CN105884748A - 一种马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体及其制备方法和应用 - Google Patents
一种马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于药物载体的技术领域，公开了一种马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体及其制备方法和应用。本发明在近红外荧光菁染料的基础上引入了马来酰亚胺结构得到所述载体，使得该载体可以有效的与含有巯基等亲核基团的小分子、多肽、蛋白质等发生迈克尔加成反应，对相应分子的进行荧光标记，实现对小分子、多肽、蛋白质的体外检测以及体内显影示踪。本发明所述荧光载体具有良好的光稳定性，与吲哚菁绿（ICG）相比具有更高的量子效率与更大的斯托克斯位移，可用于制备含亲核基团的有机小分子、多肽、蛋白质的检测试剂或试剂盒。
Description
技术领域
本发明属于药物载体的技术领域，更具体地，涉及一种马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体及其制备方法和应用。
背景技术
近红外光是指波长在700～900nm区段的电磁波，在该波长范围内，激发光对细胞造成的损伤大大减小，同时能够有效避免细胞自发荧光以及背景荧光的干扰，提高检测的准确度以及灵敏度，此外，近红外光的拥有较强的组织穿透力，理论计算近红外光渗透进入组织的深度达到7～14cm。
近红外荧光菁染料可作为荧光探针，在生物分析中应用于核酸染色或标记，氨基酸、肽和蛋白质的衍生或标记等方面。吲哚菁绿(Indocyanine Green,ICG)自1958年被FDA批准以来，已被用于检测组织灌注和清除研究，同时作为荧光素眼底造影技术(FFA)的补充，ICG广泛运用于脉络膜血管造影，随着近红外研究的深入，菁染料家族结构不断更新壮大，但到目前为止，ICG仍旧是唯一一个批准上市的NIR显影剂。
由于ICG自身不能作为其它分子的载体，同时量子效率相对低，光不稳定、斯托克斯位移小，很大程度的限制了其应用。比如，作为特定目标如药物分子的示踪检测时，需要利用其它材料进行包埋，这使得可能产生荧光物质渗漏导致的假阳性结果，以有副作用等不利情况。
发明内容
本发明要解决的技术问题在于克服现有技术的缺陷，提供一种马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体。
本发明的另一目的在于提供一种马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体的制备方法。
本发明的另一目的在于提供一种马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体的应用。
本发明通过以下技术方案实现：
一种马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体，所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体的结构式如式1所示：
其中，X-为化学上合理的任一有机酸根或无机酸根负离子。
结构式中阳离子部分为起到重要发色及结合作用的结构，X-为化学上合理的有机或无机酸负离子，不影响所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体的发色与结合性能。
本发明在近红外荧光菁染料的基础上引入了马来酰亚胺结构得到所述药物载体，使得该药物载体可以有效地与含有巯基等亲核基的有机小分子、多肽、蛋白质等物质，通过共价作用将相应分子共价结合到本发明所述药物载体上，对相应分子进行荧光标记，从而能够实现对有机小分子、多肽、蛋白质的体外检测以及体内显影示踪。
优选地，所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体为2-(-2-(-2-(4-(3-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)丙酰基)哌嗪-1-基)-3-(-2-(1-乙基-3,3-二甲基吲哚-2-叉)亚乙基)环己-1-烯-1-基)乙烯基)-1-乙基-3,3二甲基-3H-吲哚-1-碘化物，其结构式如式2所示：
一种所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体的制备方法，包括以下步骤：
S1.将氯代七甲川菁盐1与哌嗪在有机溶剂中反应制得中间体2；
