CN106188151B - 一种基于吡啶盐的离子型磷光铱配合物探针及其制备方法和生物应用 - Google Patents
一种基于吡啶盐的离子型磷光铱配合物探针及其制备方法和生物应用 Download PDFInfo
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Abstract
本发明涉及一种基于吡啶盐的离子型磷光铱配合物探针及其制备方法和生物应用，具体涉及一类用于检测还原谷胱甘肽、半胱氨酸、硫化氢等具有巯基的还原性物质的磷光铱配合物及其检测细胞内氧化还原态应用，属于有机光电功能材料技术领域。该类配合物材料由环金属配体，金属中心(铱)和修饰有吡啶盐基团的辅助配体组成，结构如下式所示。该发明所述的配合物在检测细胞内氧化还原态、磷光寿命成像及时间分辨技术方面有广阔的应用前景。
Description
技术领域
本发明涉及一种基于吡啶盐的离子型磷光铱配合物探针及其制备方法和生物应用，具体涉及一种基于吡啶盐的磷光铱配合物材料的制备方法及其在细胞内氧化还原态检测、磷光寿命成像及时间分辨技术方面的应用，属于有机光电功能材料技术领域。
背景技术
氧化还原态是一个广泛应用于自由基和氧化应激等领域的术语。细胞内的氧化还原环境由细胞内的氧化还原压力决定。在正常的细胞内，细胞内的氧化压和还原压是处于平衡的，氧化压主要由细胞内活性氧分子的含量决定而还原压主要由一些巯基蛋白和还原性的酶和氨基酸决定。细胞内的氧化还原态一旦由于氧化应激而失去平衡，会对蛋白质、DNA和脂类的结构和功能造成不可逆的破坏。因此，细胞内氧化还原态的平衡对维持生命体的正常生理活动至关重要，故开发可用于监测细胞内氧化还原态的探针具有重要意义。
相比荧光探针，基于三线态跃迁发射的磷光铱配合物具有相对较长的发光寿命，使之成为一种非常理想的可用于生物成像标记的发光探针。近年来，以磷光过渡金属铱配合物为荧光成像技术染料分子引起了人们的很大兴趣，这是由于磷光过渡金属铱配合物具有以下几大特点：具有较大的量子效率，较大的Stokes位移和较长的发射寿命。利用其发光寿命长的特点，可通过使用时间分辨技术使磷光信号与生物体内的背景荧光信号区分开，避免了其他干扰，且优越的光稳定性便于长时间观测。因此，开发具有可用于生物体检测的长寿命磷光探针具有重要意义。
发明内容
为解决上述技术问题，本发明的目的在于提供一种基于吡啶盐的离子型磷光铱配合物探针，给出它们的制备方法，并提出这类配合物在在细胞内氧化还原态检测、生物标记与细胞成像领域中的应用。
为了解决上述技术问题，本发明提出的技术方案是：一种基于吡啶盐的离子型磷光铱配合物探针，其辅助配体上含有吡啶盐，该铱配合物具有如下结构：
本发明提出的另一技术方案是：一种基于吡啶盐的离子型磷光铱配合物探针制备方法的合成路线如下：
具体是4,4’-二甲基-2,2’-联吡啶经过氧化、甲氧基化、酯的还原、氢溴酸取代四步反应得到N^N辅助配体，接着与铱二氯桥((C^N)2Ir(μ-Cl)2Ir(C^N)2)通过配位反应制备得到配合物中间体，然后将中间体与吡啶当量比为1:2在乙腈中溶解后加入过量六氟磷酸钾搅拌过夜得到目标配合物。
本发明提出的另一技术方案是：该离子型磷光铱配合物应用于细胞内氧化还原态检测。
优选的，该离子型磷光铱配合物应用于磷光寿命成像和时间分辨技术。
有益效果：
本发明其C^N配体上含有吡啶盐，可用来检测细胞内氧化还原态。
本发明合成的材料用作细胞氧化原态检测磷光探针，在还原型谷胱甘肽半胱氨酸等生物小分子的存在下磷光发射增强并蓝移，检测效果显著。
本探针材料具有低生物毒性，且容易进入细胞胞浆中，长的磷光发射寿命可使用时间分辨技术与背景荧光信号相区分以降低信噪比，使得这类探针可通过寿命成像和时间门技术成像实现细胞内氧化还原态检测，提高检测精度。
附图说明
下面结合附图对本发明的作进一步说明。
图1.为实施例2中配合物Ir1的核磁共振氢谱图；
图2.为实施例2中配合物Ir1的核磁共振碳谱图；
图3.为实施例3中磷光探针Ir1在PBS缓冲溶液中的磷光发射光谱对还原型谷胱甘肽(GSH)、半胱氨酸(Cys)、硫氢化钠的响应性；
