CN102858756A - 1,4,7-三氮杂-环壬烷的偶联物、此类偶联物的二核金属配合物、以及1,4,7-三氮杂-环壬烷和偶联物的使用方法 - Google Patents
1,4,7-三氮杂-环壬烷的偶联物、此类偶联物的二核金属配合物、以及1,4,7-三氮杂-环壬烷和偶联物的使用方法 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D255/00—Heterocyclic compounds containing rings having three nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D249/00 - C07D253/00
- C07D255/02—Heterocyclic compounds containing rings having three nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D249/00 - C07D253/00 not condensed with other rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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Abstract
将1,3-二(1,4,7-三氮杂环壬烷-1-基)-2-羟基丙烷与各种偶联部分的偶联物用于形成与磷酸酯结合的二核金属配合物。凭借其偶联的形式,将所述配合物引入色谱介质、亲和性结合试剂、以及染料中,这使得该配合物可用于各种测定、分离和纯化中。此外,将并非那么偶联的1,3-二(1,4,7-三氮杂环壬烷-1-基)-2-羟基丙烷的二核金属配合物通过MALDI-TOF质谱或染料置换用于检测生物物质的磷酸酯。
Description
相关申请的交叉参考
本申请要求2010年3月16日提交的美国临时专利申请号61/314,431和2010年7月27日提交的61/368,106的权益。这两篇临时专利申请的内容通过引用结合于本文。
技术背景
磷酸化和脱磷酸化是生理系统中代谢上重要的反应。这些反应发生在某些蛋白质的羟基、某些其它蛋白质的氮原子、以及更多蛋白质的羧基上,在各种情况下对体内这些蛋白质的活性和功能有影响。一些磷酸化/脱磷酸化反应由蛋白激酶和磷酸酶介导,这些反应的发生说明了这些酶的活性。磷酸化/脱磷酸化反应的异常与细胞癌发生、变应性紊乱、阿尔茨海默病有关。蛋白质磷酸化是所有活细胞内信号转导、凋亡、增殖、分化和代谢的必需过程。因此,确定蛋白质磷酸化的状况的方法对于探查疾病的分子起源和设计新药很有价值。
已知一些包含两种金属离子的金属酶可催化脱磷酸化反应,具体而言,可催化磷酸二酯的水解。这些反应发生机理的研究已涉及形成二金属配合物和具有相同催化功能的合成有机配体的使用。然而,人们发现大部分这些合成配体在水中具有低溶解度,这限制了它们在实验室研究中的应用。为了克服上述障碍,人们开发出了一种水溶性配体1,3-二(1,4,7-三氮杂环壬烷-1-基)-2-羟基丙烷(1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane)。Iranzo,O.等的“在用二核Zn(II)催化剂的磷酸二酯和RNA的切割中金属离子间的协同性”(“Cooperativity between Metal Ions in the Cleavage of Phosphate Diesters andRNA by Dinuclear Zn(II)Catalysts,”)(Inorganic Chemistry 42(24),7737-7746(2003))和Iranzo,O.