Detecting hypoxia in vitro using ¹⁸F-pretargeted IEDDA “click” chemistry in live cells

Abstract

Bioorthogonal IEDDA “click” can interrogate intracellular hypoxia using a radioactive reporter molecule.

Functional, Aromatic, and Fluorinated Monothiosemicarbazones: Investigations into Their Structures and Activity toward the Gallium-68 Incorporation by Microwave Irradiation

“…Preliminary experiments (under kinetic control) were carried out for the radiolabeling of the free-amine mono­(substituted) compound 4c* with the no-carried added reagent [ 18 F]­fluorobenzaldehyde ([ 18 F]­FBA, route B ). Challenges presented by this radiolabeling route remain, despite promising earlier reports − and in our hands, these mainly resulted from the fact that the desired imines hydrolyzed to some extent under the aqueous reversed-phase high-performance liquid chromatography (RP-HPLC) conditions when traces of trifluoroacetic acid (TFA) are present. Additionally, to achieve the optimum yield for the reaction under thermodynamic control, route A necessitated rather harsh conditions under conventional heating (Scheme ) or microwave irradiation (Experimental Section): these methods were not found to be compatible with the radiochemistry protocols, which were then adapted to make use of a combination of automated and manual labeling methods.…”

“…Compound 4c was further treated with an excess of formic acid to remove the protecting group, thus giving rise to the thiosemicarbazone ligand with a terminal 2-aminoethyl functional group ( 4c* ) (Scheme ). To obtain mono­(thiosemicarbazone) compounds functionalized with a 4-(fluorobenzylidene)­aminoethyl group, the intermediate amine terminal species 4c* was treated with 4-fluoro-benzaldehyde under “cold” chemistry conditions (thermodynamic control) that resemble a recent radiofluorination protocol (which has been carried out under kinetic control), − giving rise to compound 4d . An analogous method was applied for the formation of the p -propyl-benzoyl derivative 4e , a new monothiosemicarbazone functionalized with a terminal alkyne group.…”

“…The “dried” and supported 18 F-fluoride was then dissolved with anhydrous acetonitrile and transferred to a vial, and then the fluorination step was performed manually. Aliquots of dried fluoride were added by syringe to a v-bottom vial containing the precursor 4-trimethylammonium benzaldehyde triflate, a well-established precursor for the synthesis of no-carrier-added [ 18 F]­FBA by the nucleophilic substitution with [ 18 F]­fluoride. − Then the vial was heated at 90 °C for 15 min resulting in the formation of consistently >98% radiochemical purity [ 18 F]­FBA according to radio-HPLC (10.3 min, radio-HPLC Method C), and used further for the labeling reactions of 4c* . To optimize the conditions for the 18 F incorporation into compound 4c* , a variety of tests were conducted to assess the influence of temperature and solvents on labeling efficiency.…”

Recent Advances in Bioorthogonal Click Chemistry for Enhanced PET and SPECT Radiochemistry

“…The Tz-TCO ligation-mediated AZD2281 tracer was taken up more efficiently by MDA-MB-436 cells (high PARP1 protein expression) than by MDA-MB-231 cells (low PARP1 protein expression). Utilizing a TCO-modified small molecule 2-nitroimidazole that was first sequestered in tumor cells and then radiolabeled with a cell-permeable 18 F-labeled tetrazine, the pretargeted IEDDA method was effective for detecting hypoxia in cancer cells . In this strategy, the imaging result showed a higher uptake with a significant difference ( P < 0.0001) in hypoxic cells (90% ID per mg) compared with the uptake of normoxic cells (<10% ID per mg) in an incubation of 18 F-labeled tetrazine.…”

“…For PET imaging or radioimmunotherapy, numerous pretargeting applications for small molecules, metabolic sugars, and antibodies have been developed. Several studies reported that tetrazine analogues bearing various imaging modules, such as fluorescent molecules or radionuclides, were coupled with antibody macromolecules (THIO-MAB, huA33) modified with TCO in order to study the in vivo pretargeting strategy (Figure A,B), ,− while the modification of small molecules with TCO was applied through the incorporation of 18 F-labeled Tz into the small molecule-based radioactive product by Tz/TCO-based pretargeting for detecting hypoxia level (Figure C) . Moreover, Lu et al developed a metabolic pretargeting strategy by conjugating azido sugars with 64 Cu-labeled DBCO tracer using SPAAC reaction for cytotoxic T lymphocytes (CTLs) imaging, whereas a dual-receptor enhanced pretargeting (DRPT) strategy was first reported by the use of IEDDA reaction .…”