图4.为实施例4中磷光探针Ir1在PBS缓冲溶液中的寿命对还原型谷胱甘肽(GSH)、半胱氨酸(Cys)、硫氢化钠的响应性；
图5.为实施例5中磷光探针Ir1应用于活细胞内氧还原态检测的共聚焦和荧光寿命成像照片。
具体实施方式
为了更好地理解本发明专利的内容，下面通过具体的实例来进一步说明本发明的技术方案。但这些实施实例并不限制本发明。
实施例1
配合物中间体[Ir(pq)2-bpy-Br]+PF6 -的合成
(1)4,4’-二溴甲基-2,2’-联吡啶及吡啶盐配体的合成
I)取4,4’-二甲基-2,2’-联吡啶5g加入带搅拌子的250mL单口瓶，加入80mL98％的浓H2SO4置于水浴条件下剧烈搅拌，并缓慢(保证温度不会剧烈上升)加入17g K2Cr2O7，然后将其置于65℃搅拌6h，冷却至室温，加水抽滤，干燥得白色粉末。
Ⅱ)取Ⅰ所得产物2g与10mL 98％H2SO4，100mL CH3OH加入带搅拌子的单口瓶中，105℃回流过夜，反应结束加至大量水中出现白色絮状沉淀，缓慢加入NaOH溶液调pH约9.0，用CH2Cl2萃取保留有机相，无水Na2SO4干燥后，蒸干溶剂得到白色晶体，产率88％。
Ⅲ)取Ⅱ所得产物630mg与NaBH4 1g加入带搅拌子的250mL单口瓶中，加入80mL严格除水除氧的乙醇溶液，80℃回流3h，反应结束加入20mL饱和NH4Cl溶液，蒸干乙醇，用乙酸乙酯与水进行萃取，保留乙酸乙酯层，加无水Na2SO4干燥，蒸干溶剂，真空干燥得到白色粉末。
Ⅳ)取Ⅲ中所得中间产物100mg，6mL 48％HBr溶液加至带搅拌子的单口瓶，缓慢加入9mL浓H2SO4，原料溶解，置于100℃油浴搅拌锅中回流6h，反应结束，加入少量水，边搅拌边加KOH调pH至7.0，产生白色沉淀，抽滤，CH2Cl2溶解产物，用无水Na2SO4干燥，过滤后蒸干溶剂，真空干燥得到4,4’-二溴甲基-2,2’-联吡啶，产率45％。
(2)配合物中间体[Ir(pq)2-bpy-Br]+PF6 -的合成
取苯基吡啶合铱氯桥二聚体200mg，140mg 4,4’-二溴甲基-2,2’-联吡啶，六氟磷酸钾(KPF6)300mg，二氯甲烷20mL，无水甲醇10mL，依次加入带搅拌子的两口烧瓶中，氮气保护，回流搅拌24h，用二氯甲烷稀释反应液，过滤得橙红色二氯甲烷澄清液，蒸干溶剂后，用二氯甲烷/乙酸乙酯(V:V＝20:1)柱层析得橙色粉末即为配合物中间体，产率86％。
1H NMR(400MHz,CDCl3)δ(ppm):8.65(m；2H)；7.91-7.88(m；4H)；7.76(t；J＝8.00Hz；2H)；7.68(d；J＝7.60Hz；2H)；7.53-7.51(m；3H)；7.46(dd；J1＝1.20Hz；J2＝5.60Hz；1H)；7.09-7.02(m；4H)；6.92(t；J＝7.20Hz；2H)；6.27(d；J＝7.60Hz；2H)；4.80(s；2H)；4.63(s；2H)。
实施例2
目标配合物Ir1[Ir(pq)2-bpy-py]3+3PF6 -的合成
称取配合物中间体[Ir(pq)2-bpy-Br]+PF6 -(1mmol)，吡啶(2mmol)和过量六氟磷酸钾粉末加入带搅拌子的双颈瓶中，在双排管上抽真空-保氮气-抽真空，循环往复三次，最后采用氮气保护整个反应体系。注入5mL严格除水除氧的乙腈溶液，80℃搅拌回流10h。反应结束后，冷却到室温，蒸干溶剂，用柱层析法得到粗产物，展开剂为二氯甲烷和甲醇(v:v＝10:1)，将粗产物用乙腈和二氯甲烷重结晶得到红色晶体产物即为目标配合物Ir1。
1H NMR(400MHz,CD3CN-d3)δ＝8.72-8.55(m,6H)；8.34(dd,J1＝20.8,J2＝8.8Hz,4H)；8.23(d,J＝5.8Hz,2H)；8.16-8.05(m,6H)；8.02(s,2H)；7.83(d,J＝7.1Hz,2H)；7.46-7.32(m,4H)；7.22-7.11(m,4H)；6.98-7.03(m,2H)；6.85-6.75(m,2H)；6.47(d,J＝7.4Hz,2H)；5.75(s,4H).
