CN103509366B - 染料与标记生物材料的方法 - Google Patents
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- 229910019142 PO4 Inorganic materials 0.000 abstract description 5
- 125000003545 alkoxy group Chemical group 0.000 abstract description 5
- 125000004457 alkyl amino carbonyl group Chemical group 0.000 abstract description 5
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- 229910052736 halogen Inorganic materials 0.000 abstract description 5
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- 125000001072 heteroaryl group Chemical group 0.000 abstract description 5
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- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
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- C09B11/10—Amino derivatives of triarylmethanes
- C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
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Abstract
本发明提供一种染料，具有化学结构： 或上述的混合物；其中每一R1各自独立，选自氢、卤素、氰基、烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、二烷基胺基、烷基胺基羰基、二烷基胺基羰基、芳香基、杂芳基、磷酸基或磺酸基，每一R2各自独立，选自或以及每一n各自独立，选自2至10的整数。
Description
技术领域
本发明涉及染料，尤其涉及以染料标记生物材料的方法。
背景技术
染料已被广泛应用于生物学研究和医学诊断。由于染料通常具有足够的检测灵敏度、较低的价格、与较低的毒性，因此优于一般的放射性检测材料。此外，染料可于体内或体外标记生物材料(氨基酸、肽、蛋白质、抗体、生物高分子、细胞或组织)。若能大量合成染料搭配自动化检验，更有利于标记生物材料。
罗丹明(Rhodamine)系列的染料是现有的主要染料之一。然而罗丹明主体本身不溶于水，需另外接枝亲水基团如磺酸基以使其能溶于水中，以应用于标记生物材料。目前仍需兼顾染料与生物材料之间的结合浓度、染料结合至生物材料后的发光效率、以及将磺酸基接枝至罗丹明上的方法。
发明内容
本发明的一实施例提供一种染料，具有化学结构：
或上述的混合物；
其中每一R1各自独立，选自氢、卤素、氰基、烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、二烷基胺基、烷基胺基羰基、二烷基胺基羰基、芳香基、杂芳基、磷酸基或磺酸基，每一R2各自独立，选自或以及每一n各自独立，选自2至10的整数。
本发明的一实施例提供一种标记生物材料的方法，包括：混合上述染料与一生物材料，使该染料与该生物材料结合；以及提供激发光，使结合至该生物材料的该染料发光。
附图说明
图1示出本发明的一实施例中，不同浓度的不同染料在结合至BSA后对应的光密度值。
具体实施方式
本发明提供一种染料，其可以结合至氨基酸、肽、蛋白质、抗体、生物高分子、细胞或组织等具有肽键的生物材料，可用以标记上述生物材料。此染料具有末端磺酸基，可溶于水中，且具有N-酯基琥珀酰亚胺基团而易与肽键结合。在本发明的一实施例中，先将染料与待标记的生物材料混合，使染料结合至生物材料。在本发明的另一实施例中，以紫外线或可见光作为激发光照射结合染料的生物材料，使染料发光如可见光以达标记生物材料的效果。上述标记方法可用于标记体内或体外的生物材料。
在本发明某些实施例中，染料是以下述方式合成。值得注意的是，本技术领域中自可采用其它方法合成染料，而不限于下述方法。
本发明的一实施例提供一种染料，具有化学结构：
或上述的混合物；
其中每一R1各自独立，选自氢、卤素、氰基、烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、二烷基胺基、烷基胺基羰基、二烷基胺基羰基、芳香基、杂芳基、磷酸基、或磺酸基，每一R2各自独立，选自或以及每一n各自独立，选自2至10的整数。
在一实施例中，染料可通过如下举例的反应获得，但不以此为限。
首先如式1所示，进行环化反应。
接着取式1的产物与N-羟基琥珀酰亚胺反应如式2，其产物即染料。
在式1及式2中，每一R1各自独立，选自氢、卤素、氰基、烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、二烷基胺基、烷基胺基羰基、二烷基胺基羰基、芳香基、杂芳基、磷酸基或磺酸基。每一n各自独立，选自2至10的整数或3至6的整数。
在本发明的另一实施例中，取式1的产物、N-羟基琥珀酰亚胺、与氨基酸反应如式3所示，其产物即染料。
在式3中，每一R1各自独立，选自氢、卤素、氰基、烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、二烷基胺基、烷基胺基羰基、二烷基胺基羰基、芳香基、杂芳基、磷酸基或磺酸基。每一n各自独立，选自2至10的整数或3至6的整数。
为了使本发明的上述和其它目的、特征和优点能更明显易懂，下文特列举实施例，配合附图，作详细说明如下：
【实施例】
合成例1(N,N-二丁基磺酸盐-1-酚的合成)
取2.0g化合物A(0.0076mol)与1.5mL1,4-丁磺酸内酯，2.3mL三乙胺，10mL乙腈置入100mL反应瓶，加热至60℃后反应20小时，如式4所示。反应后冷却，抽除溶剂后得化合物B。化合物A的合成见Chemistry andIndustry,20,345(1974)的方法。
