CN109293698B - 一种基于苯并噻唑的线粒体pH荧光探针及其制备方法 - Google Patents
一种基于苯并噻唑的线粒体pH荧光探针及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种基于苯并噻唑的线粒体pH荧光探针及其制备方法和应用。探针具体名称为2‑(2‑(6‑羟基萘‑2‑基)乙烯基)‑3‑(6‑(三苯基膦酰基)己基)苯并噻唑‑3‑溴化物(HTBT2)。其制备方法：先将2‑甲基苯并噻唑与1,6‑二溴己烷在加热条件下反应制备3‑(6‑溴己基)‑2‑甲基苯并噻唑‑3‑溴化物(BMBI)，然后将制备的BMBI与三苯基膦和乙腈混合回流制备2‑甲基‑3‑(6‑(三苯基磷基)己基)苯并噻唑‑3‑溴化物(MTBI)，最后将MTBI和6‑羟基‑2‑萘甲醛溶于乙醇并加入少量哌啶回流后分离提纯得HTBT2。该探针pKa值为8.04±0.02，与线粒体基质(pH～8.0)非常接近。同时，该探针兼具对pH变化的高度灵敏性、良好的选择性和大的Stokes位移等优点。其可用于细胞线粒体内pH变化的监测。
Description
技术领域
本发明涉及线粒体pH荧光探针，具体为一种基于苯并噻唑的线粒体pH荧光探针及其制备方法，及其在监测活细胞线粒体中pH变化方面的应用。
背景技术
线粒体是真核细胞中一种非常重要的细胞器，与细胞分裂和凋亡，信息传递，离子调节等生理过程息息相关，尤其是细胞代谢所需的大部分能量来源于线粒体的氧化还原过程，因而线粒体也被称为细胞的“能量工厂”。正常生理条件下，线粒体基质内pH(pHmito)值约为8.0。细胞的许多生理活动，如细胞信息传递，Na+/K+/Ca2+平衡，以及活性氧的产生离不开线粒体pHmito平衡，更重要的是，研究表明线粒体自噬和凋亡过程伴随着线粒体酸化。此外，pHmito异常与人类的一些疾病和癌症密切相关，如心血管疾病、神经退行性疾病和雷伊综合征等。因此对线粒体内pHmito进行灵敏准确的实时监测具有非常重要的意义。
近年来，文献报道了一些有机小分子荧光探针用于pHmito变化的检测，但是这些探针大多数的pKa与线粒体pH(～8.0)相差较远，因而灵敏度较低。同时，这些探针大多数的发射位于可见区(<600nm)，样品本身在这一波段具有背景吸收。此外，Stokes位移较小，容易受到激发光源的干扰。因此，有必要开发一种pKa与线粒体pHmito接近的远可见-近红外pH荧光探针用于线粒体的pH变化的监测。
发明内容
本发明的目的之一是提供一种基于苯并噻唑的线粒体pH荧光探针及其制备方法；目的之二是提供该探针的用途，即在监测活细胞线粒体中pH变化方面的应用。
本发明提供的一种基于苯并噻唑的线粒体pH荧光探针，其结构式为：
其合成路线为：
本发明提供的一种基于苯并噻唑的线粒体pH荧光探针的制备方法，包括如下步骤：
(1)将2-甲基苯并噻唑和1,6-二溴己烷按摩尔比1:1.5-10混合后，140℃下于封管内反应5h；待反应冷却至室温后在CH2Cl2中用乙醚析出得到灰色固体产物3-(6-溴己基)-2-甲基苯并噻唑-3-溴化物(BMBI)；
(2)将BMBI和三苯基膦按摩尔比2:3-5溶于少量CH3CN中；将混合溶液在N2保护下回流24h；待溶液冷却后，过滤，将固体溶于CH2Cl2并用水萃取，除去水后得灰色固体产物2-甲基-3-(6-(三苯基磷基)己基)苯并噻唑-3-溴化物(MTBI)；
(3)将6-羟基-2-萘甲醛、MTBI和哌啶按摩尔比12-15:10:1溶于少量乙醇中并回流24h；除去溶剂得到2-(2-(6-羟基萘-2-基)乙烯基)-3-(6-(三苯基膦酰基)己基)苯并噻唑-3-溴化物(HTBT2)的粗品；将HTBT2粗品浓缩后，经硅胶柱分离得到纯品。
步骤(1)中所述的2-甲基苯并噻唑和1,6-二溴己烷的摩尔比优选为1:5。
步骤(2)中所述的BMBI和三苯基膦的摩尔比优选为2:3。
步骤(3)中所述的6-羟基-2-萘甲醛、MTBI和哌啶的摩尔比优选为15:10:1。
本发明的探针HTBT2具有优异的线粒体靶向定位能力，可用于线粒体内pH变化的监测。
