CN1756792A - 带有阴离子性取代基物质的捕捉剂 - Google Patents
带有阴离子性取代基物质的捕捉剂 Download PDFInfo
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- CN1756792A CN1756792A CNA2004800058577A CN200480005857A CN1756792A CN 1756792 A CN1756792 A CN 1756792A CN A2004800058577 A CNA2004800058577 A CN A2004800058577A CN 200480005857 A CN200480005857 A CN 200480005857A CN 1756792 A CN1756792 A CN 1756792A
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- trapping agent
- phosphoric acid
- anionic substituent
- polymer support
- bonded
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Abstract
指定的锌配位基直接或通过隔离基以共价结合的聚合物载体,在一定条件下,具有与阴离子性取代基(如磷酸基)相结合的特性,同时,整体表现为溶剂难溶性(优选溶剂不溶性),可以捕捉带有阴离子性取代基(如磷酸基)的物质,并且,可容易进行分离精制。
Description
技术领域
本发明涉及可用于分离精制带有阴离子性取代基(如磷酸基)的物质、由指定的锌配位基结合的聚合物载体、含有该聚合物载体的带有阴离子性取代基(如磷酸基)物质的捕捉剂,还涉及填充有这种捕捉剂的捕捉器具及其捕捉方法。
背景技术
作为用于分析带有磷酸基的物质,例如磷酸化的活体物质等的方法,在现有技术中,已知的有通过酶联免疫吸附试验法(ELISA)的方法或者使用放射性同位素的方法。
酶联免疫吸附试验法利用抗体(或者抗原亦可)与目标物质特异性结合的原理。为此,要想制作目标物质固有的抗体,存在必须大量精制、获得目标物质的问题。同时,抗体制作时利用动物的免疫反应,所以抗体的制作存在耗时的问题。还有,由于无法制作相对于数kDa(道尔顿)以下分子结构中的磷酸化部位的抗体,所以存在无法利用酶联免疫吸附试验法分析具有如此分子结构的磷酸化活体物质的问题。
另外,使用放射性同位素的方法是利用放射性同位素32P。为此,存在实验室中放射线的管理及废液的管理费事的问题。
有关降低排放水中磷酸浓度的方法,在特开平11-57695号公报中,公开了使用多金属氢氧化物的方法。另一方面,在医药领域,作为在高磷血症的治疗中使用的物质,在特表平8-506846号公报中记载有胍基结合而成的聚合物。但是,多金属氢氧化物及胍基结合而成的聚合物,对于磷酸基的结合力都弱。为此,要捕捉一定量的磷酸,就存在必须使用大量作为磷酸基结合基质的多金属氢氧化物或者胍基结合而成的聚合物的问题。
另一方面,作为与磷酸基强结合的化合物,在美国化学会志(Journal of the American Chemical Society)(美国),1991年,第113卷,第23号,第8935-8941页记载有大环状聚胺锌配位化合物。但是,大环状聚胺锌配位化合物可溶于溶剂中。为此,存在在溶液中分离精制与大环状聚胺锌配位化合物相结合的带有磷酸基的物质非常困难的问题。
从而,为了分离精制磷酸化的活体物质等带有磷酸基的物质,期望有在一定的条件下,同时具备与阴离子性取代基之一的磷酸基强力结合的特性及分离精制容易的特性,且安全又廉价的捕捉剂。
本发明的目的是提供一种在一定的条件下,与阴离子性取代基(如磷酸基)结合,同时使得分离精制带有该取代基的物质容易、安全又廉价的捕捉剂,及其填充有这种捕捉剂的简便捕捉器具及迅速且容易的捕捉方法。
发明内容
