Application of click chemistry in the synthesis of ⁶⁸Ga-labeled agents

Objectives To synthesize novel 1,2,3-triazole-linked radiolabeled agents by click chemistry

Methods Chelating agents, DO2A- and DO3A-based, were functionalized using excess of alkyne halides (3-bromo-1-propyne or 4-bromo-1-butyne) in the presence of base and were purified by either crystallization or silica gel chromatography. These novel chelating agents were used in copper-catalyzed click reactions with azide-functionalized carbohydrates (acetyl-O-protected beta-D-glucopyranosyl 1’-azide and 2’-azide) in tBuOH/H2O. Final products of click reactions were purified by silica gel chromatography. Alkynyl-modified chelating agents and their carbohydrate click products were labeled with ⁶⁸GaCl₃ in 0.5M NH₄OAc at 65⁰C for 15 min. Their radiochemical purity and yield (RCY%) were assessed by iTLC and radio-HPLC. Cellular uptake studies of click-compounds were performed in A549 lung adenocarcinoma cell line in the presence or absence of glucose.

Results Alkynyl-modified chelating agents were obtained with a total yield of 45-67%. Their structures were confirmed by ESI analysis and 1D 1H NMR spectroscopy. The radiochemical yield of ⁶⁸Ga-labeled alkynyl-tracers reached more than 98% as assessed by iTLC. Carbohydrate-click-conjugates were obtained with final yield of 34-51%. Their labeling with ⁶⁸GaCl₃ proceeded with RCY >97%. In vitro studies revealed 10-times higher uptake of the ⁶⁸Ga-labeled carbohydrate-click conjugates in A549 cancer cells compared to ⁶⁸Ga-DOTA. The results of stability tests of alkynyl-modified chelating agents and their carbohydrate-click-conjugates will be discussed.

Conclusions Novel alkynyl-containing chelating agents were synthesized by modification of two class of tetraaza-macrocyclic compounds, DO2A and DO3A-based. These new attractive tracers allow for selective attachment of azide-modified tumor targeting ligands. Purification of their conjugates is facilitated by introduction of 1,2,3-triazole ring in the “click reaction.” The results of our cellular uptake studies suggest that ⁶⁸Ga-labeled carbohydrate-click-conjugates are transported through glucose transporters