CN109134570B - C4′-氟代尿苷亚磷酰胺单体和c4′-氟代尿苷修饰rna的制备方法 - Google Patents
C4′-氟代尿苷亚磷酰胺单体和c4′-氟代尿苷修饰rna的制备方法 Download PDFInfo
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- CN109134570B CN109134570B CN201811099671.2A CN201811099671A CN109134570B CN 109134570 B CN109134570 B CN 109134570B CN 201811099671 A CN201811099671 A CN 201811099671A CN 109134570 B CN109134570 B CN 109134570B
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- BCNZYOJHNLTNEZ-UHFFFAOYSA-N tert-butyldimethylsilyl chloride Chemical compound CC(C)(C)[Si](C)(C)Cl BCNZYOJHNLTNEZ-UHFFFAOYSA-N 0.000 claims abstract description 10
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
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Abstract
Description
技术领域
本发明涉及寡聚核苷酸的化学合成领域,具体涉及C4′-氟代尿苷亚磷酰胺单体以及C4′-氟代尿苷定点修饰RNA的制备方法。
背景技术
氟代修饰的RNA在生物医学领域有广泛的应用前景[Org.Biomol.Chem.,2017,15,9552–9565]。C2′-氟代修饰的RNA成功应用于核酸适配体、小干扰RNA等,可以增强稳定性,提高效力[Nature,2011,478,404-408]。此外,C4′-氟修饰核苷类似物nucleocidin是天然存在的五种含氟天然产物之一,具有良好的抗代谢活性,C4′-氟代修饰的核苷也因此引起了关注。Guillerm[Bioorg.Med.Chem.Lett.1995,5,1455-1460]和Verdine[Org.Lett.,2007,9,5007-5009]分别发展了各自的方法合成了C4′-氟代核苷。然而C4′-氟代核苷不稳定,将C4′-氟代核苷转化为C4′-氟亚磷酰胺单体并制备C4′-氟代修饰的RNA一直没有成功,限制了对其生化性质的进一步探讨。
发明内容
本发明的目的是针对C4′-氟代核苷不稳定的实际问题,提供一种通过分阶段、选择性保护核糖核苷中羟基的策略,即C4′-氟代尿苷亚磷酰胺单体及其制备方法,以及利用C4′-氟代尿苷磷酰胺单体制备C4′-氟代尿苷定点修饰的RNA的方法。
本发明技术方案
1、为了实现上述目的,本发明第一方面提供了一种C4′-氟代尿苷亚磷酰胺单体,其中,该C4′-氟代尿苷亚磷酰胺单体的化学结构式为:
式(1)所示的化合物可以称为2′-O-(叔丁基二甲基硅烷基)-3′-[(2-氰基乙氧基)-N,N′-二异丙基亚磷酰胺]-4′-C-氟-5′-O-二甲氧基三苯甲基尿苷。
其中,在式(1)中,“DMTr-”为二甲氧基三苯甲基,“N(iPr)2-”为N,N-二异丙基胺基,“-TBDMS”为叔丁基二甲基硅烷基。
2、本发明第二方面提供了一种C4′-氟代尿苷亚磷酰胺单体的制备方法,该方法包括以下步骤:
(1)将4′-C-氟-5′-碘-5′-脱氧尿苷与硝酸银、吡啶和四氢呋喃形成第一混合溶液;之后将所述第一混合溶液与叔丁基二甲基氯硅烷进行反应,将反应后得到的溶液旋蒸除去溶剂,用二氯甲烷溶解后洗涤、过滤、干燥、柱层析得到2′-O-(叔丁基二甲基硅烷基)-4′-C-氟-5′-碘-5′-脱氧尿苷。