本发明的发明人们,为了解决上述现有技术中存在的问题,作了不懈的探讨研究,结果发现,如果聚合物载体与特定的锌配位基相结合,则该聚合物载体具有与阴离子性取代基(如磷酸基)强力结合的特性,同时,整体显示出溶剂难溶性(优选溶剂不溶性),由此,使分离精制变得极为容易,即可成为带有该取代基的物质的有用捕捉剂。同时,也确立了填充有这种捕捉剂的该物质的捕捉器具及使用这种捕捉剂的该物质的捕捉方法,从而完成了本发明。
即,本发明(1)是一种聚合物载体,为通式(1)所示锌配位基直接或通过隔离基结合的聚合物载体,
式中,R相互间可相同或不同,可以是氢原子、碳原子数为1-16的烷基、酰基、烷氧羰基、酰基烷基、烷氧羰基烷基、羧基烷基、氨基甲酰烷基、氰基烷基、羟基烷基、氨基烷基或者卤代烷基(在此,这些基团的烷基部分的碳原子数是1-16)、羧基、氨基甲酰基、氰基、羟基、氨基或者卤素基团。
还有,本发明(2)是一种带有阴离子性取代基的物质的捕捉剂,其中,含有发明(1)所述的聚合物载体或者通式(2)所示的锌配位基直接或通过隔离基结合的聚合物载体。
并且,本发明(3)是根据发明(2)所述捕捉剂,其中,阴离子性取代基为磷酸基。
还有,本发明(4)是根据发明(2)或(3)所述捕捉剂,其中,捕捉剂呈珠粒状。
并且,本发明(5)是根据发明(2)或(3)所述捕捉剂,其中,捕捉剂呈板状。
还有,本发明(6)是根据发明(2)或(3)所述捕捉剂,其中,捕捉剂呈纤维状。
并且,本发明(7)是一种带有阴离子性取代基物质的捕捉器具,其中,填充有发明(2)-(4)或(6)之一的捕捉剂,具有以过滤器进行过滤分离的功能。
还有,本发明(8)是一种带有阴离子性取代基的物质的捕捉方法,包括通过使带有阴离子性取代基的物质与发明(2)-(6)之一的捕捉剂相结合而进行捕捉的步骤。
并且,本发明(9)是根据发明(8)所述方法,其中,在该捕捉步骤之后,还包括将带有阴离子性取代基的物质从捕捉剂上解离的步骤。
附图说明
图1为使用含有由锌配位基结合而成的聚合物载体的捕捉剂,所得到的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)图的前半部分。
图2为使用含有由锌配位基结合而成的聚合物载体的捕捉剂,所得到的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)图的后半部分。
图3为使用含有由不含锌的配位基结合而成的聚合物载体的捕捉剂,所得到的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)图的前半部分。
图4为使用含有由不含锌的配位基结合而成的聚合物载体的捕捉剂,所得到的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)图的后半部分。
另外,在这些图中,1为牛血清白蛋白、2为鸡蛋白白蛋白、3为牛β-酪蛋白、4为非磷酸化型牛αs1-酪蛋白、5为牛αs1-酪蛋白、6为TRIS(三羟甲基氨基甲烷)-盐酸缓冲液(pH7.0)和0.5M氯化钠水溶液的混合液第一次、7为TRIS-盐酸缓冲液(pH7.0)和0.5M氯化钠水溶液的混合液第二次、8为TRIS-乙酸缓冲液(pH7.0)第一次、9为TRIS-乙酸缓冲液(pH7.0)第二次、10为TRIS-乙酸缓冲液(pH7.0)第三次、11为TRIS-乙酸缓冲液(pH7.0)第四次、12为10mM磷酸缓冲液(pH7.0)、13为15mM磷酸缓冲液(pH7.0)、14为20mM磷酸缓冲液(pH7.0)、15为25mM磷酸缓冲液(pH7.0)、16为30mM磷酸缓冲液(pH7.0)、17为35mM磷酸缓冲液(pH7.0)、18为40mM磷酸缓冲液(pH7.0)、19为45mM磷酸缓冲液(pH7.0)、20为50mM磷酸缓冲液(pH7.0)、21为60mM磷酸缓冲液(pH7.0)、22为70mM磷酸缓冲液(pH7.0)、23为80mM磷酸缓冲液(pH7.0)、24为90mM磷酸缓冲液(pH7.0)、25为100mM磷酸缓冲液(pH7.0)、26为200mM磷酸缓冲液(pH7.0)、27为300mM磷酸缓冲液(pH7.0)、28为400mM磷酸缓冲液(pH7.0)、29为500mM磷酸缓冲液(pH7.0)的图线。
