CN115215769A - 一种用于检测乙酰胆碱酯酶的荧光探针及其制备方法与应用 - Google Patents
一种用于检测乙酰胆碱酯酶的荧光探针及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种用于检测乙酰胆碱酯酶的荧光探针(DPHP‑AChE)及其制备方法与应用,所述DPHP‑AChE对AChE具有高选择性和敏感性,细胞毒性低,具有大的斯托克斯位移,荧光发射波长延伸到近红外区域,能够有效弥补传统荧光信号输出的不足,对于临床诊断和生物领域等方面具有市场应用与推广价值。
Description
技术领域
本发明涉及乙酰胆碱酯酶检测技术领域,特别涉及一种用于检测乙酰胆碱酯酶的荧光探针及其制备方法与应用。
背景技术
乙酰胆碱酯酶(acetylcholinesterase,AChE)具有羧肽酶和氨肽酶的活性,对动物神经传导过程起着重要的作用,它主要的生理功能是水解乙酰胆碱(ATCh),生成乙酸和胆碱(TCh),将神经系统内AChE含量控制在合理范围内,从而使得神经冲动传递受到阻碍,促进神经元发育和神经再生。研究表明,AChE可加速淀粉样蛋白β肽异常聚集,形成中枢神经系统中的阿尔茨海默氏淀粉样蛋白纤维,从而诱导阿尔兹海默症。此外,抑郁症中的异常情绪波动也可能受到AChE活动的影响。因此,了解AChE活性的变化对于深入分析这些神经系统疾病的分子机制至关重要。然而,目前尚不清楚这些疾病中AChE活性的动态调节机制。为了更深入地了解AChE在这些疾病中的作用,开发一种用于在体外和体内监测乙酰胆碱酯酶的实时原位工具显得尤为重要。目前,已有许多研究者在该领域进行了研究,开发出了许多用于AChE活性检测的分析技术,如比色法,电化学法,以及荧光方法。其中,比色法和电化学法不适用于活体生物系统中乙酰胆碱酯酶的成像,荧光探针(荧光方法)由于其优异的灵敏度和高时空分辨率已成为研究生物分子的较为理想方法。
文献J.Mater.Chem.B,2021,9,2623-2630公开了一种近红外荧光探针BD-AChE,用于实时和原位监测活细胞和活小鼠大脑中AChE水平的变化,BD-AChE的原位可视化验证了衰老模型小鼠脑内AchE水平的降低,表明BD-AChE探针具有优异的近红外荧光生物成像能力。
文献Arabian Journal of Chemistry(2022),15,103929公开了一种小分子荧光探针BDFA,通过一步反应实现了对AChE的定性和定量检测。BDFA在水介质中发出微弱的荧光,而在AChE的催化下荧光显著增强。机理研究表明,BDFA在AChE存在下消除了N,N-二甲基氨基甲酸酯的保护基团,然后自发地进行分子内环化转化生成强烈的荧光产物。
现有技术中,这些荧光探针使用氨基甲酸二甲酯单元作为AChE的识别单元,这些荧光探针容易受到聚集诱导猝灭(ACQ)的影响,并且单个荧光信号的变化也容易受到温度和pH等外部因素的干扰,在AChE活性检测方面存在一定的缺点和局限性。与之相反,具有聚集诱导发光特性(AIE)的荧光分子则不会在聚集态下发生荧光淬灭,光谱随浓度变化无漂移;具有激发态分子内质子转移(ESIPT)过程的荧光分子,可发生烯醇结构的异变,改变了荧光团的共轭体系,引起红色荧光发射和大的斯托克斯位移,有利于避免入射光的自吸收和干扰。目前现有技术中尚未报道用于AChE成像的且基于激发态分子内质子转移(ESIPT)和聚集诱导发光(AIE)活性的荧光探针,鉴于此,特提出本发明。
发明内容
本发明克服了现有技术中存在的缺陷,提供了一种用于检测乙酰胆碱酯酶的荧光探针及其制备方法与应用。
本发明的第一方面提供了一种荧光探针,所述荧光探针的结构为:
其中,X选自:-H、C1-12直链/支链烷基、C3-12环烷基、C6-12芳烷基、-F、-Cl、-Br和中的一种,R1、R2和R3各自独立地选自:-H、C1-12直链/支链烷基、C3-12环烷基、C6-12芳烷基、-F、-Cl、-Br。
优选地,R1为-H。
优选地,R2和R3均为C1-12直链/支链烷基,更优选地,R2和R3均为C1-6直链/支链烷基。
在本发明的一个实施方式中,所述的R2和R3均为乙基。
在本发明的一个实施方式中,所述荧光探针的结构为:
在本发明的一个实施方式中,所述荧光探针的结构为:
本发明的第二方面提供了如第一方面所述的荧光探针的制备方法,所述制备方法包括如下步骤:
进一步地,所述与二甲基氨基甲酰氯和三乙胺的摩尔比为0.5~5:10~30:0.5~5,优选地,所述与二甲基氨基甲酰氯和三乙胺的摩尔比为1~3:10~25:1~3,更优选地,所述与二甲基氨基甲酰氯和三乙胺的摩尔比为1~2:15~25:1~2,更进一步优选地,所述与二甲基氨基甲酰氯和三乙胺的摩尔比为1.5:18~24:1.5。