(2)将步骤(1)得到的2′-O-(叔丁基二甲基硅烷基)-4′-C-氟-5′-碘-5′-脱氧尿苷与4-二甲氨基吡啶、吡啶进行第二混合,形成第二混合溶液;再将所述第二混合溶液与乙酸酐进行反应;旋蒸除去溶剂,用二氯甲烷溶解后洗涤、过滤、干燥、柱层析得到2′-O-(叔丁基二甲基硅烷基)-3′-乙酸酯-4′-C-氟-5′-碘-5′-脱氧尿苷。
(3)将三氟乙酸与四正丁基氢氧化铵溶液混合调节pH后,加入步骤(2)得到的2′-O-(叔丁基二甲基硅烷基)-3′-乙酸酯-4′-C-氟-5′-碘-5′-脱氧尿苷、二氯甲烷进行第三混合,形成第三混合溶液;再将所述第三混合溶液与间氯过氧苯甲酸进行反应;然后将反应得到的有机相洗涤、过滤、干燥、柱层析得到2′-O-(叔丁基二甲基硅烷基)-3′-乙酸酯-4′-C-氟代尿苷。
(4)将步骤(3)得到的2′-O-(叔丁基二甲基硅烷基)-3′-乙酸酯-4′-C-氟代尿苷与氨/甲醇溶液进行反应;旋蒸除去溶剂,柱层析得到产物。
(5)将步骤(4)得到的产物与4-二甲氨基吡啶、吡啶以及4,4′-二甲氧基三苯甲基氯进行反应;旋蒸除去溶剂,用二氯甲烷溶解后然后将有机相洗涤、过滤、干燥、柱层析得到2′-O-(叔丁基二甲基硅烷基)-4′-C-氟-5′-O-二甲氧基三苯甲基尿苷。
(6)将步骤(5)得到的2′-O-(叔丁基二甲基硅烷基)-4′-C-氟-5′-O-二甲氧基三苯甲基尿苷与二氯甲烷、N,N′-二异丙基乙胺以及2-甲基咪唑进行第四混合,形成第四混合溶液;再将所述第四混合溶液与(2-氰基乙氧基)-N,N′-二异丙基亚磷酰氯进行反应;旋蒸除去溶剂,用二氯甲烷溶解后然后将有机相洗涤、过滤、干燥、柱层析得到2′-O-(叔丁基二甲基硅烷基)-3′-[(2-氰基乙氧基)-N,N′-二异丙基亚磷酰胺]-4′-C-氟-5′-O-二甲氧基三苯甲基尿苷,即本发明所述的C4′-氟代尿苷亚磷酰胺单体。
优选地,在步骤(1)中,所述4′-C-氟-5′-碘-5′-脱氧尿苷与所述硝酸银、所述吡啶、所述四氢呋喃、所述叔丁基二甲基氯硅烷用量的比为1mmol:3.5mmol:4mL:4mL:3.5mmol;以及,
所述反应的条件包括:温度为18-25℃,时间为1.5-2小时,搅拌速度为250-500转/分钟。
优选地,在步骤(2)中,所述2′-O-(叔丁基二甲基硅烷基)-4′-C-氟-5′-碘-5′-脱氧尿苷与所述4-二甲氨基吡啶、所述吡啶、所述乙酸酐用量的比为1mmol:0.3mmol:10mL:5mmol;以及,
所述乙酸酐以滴加的方式加入所述第二混合溶液中,所述乙酸酐的滴加速率为15滴/分钟;以及,
所述反应的条件包括:温度为18-25℃,时间为0.5-1小时,搅拌速度为250-500转/分钟。
优选地,在步骤(3)中,所述三氟乙酸与所述四正丁基氢氧化铵溶液、所述2′-O-(叔丁基二甲基硅烷基)-3′-乙酸酯-4′-C-氟-5′-碘-5′-脱氧尿苷、所述二氯甲烷、所述间氯过氧苯甲酸的用量比为0.8mL:10mL:1mmol:10mL:5mmol;其中所述四正丁基氢氧化铵溶液的质量浓度为55%,所述间氯过氧苯甲酸的纯度为85%;
所述三氟乙酸以滴加的方式加入所述四正丁基氢氧化铵溶液中调pH为4;所述间氯过氧苯甲酸分三次加入第三混合溶液中,间隔时间为0.5小时;以及,
所述反应的条件包括:温度为18-25℃,时间为40-46小时,搅拌速度为250-500转/分钟。
优选地,在步骤(4)中,所述2′-O-(叔丁基二甲基硅烷基)-3′-乙酸酯-4′-C-氟代尿苷与氨/甲醇溶液用量比为1mmol:8mL,其中氨/甲醇溶液的物质的量浓度为7mol/L;以及,
所述反应的条件包括:温度为18-25℃,时间为3-4小时,搅拌速度为250-500转/分钟。
优选地,在步骤(5)中,所述产物与所述4-二甲氨基吡啶、所述吡啶以及4,4′-二甲氧基三苯甲基氯的用量比为1mmol:0.3mmol:8mL:1.3mmol;以及,
