Laverman Peter, Meeuwissen Silvie A, van Berkel Sander S, Oyen Wim J G, van Delft Floris L, Rutjes Floris P J T, Boerman Otto C
Department of Nuclear Medicine, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands.
Nucl Med Biol. 2009 Oct;36(7):749-57. doi: 10.1016/j.nucmedbio.2009.05.001. Epub 2009 Jul 9.
The spontaneous copper-free tandem 1,3-dipolar cycloaddition-retro-Diels-Alder (tandem crDA) reaction between cyclic Arg-Gly-Asp-d-Phe-Orn(N(3)) [c(RGDfX)] and oxanorbornadiene-DTPA (o-DTPA) or methyloxanorbornadiene-DTPA (mo-DTPA) into two DTPA-c(RGDfX) regioisomers is characterized. Since there is no information on the stability and reaction rate of the tandem crDA reaction in biological media, we set out to characterize these reaction parameters.
The effects of concentration of the reactants, temperature, pH and reaction environment (serum, blood) on the kinetics of the reaction were determined using (111)In-labeled oxanorbornadiene-DTPA analogs. The affinity of the radiolabeled conjugate was determined in a solid-phase alpha(v)beta(3) integrin binding assay. Furthermore, the octanol-water partition coefficient was determined and, finally, the biodistribution of the labeled compounds in mice with subcutaneous alpha(v)beta(3)-expressing tumors was determined.
Fifty percent conversion was reached after 26 h. Kinetic experiments furthermore established that the reaction rate of the tandem crDA reaction follows temperature- and concentration-dependent second-order kinetics, but is independent of the pH of the medium. Affinity of the two [(111)In]DTPA-cRGDfX conjugates for alpha(v)beta(3) integrin is 191 nM. Biodistribution studies showed specific (alpha(v)beta(3)-mediated) uptake of [(111)In]DTPA-c(RGDfX) in the tumor and in alpha(v)beta(3)-expressing tissues.
The tandem crDA reaction using methyl-substituted oxanorbornadiene is a versatile method for a single-step ligation that proceeds independently of pH and also proceeds in serum and blood. Currently, we are further looking into enhancement of reaction kinetics and exploitation of tandem crDA in vivo.
对环状精氨酸-甘氨酸-天冬氨酸-d-苯丙氨酸-鸟氨酸（N(3)）[c(RGDfX)]与氧杂降冰片二烯-二乙三胺五乙酸（o-DTPA）或甲基氧杂降冰片二烯-二乙三胺五乙酸（mo-DTPA）之间自发的无铜串联1,3-偶极环加成-逆狄尔斯-阿尔德（串联crDA）反应生成两种DTPA-c(RGDfX)区域异构体进行了表征。由于尚无关于串联crDA反应在生物介质中的稳定性和反应速率的信息，我们着手对这些反应参数进行表征。
使用（111）铟标记的氧杂降冰片二烯-二乙三胺五乙酸类似物，测定反应物浓度、温度、pH值和反应环境（血清、血液）对反应动力学的影响。在固相α(v)β(3)整合素结合试验中测定放射性标记缀合物的亲和力。此外，测定了辛醇-水分配系数，最后，测定了标记化合物在皮下表达α(v)β(3)肿瘤的小鼠体内的生物分布。
26小时后达到50%的转化率。动力学实验进一步确定，串联crDA反应的反应速率遵循温度和浓度依赖性二级动力学，但与介质的pH值无关。两种[(111)铟]DTPA-cRGDfX缀合物对α(v)β(3)整合素的亲和力为191 nM。生物分布研究表明，[(111)铟]DTPA-c(RGDfX)在肿瘤和表达α(v)β(3)的组织中有特异性（α(v)β(3)介导的）摄取。
使用甲基取代的氧杂降冰片二烯的串联crDA反应是一种通用的单步连接方法，该反应与pH值无关，且在血清和血液中也能进行。目前，我们正在进一步研究提高反应动力学以及在体内利用串联crDA。
