CN108203446B - 含苯氧基丙烯酰基亚膦酰胺、制备方法及其应用 - Google Patents
含苯氧基丙烯酰基亚膦酰胺、制备方法及其应用 Download PDFInfo
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- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- CSCPPACGZOOCGX-WFGJKAKNSA-N acetone d6 Chemical compound [2H]C([2H])([2H])C(=O)C([2H])([2H])[2H] CSCPPACGZOOCGX-WFGJKAKNSA-N 0.000 description 1
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- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/22—Amides of acids of phosphorus
- C07F9/24—Esteramides
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- C07F9/2408—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyalkyl compounds
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- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/22—Amides of acids of phosphorus
- C07F9/24—Esteramides
- C07F9/2454—Esteramides the amide moiety containing a substituent or a structure which is considered as characteristic
- C07F9/2458—Esteramides the amide moiety containing a substituent or a structure which is considered as characteristic of aliphatic amines
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
- C08F220/606—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen and containing other heteroatoms
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Abstract
本发明提供了一种含苯氧基丙烯酰基亚膦酰胺、制备方法及其应用。具体地,本发明提供了一种可作为固相合成所使用的试剂,所述化合物具有式I结构,式中,R1、R2、R3、R4、R5、m的定义如说明书中所定义。通过该化合物可在核酸中引入丙烯基,并进行进一步聚合反应。本发明还公开了式I化合物的制备方法与应用。
Description
技术领域
本发明涉及一种含苯氧基丙烯酰基亚膦酰胺、制备方法及其应用,特别是涉及一种可用于核酸结构修饰的含苯氧基丙烯酰基亚膦酰胺、制备方法及其应用。
背景技术
生物大分子的核酸具有特异性识别与自组装功能,固相合成技术使得短链的寡聚核酸可通过DNA合成仪实现自动化、高效化学合成。目前寡聚核酸已被广泛应用于生物、医药与纳米技术等领域。小干扰核酸(Small interfering RNA,siRNA)可对致病基因进行沉默(Elbashir S,Tuschl T,et.al.,Duplexes of 21-nucleotide RNAs mediate RNAinterference in cultured mammalian cells.Nature,2001,411,494-988;Hamilton A,Baulcombe D,A species of small antisense RNA in posttranscriptional genesilencing in plants,Science,1999,286,950.),起到治疗疾病的作用。因此siRNA作为生物大分子药物,在临床上具有重要应用价值,目前已有多个siRNA药物进入临床试验阶段。核酸适配体是通过体外筛选技术得到的具有特定序列的寡聚单链核酸,能与靶向物高特异性与选择性结合(Ellington AD,Szostak JW,In vitro selection of RNA moleculesthat bind specific ligands,Nature,1990,346,818),具有类似于抗体的作用,被称为化学抗体,在生物检测与药物传递方面具有广泛的应用。核酸在血液循环里的稳定性差、细胞膜穿透性差,因此在生物检测、药物传递以及自身作为生物大分子药物的应用中,受到许多限制。通过化学合成在寡聚核酸的特定位置引入功能性基团,可对核酸进行改性。PCT专利申请WO2011/153493公开了Alnylam公司通过在siRNA的两端接入疏水基团,显著提高siRNA的稳定性与进入细胞能力。