S2.将中间体2与马来酰亚胺基丙酸在缩合剂作用下，发生酰胺缩合得到所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体。
所述中间体2为哌嗪七甲川菁盐。
优先地，氯代七甲川菁盐1为氯代七甲川菁碘化物。
优选地，S1中所述有机溶剂为乙腈、二氯甲烷、氯仿、四氢呋喃中的任意一种或几种；S2中所述缩合剂为N，N－二异丙基碳二亚胺、N，N－二环己基碳二亚胺、1-乙基-(3-二甲胺基丙基)碳二亚胺盐酸盐、六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷中的任意一种或几种。
一种所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体的制备方法，包括以下步骤：
S1.哌嗪用有机溶剂溶解后，调温至20～80℃，再缓慢加入氯代七甲川菁盐1的有机溶液，搅拌反应；反应完成后去有机溶剂、萃取、去萃取剂后得到中间体2；
S2.马来酰亚胺基丙酸用有机溶剂溶解后，加入缩合剂并搅拌，再加入中间体2的有机溶液，于温度为0～55℃下反应，分离得到所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体。
优选地，S1中氯代七甲川菁盐1与哌嗪的物质的量的比1:1～1:10。
优选地，S2中马来酰亚胺丙酸与缩合剂的物质的量比为1：0.5～1：5；马来酰亚胺丙酸与中间体2的物质的量的比为0.5：1～2:1。进一步优选地，马来酰亚胺丙酸、缩合剂与中间体2的物质的量比为1.2:1.2:1。
一种所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体的应用，将所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体应用于含亲核基团的有机小分子、多肽、蛋白质的共价结合、标记以及显影示踪。
一种所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体的应用，将所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体应用于作为分子载体制备含亲核基团的小分子、多肽、蛋白质与该载体的共聚物。
优选地，所述亲核基团为巯基、氨基、硒基等。
与现有技术相比，本发明具有以下有益效果：
本发明在近红外荧光菁染料的基础上创造性地引入了马来酰亚胺结构得到所述载体，使得该载体可以有效地与含有巯基、氨基等亲核基的有机小分子、多肽、蛋白质等物质发生迈克尔加成反应，将相应分子共价结合到本发明所述载体上，对相应分子的进行荧光标记，从而能够实现对有机小分子、多肽、蛋白质的体外检测以及体内显影示踪，具有快速、高效的优点，方便快速标记。
本发明通过荧光分子载体共价结合目标小分子的模式可以保障示踪显影的准度与精度，避免采用包合手段进行药物显影示踪时，荧光物质渗漏导致的假阳性结果。
本发明所药物载体与ICG相比具有更好的光稳定性，更高的量子效率与斯托克斯位移，可以高效的完成被研物质的标记，为小分子、多肽、蛋白等在体内的药代动力学性质研究提供了可靠实用的工具。
附图说明
图1为ICG和马来酰亚胺丙酰哌嗪七甲川菁碘化物的吸收光谱比较；
图2为ICG和马来酰亚胺丙酰哌嗪七甲川菁碘化物的荧光光谱比较。
具体实施方式
下面结合具体实施例进一步说明本发明。除非特别说明，本发明实施例中采用的原料、设备和方法为本领域常规市购的原料、常规使用的设备和方法。本部分内容对本发明的权利要求不作任何限定。
实施例1 所述2-(-2-(-2-(4-(3-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)丙酰基)哌嗪-1-基)-3-(-2-(1-乙基-3,3-二甲基吲哚-2-叉)亚乙基)环己-1-烯-1-基)乙烯基)-1-乙基-3,3二甲基-3H-吲哚碘化物的合成
方法一：
本方法合成例马来酰亚胺丙酰哌嗪七甲川菁碘化物包括以下步骤：
S1：将327.01mg(3.80mM)的哌嗪加入单颈瓶，加入适量的乙腈溶解，升温至40℃搅拌，随后称取240.40mg(0.38mM)的氯代七甲川菁碘化物，用适量乙腈溶解后缓慢滴加于哌嗪溶液中，薄层层析法监测反应进程，反应液逐渐由翠绿色变为蓝色，2～3h反应完全后，旋转蒸发除去溶剂，用二氯甲烷/水体系萃取，合并有机层，旋干二氯甲烷，多余的水分采用乙醇共沸蒸出，得到中间体2粗品301mg，产率93.11％。产物经低分辨质谱确证为561.1，与理论计算值相符。粗品避光保存，也可直接用于下一步反应。