进一步地,所述中,X选自:-H、C1-12直链/支链烷基、C3-12环烷基、C6-12芳烷基、-F、-Cl、-Br和中的一种,R1、R2和R3各自独立地选自:-H、C1-12直链/支链烷基、C3-12环烷基、C6-12芳烷基、-F、-Cl、-Br。
优选地,R1为-H。
优选地,R2和R3均为C1-12直链/支链烷基,更优选地,R2和R3均为C1-6直链/支链烷基。
在本发明的一个实施方式中,所述的R2和R3均为乙基。
进一步地,所述的荧光探针的合成路线如下所示:
进一步地,所述的溶剂选自:二氯甲烷、三氯甲烷、丙酮、乙酸乙酯、甲酸乙酯、甲醇、乙醇、异丙醇、丁醇、异丙胺、二甲基甲酰胺、二甲基亚砜、乙醚和石油醚中的一种。
在本发明的一个实施方式中,所述的溶剂为二氯甲烷。
进一步地,所述反应的时间为1-5天,优选为3天。
进一步地,所述反应的温度为20℃-30℃,优选为25℃。
进一步地,所述反应为在搅拌状态下反应。
进一步地,所述制备方法还包括纯化的步骤。
进一步地,所述的纯化选自:柱层析法、重结晶法、打浆法和薄层色谱法中的一种。
在本发明的一个实施方式中,所述的纯化为柱层析法。
进一步地,所述柱层析法中的流动相为单一溶剂或混合溶剂,优选地,所述柱层析法中的流动相为混合溶剂。
进一步地,所述混合溶剂为:溶剂A-溶剂B。
进一步地,所述的溶剂A选自:石油醚、氯仿和二氯甲烷中的一种,优选地,所述的A为石油醚。
进一步地,所述的溶剂B选自:乙酸乙酯、丙酮、乙醚和甲醇中的一种,优选的,所述的B为乙酸乙酯。
进一步地,所述混合溶剂可以选自:石油醚-乙酸乙酯、石油醚-丙酮、石油醚-乙醚、氯仿-甲醇和二氯甲烷-甲醇中的一种。
在本发明的一个实施方式中,所述混合溶剂为石油醚-乙酸乙酯。
进一步地,所述混合溶剂中溶剂A与溶剂B的体积比为20:1~2:1,优选地,体积比为15:1~3:1,更优选地,体积比为10:1~4:1。
本发明的第三方面提供了如第一方面所述的荧光探针在乙酰胆碱酯酶成像中的应用。
进一步地,所述的成像为在体成像或离体成像;和/或,所述成像为体式荧光显微镜成像。
进一步地,所述的成像为脑胶质瘤细胞成像,所述的脑胶质瘤选自:星型细胞瘤、少枝细胞瘤、混合胶质瘤和室管膜瘤中的一种,优选的,所述的脑胶质瘤为星型细胞瘤。
激发态分子内质子转移(ESIPT)的机理是指在激发态下分子内氢键上的质子在烯醇式和酮式之间形成互变,表现为烯醇式和酮式的双发射。当激发态以酮式发射为主时,发射波长红移,表现为较大的斯托克斯(Stokes)位移,大的Stokes位移避免了激发光谱和发射光谱重叠现象,能够有效地提高荧光分子探针的灵敏度;与传统ACQ现象相反,有机发光团(荧光团)在聚集态下表现出荧光显著增强的现象称为聚集诱导发光现象(AIE)。
本发明的荧光探针(DPHP-AChE)基于激发态分子内质子转移(ESIPT)和聚集诱导发光(AIE)的原理,可用于AChE的体内和体外成像。DPHP-AChE对AChE具有高选择性和敏感性,细胞毒性低,具有大的斯托克斯位移,荧光发射波长延伸到近红外区域,能够有效弥补传统荧光信号输出的不足,对于临床诊断和生物领域等方面具有市场应用与推广价值。
附图说明
图1为DPHP-AChE的合成路线。
图2为DPHP在氘代DMSO中的氢谱。
图3为DPHP在氘代DMSO中的碳谱。
图4为DPHP的高分辨质谱。
图5为DPHP-AChE在氘代DMSO中的氢谱。
图6为DPHP-AChE在氘代DMSO中的碳谱。
图7为DPHP-AChE的高分辨质谱。
图8为AChE(PDB:6WVO)与DPHP-AChE的结合模式,其中,(A)为复合物的3D结构图;(B)为复合物活性部位的表面图;(C)为复合物的细节绑定模式图。
图9为DPHP-AChE对AChE的光谱响应,其中,(A)为DPHP-AChE和AChE(8U/mL)混合物在120分钟时的荧光光谱;(B)为DPHP-AChE(5μM)与不同浓度(0和8U/mL)的AChE的时间依赖性荧光强度比(I638/I560)。
图10为探针DPHP-AChE对AChE的光谱响应,其中,(A)为探针DPHP-AChE(5μM)在不存在(绿色)和存在(红色)AChE(14U/mL)的情况下的吸收;(B)为相应的荧光光谱。
图11为在发射最大波长处DPHP-AChE(蓝色)和DPHP(红色)的相对发射强度(I/I0)与水/THF混合物中的水分数的关系图。
图12为DPHP在不同水体积分数的水/THF中的荧光光谱。
图13为DPHP-AChE在不同水体积分数的水/THF中的荧光光谱。