与现有的线粒体pH荧光探针相比，本发明合成的探针HTBT2具有以下优点：(1)探针HTBT2的pKa为8.04±0.02，pH线性响应范围为7.20-8.70，使其在监测线粒体pH(～8.0)波动时具有较高的灵敏度；(2)探针的最大荧光发射位于612nm附近，位于远可见-近红外区，能够有效减小对细胞的光损伤和降低生物样品自发荧光的干扰；(3)探针HTBT2具有极大的Stokes位移(～176nm)，能有效降低来自激发光的干扰；(4)探针HTBT2对pH响应具有良好的选择性，不受常见的阴、阳离子，氨基酸活性氧(ROS)和活性氮(RNS)等物质的干扰；(5)该探针具有优异的线粒体靶向定位能力，可利用激光共聚焦成像技术实现对线粒体pH的实时监测；(6)该探针合成步骤简单，成本低廉，具有潜在的商品化应用价值。
附图说明
图1.本发明探针HTBT2随pH值变化的紫外吸收光谱图。
图2.本发明探针HTBT2在自然光下识别OH-前后颜色变化，颜色由浅黄色为黄绿色。
图3.本发明探针HTBT2随pH值变化的荧光发射光谱图。
图4.本发明探针HTBT2在紫外灯下识别OH-前后颜色变化，颜色由无色变为橙红色。
图5.本发明探针HTBT2在612nm处的荧光强度随pH值变化的Boltzmann函数关系，pKa为8.04±0.02。
图6.本发明探针HTBT2的荧光强度随pH值变化线性范围为pH 7.20-8.70。
图7.本发明探针HTBT2在常见阴阳离子和生物体内一些常见氨基酸、活性氧、活性氮等物质存在下，对OH-的选择性。
图8.本发明探针HTBT2在人肝癌细胞(SMMC 7721)中与市售线粒体特异选择性染料MitoTracker Green的共定位成像图。
图9.本发明探针HTBT2分别在pH 7.00，pH 7.40，pH 8.00，pH 8.50和pH 9.00时，与SMMC 7721细胞共同孵育40min的激光共聚焦成像图。
图10.本发明探针HTBT2与SMMC7721细胞共同孵育30min后加入NH4Cl处理，分别在0min、5min、15min、20min、25min和30min时的激光共聚焦成像。
图11.本发明探针HTBT2与SMMC7721细胞共同孵育40min后，分别再加入H2O2和NAC(N-乙酰半胱氨酸)孵育1h后的激光共聚焦成像。
具体实施方式
实施例1
1，化合物3-(6-溴己基)-2-甲基苯并噻唑-3-溴化物(BMBI)、化合物2-甲基-3-(6-(三苯基磷基)己基)苯并噻唑-3-溴化物(MTBI)和探针2-(2-(6-羟基萘-2-基)乙烯基)-3-(6-(三苯基膦酰基)己基)苯并噻唑-3-溴化物(HTBT2)的制备：
(1)将2-甲基苯并噻唑(15mmol，1.90mL)，1,6-二溴己烷(75mmol，12.11mL)的混合溶液在，140℃下于封管内反应5h。待反应冷却至室温后在CH2Cl2中用乙醚析出得到灰色固体产物(5.43g，92％)。1H NMR(400MHz,CDCl3)δ8.32(d,J＝8.1Hz,1H),8.06(d,J＝8.4Hz,1H),7.81(t,J＝7.7Hz,1H),7.69(t,J＝7.7Hz,1H),5.09–4.75(m,2H),3.49(s,3H),3.40(t,J＝6.5Hz,2H),2.25–1.75(m,8H).13C NMR(101MHz,CDCl3)δ175.69,140.93,130.00,129.23,128.67,124.72,116.57,51.00,33.77,32.28,28.67,27.68,25.98,19.30.MS(ESI-MS):m/z Calcd 313.0402[M]+；found 312.0416,314.0387[M]+；
(2)将BMBI(10mmol，3.97g)和三苯基膦(15mmol，3.93g)和150mL CH3CN的混合溶液在N2保护下回流24h。待溶液冷却后，过滤，将固体溶于CH2Cl2并用水萃取，除去水后得灰色固体产物(5.96g，91％)，未经进一步纯化直接用于下一步反应。