CN110183482A - 一种监测溶酶体pH的近红外荧光探针及其制备方法和应用 - Google Patents
一种监测溶酶体pH的近红外荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本专利公开了一种监测溶酶体pH的近红外荧光探针及其制备方法和应用,属于分析化学技术领域。本发明的技术方案要点为:一种监测溶酶体pH的近红外荧光探针,其结构式如下:本发明还具体公开了该监测溶酶体pH的近红外荧光探针的制备方法及其在选择性检测水环境、生物细胞体系中pH的应用。本发明的近红外荧光探针具有近红外区的发射、选择性好、光稳定性好、可逆性好、及优异的溶酶体靶向能力等优点。
Description
技术领域
本发明涉及一种监测溶酶体pH的近红外荧光探针及其制备方法和应用,属于分析化学技术领域。
背景技术
溶酶体作为真核细胞中重要的酸性(pH约为3.8-5.5)细胞器,是蛋白质、核酸、多糖等生物大分子的分解场所,被称为细胞中“消化器官”。溶酶体在许多生命进程中起着重要的作用,如内吞、细胞生长和凋亡、自噬、离子代谢、氧化应激等。溶酶体pH的异常波动会引起溶酶体功能紊乱,与溶酶体储存疾病、癌症等疾病密切相关。因此,开发能够实时监测溶酶体pH的分析技术具有重要意义。
荧光分子探针因其操作简单、响应速度快、选择性高、灵敏度高、时空分辨率高等优点,已成为生物成像领域不可缺少的工具。近年来,科研工作者相继开发了许多用于溶酶体pH的监测的荧光分子探针。但是这些探针中的大多数探针的激发和发射波长较短(λexλem),在生物成像时存在一定的局限性,存在对生物样品的光损伤大、组织穿透深度较小、背景荧光干扰大等问题。而近红外荧光探针(near-infrared fluorescent probe)凭借其对生物样品的光损伤小、组织穿透深度较大、背景荧光干扰小等优点,成为荧光分子探针领域的研究热点。基于此,科研工作者先后开发了一些基于传统近红外菁染料(cyanine dyes)的pH荧光探针。但是上述基于近红外菁染料设计的pH近红外探针的光稳定性较差,难以在长时间、多次成像中获得准确稳定的荧光信号。而硅罗丹明染料是近年来新报道的一类具有优异光稳定性的近红外染料,其光稳定性要远好于菁染料,被广泛应用于各种分析检测物的近红外荧光探针的设计。此外,硅罗丹明与普通罗丹明染料类似,易于螺环化,方便构建针对不同检测对象的荧光探针。
发明内容
针对目前监测溶酶体pH值的近红外荧光探针所面临的问题和现状,本发明提供了一种监测溶酶体pH的近红外荧光探针,该近红外荧光探针利用吗啉基团作为溶酶体靶向定位基团,用于细胞溶酶体内pH的成像,该探针具有近红外发射、选择性好、光稳定性好、可逆性好、溶酶体靶向能力优异等特点。
本发明还提供了上述监测溶酶体pH的近红外荧光探针的制备方法及其在选择性检测水环境、生物细胞体中pH的应用。
本发明为解决上述技术问题采用如下技术方案,一种监测溶酶体pH的近红外荧光探针,其特征在于该近红外荧光探针的结构式如下:
本发明所述的监测溶酶体pH的近红外荧光探针的制备方法,其特征在于具体步骤为:
步骤S1:于-78℃,在氩气保护下,将6g 3-溴-N,N-二甲基苯胺与60毫升无水四氢呋喃加入至干燥的250毫升圆底烧瓶中,磁力搅拌5分钟使其溶解,随后将13毫升摩尔浓度为2.4mol/L的正丁基锂的正己烷溶液滴加至反应液中,滴加完毕后于0℃反应2小时,再将2.2毫升二氯二甲基硅烷溶于10毫升无水四氢呋喃中,然后滴加至上述反应液中,滴加完毕后反应至室温并搅拌过夜,加50毫升水猝灭反应,并将反应液用***萃取、无水硫酸钠干燥,减压旋干溶剂后得到粗产品,将粗产品用硅胶柱纯化得到化合物1,其结构式如下:
步骤S2:将500mg化合物1、1260mg 2-羧基苯甲醛和37.5mg溴化铜加入到100毫升玻璃厚壁耐压管中,于140℃加热搅拌反应5小时后自然冷却至室温,然后将反应混合物溶于二氯甲烷中,用质量浓度为10%的NaOH溶液洗涤三遍,回收并旋干二氯甲烷相得到粗产品,将粗产品用硅胶柱纯化得到化合物2,其结构式如下:
步骤S3:在100mL圆底烧瓶中加入443mg化合物2、20mL干燥的1,2-二氯乙烷和2mL三氯氧磷,将烧瓶中反应液加热至85℃回流4小时,减压蒸馏除去溶剂得到反应残余物,将反应残余物溶于20mL干燥的乙腈,并加入5mL三乙胺,然后向其中继续滴加含有650mg 4-(2-氨乙基)-吗啉的10mL的乙腈溶液,将反应液在室温下搅拌过夜,在减压条件下除去溶剂后,用50mL CH2Cl2溶解残余物,50mL饱和NaCl水溶液洗涤3次,无水Na2SO4干燥,旋转蒸发仪蒸除溶剂得粗产品,将粗产品用硅胶柱纯化得到化合物3,其结构式如下:
步骤S4:在室温下,将105mg化合物3溶于5mL干燥四氢呋喃中,少量多次加入76mg四氢铝锂,室温下搅拌反应6小时后,加入5mL甲醇猝灭反应,将反应液减压蒸馏除去溶剂后得到残余物,将残余物用硅胶柱纯化得到目标荧光探针化合物Lyso-NIR-pH。
本发明所述的监测溶酶体pH的近红外荧光探针在选择性检测水环境、生物细胞体系中pH的应用,其中检测包括水溶液中荧光检测、细胞成像检测。
本发明与现有技术相比具有以下有益效果:(1)该近红外荧光探针的合成相对比较容易,且后处理过程相对简单;(2)该近红外荧光探针实现了pH高选择性快速检测,具有抵抗生命体内其它物质干扰的能力;(3)该近红外荧光探针荧光探针具有近优异的溶酶体靶向能力,近红外区的发射,优异的光稳定性,能够应用于细胞溶酶体中的成像检测。该近红外荧光探针通过降低生命体内自发荧光背景干扰、降低对生物样品的光损伤、提高光稳定性等特点,以获得更加准确及稳定的光学信号及成像效果。因此,本发明中的近红外荧光探针在pH成像监测领域具有广阔的应用前景,对溶酶体pH在生物体生理和病理过程的作用机制的研究具有重要意义。
附图说明
图1是实施例1制得的荧光探针化合物Lyso-NIR-pH在不同pH条件下的荧光光谱图;
图2是实施例1制得的荧光探针化合物Lyso-NIR-pH在不同pH条件下的紫外可见吸收光谱图;
图3是实施例1制得的荧光探针化合物Lyso-NIR-pH在发射波长为675nm处的的荧光强度随pH变化的关系曲线图;
图4是实施例1制得的荧光探针化合物Lyso-NIR-pH在pH=7.4条件下对各种分析物的响应情况(从左至右):1、H+(pH=5);2、Na+;3、K+;4、Ca2+;5、Mg2+;6、Fe3+;7、Cu2+;8、Zn2+;9、Mn2+;10、Ni2+;11、Cd2+;12、Co2+;13、NH4+;14、Ac-;15、CO3 2-;16、SO4 2-;17、F-;18、Br-;19、I-;20、S2O3 -;21、NO2 -;22、H2PO4-;23、HPO4 2-;24、Glutathione;25、Arginine;26、Valine;27、Tryptophan;28、Cysteine;29、Glycine;30、Homocysteine.