图14为DPHP-AChE光稳定性测试结果图,其中测试时间为180min,DPHP-AChE=5μM,激发波长为450纳米。
图15为在37℃下与AChE孵育2小时后DPHP-AChE的高分辨质谱。
图16为DPHP-AChE的灵敏度测试结果图,其中,(A)为DPHP-AChE(5μM)与AChE(0-14U/mL)孵育120分钟后的光致发光(PL)光谱。(B)为DPHP-AChE的PL强度比(I638/I560)与AChE浓度(0-14U/mL)的关系。
图17为PL强度比(I638/I560)与AChE浓度(0-2U/mL)的线性拟合。
图18为DPHP-AChE+AChE(0U/mL)和DPHP-AChE+AChE(14U/mL)的比率成像,其中,激发波长=465纳米。
图19为DPHP-AChE(5μM,黑线)和DPHP-AChE(5μM)+AChE(14U/mL,红线)在不同pH缓冲液中的PL强度比(I638/I560),其中,激发波长=450纳米。
图20为DPHP-AChE对AChE(14U/mL)和各种其他生物分子的PL强度比,其中,激发波长=465纳米。
图21为对DPHP-AChE、DPHP的Normal形式和Keto形式进行高斯计算的示意图,其中,(A)为密度泛函理论(DFT)优化了DPHP-AChE、DPHP(Normal形式)和DPHP(Keto形式)的结构和前沿分子轨道;(B)为DPHP激发态质子转移过程示意图。
图22为DPHP在S1状态下的相对能量与O-H的键长的关系图。
图23为DPHP和DPHP-AChE对U87MG细胞的生物相容性测试。
图24为U87MG细胞的共聚焦图像。
图25为图24中的两个通道的荧光强度比(红色通道/绿色通道)。
图26为用不同浓度的谷氨酸(5mM)和LPS(1μg/mL)预孵育后U87MG细胞中DPHP-AChE的成像。
图27为图26中两个通道的荧光强度比(红色通道/绿色通道)。
图28为活体内AChE的时间依赖性比率图像。
图29为图28中PL强度比的随时间变化。
具体实施方式
为了能够更清楚地理解本发明的技术内容,特举以下实施例详细说明,其目的仅在于更好理解本发明的内容而非限制本发明的保护范围。
实施例1
1.1实验试剂和实验仪器
实验试剂:
乙酰胆碱酯酶、丁酰胆碱酯酶、脂肪酶、溶菌酶、酪氨酸酶、胰蛋白酶、乳铁蛋白、胃蛋白酶、PBS磷酸盐缓冲液、氢氧化钠、盐酸、正己烷、乙酸乙酯、溴化钠、氯化钠、4-二乙氨基苯甲醛、2'-羟基苯乙酮、无水乙醇、二甲基氨基甲酰氯、半胱氨酸、谷胱甘肽、过氧化氢、谷氨酸、脂多糖、三乙胺、高半胱氨酸、氯化钾、氯化钠、氯化钙、氯化镁、CCK-8试剂盒、U87mg细胞、DMEM培养基、特级胎牛血清、青霉素-链霉素溶液、二甲苯、胰蛋白酶(0.25%)、异氟烷、C57BL/6J小鼠。
实验仪器见下表1。
表1实验仪器
1.2DPHP和DPHP-AChE的合成
(1)将2'-羟基苯乙酮(1.36g,10mmol)和4-二乙氨基苯甲醛(1.77g,10mmol)加入到20ml乙醇中,然后加入氢氧化钠溶液(1g氢氧化钠溶解在1ml水中,25mmol)并在75℃搅拌12小时。冷却至室温后,用二氯甲烷和水萃取,用无水硫酸钠干燥,最后在乙醇中重结晶,得到蓝色固体DPHP(2.25g,71.9%)。1H NMR(400MHz,DMSO-d6)δ13.25(s,1H),8.30–8.22(m,1H),7.89–7.65(m,4H),7.51(p,J=7.8Hz,1H),6.96(p,J=8.4Hz,2H),6.76–6.63(m,2H),3.39(s,4H),1.09(dt,J=15.1,7.1Hz,6H).13C NMR(101MHz,DMSO-d6)δ193.51,162.83,150.39,147.17,136.23,132.34,130.79,121.42,120.88,119.33,118.18,114.32,111.60,44.33,39.72,12.91.质谱结果:理论值(加氢):296.16451;发现值:296.16431。