S2：称取115.9mg(0.68mM)的3-马来酰亚胺丙酸，投入于100mL的单颈瓶，加入适量乙腈溶解，取96.6mg(0.77mM)的N,N－二异丙基碳二亚胺加入单颈瓶中，室温搅拌1h。取120mg的中间体2，用乙腈溶解后缓慢加入到单颈瓶中，室温搅拌3h，薄层层析法监测反应进程，反应完毕后，旋转蒸发除去溶剂，用二氯甲烷/甲醇体系作为流动相硅胶柱层析提纯，得到113mg的终产物，产率78.51％。质谱经验证为712.1，与理论计算值相符。核磁共振氢谱：1H NMR(400MHz,CDCL3)δ7.76(d,J＝13.6Hz,2H),7.35(t,J＝7.4Hz,4H),7.18(t,J＝7.5Hz,2H),7.02(d,J＝7.8Hz,2H),6.74(s,2H),5.93(d,J＝13.5Hz,2H),4.07(q,J＝7.1Hz,4H),3.96(t,2H),3.89-3.82(m,4H),3.81-3.75(m,2H),3.63-3.54(m,2H),2.87(t,J＝7.3Hz,2H),2.54(t,J＝6.4Hz,4H),1.87(m,J＝12.8,6.4Hz,2H),1.69(s,12H),1.42(t,J＝7.2Hz,6H)。
方法二：
称取100mg的3-马来酰亚胺丙酸，投入于100mL的单颈瓶，加入适量乙腈溶解，取110mg的1-乙基-(3-二甲胺基丙基)碳二亚胺盐酸盐，加入单颈瓶中，室温搅拌1h。取102mg按实施例S1步骤中方法获得的中间体2，用乙腈溶解后缓慢加入到单颈瓶中，室温搅拌3h，薄层层析法监测反应进程，反应完毕后，旋转蒸发除去溶剂，用二氯甲烷/甲醇体系作为流动相硅胶柱层析提纯，得到89.6mg的终产物，产率72％。
方法三：
称取100mg的3-马来酰亚胺丙酸，投入于100mL的单颈瓶，加入适量乙腈溶解，冰浴降温15分钟，取370mg的六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷，加入单颈瓶中，取120mg按实施例S1步骤中方法获得的中间体2，用乙腈溶解后缓慢加入到单颈瓶中，五分钟后再加入N,N-二异丙基乙胺95mg启动反应，冰浴搅拌2h，薄层层析法监测反应进程，反应完毕后，旋转蒸发除去溶剂，用二氯甲烷/甲醇体系作为流动相硅胶柱层析提纯，得到124mg的终产物，产率85％。
实施例2 马来酰亚胺丙酰哌嗪七甲川菁碘化物与巯基十二硼烷二钠盐(BSH)共价连接
由于本发明所指马来酰亚胺丙酰哌嗪七甲川菁盐类荧光载体的阴离子部分对载体的共价化学连接性能没有本质影响，本实例主要是以马来酰亚胺丙酰哌嗪七甲川菁碘化物为代表(代号CyP)。
此实施例选取含有巯基的经典放射性硼中子捕获治疗药物巯基十二硼烷二钠盐(BSH)作为亲核分子，共价结合到所述马来酰亚胺丙酰哌嗪七甲川菁盐上，形成共聚物十二硼烷马来酰亚胺丙酰哌嗪七甲川菁硫醚盐(代号BS-CyP)。本实验证明马来酰亚胺丙酰哌嗪七甲川菁盐类荧光载体可有效地与含亲核试剂的分子进行有效的共价连接。
本实施例具体操作如下：准确称取25.2mg(0.12mM)的巯基十二硼烷二钠盐(BSH)于10mL的单颈瓶中，加入2mL的水溶解，然后加42μL(0.24mM)的N,N-二异丙基乙胺，搅拌5分钟。称取35.4mg CyP(0.042mM)，用2mL乙腈溶解后加入到上述巯基十二硼烷二钠盐溶液中，氮气保护，避光，常温搅拌，薄层层析色谱(TLC)监测反应进程，3小时后关停反应，旋干溶剂，二氯甲烷/甲醇体系柱层析提纯，得到20mg的纯品，产率52.23％。质谱测定数据为876.6，与理论计算值相符。核磁共振氢谱：1H NMR(400MHz,DMSO)δ7.70(d,J＝13.6Hz,2H),7.57(d,J＝7.3Hz,2H),7.35(t,J＝7.7Hz,2H),7.27(d,J＝7.9Hz,2H),7.16(t,J＝7.4Hz,2H),5.99(d,J＝13.6Hz,2H),4.11(q,J＝12.9,5.9Hz,4H),3.81–3.69(m,4H),3.69–3.60(m,4H),3.62–3.53(m,J＝8.1,3.1Hz,4H),3.01(dd,J＝18.5,8.1Hz,1H),2.90(d,J＝4.9Hz,2H),2.72(d,2H),2.68(m,J＝15.7,7.7Hz,2H),1.80–1.73(m,2H),1.26(t,J＝7.1Hz,6H),0.5-1.2(m,11H)。
实施例3 理化性质实验
由于本发明所指近马来酰亚胺丙酰哌嗪七甲川菁盐的阴离子部分对载体的荧光性能没有本质影响，本实例主要是以马来酰亚胺丙酰哌嗪七甲川菁碘化物(CyP)为代表。
(一)近红外菁染料ICG和CyP的吸收光谱
