CN111393430A - 一种百部生物碱类似物近红外荧光探针及其制备方法 - Google Patents
一种百部生物碱类似物近红外荧光探针及其制备方法 Download PDFInfo
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Abstract
本发明涉及光学成像领域，具体涉及一种百部生物碱类似物近红外荧光探针及其制备方法。所述近红外荧光探针应用于生物硫醇的快速检测，应用于细胞及活体内生物硫醇成像。目前报道的检测生物硫醇的荧光探针任存在一定的局限性，如复杂的合成路线，响应时间长，较短的发射波长等。上述近红外荧光探针的制备方法为5‑(羟甲基)‑7‑甲基‑3,3a‑二氢环戊[b]色满环‑1(2H)‑酮与3,7‑双(二甲基胺基)‑10H‑苯并噻嗪‑10‑羰基氯在4‑二甲氨基吡啶及吡啶作用下生成(7‑甲基‑1‑氧‑1,2,3,3a‑四氢环戊[b]色满环‑5‑基)‑甲基‑3,7‑双(二甲基胺基)‑10H‑苯并噻嗪‑10‑羧酸酯。
Description
技术领域
本发明涉及光学成像领域，具体涉及一种百部生物碱类似物近红外荧光探针及其制备方法。
背景技术
众所周知，体内的小分子生物硫醇如半胱氨酸(Cys)，同型半胱氨酸(Hcy)和谷胱甘肽(GSH)，在生理基质中起着十分重要的作用。例如，Cys和Hcy是生命系统中细胞和组织生长所必需的必需生物分子。半胱氨酸缺乏会导致各种健康问题，例如生长迟缓，脱毛，嗜睡，肝损伤，肌肉和脂肪减少以及皮肤损伤。人体血浆中Hcy水平升高是阿尔茨海默病，心血管疾病，神经管缺陷，炎症性肠病和骨质疏松症的危险因素。GSH是最丰富的细胞内非蛋白原性硫醇，在维持细胞中的还原环境中起着关键作用，并起到氧化还原调节剂的作用，因此，生物样品中含巯基分子的检测和鉴别非常重要。
目前报道的检测生物硫醇的荧光探针仍存在一定的局限性，如复杂的合成路线，响应时间长，较短的发射波长等。因此，设计并合成新型的检测生物硫醇的荧光探针具有重要的意义与价值。
发明内容
本发明的目的是提供一种新型识别生物硫醇的近红外荧光探针及其合成方法，该探针具有较好的选择特异性和灵敏性。
为了达到上述目的，本发明采用了下列技术方案：
一种百部生物碱类似物近红外荧光探针，所述百部生物碱类似物近红外荧光探针为(7-甲基-1-氧-1,2,3,3a-四氢环戊[b]色满环-5-基)-甲基-3,7-双(二甲基胺基)-10H-苯并噻嗪-10-羧酸酯，是基于色烯骨架的衍生物，结构式如下：
一种百部生物碱类似物近红外荧光探针的制备方法，包括以下步骤：
在氩气保护下，将5-(羟甲基)-7-甲基-3,3a-二氢环戊[b]色满环-1(2H)-酮(化合物2)，7-双(二甲基胺基)-10H-苯并噻嗪-10-羰基氯(化合物3)和4-二甲氨基吡啶溶于吡啶中，搅拌，反应至原料反应完，将反应液用乙醚析出，并将乙醚层干燥，滤液浓缩得粗产物，通过硅胶柱层析得到(7-甲基-1-氧-1,2,3,3a-四氢环戊[b]色满环-5-基)-甲基-3,7-双(二甲基胺基)-10H-苯并噻嗪-10-羧酸酯(化合物1)。
进一步，所述5-(羟甲基)-7-甲基-3,3a-二氢环戊[b]色满环-1(2H)-酮、3,7-双(二甲基胺基)-10H-苯并噻嗪-10-羰基氯和4-二甲氨基吡啶的摩尔比为1：1.2～3：3.38～5。
进一步，所述吡啶为0.2～0.7mol/L。
再进一步，所述搅拌的温度为45℃～80℃，搅拌的时间为12h～36h。
更进一步，所述反应至原料反应完用TLC点板监测。
本路线实现了百部生物碱类似物近红外荧光探针(7-甲基-1-氧-1,2,3,3a-四氢环戊[b]色满环-5-基)-甲基-3,7-双(二甲基胺基)-10H-苯并噻嗪-10-羧酸酯的高效合成，在反应中应用4-二甲氨基吡啶催化，能显著催化反应的进行，从而得到目标产物。
一种近红外荧光探针的应用，所述近红外荧光探针应用于生物硫醇的快速检测，应用于细胞及活体内生物硫醇成像。
与现有技术相比本发明具有以下优点：
1.本发明提供了一种荧光探针的简易合成路线，合成本探针的原料易得，成本较低；
