【作者】 杨斌；

【导师】 王博；

【作者基本信息】 吉林大学 ， 有机化学， 2024， 博士

【摘要】 生物酶是生物体中不可或缺的组成部分,在生化反应和生命活动中起到催化和调控作用,对于生物体内各种功能的正常运作至关重要。丝氨酸水解酶(Serine hydrolases,SHs)是哺乳动物体内一类重要的超家族酶。人体中存在超过200种SHs,它们不仅在基因表达、新陈代谢、神经信号传递、免疫反应等生理过程中发挥着重要作用,还与肥胖、炎症、癌症、肝损伤、糖尿病、细菌感染、阿尔茨海默病等疾病密切相关。因此,开发有效的成像工具以实现对生物体内SHs的精确、实时、可视化监测,对于SHs生物学功能的揭示及相关疾病的诊疗和病理研究均具有重要意义。有机小分子荧光探针具有分子化学结构易于调控、灵敏度高、生物安全性好等优点,基于其可通过无侵入性、无放射性的荧光成像技术实现对生物体内SHs的实时动态监测,在生物化学、医学等领域有着广阔的应用前景,受到了科研人员的广泛关注。本论文研究工作基于分子化学结构调控策略,综合考虑影响荧光团发光性能的共轭、刚性平面和取代基效应并参考理论计算和分子对接模拟结果,基于分子内电荷转移(Intramolecular charge transfer,ICT)传感模式,设计并合成了红光荧光团PFQ和近红外荧光团CHQ。针对生物体内SHs可视化检测需求,通过进一步修饰识别基团,基于上述荧光团构建了以羧酸酯酶2型(Carboxylesterase-2,CES2)和丁酰胆碱酯酶(Butyrylcholinesterase,BCh E)这两种重要的SHs为目标分析物的4种有机小分子荧光探针,在分子化学结构层面上实现了对探针分析检测性能的调控。对上述探针的光谱特性、响应机制、分析检测与成像性能、前线轨道能级分布及酶蛋白分子对接情况和生物安全性进行了详细探究,并进一步测试了其在多种疾病诊疗过程中的应用潜力。具体研究内容如下:(1)基于将强吸电子基团喹啉鎓盐和强给电子基团羟基调控于分子化学结构两端以构建ICT系统的策略,设计并合成了一种新型荧光团PFQ。PFQ具有红光发射波长(655 nm)、光稳定性好、p H适用范围广等优点,较大的斯托克斯位移(135 nm)使其能够有效避免检测时的光谱串扰和生物成像时的光散射影响。通过将作为识别基团的苯甲酰基修饰至PFQ分子骨架中,开发了一种能够检测CES2的荧光探针PFQ-E。PFQ-E自身荧光信号微弱,而CES2能够识别并“剪切”PFQ-E中的苯甲酸酯结构,从而释放荧光团PFQ,实现荧光开启型响应。PFQ-E灵敏度高、选择性好、对CES2亲和性强,具有较好的光稳定性和生物安全性,适用于生化分析和生物成像。细胞成像和生物组织成像结果表明PFQ-E能够监测细胞及组织中CES2的空间分布和活性变化。以PFQ-E为成像工具,成功揭示了扑热息痛诱导肝损伤(APAP-induced liver injury,AILI)过程中CES2的表达下调,并验证了扑热息痛诱导肝毒性的分子机制。(2)参考理论计算与分子对接模拟结果,基于荧光团PFQ和识别基团环丙甲酰基,开发了一种能够高选择性响应BCh E的荧光探针PFQ-B。该探针自身荧光信号微弱,在BCh E的催化水解作用下生成PFQ,从而产生强烈的红光信号(655 nm)。PFQ-B具有可溶于水、灵敏度与选择性高、稳定性强等优点,可通过荧光法和比色法实现对BCh E的定量检测。细胞活性实验和溶血实验结果表明PFQ-B具有较低的细胞毒性和良好的生物相容性。PFQ-B能够实现活细胞和新鲜组织切片中内源性BCh E的可视化成像,细胞亚结构定位实验表明PFQB的细胞膜穿透性能较好且在细胞中主要分布于非细胞核区域。小鼠脑部成像实验结果显示PFQ-B可区分阿尔茨海默病(Alzheimer’s disease,AD)模型小鼠与正常小鼠。(3)基于刚性环状结构可抑制分子内部单键旋转能力并增强分子的共面性,进而提高分子整体共轭程度的荧光分子设计策略,引入二氢氧杂蒽刚性环状结构,构建了一种新型近红外荧光团CHQ,实现了分子荧光性能的结构调控。该荧光团的激发和发射波长峰值均位于近红外区(715 nm/775 nm)且光稳定性好,在降低生物背景荧光干扰、提高组织穿透深度方面具有优势。通过引入识别基团苯甲酰基对荧光团CHQ做进一步的化学结构修饰,开发了一种能够快速灵敏检测CES2的近红外荧光探针CHQ-E。该探针具有响应速度极快、灵敏度与选择性高、光稳定性好、生物相容性佳等优点。生物成像实验结果表明CHQ-E可实现对Hep G2和HCT-116细胞内源性CES2的实时监测及小鼠腹腔内CES2分布与活性的可视化和药物性肝损伤过程中肝脏健康状况的评估。组织断层扫描结果显示CHQ-E具有良好的组织穿透能力,优于已报道的CES2有机小分子荧光探针,可实现生物组织内CES2的三维成像。(4)通过在荧光团CHQ分子结构上修饰环丙甲酰基作为识别基团,开发了一种灵敏的BCh E近红外荧光探针CHQ-B,并将其应用于糖尿病动物模型中BCh E表达情况的探索。CHQ-B自身荧光信号微弱,在BCh E作用下会生成荧光团CHQ,实现高达30倍的荧光信号增强。CHQ-B不易光解且能有效避免包括乙酰胆碱酯酶在内的多种物质干扰,具有优异的光稳定性和选择性,可实现BCh E荧光法和比色法双模式定量检测。细胞和生物成像实验结果表明CHQ-B适用于细胞内源性BCh E的监测和糖尿病模型小鼠中BCh E表达情况的探究,并证实了糖尿病导致的BCh E表达上调。综上所述,本论文工作基于对有机荧光团分子的光学性能调控和识别基团化学修饰策略,开发了系列酶激活有机小分子荧光探针,并将其用于丝氨酸水解酶家族成员的定量检测和细胞及组织中酶分布与活性变化的可视化,亦能进一步扩展应用于酶的生物学功能研究和相关疾病产生与修复过程的监控。本论文工作所提出的荧光分子化学结构调控策略可为后续类似研究工作提供参考,所开发的探针为相关酶活性的准确评估及相关疾病的临床诊疗和病理研究提供了新的工具。更多还原

【Abstract】 As the indispensable components of organisms,bio-enzymes are a class of natural catalysts with high efficiency and specificity for catalyzing biochemical reactions and regulating physiology activities,and play crucial roles in maintaining the body’s overall functions.Serine hydrolases(SHs)are one of the largest and most diverse enzyme classes in the mammal body.There are more than 200 SHs in the human body,that not only participate in various physiological processes,but also are found to be related to many diseases,such as obesity,inflammation,cancer,liver injury,diabetes,bacterial infection and Alzheimer’s disease(AD).Therefore,the development of effective methods for accurate,real-time and visual monitoring of the distribution and activity of SHs in vivo is significant for both exploring the physiological functions of SHs in organisms and improving diagnosis