13C NMR(100MHz，CD3CN，298K)δ＝171.23,156.80,151.12,150.32,148.48,148.42,147.09,146.38,146.22,141.50,135.39,132.36,131.88,130.51,130.37,129.19,128.86,128.76,128.06,125.67,124.70,124.42,119.32
核磁共振氢谱、碳谱如图1、图2所示。
实施例3
磷光探针Ir1的发射光谱对还原性谷胱甘肽(GSH)、半胱氨酸(Cys)、硫氢化钠的响应性
将配合物Ir1配成1.0×10-3mol/L的乙腈溶液，移取10μL溶液到990μL PBS缓冲溶液中，使其浓度稀释至1.0×10-5mol/L进行测试，分别加入50倍当量的还原型谷胱甘肽、半胱氨酸、硫氢化钠水溶液，在37℃恒温水浴中加热搅拌5分钟后在10000转速离心，使还原型谷胱甘肽等与配合物Ir1充分反应，随后取上层清液测试其荧光发射光谱，如图2所示。配合物Ir1溶液最大发射峰在635nm左右，加入还原型谷胱甘肽、半胱氨酸和硫氢化钠后，溶液的发光强度明显增强并且最大发射峰蓝移，其中，加入半胱氨酸和硫氢化钠后发光强度变化没有加入还原型谷胱甘肽明显。配合物Ir1以及加入50倍当量的还原型谷胱甘肽、半胱氨酸、硫氢化钠水溶液后的发射光谱如图3所示。
实施例4
磷光探针Ir1的寿命对还原性谷胱甘肽(GSH)、半胱氨酸(Cys)、硫氢化钠的响应性
将配合物Ir1配成1.0×10-3mol/L的乙腈溶液，移取10μL溶液到990μL PBS缓冲溶液中，使其浓度稀释至1.0×10-5mol/L进行测试，测试配合物Ir1的寿命和向Ir1的PBS缓冲液中加入100倍当量的还原性谷胱甘肽(GSH)、半胱氨酸(Cys)、硫氢化钠水溶液，在37℃恒温水浴中加热搅拌5分钟后在10000转速离心，取上层清液进行寿命测试，配合物以及加入还原剂后的寿命变化如图4所示。
实施例5：磷光探针Ir1应用于活细胞成像实验
Hela细胞按照American Type Tissue Culture Collection规定进行培养。Hela细胞用10μM配合物Ir1溶液在37℃下孵育30分钟，用培养液洗涤3次，配合物Ir1标记的Hela细胞分别用巯基活性抑制剂N-乙基顺丁烯二酰亚胺(NEM)和还原型谷胱甘肽(GSH)溶液在37℃条件下各孵育30分钟，之后均用培养基洗涤3次，用405nm波长激发细胞进行共聚焦成像和磷光寿命成像，共聚焦成像显示活细胞胞浆内有明显可见的磷光，且用GSH孵育的Hela细胞中的磷光强度比NEM孵育的细胞中的磷光强度高，磷光寿命成像显示加入NEM孵育的细胞内平均寿命小于加入GSH孵育的细胞，而细胞内GSH的含量决定了细胞内的还原态，加入GSH孵育的细胞还原态要高于加入NEM孵育的细胞，如图5所示，此结果表明磷光探针Ir1具有较好的细胞穿透性，主要分布在细胞质区域，可用于活细胞内氧化还原态检测。
Claims (4)
1.一种基于吡啶盐的离子型磷光铱配合物探针，其特征在于其辅助配体上含有吡啶盐，该铱配合物具有如下结构：
2.一种如权利要求1所述的基于吡啶盐的离子型磷光铱配合物探针的制备方法，其特征在于该制备方法的合成路线如下：
具体是4,4’-二甲基-2,2’-联吡啶经过氧化、甲氧基化、酯的还原、氢溴酸取代四步反应得到N^N辅助配体，接着与铱二氯桥((C^N)2Ir(μ-Cl)2Ir(C^N)2)通过配位反应制备得到配合物中间体，然后将中间体与吡啶当量比为1:2在乙腈中溶解后加入过量六氟磷酸钾搅拌过夜得到目标配合物。
3.一种如权利要求1所述的基于吡啶盐的离子型磷光铱配合物探针的生物应用，其特征在于该离子型磷光铱配合物应用于细胞内氧化还原态检测。
4.一种如权利要求1所述的基于吡啶盐的离子型磷光铱配合物探针的生物应用，其特征在于该离子型磷光铱配合物应用于磷光寿命成像和时间分辨技术。
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