直接取化合物B、0.9576g的LiOH．H2O、10mL甲醇与10mL水置于100mL反应瓶中，加热至60℃反应3小时。反应后以盐酸中和上述反应后的混合物以析出固体，过滤得3.04g的化合物C。上述反应如式5所示：
实施例1(染料合成)
取3g化合物C、0.9827g的1,2,4-苯三酸酐(0.0051mmol，购自TCIAMERICA)、0.1495g的对甲基苯磺酸(0.00079mol)及5mL的丙酸置入100mL的反应瓶中，加热至140℃后反应22小时如式6。将反应后的混合物降温至室温后进行管柱层析，得到化合物1A与化合物1B(总计2.5383g，产率35%)，化合物1A与1B的混合物其氢谱分析数据如下：1H NMR(MeOH-d4),δ=8.35(d,J=6Hz,1H),8.27(s,1H),7.46(d,J=6Hz,1H),7.16(d,J=6Hz,2H),7.07(d,J=6Hz,2H),7.00(s,2H),3.66(m,8H),2.88(m,8H),1.89(m,16H)。
取70mg化合物1A与1B的混合物(0.76mmol)、35.6mg的1-(3-二甲基胺基丙基)-3-乙基碳二亚胺盐酸盐(EDC-HCl，0.186mmol)、19.71mg的N-羟基琥珀酰亚胺(0.171mmol)、与4mL的甲醇置入100mL的反应瓶内，于室温下搅拌1小时如式7。将反应后的混合物进行管柱层析，得到化合物2A与2B(总计77.22mg，产率76.2%)。化合物2A与2B的混合物其氢谱分析的数据如下：1H NMR(MeOH-d4),δ=8.40(d,J=3Hz,1H),8.35(s,1H),8.02(d,J=3Hz,1H),7.08(m,6H),3.66(m,8H),2.88(m,8H),2.73(s,4H),1.89(m,16H)。经紫外线-可见光光谱仪与荧光光谱仪量测化合物2A/2B的水溶液，其最大吸收波长、最大放射波长与量子效率如表1所示。Cy3的量子效率为0.15，远低于实施例。
实施例2(染料合成)
取60mg式6产物(0.0655mmol)、15mg的1-(3-二甲基胺基丙基)-3-乙基碳二亚胺盐酸盐(EDC-HCl，0.0786mmol)、与4mL的甲醇置入100mL的反应瓶内，于室温下搅拌1小时。接着加入0.0172g(0.0172mol)的6-胺基己酸于室温下反应1小时，再加入19.71mg的N-羟基琥珀酰亚胺(0.171mmol)于室温下反应1小时如式8。将反应后的混合物进行管柱层析，得到化合物3A与3B(总计18.70mg，产率25%)。化合物3A与3B的混合物其氢谱数据如下：1H NMR(MeOH-d4),δ=8.30m,2H),7.41(m,2H),7.06(m,5H),3.68(m,8H),2.87(m,10H),2.74(s,4H),2.19(m,2H),1.85(m,18H),1.64(m,4H)。经紫外线-可见光光谱仪与荧光光谱仪量测化合物3A/3B的水溶液，其最大吸收波长、最大放射波长、与量子效率如表1所示。
实施例3(染料前驱物的合成)
取3g的N,N-二丙基磺酸盐-1-酚(0.01mol)、1.4606g(0.0076mol)的1,2,4-苯三酸酐，0.1112g的对甲基苯磺酸(0.00058mol)及5mL的丙酸置入100mL反应瓶中，加热至140℃后反应22小时如式9所示。将反应后的混合物降温至室温后进行管柱层析，得到化合物4A与4B(总计1.8972g，产率35%)。化合物4A与4B的混合物其氢谱分析的数据如下：1H NMR(MeOH-d4),δ=8.38(d,J=6Hz,1H),8.17(s,1H),7.53(d,J=6Hz,1H),7.18(m,4H),6.85(s,2H),3.84(m,8H),2.87(m,8H),2.19(m,8H)。
表1
| 化合物 | 最大吸收波长 | 最大放射波长 | 量子效率 |
| 化合物2A/2B | 562nm | 595nm | 1.45 |
| 化合物3A/3B | 560nm | 593nm | 1.40 |
实施例4
取化合物2A/2B与市售染料Cy3(购自GE Healthcare)各自溶于2μL水中后测其光密度(OD)。依据OD值，将化合物2A/2B水溶液与Cy3水溶液调整至相同浓度。取6支1.5mL的试管，各自加入10μL的5%牛血清白蛋白溶液(BSA solution)后，分别加入不同量的Cy3水溶液或化合物2A水溶液，与不同量的100mM碳酸氢钠水溶液(pH=8.5)。6支试管中的组成如表2所示：
表2
将上述6种组成各自均匀混合后，静置于室温下1小时。接着以体积排出色谱法（SEC）分离与BSA结合的染料、未与染料结合的BSA、与未与BSA结合的染料。取1μL分离后的BSA与染料结合者加入9μL水，再测量其全波长的光密度，即可知此样品的标记效果。上述6支试管中的样品其光密度对上染料用量的关系图如图1所示。如图1所示，化合物2A/2B的标记效果明显优于市售染料的标记效果，原因可能为化合物2A/2B与BSA的结合浓度高于市售染料与BSA的结合浓度，和/或化合物2A/2B比市售染料的发光效率更佳。
在实施例中，染料具有下述特性：(1)染料结合至生物材料后仍能产生很强的荧光信号，(2)染料结合至生物材料后具有稳定的吸收及放射波长，以及(3)染料与生物材料之间具有有效的结合浓度。
虽然本发明已经以数个较佳实施例揭露如上，然而其并非用以限定本发明，任何本领域技术人员，在不脱离本发明的精神和范围内，可作任意的修改与改变，因此本发明的保护范围以权利要求书所界定为准。
Claims (5)
1.一种染料，具有化学结构：
或上述的混合物；
其中R1各自是氢，每一R2各自独立，选自以及
每一n各自独立，选自2至10的整数。
2.根据权利要求1所述的染料，具有化学结构：
或上述的混合物。
3.根据权利要求2所述的染料，具有化学结构：
或上述的混合物。
4.根据权利要求1所述的染料，具有化学结构：
或上述的混合物；
其中R1各自是氢；以及
每一n各自独立，选自2至10的整数。
5.根据权利要求4所述的染料，具有化学结构：
或上述的混合物。
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