等的“在用二核Zn(II)配合物催化磷酸二酯切割中金属离子的协同作用的物理和动力学分析”(“Physical and Kinetic Analysis of theCooperative Role of Metal Ions in Catalysis of Phosphodiester Cleavage by aDinuclear Zn(II)Complex,”)(J.Am.Chem.Soc.125(7),1988-1993(2003))公开了该配体的Zn(II)配合物及其在RNA水解中的使用。Yang,M.-Y.等的“用二核Zn(II)配合物催化磷酸二酯切割的底物特异性”(“Substrate specificity forcatalysis of phosphodiester cleavage by a dinuclear Zn(II)complex,”)(Chem.Commun.2003,2832-2833)和Yang,M.-Y.等的“用小酶样金属离子配合物催化的磷酸二酯切割的过渡态类似物”(“A transition state analog for phosphatediester cleavage catalyzed by a small enzyme-like metal ion complex,”)(Bioorganic Chemistry 35,366-374(2007))报道了该分子及用磷酸酯形成的过渡态的其它研究。
发明内容
本发明部分涉及取代或未取代的1,3-二(1,4,7-三氮杂环壬烷-1-基)-2-羟基丙烷与各种偶联部分的偶联物,各偶联物能够以与上述报道中所揭示的配合物同样的方式形成二金属配合物。本发明还涉及取代或未取代的官能化的1,3-二(1,4,7-三氮杂环壬烷-1-基)-2-羟基丙烷,其官能团可与偶联部分反应。为了方便起见,所述取代或未取代的1,3-二(1,4,7-三氮杂环壬烷-1-基)-2-羟基丙烷在本文中称为“配体”,所述偶联物是与起到如下所述的各种其它功能之一的偶联部分偶联的这些配体。本发明还涉及这些偶联物的二核金属配合物,及这些配合物除了在磷酸二酯的水解中以外的各种用途。各配合物的用途源于用于形成偶联物的特定偶联部分与两种金属离子的磷酸结合活性。
本发明还涉及使用所述配体的二核金属配合物或所述配体的偶联物的二核金属配合物,通过将这样一种配合物与在合适基质中的样品进行混合以形成磷酸酯的带正电二核金属配合物,用来检测MALDI-TOF(基质辅助激光解吸/电离飞行时间)质谱样品中的磷酸酯的方法。当质谱扫描在样品和基质上进行时,由于配合物中的配体赋予的额外的质量以及配合物的电荷变化,样品中磷酸酯的带正电二核金属配合物与未配合的组分的区别增加。在本发明的这一方面中对未偶联的配体特别感兴趣。
本发明的另一方面涉及使用所述配体的二核金属配合物或所述配体的偶联物的二核金属配合物,用来检测染料置换样品中的磷酸酯的方法。在本发明的这一方面,所述二核金属配合物还可与具有两种关键特征的发色化合物配合:(1)所述发色化合物对带电金属配体(或所述配体的偶联物)的亲和性低于其对磷酸酯的亲和性,即所述磷酸酯与所述配体结合更强,并将经过接触置换所述配合物中的发色化合物;以及(2)所述发色化合物在结合时,较未结合时有不同的吸收光谱和荧光光谱。符合该描述的发色染料的一个例子是邻苯二酚紫。因此发色化合物被磷酸酯从所述配合物中置换出来而导致的光谱变化作为样品中存在磷酸酯的指示。同样在本发明的这一方面中,对未偶联的配体的使用特别感兴趣。
发明实施方式的详述
