CN111909197B - 一种三磷酸化合物和脱氧核苷酸的制备方法 - Google Patents
一种三磷酸化合物和脱氧核苷酸的制备方法 Download PDFInfo
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 45
- 239000002904 solvent Substances 0.000 claims abstract description 19
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims abstract description 16
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- QWGNYWGYVGOLOI-UHFFFAOYSA-N phosphonato phosphate tripropylazanium Chemical compound CCC[NH+](CCC)CCC.CCC[NH+](CCC)CCC.CCC[NH+](CCC)CCC.CCC[NH+](CCC)CCC.[O-]P(=O)([O-])OP(=O)([O-])[O-] QWGNYWGYVGOLOI-UHFFFAOYSA-N 0.000 claims description 7
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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
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/098—Esters of polyphosphoric acids or anhydrides
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- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
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- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
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Abstract
本发明公开了一种三磷酸化合物和脱氧核苷酸的制备方法。本发明的三磷酸化合物制备方法通过用四氢呋喃替代磷酸三甲酯/磷酸三乙酯、三正丙基胺替代三正丁基胺、乙腈替代N,N‑二甲基甲酰胺,具有产率大、副产物少、溶剂易去除、无毒安全等优点。本发明的脱氧核苷酸方法通过用吗啉‑二甲基甲酰胺溶液替代三乙胺溶液来脱除‑Fmoc基团,大幅缩短了反应时间,减少了副产物的生成,提高了产率。
Description
技术领域
本发明涉及一种三磷酸化合物和脱氧核苷酸的制备方法,属于有机合成技术领域。
背景技术
末端脱氧核苷酸转移酶是一种无需模板的DNA聚合酶,用于催化脱氧核苷酸结合到DNA分子的3'羟基端,在人类和动物的免疫反应形成过程中起重要作用。2'-脱氧核苷-5'-三磷酸是末端脱氧核苷酸转移酶的天然底物,不过有研究发现是否存在核苷片段并不影响三磷酸酯作为底物的性质,即三磷酸类化合物可以替代2'-脱氧核苷-5'-三磷酸用作某些末端脱氧核苷酸转移酶(例如:小牛胸腺末端脱氧核苷酸转移酶)的底物。然而,文献报道的三磷酸类化合物合成方法普遍的反应收率仅有7%~12%,且还需要使用三正丁基胺这种一类剧毒品,所以无法大规模推广应用。
