CN114605371B - 一种苯并吡喃衍生物bp-ⅲ及其合成方法和应用 - Google Patents
一种苯并吡喃衍生物bp-ⅲ及其合成方法和应用 Download PDFInfo
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Abstract
本发明提供了一种苯并吡喃衍生物BP‑Ⅲ及其合成方法和应用,所述苯并吡喃衍生物BP‑Ⅲ的中文名称为:(E)‑9‑(2‑羧基苯基)‑6‑(二乙氨基)‑4‑(4‑(1‑吡咯烷基)亚苄基)‑1,2,3,4‑四氢黄嘌呤。本发明将苯并吡喃衍生物BP‑Ⅲ作为荧光探针,可实现单通道检测黏度。具体基于所述苯并吡喃衍生物BP‑Ⅲ,在PBS和甘油不同的体积比混合溶液中通过荧光光谱仪检测BP‑Ⅲ随着黏度的变化而引起的荧光强度的变化。该方法还在细胞和小鼠水平实现了高灵敏度和无创性原位成像,可用于实时原位监测线粒体黏度的变化和区别脂肪肝。
Description
技术领域
本发明涉及苯并吡喃衍生物,具体属于一种苯并吡喃衍生物及其合成方法,以及该衍生物在检测黏度中的应用。
背景技术
黏度,作为细胞中的一种微环境,可以参与调节信号传导、电子传递、细胞代谢和细胞凋亡等多种重要生物过程。黏度作为一种应激反应和稳态机制,其异常将会影响细胞内复杂的系统平衡,从而导致基于黏度扩散的小分子和pH发生紊乱,以及导致细胞器发生故障和身体机能失调;更严重的将会引起炎症、脂肪肝、阿尔兹海默症和帕金森综合征等严重疾病。
分子转子荧光探针以其优异的旋转特性、显著的灵敏度、清晰的空间分辨率、无创性和原位性等独特优势,已成为活细胞多功能生物医学成像的有力工具。目前,据我们了解到的一些对于黏度检测的荧光探针,在对黏度进行检测时,虽然这些荧光探针随着黏度的增加,荧光呈现出从暗态到亮态的转变,但在灵敏度和稳定性、以及荧光强度和检测方式上的不足之处,使他们应用方面受到了很大的限制。因此开发一种高灵敏度和无创性原位成像的黏度检测工具,用于实时原位监测细胞器黏度的变化,对于了解细胞功能和阐明相关疾病的发生机制,并对与黏度相关的生物学和病理学功能进行分类将会具有重要意义。
发明内容
本发明的目的是提供一种苯并吡喃衍生物BP-Ⅲ及其合成方法,以及该衍生物在细胞水平和动物中检测黏度的应用。
本发明提供的一种苯并吡喃衍生物BP-Ⅲ,中文名称为:(E)-9-(2-羧基苯基)-6-(二乙氨基)-4-(4-(1-吡咯烷基)亚苄基)-1,2,3,4-四氢黄嘌呤,英文名称为:(E)-9-(2-carboxyphenyl)-6-(diethylamino)-4-(4-(1-Pyrrolidino)benzylidene)-1,2,3,4-tetrahydroxanthylium。其结构式为:
BP-Ⅲ的合成方法,步骤为:
1)在反应容器中,将反应温度控制在0℃,将环己酮滴加到浓硫酸中,将溶液在0℃下搅拌至环己酮滴加结束,然后加入2-(4-二乙氨基-2-羟基苯甲酰基)苯甲酸,将混合溶液在90~100℃下进一步加热回流2~4小时,冷却至室温后,将混合物缓慢加入到冰水中,充分搅拌,然后加入HClO4,搅拌至由红色固体析出,将所得悬浮液进行抽滤,并用水洗涤,真空干燥,得到化合物1;其中:环己酮、2-(4-二乙氨基-2-羟基苯甲酰基)苯甲酸、浓H2SO4和HClO4的摩尔比为2~4:1:20:3~4;
2)在反应容器中,将化合物1溶于冰乙酸中,然后加入4-(1-吡咯烷)苯甲醛,将混合物加热回流2~4小时,反应完成后,自然冷却,减压除去溶剂,得到粗产物,将粗产物进行真空干燥,柱色谱分离粗产物,用V二氯甲烷:V甲醇=20:1洗脱,得到蓝黑色粉末即BP-Ⅲ;其中:化合物1和4-(1-吡咯烷)苯甲醛的摩尔比为1:0.5~10。
上述合成的苯并吡喃衍生物BP-Ⅲ在细胞水平和小鼠中检测黏度的方法,包括如下步骤:
(1)、配制pH=7.4、浓度为10mM的PBS缓冲溶液,配制2mM的BP-Ⅲ的DMSO溶液,配制不同黏度的甘油/PBS溶液(表1);
表1配制不同黏度的甘油/PBS溶液