(2)将DPHP(444mg,1.5mmol)、二甲基氨基甲酰氯(2mL)和三乙胺(152mg,1.5mmol)溶解10mL二氯甲烷中。反应在室温下搅拌3天,粗产物用石油醚:乙酸乙酯PE/EA(10:1~4:1,v/v)混合溶剂纯化,得到黄色固体DPHP-AChE(76mg,43%)。1H NMR(400MHz,DMSO-d6)δ7.62(dd,J=7.6,1.6Hz,1H),7.58–7.49(m,3H),7.41(d,J=15.6Hz,1H),7.35(t,J=7.3Hz,1H),7.22(d,J=8.1Hz,1H),6.99(d,J=15.6Hz,1H),6.68(d,J=8.6Hz,2H),2.93(s,2H),2.68(s,8H),1.10(t,J=6.9Hz,6H).13C NMR(101MHz,DMSO-d6)δ190.83,165.03,154.03,150.00,149.30,145.86,133.98,132.19,131.33,129.73,125.96,124.20,121.06,119.63,111.60,44.25,38.69,36.71,12.89。质谱结果:理论值(加氢):367.20162;发现值:367.20163。
1.3探针DPHP-AChE母液的配制
称取一定量探针DPHP-AChE溶解在DMSO中备用(1mM),密封,避光,置于4℃保存。后续用于各类分析测试实验时,用缓冲溶液PBS(pH=7.4)稀释到所需的浓度。
1.4离子和生物活性分子的配制
将所需要离子化合物用超纯水配制成10mM的母液备用,离子化合物主要有:NaBr、NaCl、KCl、CaCl2、FeCl3、MgCl2生物活性分子:AChE(200U/mL)、BChE(50U/mL)、glutamate(50mM)、Hcy(50mM)、Cys(50mM)、GSH(50mM)、Lactoferrin、Lipase、lysozyme、Pepsine、Trypsin、Tyrosinase、Hcy、LPS(50μg/mL)溶解在超纯水中(除已标注外的浓度10mg/mL)。
2.1光谱性能测试
取1mM已配制的探针DPHP-AChE母液10μL加入到荧光皿中,接着添加不同体积的AChE母液,最后用PBS缓冲液定容至2mL,待反应后测试其吸收和荧光光谱变化。
2.2 AChE响应时间测试
取1mM的探针DPHP-AChE母液10μL于四个离心管中(内含1mL PBS),然后加入不同体积AChE母液(0μL和80μL)至该溶液中,再用PBS缓冲液定容至2mL,得到终浓度为5μM的DPHP-AChE和不同浓度(0U/mL和8U/mL)的AChE混合溶液,最后在不同时间段测试反应液的荧光光谱。
2.3选择性实验
取1mM的DPHP-AChE母液10μL于多个离心管中(内含0.5mL PBS),然后加入不同体积配制好的各种待测离子和生物活性分子母液,接着用PBS缓冲液定容2mL,在37℃反应120分钟后测试其荧光光谱。
2.4 DPHP-AChE的光稳定性实验
用荧光光谱仪的150W氙灯(450nm处)连续照射5μM的DPHP-AChE溶液180分钟。DPHP-AChE的光稳定性通过绘制I/I0与辐照时间t的曲线监测,其中I为t辐照时间后DPHP-AChE的荧光强度,I0为光照射前DPHP-AChE的荧光强度。
2.5检测限的计算
首先通过检测限计算公式LOD=3σ/κ来求探针DPHP-AChE对AChE的检测限,其中σ为3个独立的空白样品探针溶液(5μM)的638nm和560nm处荧光比值的标准偏差,κ为0-2U/mL浓度的AChE与I638/I560比值的线性关系的斜率。
2.6分子对接步骤
1.对接的化合物使用ChemDraw软件构建,然后导入Chem3D软件使用MM2模块进行优化和能量最小化,并保存为SDF格式文件作为配体分子进行分子对接。
2.AChE靶蛋白的晶体结构(PDB ID:6WVO)来自PDB数据库(https://www.rcsb.org/structure/6wvo)。蛋白质结构在Maestro11.9平台上进行处理,蛋白质结构在Maestro 11.9平台上处理,用Schrodinger Protein Preparation Wizard Protein处理,去除结晶水,添加缺失的氢原子,修复缺失的键信息和肽段,最后进行能量最小化和几何结构优化蛋白质。
3.分子对接的处理和优化由 Maestro软件中的Glide模块完成。蛋白质处理使用蛋白质制备向导模块。受体经过预处理、优化和最小化(使用OPLS3e力场进行最小化约束)。使用LigPre模块的默认设置制备复合结构。在Glide模块中筛选时,引入制备好的受体,根据蛋白质结构中天然配体与活性位点的结合,将活性位点定位在受体网格生成中。最后,通过SP方法进行分子对接和筛选。
4.分析化合物与目的蛋白的作用方式,得到化合物与蛋白质残基的相互作用。
2.7高斯计算
GaussView 5.0和Gaussian 09软件分别用于结构可视化和模拟。优化化合物的结构以获得最小的能量形式。目前研究实现了DFT的B3LYP功能。对于轨道描述,Pople基组6-31G*用于碳、氢、氮和氧原子。生成最高占据分子轨道(HOMO)和最低未占据分子轨道(LUMO)以了解所有化合物的电子密度分布。使用TD-DFT计算绘制了DPHP的苯酚氢键(在ESIPT过程中是可转移的氢)在S1状态下的势能曲线。