具体实施方式
首先,对通式(1)或者通式(2)中所示锌配位基结合的聚合物载体进行说明。所谓“聚合物载体”为可与该基团结合的聚合物,并且,只要起“载体”或者“支撑体”的功能,其他没有特殊限定。但是,优选的是,聚合物载体在使用时,对所用溶剂或者试剂有耐受性,并且,具有在进行过滤及洗净时必要的物理强度。还有,优选对带有阴离子性取代基物质的捕捉没有影响。作为具体例可列举如下:聚苯乙烯、聚乙烯、聚丙烯、聚乙炔(聚炔烃)、聚氯乙烯、聚乙烯基酯、聚乙烯基醚、聚丙烯酸酯、聚丙烯酸、聚丙烯腈、聚丙烯酰胺、聚甲基丙烯酸酯、聚甲基丙烯酸、聚甲基丙烯腈、聚甲基丙烯酰胺、聚醚、聚甲醛、聚酯、聚对苯二甲酸乙二醇酯、聚碳酸酯、聚酰胺、尼龙、聚氨酯、聚尿素、聚酰亚胺、聚咪唑、聚噁唑、聚硫化物、聚砜、聚磺酰胺、聚合物合金、纤维素、葡聚糖、琼脂糖、壳聚糖、硅石等。另外,为了确保更好的物理强度,聚合物载体亦可为具有交联结构的聚合物载体,交联剂的具体例可列举如下:二乙烯基苯、3-氯-1,2-环氧丙烷、N,N′-亚甲基双丙烯酰胺、二异氰酸4,4′-二苯甲烷等。
这种聚合物载体,与通式(1)或者通式(2)中所示的锌配位基直接或者通过隔离基相结合。在此,所谓隔离基,是指为通过使锌配位基从聚合物载体上分开而促进捕捉物质与锌配位基相结合及增加溶剂的膨润度目的而导入的聚合物,作为具体例可列举如下:聚乙二醇、聚丙烯酰胺、聚乙烯、聚酰胺、聚酯等。另外,锌配位基和聚合物载体或者隔离基相结合方式,作为具体例可列举如下:碳-碳结合、酯结合、羰基结合、酰胺结合、醚结合、硫化物结合、氨基结合、亚胺基结合等共价键结合。
本发明的通式(1)所示的锌配位基结合的聚合物载体,作为具体例可列举如下实例等:由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的TOYOPEARL(注册商标):
或者,由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的ArgoGel(注册商标):
或者,由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的TSKgel(注册商标):
或者,由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的Sepharose(商标):
本发明所述通式(2)所示锌配位基结合的聚合物载体,作为具体例可列举如下实例等:由Zn2+-四氮杂环十二烷基(サイクレン基)结合的TOYOPEARL(注册商标):
或者,由Zn2+-四氮杂环十二烷基结合的ArgoGel(注册商标):
或者,由Zn2+-四氮杂环十二烷基结合的TentaGel(注册商标):
所谓阴离子性取代基是指带负电荷的取代基,作为阴离子的具体例可列举如下:2价磷酸单酯阴离子(-OPO3 2-)、1价磷酸二酯阴离子((-O)2PO2 -)、2价膦酸阴离子(-PO3 2-)、1价膦酸阴离子((-)2PO2 -)、1价碳酸酯阴离子(-OCO2 -)、1价羧酸阴离子(-CO2 -)、1价硫酸酯阴离子(-OSO3 -)、1价磺酸阴离子(-SO3 -)等。
所谓带有磷酸基的物质是指带有2价磷酸单酯阴离子(-OPO3 2-)的物质,作为该物质的具体例可列举如下:磷酸化氨基酸、磷酸化氨基酸残基、带有磷酸化氨基酸残基的蛋白质(磷酸化蛋白质)、带有磷酸化氨基酸残基的多肽(磷酸化多肽)、带有磷酸化氨基酸残基的低聚肽(磷酸化低聚肽)、脱氧核糖核酸(DNA)、磷脂、磷酸化糖类、血中的磷酸成分、排水中的磷酸成分等。
通式(1)或者通式(2)中所示锌配位基本身与所用溶剂间的关系通常是可溶的。但是,通过将其与聚合物载体相结合,与该溶剂的关系为难溶(优选不溶)。为此,本发明的聚合物载体在优选条件下,能够捕捉溶液中带有阴离子性取代基(如磷酸基)的物质,同时,也可从溶剂中过滤或者洗净,因此,可以迅速且容易地从溶液中分离精制该物质。同时,对该物质的定量亦可使用捕捉剂。