图5是实施例1制得的荧光探针化合物Lyso-NIR-pH的pH可逆响应情况。
图6是实施例1制得的荧光探针化合物Lyso-NIR-pH在HeLa细胞中的溶酶体共定位实验;
图7是实施例1制得的荧光探针化合物Lyso-NIR-pH在细胞内的光稳定性评估实验。
图8是实施例1制得的荧光探针化合物Lyso-NIR-pH在不同pH值(pH=4,5,6)的条件下的荧光成像图。
图9是实施例1制得的荧光探针化合物Lyso-NIR-pH在HeLa细胞中加入药物氯喹前后的荧光成像情况。
图10是实施例1制得的荧光探针化合物Lyso-NIR-pH在药物***诱导的细胞凋亡过程中的荧光成像情况。
具体实施方式
以下通过实施例对本发明的上述内容做进一步详细说明,但不应该将此理解为本发明上述主题的范围仅限于以下的实施例,凡基于本发明上述内容实现的技术均属于本发明的范围。
实施例1
荧光探针化合物Lyso-NIR-pH的合成
(1)化合物1的合成
于-78℃,在氩气保护下,将6g 3-溴-N,N-二甲基苯胺与60毫升无水四氢呋喃加入至干燥的250毫升圆底烧瓶中,磁力搅拌5分钟使其溶解,随后将13毫升摩尔浓度为2.4mol/L的正丁基锂的正己烷溶液滴加至反应液中,滴加完毕后于0℃反应2小时,再将2.2毫升二氯二甲基硅烷溶于10毫升无水四氢呋喃中,然后滴加至上述反应液中,滴加完毕后反应至室温并搅拌过夜,加50毫升水猝灭反应,并将反应液用***萃取(50毫升×2),将萃取的***溶液用饱和NaCl水溶液洗涤(50毫升×3),无水Na2SO4干燥,并用旋转蒸发仪蒸除溶剂得粗产品,将粗产品用硅胶柱纯化,硅胶颗粒大小为200-300目,洗脱剂体积配比为石油醚/乙酸乙酯=80:1,得到化合物1,黄色油状物,3.35g,产率75%,其合成路线如下:
(2)化合物2的合成
将500mg化合物1(1.68mmol)、1260mg 2-羧基苯甲醛(8.4mmol)和37.5mg溴化铜(0.168mmol)加入到100毫升玻璃厚壁耐压管中,封管后放置到油浴锅中于140℃加热搅拌5小时后自然冷却至室温,然后将反应混合物溶解在50毫升二氯甲烷中,用质量浓度为10%的NaOH溶液洗涤(50毫升×3),除去未反应的2-羧基苯甲醛等酸性副产物,将得到的二氯甲烷相用无水Na2SO4干燥,并用旋转蒸发仪蒸除溶剂得粗产品,将粗产品用硅胶柱纯化,硅胶颗粒大小为200-300目,洗脱剂体积配比为石油醚/乙酸乙酯=2:1,得到化合物2,绿色固体,0.33g,产率45%,其合成路线如下:
(3)化合物3的合成
在100mL圆底烧瓶中,先加入443mg化合物2、20mL干燥的1,2-二氯乙烷、2mL三氯氧磷。将烧瓶中反应液加热至85℃回流4小时,减压蒸馏除去溶剂,得到反应残留物。将反应残余物溶于20mL干燥的乙腈,并加入5mL三乙胺,然后向其中继续滴加含有650mg 4-(2-氨乙基)-吗啉的10mL的乙腈溶液。将反应液在室温下搅拌过夜,在减压条件下除去溶剂后,用50mL CH2Cl2溶解残余物,并用饱和NaCl水溶液洗涤(50毫升×3),无水Na2SO4干燥,随后用旋转蒸发仪蒸除溶剂得粗产品。将粗产品用硅胶柱纯化,硅胶颗粒大小为200-300目,洗脱剂配比为二氯甲烷/甲醇=25:1,得到化合物3,灰绿色固体化合物,277.0mg,产率51.3%。其合成路线如下:
(4)化合物4的合成