1H NMR(400MHz,CDCl3)δ8.54(d,J＝8.6Hz,1H),8.19(d,J＝8.0Hz,1H),8.03–7.54(m,17H),5.20–5.01(m,2H),3.78(t,J＝14.4Hz,2H),3.53(s,3H),2.04(d,J＝19.0Hz,2H),1.87(s,4H),1.73(s,2H).13CNMR(101MHz,CDCl3)δ175.31(s),141.10(s),135.05(d,4J(C,P)＝3.0Hz,ArC),133.75(d,J＝10.0Hz,2J(C,P),ArC),130.55(d,3J(C,P)＝12.5Hz,ArC),130.34(s),128.78(s),128.70(s),123.86(s),118.31(d,1J(C,P)＝71.2Hz,ArC),117.81(s),50.83(s),29.11(d,3J(C,P)＝16.8Hz,PCH2CH2CH2),28.35(s),25.13(s),22.46(d,1J(C,P)＝50.4Hz,PCH2),21.93(d,2J(C,P)＝4.4Hz,PCH2 CH2),19.34(s).31P NMR(162MHz,CDCl3)δ24.45.MS(ESI-MS):m/z Calcd247.6069[M]2+；found 247.6070[M]2+
(3)将6-羟基-2-萘甲醛(1.29g,7.5mmol)，MTBI(3.28g,5mmol)和哌啶(0.5mmol)的混合物在30mL乙醇溶液中回流24h。然后将除去溶剂后的初产物并通过柱色谱纯化，含有10％的甲醇的二氯甲烷为洗脱剂得到黄色固体产物(0.93g,23％)。1H NMR(400MHz,DMSO-d6)δ10.02(s,1H),8.39(d,J＝22.8Hz,2H),7.95–7.69(m,3H),7.69–7.49(m,24H),7.24(ddd,J＝84.1,56.2,31.5Hz,1H),3.14(d,J＝14.7Hz,1H),3.06–2.93(m,3H),1.63(dt,J＝11.2,5.6Hz,3H),1.53(dt,J＝10.7,5.4Hz,2H),1.42–0.96(m,1H).31P NMR(162MHz,CDCl3)δ24.45.MS(ESI-MS):m/z Calcd 324.6279[M]2+；found 324.6273[M]2+
实施例2
将实施例1中的探针HTBT2浓度保持在200μM，在不同pH的Tris–盐酸缓冲液(VDMSO:VH2O＝2:1,0.05M)体系中测量其吸收光谱(图1)。随着pH值的从9.30降低至6.40，566nm处的吸收峰逐渐降低，436nm处的吸收峰相应增强，并且在479nm处存在一个等吸收点。溶液的颜色也由原来的黄色变为了黄绿色(图2)。
实施例3
将实施例1中的探针浓度保持在10μM，在不同pH的Tris–盐酸缓冲液(VDMSO:VH2O＝2:1,0.05M)体系中测量其荧光发射光谱，固定激发波长为436nm(图3)。随着pH值由9.30降低到6.40，溶液在612nm处新的荧光发射峰并逐渐增强。紫外灯下溶液的颜色由无色变为橙红色(图4)。通过HTBT2在612nm处的荧光强度值与pH值的Boltzmann函数拟合，计算pKa值为8.04±0.02(图5)，pH响应线性范围为7.20-8.70。线性回归方程为F＝764314.81192-79080.76184×pH,相关系数R2＝0.9991(图6)。
实施例4
将实施例1中的探针浓度保持在10μM，分别考察该探针和常见的阴、阳离子以及生命体中一些氨基酸、ROS和RNS等物质的响应情况。如图7所示，该探针对上述物质几乎没有响应，证明本探针对OH-具优异的选择性。图7中物质的顺序和浓度依次为：1,probe；2,F-(1mM)；3,Cl-(10mM)；4,Br-(1mM)；5,I-(1mM)；6,SO4 2-(1mM)；7,S2O3 2-(1mM)；8,SO3 2-(1mM)；9,HS-(1mM)；10,NO3 -(1mM)；11,NO2 -(1mM)；12,Ac-(1mM)；13,HCO3 -(1mM)；14,ClO4 -(1mM)；15,K+(140mM)；16,Cd2+(1mM)；17,Mg2+(1mM)；18,Li+(1mM)；19,Co2+(1mM)；20,Hg2+(1mM)；21,Ba2+(1mM)；22,Ni2+(1mM)；23,H2O2(1mM)；24,O2 -(1mM)；25,HClO(1mM)；26,ONOO-(100μM)；27,L-GSH(1mM)；28,Hcy(1mM)；29,Cys(1mM)
实施例5
