CN112940104A - 一种特立帕肽杂质f - Google Patents
一种特立帕肽杂质f Download PDFInfo
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- CN112940104A CN112940104A CN201911259992.9A CN201911259992A CN112940104A CN 112940104 A CN112940104 A CN 112940104A CN 201911259992 A CN201911259992 A CN 201911259992A CN 112940104 A CN112940104 A CN 112940104A
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Abstract
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。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,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%。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。
Description
技术领域
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。
背景技术
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EliLilly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-Asp30-Val31-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。
发明内容
有鉴于此,本发明提供了一种特立帕肽杂质F。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%。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种特立帕肽杂质F,其结构式如式I所示:
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:
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。
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:
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;
步骤B:脱除Asp(PG)的PG保护基,关环,得到肽树脂;
步骤C:将肽树脂进行裂解,得到特立帕肽杂质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。
作为优选,步骤A中,树脂为Wang Resinn,替代度为0.1~3.0mmol/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。
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。
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。
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。
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。
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。
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。
作为优选,步骤B中,关环采用叠氮磷酸二苯酯关环。
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、H2O、TIS、PhOMe中的一种或几种。
优选地,捕捉剂为TIS。
作为优选,裂解液中三氟乙酸与TIS的体积比为(90~99):(1~10)。
优选地,裂解液中三氟乙酸与TIS的体积比为95:5。
本发明提供了一种特立帕肽杂质F。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:
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;
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。
附图说明
图1:杂质F的合成路线图;
图2:杂质F粗肽的HPLC图谱(实施例3);
图3:杂质F精肽的HPLC图谱(实施例8);
图4:杂质F的一级质谱图;
图5:杂质F酶切后的一级质谱图;
图6:杂质F酶切后母离子M/Z为1455的片段的二级质谱图;
图7:杂质F酶切后母离子M/Z为886的片段的二级质谱图;
图8:杂质F酶切后母离子M/Z为702的片段的二级质谱图;
图9:杂质F酶切后母离子M/Z为872的片段的二级质谱图;
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;
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;
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;
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。
具体实施方式
本发明公开了一种特立帕肽杂质F,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。
缩写及英文含义如下:
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:
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。
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。
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、H2O、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。
具体路线见图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。
下面结合实施例,进一步阐述本发明:
实施例1:氨基酸的偶联
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。
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。
实施例2:氨基酸的偶联
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。
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。
实施例3:杂质F的合成
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。
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乙醚中沉降,离心洗涤,真空干燥得到杂质F粗肽90g,纯度42.96%(图2、表1)。
表1
实施例4:杂质F的合成
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。
加入裂解液(TFA:H2O=95:5,体积比)1.6L室温裂解2小时,过滤,滤液倒入16L乙醚中沉降,离心洗涤,真空干燥得到杂质F粗肽120g,纯度40.35%。
实施例5:杂质F的合成
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乙醚中沉降,离心洗涤,真空干燥得到杂质F粗肽9g,纯度约1%。
实施例6:杂质F的合成
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乙醚中沉降,离心洗涤,真空干燥得到杂质F粗肽2g,纯度0.4%。
实施例7:杂质F的合成
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%。
实施例8:杂质F的纯化
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)。
1、HPLC制备纯化色谱条件如下:
(1)流动相A1:0.1%TFA溶液,流动相B1:纯乙腈;
(3)监测波长:220nm;
(4)流速:500mL/min
(5)纯化梯度:
时间(min) | 流动相A1(%) | 流动相B1(%) |
0 | 90 | 10 |
5 | 75 | 25 |
105 | 65 | 35 |
115 | 30 | 70 |
2、HPLC分析检测色谱条件如下:
(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);
(2)色谱柱:Waters BEHPeptide 300-C18-1.7μm 2.1*100mm;
(3)流速:0.4mL/min;
(4)监测波长:214nm;
(5)柱温:60℃;
(6)检测梯度:
时间(min) | 流动相A(%) | 流动相B(%) |
0 | 100 | 0 |
5 | 65 | 35 |
29 | 61 | 39 |
30 | 0 | 100 |
35 | 0 | 100 |
35.1 | 100 | 0 |
40 | 100 | 0 |
表2
实施例9:杂质F的结构确认
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。
1、实验条件:
(1)仪器型号:MALDI-TOF-TOF,Autoflex Speed;
(2)一级质谱
激光器波长和频率:ND:YAG 355nm,1000Hz;
极性:Positive;
操作模式:RP_700-3500;
检测器电压:2000V;
分析软件:FlexAnalysis;
靶型号:MTP 384ground steel;
基质:DHB用30%的乙腈水溶液溶解成20mg/mL;
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。
(3)二级质谱
激光器波长和频率:ND:YAG 355nm,1000Hz;
极性:Positive reflector;
操作模式:LIFT;
检测器电压:2000V;
分析软件:FlexAnalysis、BioTools、Sequence Editor;
靶型号:MTP 384ground steel;
基质:DHB用30%的乙腈水溶液溶解成20mg/mL;
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。
(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。
(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。
(6)结果如下:
1)、杂质F的一级质谱结果见图4。
2)、杂质F酶切后的一级质谱结果见图5。
3)、杂质F酶切后二级质谱结果见图6-9。
4)、用BioTools软件处理MALDI-TOF-TOF二级质谱的结果见图10-13。
(7)数据分析
表3:杂质F实测一级质谱与理论值比较
样品 | 一级质谱理论值 | 一级质谱实测值 |
杂质F | 4100.727 | 4100.824 |
表4:杂质F酶切后实测一级质谱与理论值比较
片段肽序 | 一级质谱理论值 | 一级质谱实测值 |
SVSEIQLMHNLGK | 1455.762 | 1455.732 |
HLNSMER | 886.420 | 886.461 |
VEWLR | 702.393 | 702.387 |
LQDVHNF | 872.426 | 872.406 |
A、从表3可见,杂质F的一级质谱与理论值一致。
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。
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)。
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。
序列表
<110> 深圳翰宇药业股份有限公司
<120> 一种特立帕肽杂质F
<130> S19P2976
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 34
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> UNSURE
<222> (30)..(30)
<223> Xaa(30)=Asu
<220>
<221> UNSURE
<222> (30)..(30)
<223> The 'Xaa' at location 30 stands for Gln, Arg, Pro, or Leu.
<400> 1
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Xaa Val His
20 25 30
Asn Phe
Claims (10)
2.根据权利要求1所述的特立帕肽杂质F,其特征在于,其合成方法包括如下步骤:
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;
步骤B:脱除Asp(PG)的PG保护基,关环,得到肽树脂;
步骤C:将肽树脂进行裂解,得到特立帕肽杂质F。
3.根据权利要求2所述的特立帕肽杂质F,其特征在于,步骤A中,所述树脂为WangResinn,替代度为0.1~3.0mmol/g。
4.根据权利要求2所述的特立帕肽杂质F,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。
5.根据权利要求2所述的特立帕肽杂质F,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。
6.根据权利要求2所述的特立帕肽杂质F,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。
7.根据权利要求2所述的特立帕肽杂质F,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。
8.根据权利要求2所述的特立帕肽杂质F,其特征在于,步骤B中,所述关环采用叠氮磷酸二苯酯关环。
9.根据权利要求2至8中任一项所述的特立帕肽杂质F,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、H2O、TIS、PhOMe中的一种或几种。
10.根据权利要求9所述的特立帕肽杂质F,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)。
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CN116120426A (zh) * | 2022-12-29 | 2023-05-16 | 江苏诺泰澳赛诺生物制药股份有限公司 | 一种纯化利拉鲁肽氧化杂质的方法 |
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