Bioorthogonal Catalysis: A General Method To Evaluate Metal-Catalyzed Reactions in Real Time in Living Systems Using a Cellular Luciferase Reporter System

Abstract

The development of abiological catalysts that can function in biological systems is an emerging subject of importance with significant ramifications in synthetic chemistry and the life sciences. Herein we report a biocompatible ruthenium complex [Cp(MQA)Ru(C3H5)]+PF6–2 (Cp = cyclopentadienyl, MQA = 4-methoxyquinoline-2-carboxylate) and a general analytical method for evaluating its performance in real time based on a luciferase reporter system amenable to high throughput screening in cells and by extension to evaluation in luciferase transgenic animals. Precatalyst 2 activates alloc-protected aminoluciferin 4b, a bioluminescence pro-probe, and releases the active luminophore, aminoluciferin (4a), in the presence of luciferase-transfected cells. The formation and enzymatic turnover of 4a, an overall process selected because it emulates pro-drug activation and drug turnover by an intracellular target, is evaluated in real time by photon counting as 4a is converted by intracellular luciferase to oxyaminoluciferin and light. Interestingly, while the catalytic conversion (activation) of 4b to 4a in water produces multiple products, the presence of biological nucleophiles such as thiols prevents byproduct formation and provides almost exclusively luminophore 4a. Our studies show that precatalyst 2 activates 4b extracellularly, exhibits low toxicity at concentrations relevant to catalysis, and is comparably effective in two different cell lines. This proof of concept study shows that precatalyst 2 is a promising lead for bioorthogonal catalytic activation of pro-probes and, by analogy, similarly activatable pro-drugs. More generally, this study provides an analytical method to measure abiological catalytic activation of pro-probes and, by analogy with our earlier studies on pro-Taxol, similarly activatable pro-drugs in real time using a coupled biological catalyst that mediates a bioluminescent readout, providing tools for the study of imaging signal amplification and of targeted therapy.

The development of anticancer ruthenium(ii) complexes: from single molecule compounds to nanomaterials

“…This approach provides a set of critical metrics to observe the performance of biological catalysts and caging strategies for analogously cleavable pro-drugs. 321 Similarly, Mascarenas et al . developed a Ru(II) catalyst in the mitochondria of living cells by integrating the phosphonium-targeting moieties to Ru(II) complexes.…”

“…The rate of the probe release and enzymatic turnover could be evaluated in 4T1 cells using a luciferase reporter system. 321 With this method, researchers could measure the catalytic cleavage of a pro-probe and intracellular enzyme-mediated turnover of the released probe. This approach provides a set of critical metrics to observe the performance of biological catalysts and caging strategies for analogously cleavable pro-drugs.…”

Chemical Activation in Blood Serum and Human Cell Culture: Improved Ruthenium Complex for Catalytic Uncaging of Alloc‐Protected Amines

“…Several applications based on these four ruthenium complexes have been reported . For example, Waymouth, Wender and co‐workers demonstrated the bioorthogonal catalytic activation of cellular bioluminescence; Mascareñas and co‐workers developed organelle‐selective reactions within human mitochondria; and we demonstrated the catalytic activation of a caged anticancer drug that induced apoptosis in HeLa cells (Scheme ).…”

Intracellular Chemistry: Integrating Molecular Inorganic Catalysts with Living Systems

“…In a separate study, Mascareñas and his group were able to tether organelle‐targeting units to Ru1 to direct it toward the mitochondria, where it could do selective chemistry . Finally, Waymouth/Wender and co‐workers have also taken advantage of biocompatible allylcarbamate cleavage to devise a bioluminescent method to evaluate the efficiency of metal‐catalyzed reactions in living systems . With the development of more active generations of ruthenium SIMCats in recent years, such as Ru3 , we anticipate that this SIMCat‐promoted chemistry will have even greater utility to practitioners working in the life sciences.…”