【作者】 杨欣；

【导师】 钱鹰；

【作者基本信息】 东南大学 ， 有机化学， 2019， 硕士

【摘要】 近年来,荧光成像探针作为一种新兴的检测手段而吸引了研究者的注意。近红外荧光成像技术具有较强的组织穿透能力、对样品光损伤小、可避免背景荧光干扰等优点而表现出较广阔的发展空间。荧光探针是实现生物成像的分子工具,核心就是荧光团的选择。罗丹明荧光团具有摩尔消光系数大、量子产率高及独特的螺环结构控制荧光开关等优点,被广泛用于荧光探针的构建中,但其吸收及发射波长均位于可见光区域,这严重限制了这类染料在生物成像中的应用。花菁类荧光染料吸收及发射均位于近红外区域,但用于荧光探针构建时有较强的背景荧光和较弱的水溶性。基于上述问题,我们设计合成了三种近红外探针分子,具有长波长发射的谷胱甘肽荧光探针Rh NM,完全应用于水溶液中检测半胱氨酸的荧光探针Ay SA,用于光动力治疗中的近红外光敏剂Cy OH并对三种探针分子的结构进行了核磁共振氢谱、碳谱及高分辨质谱表征。(1)本文通过knoevenagel缩合反应设计合成了氧杂蒽类近红外谷胱甘肽(GSH)探针Rh NM;通过Retro-knoevenagel缩合反应设计合成了近红外全水溶性半胱氨酸(Cys)探针;最后设计合成了一种光动力治疗中的近红外光敏剂Cy OH。(2)谷胱甘肽(GSH)在人体生理活动扮演着重要角色,其不正常的含量与人体的某些疾病密切相关,因此对它们的检测也至关重要。故本文基于氧杂蒽结构,重新修饰罗丹明结构以延长波长,通过knoevenagel缩合反应,将费歇尔醛连接至罗丹明O原子活泼α-H位置用来延长波长,马来酰亚胺基苯甲酰氯通过亲核取代反应和罗丹明乙二胺相连,构筑了长波长发射的谷胱甘肽荧光探针。我们对其性能进行了研究,结果表明探针可以实现对GSH的专一性识别及裸眼识别,识别机理是两级联动反应:迈克尔加成反应及螺酰胺环打开,发射波长为746 nm、量子产率为0.36、响应时间快、p H范围广,并成功进行了Hep G-2细胞内外源性GSH成像。总之,探针Rh NM可用作生物体中GSH传感器,定量检测生理活动中的GSH含量。(3)半胱氨酸(Cys),可以促进细胞内氧化还原活性、异生物质代谢和细胞内信号转导等过程。当生物体内缺乏半胱氨酸时会出现生长迟缓,肝脏受损,甚至皮肤病变。因此,对生物体内半胱氨酸的检测具有重要的意义。故本章设计合成了一种新颖的近红外荧光探针,用于在水溶液中检测半胱氨酸。我们在花菁荧光母核上引入长链磺酸基团用作水溶性官能团,丙烯酰氯基团作为半胱氨酸的识别受体,通过共轭加成及分子内环化的机制专一性识别半胱氨酸。结果表明,在100%水溶液中,探针在五分钟内显示出对Cys的高辨别力,并且发射波长为701 nm。其具有较广的p H范围及可以在5分钟内实现对Cys的检测,并对Bel-7402细胞内外源性的Cys进行了荧光成像;故探针Ay SA在检测组织及动物中的半胱氨酸具有很好的应用前景。(4)光动力治疗,是一种利用光敏剂和激光激活治疗肿瘤的新方法。故本章合成了一种独特的水溶性七甲川花菁染料作为近红外光敏剂。使用长链磺酸基作为水溶性官能团,4-氨基-2,2,6,6-四甲基哌啶-N-氧基部分(TEMPOH)作为氮氧自旋标记,以获得体内生化反应信息。正如所料,光敏剂在体外光动力疗法中表现良好,具有较低的暗毒性及较高的光毒性及较高的单线态氧量子产率(φ_△=16.96%)。它具有出色的细胞摄取能力和较好的细胞相容性。将其成功应用于近红外荧光成像和AO/EB染色。总之,基于七甲川菁类染料的光敏剂在体内癌症治疗中具有很大的潜力,为癌症的治疗提供了新的选择。更多还原