(2)、将配制的不同黏度的甘油/PBS溶液,分别加入10μL BP-Ⅲ的DMSO溶液于荧光比色皿中,充分混合均匀后,在荧光分光光度仪上检测BP-Ⅲ随着黏度的增加荧光强度在740nm处的变化;
(3)、取2000μL PBS缓冲溶液、10μL BP-Ⅲ的DMSO溶液于比色皿中,在荧光分光光度仪上检测BP-Ⅲ随着时间的增加(0-60min)在740nm处的荧光强度变化;
(4)、取2000μL甘油缓冲溶液、10μL BP-Ⅲ的DMSO溶液于比色皿中,在荧光分光光度仪上检测BP-Ⅲ随着时间的增加(0-60min)在740nm处的荧光强度变化。
与现有技术相比,本发明的有益效果:
1、本发明苯并吲哚衍生物BP-Ⅲ的合成只需两步,合成方法简单,操作方便;
2、探针BP-Ⅲ对黏度的检测,斯托克斯位移较大,近红外发射(740nm),具有高穿透性、低背景荧光、便于活体成像等优点;
3、检测手段简单,通过红色通道,可实现实时观察细胞中黏度的变化,实现对脂肪肝的检测;
4、苯并吡喃鎓盐中的正离子,可以精确的靶向线粒体,从而实现对线粒体黏度的检测。
附图说明
图1实施例1制备的BP-Ⅲ的核磁氢谱
图2实施例1制备的BP-Ⅲ的核磁碳谱
图3BP-Ⅲ在不同有机溶液中的荧光光谱图
图4BP-Ⅲ在不同比例甘油/PBS溶液中的荧光光谱图
图5BP-Ⅲ在不同比例甘油/PBS溶液中的荧光强度与黏度的工作曲线
图6BP-Ⅲ在不同pH体系下的荧光光谱图
图7BP-Ⅲ与各种分析物的荧光柱状图
图8BP-Ⅲ在PBS和甘油中60min内740nm处的荧光光谱图
图9BP-Ⅲ检测正常肝细胞HL-7702黏度细胞成像图
图10BP-Ⅲ检测肝癌细胞HepG-2黏度细胞成像图
图11BP-Ⅲ线粒体定位成像
图12BP-Ⅲ在正常肝和脂肪肝中的成像图
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。
实施例1BP-Ⅲ的制备和表征:
BP-Ⅲ的合成路线:
BP-Ⅲ的合成方法,步骤为:
1)在250mL的圆底烧瓶中,将反应温度控制在0℃,将6mL环己酮滴加到80mL浓硫酸中,将溶液在0℃下搅拌直至结束,然后加入5.89g 2-(4-二乙氨基-2-羟基苯甲酰基)苯甲酸,将混合溶液在90℃下进一步加热回流2小时,冷却至室温后,将混合物缓慢加入到500mL冰水中,充分搅拌,然后加入7.5mL HClO4,搅拌至由红色固体析出,将所得悬浮液进行抽滤,并用水洗涤,真空干燥,得到化合物1,无需进一步纯化,即可用于下一步。
2)在100mL的圆底烧瓶中,将1.0665g化合物1溶于35ml冰乙酸中,然后加入0.804g4-(1-吡咯烷)苯甲醛,将混合物在110℃下加热回流2小时,反应完成后,自然冷却,减压除去溶剂,得到粗产物,将粗产物真空干燥后通过柱色谱纯化(二氯甲烷和甲醇的体积比为20:1),得到蓝黑色粉末即BP-Ⅲ。(600MHz,DMSO)δ8.19(d,J=7.4Hz,2H),7.85(t,J=7.5Hz,1H),7.75(t,J=7.5Hz,1H),7.68(d,J=7.4Hz,2H),7.37(d,J=7.1Hz,1H),7.28(s,1H),7.15(s,1H),6.87(d,J=9.1Hz,1H),6.73(d,J=8.0Hz,2H),3.62(d,J=6.2Hz,4H),3.34(s,6H),2.92(s,2H),2.00(s,4H),1.75(d,J=52.9Hz,2H),1.22(s,6H).(见图1)。13CNMR(151MHz,DMSO)δ166.91(s),157.73(s),154.70(s),152.40(s),147.11(s),141.30(s),133.51(s),131.34(s),131.03(s),130.52(s),130.26(s),129.53(d,J=12.7Hz),124.47(s),124.13(s),120.59(s),117.13–117.04(m),116.24(s),112.62(s),95.94(s),45.63(s),26.36(s),25.06(s),20.94(s),12.97(s).(见图2)。