2.8pH对探针的影响
不同pH的溶液配制以1M盐酸溶液和1M氢氧化钠溶液在PBS缓冲液中配置,利用pH计来标定最终pH值,接下测试了5μMDPHP-AChE和5μM DPHP-AChE加14U/mL AChE混合溶液在不同pH溶液中的荧光强度,每组测试三次平行实验。
2.9细胞培养
U87mg细胞培养于DMEM(含10%热灭活FBS,100mg·mL-1青霉素和100mg·mL-1链霉素)的培养基中,置于37℃含5%CO2的加湿培养箱。
2.10细胞毒性
将U87MG细胞传到96孔板中并用200μL培养基来培养,在培养24h后,替用包含不同浓度的DPHP-AChE(0μM,5μM,10μM,15μM,20μM)的培养基培养,继续培养24h,到时间后除去96孔板中的溶剂,加100μL含10%的CCK8的培养基,接下来孵育3h待溶液变为橙色,用酶标仪测试450nm处的吸光值(OD)。
2.11细胞成像
将培养U87MG细胞的八孔板分组设置为:空白组不加探针DPHP-AChE,实验组加入DPHP-AChE(1μM),Donepezil:多奈哌齐(50μM)对LO2细胞预先孵育5小时,然后用1μM DPHP-AChE在37℃下孵育3小时,通过使用共聚焦显微镜分别收取通道I(520-590nm)和通道II(600-700nm)的荧光信号。
2.12活体比率成像
动物实验是经中国科学院深圳先进技术研究院动物保育与使用委员会批准后进行。将两组Balb/C小鼠腹部脱毛,接着皮下注入DPHP-AChE(100μM,25μL)溶液,随后两组分别注入PBS缓冲液(100μL)和等体积AChE(200U/mL,100μL)溶液。在异氟烷持续麻醉的条件下使用小动物三维成像系统收集不同时间段520-590nm和600-700nm的荧光信号,激发为465nm,Ratio通道由ImageJ软件得到。
实施例2
1.1探针DPHP-AChE的表征
合成路线如图1所示。图2-7是DPHP-AChE的核磁和质谱表征结果。
1.2分子对接
图8为AChE(PDB:6WVO)与DPHP-AChE的结合模式,其中,(A)为复合物的3D结构;(B)为活性部位的表面;(C)为复合物的细节绑定模式。如图8所示,DPHP-AChE与AChE结合效果好且结合力强(结合能为-8.3kcal/mol)。对接后DPHP-AChE与AChE形成的复合物通过Pymol2.1软件可视化,得到DPHP-AChE与AChE的结合模式。根据结合模式,可以清楚地看到DPHP-AChE和AChE蛋白的活性口袋中的许多氨基酸残基相互结合。具体来说,DPHP-AChE与AChE结合的活性氨基酸残基主要为TYR-124、HIS-287、TRP-286、PHE-297、PHE-295、VAL-294、PHE-338、TYR-341、TYR-72,VAL-282。发明人发现AChE蛋白的活性口袋主要由疏水性氨基酸组成,而DPHP-AChE具有很强的疏水性,因此DPHP-AChE和AChE主要存在于疏水相互作用。特别是TYR-124和HIS-287氨基酸可以与DPHP-AChE的苯环形成π-π共轭,这对稳定蛋白质袋中的小分子有重要贡献。AChE蛋白的活性口袋具有高度疏水性,与高度疏水的DPHP-AChE具有高度的匹配性,使得DPHP-AChE的各个部分都能与AChE的凹槽形成良好的相互作用。这些强相互作用和优良的匹配模式对稳定DPHP-AChE起到了重要作用,促使DPHP-AChE与AChE形成稳定的复合物,有利于AChE水解DPHP-AChE。
1.3 DPHP-AChE对AChE的光谱响应
图9中,(A)为DPHP-AChE和AChE(8U/mL)混合物在120分钟时的荧光光谱;(B)为DPHP-AChE(5μM)与不同浓度(0和8U/mL)的AChE的时间依赖性荧光强度比(I638/I560)。如图9所示,DPHP-AChE和AChE混合物的荧光强度比在120分钟内达到一个平台期。因此,发明人使用120分钟作为后续研究的孵育时间。图10中,(A)为探针DPHP-AChE(5μM)在不存在(绿色)和存在(红色)AChE(14U/mL)的情况下的吸收;(B)为对应的荧光光谱。如图10所示,DPHP-AChE在454nm处具有最大的紫外吸收,在560nm处具有相应的最大荧光发射。由于酯基保护抑制了ESIPT过程,因此DPHP-AChE的荧光发射相对较短。加入AChE后,出现了一个较大的红移宽峰,相应的荧光发射光谱显示出从600nm到700nm的红移。发射最大值位于638nm,相对于450nm的激发,表现出较大的斯托克斯位移(188nm)。
