CN107098934B - 一种邻苯二醛衍生物亚磷酰胺单体、合成方法及dna快速偶联蛋白质的方法 - Google Patents
一种邻苯二醛衍生物亚磷酰胺单体、合成方法及dna快速偶联蛋白质的方法 Download PDFInfo
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Abstract
一种邻苯二醛衍生物亚磷酰胺单体、合成方法和及DNA快速偶联蛋白质的方法,涉及DNA快速偶联蛋白质。所述邻苯二醛衍生物亚磷酰胺单体的分子式为C28H46N3O6P。首先邻苯二醛羧酸衍生物(OPA‑COOH)与6‑氨基‑1‑己醇反应制备邻苯二醛羟基衍生物(OPA‑OH),然后在氮气保护及二氯甲烷溶剂和N,N‑二异丙基乙胺条件下,与2‑氰乙基‑N,N‑二异丙基氯代亚磷酰胺室温条件下反应2h,经硅胶柱层析分离,得到邻苯二醛衍生物亚磷酰胺单体。利用邻苯二醛衍生物的相邻醛基能够与蛋白质赖氨酸残基上的氨基进行快速、高效生成苄甲内酰胺的反应特点,最终实现OPA‑DNA与天然蛋白质快速偶联的目的。与其他偶联方法相比,该方法具有选择性、快速、高效的优点。
Description
技术领域
本发明涉及一种邻苯二醛衍生物亚磷酰胺单体及其合成方法,尤其涉及DNA与蛋白质快速偶联的方法。
背景技术
天然蛋白质的修饰,对于生物分析研究领域具有非常重要的意义。比如,在生物传感器方面,通常需要DNA与蛋白质的偶联。传统的DNA与蛋白质的偶联方法,主要分为两类,即非共价偶联和共价偶联。通过生物素和链霉亲和素的非共价结合时最为常见的非共价偶联方式,由于二者非常强的非共价结合能力而被广泛应用于生物分析领域,如酶联免疫吸附测定(ELISA)。然而,上述偶联体系比较繁琐,需要借助于分子量比较大的链霉亲和素,且修饰步骤复杂、耗时,一定程度上限制了其应用。常见的DNA与蛋白质的共价偶联方法包括(1)巯基修饰的DNA(SH-DNA)通过4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐(sulfo-SMCC)或M-马来酰亚胺苯甲酸琥珀酰亚胺酯(sulfo-MBS)和蛋白质上的氨基进行偶联;(2)氨基修饰的DNA(NH2-DNA)通过sulfo-SMCC或sulfo-MBS和蛋白质上的巯基进行偶联;(3)NH2-DNA通过辛二酸二琥珀酰亚胺酯(DSS)和蛋白质上的氨基进行偶联;(4)NH2-DNA通过1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC·HCl)及N-羟基琥珀酰亚胺磺酸钠盐(sulfo-NHS)和蛋白质上的羧基进行偶联;(5)NH2-DNA通过戊二醛和蛋白质上的氨基进行偶联(1、Greg T.Hermanson,Bioconjugate Techniques[B].USA,PierceBiotechnology&Thermo Fisher Scientific,2008,1-1202)。然而,上述共价偶联方法,所需时间较长,或操作处理繁琐,而且大部分偶联效率较低。如何实现快速、高效的DNA与蛋白质的共价偶联仍是亟待解决的问题。
发明内容
本发明的目的是针对现有的DNA与蛋白质共价偶联的难题,提供一种DNA修饰单体及其合成方法,以及提供一种DNA与蛋白质选择性、快速、高效偶联的方法。
本发明的技术方案如下:
一种邻苯二醛衍生物亚磷酰胺单体,其分子式为C28H46N3O6P,其结构式如下:
本发明的又一技术方案为:
一种邻苯二醛衍生物亚磷酰胺单体的合成方法,包括以下步骤:
1)将邻苯二醛羧酸衍生物(OPA-COOH)、6-氨基-1-己醇、二环己基碳二亚胺(DCC)、1-羟基苯并三氮唑(HOBT)按照摩尔1:1.5-2.5:1-2:1-2混合,抽排除去空气,加入溶剂四氢呋喃搅拌反应而后过滤除去沉淀,再用硅胶柱层析分离纯化,得到邻苯二醛羟基衍生物(OPA-OH);反应的时间可为12~24h;
2)在氮气保护下,将OPA-OH和N,N-二异丙基乙胺(DIPEA)混合,用二氯甲烷溶解,再加入2-氰乙基-N,N-二异丙基氯代亚磷酰胺,搅拌反应。而后用硅胶柱层析分离纯化,得到邻苯二醛衍生物亚磷酰胺单体,其中,所述OPA-OH、2-氰乙基-N,N-二异丙基氯代亚磷酰胺、N,N-二异丙基乙胺的摩尔比为1:1-2:2-5;
在步骤2)中,所述反应温度可以为25℃,反应的时间可为3-5h。
在本发明的较佳实施例中,步骤1),加入的溶剂的体积为溶质的1-20倍。
在本发明的较佳实施例中,步骤2),所述反应温度为20-30℃,反应时间为1-5h。
本发明的再一技术方案为:
一种DNA快速偶联蛋白质的方法,包括如下步骤:
1)合成权利要求1所述的邻苯二醛衍生物亚磷酰胺单体;
2)在DNA的5’端修饰邻苯二醛衍生物亚磷酰胺单体;
3)在缓冲液的条件下,将5’端修饰邻苯二醛衍生物亚磷酰胺单体的DNA和蛋白质室温孵育0.5-3h。
在本发明的较佳实施例中,缓冲溶液为磷酸盐缓冲溶液。
在本发明的较佳实施例中,步骤2)包括以下步骤:
(1)CPG作为固相载体,以正常的DNA单体碱基作为原料,在DNA合成仪上由3’端到5’端合成DNA序列,然后在DNA的5’端修饰邻苯二醛衍生物亚磷酰胺单体;
(2)加入甲胺/氨水(1:1),65℃条件下孵育30min将OPA-DNA产物从CPG上切割下来。
(3)利用反相色谱(C18柱)进行分离纯化,所用流动相为0.1mol/L醋酸三乙胺(TEAA)和乙腈。
(4)利用20%三氟乙酸/二氯甲烷(v/v),脱去醛基保护,而后抽干去除。
在步骤4)中,所述20%三氟乙酸/二氯甲烷(v/v)脱去醛基保护之前,先用脱盐柱脱盐纯化。
所述一种邻苯二醛衍生物亚磷酰胺单体的合成路线如下:
本发明中所合成的邻苯二醛衍生物亚磷酰胺单体,可以通过DNA合成仪修饰到DNA5’末端,后续经过纯化得到具有高反应活性的OPA-DNA。所合成的OPA-DNA可以在温和的反应条件(磷酸盐缓冲溶液)下,选择性、快速、高效的与天然蛋白质实现共价偶联。和传统的DNA及蛋白质的偶联方法相比,有效的解决了所需时间较长,或操作处理繁琐,而且大部分偶联效率较低等困难。
本发明具有以下优点:
1、合成原料廉价,步骤简单可行;
2、OPA-DNA可以在温和的反应条件(磷酸盐缓冲溶液)下,选择性、快速、高效的与天然蛋白质实现共价偶联。
附图说明
图1为邻苯二醛衍生物亚磷酰胺单体的1H NMR谱图。
图2为邻苯二醛衍生物亚磷酰胺单体的31P NMR谱图。在图2中,147.25ppm为phosphoramidite的特征峰。
图3为邻苯二醛衍生物亚磷酰胺单体的ESI-MS质谱分子图。在图3中,理论分子量为552.7([M+H]+),实际分子量为552.2([M+H]+)、574.3([M+Na]+)、590.3([M+K]+)。
图4为HPLC分离纯化结果。用260nm和490nm吸收峰同时进行监测,从结果可以看出,在保留时间为13min时,产物被分离出来。
图5为核酸序列strand A的分子量分析谱图。用MALDI-TOF-MS技术对序列的分子量进行分析,理论分子量为3940,实际分子量为3946,说明邻苯二醛衍生物亚磷酰胺单体成功修饰到DNA上。
