CN114560850A - 一种近红外荧光分子探针、其制备方法及其应用 - Google Patents
一种近红外荧光分子探针、其制备方法及其应用 Download PDFInfo
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Abstract
本发明提供一种近红外荧光分子探针、其合成方法及其应用，近红外荧光分子探针的分子结构式为
，其中，R1为氢或者具有1‑6个C的正构烷基；R2为氢、甲烷基、乙烷基、苯基或苄基。本发明的近红外荧光分子用于B型单胺氧化酶检测，具有信噪比高和选择性好的优点。
Description
技术领域
本发明属于荧光分子探针技术领域，具体涉及一种用于检测B型单胺氧化酶的近红外荧光分子探针及其制备方法和应用。
背景技术
单胺氧化酶是位于线粒体内膜上的一种黄素酶，多存在于人体脑部和肝部，可以与单胺类生物分子反应与其他酶一起维持人体内的胺类物质平衡。单胺氧化酶有A型和B型两种亚型，其中，B型单胺氧化酶与神经退行性类疾病如帕金森综合征有紧密的联系，科研工作者发现该种疾病病患的B型单胺氧化酶高于正常人的水平。B型单胺氧化酶抑制剂类药物可用于帕金森综合征早期治疗，且不会引起“奶酪效应”，而B型单胺氧化酶在此类疾病中起到的作用尚不十分明确，需要进一步研究发现。已有一些研究工作通过敲除该种酶的小鼠模型研究该酶的病理机制，但基因敲除小鼠会引起高水平底胺的积累，导致其他受体和蛋白质发生复杂的变化。探针通过成像的方式可追踪显示该种酶的变化，能够运用探针精准检测并对B型单胺氧化酶进行成像是十分有必要的，选择性好的探针还可用于B型单胺氧化酶抑制剂类药物的大通量的筛选。
目前已公布发表的B型单胺氧化酶探针多处在可见光区，都是传统下转换发光型探针，该类探针在应用于生物组织时受生物自发荧光，激发发射被散射或吸收的影响。用于B型单胺氧化酶的信噪比高、选择性好的近红外区荧光分子探针亟待开发和研究。
发明内容
本发明的目的是针对现有技术的不足，从而提供一种用于B型单胺氧化酶的信噪比高、选择性好的近红外区荧光分子探针。
为了达到上述发明目的，本发明采用以下技术方案：
一种近红外荧光分子探针，其分子结构式为
其中，R1为氢或者具有1-6个C的正构烷基；R2为氢、甲烷基、乙烷基、苯基或苄基。
作为技术方案的进一步改进，为了提高探针的上转换发光能力，降低合成难度，R2为乙烷基。在同等浓度，同样激光激发条件下，随着R2基团给电子能力增强，探针的上转换发光能力更强，如图7所示，在同等浓度，同样激光激发条件下，R2为乙烷基的探针上转换发光能力更强。
本发明的荧光分子的结构由半花菁荧光团、氨基甲酸酯和丙烷伯胺组成，其中，氨基甲酸酯为连接基团，末端的丙烷伯胺作为响应基团。半花菁荧光团能吸收近红外的光并发出波长更短的荧光，不同R2取代基荧光团得吸收波长范围为715-730 nm，发射波长范围为734-750 nm连接氨基甲酸酯基团时被邻近的缺电子氨基甲酸酯键显著猝灭失去荧光效应。在B型单胺氧化酶催化氨基氧化成醛(通过亚胺中间体)时，丙醛部分由β-消除自发释放，随后释放出电子丰富、高度荧光的半花菁荧光团。
所述近红外荧光分子探针可以精准的检测B型单胺氧化酶，在成像溶液中、细胞中或者活体中B型单胺氧化酶中的具有信噪比高和选择性好的优点，可用于制备检测B型单胺氧化酶的工具或者试剂中，作为检测工具或者试剂的组成，其在检测工具或者检测试剂中的量满足检测有效量即可。
所述近红外荧光分子探针在成像溶液中、细胞中或者活体中B型单胺氧化酶中的应用，该应用基于非诊断或者治疗疾病的目的：如构建研究神经退行性类疾病的细胞或者动物模型，所述近红外荧光分子探针还可用于筛选B型单胺氧化酶抑制剂类的药物。
本发明还公开一种所述的近红外荧光分子探针的制备方法，包括如下步骤：
步骤一、在惰性气氛和冰浴条件下，将化合物1和三光气溶解到溶剂中，然后加缚酸剂触发反应；
步骤二、步骤一得到的反应液旋蒸除去溶剂后再加入新鲜的溶剂，在冰浴条件下溶解步骤一得到的产物；
步骤三、将步骤二得到的溶液撤去冰浴，在室温下加入3-Boc氨基丙醇并滴加缚酸剂进行反应；
步骤四、在室温下对步骤三得到的反应液加入过量盐酸脱去Bｏｃ基团，然后加入碱中和多余的盐酸；其中，所述化合物1的结构式为
。
化合物1的合成方法可参考公开文献，例如文献：Aliya Tiemuer 1 , Hui Yu 1 ,Chao Zhao, Wanlu Sun, Yuanyuan Zhang, Yiming Jiang, Yueqing Gu , Yi Liu.Nitroso-caged upconversion luminescent prodrug: Near infrared light-activatable NO nano-donor for gas therapy, Chemical Engineering Journal, 430(2022) 132858.
