Stability analysis of glutamic acid linked peptides coupled to NOTA through different chemical linkages

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.