【作者】 贾丽娜；

【导师】 张岚；

【作者基本信息】 中国科学院研究生院（上海应用物理研究所） ， 无机化学， 2014， 博士

【摘要】 点击化学（Click Chemistry）是由2001年的诺贝尔奖获得者Sharpless教授提出的，它是一种模块化的合成方法，在生物偶联、药物筛选、材料科学等领域得到了广泛的应用。其中的Cu（I）催化的叠氮与炔的1，3-偶极环加成（CuAAC）反应，几乎成了点击化学的代名词。该反应条件温和，对溶剂和pH不敏感，有优越的区域选择性和极高的化学选择性，并且炔基和叠氮基体积小，容易通过亲核反应或亲电反应引入有机物，这些特点顺应了放射性药物，尤其是短半衰期的正电子药物（PET探针）制备中的种种严格要求，因此本研究希望借助点击化学的众多优势，实现对目标分子的标记，建立简单、高效的PET探针制备技术，并通过生物学评价对其进行筛选，探索其临床应用价值。法尼酯X受体（FXR）在肝再生、肝癌形成、肠道疾病、肠癌形成、胆石病、胆汁淤积和动脉粥样硬化等疾病中扮演着重要角色。因此，本文应用点击化学的标记方法制备评价了一种靶向FXR的胆汁酸类18F-PET探针-18F-CDCA。该PET探针具有较高的标记率（>96%）、放化产率（74±2%，衰变校正）、放化纯度（>99%）和比活度（>320GBq/μmol）。总合成时间（含HPLC纯化时间）为70-80min。18F-CDCA具有良好的体内外稳定性。裸鼠的动态PET/CT显像评价显示肝脏对18F-CDCA摄取迅速，在肝脏几乎均匀分布，并具有适宜的清除时间，这对于肝脏的PET显像极为有利。18F-CDCA在肝脏异常的早期诊断和肿瘤分期中具有重要潜力。G-蛋白偶联受体（TGR5）是一类位于细胞膜上的胆汁酸受体，在多种肿瘤组织中过量表达，并与患者的预后相关，是肿瘤诊断、治疗和预后评估的一个潜在靶点。因此，本文应用点击化学的标记方法制备评价了一种靶向TGR5的胆汁酸类化合物的18F-PET探针-18F-LCA。该探针具有较高的放化产率（66～74%）、比活度（>300GBq/μmol）和放化纯度（>99%）。18F-LCA具有其母体化合物LCA相似的亲脂性，并具有良好的体外稳定性。本工作为进一步研究18F-LCA作为TGR5过量表达的肿瘤的PET探针奠定了良好的基础。F-18标记的生物素是基于生物素-亲和素系统（biotin-avidin system，BAS）的预定位PET显像技术的关键，具有高亲和性和低背景吸收等优势。为了克服以往的生物素的F-18标记方法中存在的合成步骤繁琐，制备耗时，放射化学产率低，标记产物不稳定等缺点，本文应用点击化学的方法制备评价了一种新颖的F-18标记的生物素-18F-Biotin。该PET探针制备方便快捷，具有相对较高的放化产率（67±3%，n=3）、比活度（98±2GBq/μmol，n=3）和放化纯度（>99%），总合成时间为75～80min（含HPLC纯化时间），较文献报道的结果均有所改善。18F-Biotin具有良好的亲水性和体外稳定性，无明显的脱氟或降解现象，为该PET探针应用于进一步的体内评价奠定了良好的基础。本文建立了一种应用一锅法制备多个18F-PET探针以用于对PET探针筛选和临床前研究的策略。具体是以2-[18F]氟叠氮乙烷（[18F]FEA）为Click合成子，反应中以CuSO4/Sodium L-ascorbate为催化体系，在反应体系中同时加入等物质量的几种不同的端炔前体，经过点击化学反应后产物通过一次优化的HPLC分离，即可以在温和条件下一锅制备多个PET探针。若该方法与高通量筛选相结合，有望加速PET探针的发现。更多还原

