CN109134615B - 一种比伐芦定的制备方法 - Google Patents
一种比伐芦定的制备方法 Download PDFInfo
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- CN109134615B CN109134615B CN201811101059.4A CN201811101059A CN109134615B CN 109134615 B CN109134615 B CN 109134615B CN 201811101059 A CN201811101059 A CN 201811101059A CN 109134615 B CN109134615 B CN 109134615B
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- gly
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- bivalirudin
- pro
- resin
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- 108010055460 bivalirudin Proteins 0.000 title claims abstract description 67
- OIRCOABEOLEUMC-GEJPAHFPSA-N bivalirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)CNC(=O)CNC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 OIRCOABEOLEUMC-GEJPAHFPSA-N 0.000 title claims abstract description 63
- 229960001500 bivalirudin Drugs 0.000 title claims abstract description 57
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 42
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 27
- 238000000746 purification Methods 0.000 claims abstract description 24
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 18
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- 150000001413 amino acids Chemical group 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 11
- FBKUOPULLUJMOC-UHFFFAOYSA-N 2-[[2-(9h-fluoren-9-ylmethoxycarbonylamino)acetyl]amino]acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 FBKUOPULLUJMOC-UHFFFAOYSA-N 0.000 claims abstract description 10
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- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000005695 Ammonium acetate Substances 0.000 claims abstract description 6
- 229940043376 ammonium acetate Drugs 0.000 claims abstract description 6
- 235000019257 ammonium acetate Nutrition 0.000 claims abstract description 6
- 239000011347 resin Substances 0.000 claims description 41
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- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 25
- 239000012071 phase Substances 0.000 claims description 22
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- 238000010511 deprotection reaction Methods 0.000 claims description 18
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 16
- 239000003795 chemical substances by application Substances 0.000 claims description 15
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 11