以珠粒状使用的捕捉剂,其用途的具体例可列举如下:金属固定亲和层析(IMAC)柱等的柱载体、用于精制或者浓缩磷酸化蛋白质或者脱氧核糖核酸(DNA)等带有磷酸基的物质的柱填充剂、用于分离或者精制带有磷酸基的物质的磁性珠粒、用于高磷血症治疗的捕捉血中磷酸成分的制剂等。
以板状使用的捕捉剂,其用途的具体例可列举如下:用于基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF MS)的试样基板、用于精制或者检出蛋白质或者脱氧核糖核酸(DNA)等的薄片等。
以纤维状使用的捕捉剂,其用途的具体例可列举如下:用于分离带有磷酸基的物质和不带有磷酸基的物质的分离膜、中空纤维膜过滤器等。
本发明的带有阴离子性取代基(如磷酸基)物质的捕捉器,填充含有上述聚合物载体的捕捉剂或者是珠粒或纤维状捕捉剂,具有通过过滤器进行过滤分离的功能。在此,所谓过滤器是指,将溶解有这些捕捉剂及与这些捕捉剂相结合的带有该取代基的物质和不与这些捕捉基相结合的不带有该取代基的物质的溶液,或者,溶解有这些捕捉剂和从这些捕捉剂上解离的带有该取代基的物质的溶液,通过简便的固液分离达到过滤分离的目的,而导入的分离材料,作为具体例可列举如下:玻璃滤器、金属烧结滤器、膜滤器、超滤膜、石棉、玻璃棉、聚乙烯、聚丙烯、聚四氟乙烯、聚醚砜、纤维素等。另外,此过滤器不仅是安置在捕捉剂的下方,上下都可安置,通过采用2个过滤器夹持这些捕捉剂,也一并有抑制这些捕捉剂流动范围的效果。
本发明的带有阴离子性取代基(特别是磷酸基)物质的捕捉器具,作为具体例可列举如下:由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的TOYOPEARL(注册商标)柱、由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的Sepharose(商标)柱、由Zn2+ -四氮杂环十二烷基结合的TOYOPEARL(注册商标)柱、由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的Sepharose(商标)离心式过滤设备等。
本发明的带有阴离子性取代基(如磷酸基)物质的捕捉方法,利用了与聚合物载体相结合的锌配位基与该物质相结合的原理。而且,根据捕捉该物质时的规模或者状态,可以选择诸如珠粒、板、纤维等的聚合物载体,根据用途不同进行对应的物质的捕捉。
另外,本发明的带有阴离子性取代基(如磷酸基)物质的捕捉方法为,例如使该物质在为生理条件的中性条件下等与本发明的聚合物载体相结合,随后,例如通过改变溶液的pH值、具有缓冲作用的酸和其盐或者碱和其盐的种类、缓冲液或者溶液所含盐浓度的变化等,使结合的该物质从本发明的聚合物载体上解离。之所以可以捕捉该物质,进而释放捕捉的该物质,是由于该物质与本发明的聚合物载体的结合因例如溶液的pH值、具有缓冲作用的酸和其盐或者碱和其盐的种类、缓冲液或者溶液所含盐的浓度等的缘故而改变。
同时,本发明的带有阴离子性取代基(如磷酸基)物质的捕捉方法,也包含通过添加例如乙二胺四乙酸(EDTA)等螯合剂,使结合的该物质从本发明的聚合物载体上解离。之所以可释放捕捉的该物质,是由于例如乙二胺四乙酸(EDTA)等螯合剂将锌离子从该物质与本发明的聚合物载体的结合体中抽出,从而解离结合的该物质。
这样,使用迅速且容易的捕捉方法,对带有阴离子性取代基(如磷酸基)物质进行分离精制及回收,可以通过改变例如溶液的pH值、具有缓冲作用的酸和其盐或者碱和其盐的种类、缓冲液或者溶液所含盐的浓度,或者,通过添加例如乙二胺四乙酸(EDTA)等螯合剂,而随意进行。
实施例
以下,通过列举实施例对本发明进行更具体的说明,但本发明不仅限于下述所记载的实施例。另外,这些实施例中的锌离子(Zn2+)担载量(单位mmol Zn2+/g或者μmol Zn2+/ml),表示为相对于锌配位基结合的聚合物载体总量的锌离子含量值。
实施例1
将TOYOPEARL(注册商标)AF-Epoxy-650M 10g和四氮杂环十二烷6.9g和碳酸钾2.2g的乙醇溶液75ml,在40℃搅拌4天。反应液过滤洗净后,将所得珠粒和硝酸锌六水合物12g的水溶液75ml,在40℃搅拌2天。反应液过滤洗净后,进行减压干燥,得到由Zn2+-四氮杂环十二烷基结合的TOYOPEARL(注册商标)12g(0.3mmol Zn2+/g)。