【Abstract】 In recent years,fluorescent imaging probes have attracted the attention of researchers as an emerging detection method.Near-infrared fluorescence imaging technology has great space for development due to its strong tissue penetration ability,smaller damage to the sample,little background fluorescence interference and so on.Fluorescent probes were as molecular tools for bioimaging,and the core is the choice of fluorophores.The rhodamine fluorophore has the advantages of large molar extinction coefficient,high quantum yield and unique spiro structure control fluorescent switch.It is widely used in the construction of fluorescent probes,but its absorption and emission are located in the visible region,which is severely limited its application in bioimaging.The absorption and emission wavelengths of the cyanine-based fluorescent dyes are all located in the near-infrared region,but it has strong background fluorescence and poor water solubility when they used as fluorescence probes.Based on the above question,we designed and synthesized three near-infrared probe molecules,a glutathione fluorescent probe named Rh NM with long wavelength emission;cysteine fluorescent probe Ay SA applied in pure water solution;A near-infrared photosensitizer Cy OH for used as photosentizer in photodynamic therapy;the compound of three dyes were characterized by ¹H NMR,¹³C NMR,MS.1)In this paper,the near-infrared GSH probe Rh NM was designed and synthesized by the knoevenagel condensation reaction based on xanthene;the near-infrared water-soluble Cys probe was synthesized by retro-knoevenagel condensation reaction.Finally,near-infrared photosensitizer Cy OH in photodynamic therapy was synthesized.2)Glutathione plays an important role in human physiological activities,and its abnormal content is closely related to certain diseases of the human body,it is also crucial for their detection.Therefore,in this paper,based on the structure of xanthene,the rhodamine structure was re-modified to prolong the absoption and emmission wavelength to NIR region.2,2-（1,3,3-trimethylindolin-2-ylidene）-acetaldehyde was conjugated with rhodamine by Knoevenagel condensation to shift the wavelength to NIR region,The maleimidobenzoyl chloride was linked to rhodamine ethylenediamine by nucleophilic substitution reaction,then long wavelength glutathione fluorescent probe was constructed.The recognizition performance of probe was investigated and the results show that the probe can selectively recognize GSH with nake eye.The recognition mechanism is based Michael addition reaction and the GSH induced spiro ring opening,the emission wavelength reached to 746 nm and the quantum yield for 0.36.It can react with GSH in few minites,the p H range is wide,and the probe was also successfully applied in Hep G-2 cells for exogenous GSH imaging.In summary,the probe Rh NM can be used as a GSH sensor in an organism to quantitatively detect GSH content in physiological activities.3)Cysteine（Cys）can promote intracellular redox activity,heterogeneous metabolism and intracellular signal transduction.When the body lacks cysteine,it may accured growth retardation,liver damage,and even skin lesions et al.So the detection of cysteine in living organisms is of great significance.Therefore,this chapter designed and synthesized a novel near-infrared fluorescent probe for the detection of cysteine in pure aqueous solution.Sulfonic acid group was linked to cyanine fluorescent core used as water-soluble functional group,and an acryloyl chloride group as a recognition receptor for cysteine based on conjugate addition and intramolecular cyclization.The results showed that in a 100%aqueous solution,the probe showed a high discrimination against Cys in a few minutes and the emission wavelength reach to 701 nm.It has a wide p H range and fast response time for 5minites,and was applied in Bel-7402 cells for exogenous Cys imaging.Ay SA has a good application in detecting cysteine in tissues and animals.4)Photodynamic therapy is a new method for the treatment of tumors using photosensitizers and laser activation.Therefore,this chapter synthesizes a unique water-soluble heptamethine cyanine dye as a near-infrared PDT photosensitizer.Sulfonic acid was used as a water-soluble functional group,4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl moiety（TEMPOH）is used as a nitrogen-oxygen spin label to obtain biochemical reaction information in vivo.As expected,photosensitizers performed well in in vitro photodynamic therapy.It has low dark toxicity and large phototoxicity and high singlet oxygen quantum yield（φ△=16.96%）.It has excellent cell uptake and good cell compatibility.It was successfully applied to near-infrared fluorescence imaging and AO/EB staining.In summary,Heptamethine cyanine dye used as a photosensitizer have great potential for cancer treatment in vivo,providing a new choice for the treatment of cancer.更多还原