2.本发明提供了荧光探针的用途，可快速荧光检测生物硫醇，具有较好的选择性及其灵敏性；
3.本探针具有较好的生物相容性，较长的发射波长(690nm),可应用于细胞及活体内生物硫醇成像。
附图说明
图1是本发明荧光探针的核磁共振的氢谱图
图2是本发明荧光探针的核磁共振的碳谱图
图3是本发明荧光探针检测140μM Cys不同时间的紫外光谱图；
图4是本发明荧光探针检测140μM Cys不同时间的荧光光谱图；
图5是本发明荧光探针及其加入Cys的动力学分析曲线图；
图6是本发明荧光探针在加入小分子生物硫醇以及不同氨基酸作用后的荧光光谱图。
具体实施方式
实施例1：本发明荧光探针的具体合成过程
(1)化合物2的具体合成步骤如下：
将2，6-双(羟甲基)-对甲酚(5g，29mmol)和MnO2(20g，229mmol)在400ml丙酮中的混合物在室温下搅拌过夜，过滤，滤液浓缩所得残余物。经柱色谱分离(PE：EA＝2∶1)，得到化合物5(53％)，为白色固体。
将化合物5(278mg，1.67mmol)，环戊-2-烯-1-酮(化合物6)(0.280mL，3.35mmol)和1-H-咪唑(120mg，1.67mmol)溶于四氢呋喃(THF，2.5mL)与蒸馏水(2.5mL)。将混合物在室温搅拌96h，最终混合物用10mL HCl溶液(1mol/L)稀释，乙酸乙酯(3×15.27mL)萃取。滤液浓缩所得残余物，通过硅胶柱层析分离(PE:EA＝1:2)，以50％的产率获得化合物2(193mg)。
(2)化合物3的具体合成步骤如下：
在氩气保护下,将亚甲基蓝(化合物7)(1.00g，3.13mmol)溶解在16mL二氯甲烷置于100mL圆底烧瓶中，用注射器分别加入溶解在10mL水中的NaHCO3(525mg，6.25mmol)，13mL水中的连二亚硫酸钠(1.1g，6.3mmol)，直至溶液变为黄色。将混合物用冰水浴冷却，先向其中加入三乙胺0.52ml，后逐滴加入溶解在2mL二氯甲烷中的双(三氯甲基)碳酸酯(241mg，0.81mmol)。混合物搅拌2h，溶液在搅拌下倒入冰水中，并将混合物用二氯甲烷萃取，合并的萃取物用盐水洗涤，经无水硫酸钠干燥，滤液浓缩所得残余物，通过硅胶柱层析分离(乙酸乙酯/石油醚＝1:4)得到白色固体产物(化合物3)370mg(35％)。
实施例2.荧光探针的具体合成步骤如下：
将化合物2(32mg，139μmol，1当量)，化合物3(58mg，167μmol，1.2当量)和DMAP(57mg，470μmol，3.38当量)溶于0.64mL吡啶中，在氩气下于45℃搅拌过夜。TLC点板监测反应至原料反应完，将反应液用乙醚析出，并将乙醚层干燥，滤液浓缩所得残余物，通过硅胶柱层析分离(PE：EA＝2：1，5％三乙胺)得到荧光探针49mg。1H NMR(600MHz,CDCl3)δ7.40(s,2H),7.18(d,J＝2.5Hz,1H),7.08(d,J＝2.2Hz,1H),7.00(d,J＝2.2Hz,1H),6.69(s,2H),6.63(s,2H),5.29(s,1H),5.21(ddd,J＝9.6,7.5,2.5Hz,2H),2.95(s,12H),2.70(dt,J＝12.3,8.1Hz,1H),2.61(dd,J＝18.5,9.0Hz,1H),2.35(ddd,J＝18.6,12.7,8.7Hz,1H),2.26(s,3H),2.16(ddd,J＝12.5,9.1,3.5Hz,1H)。13C NMR(151MHz,CDCl3)δ201.6,171.2,163.4,154.4,150.7,148.8,134.6,132.8,132.7,131.7,131.1,130.2,127.8,127.1,124.2,121.5,111.0,110.3,76.8,75.8,62.9,60.4,58.5,40.8,37.2,29.7,28.1,21.1,20.5,18.5,14.2。
实施例3：本发明荧光探针和Cys反应前后紫外、荧光光谱图:
在本发明的荧光探针溶液(10μM，PBS：DMSO＝1:1)中加入140μM的Cys(搅拌)，10min后，测定紫外和荧光强度变化。结果可参见图3和图4，所有的紫外和荧光强度均进行了归一法。