所述配体,其是取代或未取代的1,3-二(1,4,7-三氮杂环壬烷-1-基)-2-羟基丙烷,由以下通式(I)表示:
在式(I)中,所述基团R1、R2、R3、R4、R5和R6是相同或不同的(一些相同,其它不同),其是H或低级烷基。术语“低级烷基”定义为具有小于或等于6个碳原子的烷基。优选的R1至R6的基团是H、C1-C3烷基、或选自H和C1-C3烷基的组合,且最优选为H。本文中术语“取代的”指R1、R2、R3、R4和R6中的一个或多个是烷基,而不是H原子。
本文所述的未经偶联的配体是其中R1、R2、R3、R4、R5和R6是相同或不同的,且各自为H或低级烷基。所述配体的偶联物是式(I)的这类化合物,其中偶联部分占据R1、R2、R3、R4、R5和R6所示的任意位置,通过连接基团将所述偶联部分接到所述结构的其余部分上。如上所述,不表示所述偶联部分的任意这些R基团是H或低级烷基。对于偶联物,R1至R6中的一个或多个因此为L-R7,其中L表示连接基团,R7是偶联部分;并且R1至R6的其余基团(其是相同或不同的(一些相同,其它不同))是H或C1-C6烷基。对于官能化配体,R1至R6中的一个或多个是L’,其中L’表示未反应的可与偶联部分反应的连接基团。在作为偶联物中的官能化的配体中,所述R1至R6中的其余基团(其可以是相同或不同的(一些相同,其它不同))各自是H或C1-C6烷基。在优选的官能化的配体或偶联物中,R1至R6中仅一个分别是L’或L-R7;在更优选的配体或偶联物中,仅R5、仅R6、或仅R2是–L’或–L-R7。在未被–L’或–L-R7取代的R1至R6基团中,这些基团优选为H、C1-C3烷基、或选自H和C1-C3烷基的组合;最优选为H。所述偶联部分R7是丙烯酰胺基、荧光染料、亲和型结合部分、或固体色谱载体。所述连接基团L’或L是具有两个结合位点的任意常规连接体,其中一个与偶联部分R7形成共价键,另一个与式(I)的其余部分上相邻碳原子形成共价键。除了氢原子填充可用价位以外,所述连接基团通常具有3-30个选自C、N、O、P、和S的原子,并且其是环状、非环状、芳族或环状、非环状和芳族部分的组合。连接基团的例子是胺基烷基键、烯基键、酰胺键或含酰胺键、酯键或含酯键、醚键或含醚键。优选的L’连接基团是-(C1-C4烷基)-NH2、–(C1-C4烷基)-C(=O)-NH2、–(C1-C4烷基)-NH-CO2H、-(C1-C4烷基)-C(=O)-NH-(C1-C4烷基)-NH2、和–(C1-C4烷基)-NH-C(=O)-(C1-C4)-NH2,特别优选为–CH2CH2-NH-、–CH2-C(=O)-NH2、–CH2CH2-NH-CO2H、-CH2-C(=O)-NH-CH2CH2-NH2和-CH2CH2--NH-C(=O)-CH2CH2-NH2。优选的L 基团为(C1-C4烷基)-C(=O)-NH-(C1-C4烷基)-,–(C1-C4烷基)-NH-C(=O)-(C1-C4烷基)-NH-和-(C1-C4烷基)-NH-;特别优选为–CH2-C(=O)-NH-CH2-CH2-、-CH2-CH2-NH-C(=O)-CH2-CH2-NH-和-CH2-CH2-NH-。
当R7为丙烯酰胺基时,-L-R7优选为
-CH2-C(O)-NH-(CH2)n-NH-C(O)-CH=CH2或–(CH2)2-NH-C(O)-(CH2)n-NH-C(O)-CH=CH2
其中n为1-6;以及最优选为
-CH2-C(O)-NH-(CH2)2-NH-C(O)-CH=CH2和-(CH2)2-NH-C(O)-(CH2)2-NH-C(O)-CH=CH2。
当该特定L-R7取代R5,其余R’都是H时,所述偶联物具有以下结构式:
官能化聚丙烯酰胺凝胶可根据本发明由偶联物和二核金属配合物形成,其中所述偶联部分是丙烯酰胺。这些凝胶中的所述官能团是含丙烯酰胺偶联物的二金属配合物,并且所产生的凝胶可用于将蛋白质、肽、或核酸的磷酸单酯从这些物质的非磷酸化形式中电泳分离。在电泳过程中由于凝胶上磷酸酯与二金属配合物官能团之间的相互作用,混合物中蛋白质、肽、或核酸的磷酸单酯将迁移更慢。由此可将混合物的磷酸化组分与它们的未磷酸化对应物进行区分。所述官能化凝胶通过在单体混合物中包括二金属配合物形成,所述单体混合物通常还包括非官能化丙烯酰胺、交联剂和引发剂。合适的交联剂和引发剂在本领域是众所周知的。交联剂的例子是双丙烯酰胺和二丙烯酸乙二酯(ethylene diacrylate),引发剂的例子是核黄素、过硫酸铵和四甲基乙二胺(TEMED)。单体的聚合条件和单体混合物的各种组分的浓度与形成聚丙烯酰胺凝胶的现有技术方法中所用的相同。
用于实施本发明的式(I)配体的二核金属配合物具有以下通式(II):
在式(II)中,R1至R6如上定义,且M为二价金属。一组优选的二价金属为Ca、Zn、Cr、Mn、Fe、Co、Ni和Cu(均以二价形式)。特别优选的二价金属为Zn(II)和Mn(II)。
当用于本发明的各种方法时,式(II)的二核金属配合物形成式(III)的磷酸盐结合配合物(phosphate association complexes):
在式(III)中,X是肽、蛋白质、核酸、或一般而言(出于任何本文所述的各种目的)准备将本发明的配合物应用于其上的任意物质的残基。
当R7是荧光染料时,所述染料可为任意已知用于标记生物物质的各种荧光染料。例子是:
荧光素和荧光素衍生物,例如荧光素异硫氰酸酯;
若丹明衍生物,例如四甲基若丹明、若丹明B。若丹明6G、磺基若丹明B、若丹明101(德克萨斯红)和若丹明110;
4,4-二氟-4-硼-3a,4a-二氮杂-s-二环戊二烯并苯(indacene)(BODIPY)及其衍生物;
芘和芘衍生物,例如8-甲氧基芘-1,3,6-三磺酸;
吡啶基噁唑衍生物;
达泼西(dapoxyl)衍生物;
伞形酮;
1-苯胺基-8-萘磺酸;
3,6-二磺酸根-4-氨基-萘酰亚胺(naphthalimide);
三-、五-、和七-次甲基花青染料;和
发光过渡金属配合物,例如三(2,2’-联吡啶)钌(II)或Ir(III)的环金属化配合物。
当例如R7为四甲基若丹明时,L-R7优选为下式:
其中,星号表示连接到1,3-二(1,4,7-三氮杂壬烷-1-基)-2-羟基丙烷上的位点。当该连接体和染料取代R5,且R1至R4都为H时,所得的偶联物具有下式:
由包含荧光染料的偶联物形成的本发明二核金属配合物例如通常在生物流体的样品和测定介质中可用于凝胶或印迹的染色以对磷酸酯(例如磷蛋白)进行鉴定和定量。
当R7是亲和型结合部分时,此类结合部分的例子是生物素、亲和素、链霉亲和素、抗体和抗体片段。当R7为生物素时,L-R7优选为下式:
其中,星号表示连接到1,3-二(1,4,7-三氮杂壬烷-1-基)-2-羟基丙烷上的位点。当该官能团取代R5,且R1至R4都为H时,所得的偶联物具有下式:
其中R6是亲和性结合部分的本发明二核金属配合物可用于检测结合于或固定在固体载体上的磷蛋白和其它磷酸酯。亲和性结合部分与第二亲和性结合部分特异性结合以形成亲和性结合对的能力使(在所述配合物已与磷酸酯结合之前或之后)报告基团能通过结合对连接到所述配合物上。这使得能通过任何类型的可与亲和性结合部分偶联的报告基团来检测。亲和性结合对是众所周知的,例如生物素与亲和素、生物素与链霉亲和素、以及任意的各种抗体或抗体片段与抗原。报告基团的例子是放射性标记、化学发光标记和酶。酶的例子是辣根过氧化物酶、氯霉素乙酰基转移酶、β-半乳糖苷酶、碱性磷酸酶和荧光素酶。为了说明包含亲和性结合部分的二核金属配合物的使用,捕获磷酸酯的固体载体(例如印迹膜)首先与所述配合物接触。如果例如所述配合物上的偶联部分是生物素,所述载体则与链霉亲和素-酶偶联物接触。然后将所述载体与酶底物进行孵育,并检测变化,最常见为底物中由酶介导的颜色变化。接触和孵育的最佳条件对本领域的技术人员是显而易见的。