制备方法中涉及的缚酸剂可选用常见的三乙胺、吡啶等有机碱，步骤四中的碱优选无机弱碱，如碳酸氢钠、氨水、碳酸氢钾等，步骤一和步骤二的溶剂可选用二氯甲烷，本制备方法的反应路线如下，其具有合成工艺简单、原料易得、成本低和收率高的特点。
作为技术方案的进一步改进，基于易得性和成本考虑，步骤一和二中的惰性气氛为氮气或者氩气。
作为技术方案的进一步改进，步骤四制得的混合物通过柱层析法分离得到探针，柱层析法选用二氯甲烷和甲醇作为流动相，流动相梯度从200：1到100：1。
本发明相对现有技术具有突出的实质性特点和显著的进步，具体的说，第一、本发明的近红外荧光分子探针因生物相容性好易于进入细胞，减
少响应时间短，且具有灵敏度高的优点，使得探针细胞毒性低。
第二、本发明的探针的近红外荧光团具有上转换特性，可在长波长激发
下发出近红外光，减小了激发光的影响，提高信噪比。
第三、本发明的近红外荧光分子探针吸收和发射均在近红外区，避免了
生物组织自发荧光的干扰，且较强的生物体穿透性。
第四、本发明的近红外荧光分子探针选择性强，可分辨单胺氧化酶的两
种亚型。
第五、本发明近红外荧光分子探针的制备方法具有合成工艺简单、原料
易得、成本低和收率高优点。
附图说明
图1是近红外荧光分子探针与B型单胺氧化酶响应的荧光变化图。
图2是近红外荧光分子探针的选择性实验数据图。
图3是近红外荧光分子探针的共聚焦显微镜细胞成像图。
图4是近红外荧光分子探针的对单胺氧化酶的两种亚型选择性实验数据图。
图5是在不同浓度近红外荧光分子探针下的细胞存活结果图。
图6是B型单胺氧化酶被抑制后探针的荧光强度变化图。
图7是具有不同R2基团的近红外荧光分子探针的发射荧光强度图。
具体实施方式
下面通过具体实施方式，对本发明的技术方案做进一步的详细描述。
本发明中实验所用试剂、耗材等，若无特殊说明，都可以经商业途径购买获得。实验方法若无特殊说明都为常规方法。实施例中所选用的以下所有试剂皆为市售分析纯或化学纯。
3-boc氨基丙醇选购自乐研，三光气和帕吉林（PA）选购自阿拉丁试剂(上海)有限公司。
实施例1 近红外荧光分子探针的制备实验
将化合物1（50 mg 0.105mmol）溶于5mL二氯甲烷中，在冰浴条件下，加入三光气（15.58 mg 0.0525 mmol），向装置中充入氮气，向体系中滴加三乙胺（2.121mg,0.021mmol）反应液颜色由绿色变蓝色。滴加后该反应在常温反应30分钟后，除去溶剂加入新鲜二氯甲烷作为溶剂。称取3-boc氨基丙醇（22.08mg 0.126mmol）加入烧瓶中，取三乙胺（4.242mg 0.042mmol）溶解在二氯甲烷中滴加。反应在常温下搅拌过夜，加入稀释的盐酸（盐酸：甲醇（V/V）=1：4）常温下搅拌2小时后加入碳酸氢钠除去多余的盐酸，旋干溶剂通过柱层析纯化产物最终得到产物为黑色固体6.5 mg, 产率约为12%。
实施例1制备的探针质谱图通过质谱仪（型号：LCQ-Advantages）以及核磁共振波谱仪（型号：Bruker 300MHz）确认，相关参数为 1H NMR (300 MHz, Methanol-d4) δ 8.90(d, J = 15.3 Hz, 1H), 8.41 (d, J = 8.5 Hz, 1H), 8.17 (d, J = 8.9 Hz, 1H),8.11 (d, J = 8.2 Hz, 1H), 7.86 (d, J = 8.9 Hz, 1H), 7.76 (t, J = 7.4 Hz, 1H),7.64 (t, J = 7.5 Hz, 1H), 7.53 (s, 1H), 7.49 (d, J = 4.6 Hz, 1H), 7.30 (s,1H), 7.24 (d, J = 8.3 Hz, 1H), 6.69 (d, J = 15.3 Hz, 1H), 4.60 (q, J = 7.2Hz, 2H), 4.28 (t, J = 6.0 Hz, 2H), 3.87 (q, J = 7.1 Hz, 2H), 2.93 (t, J = 7.3Hz, 2H), 2.80 (dt, J = 11.6, 5.8 Hz, 4H), 2.11 (s, 6H), 2.04 – 1.92 (m, 4H),1.58 (t, J = 7.2 Hz, 3H), 1.24 (t, J = 7.1 Hz, 3H). MS(ESI+):calcd forC37H42N3O3 +, 576 .3221[M]+；found [M]+576.32195.