- NTFTULBKHJJQAW-HNNXBMFYSA-N 9h-fluoren-9-ylmethyl n-[(2s)-4-methyl-1-oxopentan-2-yl]carbamate Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C=O)C3=CC=CC=C3C2=C1 NTFTULBKHJJQAW-HNNXBMFYSA-N 0.000 claims description 10
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 10
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical group [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 claims description 10
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- JAUKCFULLJFBFN-VWLOTQADSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid Chemical compound C1=CC(OC(C)(C)C)=CC=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JAUKCFULLJFBFN-VWLOTQADSA-N 0.000 claims description 8
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 8
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- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 claims description 8
- ZPGDWQNBZYOZTI-SFHVURJKSA-N (2s)-1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-SFHVURJKSA-N 0.000 claims description 7
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- OTKXCALUHMPIGM-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 OTKXCALUHMPIGM-FQEVSTJZSA-N 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 6
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- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 claims description 5
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 5
- 230000009089 cytolysis Effects 0.000 claims description 5
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- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 5
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- ZYJPUMXJBDHSIF-LLVKDONJSA-N (2r)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-phenylpropanoic acid Chemical compound CC(C)(C)OC(=O)N[C@@H](C(O)=O)CC1=CC=CC=C1 ZYJPUMXJBDHSIF-LLVKDONJSA-N 0.000 claims description 4
- SJVFAHZPLIXNDH-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=CC=C1 SJVFAHZPLIXNDH-QFIPXVFZSA-N 0.000 claims description 4
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 claims description 4
- KJYAFJQCGPUXJY-UMSFTDKQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-(tritylamino)butanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(=O)NC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KJYAFJQCGPUXJY-UMSFTDKQSA-N 0.000 claims description 4
- QXVFEIPAZSXRGM-DJJJIMSYSA-N (2s,3s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@@H](C)CC)C(O)=O)C3=CC=CC=C3C2=C1 QXVFEIPAZSXRGM-DJJJIMSYSA-N 0.000 claims description 4
