Selvaraj Ramajeyam, Giglio Benjamin, Liu Shuanglong, Wang Hui, Wang Mengzhe, Yuan Hong, Chintala Srinivasa R, Yap Li-Peng, Conti Peter S, Fox Joseph M, Li Zibo
†Brown Laboratories, Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19803, United States.
‡Department of Radiology and Biomedical Research Imaging Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
Bioconjug Chem. 2015 Mar 18;26(3):435-42. doi: 10.1021/acs.bioconjchem.5b00089. Epub 2015 Mar 4.
The fast kinetics and bioorthogonal nature of the tetrazine trans-cyclooctene (TCO) ligation makes it a unique tool for PET probe construction. In this study, we report the development of an (18)F-labeling system based on a CF3-substituted diphenyl-s-tetrazine derivative with the aim of maintaining high reactivity while increasing in vivo stability. c(RGDyK) was tagged by a CF3-substituted diphenyl-s-tetrazine derivative via EDC-mediated coupling. The resulting tetrazine-RGD conjugate was combined with a (19)F-labeled TCO derivative to give HPLC standards. The analogous (18)F-labeled TCO derivative was combined with the diphenyl-s-tetrazine-RGD at μM concentration. The resulting tracer was subjected to in vivo metabolic stability assessment, and microPET studies in murine U87MG xenograft models. The diphenyl-s-tetrazine-RGD combines with an (18)F-labeled TCO in high yields (>97% decay-corrected on the basis of TCO) using only 4 equiv of tetrazine-RGD relative to the (18)F-labeled TCO (concentration calculated based on product's specific activity). The radiochemical purity of the (18)F-RGD peptides was >95% and the specific activity was 111 GBq/μmol. Noninvasive microPET experiments demonstrated that (18)F-RGD had integrin-specific tumor uptake in subcutaneous U87MG glioma. In vivo metabolic stability of (18)F-RGD in blood, urine, and major organs showed two major peaks: one corresponded to the Diels-Alder conjugate and the other was identified as the aromatized analog. A CF3-substituted diphenyl-s-tetrazine displays excellent speed and efficiency in (18)F-PET probe construction, providing nearly quantitative (18)F labeling within minutes at low micromolar concentrations. The resulting conjugates display improved in vivo metabolic stability relative to our previously described system.
四嗪-反式环辛烯（TCO）连接反应的快速动力学和生物正交性质使其成为构建PET探针的独特工具。在本研究中，我们报道了一种基于CF3取代的二苯基-s-四嗪衍生物的（18）F标记系统的开发，目的是在提高体内稳定性的同时保持高反应活性。通过EDC介导的偶联反应，用CF3取代的二苯基-s-四嗪衍生物标记c（RGDyK）。将所得的四嗪-RGD缀合物与（19）F标记的TCO衍生物结合，得到HPLC标准品。将类似的（18）F标记的TCO衍生物与二苯基-s-四嗪-RGD以微摩尔浓度混合。对所得示踪剂进行体内代谢稳定性评估，并在小鼠U87MG异种移植模型中进行微型PET研究。相对于（18）F标记的TCO（根据产物比活计算浓度），仅使用4当量的四嗪-RGD，二苯基-s-四嗪-RGD就能以高产率（基于TCO的衰变校正后>97%）与（18）F标记的TCO结合。（18）F-RGD肽的放射化学纯度>95%，比活为111 GBq/μmol。非侵入性微型PET实验表明，（18）F-RGD在皮下U87MG胶质瘤中具有整合素特异性肿瘤摄取。（18）F-RGD在血液、尿液和主要器官中的体内代谢稳定性显示出两个主要峰：一个对应于狄尔斯-阿尔德共轭物，另一个被鉴定为芳构化类似物。CF3取代的二苯基-s-四嗪在（18）F-PET探针构建中显示出优异的速度和效率，在低微摩尔浓度下几分钟内就能实现几乎定量的（18）F标记。相对于我们之前描述的系统，所得缀合物的体内代谢稳定性有所提高。