图6为OPA-DNA与细胞色素C偶联的电泳分析结果。其中,(A)为凝胶成像仪分析结果,(B)为考马斯亮蓝染色结果。
图7为不同浓度OPA-DNA与细胞色素C偶联的电泳分析结果。其中,(A)为凝胶成像仪分析结果,(B)为考马斯亮蓝染色结果。
具体实施方式
实施例1邻苯二醛衍生物亚磷酰胺单体的合成,步骤如下:
步骤一:合成中间产物2(OPA-OH),合成路线如下图所示:
在一个25mL圆底烧瓶中,依次加入1(252mg,1mmol),6-氨基-1-己醇(234mg,2mmol),DCC(247.2mg,1.2mmol),HOBT(162mg,1.2mmol),10mL溶剂四氢呋喃,在氮气保护下,室温搅拌反应24h。反应结束后,旋转蒸发除去溶剂,用硅胶柱进行分离纯化,核磁和质谱表征。1H NMR(500MHz,Methanol-D4):δ=7.34-7.28(m,3H),6.29(s,1H),6.03(s,1H),3.56(t,2H),3.44-3.39(m,6H),3.15(m,2H),3.01(t,2H),2.52(t,2H),1.53(m,2H),1.45(m,2H),1.35(m,2H),1.29(m,2H)ppm.13C NMR(500MHz,CDCl3):δ=174.82,144.31,140.10,137.77,131.20,124.01,123.88,107.85,106.77,62.87,54.72,54.63,40.42,38.81,33.46,32.73,30.31,27.68,26.53ppm.ESI-MS calculated for C19H29NO5Na:274.2([M+Na]+),found:374.1.
步骤二:合成产物3,合成路线如下图所示:
在一个10mL圆底烧瓶中,加入2(126mg,0.5mmol),抽排除去空气,在氮气保护下加入5mL二氯甲烷。在室温条件下逐滴加入DIPEA(194mg,1.5mmol),搅拌均匀后,滴加2-氰乙基-N,N-二异丙基氯代亚磷酰胺(142mg,0.6mmol),室温搅拌反应3h。反应结束后,旋转蒸发除去溶剂,用硅胶柱进行分离纯化,核磁和质谱表征,如图1-3。1H NMR(500MHz,Methanol-D4):δ=7.34-7.27(m,3H),6.29(s,1H),6.02(d,1H),3.85-3.83(m,2H),3.76(t,1H),3.68-3.65(m,4H),3.43(q,6H),3.14-3.11(m,2H),3.00(t,2H),2.74(t,2H),2.63(t,1H),2.50(t,2H),2.62-2.59(m,2H),1.43-1.39(m,4H),1.22(t,12H).13C NMR(500MHz,Methanol-D4):δ=174.67,140.10,137.77,131.34,131.21,124.00,123.89,119.53,107.83,106.75,64.61,59.74,59.59,58.34,54.73,54.29,44.28,44.18,40.29,38.76,32.74,32.17,30.27,27.54,26.71,25.05,24.99,21.83ppm.31P NMR(202MHz,Methanol-D4)δ147.25ppm.ESI-MS calculated for C28H46N3O6PNa:574.3([M+Na]+),found:574.3.