在室温下,将105mg化合物3溶于5mL干燥四氢呋喃中,少量多次加入总量为76mg四氢铝锂。室温下搅拌反应6小时后,加入5mL甲醇猝灭反应。将反应液减压蒸馏除去溶剂后得到残余物,将残余物用硅胶柱纯化,得到目标荧光探针化合物Lyso-NIR-pH,38.0mg,产率36.2%。其合成路线如下:
实施例2
荧光探针化合物Lyso-NIR-pH在不同pH条件下荧光光谱图的测定
荧光光谱的测量在40mM Britton-Robinson缓冲溶液(含1%DMSO)中测定。将Lyso-NIR-pH荧光探针溶于二甲基亚砜(DMSO)中,制成500μM储备液。测试溶液Lyso-NIR-pH(5.0μM)由上述500μM储备液稀释制得。不同的pH通过加入微量等浓度的HCl或NaOH获得。试验液在室温下保存30min,在620nm激发波长下测定荧光光谱。荧光发射光谱范围为640~800nm,激发缝为3nm,发射缝为3nm。荧光光谱如图1所示,当BR缓冲液的pH值大于7.4时,探针Lyso-NIR-pH几乎是没有荧光的,因为它是稳定的非荧光螺环结构。当pH从7.4下降到3时,在675nm处出现了显著增强的近红外荧光信号,这是由于H+诱导的螺旋环打开,荧光强度增加1400多倍。所用的荧光测定仪器为Perkin Elmer LS55荧光分光光度计。
实施例3
荧光探针化合物Lyso-NIR-pH在不同pH条件下紫外可见吸收光谱图的测定
图2为探针Lyso-NIR-pH(5μM)在pH 7.4-pH 3条件下的紫外可见吸收光谱图。从图2可以看出,随着pH的减小,在655nm处可以观察到一个逐渐增加的吸收峰。这表明探针Lyso-NIR-pH对pH产生了响应,其螺环结构在质子H+作用下被打开,吸收增大。紫外可见吸收光谱测定用的仪器为TU-1900型紫外可见分光光度计(Beijing Purkinje GeneralInstrument Co.,Ltd.)。
实施例4
荧光探针化合物Lyso-NIR-pH的pH滴定曲线
图3为探针Lyso-NIR-pH的pH滴定曲线。从图3可以看出,随着pH的减小(pH 7.4-pH3),探针在675nm处的荧光强度逐渐增强。我们将探针的荧光强度与pH按照Henderson-Hasselbalch equation作图得到图3,并进一步求得探针Lyso-NIR-pH对pH的pKa为4.63。溶酶体的pH一般为3.8-5.5,而探针Lyso-NIR-pH的pKa刚好处于这个范围内,有利于溶酶体pH的荧光监测成像。
实施例5
荧光探针化合物Lyso-NIR-pH的选择性考察
选择性是评估荧光探针性能的一个重要指标。如图4所示,Lyso-NIR-pH(5μM)在pH=7.4时的荧光强度显示,在加入20倍当量常见阳离子(Na+、K+、Ca2+、Mg2+、Fe3+、Cu2+、Zn2+、Al3+、Mn2+、Ni2+、Cd2+、Co2+、NH4 +)后,荧光无明显增加。此外,当分别加入20倍当量常见阴离子后(Ac-、CO3 2-、SO4 2-、F-、I-、S2O3 -、NO2-、Cl-、Br-、H2PO4-、HPO4 2-),Lyso-NIR-pH的荧光强度增加也可忽略不计。200倍当量各种氨基酸(缬氨酸、色氨酸、半胱氨酸、甘氨酸、高半胱氨酸)与谷胱甘肽,对Lyso-NIR-pH的荧光干扰也可以忽略不计。这些结果表明,Lyso-NIR-pH对酸性pH有特异性的荧光响应,其它分析物对探针无明显干扰,可以满足实际生物样品中pH监测的需求。
实施例6