所述反应的条件包括:温度为40-50℃,时间为3-5小时,搅拌速度为250-500转/分钟。
优选地,在步骤(6)中,所述2′-O-(叔丁基二甲基硅烷基)-4′-C-氟-5′-O-二甲氧基三苯甲基尿苷与所述二氯甲烷、所述N,N′-二异丙基乙胺、所述2-甲基咪唑、所述(2-氰基乙氧基)-N,N′-二异丙基亚磷酰氯的用量比为1mmol:20mL:10mmol:4mmol:5mmol;以及,
所述反应的条件包括:温度为18-25℃,时间为15分钟,搅拌速度为250-500转/分钟。
3、本发明第三方面提供了一种C4′-氟代尿苷定点修饰RNA的制备方法,其中,该制备方法中的合成试剂包括以上所述的制备方法制备得到的C4′-氟代尿苷亚磷酰胺单体。
优选地,该制备方法在ABI 394DNA/RNA合成仪上进行以得到寡聚核苷酸,其中:
优选地,所述合成试剂还包括叔丁基二甲基氯硅烷保护2′-OH的Ac-rC、Bz-rA、Ac-rG和rU;
优选地,三氯乙酸/二氯甲烷用于脱保护;
优选地,CapA试剂为乙酸酐、吡啶和四氢呋喃的组合物,且所述乙酸酐、所述吡啶和所述四氢呋喃的体积比为10体积%:(5-15)体积%:(75-85)体积%;
优选地,Cap B试剂为N-甲基咪唑和四氢呋喃的组合物,且所述N-甲基咪唑和所述四氢呋喃的体积比为10体积%:90体积%;
优选地,活化剂为乙硫基四唑和乙腈的组合物;
优选地,氧化剂为碘、水、吡啶和四氢呋喃的组合物,且所述碘、所述水、所述吡啶和所述四氢呋喃的用量比为0.02M:(1-2)体积%:(10-30)体积%:(75-85)体积%;以及所述,
优选地,所述C4’-氟代RNA亚磷酰胺单体的偶合时间为0.5-1小时;
优选地,将上述固相合成得到的寡聚核苷酸用0.75mL浓度为28重量%的氨水混合0.25mL无水乙醇在55℃下处理10-12h,然后,取出上清液离心浓缩后,加入0.5mL三乙胺三氢氟酸盐处理20-24h,经C18柱脱盐处理后,离心浓缩,采用20重量%的变性聚丙烯凝胶电泳纯化。
本发明的优点和有益效果是:(1)通过分阶段、选择性保护尿苷中羟基的策略,成功制备了C4′-氟代尿苷亚磷酰胺单体;(2)C4′-氟代尿苷亚磷酰胺单体成功应用于制备C4′-氟代尿苷定点修饰的RNA。本发明所述的C4′-氟代尿苷定点修饰的RNA的制备方法,在研究C4′-氟代RNA的生化性质等方面有着广泛的应用前景。
附图说明:
图1为UPLC-MS分析C4′-氟代尿嘧啶核苷修饰RNA分子(7)的纯度。
具体实施方式
以下对本发明的具体实施方式进行详细说明。此处所描述的具体实施方式仅用于说明和解释本发明,并不用于限制本发明。
4′-C-氟-5′-碘-5′-脱氧尿苷(自制);二氯甲烷(天津市河东区广达服务部,货号1220);Na2CO3·10H2O晶体(天津市河东区广达服务部,货号2093);氯化钠(天津市化学试剂供销公司,货号017);乙酸酐(天津市化学试剂供销公司,货号282);吡啶[阿拉丁试剂(上海)有限公司,货号P111511];四氢呋喃(北京华威锐科化工有限公司,货号HWMT818767);甲醇(北京百灵威科技有限公司,货号980290-500ML)、55%(w/w)四正丁基氢氧化铵水溶液(北京百灵威科技有限公司,货号16198-50g);氨水/甲醇(2N)溶液(Alfa,货号H27080);氨水/甲醇(7N)溶液(Alfa公司,货号H30382);2-甲基咪唑[梯希爱(上海)化成工业发展有限公司,货号M0508]、N,N-二异丙基乙胺[梯希爱(上海)化成工业发展有限公司,货号D1599];叔丁基二甲基氯硅烷[梯希爱(上海)化成工业发展有限公司,货号B0995];硝酸银(安耐吉,货号A01W5100190250)、间氯过氧苯甲酸(安耐吉,货号A01W520002-100g)、2-氰乙基N,N-二异丙基氯亚磷酰胺(安耐吉,货号A07M901005-01);4-二甲氨基吡啶(Adamas,货号14766B)、4,4”-双甲氧基三苯甲基氯(Adamas,货号55311C);三氟乙酸(Acros,货号293810250)等原料为市售品。
