Enzymatic Conversion of 6-Nitroquinoline to the Fluorophore 6-Aminoquinoline Selectively under Hypoxic Conditions

Abstract

There is substantial interest in small molecules that can be used to detect or kill the hypoxic (low oxygen) cells found in solid tumors. Nitroaryl moieties are useful components in the design of hypoxia-selective imaging agents and prodrugs because one-electron reductases can convert the nitroaryl group to nitroso, hydroxylamino, and amino metabolites selectively under low oxygen conditions. Here we describe the in vitro, cell free metabolism of a pro-fluorescent substrate, 6-nitroquinoline (1) under both aerobic and hypoxic conditions. Both LC-MS and fluorescence spectroscopic analysis provided evidence that the one-electron reducing enzyme system, xanthine/xanthine oxidase, converted the nonfluorescent parent compound 1 to the known fluorophore 6-aminoquinoline (2) selectively under hypoxic conditions. The presumed intermediate in this reduction process, 6-hydroxylaminoquinoline (6) is fluorescent and can be efficiently converted by xanthine/xanthine oxidase to 2 only under hypoxic conditions. This finding provides evidence for multiple oxygen-sensitive steps in the enzymatic conversion of nitroaryl compounds to the corresponding amino derivatives. In a side reaction that is separate from the bioreductive metabolism of 1, xanthine oxidase converted 1 to 6-nitroquinolin-2(1H)-one (5). These studies may enable the use of 1 as a fluorescent substrate for the detection and profiling of one-electron reductases in cell culture or biopsy samples. In addition, the compound may find use as a fluorogenic probe for detection of hypoxia in tumor models. The occurrence of side products such as 5 in the enzymatic bioreduction of 1 underscores the importance of metabolite identification in the characterization of hypoxia-selective probes and drugs that employ nitroaryl units as oxygen sensors.

Time Course of Aldehyde Oxidase and Why It Is Nonlinear

“…5NQ is reduced by AOX to 5-aminoquinoline. Unlike its isomer, 6-nitroquinoline, 5NQ can be both oxidized and reduced by AOX under normal oxygen condition (Rajapakse et al, 2013). Time-course results indicate that we do see a change in activity with alternate reducing substrate as one would expect if electrons are transferred more slowly (Fig.…”

Conversion of a Dehalogenase into a Nitroreductase by Swapping its Flavin Cofactor with a 5‐Deazaflavin Analogue

“…Enthusiasm for engineering new enzymes to reduce nitroaromatic compounds has grown with their increasing contributions to produce fine chemicals, facilitate medical imaging, activate prodrugs, silence genes, and remove toxins and pollutants from the environment . The most direct method for generating the necessary catalysts involves either mutation of existing nitroreductases (NR) or mining genomic databases for new candidates .…”

Physicochemical Considerations of Tumor Selective Drug Delivery and Activity Confinement with Particular Reference to 1,2-Bis(Sulfonyl)-1- Alkylhydrazines Delivery

“…(b) The subsequent stepwise reduction to give the unstable hydroxylamino and amino products is very rapid. This process is presently not fully understood but appears to involve disproportionation and a series of radical reactions/one electron reductions [ 52 ]. Thus, cytochrome P450 reductase (and xanthine/xanthine oxidase) reduction of nitrobenzene (a model of the nitrobenzylcarbamate trigger/linker) results in mixtures of untouched starting material and hydroxylamino and amino products, but no intermediately reduced species are detected [ 41 ].…”