or therapy of diseases.Organic fluorophore-based probes have many advantages,including easy regulation of molecular chemical structure,high sensitivity and biosecurity,by which the non-invasive,non-radioactive and real-time fluorescence imaging of SHs in vivo can be performed.Due to their great application prospects in biochemical and clinical researches,organic small-molecule fluorescent probes have attracted much attention from researchers.In this work,two kinds of fluorophores with intramolecular charge transfer(ICT)effect were designed and synthesized based on chemical structure regulation strategy and results of theoretical calculation and molecular docking simulations.To meet the requirements of fluorescent visualization of SHs,four enzyme-activated fluorescent probes were developed by modifying corresponding recognition groups on the above fluorophores for sensing carboxylesterase-2(CES2)and butyrylcholinesterase(BCh E)in vivo.Fluorescent properties,sensing mechanisms,biocompatibility,analytical performance and application potential of the developed probes were investigated and evaluated.The detailed research works are listed as follows:(1)A fluorophore PFQ was designed and synthesized by regulating the electronwithdrawing group and the electron-donating group at opposite sides of the molecule.The intramolecular charge transfer(ICT)was attributed as the photoluminescence mechanism of PFQ.PFQ exhibits a red maximum emission wavelength(655 nm),good photostability and a wide application range of environmental p H.A large Stokes shift(135 nm)facilitates the suppression of spectral interference induced by the bandwidth and the scattering of excitation light during the analyses of biosamples.A sensitive CES2-activatable fluorescent probe PFQ-E was developed by modifying PFQ with benzoyl group as the recognition group.PFQ-E exhibits a weak fluorescence signal.After the cleavage of the benzoyl group by CES2-catalyzed hydrolysis reaction,the fluorophore PFQ will be generated and result in a fluorescence "turn-on" response.PFQ-E exhibits high sensitivity,good selectivity and strong affinity towards CES2,and displays good photostability and biocompatibility,which make it suitable for bioanalysis and bioimaging.The spatial distribution and activity variation of endogenous CES2 in tissues as well as living cells were monitored by PFQ-E,and the down-regulation of CES2 activity in acetaminophen-induced liver injury(AILI).The production and remediation pathways of AILI and its underlying molecular mechanism were revealed.(2)A highly sensitive fluorescent probe PFQ-B was also developed by linking cyclopropanecarbonyl group as the recognition group to PFQ.The fluorescence signal of PFQ-B is very weak,while BCh E can catalyze the hydrolysis of PFQ-B to release the fluorophore PFQ,resulting in a bright red fluorescence signal.PFQ-B exhibits excellent performance,including good water solubility,high sensitivity,good photostability,good selectivity and a wide application range of p H,and can be employed for the quantitative detection of BCh E by fluorometry and colorimetry.Results of cell viability and hemolysis experiments demonstrated the low cytotoxicity and good biocompatibility of PFQ-B.PFQ-B can also be utilized to visualize endogenous BCh E in living cells and tissues.Results of the cell substructure localization experiments revealed the good cell-penetrating ability and the non-nucleus distribution of PFQ-B.PFQ-B was applied to imaging BCh E in brain tissues of AD mouse models and normal mice.Results demonstrated the excellent performance of PFQ-B for discriminating AD tissues.