在核酸中引入丙烯酰基,可通过聚合反应,方便地将核酸接到高分子聚合物中,并应用于制备水凝胶、药物传递等领域。针对已有技术中化学交联DNA水凝胶制备与纯化困难,中国发明专利CN 103819501A公开了一种甲基丙烯基亚磷酰胺单体及合成方法,该发明通过在亚磷酰胺单体中引入具有较大疏水性的三苯基甲基基团,使得相应的DNA在高效液相色谱过程中,得到纯度更高的带有甲基丙烯基的DNA单链。该发明通过将2-位取代-1,3-丙二醇与4,4’-二甲氧基三苯基氯甲烷反应,将一个羟基保护得到单一保护的三苯基甲基醚。由于1-位羟基和3-位羟基都是对称的伯羟基,容易生成双取代的副产物,导致合成效率不高。另外,通过柔性结构的脂肪链与DNA分隔的丙烯基,疏水性弱、刚性不够,可能不利于聚合反应。现有的丙烯酰基亚磷酰胺的合成产率低,相应的丙烯基与DNA之间的链接为脂肪链,品类单一。
因此本领域尚需研发具有结构多元的丙烯酰基亚膦酰胺。
发明内容
本发明的目的在于提供一种含苯氧基丙烯酰基亚膦酰胺。
本发明的另一目的在于提供该化合物的制备方法。
本发明的再一目的在于提供该化合物的应用。
本发明的第一方面,提供了一种含苯氧基丙烯酰基亚膦酰胺,所述化合物具有式I所示结构:
式中,R1选自:H、F、Cl、Br、I、C1-C6烷基、C6-C20芳基、C3-C8环烷基、C1-C10杂环烷基、C5-C20杂芳基;
各R2、R3、R4、R5独立地选自:H、F、Cl、Br、I、C1-C6烷基、C1-C6烷氧基、C6-C20芳基;
m为CH2基团的个数,m=1、2、3、4或5。
在另一优选例中,该R1为甲基(-CH3)取代基团,且具有一个或多个选自下组的特征:
(a)各R2、R3、R4、R5独立地选自:H、F、Cl、Br、I、C1-C6烷基、C1-C6烷氧基、C6-C20芳基;
(b)m为CH2基团的个数,m=1、2、3、4或5。
在另一优选例中,各R2、R3、R4、R5独立地选自:H、C1-C3烷氧基、C1-C3烷基。
在另一优选例中,该R2为氢(-H)取代基,且该CH2基团的个数m=2。
在另一优选例中,各R3、R4、R5独立地选自:H、-OCH3。
在另一优选例中,各R3为H,R4、R5为-OCH3。
本发明的第二方面,提供式I化合物的制备方法,包括以下步骤:
(a)提供具有式II结构的双羟基化合物;
其中,R1、R2、m的定义如第一方面所述;
(b)将式II化合物进行两步通用的固相合成试剂的制备反应,得到如第一方面所述化合物。
在另一优选例中,提供式II化合物的一种制备方法,包括以下步骤:
(a)提供具有式III结构的化合物;
其中,R1、R2、m的定义如第一方面所述;
(b)将式III化合物与缩水甘油(HOCH2CH(O)CH2)反应,得到如第二方面所述的式II化合物。
在另一优选例中,式II化合物的合成方法包括以下步骤:
(a)提供具有式IV结构的化合物;
其中,X=F、Cl、Br、I、OH,R1、R2、m的定义如第一方面所述;
(b)将式IV化合物与甘油(HOCH2CHOHCH2OH)反应,得到如第二方面所述的式II化合物。
在另一优选例中,式II化合物的合成方法包括以下步骤:
(a)提供具有式IV-a结构的化合物;
其中,X=F、Cl、Br、I,R1、m的定义如第一方面所述;
(b)将式IV-a化合物与甘油(HOCH2CHOHCH2OH)反应,得到如第二方面所述的式II化合物。
在另一优选例中,提供式III化合物的一种制备方法:
由商品化的或合成得到的具有式V结构的化合物,与R1取代的丙烯酰氯反应,得到具有式III结构的化合物。
在另一优选例中,提供式III化合物的一种制备方法:
由商品化的或合成得到的具有式V结构的化合物,与R1取代的丙烯酸反应,得到具有式III结构的化合物。
在另一优选例中,提供式IV化合物的一种制备方法:
由商品化的或合成得到的具有式VI结构的化合物,与R1取代的丙烯酰氯反应,得到具有式IV结构的化合物。
在另一优选例中,提供式IV化合物的一种制备方法:
由商品化的或合成得到的具有式VI结构的化合物,与R1取代的丙烯酸反应,得到具有式IV结构的化合物。
本发明的第三方面,提供式I化合物的应用,用于合成含有苯氧基-丙烯酰基的核酸。
在另一优选例中,应用式I化合物可在核酸3’-端或5’-端高效接入苯氧基-丙烯酰基,该含有苯氧基-丙烯酰基的核酸可应用于聚合反应单体,顺利进行聚合反应得到水溶性聚合物。