核苷酸是一类由嘌呤碱或嘧啶碱、核糖或脱氧核糖以及磷酸三种物质组成的化合物。核苷酸最常见的修饰手段是在5位标记荧光染料分子或者在7位碱基上修饰一些可用于检测的基团,用于各种基因组学,包括DNA标记和测序,而7位碱基上修饰的分子在与DNA聚合酶发生作用后,这些带标记核苷酸就会***延伸的DNA链当中,导致新的DNA结构发生变化。近些年,研究人员在研究单分子测序时发现5位修饰亦可用于DNA测序,而此类修饰一般是延伸5位磷酸酯键并甩出一个容易与一些标记分子连接的官能团,例如-NH2等,因此这类化合物的合成研究对单分子测序至关重要,其多样性能够增加单分子测序底物筛选的样本量,为优化筛选测序底物提供更多的可能性。然而,文献报道的通过三磷酸类化合物合成核苷酸的方法普遍存在目标产物收率较低、副产物多、制备周期长、溶剂难去除等问题,无法大规模推广应用。
因此,有必要开发一种收率更高、更加安全的三磷酸化合物合成方法,并进一步利用三磷酸化合物来合成脱氧核苷酸。
发明内容
本发明的目的在于提供一种三磷酸化合物和脱氧核苷酸的制备方法。
本发明所采取的技术方案是:
一种三磷酸化合物的制备方法,包括以下步骤:
4)将三乙胺-碳酸缓冲液加入步骤3)的反应液中,室温反应,浓缩,得到三磷酸化合物粗品;
优选的,步骤1)所述溶剂为1,4-二氧六环、乙腈、二甲基甲酰胺、二氯甲烷中的至少一种。
优选的,步骤1)所述Na2CO3溶液的质量分数为5%~15%。
优选的,步骤1)所述反应的时间为10~15h。
优选的,步骤2)所述反应的时间为3~5h。
优选的,步骤3)所述反应的时间为1~2h。
优选的,步骤4)所述反应的时间为20~50h。
一种脱氧核苷酸的制备方法,包括以下步骤:
4)通过反相高效液相色谱对脱氧核苷酸粗品进行纯化,得到脱氧核苷酸
优选的,步骤1)所述溶剂为二甲基甲酰胺、乙腈、二甲基亚砜中的至少一种。
优选的,步骤1)中第一次反应的时间为3~5h,第二次反应的时间为20~40min。
优选的,步骤2)所述反应的时间为10~20h。
优选的,步骤3)所述反应的时间为2~3h。
本发明的有益效果是:本发明的三磷酸化合物制备方法具有产率高、副产物少、溶剂易去除、无毒安全等优点,由该三磷酸化合物制备脱氧核苷酸的方法具有反应时间短、副产物少、产率高等优点,均适合进行大规模推广应用。
具体来说:
1)本发明的三磷酸化合物制备方法的收率大于40%,约为传统方法的4倍,本发明的脱氧核苷酸制备方法的收率大于80%,而传统方法低于60%;
4)本发明用乙腈替代N,N-二甲基甲酰胺来分散焦磷酸三正丙基铵盐,反应结束后溶剂更加容易去除;
5)本发明用吗啉-二甲基甲酰胺溶液替代三乙胺溶液来脱除-Fmoc基团,大幅缩短了反应时间,减少了副产物的生成,提高了脱氧核苷酸的产率。
附图说明
图1为实施例1步骤4)中的三磷酸化合物粗品的液相色谱图。
图2为实施例1中的N-(9-芴基甲氧羰基)-6-氨基己基三磷酸的核磁共振氢谱图。
图3为实施例1中的N-(9-芴基甲氧羰基)-6-氨基己基三磷酸的核磁共振磷谱图。
图4为实施例1中的dA6P-NH2-Nucleotides的核磁共振氢谱图。
图5为实施例1中的dA6P-NH2-Nucleotides的核磁共振磷谱图。
图6为实施例2中的dA4P-NH2-Nucleotides的核磁共振氢谱图。
图7为实施例2中的dA4P-NH2-Nucleotides的核磁共振磷谱图。
图8为实施例3中的dT4P-NH2-Nucleotides的核磁共振氢谱图。
图9为实施例3中的dT4P-NH2-Nucleotides的核磁共振磷谱图。
图10为实施例4中的N-(9-芴基甲氧羰基)-2-氨基乙基三磷酸的核磁共振氢谱图。
图11为实施例4中的N-(9-芴基甲氧羰基)-2-氨基乙基三磷酸的核磁共振磷谱图。