当R7是固体色谱载体时,本文所用的术语“固体”将包括半固体,例如凝胶;挠性固体,例如膜(membranes)和薄膜(films);和刚性固体,例如用于形成不可压缩的珠、颗粒、和柱或管壁的刚性固体。这些固体的例子是聚丙烯酰胺、交联的硅聚合物、硅胶、琼脂糖、聚乙烯醇、纤维素和硝化纤维素。承载本发明二核金属配合物的色谱载体可用于从流体(例如生物流体、测定介质、或包含磷酸化化合物的任何流体)中提取磷酸化化合物(磷酸酯),用来纯化或富集。
本发明的偶联物可通过常规方法来制备,由1,4,7-三氮杂环壬烷衍生物与一种或多种(用合适的保护基团或通过形成1,4,7-三氮杂环壬烷三环邻酰胺进行保护的)仲胺基开始。可将合适的官能团选择性地置于一个或多个未保护的R1至R4位置。还可通过选择用三碳链连接各大环中的一个环氮从而将两个1,4,7-三氮杂环壬烷基连接起来的合适的合成中间体将合适的官能团置于R5位置。官能团的选择将根据所选择的连接基团以及连接基团的连接方式而改变,所述连接可通过现有技术中已知的任何各种连接反应来实现。官能团的突出例子是可与加到所述偶联部分上的胺基官能团反应的羧酸或羧酸的酯、或可与活性羧酸酯或酰氯反应的氨基,两者都将形成酰胺键。如本领域已知,羧酸官能化的配体与带胺基的偶联配对物(partner)的反应可直接或在活化的酸(例如酰氯、酸酐或琥珀酰亚胺基酯)存在下容易实现。如本领域已知,胺基官能化的配体与带有活化的酯或酰氯的偶联配对物的反应可容易实现。
官能化的配体的制备通过其中甲酯占据R5位置、其它所有R-基团为H的配体进行说明。此类制备的一个例子是用1,4,7-三氮杂环壬烷开始并与二甲基甲酰胺二甲基缩醛反应形成1,4,7-三氮杂环壬烷三环邻酰胺,其再与3,5-二溴乙酰丙酸甲酯反应,形成3,5-二(4-甲酰基-1,4,7-三氮杂壬烷-1-基)-4-氧代戊酸甲酯(methyl3,5-bis(4-formyl-1,4,7-triazonan-1-yl)-4-oxopentanoate)。后者再与硼氢化钠反应形成3,5-二(4-甲酰基-1,4,7-三氮杂壬烷-1-基)-4-羟基戊酸甲酯,其再与盐酸反应,生成4-羟基-3,5-二(1,4,7-三氮杂壬烷-1-基)戊酸甲酯。这一系列的反应如下所示:
在分子上的其它位置放置甲酯基以及用其他取代基获得官能化的配体的该方法的变化对于有机化学合成领域的技术人员来说是显而易见的。一个例子如下所示,其放置了胺基,从在两个N原子的位置上用丁氧基羰基保护的1,4,7-三氮杂环壬烷开始,与1,3-二溴-2-丙醇反应,形成7-(3-溴-2-羟基丙烷)-1,4,7-三氮杂壬烷-1,4-二羧酸二叔丁酯(步骤1)。分开地,将未保护的1,4,7-三氮杂环壬烷与N,N-二甲基甲酰胺二甲基缩醛反应,形成相应的三环结构,其再与(2-溴乙基)氨基甲酸叔丁酯反应并水解,得到(2-(4-甲酰基-1,4,7-三氮杂壬烷-1-基)-3-)1,4,7-三氮杂壬烷-1-基)乙基氨基甲酸叔丁酯。再将这两种产物反应,形成1-(4-(2-氨基乙基)-1,4,7-三氮杂壬烷-1-基)-3-(1,4,7-三氮杂壬烷-2-基)丙-2-醇,然后脱保护(步骤3)。
步骤1:
步骤2:
步骤3:
