Lang Lixin, Ma Ying, Kiesewetter Dale O, Chen Xiaoyuan
Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH) , 31 Center Drive, Building 31, 1C22, Bethesda, Maryland 20892-2281, United States.
Mol Pharm. 2014 Nov 3;11(11):3867-74. doi: 10.1021/mp400706q. Epub 2014 Feb 24.
Glutamic acid is a commonly used linker to form dimeric peptides with enhanced binding affinity than their corresponding monomeric counterparts. We have previously labeled NOTA-Bn-NCS-PEG3-E[c(RGDyK)]2 (NOTA-PRGD2) [1] with [(18)F]AlF and (68)Ga for imaging tumor angiogenesis. The p-SCN-Bn-NOTA was attached to E[c(RGDyK)]2 [2] through a mini-PEG with a thiourea linkage, and the product [1] was stable at radiolabeling condition of 100 °C and pH 4.0 acetate buffer. However, when the same p-SCN-Bn-NOTA was directly attached to the α-amine of E[c(RGDfK)]2 [3], the product NOTA-Bn-NCS-E[c(RGDfK)]2 [4] became unstable under similar conditions and the release of monomeric c(RGDfK) [5] was observed. The purpose of this work was to use HPLC and LC-MS to monitor the decomposition of glutamic acid linked dimeric peptides and their NOTA derivatives. A c(RGDyK) [6] and bombesin (BBN) [7] heterodimer c(RGDyK)-E-BBN [8], and a dimeric bombesin E(BBN)2 [9], both with a glutamic acid as the linker, along with a model compound PhSCN-E[c(RGDfK)] [10] were also studied. All the compounds were dissolved in 0.5 M pH 4.0 acetate buffer at the concentration of 1 mg/mL, and 0.1 mL of each sample was heated at 100 °C for 10 min and the more stable compounds were heated for another 30 min. The samples at both time points were analyzed with analytical HPLC to monitor the decomposition of the heated samples. The samples with decomposition were further analyzed by LC-MS to determine the mass of products from the decomposition for possible structure elucidation. After 10 min heating, the obvious release of c(RGDfK) [5] was observed for NOTA-Bn-NCS-E[c(RGDfK)]2 [4] and Ph-SCN-E[c(RGDfK)] [10]. Little or no release of monomers was observed for the remaining samples at this time point. After further heating, the release of monomers was clearly observed for E[c(RGDyK)]2 [2], E[c(RGDfK)]2 [3], c(RGDyK)-E-BBN [8], and E(BBN)2 [9]. No decomposition or little decomposition was observed for NOTA-Bn-NCS-PEG3-E[c(RGDyK)]2 [1], PEG3-E[c(RGDyK)]2 [11], NOTA-E[c(RGDyK)]2 [12], and NOTA-PEG3-E[c(RGDyK)]2 [13]. The glutamic acid linked dimeric peptides with a free α-amine are labile due to the neighboring amine participation in the hydrolysis. The stability of peptides could be increased by converting the free amine into amide. The instability of thiourea derivatives formed from α-amine was caused by participation of thiol group derived from thiourea.
谷氨酸是一种常用的连接基，用于形成二聚体肽，其结合亲和力比相应的单体肽更高。我们之前已用[(18)F]AlF和(68)Ga标记NOTA-Bn-NCS-PEG3-E[c(RGDyK)]2（NOTA-PRGD2）[1]用于肿瘤血管生成成像。对-SCN-Bn-NOTA通过带有硫脲键的微型聚乙二醇连接到E[c(RGDyK)]2 [2]上，产物[1]在100°C和pH 4.0醋酸盐缓冲液的放射性标记条件下稳定。然而，当相同的对-SCN-Bn-NOTA直接连接到E[c(RGDfK)]2 [3]的α-氨基上时，产物NOTA-Bn-NCS-E[c(RGDfK)]2 [4]在类似条件下变得不稳定，并观察到单体c(RGDfK) [5]的释放。这项工作的目的是使用高效液相色谱法（HPLC）和液相色谱-质谱联用（LC-MS）来监测谷氨酸连接的二聚体肽及其NOTA衍生物的分解情况。还研究了一种c(RGDyK) [6]和蛙皮素（BBN）[7]的异二聚体c(RGDyK)-E-BBN [8]，以及一种以谷氨酸为连接基的二聚体蛙皮素E(BBN)2 [9]，还有一种模型化合物PhSCN-E[c(RGDfK)] [10]。所有化合物都溶解在0.5 M pH 4.0的醋酸盐缓冲液中，浓度为1 mg/mL，每个样品取0.1 mL在100°C加热10分钟，较稳定的化合物再加热30分钟。在两个时间点的样品都用分析型HPLC进行分析，以监测加热样品的分解情况。对有分解的样品进一步用LC-MS分析，以确定分解产物的质量，以便可能阐明结构。加热10分钟后，观察到NOTA-Bn-NCS-E[c(RGDfK)]2 [4]和Ph-SCN-E[c(RGDfK)] [10]有明显的c(RGDfK) [5]释放。在这个时间点，其余样品几乎没有或没有观察到单体释放。进一步加热后，明显观察到E[c(RGDyK)]2 [2]、E[c(RGDfK)]2 [3]、c(RGDyK)-E-BBN [8]和E(BBN)2 [9]有单体释放。对于NOTA-Bn-NCS-PEG3-E[c(RGDyK)]2 [1]、PEG3-E[c(RGDyK)]2 [11]、NOTA-E[c(RGDyK)]2 [12]和NOTA-PEG3-E[c(RGDyK)]2 [13]，未观察到分解或几乎没有分解。由于相邻氨基参与水解，带有游离α-氨基的谷氨酸连接的二聚体肽不稳定。通过将游离胺转化为酰胺可以提高肽的稳定性。由α-氨基形成的硫脲衍生物的不稳定性是由硫脲衍生的硫醇基团参与引起的。