实施例2
向带有聚乙烯制过滤器的柱内填充由Zn2+-四氮杂环十二烷基结合的TOYOPEARL(注册商标)100mg(0.3mmol Zn2+/g),制成由Zn2+-四氮杂环十二烷基结合的TOYOPEARL(注册商标)柱。
实施例3
用50mM TRIS-盐酸缓冲液(pH7.0)使由Zn2+-四氮杂环十二烷基结合的TOYOPEARL(注册商标)柱膨润。向柱内添加以50mM TRIS-盐酸缓冲液(pH7.0)调节过的10mM 4-硝基苯甲酸钠溶液1.0ml,之后,添加50mM TRIS-盐酸缓冲液(pH7.0)2.0ml并使其流出。收集流出液,使用紫外可见光分光光度计对流出液中的1价4-硝基苯甲酸阴离子的捕捉率进行测量,结果为100%。
实施例4
用50mM TRIS-盐酸缓冲液(pH7.0)使由Zn2+-四氮杂环十二烷基结合的TOYOPEARL(注册商标)柱膨润。向柱内添加以50mM TRIS-盐酸缓冲液(pH7.0)调节过的10mM 4-硝基苯磷酸二钠溶液1.0ml,之后,添加50mM TRIS-盐酸缓冲液(pH7.0)2.0ml并使其流出。收集流出液,使用紫外可见光分光光度计对流出液中的2价4-硝基苯磷酸阴离子的捕捉率进行测量,结果为98%。
实施例5
将1,3-二氨基-2-丙醇33g和2-吡啶羧基醛116g和氰基三羟基硼酸(シアノトリヒドロほう酸)钠50g的甲醇溶液500ml,在室温搅拌3天。经过后处理后,用柱层析法精制,得到合成中间体34g。
将合成中间体18g和6-溴甲基烟酸甲酯12g和碳酸钾14g的N,N-二甲基甲酰胺溶液225ml,在50℃搅拌1小时。经过后处理后,用柱层析法精制,得到甲酯体22g。
将甲酯体17g和1.0M氢氧化钠水溶液83ml的甲醇溶液40ml,在室温搅拌1小时。中和后,用柱层析法精制,得到N-(5-羧基-2-吡啶基)甲基-N,N’,N’-三[(2-吡啶基)甲基]-1,3-二氨基-2-丙醇13g。
实施例6
将TOYOPEARL(注册商标)AF-Amino-650M 1.2g和N-(5-羧基-2-吡啶基)甲基-N,N’,N’-三[(2-吡啶基)甲基]-1,3-二氨基-2-丙醇450mg和双环己基碳二亚胺210mg和1-羟苯并三唑150mg的N,N-二甲基甲酰胺溶液10ml,在40℃搅拌18小时。反应液过滤洗净后,将所得珠粒和乙酸锌二水合物480mg和10M氢氧化钠水溶液0.10ml的乙醇溶液7.0ml,在40℃搅拌18小时。反应液过滤洗净后,将所得珠粒和1.0M高氯酸钠水溶液3.0ml的水溶液7.0ml,在40℃搅拌18小时。反应液过滤洗净后,进行减压干燥,得到由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的TOYOPEARL(注册商标)1.0g(0.5mmol Zn2+/g)。
实施例7
向带有聚乙烯制过滤器的柱内填充由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的TOYOPEARL(注册商标)100mg(0.5mmol Zn2+/g),制成由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的TOYOPEARL(注册商标)柱。
实施例8
用50mM TRIS-盐酸缓冲液(pH7.0)/乙腈(1/1)溶液使由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的TOYOPEARL(注册商标)柱膨润。向柱内添加以50mM TRIS-盐酸缓冲液(pH7.0)/乙腈(1/1)溶液调节过的10mM 4-硝基苯甲酸钠溶液1.0ml,之后,添加50mM TRIS-盐酸缓冲液(pH7.0)/乙腈(1/1)溶液2.0ml并使其流出。收集流出液,使用紫外可见光分光光度计对流出液中的1价4-硝基苯甲酸阴离子的捕捉率进行了测量,结果为100%。随后,添加50mM磷酸缓冲液(pH3.0)/乙腈(1/1)溶液50ml并使其流出。收集流出液,使用紫外可见光分光光度计对流出液中的1价4-硝基苯甲酸阴离子的回收率进行了测量,结果为100%。