另一个例子如下所示,从N-(3-丁炔基)邻苯二甲酰胺的水合开始,形成N-(3-氧代丁基)邻苯二甲酰胺。随后与溴反应,再与维悌希试剂反应,再与过氧乙酸反应、再与两个N原子被保护的1,4,7-三氮杂环壬烷反应、接着脱保护,形成2-(1,4,7-三氮杂壬烷-1-基)甲基)-4-1-(1,4,7-三氮杂壬烷-1-基)丁-2-醇。
(注:Base为碱。)
本发明使用的配体或偶联物的配合可在配体、官能化的配体(偶联之前)、或偶联物本身上进行。在任一种情况下,配合可通过将合适的金属盐(例如Zn(NO3)2,其中将形成Zn配合物)与所述配体的氢溴酸盐或盐酸盐以合适的摩尔比混合,且将pH调至6.5-7.0来实现。
在所附的权利要求书中,术语“一个”或“一种”是用来表示“一个(种)或多个(种)”。当术语“包含”及其各种变体例如“包括”和“含有”位于叙述步骤或要素之前的时候,是用来表示添加其它的步骤或要素是任选的,并且是非排它性的。本说明书中引用的所有专利、专利申请和其它公开的参考材料都通过引用全文结合入本文中。当本说明书的内容与本发明引用的任何参考材料以及任何现有技术之间存在矛盾之处的时候,以本说明书为准。所述矛盾之处包括现有技术对词或词组的定义与本说明书对相同的词或词组明确给出的定义之间的差异。
Claims (45)
1.一种具有以下结构式的化合物:
在该式中:
R1至R6中的一个或多个是-L’,其中L’是可与选自下组的偶联部分反应以形成所述化合物与所述偶联部分的偶联物的连接基团,所述偶联部分包括:丙烯酰胺基、荧光染料、亲和型结合部分和固体色谱载体;以及
R1至R6的其余基团独立地选自H和C1-C6烷基。
2.如权利要求1所述的化合物,其特征在于,L’是选自下组的部分:-(C1-C4烷基)-NH2、–(C1-C4烷基)-C(=O)-NH2、–(C1-C4烷基)-NH-CO2H、-(C1-C4烷基)-C(=O)-NH-(C1-C4烷基)-NH2、和–(C1-C4烷基)-NH-C(=O)-(C1-C4)-NH2。
3.如权利要求1所述的化合物,其特征在于,L’是选自下组的部分:-CH2CH2-NH-、-CH2-C(=O)-NH2、-CH2CH2-NH-CO2H、-CH2-C(=O)-NH-CH2CH2-NH2和-CH2CH2--NH-C(=O)-CH2CH2-NH2。
5.如权利要求4所述的化合物,其特征在于,R5是L-R7,R1、R2、R3、R4和R6独立地选自H和C1-C6烷基。
6.如权利要求4所述的化合物,其特征在于,R5是–L-R7,R1、R2、R3、R4和R6是H。
7.如权利要求4所述的化合物,其特征在于,R2是–L-R7,R1、R3、R4、R5和R6独立地选自H和C1-C6烷基。
8.如权利要求4所述的化合物,其特征在于,R2是–L-R7,R1、R3、R4、R5和R6是H。
9.如权利要求4所述的化合物,其特征在于,R6是–L-R7,R1、R2、R3、R4和R5独立地选自H和C1-C6烷基。
10.如权利要求4所述的化合物,其特征在于,R6是–L-R7,R1、R2、R3、R4和R5是H。
11.如权利要求4所述的化合物,其特征在于,L是选自下组的部分:–CH2-C(=O)-NH-(C1-C4烷基)-、–CH2-C(=O)-NH-(C1-C4烷基)-NH-和-(C1-C4烷基)-NH-。
12.如权利要求4所述的化合物,其特征在于,L是选自下组的部分:-CH2-C(=O)-NH-CH2-CH2-、–CH2-C(=O)-NH-CH2-CH2-NH-和-C2H5-NH-。
13.如权利要求4所述的化合物,其特征在于,R7是丙烯酰胺基。
14.如权利要求4所述的化合物,其特征在于,R5是-CH2-C(=O)-NH-CH2-CH2NH-C(=O)-CH=CH2,R1、R2、R3、R4和R6是H。