图12为对比例步骤4)中的三磷酸化合物粗品的液相色谱图。
具体实施方式
下面结合具体实施例对本发明作进一步的解释和说明。
实施例1:
N-(9-芴基甲氧羰基)-6-氨基己基三磷酸的合成:
1)将5.3g的芴甲氧羰酰氯溶于40mL的1,4-二氧六环,在0℃下加入2g的6-氨基-1-己醇,充分溶解,再缓慢加入20mL浓度10%的Na2CO3溶液,25℃反应12h,用甲醇-二氯甲烷(甲醇、二氯甲烷体积比1:19)萃取产物,合并有机相,在旋转蒸发仪上旋干溶剂,用混合溶剂(由二氯甲烷、乙酸乙酯和石油醚按照体积比1:3:6组成)打浆,得到(产率:98%);
4)将50mL浓度0.1mol/L的三乙胺-碳酸缓冲液加入步骤3)的反应液中,调节pH至7.5左右,25℃反应48h,浓缩,得到三磷酸化合物粗品;
5)通过反相高效液相色谱对三磷酸化合物粗品进行纯化,得到N-(9-芴基甲氧羰基)-6-氨基己基三磷酸(产率:42%);
通过反相高效液相色谱对三磷酸化合物粗品进行纯化的具体操作为:
a)将三磷酸化合物粗品溶解在甲醇中,再用滤膜过滤;
b)采用反相高效液相色谱对滤液进行纯化,色谱柱规格为C18(Innoval ODS-2 50×250mm,5μm),流动相A为0.1mol/L的三乙胺-碳酸缓冲液,流动相B为甲醇,将色谱柱用流动相B:62%平衡后,用流动相B:62%~75%梯度洗脱,收集纯化后的化合物,冻干。
性能测试:
步骤4)中的三磷酸化合物粗品的液相色谱图如图1所示。
N-(9-芴基甲氧羰基)-6-氨基己基三磷酸的核磁共振氢谱图如图2所示,核磁共振磷谱图如图3所示,解谱分析如下:
1H NMR(D2O,400MHz):δ=7.47(s,2H),7.30(s,2H),7.14-7.08(m,4H),4.08(s,2H),3.81-3.73(m,3H),2.71(s,2H),1.37-0.71(m,8H)。
31P NMR(D2O):δ=-11.0(m,2P),-23.6(t,1P)。
N-(9-芴基甲氧羰基)-6-氨基己基三磷酸的结构式:
dA6P-NH2-Nucleotides的合成:
1)将0.1g的N-(9-芴基甲氧羰基)-6-氨基己基三磷酸加入10mL的无水乙腈,用旋转蒸发仪减压蒸干,再重复一次操作,除去化合物中的水分,再将N-(9-芴基甲氧羰基)-6-氨基己基三磷酸溶于3mL的二甲基甲酰胺,再加入0.7mmol的N,N'-羰基二咪唑,25℃搅拌4h,再加入1.05mmol的甲醇,继续搅拌30min,得到
4)通过中压制备色谱对脱氧核苷酸粗品进行初步分离纯化,产物出峰位置为2min左右,梯度条件为:0~100甲醇,15min,再通过反相高效液相色谱对初步分离纯化的产品进行进一步纯化,流动相用0.1mol/L的三乙胺-碳酸缓冲液/乙腈,制备柱填料为反向C18,制备梯度3%~15%,产品出峰位置为20min左右,得到脱氧核苷酸dA6P-NH2-Nucleotides(产率:83%)。
性能测试:
dA6P-NH2-Nucleotides的核磁共振氢谱图如图4所示,核磁共振磷谱图如图5所示,解谱分析如下:
1H NMR(D2O,400MHz):δ=8.37(s,1H),8.12(s,H),6.39(t,H),4.17-4.44–4.00(m,3H),3.85(t,2H),2.69(m,H),2.46(m,H),1.53(m,4H),1.53(m,H),1.29(m,4H)。
31P NMR(D2O):δ=-10.91(bs,P),-10.45(bs,P),-23.35(bm,4P)。
dA6P-NH2-Nucleotides的结构式:
实施例2:
dA4P-NH2-Nucleotides的合成:
1)将0.1g的N-(9-芴基甲氧羰基)-6-氨基己基三磷酸加入10mL的无水乙腈,用旋转蒸发仪减压蒸干,再重复一次操作,除去化合物中的水分,再将N-(9-芴基甲氧羰基)-6-氨基己基三磷酸溶于3mL的二甲基甲酰胺,再加入0.7mmol的N,N'-羰基二咪唑,25℃搅拌4h,再加入1.05mmol的甲醇,继续搅拌30min,得到
4)通过中压制备色谱对脱氧核苷酸粗品进行初步分离纯化(操作同实施例1),再通过反相高效液相色谱对初步分离纯化的产品进行进一步纯化(操作同实施例1),得到脱氧核苷酸dA4P-NH2-Nucleotides(产率:85%)。
性能测试:
dA4P-NH2-Nucleotides的核磁共振氢谱图如图6所示,核磁共振磷谱图如图7所示,解谱分析如下:
1H NMR(D2O,400MHz):δ=8.36(s,1H),8.11(s,H),6.39(t,H),4.21-4.08(m,3H),3.85(m,2H),2.88(m,2H),2.73(m,H),2.51(m,H),1.53(m,4H),1.53(m,H),1.29(m,4H)。
31P NMR(D2O):δ=-10.84(bs,P),-11.49(bs,P),-21.98(bm,2P)。
dA4P-NH2-Nucleotides的结构式:
实施例3:
dT4P-NH2-Nucleotides的合成:
1)将0.1g的N-(9-芴基甲氧羰基)-6-氨基己基三磷酸加入10mL的无水乙腈,用旋转蒸发仪减压蒸干,再重复一次操作,除去化合物中的水分,再将N-(9-芴基甲氧羰基)-6-氨基己基三磷酸溶于3mL的二甲基甲酰胺,再加入0.7mmol的N,N'-羰基二咪唑,25℃搅拌4h,再加入1.05mmol的甲醇,继续搅拌30min,得到
4)通过中压制备色谱对脱氧核苷酸粗品进行初步分离纯化(操作同实施例1),再通过反相高效液相色谱对初步分离纯化的产品进行进一步纯化(操作同实施例1),得到脱氧核苷酸dT4P-NH2-Nucleotides(产率:80%)。
性能测试:
dT4P-NH2-Nucleotides的核磁共振氢谱图如图8所示,核磁共振磷谱图如图9所示,解谱分析如下:
1H NMR(D2O,400MHz):δ=7.65(s,1H),6.29(t,H),4.56(t,H),4.15(m,3H),3.93(m,2H),2.94(m,2H),2.30(m,2H),1.86(s,3H),1.61(m,4H),1.36(m,4H)。
31P NMR(D2O):δ=-10.79(bs,P),-11.51(bs,P),-21.92(bm,2P)。
dT4P-NH2-Nucleotides的结构式:
实施例4:
N-(9-芴基甲氧羰基)-2-氨基乙基三磷酸的合成:
1)将5.1g的芴甲氧羰酰氯溶于40mL的1,4-二氧六环,在0℃下加入1g的2-羟基乙胺,充分溶解,再缓慢加入20mL浓度10%的Na2CO3溶液,25℃反应12h,用甲醇-二氯甲烷(甲醇、二氯甲烷体积比1:19)萃取产物,合并有机相,在旋转蒸发仪上旋干溶剂,用混合溶剂(由二氯甲烷、乙酸乙酯和石油醚按照体积比1:3:6组成)打浆,得到(产率:98%);
4)将50mL浓度0.1mol/L的三乙胺-碳酸缓冲液加入步骤3)的反应液中,调节pH至7.5左右,25℃反应48h,浓缩,得到三磷酸化合物粗品;
5)通过反相高效液相色谱对三磷酸化合物粗品进行纯化,得到N-(9-芴基甲氧羰基)-2-氨基乙基三磷酸(产率:41%);
通过反相高效液相色谱对三磷酸化合物粗品进行纯化的具体操作为:
a)将三磷酸化合物粗品溶解在甲醇中,再用滤膜过滤;
