A method for site-specific radiolabeling of the serine protease active site inhibited factor seven (FVIIai) with ⁶⁴Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in hemostasis, angiogenesis, proliferation, cell migration, and survival of cancer cells. First a single azide moiety was introduced in the active site of this 50 kDa protease. Then a NOTA moiety was introduced via a strain promoted azide–alkyne reaction and the corresponding conjugate was labeled with ⁶⁴Cu. Binding to TF and the stability was evaluated in vitro. TF targeting capability of the radiolabeled conjugate was tested in vivo by positron emission tomography (PET) imaging in pancreatic human xenograft cancer mouse models with various TF expressions. The conjugate showed good stability (>91% at 16 h), an immunoreactivity of 93.5%, and a mean tumor uptake of 2.1 ± 0.2%ID/g at 15 h post injection. In conclusion, FVIIai was radiolabeled with ⁶⁴Cu in single well-defined position of the protein. This method can be utilized to prepare conjugates from serine proteases with the label at a specific position.

中文翻译：

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使用无铜点击化学法对丝氨酸蛋白酶，活性位点抑制因子七叠氮化物（FVIIai-N ₃）进行位点特异性⁶⁴ Cu标记

已经使用双正交点击反应应用了一种用⁶⁴ Cu进行丝氨酸蛋白酶活性位点抑制因子七（FVIIai）的位点特异性放射性标记的方法。FVIIai与组织因子（TF）结合，后者是一种涉及止血，血管生成，增殖，细胞迁移和癌细胞存活的跨膜蛋白。首先，将单个叠氮化物部分引入该50kDa蛋白酶的活性位点。然后通过促进叠氮化物-炔反应的应变引入NOTA部分，并用⁶⁴ Cu标记相应的结合物。结合TF和稳定性进行了体外评价。在体内测试了放射性标记缀合物的TF靶向能力通过正电子发射断层扫描（PET）成像在具有各种TF表达的胰腺人类异种移植癌小鼠模型中。该结合物显示出良好的稳定性（在16小时时＞ 91％），免疫反应性为93.5％，并且在注射后15小时时平均肿瘤摄取为2.1±0.2％ID / g。总之，FVIIai在该蛋白的单个明确定义的位置进行了⁶⁴ Cu的放射性标记。该方法可用于由丝氨酸蛋白酶制备结合物，其标记物位于特定位置。