15.如权利要求4所述的化合物,其特征在于,R5是-CH2-CH2-NH-C(=O)-CH=CH2,R1、R2、R3、R4和R6是H。
16.如权利要求4所述的化合物,其特征在于,R2是-CH2-CH2-NH-C(=O)-CH=CH2,R1、R3、R4、R5和R6是H。
17.如权利要求4所述的化合物,其特征在于,R6是-CH2-CH2NH-C(=O)-CH=CH2,R1、R2、R3、R4和R5是H。
18.如权利要求4所述的化合物,其特征在于,R7是荧光染料。
19.如权利要求4所述的化合物,其特征在于,R7是生物素。
20.如权利要求4所述的化合物,其特征在于,R7是固体色谱载体。
22.如权利要求21所述的化合物,其特征在于,M是选自下组的金属:Ca、Zn、Cr、Mn、Fe、Co、Ni和Cu。
23.如权利要求21所述的化合物,其特征在于,M是选自下组的金属:Zn和Mn。
24.如权利要求21所述的化合物,其特征在于,R5是-L-R7,R1、R2、R3、R4和R6独立地选自H和C1-C6烷基。
25.如权利要求21所述的化合物,其特征在于,R5是L-R7,R1、R2、R3、R4和R6是H。
26.如权利要求21所述的化合物,其特征在于,R2是–L-R7,R1、R3、R4、R5和R6独立地选自H和C1-C6烷基。
27.如权利要求21所述的化合物,其特征在于,R2是L-R7,R1、R3、R4、R5和R6是H。
28.如权利要求21所述的化合物,其特征在于,R6是L-R7,R1、R2、R3、R4和R5独立地选自H和C1-C6烷基。
29.如权利要求21所述的化合物,其特征在于,R6是L-R7,R1、R2、R3、R4和R5是H。
30.如权利要求21所述的化合物,其特征在于,L是选自下组的部分:-CH2-C(=O)-NH-(C1-C4烷基)-、-CH2-C(=O)-NH-(C1-C4烷基)-NH-和-(C1-C4烷基)-NH-。
31.如权利要求21所述的化合物,其特征在于,L是选自下组的部分:-CH2-C(=O)-NH-CH2-CH2-、–CH2-C(=O)-NH-CH2-CH2-NH-和-C2H5-NH-。
32.如权利要求21所述的化合物,其特征在于,R7是丙烯酰胺基。
33.如权利要求21所述的化合物,其特征在于,R5是-CH2-C(=O)-NH-CH2-CH2-NH-C(=O)-CH=CH2,R1、R2、R3、R4和R6是H。
34.如权利要求21所述的化合物,其特征在于,R5是-CH2-CH2-NH-C(=O)-CH=CH2,R1、R2、R3、R4和R6是H。
35.如权利要求21所述的化合物,其特征在于,R2是-CH2-CH2-NH-C(=O)-CH=CH2,R1、R3、R4、R5、和R6是H。
36.如权利要求21所述的化合物,其特征在于,R6是-CH2-CH2-NH-C(=O)-CH=CH2,R1、R2、R3、R4和R5是H。
37.如权利要求21所述的化合物,其特征在于,R7是荧光染料。
38.如权利要求21所述的化合物,其特征在于,R7是生物素。
39.如权利要求21所述的化合物,其特征在于,R7是固体色谱载体。
43.一种从液体溶液中提取磷酸化物质的方法,所述方法包括使所述液体溶液通过提取介质,所述提取介质包括偶联于固体载体的以下化合物:
在该式中:
R1至R6中的一个是–L-*,其中L是连接基团,*是与所述固体载体连接的位点;
R1至R6的其余基团独立地选自H和C1-C6烷基;以及
M是二价金属。
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