Ruthenium-Catalyzed Redox Isomerizations inside Living Cells

Abstract

Tailored ruthenium(IV) complexes can catalyze the isomerization of allylic alcohols into saturated carbonyl derivatives under physiologically relevant conditions, and even inside living mammalian cells. The reaction, which involves ruthenium-hydride intermediates, is bioorthogonal and biocompatible, and can be used for the "in cellulo" generation of fluorescent and bioactive probes. Overall, our research reveals a novel metal-based tool for cellular intervention, and comes to further demonstrate the compatibility of organometallic mechanisms with the complex environment of cells.

Figure 1

(a) Transfer hydrogenations have been developed to either alter the redox status of cells or to reduce abiotic substrates; (b) this work, and ruthenium-hydride intermediates that are likely involved in the reaction.

Figure 2

Reactivity of ruthenium complex in living cells. (a) Ruthenium catalyzed isomerization of allylic alcohol 1g; (b) Fluorescence micrographies of HeLa cells (confocal): (A) cells incubated with substrate 1g (brightfield and green channel); (B) cells incubated with [Ru], washed and treated with substrate 1g (brightfield and green channel); (C) cells incubated with product 2g (brightfield and green channel); (c) CTFC measurements in HeLa, Vero and A549 cells. Reaction conditions: cells were incubated with [Ru] (10 μM) for 30 min, followed by two washings with DMEM and treatment with substrate 1g (100 μM) for 1 h. Error bars represent the standard error of three independent experiments. λ_ex = 385 nm, λ_em = 520–700 nm. Scale bar: 12.5 μm.

Figure 3

(a) Extracted ion chromatogram of the product 2g generated intracellularly (methanolic extract). HeLa cells pretreated with 10 μM of [Ru] for 50 min were washed twice and incubated with 100 μM of substrate 1g for 30 min/6 h; (b) turnover numbers of intracellular catalysis at 10 and 25 μM of catalyst loading (the quantification was performed considering the total crude material after methanolic extract and two washing steps (DMEM and PBS)); (inset) ICP-MS values of the intracellular accumulation of ruthenium after incubation of cells in DMEM with 10 or 25 μM (in DMSO) for 50 min, double washing with PBS and digestion with HNO₃; (c) cytotoxicity studies in HeLa cells. Reaction conditions: cells were incubated with either substrate 1g (100 μM) or product 2g (100 μM) for 6/24 h. Alternatively, cells were mixed with [Ru] (50 μM) for 30 min, washed twice with DMEM and treated with substrate 1g (100 μM) for 6/24 h (labeled as intracellular reaction). Right bar: cells were incubated with [Ru] (50 μM) for 30 min, followed by two washings with DMEM and the toxicity checked after 24 h. Error bars represent the standard error of three independent experiments.

Figure 4

Generation of α,β-unsaturated ketones. (a) Scope of the ruthenium-catalyzed isomerization. ^aPerformed using 3 (0.2 mmol), solvent (1.0 mL) and [Ru] (2 mol %). ^b[Ru] (5 mol %), cells lysates 7 mg/mL. (b) Selected biological studies of GSH consumption using the transformation 3b→4b as a model. Reaction conditions: for intracellular catalysis, cells were incubated with [Ru] (50 μM) for 30 min, followed by two washings with DMEM and treatment with substrate 3b (100 μM), for 6 h. For control experiments, cells were incubated with substrate 3b or product 4b (100 μM) for 6 h. Error bars represent the standard error of three independent experiments.