Bioorthogonal Catalysis: A General Method To EvaluateMetal-Catalyzed Reactions in Real Time in Living Systems Using a CellularLuciferase Reporter System

摘要

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 providesalmost exclusively luminophore >4a. Our studies show thatprecatalyst >2 activates >4b extracellularly,exhibits low toxicity at concentrations relevant to catalysis, andis comparably effective in two different cell lines. This proof ofconcept study shows that precatalyst >2 is a promisinglead for bioorthogonal catalytic activation of pro-probes and, byanalogy, similarly activatable pro-drugs. More generally, this studyprovides an analytical method to measure abiological catalytic activationof pro-probes and, by analogy with our earlier studies on pro-Taxol,similarly activatable pro-drugs in real time using a coupled biologicalcatalyst that mediates a bioluminescent readout, providing tools forthe study of imaging signal amplification and of targeted therapy.