实施例1
C4′-氟代尿嘧啶亚磷酰胺单体的合成。
其合成步骤如下:
步骤一、化合物(3)的制备:在氩气保护下,将式(2)对应单体(371.9mg,1mmol,1.0eq.)溶于4mL干燥的四氢呋喃,加入4mL干燥的吡啶和527.5mg(3.5mmol,3.5eq.)叔丁基二甲基氯硅烷,594.5mg(3.5mmol,3.5eq.)硝酸银。室温下搅拌1.5小时。旋干溶剂,加入20mL二氯甲烷稀释,依次用20mL饱和碳酸氢钠水溶液、饱和氯化钠水溶液清洗,有机层用无水硫酸镁干燥,用乙酸乙酯/石油醚(梯度,v/v,1:4~1:2~1:1)柱层析,得白色固体式(3)对应单体338.5mg(0.7mmol,两步产率70重量%)。
1H NMR(400MHz,DMSO-d6)δ(ppm)11.52(s,1H,NH),7.69(d,J=7.87Hz,1H,H-6,=CH),5.95(s,1H,H-1’),5.71(d,J=7.55Hz,1H,H-5),5.34(d,J=8.52Hz,1H,3’-OH),4.42(m,H-3’),4.34(m,H-2’),3.56(m,2H,H-5’,H-5”),0.84(s,9H,tBu),0.05(s,3H,-CH 3),0.04(s,3H,-CH 3);13C NMR(100.6MHz,CDCl3)δ(ppm)163.23,149.52,142.57,114.18(d,JH-F=229.69Hz,1C,C-4’),103.31,97.49,73.22,71.22(d,JC-F=23.24Hz,1C,C-3’),25.56,17.99,2.29(d,JC-F=33.48Hz,1C,C-5’),-4.87,-5.22;19F NMR(376MHz,DMSO-d6)δ(ppm)-110.27.m/z:calcd for C15H24FIN2O5Si[M+H]+,487.0561;found,487.0572.
步骤二、化合物(4)的制备:称取48.5mg(0.1mmol,1.0eq.)式(3)所示的单体溶于1mL干燥的吡啶。在氩气保护下,加入4mg(0.03mmol,0.3eq.)4-二甲氨基吡啶。在冰浴条件(0℃)及搅拌下,将0.05mL(0.5mmol,5.0eq.)乙酸酐缓慢滴加至反应体系,升至室温搅拌1小时。旋干溶剂,加入5mL二氯甲烷稀释,依次用5mL饱和碳酸氢钠水溶液、饱和氯化钠水溶液清洗,有机层用无水硫酸镁干燥,旋干溶剂,用乙酸乙酯/石油醚(梯度,v/v,1:8~1:4~1:2)柱层析,得白色固体式(4)对应单体52.1mg(0.098mmol,产率98重量%)。
1H NMR(400MHz,DMSO-d6)δ(ppm)9.87(s,1H,NH),7.30(d,J=7.96Hz,1H,H-6,=CH),5.82(m,2H,H-1’and H-5),5.39(d,J=6.70Hz,JH-F=14.48Hz,1H,H-3’),4.67(m,H-2’),3.58(dd,1H,J=7.95Hz),JH-F=11.16Hz,H-5”),2.17(s,3H,-CH 3),0.86(s,9H,tBu),0.01(s,3H,-CH 3),0.003(s,3H,-CH 3);13C NMR(100.6MHz,DMSO-d6)δ(ppm)169.8,163.1,149.8,141.3,113.6(d,1C,JC-F=237.38Hz,C-4’),103.5,94.67,72.3,71.6(d,JC-F=19.59Hz,1C,C-3’),25.36,20.53,17.78,3.26(d,JC-F=35.30Hz,1C,C-5’),-4.99,-5.31.m/z:calcd for C17H26FIN2O6Si[M+Na]+,529.0667;found,529.0639.