b)采用反相高效液相色谱对滤液进行纯化,色谱柱规格为C18(Innoval ODS-2 50×250mm,5μm),流动相A为0.1mol/L的三乙胺-碳酸缓冲液,流动相B为甲醇,将色谱柱用流动相B:62%平衡后,用流动相B:62%~75%梯度洗脱,收集纯化后的化合物,冻干。
性能测试:
N-(9-芴基甲氧羰基)-2-氨基乙基三磷酸的核磁共振氢谱图如图10所示,核磁共振磷谱图如图11所示,解谱分析如下:
1H NMR(D2O,400MHz):δ=7.82(d,J=7.5Hz,2H),7.62(d,J=7.4Hz,2H),7.41(m,2H),7.38-7.31(m,2H),4.39(d,J=6.0Hz,2H),4.22(d,J=5.6Hz,1H),3.89(d,J=5.7Hz,2H),3.26(d,J=5.8Hz,2H)。
31P NMR(D2O):δ=-9.88(m,1P),-10.97(m,1P),-23.06(t,1P)。
N-(9-芴基甲氧羰基)-2-氨基乙基三磷酸的结构式:
对比例:
N-(9-芴基甲氧羰基)-6-氨基己基三磷酸的合成:
1)将5.3g的芴甲氧羰酰氯溶于40mL的1,4-二氧六环,在0℃下加入2g的6-氨基-1-己醇,充分溶解,再缓慢加入20mL浓度10%的Na2CO3溶液,25℃反应12h,用甲醇-二氯甲烷(甲醇、二氯甲烷体积比1:19)萃取产物,合并有机相,在旋转蒸发仪上旋干溶剂,用混合溶剂(由二氯甲烷、乙酸乙酯和石油醚按照体积比1:3:6组成)打浆,得到
4)将50mL浓度0.1mol/L的三乙胺-碳酸缓冲液加入步骤3)的反应液中,调节pH至7.5左右,25℃反应48h,浓缩,得到三磷酸化合物粗品;
5)通过反相高效液相色谱对三磷酸化合物粗品进行纯化(操作同实施例1),得到N-(9-芴基甲氧羰基)-6-氨基己基三磷酸(产率:29%)。
性能测试:
步骤4)中的三磷酸化合物粗品的液相色谱图如图12所示。
由图1可知:步骤2)中以四氢呋喃为溶剂,液相色谱图中9.15min为N-(9-芴基甲氧羰基)-6-氨基己基三磷酸,8.90min为五磷酸副产物。
由图12可知:步骤2)中以磷酸三甲酯为溶剂,液相色谱图中9.13min为N-(9-芴基甲氧羰基)-6-氨基己基三磷酸,8.87min为五磷酸副产物,11.1min为二磷酸二甲酯副产物。
由此可见,步骤2)中以四氢呋喃为溶剂可以显著降低制备得到的三磷酸产品的杂质含量,进而可以提高三磷酸产品的产率。
dA6P-NH2-Nucleotides的合成:
1)将0.1g的N-(9-芴基甲氧羰基)-6-氨基己基三磷酸加入10mL的无水乙腈,用旋转蒸发仪减压蒸干,再重复一次操作,除去化合物中的水分,再将N-(9-芴基甲氧羰基)-6-氨基己基三磷酸溶于3mL的二甲基甲酰胺,再加入0.7mmol的N,N'-羰基二咪唑,25℃搅拌4h,再加入1.05mmol的甲醇,继续搅拌30min,得到
4)通过中压制备色谱对脱氧核苷酸粗品进行初步分离纯化(操作同实施例1),再通过反相高效液相色谱对初步分离纯化的产品进行进一步纯化(操作同实施例1),得到脱氧核苷酸dA6P-NH2-Nucleotides(产率:55%)。
由此可见,步骤3)中采用吗啉-二甲基甲酰胺溶液替代三乙胺溶液可以大幅缩短反应时间,且可以大幅提高产物收率。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
3.根据权利要求1或2所述的三磷酸化合物的制备方法,其特征在于:步骤1)所述溶剂为1,4-二氧六环、乙腈、二甲基甲酰胺、二氯甲烷中的至少一种。
9.根据权利要求7或8所述的脱氧核苷酸的制备方法,其特征在于:步骤1)所述溶剂为二甲基甲酰胺、乙腈、二甲基亚砜中的至少一种。
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