【Abstract】 Click chemistry introduced by2001Nobel Laureate K. Barry Sharpless is apowerful modular synthesis approach that has been widely applied inbioconjugation chemistry, drug screening and material science. The copper (I)catalyzed cycloaddition between azides and alkynes (CuAAC) to form1,2,3-triazoles is the most well-known ‘click reaction’ and nearly becamesynonymous with the concept of click chemistry. The click reaction meets theneed for requirements in the preparation of radiopharmaceuticals, especially forradiopharmaceuticals with short half-life nuclides (e.g. PET probes) because ofits mild reaction conditions, tolerance of solvents and pH, high chemoselectivityand perfect regioselectivity, meanwhile, azide and alkyne groups are small insize and easy to be introduced into organic compounds by both nucleophilic andelectrophilic processes. Therefore, this paper aimed for radiolabeing of targetmolecules with the advantages of click chemistry and developing simple andefficient preparation techniques of PET probes. Furthermore, the screening ofPET probes was conducted through biological evaluation for the investigation oftheir clinical value.The farnesoid-X-receptor (FXR) plays important roles in liver regeneration,liver tumorigenesis, intestinal diseases, intestinal tumorigenesis, gallstonedisease, cholestasis, and atherosclerosis. Therefore, the PET probe18F-CDCAtargeting FXR which was an18F-labeled bile acid compound was synthesizedwith the click chemistry and biologically evaluated. The PET probe showed ahigh radiolabeling yield (>96%), high decay-corrected radiochemical yield (74±2%), high radiochemical purity (>99%), and high specific activity (>320GBq/μmol) and the overall synthesis time, including HPLC purification, was70-80min.18F-CDCA had a high metabolic stability in vitro and in vivo. PETimaging studies in nude mice indicated a rapid uptake of18F-CDCA into livertissue with uniform distribution of radioactivity and a suitable clearance time in the liver which was beneficial for PET imaging in the liver. Thus, it is potentialfor early detection of abnormalities in the liver and staging of neoplasms with18F-CDCA.G protein-coupled receptor (TGR5) is a cell-surface bile acid receptor whichis a potential target for diagnosis, treatment and prognosis evaluation of thetumors, whose expression is strongly upregulated in many tumors compared toexpression levels in normal tissues and is associated with the prognosis of thepatients. Therefore, the18F-PET probe18F-LCA was prepared and evaluatedusing the click chemistry method with a high radiochemical yield (66～74%),high radiochemical purity (>99%), and high specific activity (>300GBq/μmol).18F-LCA had a similar lipophilicity to the parental bile acid lithocholic acid anda high metabolic stability in vitro. This work made a solid foundation for furtherstudy the18F-LCA as the PET probe for the tumors of TGR5-overexpression.F-18labeled biotin was the key of pretargeting PET imaging based onbiotin-avidin system (BAS) with higher binding affinity and lower backgroundactivity. In order to overcome the disadvantages of tedious multi-step procedure,time-consuming preparation, low radiochemical yield and instability ofradiolabeling product as previous reports, the18F-labeled biotin was preparedand evaluated with the click chemistry method. It’s convenient and efficient tosynthesize18F-biotin with a high radiochemical yield (67±3%), and high specificactivity (98±2GBq/μmol), high radiochemical purity (>99%) and the overallsynthesis time, including HPLC purification, was75-80min, which wereimproved compared with previous reports.18F-biotin had good hydrophilicityand good stability in vitro, defluorination or radiometabolites were not detectedafter long-term incubation, which provided good foundation for further study invivo.A novel strategy for labeling multiple18F-PET probes in one pot has beendeveloped for PET probes screening and preclinical studies. In this study,2-[18F]fluoroethylazide was selected as the prosthetic group, CuSO4/Sodium L-ascorbate was selected as the catalytic system. It simply requires the additionof equal amount of multiple terminal alkynyl precursors to the click reactionsystem, and then multiple PET probes could be prepared in one pot under mildreaction conditions after separation with appropriate HPLC gradients in a singlerun. If combined with high throughout screening, the discovery ofhigh-efficiency PET probes may be accelerated.更多还原