实施例2
近红外荧光分子探针与B型单胺氧化酶体外响应的荧光发射测量实验
配置1 mM以DMSO为溶剂的探针溶液，并用PBS稀释至10 μM。为模拟生物环境下的探针的响应情况，选取B型单胺氧化酶高表达的HepG2细胞，将细胞裂解取后取裂解液，每500 uL裂解液含约106个细胞。将稀释后的探针溶液加入比色皿中，每次取1 μL加入其中，37°C孵育10分钟左右，用荧光分光光度计测试其荧光发射的相对强度。所得数据经origin软件处理得图1，如图1所示，随着酶浓度的增加，其荧光信号不断增加，表明探针在溶液中单胺氧化酶的响应效果好。
此外，探针的近红外荧光的发射波长在近红外区域，组织穿透能力强，在近红外通道的成像时可以有效地避免生物体自发荧光的干扰。
实施例3 近红外荧光分子探针的选择性实验：
通过与浓度均为100μM的不同底物（Co2+，Mg2+，Cu2+，Na+，Fe2+，K+，ClO-，H2O2，VC，NONOate，HepG2裂解液）孵育来检测实施例1制备的探针(10μM)对B型单胺氧化酶的选择性。用荧光分光光度计分别测试探针在不同底物中荧光发射的相对强度。如图2所示，只有含有B型单胺氧化酶的HepG2裂解液具有诱导荧光信号显著增强的能力，而其他物质不能诱导荧光信号显著变化。结果明显表明，本发明探针是一个高度特异的探针，在复杂的生物环境中对的B型单胺氧化酶响应。
实施例4 近红外荧光分子探针与HepG2细胞进行时间孵育实验
将约500个HepG2细胞（B型单胺氧化酶高表达细胞）均匀的铺在共聚焦皿上，孵育24小时后，加入探针，使浓度达到5μM并在37°C恒温培养箱中孵育不同时间，取出后用PBS洗涤三遍，并加入20μL DAPI摇晃使其均匀铺在共聚焦皿上，静置5分钟后，用PBS洗涤三遍，加入1mL 4%多聚甲醛固定，静置5分钟后。利用激光聚焦荧光显微镜（型号：LSM８００）成像，如图3所示。
实施例5 特异响应性实验
将500个HepG2细胞（B型单胺氧化酶高表达细胞）和SH-SY5Y细胞（A型单胺氧化酶高表达细胞）均匀铺在共聚焦皿上，孵育24小时后，使浓度达到5μM并在37°C 恒温培养箱中孵育不同时间，取出后用PBS洗涤三遍，并加入20μL DAPI摇晃使其均匀铺在共聚焦皿上，静置5分钟后，用PBS洗涤三遍，加入1mL 4%多聚甲醛固定，静置5分钟后。利用激光聚焦荧光显微镜成像，如图4所示。HepG2为B型单胺氧化酶高表达的细胞，而SH-SY5Y为A型单胺氧化酶高表达的细胞，该实验可确定本发明的近红外荧光分子探针可区分单胺氧化酶的两种亚型。
实施例6 细胞毒性实验
取约105个HepG2细胞均匀铺在96孔板中，在最外圈孔中各加入200 μL PBS减弱培养基蒸发。在37℃下培养24小时后，向各孔中加入探针使最终浓度达到0，5，10，15，20 μM培养24小时。向各孔加入MTT试剂摇晃均匀，３７°Ｃ 下静置4小时后，去除上层液体，向孔中加入100 μL二甲基亚砜，溶解孔底的甲臜。通过酶标仪检测各孔在490 nm处的吸光度，结果如图5所示，在0-20 μM浓度区间活细胞达75%以上，表明该探针的细胞毒性小，生物适用性好。
实施例7 近红外荧光分子探针与B型单胺氧化酶抑制剂作用的实验
配置1 mM以DMSO为溶剂的探针溶液，并用PBS稀释至10μM。选取B型单胺氧化酶高表达的HepG2细胞，将细胞裂解取后取裂解液，每500 uL裂解液含约106个细胞。
取2只离心管各加入200ul细胞裂解液，其中1只加入100ul浓度是200uM的PA,另一只加入等量PBS。
在三个比色皿中分别加入稀释后的探针溶液2mL,取2只离心管中孵育两小时的裂解液各10μL分别加入两个比色皿中，测试三个比色皿中溶液的荧光强度。所得数据经origin软件处理得图6，如图6所示，由于B型单胺氧化酶活性受到抑制，与不加抑制剂的组别相比，加入抑制剂后发出的荧光强度大大降低。该探针表现出在B型单胺氧化酶抑制剂筛选应用中的潜力。