1.4 DPHP的AIE性质
在水/THF混合溶液中对DPHP的AIE特征进行表征,图11为在发射最大波长处DPHP-AChE(蓝色)和DPHP(红色)的相对发射强度(I/I0)与水/THF混合物中的水分数的关系图,图12为DPHP在不同水体积分数的水/THF中的荧光光谱,图13为DPHP-AChE在不同水体积分数的水/THF中的荧光光谱。如图11和图12所示,DPHP在纯THF中具有弱荧光发射,随着THF/水混合物中的水含量从0%增加到90%,荧光强度逐渐增加,发射波长发生红移。在含水量为90vol%时,发射最大峰移至639nm,红色荧光强度比纯THF溶液高近11倍。然而,DPHP-AChE在水/THF混合溶液中表现出相反的现象(图11和图13)。
1.5光稳定性测试
如图14所示,DPHP-AChE光稳定性测试180min,DPHP-AChE=5μM,激发波长为450纳米。用150W氙灯照射3小时,DPHP-AChE也表现出较高的稳定性。
1.6 AChE对DPHP-AChE的水解
在37℃下与AChE孵育2小时后DPHP-AChE的高分辨质谱如图15所示(DPHP([M++H]计算:296.16451,发现:296.16434)。DPHP-AChE([M++Na]计算:389.18356,发现:389.18332)),图中除了存在DPHP-AChE的质谱峰([M++Na]=389.18332)外,AChE和DPHP-AChE的混合物中还出现了DPHP的产物峰([M++H]=296.16434)。
1.7灵敏度
图16中(A)为DPHP-AChE(5μM)与AChE(0-14U/mL)孵育120分钟后的光致发光(PL)光谱;(B)为DPHP-AChE的PL强度比(I638/I560)与AChE浓度(0-14U/mL)的关系;插图B:探针与不同浓度BChE(0U/mL和14U/mL)反应的比值图像,图17为PL强度比(I638/I560)与AChE浓度(0-2U/mL)的线性拟合,图18为DPHP-AChE+AChE(0U/mL)和DPHP-AChE+AChE(14U/mL)的比率成像,其中,激发波长=465。如图16和图17所示,DPHP-AChE与不同浓度(0-14U/mL)的AChE孵育后,560nm处的荧光逐渐降低,而在638nm处的荧光急剧增加,荧光强度比(I638/I560)在0-2U/mL范围内呈现出极好的线性关系(Y=0.23279X+0.27307,R2=0.99292)。计算的检测限为0.10993U/mL,低于生理活性(约5U/mL)。此外,两个不同浓度AChE(0U/mL和14U/mL)的荧光信号比值图像也显示出高度对比的变化,只有探针的离心管中几乎没有信号,而和AChE孵育的探针中显著增加的信号(图18和图16)。
1.8特异性和pH研究
图19为DPHP-AChE(5μM,黑线)和DPHP-AChE(5μM)+AChE(14U/mL,红线)在不同pH缓冲液中的PL强度比(I638/I560),其中,激发波长=450纳米。如图19所示,DPHP-AChE仅对AChE的荧光强度比(I638/I560)有显著变化,而对其他生物活性分子和离子几乎没有影响。此外,DPHP-AChE在生理环境的pH范围(6.8-7.4)中表现出优异的性能(如图20所示,DPHP-AChE对AChE(14U/mL)和各种其他生物分子的PL强度比(I638/I560):a:K+(1mM);b:Na+(1mM);c:Ca2+(1mM);d:Fe3+(1mM);e:Mg2+(1mM);f:Cl-(1mM);g:Br-(1mM);h:Lipase(1mg/mL);i:Lysozyme(1mg/mL);j:Pepsine(1mg/mL);k:Trypsin(1mg/mL);l:Tyrosinase(1mg/mL);m:LPS(10μg/mL);n:glutamate(5mM);o:GSH;p:Hcy(1mM);q:butyrylcholinesterase(50U/mL);r:AChE(14U/mL);激发波长=465纳米)。因此,DPHP-AChE是检测AChE活性的有力工具。
1.9 DPHP的ESIPT性质
