CN109293698A - 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。
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荧光探针用于细胞线粒体内pH变化的监测。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113234040A (zh) * | 2021-05-28 | 2021-08-10 | 中国科学院新疆理化技术研究所 | 一种用于检测pH荧光探针分子及其制备方法 |
CN114133413A (zh) * | 2021-11-04 | 2022-03-04 | 广东工业大学 | 一种苯并噻唑-三苯胺化合物及其制备方法与应用 |
WO2023045909A1 (zh) * | 2021-09-22 | 2023-03-30 | 杭州天玑济世生物科技有限公司 | 一类具有萘胺结构的小分子化合物及其应用 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1282334A (zh) * | 1997-11-25 | 2001-01-31 | 奥塔戈大学 | 线粒体靶向的抗氧剂 |
CN104531139A (zh) * | 2015-01-06 | 2015-04-22 | 山西大学 | 一种咔唑类的pH荧光探针及其制备方法和应用 |
CN106565596A (zh) * | 2016-10-28 | 2017-04-19 | 山西大学 | 一种萘基衍生物作为线粒体靶向型pH荧光探针的应用 |
CN106632138A (zh) * | 2016-09-23 | 2017-05-10 | 济南大学 | 一种识别肼的小分子荧光探针及其应用 |
CN108485653A (zh) * | 2018-05-10 | 2018-09-04 | 河南科技大学 | 同时检测过氧化氢和过氧化亚硝基阴离子的近红外荧光探针及其合成方法和应用 |
-
2018
- 2018-10-15 CN CN201811194600.0A patent/CN109293698B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1282334A (zh) * | 1997-11-25 | 2001-01-31 | 奥塔戈大学 | 线粒体靶向的抗氧剂 |
CN104531139A (zh) * | 2015-01-06 | 2015-04-22 | 山西大学 | 一种咔唑类的pH荧光探针及其制备方法和应用 |
CN106632138A (zh) * | 2016-09-23 | 2017-05-10 | 济南大学 | 一种识别肼的小分子荧光探针及其应用 |
CN106565596A (zh) * | 2016-10-28 | 2017-04-19 | 山西大学 | 一种萘基衍生物作为线粒体靶向型pH荧光探针的应用 |
CN108485653A (zh) * | 2018-05-10 | 2018-09-04 | 河南科技大学 | 同时检测过氧化氢和过氧化亚硝基阴离子的近红外荧光探针及其合成方法和应用 |
Non-Patent Citations (3)
Title |
---|
BO LIN等: "A naphthalene-based fluorescent probe with a large Stokes shift for mitochondrial pH imaging", 《ANALYST》 * |
HAO WANG等: "A dual-site fluorescent probe for separate detection of hydrogen sulfide and bisulfide", 《DYES AND PIGMENTS》 * |
XIUQIONG CHEN等: "A ratiometric fluorescent probe for palladium detection based on an allyl carbonate group functionalized hemicyanine dye", 《TETRAHEDRON LETTERS》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113234040A (zh) * | 2021-05-28 | 2021-08-10 | 中国科学院新疆理化技术研究所 | 一种用于检测pH荧光探针分子及其制备方法 |
CN113234040B (zh) * | 2021-05-28 | 2022-03-25 | 中国科学院新疆理化技术研究所 | 一种用于检测pH荧光探针分子及其制备方法 |
WO2023045909A1 (zh) * | 2021-09-22 | 2023-03-30 | 杭州天玑济世生物科技有限公司 | 一类具有萘胺结构的小分子化合物及其应用 |
CN114133413A (zh) * | 2021-11-04 | 2022-03-04 | 广东工业大学 | 一种苯并噻唑-三苯胺化合物及其制备方法与应用 |
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