荧光探针化合物Lyso-NIR-pH的可逆性考察
随后,我们考察了Lyso-NIR-pH的可逆性(图5)。从图中可知,探针Lyso-NIR-pH(5μM)所处缓冲体系的pH经过三次循环切换后(pH 7到pH 3.5),其荧光强度值仍能维持在初始值的90%以上,之歌结果表明了探针Lyso-NIR-pH对pH具有较好的可逆性,能够用于pH的多次测定。
实施例7
荧光探针化合物Lyso-NIR-pH的溶酶体共定位实验
为了考察Lyso-NIR-pH探针在细胞中对溶酶体的靶向能力,我们利用探针Lyso-NIR-pH(5μM)与商业化溶酶体染料LysoTracker Green DND-26(500nM)和线粒体染料MitoTracker Green FM(1μM)对比,进行了共定位实验。首先,我们在对HeLa细胞进行了溶酶体共定位实验。如图6a-6d所示,在绿色通道可以观察到有绿色荧光(510-550nm)发出(图6a),此为LysoSensor Green DND-26发射的光,而在红色通道可以观察到有红色荧光(650-720nm)发出(图6b),此为探针Lyso-NIR-pH发出的红光,将两者用软件进行处理可以得出两种染料的共定位系数为0.90(图6c)。同时,探针荧光与商业化溶酶体染料的荧光的线性分布呈现出明显的同步性(图6d)。这说明探针Lyso-NIR-pH主要聚集在溶酶体中,可以定位到溶酶体中。随后,我们进行了探针与商业化线粒体染料的共定位实验(6e-6h)。如图所示,探针与商业化线粒体定位剂荧光无明显重叠(图6g),且线性分布不同步(图6h),由图6g经软件处理得到两者间的共定位系数只有0.43,这从进一步证实Lyso-NIR-pH具有溶酶体靶向性,而非线粒体靶向性。
实施例8
荧光探针化合物Lyso-NIR-pH的光稳定性考察
通过与传统的近红外染料Cy5-N3对比,我们考察了荧光探针化合物Lyso-NIR-pH的光稳定性(图7)。如图所示,经过50次的持续激发成像,荧光探针化合物Lyso-NIR-pH的荧光强度仍然维持在初始值的88%,而传统的近红外染料Cy5-N3的荧光强度则呈现出明显的下降(为初始值的10%)。这个结果表明荧光探针化合物Lyso-NIR-pH具有较好的光稳定性,能够满足体内长时间成像的需求,获得稳定的荧光信号。激发波长为635nm,功率为20%。
实施例9
荧光探针化合物Lyso-NIR-pH对不同pH缓冲溶液作用的细胞溶酶体pH值的成像研究
在HeLa细胞中,考察探针Lyso-NIR-pH对细胞溶酶体pH值变化的荧光成像情况。具体操作步骤如下:用Lyso-NIR-pH(5μM)在PBS缓冲液(pH7.4)中37℃孵育A549细胞和HeLa细胞30min,然后在含10μM尼日利亚菌素(Nigericin,一种H+/K+离子载体,使细胞内和细胞外的pH均一化)的不同pH值(4.0、5.0和6.0)的缓冲液中,在37℃下,孵育30min。然后用共聚焦显微镜进行成像,成像结果如图8所示。从图中可知,荧光信号随着体系pH的升高而逐渐减小。激发波长为635nm,红色通道波长收集范围为650-720nm。
实施例10
荧光探针化合物Lyso-NIR-pH对药物氯喹诱导的溶酶体pH变化的成像情况
荧光探针化合物Lyso-NIR-pH(5μM)被应用于碱性药物氯喹诱导的溶酶体pH变化的监测(图9)。如图所示,当氯喹(100μM)加入后150秒内,细胞的荧光强度明显下降,这个结果说明碱性药物氯喹诱导了溶酶体pH的升高,导致探针的呈螺环结构,荧光显著下降,也表明了探针实时监控溶酶体内pH的能力。