在另一优选例中,应用式I化合物可在核酸3’-端与5’-端高效接入1-5个苯氧基-丙烯酰基,该含有多个苯氧基-丙烯酰基的核酸具有显著提高的生物稳定性。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一赘述。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1
中间产物1的合成:
方法A:0℃下,向装有4-(2-氨基乙基)苯酚(13.7g,100mmol)的吡啶(100mL)溶液的圆底烧瓶中滴加甲基丙烯酰氯(11.4g,110mmol),升至室温反应2小时。加入饱和碳酸氢钠溶液终止反应,减压蒸馏除去吡啶,得到的残渣经柱层析得到化合物1(16.8克,82%),为淡黄色固体。
方法B:将甲基丙烯酸(0.86克,10mmol)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC.HCL,1.91克,10mmol)、N-羟基丁二酰亚胺(NHS,1.15克,10mmol)、4-(2-氨基乙基)苯酚、(1.37g,10mmol)的二氯甲烷(30mL)溶液在室温反应12小时。反应液用碳酸氢钠水溶液与饱和食盐水洗,有机相用硫酸钠干燥后减压蒸除溶剂,得到的残渣柱层析得到化合物1(1.78克,87%)。
1H NMR(400MHz,DMSO)δ9.20(s,1H),7.98(t,J=5.3Hz,1H),6.98(d,J=8.3Hz,2H),6.68(d,J=8.3Hz,2H),5.61(s,1H),5.30(s,1H),3.25(dd,J=14.4,6.3Hz,2H),2.63(t,J=7.2Hz,2H),1.84(s,3H);13C NMR(101MHz,DMSO)δ166.13,155.03,136.41,131.39,130.50,121.30,116.07,43.22,35.93,16.43.MS(ESI+):m/e=206(M+1)。
实施例2
中间产物2的合成:
方法A:0℃下,向装有2-(4-碘苯基)乙胺(2.47g,10mmol)的吡啶(10mL)溶液的圆底烧瓶中滴加甲基丙烯酰氯(1.2g,11mmol),升至室温反应5小时。加入饱和碳酸氢钠溶液终止反应,减压蒸馏除去吡啶,得到的残渣经柱层析得到化合物2(2.8克,89%),为淡黄色固体。
1H NMR(400MHz,DMSO)δ9.20(s,1H),8.02(t,J=5.3Hz,1H),7.61(d,J=8.3Hz,2H),7.02(d,J=8.3Hz,2H),5.63(s,1H),5.31(s,1H),3.24(dd,J=14.4,6.3Hz,2H),2.66(t,J=7.2Hz,2H),1.83(s,3H);13C NMR(101MHz,DMSO)δ167.09,155.13,137.41,130.95,130.13,121.12,115.32,43.21,36.32,16.03.MS(ESI+):m/e=316(M+1)。
实施例3
中间产物3的合成:
将缩水甘油(6.5克,88mmol)、N-(4-羟基苯乙基)甲基丙烯酰胺(16.4克,80mmol)与三乙胺(8mL)的乙醇(80mL)溶液回流反应10小时。减压蒸馏除去溶剂,得到的残渣经柱层析得到化合物2(17.6克,79%),为白色固体。
1H NMR(400MHz,DMSO)δ7.99(s,1H),7.10(d,J=8.4Hz,2H),6.85(d,J=8.4Hz,2H),5.61(s,1H),5.30(s,1H),4.95(d,J=4.7Hz,1H),4.68(t,J=5.6Hz,1H),3.95(dd,J=9.3,3.7Hz,1H),3.83-3.75(m,2H),3.43(t,J=5.4Hz,2H),3.27(dd,J=13.9,6.7Hz,2H),2.68(t,J=7.4Hz,2H),1.83(s,3H);13C NMR(101MHz,DMSO)δ167.32,157.11,140.00,131.27,129.52,118.80,114.24,69.91,69.4562.68,40.76,34.16,18.63.MS(ESI+):m/e=280(M+1)。
实施例4
中间产物3的合成:
往甘油(1.01克,11mmol)、化合物2(3.15克,10mmol)的二甲基甲酰胺(30mL)溶液中加入碳酸钾(2.75克,20mmol),110℃下反应10小时。减压蒸馏除去溶剂,得到的残渣经柱层析得到化合物3(1.70克,61%),为白色固体。
实施例5
中间产物4的合成:
在化合物3(13.9克,50mmol)的吡啶(50mL)溶液中分批加入4,4’-二甲基三苯基氯甲烷(18.6克,55mmol),室温反应5小时后加入甲醇终止反应。减压蒸馏除去吡啶,得到的残余物经柱层析得到化合物4(24.7克,85%),为白色固体。