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- JZBHVBQPWXZALQ-FQEVSTJZSA-N 2-o-(2,5-dioxopyrrolidin-1-yl) 1-o-(9h-fluoren-9-ylmethyl) (2s)-pyrrolidine-1,2-dicarboxylate Chemical compound O=C([C@H]1N(CCC1)C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)ON1C(=O)CCC1=O JZBHVBQPWXZALQ-FQEVSTJZSA-N 0.000 claims 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 4
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Abstract
本发明涉及多肽合成领域,特别涉及一种比伐芦定的制备方法。发明采用Fmoc‑Pro‑Gly‑Gly‑OH与Fmoc‑Gly‑Gly‑OH不存在侧链保护的氨基酸片段为原料,工艺简单,质量可控,易工业化生产,且通过Fmoc‑Pro‑Gly‑Gly‑OH更好的避免了Fmoc‑Pro‑Gly‑Gly‑Gly‑Gly‑OH溶解性差、重结晶困难等难点,同时避免了Bivalirudin±1Gly和Bivalirudin±2Gly的杂质产生。固相合成比伐芦定操作简单,易工业生产,采用TFA裂解,避免HF使用,安全性高,合成比伐芦定粗品纯度在93%以上,产率99%以上。采用乙酸铵溶液和乙腈进行一步纯化,TFA/水和乙腈完成二步纯化,冻干即可得到精肽纯度99.8%以上,收率58%以上,纯化工艺简单,更易于工业化大规模生产。
Description
技术领域
本发明涉及多肽原料药制备领域,特别涉及一种比伐芦定的制备方法。
背景技术
比伐芦定,英文通用名称为Bivalirudin,商品名为Angiomax,是一种直链线性二十肽,其分子结构式如下:
氨基酸序列如下:
D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ilu-Pro-Glu-G lu-Tyr-Leu
分子式:C98H138N24O33,分子量:2180.32,CAS:128270-60-0。
注射用比伐芦定(商品名:Angiomax)是满足FDA要求的多肽类产品,比伐芦定为直接的、特异的、可逆的凝血酶抑制剂,作为抗凝剂用于成人择期经皮冠状动脉介入治疗(PCI)。比伐芦定(bivalirudin),由美国制药公司(The Medicines Company)出品。2000年12月经FDA批准上市。
比伐芦定是凝血酶的直接抑制剂,与游离及血栓上凝血酶的催化位点和阴离子外结合位点特异结合起抑制作用。凝血酶是一种丝氨酸蛋白酶,在血栓形成过程中起重要的作用,它首先将纤维蛋白原分解为纤维蛋白单体,然后将凝血酶因子XIII激活为XIIIa,使纤维蛋白之间共价连接成为稳定的网架,形成血栓。凝血酶同时还可激活凝血酶因子V和VIII,进一步促进凝血酶的形成,还可激活血小板导致血小板凝聚,释放血小板聚集物。比伐芦定与凝血酶的结合过程是可逆的,凝血酶通过缓慢的酶解比伐芦定Arg3-Pro4之间的肽键可使凝血酶恢复原来的生物活性。体外研究表明,比伐芦定不仅能抑制游离的凝血酶,还能抑制与血块结合的凝血酶而不会被血小板释放出的物质中和,它能延长正常人血浆激活的部分促凝血酶原激酶时间(aPTT)、凝血酶时间(TT)和凝血酶原时间(PT),并与比伐芦定的浓度呈线性关系,但临床应用是否存在这种相关性尚不清楚。
比伐芦定结构中含有-Gly-Gly-Gly-Gly-片段,固相逐步合成过程中,由于Gly自身特性,极易在主峰前后产生Bivalirudin±1Gly和Bivalirudin±2Gly的杂质,这些杂质在后续分离中很难去除,专利CN102260323A先在液相条件下合成Fmoc-Gly-Gly-Gly-Gly-OH四肽片段,然后将其接到肽树脂上,再逐步接上剩余氨基酸,但是Fmoc-Gly-Gly-Gly-Gly-OH四肽片段溶解性较差,重结晶极为困难,不易得到高纯度Fmoc-Gly-Gly-Gly-Gly-OH四肽片段。成都圣诺生物制药有限公司专利CN102286076A和CN102532274A通过Fmoc-Gly-Gly-Gly-Gly-OH、片段Fmoc-Arg(Pbf)-Pro-OH与Fmoc-Gly-Gly-Gly-Gly-Asn(R)-Gly-Asp(OtBu)-OH作为比伐卢定合成片段原料,减少了Bivalirudin±1Gly、Bivalirudin±2Gly和Bivalirudin-Arg杂质,但是,该多片段合成工艺复杂,质量可控性差,不易工业生产。专利CN102731624A先用酸不稳定CTC树脂合成Fmoc-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-OH,然后两倍投料条件下接到Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(Ot Bu)-Tyr(tBu)-Leu-Wang Resins,然后TFA组分裂解得到目标肽,此方法针对Bivalirudin±1Gly、Bivalirudin±2Gly控制效果有限,且片段偶联效果欠佳,成本较高。齐鲁制药有限公司专利CN103242431A采取液相中Fmoc-Pro-OH与H-Gly-Gly-Gly-Gly-OH合成Fmoc-Pro-Gly-Gly-Gly-Gly-OH五肽片段为原料,Fmoc-Leu-Wang树脂为固相载体合成比伐芦定,有效避免Bivalirudin±1Gly和Bivalirudin±2Gly的杂质产生。