实施例2
配制2.0mL不同的有机溶剂(N,N-二甲基甲酰胺、二甲基亚砜、二氯甲烷、PBS、甲醇、水、四氢呋喃、乙醇、乙腈、甘油)。然后分别与10μL浓度为2.0mmol/L的探针BP-Ⅲ溶液混合于比色皿中,并且充分混合均匀后,测试它们的荧光强度。在650nm的激发下,探针BP-Ⅲ在甘油中的荧光强度远远大于在其他溶剂中的荧光强度(见图3)。
实施例3
配制2.0mL甘油和PBS(甘油体积含量比例为0%、10%、20%、30%、40%、50%、60%、70%、80%、90%、100%)不同黏度溶液,充分混合均匀,并且超声半小时,除去溶液中的气泡。然后分别与10μL浓度为2.0mmol/L的探针BP-Ⅲ溶液混合于比色皿中,并且充分混合均匀后,测试它们的荧光强度。随着黏度的增加(甘油体积比增加),探针BP-Ⅲ在740nm处的荧光逐渐增强(Ex=650nm)(见图4)。
实施例4
配制2.0mL甘油和PBS(甘油体积含量比例为0%、10%、20%、30%、40%、50%、60%、70%、80%、90%、100%)不同黏度溶液,充分混合均匀,并且超声半小时,除去溶液中的气泡。然后分别与10μL浓度为2.0mmol/L的探针BP-Ⅲ溶液混合于比色皿中,并且充分混合均匀后,同时在荧光光谱仪上测定740nm处荧光强度为38.72、53.59、80.37、93.02、132.8、188.8、289.1、475、773.6、1224、2078,以黏度为横坐标,以荧光强度F为纵坐标绘制图,得到BP-Ⅲ的荧光强度与黏度的工作曲线;线性回归方程为:y740=0.5614c+1.4711,c的单位为10-6mol/L(见图5)。
实施例5
1)配置pH=2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0的PBS缓冲溶液,分别向9个比色皿中加入10μL BP-Ⅲ的DMSO溶液,混合均匀后分别记录各pH环境下探针BP-Ⅲ在740nm处的荧光强度。
2)分别向9个比色皿中加入1000μL不同pH的PBS缓冲溶液,再向其加入1000μL甘油和10μL BP-Ⅲ的DMSO溶液,混合均匀后分别记录各pH环境下探针BP-Ⅲ在740nm处的荧光强度。结果显示,探针BP-Ⅲ在pH=4.0~10.0范围内荧光强度无明显变化(见图6)。
实施例6
1)配置pH=7.4、浓度为10mM的PBS缓冲溶液,分别向每个比色皿中加入2.0mL PBS溶液和10μL BP-Ⅲ的DMSO溶液,然后再加入100当量的其它分析物:1.NaI、2.Ca2+、3.MgCl、4.NaBr、5.KI、6.Na2SO4、7.NO2-、8.H2O2、9.H2S、10.HClO、11.SO2、12.L-赖氨酸、13.L-脯氨酸、14.L-谷氨酸、15.硫醇半胱氨酸、16.谷胱甘肽、17.同型半胱氨酸、18.PBS,混合均匀后分别记录加入不同分析物后探针BP-Ⅲ在740nm处的荧光强度。
2)配置甘油/PBS体积比为1:1的缓冲溶液,分别在每个比色皿中加入2mL的混合缓冲溶液和10μL BP-Ⅲ的DMSO溶液,然后再加入100当量的其它分析物:1.NaI、2.Ca2+、3.MgCl、4.NaBr、5.KI、6.Na2SO4、7.NO2-、8.H2O2、9.H2S、10.HClO、11.SO2、12.L-赖氨酸、13.L-脯氨酸、14.L-谷氨酸、15.硫醇半胱氨酸、16.谷胱甘肽、17.同型半胱氨酸、18.甘油:PBS(1:1),混合均匀后分别记录加入不同分析物后探针BP-Ⅲ在740nm处的荧光强度。结果显示,各种分析物基本上没有引起检测体系中荧光强度的变化(见图7)。
实施例7
配制2mL pH=7.4、浓度为10mM的PBS缓冲溶液和2mL 100%的甘油溶液于比色皿中,配制2mMBP-Ⅲ的DMSO溶液,分别在PBS缓冲溶液的比色皿和甘油的比色皿中加入10μLBP-Ⅲ的DMSO溶液,混合均匀后分别记录两个比色皿中探针BP-Ⅲ在740nm处0-60min内的荧光强度变化。结果显示,60min内BP-Ⅲ在740nm处的荧光强度变化不大(见图8)。