实施例2邻苯二醛衍生物修饰的核酸分子的合成与纯化。
以6-FAM标记的CPG作为固相载体,以正常的DNA单体碱基作为原料,在DNA合成仪上由3’端到5’端合成DNA序列,最后在5’端修饰邻苯二醛衍生物亚磷酰胺单体。具体合成的序列如下:5’-X(T)10Y-3’,其中X为产物3,Y为6-FAM。DNA合成结束后,将上述CPG转移至1.5mL Eppendorf管中,加入0.4mL甲胺/氨水(v/v=1:1),65℃孵育30min,将DNA从CPG上切割下来。而后真空抽除甲胺/氨水,再用0.5mL 0.1mol/L醋酸三乙胺(TEAA)溶解,使用C18反相高效液相色谱进行纯化,如图4。将得到的产品进行真空干燥,溶液超纯水后,再用凝胶过滤柱进行脱盐纯化并真空干燥。然后,用适量三氟乙酸/二氯甲烷(v/v=1:4)溶解上述产物,室温条件下孵育30min,再真空干燥。最后再用二次去离子水溶解DNA(即为OPA-DNA),并用紫外-可见分光光度计测定260nm处核酸的吸光度,根据DNA的摩尔消光系数,计算出相应物质的量浓度。定量后真空浓缩,并用MALDI-TOF表征其分子量,如图5。
实施例3反相高效液相色谱进行纯化DNA。
实验采用梯度洗脱程序,以0.1mol/L TEAA(A相)和95%乙腈(B相)为流动相,利用C18反相色谱柱纯化DNA。梯度洗脱程序如下:0-3min,100%A;3-35min,A由100%逐渐降低至50%。由于产物DNA 3’端标记荧光素,所以产物用260nm和490nm吸收峰同时进行监测。从图4结果可以看出,在保留时间为13min时,产物被分离出来。
实施例4 OPA-DNA与细胞色素C偶联的电泳分析结果
磷酸盐缓冲液:(137mmol/L NaCl,2.7mmol/L KCl,10mmol/L Na2HPO4,2mmol/LKH2PO4,pH 7.4)。
以细胞色素C(Cyto C)为例,尝试OPA-DNA与蛋白质的偶联,并用20%SDS-PAGE胶进行电泳分析。偶联反应具体操作如下:10uL反应体系中,40uM Cyto C与40uM OPA-DNA室温孵育,反应时间分别为0,0.5,1,2和3h,而后加入1uL饱和甘氨酸水溶液室温孵育0.5h进行猝灭。对照组DNA(ctrl DNA)为标记有荧光素但是没有OPA修饰的DNA(5’-(T)10-FAM-3’)。对照组分别为40uM Cyto C,40uM ctrl DNA,40uM ctrl DNA+40uM Cyto C以及40uM OPA-DNA。SDS-PAGE胶分析的详细步骤如下,首先配制分离胶为20%SDS-PAGE胶(浓缩胶为5%),每个样品加入5uL 4×loading buffer,95℃孵育10min,而后放置冰上冷却。用微量上样针将样品加入到胶孔中,电泳条件:60V电泳0.5h,120V电泳1.5h。电泳结束后,先用凝胶成像仪成像在紫外模式下拍照,再用考马斯亮蓝染色进行分析。凝胶成像仪分析如图6A所示,3泳道与4泳道中DNA只有相同位移的DNA条带,说明DNA不能通过静电吸附作用与Cyto C结合而在胶图上产生新的条带;6-10泳道与5泳道相比,游离DNA的荧光条带都明显变弱,且DNA与蛋白偶联的产物条带随着反应时间的延长,强度逐渐增强且偶联多条DNA的条带也增多,说明DNA可以快速偶联到蛋白质分子上,且随着反应时间的延长,多条DNA偶联产物逐渐增多。从图6B考染结果来看,偶联一条DNA及两条的Cyto C有考染出蛋白条带而其他荧光条带均没有,说明偶联产物(蛋白质和DNA)主要是一对一和一对二的偶联。通过对DNA条带的灰度分析,当反应时间为1h及其以上时,DNA反应效率都达到50%以上,该方法实现了DNA与蛋白质的快速、高效偶联。
实施例5不同浓度OPA-DNA与细胞色素C偶联的电泳分析结果