将ICG和实施例1中制备得到的马来酰亚胺丙酰哌嗪七甲川菁碘化物分别溶于甲醇中，制备浓度为1mM的储备液，稀释至3.13μM，分别扫描紫外吸收光谱，测得其紫外可见光谱如图1所示。由图1可见ICG在甲醇中的最大吸收波长是790nm，在710nm处含一小肩峰。CyP呈现明显的双吸收峰，最大吸收波长为762nm和703nm，相较ICG最大吸收发生明显的蓝移。同时相同浓度的CyP的吸光强度只有ICG的一半左右，因而CyP的吸光系数低于ICG的吸光系数。CyP在近红外区的宽吸收峰意味着能够选取最合适的激发波长。
(二)近红外菁染料ICG和CyP的荧光光谱
将ICG和实施例1中配制的荧光载体CyP的倍半稀释液用于扫描荧光光谱，激发波长700nm，检测波长为750-850nm，测得的荧光光谱如图2所示。由图2可知，在选择700nm激发波长情况下，CyP的最大发射波长为792nm，斯托克斯位移为89nm；ICG的最大发射波长为825nm，与其最大吸收峰相比，斯托克斯位移为35nm。同时，相同62.5μM浓度条件下，CyP的荧光强度明显高于ICG。这些数据说明CyP具有更好的荧光光学性质。
Claims (8)
1.一种马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体，其特征在于，结构式如式1所示：
其化学名为：
2-(-2-(-2-(4-(3-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)丙酰基)哌嗪-1-基)-3-(-2-(1-乙基-3,3-二甲基吲哚-2-叉)亚乙基)环己-1-烯-1-基)乙烯基)-1-乙基-3,3二甲基-3H-吲哚-1-盐。
2.根据权利要求1所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体，其特征在于，X-为化学上合理的任一有机酸根或无机酸根负离子。
3.根据权利要求2所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体，其特征在于，X-为碘离子。
4.一种如权利要求1～3任意一项所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体的制备方法，其特征在于，包括以下步骤，如式2所示：
S1.将氯代七甲川菁盐1与过量哌嗪在有机溶剂中反应制得中间体2；
S2.将中间体2与马来酰亚胺基丙酸在缩合剂作用下，发生酰胺缩合得到所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体。
5.根据权利要求4所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体的制备方法，其特征在于，S1中所述有机溶剂为乙腈、二氯甲烷、氯仿、四氢呋喃中的任意一种或几种；S2中所述缩合剂为N，N－二异丙基碳二亚胺、N，N－二环己基碳二亚胺、1-乙基-(3-二甲胺基丙基)碳二亚胺盐酸盐、六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷中的任意一种或几种。
6.根据权利要求4所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体的制备方法，其特征在于，包括以下步骤：
S1.哌嗪用有机溶剂溶解后，调温至20～80℃，再缓慢加入氯代七甲川菁盐1的有机溶液，搅拌反应；反应完成后去有机溶剂、萃取、去萃取剂后得到中间体2；
S2.马来酰亚胺基丙酸用有机溶剂溶解后，加入缩合剂并搅拌，再加入中间体2的有机溶液，于温度为0～55℃下反应，分离得到所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体。
7.一种如权利要求1～3任意一项所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体的应用，其特征在于，将所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体应用于含亲核基团的有机小分子、多肽、蛋白质的共价结合、标记以及显影示踪。
8.一种如权利要求1～3任意一项所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体的应用，其特征在于，将所述马来酰亚胺丙酰哌嗪七甲川菁盐荧光载体应用于作为分子载体制备含亲核基团的小分子、多肽、蛋白质与该载体的共聚物。
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