步骤三、化合物(5)的制备:在反应容器中,滴加三氟乙酸到496μL(1.86mmol,20eq.)四正丁基氢氧化铵(体积浓度55%)中调pH至4,将49.3mg(0.093mmol,1.0eq.)式(4)对应的单体溶于2mL二氯甲烷,加入至反应体系,分三次加入80mg(0.46mmol,5.0eq.)间氯过氧苯甲酸(间隔30min),常温搅拌46h。加入2mL饱和硫代硫酸钠水溶液淬灭反应,依次用5mL饱和碳酸氢钠水溶液溶液、5mL饱和氯化钠水溶液清洗,用丙酮/乙酸乙酯/石油醚(v/v/v,1:1:5)柱层析,得白色固体式(5)所对应的单体29.5mg(0.070mmol,产率75.8重量%)。
1H NMR(400MHz,DMSO)δ(ppm)11.54(s,1H,-NH),7.80(d,J=8.12Hz,1H,H-6,=CH),6.01(d,J=4.33Hz 1H,H-1’),5.75(m,1H,H-5),5.33(dd,J=5.33Hz,JH-F=13.37Hz1H,H-3’),4.58(d,J=4.41Hz,J=6.23Hz,1H,H-2’),3.61(m,2H,H-5’,H-5”),2.11(s,3H,OMe),0.81(s,9H,tBu),-0.02(s,3H,-CH 3),-0.01(s,3H,-CH 3);13C NMR(100.6MHz,DMSO-d6)δ(ppm)169.72,163.33,150.78,141.15,116.40(d,1C,JH-F=235.53Hz,C-4’),103.18,91.33,71.79,70.27(d,JC-F=17.37Hz,1C,C-3’),61.33(d,JC-F=39.38Hz,1C,C-5’),25.77,20.88,17.99,-4.85,-5.05.m/z:calcd for C17H27FIN2O7Si[M+H]+,419.1650;found,419.1641.
步骤四、化合物(6)的制备:在氩气保护下,将144.3mg(0.34mmol,1.0eq.)式(5)所对应的单体加入到3mL(7N)的氨/甲醇中,常温搅拌3h。旋干除去溶剂,用乙酸乙酯/石油醚(梯度,v/v,1:4~1:2~1:1)柱层析,将得到Rf=0.34(丙酮/乙酸乙酯/石油醚=1:1:4)的组分溶于3mL无水吡啶中,加入11mg(0.09mmol,0.3eq.)4-二甲氨基吡啶、132.1mg(0.4mmol,1.3eq.)二甲氧基三苯甲基,40℃搅拌4小时,1mL甲醇淬灭反应,旋干溶剂,二氯甲烷溶解后饱和碳酸氢钠水溶液清洗,用乙酸乙酯/石油醚(梯度,v/v,1:8~1:4~1:2~1:1,加入0.5体积%的三乙胺)柱层析,得式(6)对应的单体140.5mg(0.204mmol,产率68重量%)。
1HNMR(400MHz,DMSO-d6)δ(ppm)11.56(s,1H,-NH),7.82(d,J=8.06Hz,1H,H-6,=CH),7.41-7.26(m,9H,DMTr-ArH),6.92(s,2H,DMTr-ArH),6.90(s,2H,DMTr-ArH),6.00(s,1H,H-1’),5.46(d,J=8.01Hz,1H,H-5),5.14(d,J=9.07Hz,1H,-OH),4.61(m,1H,H-3’),4.56(d,J=6.08Hz,1H,H-2’),3.74(s,6H,-OMe),3.30(m,1H,H-5’),3.22(m,1H,H-5”),0.88(s,9H,tBu),0.11(s,3H,-CH 3),0.10(s,3H,-CH 3);13C NMR(100.6MHz,DMSO-d6)δ(ppm)162.94,158.21,150.06,144.23,141.29,134.80,134.68,129.75,127.89,127.63,126.84,116.67(d,JH-F=229.69Hz,1C,C-4’),113.24,102.05,92.79,86.10,72.92,69.40(d,JC-F=20.57Hz,1C,C-3’),63.42,54.97,29.98,25.55,-4.87,-5.22。m/z:calcd forC36H43FN2NaO8Si[M+Na]+,701.2670;found,701.2670.