实验尝试不同浓度OPA-DNA与细胞色素C(Cyto C)的偶联,并用20%SDS-PAGE胶进行电泳分析。偶联反应具体操作如下:10uL反应体系中,40uM Cyto C分别与20uM,40uM和80uM OPA-DNA室温孵育2h,而后加入1uL饱和甘氨酸水溶液室温孵育0.5h进行猝灭。对照组DNA(ctrl DNA)为标记有荧光素但是没有OPA修饰的DNA(5’-(T)10-FAM-3’)。对照组分别为40uM Cyto C,80uM ctrl DNA,80uM ctrl DNA+40uM Cyto C以及不同浓度(20uM,40uM和80uM OPA-DN)的OPA-DNA溶液。SDS-PAGE胶分析的详细步骤如下,首先配制分离胶为20%SDS-PAGE胶(浓缩胶为5%),每个样品加入5uL 4×loading buffer,95℃孵育10min,而后放置冰上冷却。用微量上样针将样品加入到胶孔中,电泳条件:60V电泳0.5h,120V电泳1.5h。电泳结束后,先用凝胶成像仪成像在紫外模式下拍照,再用考马斯亮蓝染色进行分析。凝胶成像仪分析如图7A所示,6泳道与5泳道相比,OPA-DNA荧光条带大部分明显上移,而且上移条带与蛋白能够共定位,说明OPA-DNA能够高效率的与细胞色素C进行共价偶联。结合8泳道与7泳道及10泳道与9泳道结果,发现随着OPA-DNA浓度的增加,多条OPA-DNA与细胞色素C偶联产物逐渐增多,但是OPA-DNA的反应效率随着其浓度的增加而明显下降。从图7B考染结果来看,当OPA-DNA浓度较低时,主要为一条OPA-DNA与细胞色素C的偶联产物,随着OPA-DNA浓度的增加,偶联两条及三条OPA-DNA的蛋白条带明显增加,说明多条OPA-DNA与细胞色素C偶联产物逐渐增多。该方法实现了DNA与蛋白质的快速高效偶联。
Claims (9)
1.邻苯二醛衍生物亚磷酰胺单体,分子式为C28H46N3O6P,其结构式如下:
2.如权利要求1所述的邻苯二醛衍生物亚磷酰胺单体的用途,用于DNA与蛋白质的偶联。
3.如权利要求1所述的邻苯二醛衍生物亚磷酰胺单体的合成方法,包括以下步骤:
1)1)将邻苯二醛羧酸衍生物OPA-COOH、6-氨基-1-己醇、二环己基碳二亚胺、1-羟基苯并三氮唑按照摩尔比1:1.5-2.5:1-2:1-2混合,抽排除去空气,加入溶剂四氢呋喃搅拌反应,反应时间为12~24h;而后过滤除去沉淀,再分离纯化,得到邻苯二醛羟基衍生物OPA-OH;其中,OPA-COOH及OPA-OH结构式分别如下:
2)在氮气保护下,将OPA-OH和N,N-二异丙基乙胺混合,用二氯甲烷溶解,再加入2-氰乙基-N,N-二异丙基氯代亚磷酰胺,搅拌反应后用硅胶柱层析分离纯化,得到邻苯二醛衍生物亚磷酰胺单体,其中,所述OPA-OH、2-氰乙基-N,N-二异丙基氯代亚磷酰胺、N,N-二异丙基乙胺的摩尔比为1:1-2:2-5。
4.如权利要求3所述一种邻苯二醛衍生物亚磷酰胺单体的合成方法,其特征在于步骤1),加入的溶剂的体积为溶质的1-20倍。
5.如权利要求3所述一种邻苯二醛衍生物亚磷酰胺单体的合成方法,其特征在于步骤2),所述反应温度为20-30℃,反应时间为1-5h。
6.DNA快速偶联蛋白质的方法,包括如下步骤:
1)合成权利要求1所述的邻苯二醛衍生物亚磷酰胺单体;
2)在DNA的5’端修饰邻苯二醛衍生物亚磷酰胺单体;
3)在缓冲液的条件下,将5’端修饰邻苯二醛衍生物亚磷酰胺单体的DNA和蛋白质室温孵育0.5-3小时。
7.如权利要求6所述DNA快速偶联蛋白质的方法,其特征在于缓冲溶液为磷酸盐缓冲溶液。
8.如权利要求6所述的DNA快速偶联蛋白质的方法,其特征在于:
步骤2)包括以下步骤:
1)CPG作为固相载体,以正常的DNA单体碱基作为原料,在DNA合成仪上由3’端到5’端合成DNA序列,然后在DNA的5’端修饰邻苯二醛衍生物亚磷酰胺单体;
2)加入甲胺/氨水,65℃条件下孵育30min将OPA-DNA产物从CPG上切割下来;
3)利用反相色谱进行分离纯化,所用流动相为0.1M醋酸三乙胺和乙腈;
4)利用20%三氟乙酸/二氯甲烷(v/v),脱去醛基保护,而后抽干去除。
9.如权利要求8所述邻苯二醛衍生物亚磷酰胺单体修饰合成OPA-DNA,其特征在于步骤4),所述20%三氟乙酸/二氯甲烷(v/v)脱去醛基保护之前,先用脱盐柱脱盐纯化。
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