实施例9
用50mM TRIS-盐酸缓冲液(pH7.0)使由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的TOYOPEARL(注册商标)柱膨润。向柱内添加以50mM TRIS-盐酸缓冲液(pH7.0)调节过的10mM 4-硝基苯磷酸二钠溶液1.0ml,之后,添加50mM TRIS-盐酸缓冲液(pH7.0)2.0ml并使其流出。收集流出液,使用紫外可见光分光光度计对流出液中的2价4-硝基苯磷酸阴离子的捕捉率进行了测量,结果为100%。随后,添加作为淋洗液的50mM磷酸缓冲液(pH3.0)50ml并使其流出。收集流出液,使用紫外可见光分光光度计对流出液中的2价4-硝基苯磷酸阴离子的回收率进行了测量,结果为89%,然而,添加作为淋洗液的50mM TRIS-盐酸缓冲液(pH7.0)50ml并使其流出,收集流出液,使用紫外可见光分光光度计对流出液中的2价4-硝基苯磷酸阴离子的回收率进行了测量,结果为0%。
实施例10
将1,3-二氨基-2-丙醇33g和2-吡啶羧基醛116g和氰基三羟基硼酸钠50g的甲醇溶液500ml,在室温搅拌3天。经过后处理后,用柱层析法精制,得到合成中间体34g。
将合成中间体18g和6-溴甲基烟酸甲酯12g和碳酸钾14g的N,N-二甲基甲酰胺溶液225ml,在50℃搅拌1小时。经过后处理后,用柱层析法精制,得到甲酯体22g。
将甲酯体10g和乙二胺23g的甲醇溶液100ml,在室温搅拌3天。经过后处理后,用柱层析法精制,得到N-{5-[N-(2-氨基)乙基]氨基甲酰基-2-吡啶基}甲基-N,N’,N’-三[(2-吡啶基)甲基]-1,3-二氨基-2-丙醇10g。
实施例11
将NHS-活化的Sepharose(商标)4FF的20mM乙腈溶液5.0ml和N-{5-[N-(2-氨基)乙基]氨基甲酰基-2-吡啶基}甲基-N,N’,N’-三[(2-吡啶基)甲基]-1,3-二氨基-2-丙醇的5.0mM乙腈溶液5.0ml,在50℃搅拌1小时。反应液过滤洗净后,再经过20mM碳酸钠水溶液的洗净,得到由N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙醇基结合的Sepharose(商标)5.0ml。
实施例12
向带有聚丙烯制过滤器的柱内填充由N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙醇基结合的Sepharose(商标)1.0ml,用100mM2-(N-吗啉代)乙磺酸(MES)缓冲液(pH6.0)和20mM乙酸锌水溶液的混合液5.0ml、100mM 2-(N-吗啉代)乙磺酸(MES)缓冲液(pH6.0)和0.1mM乙酸锌水溶液的混合溶液5.0ml、TRIS-盐酸缓冲液(pH7.0)和0.5M氯化钠水溶液的混合液5.0ml平衡化,制成由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的Sepharose(商标)柱。
实施例13
向由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的Sepharose(商标)柱内,添加牛血清白蛋白(分子量66,000磷酸化丝氨酸残基×0)50μg和鸡蛋白白蛋白(分子量45,000磷酸化丝氨酸残基×2)50μg和牛αs1-酪蛋白(分子量24,000磷酸化丝氨酸残基×8)50μg和牛αs1-非磷酸化型酪蛋白(分子量24,000磷酸化丝氨酸残基×0)50μg和牛β-酪蛋白(分子量25,000磷酸化丝氨酸残基×5)50μg。