1H NMR(400MHz,DMSO)δ7.97(t,J=5.5Hz,1H),7.40(d,J=7.6Hz,2H),7.29(d,J=7.4Hz,1H),7.25(dd,J=8.7,1.3Hz,4H),7.21(t,J=7.2Hz,1H),7.11(d,J=8.4Hz,2H),6.86(dd,J=8.8,1.8Hz,4H),6.82(d,J=8.5Hz,2H),5.61(s,1H),5.29(s,1H),5.14(d,J=4.9Hz,1H),4.01-3.88(m,3H),3.73(s,6H),3.28(dd,J=14.0,6.5Hz,2H),3.06(d,J=4.4Hz,2H),2.68(t,J=7.4Hz,2H),1.83(s,3H);13C NMR(101MHz,DMSO)δ167.82,158.48,157.45,145.51,140.53,136.24,131.90,130.20,130.02,128.23,127.05,119.23,114.81,113.58,85.73,69.89,68.62,64.93,55.48,41.24,34.67,19.12.MS(ESI+):m/e=582(M+1)。
实施例6
目标产物苯氧基丙烯酰基亚磷酰胺5的合成
将装有化合物4(5.81克,10mmol)的二氯甲烷(30mL)溶液的圆底烧瓶置于氮气下保护并冷却到0℃。滴加入N,N-二异丙基乙基胺与2-氰乙基-N,N-二异丙基氯代亚磷酰胺。反应两小时后反应液用碳酸氢钠水溶液与饱和食盐水洗,有机相用硫酸钠干燥后减压蒸除溶剂,得到的残渣柱层析得到产物5。1H NMR(400MHz,acetone-d6):δ7.44-7.53(m,4H),7.26-7.34(m,7H),7.05-7.10(m,2H),6.84-6.87(m,J=7.5Hz,4H),5.61(s,1H),5.29(s,1H),4.10-4.39(m,2H),3.76(s,6H),3.56-3.88(m,2H),3.29-3.40(m,2H),2.86(m,2H),2.72-2.74(m,1H),2.60-2.63(m,1H),1.83(s,3H),1.05-1.28(m,12H);31P NMRδ:149.51,149.23.MS(ESI+):m/e=782(M+1)。
实施例7
含苯氧基-丙烯酰基核酸的合成:
应用化合物5配制成0.1摩尔/升的无水乙腈溶液,装载到DNA合成仪。以CPG作为固相载体,通过计算机程序化设计,合成得到具有任意序列的功能化核酸。本实施例合成两种典型的功能化核酸用于示例说明应用研究:核酸一:5’-XTTTTAGTCCTTTAC,核酸二:5’-XXTTTTAGTCCTTTAXX-3’;其中X代表应用化合物5合成得到的单元。固相合成得到的核酸经过标准的脱保护、高效液相色谱纯化,得到接有1个丙烯酰基基团的核酸一与4个丙烯酰基基团的核酸二。
实施例8
通过核酸一制备高分子核酸聚合物:将核酸一溶解于超纯水,配制成浓度为1mM的溶液,往该溶液中加入丙烯酰胺,配制成含3-6%丙烯酰胺的混合溶液。加入聚合反应引发剂,在室温反应10-30分钟,得到高分子核酸聚合物,可用于水凝胶的制备。
实施例9
制备得到的核酸二与相应的全部由天然碱基构成的核酸三(序列:5’-TTTTTTAGTCCTTTATT-3’)分别添加到以下生物溶液中:血清溶液、细胞裂解液中,并放置于37℃细胞培养箱中。按1、2、4、8、16小时的梯度进行取样检测,PAGE胶结果显示核酸二在上述环境可稳定存在8小时以上,而天然的核酸三在血清溶液、细胞裂解液等生物环境下迅速降解。本实施例说明,通过本发明提供的亚磷酰胺在核酸的两端接入疏水性基团,可显著提高核酸的稳定性。
Claims (7)
2.如权利要求1所述的化合物,其特征在于,该R1为甲基(-CH3)取代基团,且具有一个或多个选自下组的特征:
(a)各R2、R3、R4、R5独立地选自:H、F、Cl、Br、I、C1-C6烷基、C1-C6烷氧基、C6-C20芳基;
(b)m为CH2基团的个数,m=1、2、3、4或5。
3.如权利要求2所述的化合物,其特征在于,各R2、R3、R4、R5独立地选自:H、C1-C3烷氧基、C1-C3烷基。
4.如权利要求3所述的化合物,其特征在于,该R2为氢(-H)取代基,且该CH2基团的个数m=2。
5.如权利要求4所述的化合物,其特征在于,各R3、R4、R5独立地选自:H、-OCH3。
6.如权利要求1所述的式I化合物的应用,其特征在于,通过式I化合物在核酸中任意位置引入丙烯酰基,该丙烯酰基进行聚合反应,顺利实现将核酸接入到聚合物中。
7.如权利要求1所述的式I化合物的应用,其特征在于,通过式I化合物在核酸末端中引入包含苯氧基丙烯酰基取代基,以提高核酸的生物稳定性。
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