Abstract
1104
Objectives: Commercially available chelates based on DOTA offer convenient routes for assembling new theranostic agents. A peptide-DOTA conjugate made with one of the commercially available bifunctional chelates, isothiocyanatobenzyl-DOTA (SCN-Bn-DOTA), resulted in more than one product peak on HPLC after incubation with non-radioactive lutetium. While benzyl-DOTA isomerism has been described with lanthenizes (particularly Gd), there has been no description and characterization of gallium labeled Bn-DOTA-peptides in the literature. The purpose of this study is to generate and characterize potential isomers of our peptide with LC-MS.
Methods: A peptide with sequence of E’EC6EEEYK which targets glutamate carboxypeptidase II (GCPII) was synthesized with standard Fmoc peptide synthesis with N-terminal urea incorporated using the method discovered by Kozikowski et. al. Purified E’EC6EEEYK was then reacted with SCN-Bn-DOTA in DMSO with diisopropylethylamine. The resulting peptide was purified with HPLC and lyophilized. For gallium labeling, the peptide was dissolved in ultrapure water and incubated with molar excess [natGa]Ga(NO3)3 at 95 deg C for 10 minutes. For radiolabeling, the peptide was incubated with [68Ga]GaCl3 from 68Ge/68Ga generator in a sodium acetate buffer at pH4 at 95 deg C for 10 minutes.
Results: The reverse phase HPLC trace of apo E'EC6EEEYK-Bn-DOTA with C18 analytical column was a single peak with retention time of 10.7 minutes and mass of 1664 (m+1/z) by LC-MS. The natGa labeled peptide exhibited 2 peaks with retention times of 11.2 and 12.2 minutes. Both peaks have the exact same mass of 1731 (m+1/z) which corresponds to the atomic weight of natGa - 3 protons. Radiolabeling with 68Ga resulted in 2 radioactive peaks corresponding to peaks of natGa labeled peptide. The peaks could be purified and exhibited no equilibration at room temperature. Findings indicate gallium incorporated E’EC6EEEYK-Bn-DOTA has two structurally unique species that are identical in charge.
Conclusions: natGa and 68Ga labeling of the E’EC6EEEYK-Bn-DOTA conjugate resulted in two structurally unique species. This is possibly related to TSAP/SAP isomerization of Gadolinium bound chiral-DOTA species described in the literature. Further investigation is worthwhile, as one of the conformations may have enhanced stability for the chelated atom.