实施例8
先将HL-7702细胞中加入2mL的PBS,再将探针BP-Ⅲ溶液加入HL-7702细胞中,使得其浓度为10μM,将HL-7702细胞在37℃下放置20min后,用PBS冲洗细胞三次,再在荧光共聚焦显微镜下进行成像拍照,探针呈现红色荧光。接着,将孵过探针的细胞加入10uM的制霉菌素,并在荧光共聚焦显微镜下观察20min内红色通道的荧光强度变化(见图9)。在荧光成像仪下显示红色通道荧光明显增强。
实施例9
先将HepG-2细胞中加入2mL的PBS,再将探针溶液加入HepG-2细胞中,使得其浓度为10μM,将HepG-2细胞在37℃下放置20min后,用PBS冲洗细胞三次,再在荧光共聚焦显微镜下进行成像拍照,探针呈现红色荧光。将孵过探针的细胞加入10uM的制霉菌素,并在荧光共聚焦显微镜下观察20min内红色通道的荧光强度变化(见图10)。在荧光成像仪下显示红色通道荧光明显增强。
实施例10
将HL-7702细胞中加入500uM Mito-Tracker Green,在37℃的恒温箱放置20min后,用PBS冲洗三次,再将探针DMSO溶液加入HL-7702细胞中,使得其浓度为10μM,放置20min后,用PBS冲洗细胞三次,再在荧光共聚焦显微镜下进行成像拍照(见图11)。红色通道为探针,绿色通道为Mito-Tracker Green,结果表明探针对线粒体具有良好的靶向性(皮尔逊系数0.91),说明探针能够特异性染色活细胞中的线粒体。
实施例11
首先,通过给小鼠喂食高脂食品六周,建立脂肪肝小鼠模型。然后,将小鼠的肝脏器官分离出来进行实验。肉眼观察,正常组肝脏呈均匀的暗红色,边缘锐利。反之,模型组肝的颜色呈黄色,肝表面油腻,说明脂肪肝模型建立成功。随后,将分离的器官在探针DMSO溶液(浓度为50μM)中孵育5分钟,用PBS冲洗三次后进行成像拍照,模型组脂肪肝小鼠肝脏比正常小鼠肝脏呈现出更亮的荧光(见图12),这些结果表明,BP-Ⅲ可用于正常肝和脂肪肝的区别可视化。
上述实验结果说明BP-Ⅲ是单通道实时原位监测线粒体黏度变化和区别脂肪肝的良好的候选者。
Claims (7)
1.一种苯并吡喃衍生物BP-Ⅲ,其特征在于,结构式为:
2.如权利要求1所述的一种苯并吡喃衍生物BP-Ⅲ的合成方法,其特征在于,包括如下步骤:
1)在反应容器中,将反应温度控制在0℃,将环己酮滴加到浓硫酸中,将溶液在0℃下搅拌至环己酮滴加结束,然后加入2-(4-二乙氨基-2-羟基苯甲酰基)苯甲酸,将混合溶液在90~100℃下进一步加热回流2~4小时,冷却至室温后,将混合物缓慢加入到冰水中,充分搅拌,然后加入HClO4,搅拌至由红色固体析出,将所得悬浮液进行抽滤,并用水洗涤,真空干燥,得到化合物1;其中:环己酮、2-(4-二乙氨基-2-羟基苯甲酰基)苯甲酸、浓H2SO4和HClO4的摩尔比为2~4:1:20:3~4;
2)在反应容器中,将化合物1溶于冰乙酸中,然后加入4-(1-吡咯烷)苯甲醛,将混合物加热回流2~4小时,反应完成后,自然冷却,减压除去溶剂,得到粗产物,将粗产物进行真空干燥,柱色谱分离粗产物,用V二氯甲烷:V甲醇=20:1洗脱,得到蓝黑色粉末即BP-Ⅲ;其中:化合物1和4-(1-吡咯烷)苯甲醛的摩尔比为1:0.5~10。
3.如权利要求2所述的一种苯并吡喃衍生物BP-Ⅲ的合成方法,其特征在于,所述步骤1)中环己酮、2-(4-二乙氨基-2-羟基苯甲酰基)苯甲酸、浓H2SO4和HClO4的摩尔比为3:1:20:3~4。
4.如权利要求2所述的一种苯并吡喃衍生物BP-Ⅲ的合成方法,其特征在于,所述步骤2)中化合物1和4-(1-吡咯烷)苯甲醛的摩尔比为1:2。
5.如权利要求1所述的苯并吡喃衍生物BP-Ⅲ在制备单通道检测黏度的荧光探针中的应用。
6.如权利要求1所述的苯并吡喃衍生物BP-Ⅲ在制备细胞成像试剂中的应用。
7.如权利要求1所述的苯并吡喃衍生物BP-Ⅲ在制备区别脂肪肝探针中的应用。
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