利要求1所述的合成方法,其特征在于,步骤A中,所述偶联采用的脱除Fmoc保护基团的试剂为10%~30%哌啶溶液,溶解氨基酸和哌啶溶液所用的溶剂为NMP、THF、DCM、DMF或DMSO中的一种或几种。The synthesis method according to claim 1, characterized in that, in step A, the reagent for removing the Fmoc protective group used in the coupling is a 10%-30% piperidine solution, which is used to dissolve the amino acid and the piperidine solution The solvent is one or more of NMP, THF, DCM, DMF or DMSO.
- 根据权利要求1所述的合成方法,其特征在于,步骤B中,所述Asp(PG)中的PG保护基为All保护基,所述脱除采用的催化剂为四三苯基膦钯,清除剂为苯硅烷或吗啉。The synthesis method according to claim 1, characterized in that, in step B, the PG protecting group in the Asp(PG) is an All protecting group, and the catalyst used for the removal is tetrakistriphenylphosphine palladium. The agent is phenylsilane or morpholine.
- 根据权利要求1所述的合成方法,其特征在于,步骤B中,所述Asp(PG)中的PG保护基为Dmab保护基,所述脱除采用的清除剂为1%~5%水合肼-DMF溶液。The synthesis method according to claim 1, wherein in step B, the PG protecting group in the Asp(PG) is a Dmab protecting group, and the scavenger used for the removal is 1% to 5% hydrazine hydrate -DMF solution.
- 根据权利要求1所述的合成方法,其特征在于,步骤B中,所述关环采用叠氮磷酸二苯酯关环。The synthesis method according to claim 1, wherein in step B, the ring closure adopts diphenyl azide phosphate ring closure.
- 根据权利要求1至7中任一项所述的合成方法,其特征在于,步骤C中,所述裂解采用的裂解液为含有捕捉剂的三氟乙酸溶液,所述捕捉剂为PhSMe、PhOH、EDT、H 2O、TIS、PhOMe中的一种或几种。 The synthesis method according to any one of claims 1 to 7, characterized in that, in step C, the lysis solution used in the lysis is a trifluoroacetic acid solution containing a capture agent, and the capture agent is PhSMe, PhOH, One or more of EDT, H 2 O, TIS, PhOMe.
- 根据权利要求8所述的合成方法,其特征在于,所述捕捉剂为TIS,裂解液中三氟乙酸与TIS的体积比为(90~99):(1~10)。The synthesis method according to claim 8, wherein the capture agent is TIS, and the volume ratio of trifluoroacetic acid to TIS in the lysate is (90-99): (1-10).
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1557176A1 (en) * | 2004-01-26 | 2005-07-27 | Ferring B.V. | Treatment of bone metastases by means of PTH receptor agonists |
US20060069021A1 (en) * | 2004-08-13 | 2006-03-30 | Nastech Pharmaceutical Company Inc. | Compositions and methods for intranasal administration of inactive analogs of PTH or inactivated preparations of PTH or PTH analogs |
CN103467595A (en) * | 2013-09-06 | 2013-12-25 | 深圳翰宇药业股份有限公司 | Method for preparing teriparatide |
CN104017064A (en) * | 2014-06-13 | 2014-09-03 | 杭州诺泰制药技术有限公司 | Method for preparing teriparatide |
CN104530218A (en) * | 2015-01-07 | 2015-04-22 | 哈尔滨吉象隆生物技术有限公司 | Solid-phase synthesis method of teriparatide |
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1557176A1 (en) * | 2004-01-26 | 2005-07-27 | Ferring B.V. | Treatment of bone metastases by means of PTH receptor agonists |
US20060069021A1 (en) * | 2004-08-13 | 2006-03-30 | Nastech Pharmaceutical Company Inc. | Compositions and methods for intranasal administration of inactive analogs of PTH or inactivated preparations of PTH or PTH analogs |
CN103467595A (en) * | 2013-09-06 | 2013-12-25 | 深圳翰宇药业股份有限公司 | Method for preparing teriparatide |
CN104017064A (en) * | 2014-06-13 | 2014-09-03 | 杭州诺泰制药技术有限公司 | Method for preparing teriparatide |
CN104530218A (en) * | 2015-01-07 | 2015-04-22 | 哈尔滨吉象隆生物技术有限公司 | Solid-phase synthesis method of teriparatide |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116120426A (en) * | 2022-12-29 | 2023-05-16 | 江苏诺泰澳赛诺生物制药股份有限公司 | Method for purifying liraglutide oxidized impurities |
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