随后,按下列顺序添加各缓冲液并使其流出:TRIS-盐酸缓冲液(pH7.0)和0.5M氯化钠水溶液的混合溶液1.0ml2次、TRIS-乙酸缓冲液(pH7.0)1.0ml 4次、10mM磷酸缓冲液(pH7.0)1.0m l、15mM磷酸缓冲液(pH7.0)1.0ml、20mM磷酸缓冲液(pH7.0)1.0m l、25mM磷酸缓冲液(pH7.0)1.0ml、30mM磷酸缓冲液(pH7.0)1.0ml、35mM磷酸缓冲液(pH7.0)1.0ml、40mM磷酸缓冲液(pH7.0)1.0ml、45mM磷酸缓冲液(pH7.0)1.0ml、50mM磷酸缓冲液(pH7.0)1.0ml、60mM磷酸缓冲液(pH7.0)1.0ml、70mM磷酸缓冲液(pH7.0)1.0ml、80mM磷酸缓冲液(pH7.0)1.0ml、90mM磷酸缓冲液(pH7.0)1.0ml、100mM磷酸缓冲液(pH7.0)1.0ml、200mM磷酸缓冲液(pH7.0)1.0ml、300mM磷酸缓冲液(pH7.0)1.0ml、400mM磷酸缓冲液(pH7.0)1.0ml、500mM磷酸缓冲液(pH7.0)1.0ml。将流出液浓缩后,进行斑点实验(スポツトレ),以十二烷基硫酸纳一聚丙烯酰胺凝胶电泳(SDS-PAGE)进行分离,之后,用科麦西亮蓝(クマシ一ブリリアントブル一)(CBB)染色。电泳图的前半部分如图1所示、后半部分如图2所示。在图1及图2中,可得到确认,按照最初流出的是没有磷酸化丝氨酸残基的牛血清白蛋白,随后是磷酸化丝氨酸残基数量由少到多的顺序,鸡蛋白白蛋白、牛β-化酪蛋白、牛αs1-酪蛋白流出。
实施例14
向微量吸移管端头中填入纤维素制滤纸,填充从由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的Sepharose(商标)柱中取出的由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的Sepharose(商标)10μl,之后,用纤维素制滤纸盖住,制成由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的Sepharose(商标)的端头。
实施例15
采用装上由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的Sepharose(商标)端头的微量吸移管,吸入以含有0.50M硝酸钠的100mM TRIS-乙酸缓冲剂(pH7.4)调节过的0.22mMp60c-src肽521-533和0.18mM磷酸化p60c-src肽521-533的混合溶液10μl,进行5分钟平衡化后排出。之后,用含有0.50硝酸钠的100mMTRIS-乙酸缓冲液(pH7.4)10μl进行5次洗净。随后,用10mM磷酸缓冲液(pH7.0)10μl进行6次洗净,回收洗液,用高速液相色谱仪进行纯度测定,其结果为磷酸化p60c-src肽521-533 100%,p60c-src肽521-5330%。
实施例16
向PS20 ProteinChip(注册商标)Array(阵列)中加入以100mM碳酸氢钠水溶液/乙腈(1/3)溶液调节的0.38M N-{5-[N-(2-氨基)乙基]氨基甲酰基-2-吡啶基}甲基-N,N’,N’-三[(2-吡啶基)甲基]-1,3-二氨基-2-丙醇溶液3.0μl,在室温反应4小时。进而,加入以100mM碳酸氢钠水溶液调节的0.50M 2-氨基乙醇溶液3.0μl,在室温反应4小时。洗净后,用以100mM 2-(N-吗啉代)乙磺酸(MES)缓冲液(pH6.0)调节的0.50M乙酸锌溶液洗净,得到由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的ProteinChip(注册商标)Array。
实施例17
向由Zn2+ 2-N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙氧基结合的ProteinChip(注册商标)Array中加入以100mM TRIS-乙酸缓冲液(pH7.4)调节的0.94mM 4-甲基繖形基(ウンベリフェリル)磷酸二钠溶液3.0μl,在室温进行1小时平衡化后回收。收集回收液,使用紫外可见光分光光度计对回收液中的2价4-甲基繖形基磷酸阴离子的捕捉率进行测定,为16%。
比较例1