步骤五、化合物(1)的制备:称取2.16g(3.16mmol,1.0eq.)式(6)所对应的单体,在氩气保护下溶于60mL二氯甲烷中。依次加入4.5mL(31.6mmol,10eq.)N,N’-二异丙基乙胺,1mL(12.64mmol,4eq.)2-甲基咪唑,2.4mL(15.8mmol,5eq.)(2-氰基乙氧基)-N,N’-二异丙基亚磷酰氯,常温下搅拌15分钟。反应结束后,在氩气保护下将体系减压蒸干,然后用少量二氯甲烷溶解,快速柱层析丙酮/乙酸乙酯/石油醚(v/v/v,1:1:5,+0.5体积%三乙胺)纯化得到浅黄色固体式(1)所对应的单体2.686g(3mmol,产率95重量%)。产物有2个异构体,比例为:2.25:1。
1H NMR(400MHz,CDCl3)δ(ppm)7.81(d,J=8.07Hz,0.64H,H-6,=CH),7.66(d,J=8.12Hz,0.26H,H-6,=CH)7.26-7.10(m,9H,DMTr-ArH),6.72(s,2H,DMTr-ArH),6.70(s,2H,DMTr-ArH),6.09(s,0.26H,H-1’),5.96(s,0.61H,H-1’)5.07(d,J=8.01Hz,1H,H-5),4.40(m,1H,H-3’),4.28(m,0.28H,H-2’),4.19(d,J=4.80Hz 0.63H,H-2’),3.66(s,6H,-OMe),3.46(m,4H),2.52(m,0.68H),2.37(m,1.3H),1.02(m,12H,4CH3),0.89(d,J=6.60Hz,2H)0.93(s,9H,tBu),0.05-0.00(m,6H,-CH 3);31P NMR(162MHz,CD3Cl)δ:152.2(s,0.27P),149.6(s,0.64P)。m/z:calcd for C45H61FN4O9PSi[M+H]+,879.3929;found,879.3942.
实施例2
本实施例在于说明由实施1制备的C4′-氟代尿嘧啶核苷亚磷酰胺单体(1)进行RNA固相合成RNA序列(7):5′-CCAUXAUAGC。在序列(7)中,X为C4′-氟代尿嘧啶核苷。
在ABI 394DNA/RNA合成仪上通过常规程序和试剂进行1μmol寡聚核苷酸合成。合成试剂包括Ac-rC,Bz-rA,Ac-rG,rU和实施例1制备的C4’-氟代尿嘧啶亚磷酰胺单体(1);3重量%三氯乙酸/二氯甲烷用于脱保护;CapA试剂:10体积%乙酸酐/10体积%吡啶/80体积%四氢呋喃(v/v/v),Cap B试剂:10体积%N-甲基咪唑/90体积%四氢呋喃(v/v);活化剂:0.25M乙硫基四唑/乙腈;氧化剂:0.02M碘/2体积%水/20体积%吡啶/78体积%四氢呋喃(v/v/v)。C4’-氟代尿嘧啶核苷亚磷酰胺单体的偶合时间是1小时。
将上述固相合成得到的RNA分子用0.75mL(28重量%)氨水混合0.25mL无水乙醇在55℃下处理12h,然后,取出上清液离心浓缩后,加入0.5mL三乙胺三氢氟酸盐处理24h,经C18柱脱盐处理后,离心浓缩,采用20重量%的变性聚丙烯凝胶电泳纯化。得到纯的RNA(7)经过了UPLC-MS分析鉴定(图1)。
可见,实施例1制备的C4’-氟代尿嘧啶亚磷酰胺单体(1)可以用于常规的RNA固相合成与纯化。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于此。在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,包括各个技术特征以任何其它的合适方式进行组合,这些简单变型和组合同样应当视为本发明所公开的内容,均属于本发明的保护范围。
