Wurzer Alexander, Vágner Adrienn, Horváth Dávid, Fellegi Flóra, Wester Hans-Jürgen, Kálmán Ferenc K, Notni Johannes
Pharmaceutical Radiochemistry, Technische Universität München, Munich, Germany.
Department of Inorganic and Analytical Chemistry, University of Debrecen, Debrecen, Hungary.
Front Chem. 2018 Apr 10;6:107. doi: 10.3389/fchem.2018.00107. eCollection 2018.
Due to its 4 carbonic acid groups being available for bioconjugation, the cyclen tetraphosphinate chelator DOTPI, 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetrakis[methylene(2-carboxyethylphosphinic acid)], represents an ideal scaffold for synthesis of tetrameric bioconjugates for labeling with radiolanthanides, to be applied as endoradiotherapeuticals. We optimized a protocol for bio-orthogonal DOTPI conjugation via Cu(I)-catalyzed Huisgen-cycloaddition of terminal azides and alkynes (CuAAC), based on the building block DOTPI(azide). A detailed investigation of kinetic properties of Cu(II)-DOTPI complexes aimed at optimization of removal of DOTPI-bound copper by transchelation. Protonation and equilibrium properties of Ca(II)-, Zn(II), and Cu(II)-complexes of DOTPI and its tetra-cyclohexylamide DOTPI(Chx) (a model for DOTPI conjugates) as well as kinetic inertness (transchelation challenge in the presence of 20 to 40-fold excess of EDTA) were investigated by pH-potentiometry and spectrophotometry. Similar stability constants of Ca-, Zn, and Cu-complexes of DOTPI (log = 8.65, log = 15.40, log = 20.30) and DOTPI(Chx) (log = 8.99, log = 15.13, log = 20.42) were found. Transchelation of Cu(II)-complexes occurs via proton-assisted dissociation, whereafter released Cu(II) is scavenged by EDTA. The corresponding dissociation rates [ = 25 × 10 and 5 × 10 s for Cu(DOTPI) and Cu(DOTPI(Chx)), respectively, at pH 4 and 298 K] indicate that conjugation increases the kinetic inertness by a factor of 5. However, demetallation is completed within 4.5 and 7.2 h at pH 2 and 25°C, respectively, indicating that Cu(II) removal after formation of CuAAC can be achieved in an uncomplicated manner by addition of excess HEDTA. For proof-of-principle, tetrameric DOTPI conjugates of the prostate-specific membrane antigen (PSMA) targeting motif Lys-urea-Glu (KuE) were synthesized via CuAAC as well as dibenzo-azacyclooctine (DBCO) based, strain-promoted click chemistry (SPAAC), which were labeled with Lu-177 and subsequently evaluated and in SCID mice bearing subcutaneous LNCaP tumor (PSMA+ human prostate carcinoma) xenografts. High affinities (3.4 and 1.4 nM, respectively) and persistent tumor uptakes (approx. 3.5% 24 h after injection) confirm suitability of DOTPI-based tetramers for application in targeted radionuclide therapy.
由于环轮烯四膦酸酯螯合剂DOTPI（1,4,7,10 - 四氮杂环十二烷 - 1,4,7,10 - 四[亚甲基（2 - 羧乙基膦酸）]）有4个碳酸基团可用于生物共轭，它是合成用于放射性镧系元素标记的四聚体生物共轭物的理想支架，可作为体内放射治疗剂应用。我们基于构建模块DOTPI(叠氮化物)，通过铜（I）催化的末端叠氮化物和炔烃的胡伊斯根环加成反应（CuAAC），优化了一种用于生物正交DOTPI共轭的方案。对Cu（II） - DOTPI配合物的动力学性质进行了详细研究，旨在通过转螯合作用优化去除与DOTPI结合的铜。通过pH电位滴定法和分光光度法研究了DOTPI及其四环己基酰胺DOTPI(Chx)（DOTPI共轭物的模型）的Ca（II） - 、Zn（II） - 和Cu（II） - 配合物的质子化和平衡性质，以及动力学惰性（在20至40倍过量的EDTA存在下的转螯合挑战）。发现DOTPI（log = 8.65，log = 15.40，log = 20.30）和DOTPI(Chx)（log = 8.99，log = 15.13，log = 20.42）的Ca - 、Zn - 和Cu - 配合物具有相似的稳定常数。Cu（II） - 配合物的转螯合作用通过质子辅助解离发生，之后释放的Cu（II）被EDTA清除。相应的解离速率[在pH 4和298 K时，Cu(DOTPI)和Cu(DOTPI(Chx))分别为25×10和5×10 s]表明共轭使动力学惰性增加了5倍。然而，在pH 2和25°C下，脱金属分别在4.5小时和7.2小时内完成，这表明在形成CuAAC后，通过加入过量的HEDTA可以以简单的方式实现Cu（II）的去除。为了进行原理验证，通过CuAAC以及基于二苯并氮杂环辛炔（DBCO）的应变促进点击化学（SPAAC）合成了靶向前列腺特异性膜抗原（PSMA）基序Lys - 脲 - Glu（KuE）的四聚体DOTPI共轭物，用Lu - 177进行标记，随后在携带皮下LNCaP肿瘤（PSMA + 人前列腺癌）异种移植瘤的SCID小鼠中进行了评估。高亲和力（分别为3.4和1.4 nM）和持续的肿瘤摄取（注射后24小时约为3.5%）证实了基于DOTPI的四聚体适用于靶向放射性核素治疗。