向带有聚乙烯制过滤器的柱内填充TOYOPEARL(注册商标)AF-Epoxy-650M 100mg,用50mM TRIS-盐酸缓冲液(pH7.0)进行膨润。向柱内添加以50mM TRIS-盐酸缓冲液(pH7.0)调节过的10mM 4-硝基苯磷酸二钠溶液1.0ml,之后添加50mM TRIS-盐酸缓冲液(pH7.0)2.0ml并使其流出。收集流出液,使用紫外可见光分光光度计对流出液中的2价4-硝基苯磷酸阴离子的捕捉率进行测定,为0.5%。
比较例2
向带有聚丙烯制过滤器的柱内填充由N,N,N’,N’-四[(2-吡啶基)甲基]-1,3-二氨基-2-丙醇基结合的Sepharose(商标)1.0ml,以TRIS-盐酸缓冲液(pH7.0)和0.5M氯化钠水溶液的混合液5.0ml平衡化。向柱内添加牛血清白蛋白(分子量66,000磷酸化丝氨酸残基×0)50μg和鸡蛋白白蛋白(分子量45,000磷酸化丝氨酸残基×2)50μg和牛αs1-酪蛋白(分子量24,000磷酸化丝氨酸残基×8)50μg和牛αs1-非磷酸化型酪蛋白(分子量24,000磷酸化丝氨酸残基×0)50μg和牛β-酪蛋白(分子量25,000磷酸化丝氨酸残基×5)50μg。随后,按下列顺序添加各缓冲液并使其流出:TRIS-盐酸缓冲液(pH7.0)和0.5M氯化钠水溶液的混合液1.0ml 2次、TRIS-乙酸缓冲液(pH7.0)1.0ml 4次、10mM磷酸缓冲液(pH7.0)1.0ml、15mM磷酸缓冲液(pH7.0)1.0ml、20mM磷酸缓冲液(pH7.0)1.0ml、25mM磷酸缓冲液(pH7.0)1.0ml、30mM磷酸缓冲液(pH7.0)1.0ml、35mM磷酸缓冲液(pH7.0)1.0ml、40mM磷酸缓冲液(pH7.0)1.0ml、45mM磷酸缓冲液(pH7.0)1.0ml、50mM磷酸缓冲液(pH7.0)1.0ml、60mM磷酸缓冲液(pH7.0)1.0ml、70mM磷酸缓冲液(pH7.0)1.0ml、80mM磷酸缓冲液(pH7.0)1.0ml、90mM磷酸缓冲液(pH7.0)1.0ml、100mM磷酸缓冲液(pH7.0)1.0ml、200mM磷酸缓冲液(pH7.0)1.0ml、300mM磷酸缓冲液(pH7.0)1.0ml、400mM磷酸缓冲液(pH7.0)1.0ml、500mM磷酸缓冲液(pH7.0)1.0ml。将流出液浓缩后,进行斑点实验(スポツトレ),以十二烷基硫酸纳一聚丙烯酰胺凝胶电泳(SDS-PAGE)进行分离,之后,用科麦西亮蓝(CBB)染色。电泳图的前半部分如图3所示、后半部分如图4所示。在图3及图4中,可以得到确认的是,从最初开始是牛血清白蛋白、鸡蛋白白蛋白、牛β-酪蛋白、牛αs1-酪蛋白流出。
产业上的可利用性
本发明的由指定的锌配位基结合的聚合物载体,在一定条件下,与阴离子性取代基(如磷酸基)结合,同时,由于整体具有溶剂难溶性(优选溶剂不溶性),所以成为使分离精制带有该取代基物质容易、安全且廉价的捕捉剂。同时,通过使用此聚合物载体,提供一种以过滤器可以简单分离精制的捕捉器具及在中性条件下使其结合,随后,在一定条件下使其解离,迅速且容易的捕捉方法。
Claims (9)
2、一种带有阴离子性取代基的物质的捕捉剂,其中,含有权利要求1的聚合物载体或通式(2)所示的锌配位基直接或通过隔离基结合的聚合物载体。
3、权利要求2的捕捉剂,其特征在于,阴离子性取代基为磷酸基。
4、权利要求2或3的捕捉剂,其特征在于,捕捉剂呈珠粒状。
5、权利要求2或3的捕捉剂,其特征在于,捕捉剂呈板状。
6、权利要求2或3的捕捉剂,其特征在于,捕捉剂呈纤维状。
7、一种带有阴离子性取代基的物质的捕捉器具,其中,填充有权利要求2-4或6之一的捕捉剂,具有以过滤器进行过滤分离的功能。
8、一种带有阴离子性取代基的物质的捕捉方法,其中,包括通过使带有阴离子性取代基物质与权利要求2-6之一的捕捉剂相结合而进行捕捉的步骤。
9、权利要求8的方法,其特征在于,在该捕捉步骤之后,还包括将带有阴离子性取代基的物质从捕捉剂上解离的步骤。
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