Claims (10)
2.一种权利要求1所述C4′-氟代尿苷亚磷酰胺单体的制备方法,其特征在于,该方法包括以下步骤:
(1)将4′-C-氟-5′-碘-5′-脱氧尿苷与硝酸银、吡啶和四氢呋喃形成第一混合溶液;之后将所述第一混合溶液与叔丁基二甲基氯硅烷进行反应,且所述4′-C-氟-5′-碘-5′-脱氧尿苷与所述硝酸银、所述吡啶、所述四氢呋喃和所述叔丁基二甲基氯硅烷的用量比为1mmol:3.5mmol:4mL:4mL:3.5mmol,将反应后得到的溶液旋蒸除去溶剂,再用二氯甲烷溶解后洗涤、过滤、干燥、柱层析得到2′-O-(叔丁基二甲基硅烷基)-4′-C-氟-5′-碘-5′-脱氧尿苷;
(2)将步骤(1)得到的2′-O-(叔丁基二甲基硅烷基)-4′-C-氟-5′-碘-5′-脱氧尿苷与4-二甲氨基吡啶、吡啶进行第二混合,形成第二混合溶液;再将所述第二混合溶液与乙酸酐进行反应,所述2′-O-(叔丁基二甲基硅烷基)-4′-C-氟-5′-碘-5′-脱氧尿苷与所述4-二甲氨基吡啶、所述吡啶、所述乙酸酐用量的比为1mmol:0.3mmol:10mL:5mmol;旋蒸除去溶剂,用二氯甲烷溶解后洗涤、过滤、干燥、柱层析得到2′-O-(叔丁基二甲基硅烷基)-3′-乙酸酯-4′-C-氟-5′-碘-5′-脱氧尿苷;
(3)将三氟乙酸与四正丁基氢氧化铵溶液混合调节pH为4后,加入步骤(2)得到的2′-O-(叔丁基二甲基硅烷基)-3′-乙酸酯-4′-C-氟-5′-碘-5′-脱氧尿苷、二氯甲烷进行第三混合,形成第三混合溶液;再将所述第三混合溶液与间氯过氧苯甲酸进行反应,且所述三氟乙酸与所述四正丁基氢氧化铵溶液、所述2′-O-(叔丁基二甲基硅烷基)-3′-乙酸酯-4′-C-氟-5′-碘-5′-脱氧尿苷、所述二氯甲烷、所述间氯过氧苯甲酸的用量比为0.8mL:10mL:1mmol:10mL:5mmol;然后将反应得到的有机相洗涤、过滤、干燥、柱层析得到2′-O-(叔丁基二甲基硅烷基)-3′-乙酸酯-4′-C-氟代尿苷;
(4)将步骤(3)得到的2′-O-(叔丁基二甲基硅烷基)-3′-乙酸酯-4′-C-氟代尿苷与氨/甲醇溶液进行反应,且所述2′-O-(叔丁基二甲基硅烷基)-3′-乙酸酯-4′-C-氟代尿苷与氨/甲醇溶液用量比为1mmol:8mL;旋蒸除去溶剂,柱层析得到产物;
(5)将步骤(4)得到的产物与4-二甲氨基吡啶、吡啶以及4,4′-二甲氧基三苯甲基氯进行反应,且所述产物与所述4-二甲氨基吡啶、所述吡啶以及4,4′-二甲氧基三苯甲基氯的用量比为1mmol:0.3mmol:8mL:1.3mmol;旋蒸除去溶剂,用二氯甲烷溶解后然后将有机相洗涤、过滤、干燥、柱层析得到2′-O-(叔丁基二甲基硅烷基)-4′-C-氟-5′-O-二甲氧基三苯甲基尿苷;
(6)将步骤(5)得到的2′-O-(叔丁基二甲基硅烷基)-4′-C-氟-5′-O-二甲氧基三苯甲基尿苷与二氯甲烷、N,N′-二异丙基乙胺以及2-甲基咪唑进行第四混合,形成第四混合溶液;再将所述第四混合溶液与(2-氰基乙氧基)-N,N′-二异丙基亚磷酰氯进行反应,且所述2′-O-(叔丁基二甲基硅烷基)-4′-C-氟-5′-O-二甲氧基三苯甲基尿苷与所述二氯甲烷、所述N,N′-二异丙基乙胺、所述2-甲基咪唑、所述(2-氰基乙氧基)-N,N′-二异丙基亚磷酰氯的用量比为1mmol:20mL:10mmol:4mmol:5mmol;旋蒸除去溶剂,用二氯甲烷溶解后然后将有机相洗涤、过滤、干燥、柱层析得到2′-O-(叔丁基二甲基硅烷基)-3′-[(2-氰基乙氧基)-N,N′-二异丙基亚磷酰胺]-4′-C-氟-5′-O-二甲氧基三苯甲基尿苷,即权利要求1所述的C4′-氟代尿苷亚磷酰胺单体。