虽然Fmoc-Pro-Gly-Gly-Gly-Gly-OH溶解性比Fmoc-Gly-Gly-Gly-Gly-OH好些,但是五肽片段Fmoc-Pro-Gly-Gly-Gly-Gly-OH制备仍无法通过萃取得到片段五肽产物,而是从水中获得,不易工业生产,另一方面重结晶困难,仍存在Fmoc-Pro-Gly-Gly-Gly-Gly-Gly-OH、Fmoc-Pro-Gly-Gly-Gly-Gly-Gly-OH、Fmoc-Pro-Gly-Gly-Gly-OH的杂质,需用DMF溶解后重结晶质量可控性较差。专利201510754607.3中采用片段Pro-Gly-Gly与Gly-Gly-Asn(Trt)为原料固相合成,其中片段Gly-Gly-Asn(Trt)存在侧链保护基,液相调酸或固相裂解的方式均可能导致保护基的脱落,质量可控性差,为大规模生产带来不便。专利201510005428.X中采用Fmoc-Gly-Gly-OH为固相合成原料,进行比伐芦定固相合成,但是研究发现原料Fmoc-Gly-Gly-OH中存在游离的H-Gly-OH及H-Gly-Gly-OH氨基酸,将增加产生Bivalirudin±1Gly、Bivalirudin±2Gly杂质产生的风险。综上所述,需要一种易工业生产、质量可控的一种比伐芦定制备方法。
发明内容:
本发明旨在针对现有技术的不足,对比伐芦定制备方法进行总结,发明了一种既能有效避免Bivalirudin±1Gly和Bivalirudin±2Gly的杂质产生,又具有质量易控,易工业化的一种比伐芦定的制备方法。
本发明的技术方案如下:
一种比伐芦定的制备方法,该方法具体步骤如下:
(1)采用Fmoc-Leu-Wang树脂或Fmoc-Leu-CTC树脂作为固相载体,加入脱保护剂,脱除树脂上的Fmoc保护基;
(2)将步骤(1)中脱除Fmoc保护基的树脂在活化剂存在下,按比伐芦定序列依次偶联Fmoc保护氨基酸,获得侧链保护的比伐芦定肽树脂:
R1-D-Phe-Pro-Arg(Pbf)-X-Y-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-树脂
其中,R1为Fmoc或Boc;
X为Pro-Gly-Gly;
Y为Gly-Gly;
(3)将步骤(2)得到的侧链保护的比伐芦定肽树脂进行裂解,沉降得比伐芦定粗品;
(4)将步骤(3)中的比伐芦定粗品纯化、冻干,得比伐芦定精品。
上述步骤(1)所述Fmoc-Leu-Wang树脂或Fmoc-Leu-CTC树脂替代度范围为0.40~0.80mmol/g,优选0.6~0.7mmol/g;
步骤(1)中,所述脱保护剂是20%v/v哌啶/N,N-二甲基甲酰胺(DMF)溶液,树脂与脱保护试剂质量体积比是1:6~12,脱保护反应时间5~20min,优选的,脱保护试剂用量为8~10倍树脂质量,脱保护反应时间8~12min。
所述步骤(2)中Fmoc保护氨基酸形式为:
Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Pro-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gly-Gly-OH、Fmoc-Pro-Gly-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-D-Phe-OH或Boc-D-Phe-OH,投料倍数优选合成规模的2.0~3.0倍;
所述步骤(2)中活化剂为HOBT/DIC、HOAT/DIC、HBTU/HOBT/DIEA、HATU/HOAT/DIEA中的一种,优选的,活化剂投料量为所述树脂的2.2~3.3倍;偶联时间1~5h,优选2~3h。
Fmoc-Pro-Gly-Gly-OH片段的合成步骤为:采用液相合成法,以H-Gly-Gly-OH、Fmoc-Pro-Osu、碳酸钠为原料,1,4-二氧六环、水为溶剂,TLC检测反应,加入Fmoc-Pro-Osu投料倍数的5~8倍乙酸乙酯,萃取浓缩析晶后重结晶,过滤干燥得Fmoc-Pro-Gly-Gly-OH。
所述H-Gly-Gly-OH与碳酸钠的摩尔比优选1:0.8~1.0,Fmoc-Pro-OSu与双甘肽的摩尔比优选1:1.2~1.4。
所述步骤(3)中,裂解用到的裂解液为90~95%的TFA和苯甲硫醚、乙二硫醇、苯酚、水、三异丙基硅烷中的一种或几种,优选TFA/H2O/TIS/苯甲硫醚;裂解液用量优选肽树脂质量的6~8倍;裂解反应时间优选2.0~3.0h。
步骤(4)中,纯化采用反相C18或C8制备柱,流动相A相:50~100mmol/L乙酸铵溶液,B相乙腈,梯度洗脱,进行一步纯化,得到纯度96%以上的一纯合格液;再采用流动相A相0.01~0.05%TFA/水,B相乙腈,流速600ml/min,梯度洗脱,完成转盐纯化(二步纯化),获到二纯合格液,合格溶液浓缩冻干,最终得到纯度大于99.5%的精肽样品。
本发明的有益效果:
1、本发明采用Fmoc-Pro-Gly-Gly-OH与Fmoc-Gly-Gly-OH不存在侧链保护的氨基酸片段为原料,工艺简单,质量可控,易工业化生产,以Fmoc-Pro-Osu与H-Gly-Gly-OH原料液相合成Fmoc-Pro-Gly-Gly-OH,通过重结晶纯度可达99.9%以上,产率92%以上,且通过Fmoc-Pro-Gly-Gly-OH更好的避免了Fmoc-Pro-Gly-Gly-Gly-Gly-OH溶解性差、重结晶困难等难点,同时避免了Bivalirudin±1Gly和Bivalirudin±2Gly的杂质产生。
2、本发明固相合成比伐芦定操作简单,易工业生产,采用TFA裂解,避免HF使用,安全性高;同时采用Fmoc-Pro-Gly-Gly-OH和Fmoc-Gly-Gly-OH为原料,减少了单独Fmoc-Gly-Gly-OH为原料产生杂质的风险,有效避免了Bivalirudin±1Gly和Bivalirudin±2Gly的杂质产生,比伐芦定粗品纯度在95%以上,产率99%以上。