对DPHP-AChE、DPHP的Normal形式和Keto形式进行高斯计算。图21中,(A)为密度泛函理论(DFT)优化了DPHP-AChE、DPHP(Normal形式)和DPHP(Keto形式)的结构和前沿分子轨道。计算基于DFT在B3LYP/6-31G*基组;(B)为DPHP激发态质子转移过程示意图。如图21所示,DPHP-AChE的能隙为3.47371eV,经AChE水解后生成烯醇型(能隙=3.31150Ev)。DPHP(Normalform)的质子供体(羟基)在光激发作用下通过分子内氢键转移到质子受体(羰基)上。具有更强共轭结构的酮式结构比正常结构具有更小的能隙(2.71075eV)。DPHP(Normalform)LUMO轨道上羰基氧的电子密度增加,羟基氧的电子密度略有下降,羰基氧的电子密度明显大于羟基氧,有利于质子从羟基转移到羰基。图22为通过在B3LYP/6-31G水平上使用TD-DFT计算改变O-H的键长,DPHP在S1状态下的相对能量。根据图21和图22,发明人使用TD-DFT在S1状态下计算DPHP随苯酚的氢距离变化(在ESIPT过程中是可转移的氢)的势能曲线(PEC)。PEC表明S1态质子转移需要0.55221kcal/mol的活化能。这些数据表明ESIPT过程在S1状态下的可行性。激发态(纳秒级)的质子转移过程远高于烯醇激发态(皮秒级)的失活速度,DPHP-AChE和AChE反应产生的红色荧光发射是ESIPT造成的。
1.10细胞毒性评估
在进行细胞成像之前,我们使用CCK8方法来评估DPHP和DPHP-AChE对U87MG细胞的生物相容性。图23为使用CCK8方法在不同浓度的DPHP-AChE和DPHP下U87MG细胞的细胞活力。如图23所示,结果表明DPHP和DPHP-AChE在一个较大的工作浓度范围内对U87MG细胞的存活率影响较小。
1.11细胞成像
选择U87MG细胞作为DPHP-AChE的体外成像对象。对照组:用DPHP-AChE(1μM)在37℃下处理3小时的细胞;多奈哌齐组:细胞用50μM多奈哌齐预处理5小时,然后与DPHP-AChE(1μM)在37℃下孵育3小时。其中,红色通道的采集范围为600-700nm;绿色通道在520-590nm范围内采集。图24为U87MG细胞的共聚焦图像,其中,对照组:用DPHP-AChE(1μM)在37℃下处理3小时的细胞;多奈哌齐组:细胞用50μM多奈哌齐预处理5小时,然后与DPHP-AChE(1μM)在37℃下孵育3小时;红色通道的采集范围为600-700nm;绿色通道在520-590nm范围内采集。如图24所示,用DPHP-AChE处理的细胞在红色通道中显示出明亮的荧光,而在绿色通道中的荧光相对较弱。用AChE抑制剂多奈哌齐预处理的细胞在绿色通道中显示出显着的荧光信号,而红色通道显示出较弱的信号发射。图25为图24两个通道的荧光强度比(红色通道/绿色通道),发明人发现用多奈哌齐处理的细胞中荧光信号的比率(红色通道/绿色通道)比未用多奈哌齐预处理的细胞低5.10倍(图25)。这些结果表明探针DPHP-AChE可用于对活细胞中的AChE进行成像。此外,研究表明,在氧化应激的作用下,AChE的活性得到了过度激活。发明人用谷氨酸和脂多糖(LPS)对U87MG细胞进行了预处理。图26为用不同浓度的谷氨酸(5mM)和LPS(1μg/mL)预孵育后U87MG细胞中DPHP-AChE的成像,图27为图26中两个通道的荧光强度比(红色通道/绿色通道)。在用谷氨酸盐和LPS处理后,这些细胞的荧光信号比(红色通道/绿色通道)分别增强了1.80倍和2.01倍(图26和图27)。这些数据阐明氧化应激调节AChE在脑肿瘤中的作用提供了一个新的和有益的视角。
1.12活体比率成像
发明人进一步评估了DPHP-AChE体内成像的潜力,图28为活体内AChE的时间依赖性比率图像,图29为图28中PL强度比的随时间变化。如图28和图29所示,实验组小鼠腹部荧光红通道的强度显着高于对照组小鼠脑区荧光红通道的强度。对照组小鼠,而相应的绿色通道荧光信号显着低于对照组小鼠,实验组荧光比值信号(红色通道/绿色通道)是对照组小鼠的9.47倍。显然,这些直接可视化结果也表明存在DPHP-AChE用于体内AChE成像的可能性。
2.1小结
发明人开发了一个比率AIE探针DPHP-AChE,用于AChE的体内和体外成像。DPHP-AChE对AChE具有高选择性和敏感性,细胞毒性低,具有大的斯托克斯位移,荧光发射波长延伸到近红外区域。正是因为具有这些优点,DPHP-AChE可进一步用于U87MG癌细胞、活体内AChE的实时比率成像。这些结果表明,DPHP-AChE可用于详细了解AChE在复杂生理和病理过程中的作用。
Claims (10)
3.权利要求1所述的荧光探针,其特征在于,所述的R2和R3均为C1-12直链/支链烷基,优选地,R2和R3均为C1-6直链/支链烷基,更优选地,所述的R2和R3均为乙基。