3.根据权利要求2所述的制备方法,其特征在于,在步骤(1)中,所述反应的条件包括:温度为18-25℃,时间为1.5-2小时,搅拌速度为250-500转/分钟。
4.根据权利要求2所述的制备方法,其特征在于,在步骤(2)中,所述乙酸酐以滴加的方式加入所述第二混合溶液中,所述乙酸酐的滴加速率为15滴/分钟;以及,
所述反应的条件包括:温度为18-25℃,时间为0.5-1小时,搅拌速度为250-500转/分钟。
5.根据权利要求2所述的制备方法,其特征在于,在步骤(3)中,所述四正丁基氢氧化铵溶液的质量浓度为55%,所述间氯过氧苯甲酸的纯度为85%;
所述三氟乙酸以滴加的方式加入所述四正丁基氢氧化铵溶液中调pH为4;所述间氯过氧苯甲酸分三次加入第三混合溶液中,间隔时间为0.5小时;以及,
所述反应的条件包括:温度为18-25℃,时间为40-46小时,搅拌速度为250-500转/分钟。
6.根据权利要求2所述的制备方法,其特征在于,在步骤(4)中,所述氨/甲醇溶液的物质的量浓度为7mol/L;以及,
所述反应的条件包括:温度为18-25℃,时间为3-4小时,搅拌速度为250-500转/分钟。
7.根据权利要求2所述的制备方法,其特征在于,在步骤(5)中,所述反应的条件包括:温度为40-50℃,时间为3-5小时,搅拌速度为250-500转/分钟。
8.根据权利要求2所述的制备方法,其特征在于,在步骤(6)中,所述反应的条件包括:温度为18-25℃,时间为15分钟,搅拌速度为250-500转/分钟。
9.一种含有C4’-氟代尿苷定点修饰的RNA的制备方法,其特征在于,该制备方法中的合成试剂包括权利要求1所述的C4’-氟代尿苷亚磷酰胺单体。
10.根据权利要求9所述的制备方法,其中,该制备方法在ABI 394DNA/RNA合成仪上进行固相合成以得到寡聚核苷酸,其中:
所述合成试剂还包括叔丁基二甲基氯硅烷保护2′-OH的Ac-rC、Bz-rA、Ac-rG和rU;
三氯乙酸/二氯甲烷用于脱保护;
Cap A试剂为乙酸酐、吡啶和四氢呋喃的组合物,且所述乙酸酐、所述吡啶和所述四氢呋喃的体积比为10体积%:(5-15)体积%:(75-85)体积%;
Cap B试剂为N-甲基咪唑和四氢呋喃的组合物,且所述N-甲基咪唑和所述四氢呋喃的体积比为10体积%:90体积%;
活化剂为乙硫基四唑和乙腈的组合物;
氧化剂为碘、水、吡啶和四氢呋喃的组合物,且所述碘、所述水、所述吡啶和所述四氢呋喃的用量比为0.02M:(1-2)体积%:(10-30)体积%:(75-85)体积%;以及,
所述C4’-氟代RNA亚磷酰胺单体的偶合时间为0.5-1小时;
将上述固相合成得到的寡聚核苷酸用0.75mL浓度为28重量%的氨水混合0.25mL无水乙醇在55℃下处理10-12h,然后,取出上清液离心浓缩后,加入0.5mL三乙胺三氢氟酸盐处理20-24h,经C18柱脱盐处理后,离心浓缩,采用20重量%的变性聚丙烯凝胶电泳纯化。
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