最后应当说明的是:以上实施例仅用以说明本发明的技术方案而非对其限制；尽管参照较佳实施例对本发明进行了详细的说明，所属领域的普通技术人员应当理解，依然可以对本发明的具体实施方式进行修改或者对部分技术特征进行等同替换；而不脱离本发明技术方案的精神，其均应涵盖在本发明请求保护的技术方案范围当中。
Claims (9)
1.一种近红外荧光分子探针，其特征在于，其分子结构式为
其中，R1为氢或者具有1-6个C的正构烷基；R2为氢、甲烷基、乙烷基、苯基或苄基。
2.根据权利要求1所述的近红外荧光分子探针，其特征在于，R2为乙烷基。
3.一种根据权利要求1或2所述的近红外荧光分子探针的制备方法，其特征在于，包括如下步骤：
步骤一、在惰性气氛和冰浴条件下，将化合物1和三光气溶解到溶剂中，然后加缚酸剂触发反应；
步骤二、步骤一得到的反应液旋蒸除去溶剂后再加入新鲜的溶剂，在冰浴条件下溶解步骤一得到的产物；
步骤三、将步骤二得到的溶液撤去冰浴，在室温下加入3-Boc氨基丙醇并滴加缚酸剂进行反应；
步骤四、在室温下对步骤三得到的反应液加入过量盐酸脱去Bｏｃ基团，然后加入碱中和多余的盐酸；其中，所述化合物1的结构式为
。
4.根据权利要求3所述的制备方法，其特征在于，步骤一中的惰性气氛为氮气或者氩气。
5.根据权利要求3所述的制备方法，其特征在于，步骤四制得的反应液通过柱层析法分离得到探针，柱层析法选用二氯甲烷和甲醇作为流动相，流动相梯度从200：1到100：1。
6.一种包含权利要求1或2所述近红外荧光分子探针的工具或者试剂，其特征在于，所述工具或者试剂用来检测B型单胺氧化酶。
7.一种权利要求1或2所述近红外荧光分子探针的应用，其特征在于，所述近红外荧光分子探针用于制备检测B型单胺氧化酶的工具或者试剂。
8.一种权利要求1或2所述近红外荧光分子探针在成像溶液中、细胞中或者活体中B型单胺氧化酶中的应用，其特征在于，该应用是基于非诊断或者治疗疾病的目的。
9.根据权利要求8所述近红外荧光分子探针的应用，其特征在于，所述近红外荧光分子探针用于筛选B型单胺氧化酶抑制剂类的药物。
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| CN107722057A (zh) * | 2017-11-08 | 2018-02-23 | 中国科学院烟台海岸带研究所 | 基于花菁的有机化合物及其应用 |
| CN109879821A (zh) * | 2019-03-18 | 2019-06-14 | 济南大学 | 一种检测单胺氧化酶b的基于激发态分子内质子转移的荧光探针的制备 |
| CN110218215A (zh) * | 2019-07-30 | 2019-09-10 | 济南大学 | 一种双光子比率型荧光探针在检测单胺氧化酶b中的应用 |
| CN110684523A (zh) * | 2019-10-11 | 2020-01-14 | 中国药科大学 | 一种用于检测硫化氢的近红外荧光分子探针及其制备方法和应用 |
| CN112142718A (zh) * | 2020-09-25 | 2020-12-29 | 中国药科大学 | 用于检测次氯酸的近红外荧光分子探针、制备方法及用途 |
| CN112159396A (zh) * | 2020-09-28 | 2021-01-01 | 中国药科大学 | 一种检测γ-谷氨酰转肽酶近红外荧光分子探针及其制备方法与应用 |
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