如图3所示，本发明荧光探针溶液中不加入(左边)和加入(右边)Cys后的肉眼可见颜色变化图片。加入Cys后，荧光发生明显的变化，由无色变为蓝色，说明本探针与Cys发生反应，可用于示踪Cys。
如图3所示，本发明荧光探针溶液在加入Cys前后的紫外-可见吸收光谱。可以发现，在加入Cys后，本发明探针在550nm至700nm的波长范围内显示出主要的吸收带，且该吸收带随时间增加；并在666nm处出现最大吸收峰，以上变化表明本发明探针在加入Cys后其化学结构发生了明显变化。
如图4所示，本发明荧光探针溶液在加入Cys前后的荧光发射光谱的变化(600nm激发，缝隙：2.5nm/5nm)。可以发现，在加入Cys后，本发明荧光探针显示出对Cys的荧光开启响应，其荧光发射范围为635nm至800nm，且主要是在698nm处出现了一个新的明显吸收峰，说明Cys与探针发生反应，释放出荧光团。
结合图3，4光谱图所示，说明本荧光探针在加入Cys后，会和Cys发生化学反应致使其化学键的断裂与生成，引发其结构发生明显变化，从而释放出亚甲蓝染料，产生一系列颜色的变化。
实施例4：本发明荧光探针和Cys的动力学分析
如图5所示为本发明荧光溶液中加入Cys荧光发射光谱在698nm处随时间变化光谱图。荧光探针溶液中加入Cys后，荧光发射光谱随时间的变化在400s内基本完成，说明本探针具有快速响应的能力；在纯的探针溶液中，400s内，本发明荧光探针的荧光强度基本保持不变，说明本发明荧光探针具有很好的稳定性。
实施例5：本发明荧光探针在加入小分子生物硫醇以及不同氨基酸作用后的荧光光谱图
在本发明的荧光探针溶液(5μM，PBS：DMSO＝1:1)中分别加入140μM Cys、Hcy、GSH、H2S、L-亮氨酸、L-苏氨酸、L-蛋氨酸、L-脯氨酸、L-天冬氨酸、L-缬氨酸、反式-4-羟基-L-脯氨酸、L-天冬酰胺、L-谷氨酸、L-苯丙氨酸、L-精氨酸、L-异亮氨酸(搅拌)，10分钟后测试其荧光发射。如图6所示，只有小分子生物硫醇能够引起较强的荧光增强，而其他氨基酸则没有明显变化。说明本发明荧光探针对于生物硫醇Cys、Hcy以及GSH具有较高的选择性，且在反应前后荧光强度有明显的区别，因此，该荧光探针能够被应用于生物系统中进行某些小分子生物硫醇的荧光检测和成像。
Claims (7)
1.一种百部生物碱类似物近红外荧光探针，其特征在于，所述百部生物碱类似物近红外荧光探针为(7-甲基-1-氧-1,2,3,3a-四氢环戊[b]色满环-5-基)-甲基-3,7-双(二甲基胺基)-10H-苯并噻嗪-10-羧酸酯，结构式如下：
2.一种百部生物碱类似物近红外荧光探针的制备方法，其特征在于，包括以下步骤：
在氩气保护下，将5-(羟甲基)-7-甲基-3,3a-二氢环戊[b]色满环-1(2H)-酮，7-双(二甲基胺基)-10H-苯并噻嗪-10-羰基氯和4-二甲氨基吡啶溶于吡啶中，搅拌，反应至原料反应完，将反应液用乙醚析出，并将乙醚层干燥，滤液浓缩得粗产物，通过硅胶柱层析得到(7-甲基-1-氧-1,2,3,3a-四氢环戊[b]色满环-5-基)-甲基-3,7-双(二甲基胺基)-10H-苯并噻嗪-10-羧酸酯。
3.根据权利要求2所述的一种近红外荧光探针的制备方法，其特征在于，所述5-(羟甲基)-7-甲基-3,3a-二氢环戊[b]色满环-1(2H)-酮、3,7-双(二甲基胺基)-10H-苯并噻嗪-10-羰基氯和4-二甲氨基吡啶的摩尔比为1：1.2～3：3.38～5。
4.根据权利要求2所述的一种近红外荧光探针的制备方法，其特征在于，所述吡啶为0.2～0.7mol/L。
5.根据权利要求2所述的一种近红外荧光探针的制备方法，其特征在于，所述搅拌的温度为45℃～80℃，搅拌的时间为12h～36h。
6.根据权利要求2所述的一种近红外荧光探针的制备方法，其特征在于，所述反应至原料反应完用TLC点板监测。
7.一种近红外荧光探针的应用，其特征在于，所述近红外荧光探针应用于生物硫醇的快速检测，应用于细胞及活体内生物硫醇成像。
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