为了确认实施例1中的探针HTBT2是否具有线粒体靶向定位能力，我们首先将探针HTBT2与市售线粒体特异选择性染色MitoTracker Green FM进行共定位实验。在pH 7.40的条件下，将贴壁的SMMC 7721细胞与探针HTBT2(终浓度40μM)在37℃，5％CO2的孵育箱中共同孵育40min，然后用磷酸盐缓冲液(pH 7.40)轻轻洗涤3次，移除多余的探针，再加入MitoTracker Green FM(终浓度2μM)继续孵育5min后，在激光共聚焦显微镜下观察二者的共定位情况。其中，HTBT2固定激发波长为458nm，收集绿色发射范围560-660nm；MitoTracker Green FM固定激发波长为488nm，收集绿色发射范围505-540nm。由图8c可知，HTBT2的荧光(红色)分布于细胞质区域，说明探针具有良好的细胞膜通透性。此外，HTBT2的红色荧光与MitoTracker Green FM的绿色荧光(图8a)能够很好地重叠，经软件处理得到黄色荧光(图8d)，表明HTBT2与MitoTracker Green FM具有显著的共定位成像，能够靶向定位于线粒体中。明场成像进一步证实了经HTBT2孵育后细胞的存活性(图8c)，说明HTBT2对细胞具有低毒性。
实施例6
将贴壁的SMMC 7721细胞与实施例1中的探针HTBT2在pH 7.40的条件下，于37℃、5％CO2的孵育箱中共同孵育40min，然后用磷酸盐缓冲液(pH 7.40)轻轻洗涤3次，除去多余的HTBT2，再分别利用pH 7.00、7.40、8.00、8.50和9.00的高K+缓冲液(30mM NaCl、120mmKCl、1mM CaCl2、0.5mM MgSO4、1mM NaH2PO4、5mM葡萄糖、20mM HEPES和20mM NaOAC)和H+/K+离子载体--尼日尼亚菌素继续处理10min，在激光共聚焦显微镜下观察。固定激发波长为458nm，收集红色荧光发射范围560-660nm。pH 9.00时红色通道几乎观察不到荧光(图9o)；随着pH降至7.00时，红色通道逐渐观察到明亮的荧光(图9l,i,f,c)。明场成像进一步证实了经HTBT2孵育后细胞的存活性(图9a,d,g,j,m)。
实施例7
为了证明实施案例1中的探针HTBT2具有对细胞内线粒体pH变化的快速响应能力，我们分别使用NH4Cl(5mM)、H2O2(0.1mM)和NAC(0.5mM)处理被HTBT2染色的SMMC 7721细胞。用NH4Cl处理后的HTBT2细胞荧光逐渐减弱，说明NH4Cl处理后的SMMC7721细胞的线粒体pH逐渐升高(图10)。如图11所示，经H2O2处理后的SMMC7721细胞相较于未处理的细胞pH明显降低，而经NAC处理的细胞pH明显增加。这些结果表明HTBT2能对细胞内线粒体pH的变化做出快速响应。
Claims (6)
1.一种基于苯并噻唑的线粒体pH荧光探针，其特征在于结构式为：
2.如权利要求1所述的pH荧光探针的制备方法，其特征在于包括如下步骤：
（1） 将2-甲基苯并噻唑和1,6-二溴己烷按摩尔比1 : 1.5-10混合后，140℃下于封管内反应5 h；待反应冷却至室温后在CH2Cl2中用乙醚析出得到灰色固体产物3-（6-溴己基）-2-甲基苯并噻唑-3-溴化物BMBI；
（2）将BMBI和三苯基膦按摩尔比2 : 3-5溶于少量CH3CN中；将混合溶液在N2保护下回流24h；待溶液冷却后，过滤，将固体溶于CH2Cl2并用水萃取，除去水后得灰色固体产物2-甲基-3-（6-（三苯基磷基）己基）苯并噻唑-3-溴化物MTBI；
（3）将6-羟基-2-萘甲醛、MTBI和哌啶按摩尔比12-15 : 10 : 1溶于少量乙醇中并回流24 h；除去溶剂得到2-（2-（6-羟基萘-2-基）乙烯基）-3-（6-（三苯基膦酰基）己基）苯并噻唑-3-溴化物HTBT2的粗品；将HTBT2粗品浓缩后，经硅胶柱分离得到纯品。
3.如权利要求2所述的pH荧光探针的制备方法，其特征在于步骤（1）中所述的2-甲基苯并噻唑和1,6-二溴己烷的摩尔比为1 : 5。
4.如权利要求2所述的pH荧光探针的制备方法，其特征在于步骤（2）中所述的BMBI和三苯基膦的摩尔比为2 : 3。
5.如权利要求2所述的pH荧光探针的制备方法，其特征在于步骤（3）中所述的6-羟基-2-萘甲醛、MTBI和哌啶的摩尔比为15 : 10 : 1。
6.如权利要求1所述的pH荧光探针在制备用于细胞线粒体内pH变化的监测试剂中的应用。
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