5.如权利要求1所述的荧光探针的制备方法,其特征在于,所述制备方法包括如下步骤:
将与二甲基氨基甲酰氯和三乙胺在溶剂中混合,反应,其中,X选自:-H、C1-12直链/支链烷基、C3-12环烷基、C6-12芳烷基、-F、-Cl、-Br和中的一种,R1、R2和R3各自独立地选自:-H、C1-12直链/支链烷基、C3-12环烷基、C6-12芳烷基、-F、-Cl、-Br;
优选地,所述反应的时间为1-5天;
优选地,所述反应的温度为20℃-30℃;
优选地,所述制备方法还包括纯化的步骤;
优选地,所述的纯化选自:柱层析法、重结晶法、打浆法和薄层色谱法中的一种。
6.权利要求5所述的制备方法,其特征在于,所述柱层析法中的流动相为单一溶剂或混合溶剂;
优选地,所述柱层析法中的流动相为混合溶剂;
优选地,所述混合溶剂为:溶剂A-溶剂B,所述的溶剂A选自:石油醚、氯仿和二氯甲烷中的一种,所述的溶剂B选自:乙酸乙酯、丙酮、乙醚和甲醇中的一种;
优选地,所述混合溶剂选自:石油醚-乙酸乙酯、石油醚-丙酮、石油醚-乙醚、氯仿-甲醇和二氯甲烷-甲醇中的一种;
优选地,所述混合溶剂为石油醚-乙酸乙酯;
优选地,所述混合溶剂中溶剂A与溶剂B的体积比为20:1~2:1。
8.权利要求5所述的制备方法,其特征在于,所述的R2和R3均为C1-12直链/支链烷基,优选地,R2和R3均为C1-6直链/支链烷基,更优选地,所述的R2和R3均为乙基。
10.如权利要求1所述的荧光探针在乙酰胆碱酯酶成像中的应用,其特征在于,所述的成像为在体成像或离体成像;
优选地,所述成像为体式荧光显微镜成像;
优选地,所述的成像为脑胶质瘤细胞成像;
优选地,所述的脑胶质瘤选自:星型细胞瘤、少枝细胞瘤、混合胶质瘤和室管膜瘤中的一种。
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040067981A1 (en) * | 2001-01-26 | 2004-04-08 | Sankyo Company, Limited | Benzylamine analogues |
CN105481796A (zh) * | 2014-09-19 | 2016-04-13 | 四川大学 | 一类氨基甲酸查尔酮酯类化合物、其制备方法和用途 |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040067981A1 (en) * | 2001-01-26 | 2004-04-08 | Sankyo Company, Limited | Benzylamine analogues |
CN105481796A (zh) * | 2014-09-19 | 2016-04-13 | 四川大学 | 一类氨基甲酸查尔酮酯类化合物、其制备方法和用途 |
Non-Patent Citations (3)
Title |
---|
XIANG CHUNBAI等: ""A responsive AIE-active fluorescent probe for visualization of acetylcholinesterase activity in vitro and in vivo"", MATER. CHEM. FRONT., vol. 6, pages 1515 - 1521 * |
XIAO GANYUAN等: ""Design, synthesis and biological evaluation of 4’-aminochalcone-rivastigmine hybrids as multifunctional agents for the treatment of Alzheimer’s disease"", BIOORGANIC & MEDICINAL CHEMISTRY, vol. 25, pages 1030 - 1041, XP029902200, DOI: 10.1016/j.bmc.2016.12.013 * |
YAO MENG等: ""A sensitive and selective fluorescent probe for acetylcholinesterase: Synthesis, performa nce, mechanism and application"", ARABIAN JOURNAL OF CHEMISTRY, vol. 15, pages 1 - 9 * |
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