3、本发明纯化步骤采用乙酸铵溶液和乙腈进行一步纯化,TFA/水和乙腈完成二步纯化,冻干即可得到精肽纯度99.8%以上,收率59%以上,纯化工艺简单,更易于工业化大规模生产。
具体实施方式:
以下结合具体实施方式对本发明作进一步描述,下述说明仅是为了解释本发明,本发明的保护范围并不限于这些实施例,本领域的技术人员应理解,对本发明内容所作的等同替换,或相应的改进,仍属于本发明的保护范围之内。
权利书与说明书中所提原料与试剂缩写含义:
CTC树脂 2-氯三苯甲基氯树脂
Wang Resins 王树脂
Fmoc 9-芴甲氧羰基
tBu 叔丁基
Boc 叔丁氧羰基
Pbf 2,2,4,6,7-五甲基苯并呋喃-5-磺酰基
Trt 三苯甲基
DCM 二氯甲烷
DMF N,N-二甲基甲酰胺
DMAP 4-二甲氨基吡啶
DIPEA N,N-二异丙基乙胺
DIC N,N-二异丙基碳二亚胺
HBTU 苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐
HATU 2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯
TBTU O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸
HOBT 1-羟基苯并三唑
HOAT 1-羟基-7-偶氮苯并三氮唑
DIEA N,N-二异丙基乙胺
TFA 三氟乙酸
TIS 三异丙基硅烷
HOSu N-羟基琥珀酰亚胺
Kaiser test检测试剂 茚三酮
实施例1:Fmoc-Pro-Gly-Gly-OH的合成
称取Fmoc-Pro-OSu(43.45g,100mmol)加入200ml溶剂1,4-二氧六环溶解,称取H-Gly-Gly-OH(18.48g,140mmol)与碳酸钠(20.14g,190mmol)加入200ml水中溶解,缓慢加入Fmoc-Pro-OSu溶液,采用TLC检测反应,室温反应3.0小时后,减压浓缩除去1,4-二氧六环,加入300ml乙酸乙酯,以2N稀盐酸调节pH至2~3,萃取保留乙酸乙酯相,浓缩析晶,过滤,干燥,加入360ml乙酸乙酯复溶,继续使用乙酸乙酯重结晶,浓缩,冷冻析晶,抽滤,干燥,得Fmoc-Pro-Gly-Gly-OH,称重41.84g,总收率92.77%,纯度99.98%。
实施例2:侧链全保护比伐芦定肽树脂的合成
称取Fmoc-Leu-Wang树脂25.0g(替代度0.6mmol/g),合成摩尔数为15.0mmol,加入至多肽合成反应器中,加入250.0ml DMF溶胀树脂30min后,抽掉DMF,使用脱保护试剂20%v/v哌啶/N,N-二甲基甲酰胺(DMF)溶液脱保护,每次200ml,脱保护反应10min,重复一次后,用250ml DMF洗涤树脂,洗涤6次,每次3min。
称取Fmoc-Tyr(tBu)-OH(20.67g,45.0mmol)和HOBt(6.69g,49.5mmol)于锥形瓶中加入100.0ml DMF溶解,冰水浴中加入DIC(7.76ml,49.5mmol)活化后加入至多肽合成反应器中,室温反应2.0h,采用Kaiser test检测反应终点(阴性无色)。重复上述Fmoc-Tyr(tBu)-OH偶联步骤(投料倍数及缩合剂用量一致),依次与比伐芦定序列对应Fmoc保护氨基酸偶联,对应保护氨基酸为:Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Pro-OH、Fmoc-Ile-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Phe-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gly-Gly-OH、Fmoc-Pro-Gly-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Pro-OH、Fmoc-D-Phe-OH,脱除Fmoc保护后得到侧链全保护比伐芦定肽树脂,结构如下:
H-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-树脂。
实施例3:侧链全保护比伐芦定肽树脂的合成
称取Fmoc-Leu-Wang树脂25.0g(替代度0.6mmol/g),合成摩尔数为15.0mmol,加入至多肽合成反应器中,加入250.0ml DMF溶胀树脂30min后,抽掉DMF,使用脱保护试剂20%v/v哌啶/N,N-二甲基甲酰胺(DMF)溶液脱保护,每次200ml,脱保护反应10min,重复一次后,用250ml DMF洗涤树脂,洗涤6次,每次3min。
称取Fmoc-Tyr(tBu)-OH(20.67g,45.0mmol)和HOBt(6.69g,49.5mmol)于锥形瓶中加入100.0ml DMF溶解,冰水浴中加入DIC(7.76ml,49.5mmol)活化后加入至多肽合成反应器中,室温反应2.0h,采用Kaiser test检测反应终点(阴性无色),重复上述Fmoc-Tyr(tBu)-OH偶联步骤(投料倍数及缩合剂用量一致),依次与比伐芦定序列对应Fmoc保护氨基酸偶联,其中,1位D-Phe采用Boc-D-Phe-OH,其余对应保护氨基酸为:Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Pro-OH、Fmoc-Ile-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Phe-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gly-Gly-OH、Fmoc-Pro-Gly-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Pro-OH、Boc-D-Phe-OH,得到侧链全保护比伐芦定肽树脂,结构如下:
Boc-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-树脂。
实施例4:比伐芦定粗品的合成
称取实施例2所得比伐卢定肽树脂50g置于1000ml圆底烧瓶中,冰浴下加入300ml裂解试剂(优选三氟醋酸/H2O/苯甲硫醚/TIS=90.0/5/2.5/2.5,体积比),反应3h后,抽滤,树脂用16ml TFA洗涤3次,合并滤液和洗液,倾倒入2.0L冷的甲叔醚中,沉降,抽滤得固体,甲叔醚洗涤6次后真空干燥得粗肽26.67g,纯度95.03%,收率99.40%。
实施例5:比伐芦定粗品的合成
称取实施例3所得比伐卢定肽树脂50g置于1000ml圆底烧瓶中,冰浴下加入300ml裂解试剂(优选三氟醋酸/H2O/苯甲硫醚/TIS=90.0/5/2.5/2.5,体积比),反应3h后,抽滤,树脂用15ml TFA洗涤3次,合并滤液和洗液,倾倒入2.0L冷的甲叔醚中,沉降,抽滤得固体,甲叔醚洗涤6次后真空干燥得粗肽26.55g,纯度96.26%,收率99.44%。
实施例6:比伐芦定粗肽纯化
将实施案例4中比伐芦定粗肽溶于乙腈和水的混合溶液中,抽滤,滤液采用C18或者C8反相色谱柱子上样纯化,流动相采用50~100mmol/L乙酸铵溶液,B相乙腈,梯度洗脱,进行一步纯化,得到纯度96%以上的一纯合格液;继续进行二次纯化,流动相采用三氟乙酸:乙腈(0.1-100:100-0.1,v/v),检测波长为220nm,收集样品峰并合并合格样品后,进行脱盐,冻干,得到比伐芦定精肽,纯化后精肽收率59.06%,纯度为99.82%。
实施例7:比伐芦定粗肽纯化
将实施案例5中比伐芦定粗肽溶于乙腈和水的混合溶液中,抽滤,滤液采用C18或者C8反相色谱柱子上样纯化,流动相采用50~100mmol/L乙酸铵溶液,B相乙腈,梯度洗脱,进行一步纯化,得到纯度96%以上的一纯合格液;继续进行二次纯化,流动相采用三氟乙酸:乙腈(0.1-100:100-0.1,v/v),检测波长为220nm,收集样品峰并合并合格样品后,进行脱盐,冻干,得到比伐芦定精肽,纯化后精肽收率59.13%,纯度为99.85%。
Claims (6)
1.一种比伐芦定的制备方法,其特征在于,该方法具体步骤为:
(1)采用Fmoc-Leu-Wang树脂或Fmoc-Leu-CTC树脂作为固相载体,加入脱保护剂,脱除树脂上的Fmoc保护基;
(2)将步骤(1)中脱除Fmoc保护基的树脂在活化剂存在下,按比伐芦定序列依次偶联Fmoc保护氨基酸:Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Pro-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gly-Gly-OH、Fmoc-Pro-Gly-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-D-Phe-OH或Boc-D-Phe-OH,获得侧链保护的比伐芦定肽树脂:
R1-D-Phe-Pro-Arg(Pbf)-X-Y-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-树脂
其中,R1为Fmoc或Boc;
X为Pro-Gly-Gly;
Y为Gly-Gly;
所述Fmoc-Pro-Gly-Gly-OH的合成步骤为:采用液相合成法,以H-Gly-Gly-OH、Fmoc-Pro-Osu、碳酸钠为原料,1,4-二氧六环、水为溶剂,TLC检测反应,加入Fmoc-Pro-Osu投料倍数的5~8倍乙酸乙酯,萃取浓缩析晶后重结晶,过滤干燥,所述H-Gly-Gly-OH与碳酸钠的摩尔比为1:0.8~1.0,Fmoc-Pro-OSu与双甘肽的摩尔比为1:1.2~1.4;
(3)将步骤(2)得到的侧链保护的比伐芦定肽树脂进行裂解,沉降得比伐芦定粗品;
(4)将步骤(3)中的比伐芦定粗品纯化、冻干,得比伐芦定精品,所述纯化采用反相C18或C8制备柱,流动相A相:50~100mmol/L乙酸铵溶液,B相乙腈,梯度洗脱,进行一步纯化,得到纯度96%以上的一纯合格液;将一纯合格液再采用流动相A相0.01~0.05%TFA/水,B相乙腈,流速600ml/min,梯度洗脱,完成转盐纯化,获到二纯合格液,合格溶液浓缩冻干,得比伐芦定精肽样品。
2.根据权利要求1所述的比伐芦定的制备方法,其特征在于,步骤(1)所述Fmoc-Leu-Wang树脂或Fmoc-Leu-CTC树脂替代度范围为0.40~0.80mmol/g;所述脱保护剂是20%v/v哌啶/N,N-二甲基甲酰胺(DMF)溶液,脱保护试剂用量为6~12倍树脂质量。
3.根据权利要求2所述的比伐芦定的制备方法,其特征在于,所述Fmoc-Leu-Wang树脂或Fmoc-Leu-CTC树脂替代度范围为0.6~0.7mmol/g,脱保护试剂用量为8~10倍树脂质量。
4.根据权利要求1所述的比伐芦定的制备方法,其特征在于,所述步骤(2)中Fmoc保护氨基酸投料倍数优选合成规模的2.0~3.0倍;活化剂为HOBT/DIC、HOAT/DIC、HBTU/HOBT/DIEA、HATU/HOAT/DIEA中的一种,活化剂投料量为所述树脂的2.2~3.3倍;偶联时间1~5h。
5.根据权利要求1所述的比伐芦定的制备方法,其特征在于,所述步骤(3)中,裂解用到的裂解液为90~95%的TFA和苯甲硫醚、乙二硫醇、苯酚、水、TIS中的一种或几种,裂解液用量为肽树脂质量的6~8倍,裂解反应时间2.0~3.0h。
6.根据权利要求5所述的比伐芦定的制备方法,其特征在于,所述裂解液为TFA/H2O/TIS/苯甲硫醚。
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