实施例11
荧光探针化合物Lyso-NIR-pH对细胞凋亡诱导的的溶酶体pH变化的成像情况
最后,探针Lyso-NIR-pH(5μM)被用于细胞凋亡过程中溶酶体pH变化的监测(图10)。在细胞凋亡过程中,溶酶体pH会发生质子泄露,导致pH上升。如图所示,当加入2μM***(诱导细胞凋亡的药物)后2小时内,细胞形貌发生了明显的变化,表明细胞确实发生了细胞凋亡进程。与此同时,细胞的荧光也逐渐下降。以上结果表明探针Lyso-NIR-pH成功地实现了细胞凋亡过程中溶酶体pH上升过程的监测。
以上实施例描述了本发明的基本原理、主要特征及优点,本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明原理的范围下,本发明还会有各种变化和改进,这些变化和改进均落入本发明保护的范围内。
Claims (3)
1.一种监测溶酶体pH的近红外荧光探针,其特征在于该近红外荧光探针的结构式如下:
2.一种权利要求1所述的监测溶酶体pH的近红外荧光探针的制备方法,其特征在于具体步骤为:
步骤S1:于-78℃,在氩气保护下,将6g3-溴-N,N-二甲基苯胺与60毫升无水***加入至干燥的250毫升圆底烧瓶中,磁力搅拌5分钟使其溶解,随后将13毫升摩尔浓度为2.4mol/L的正丁基锂的正己烷溶液滴加至反应液中,滴加完毕后于0℃反应2小时,再将2.2毫升二氯二甲基硅烷溶于10毫升无水***中,然后滴加至上述反应液中,滴加完毕后反应至室温并搅拌过夜,加50毫升水猝灭反应,并将反应液用***萃取、无水硫酸钠干燥,减压旋干溶剂后得到粗产品,将粗产品用硅胶柱纯化得到化合物1,其结构式如下:
步骤S2:将500mg化合物1、1260mg2-羧基苯甲醛和37.5mg溴化铜加入到100毫升玻璃厚壁耐压管中,于140℃加热搅拌反应5小时后自然冷却至室温,然后将反应混合物溶于二氯甲烷中,用质量浓度为10%的NaOH溶液洗涤三遍,回收并旋干二氯甲烷相得到粗产品,将粗产品用硅胶柱纯化得到化合物2,其结构式如下:
步骤S3:在100mL圆底烧瓶中,先加入443mg化合物2、20mL干燥的1,2-二氯乙烷和2mL三氯氧磷,将烧瓶中反应液加热至85℃回流4小时,减压蒸馏除去溶剂得到反应残余物,将反应残余物溶于20mL干燥的乙腈,并加入5mL三乙胺,然后向其中继续滴加含有650mg4-(2-氨乙基)-吗啉的10mL的乙腈溶液,将反应液在室温下搅拌过夜,在减压条件下除去溶剂后,用50mL CH2Cl2溶解残余物,50mL饱和NaCl水溶液洗涤3次,无水Na2SO4干燥,旋转蒸发仪蒸除溶剂得粗产品,将粗产品用硅胶柱纯化得到灰绿色固体化合物3,其结构式如下:
步骤S4:在室温下,将105mg化合物3溶于5mL干燥四氢呋喃中,多次加入总量为76mg四氢铝锂,室温下搅拌反应3小时后加入5mL甲醇猝灭反应,将反应液用旋转蒸发仪除去溶剂后得到残余物,将残余物用硅胶柱纯化得到目标荧光探针化合物Lyso-NIR-pH。
3.权利要求1所述的监测溶酶体pH的近红外荧光探针在选择性检测水环境、生物细胞体系中pH的应用,其中检测包括水溶液中荧光检测和细胞成像检测。
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