(3)A near-infrared(NIR)fluorophore CHQ was designed and synthesized by introducing dihydroxanthene group to PFQ.The rigid ring chemical structure of dihydroxanthene group will inhibit the rotation of single bonds,enhance the planarity,and improve the overall conjugation degree of the CHQ molecule.Both the excitation and the emission maxima of CHQ are in the NIR region(715 nm and 775 nm,respectively),which improves tissue penetration depth,and eliminates autofluorescence and photodamage of biosamples during analyses.A novel NIR fluorescent probe CHQ-E was developed for the rapid and sensitive detection of CES2 by linking a recognition group(benzoyl)to CHQ.The benzoyl group can be easily cleaved from CHQ-E by CES2,resulting in the "turn-on" fluorescence.CHQ-E displayed superior analytical performance for sensing endogenous CES2 in living Hep G2 and HCT-116 cells,including high sensitivity,good photostability and biosafety.Results of tomographic scanning experiment using hepatic tissue of mouse revealed the three-dimensional imaging capability of CHQ-E.CHQ-E was applied to monitoring in vivo CES2 activity in drug-induced hepatotoxicity and remediation models,demonstrating its broad potential for the assessment of liver health status.(4)By respectively employing CHQ and cyclopropanecarbonyl group as the fluorophore and the recognition group,a sensitive NIR fluorescent probe CHQ-B was also developed to investigate the expression of BCh E in tissues of diabetic mouse models.The fluorescence signal of CHQ-B is very weak due to the inhibition of ICT process induced by the cyclopropanecarbonyl group.After the cyclopropanecarbonyl group is specifically cleaved from CHQ-B by the BCh E-catalyzed hydrolysis reaction,and the consequent product CHQ will cause 30-fold fluorescence "turn-on" response.The probe CHQ-B exhibits excellent photostability,good water solubility,and highly sensitive and selective responses towards BCh E,and is suitable for quantitative detection of BCh E by fluorescent and colorimetric modes.Additionally,the good biocompatibility of CHQ-B facilitates its application in fluorescent imaging of BCh E in vivo.Experimental results of fluorescence imaging diabetic mouse models confirm the up-regulation of BCh E expression induced by diabetes.In summary,the chemical structure regulation strategy as well as the recognition group modification strategy were proved to be effective for the development of organic small-molecule fluorescent probes with enzyme-activated response.All the organic small-molecule fluorescent probes developed in this work are adequate for both quantitative detection of the activity and visual imaging of the distribution of corresponding SHs in living cells or tissues,and are easy to be applied to related research processes of enzymatic functions or the production and remediation pathways of enzyme-related diseases and their underlying molecular mechanism.The strategies of chemical structure regulation and modification of fluorophore molecules proposed in this work can provide effective reference for similar research works in the future.The probes developed in this work provide novel tools for clinical diagnosis and therapy.更多还原