Ruthenium-Catalyzed Redox Isomerizations inside Living Cells

Article abstract of DOI:10.1021/jacs.9b00837

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.

Full text of DOI:10.1021/jacs.9b00837

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Communication
Ruthenium-catalyzed Redox Isomerizations inside Living Cells
Cristian Vidal, Maria Tomás-Gamasa, Alejandro Gutiérrez González, and José Luis Mascareñas
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b00837 • Publication Date (Web): 20 Mar 2019
Downloaded from http://pubs.acs.org on March 20, 2019
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Ruthenium-catalyzed Redox Isomerizations inside Living Cells
Cristian Vidal,† María Tomás-Gamasa,† Alejandro Gutiérrez-González, José L. Mascareñas*
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Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS), and Departamento de
Química Orgánica. Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
KEYWORDS. bioorthogonal chemistry, isomerizations, ruthenium, fluorescence, intracellular catalysis
Gimeno and coworkers on
a
ruthenium-mediated
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.
isomerization of allylic alcohols to ketones in water,9 we
questioned whether this type of redox-neutral processes,
which likely involve ruthenium hydride species, could be
achieved in biorelevant media or even in intracellular
settings. Do the ruthenium intermediates survive to the
stringent conditions of a complex biological buffer or a
living cell? Are the hydride intermediates compatible with
biological components? Can this “non-natural” metal
catalysis promote biological responses?
Enzymes catalyze a myriad of chemical transformations
that are essential to life, including hydrolysis, ligations,
isomerizations or redox processes.1 Chemists are not
constrained to generate life-sustaining reactions, and along
the years have invented many catalytic processes that do
not occur in nature. Especially appealing are those
transformations involving metal catalysis.2 While these
reactions are less efficient than those promoted by
enzymes, they present a broader scope, and are more
versatile and mechanistically diverse. Therefore, exporting
the power of “non-natural” metal catalysis to the biological
arena might open unforeseen opportunities for metabolic
or genetic intervention, and unveil new tools in therapy and
diagnosis.
Unfortunately, most transition metal-catalyzed processes
occur under conditions that are not compatible with
aqueous and biological settings, and therefore, performing
these reactions under physiological environments is
certainly challenging. Nonetheless, along recent years
several groups have demonstrated the viability of
promoting bioorthogonal metal-mediated reactions in
biological buffers, and even in living settings.3 While the
area is yet in its infancy, there have been reports on
biocompatible metal-promoted deprotections (Pd, Ru),4
click-like annulations (Cu or Ru)5, cross-couplings (Pd),6, 4b,
4i and even gold-promoted carbocyclizations.7
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.
Herein we provide some answers to these questions, by
reporting the first metal-catalyzed isomerization reaction
that can be carried out under biological conditions, and
more importantly, in the interior of living mammalian cells
(Figure 1b). The reaction is promoted by a ruthenium(IV)
catalyst that combines water solubility with some
lipophilicity. The redox-neutral isomerization allows to
transform non-functional, abiotic allyl alcohols into
fluorescent or bioactive ketones, in the interior of living
mammalian cells.
Sadler and Do have independently reported the use of
osmium or iridium complexes to alter NADH/NAD+ cellular
equilibriums, or promote reductions of specific aldehydes
or ketones (Figure 1a).8 Albeit the efficiency of these
processes was low, these studies suggest that metal-hydride
species can be generated inside cells. In light of these
observations, and considering previous contributions by
The viability of the isomerization was first explored using
1-phenylprop-2-en-1-ol (1a) as substrate, and the bis-allyl
Ru(IV) complex [Ru] as catalyst (Table 1 and S1).10 The
reaction is very efficient in water and PBS at physiological
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temperatures (37 °C), even using only 1 mol% of the metal
therefore we designed the substrate 1g, which displays very
low fluorescence above 500 nm when excited at 385 nm.
However, the isomeric ketone product 2g is highly emissive
at the same wavelength of excitation (Figures S1-S3).
1
2
3
4
complex (92%, 1.5 h). More importantly, the transformation
can be efficiently accomplished in complex cell culture
media, such as DMEM (Dubelcco’s modified Eagle´s
medium), and even in cell lysates (using 5 mol% of catalyst).
The intracellular reactions were explored in human (A549
and HeLa), or animal (Vero) cell lines. Confocal microscopy
of live cells treated as indicated in the caption of Figure 2c,
revealed that there is a considerable intracellular build-up
of green fluorescence after 1 h, mainly located in the cytosol,
even using only 10 μM of [Ru] and 100 μM of substrate
(Figure 2b, panel B). Considering the mean value of
corrected total cell fluorescence (CTCF) there is an increase
of up to 38-fold in the emission of cells treated with 1g,
when they had been previously pre-incubated with the
ruthenium complex (Figure 2c). Control experiments of
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The efficiency and orthogonality of the reaction can be
replicated with other substrates containing modified aryl or
naphthyl substituents to give the expected ketones (Table 1,
2a-d and 2f-g, respectively), as well as with aliphatic
derivatives (Table 1, product 2e). Other ruthenium
catalysts like [RuCp*Cl(COD)], [RuCp(MeCN)3][PF6] or
[RuCl2(p-cymene)]2 do work in water, but perform very
poorly in cell lysates. We also tested the iridium complex
employed by Do and coworkers for the reduction of
carbonyl compounds, as well as [IrCl2Cp*]2,11 but
isomerization yields were also very low (Table S1).
Therefore, complex [Ru] seems to present the best balance
between reactivity and stability, when used in biologically-
relevant media.
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untreated cells confirmed that such increase is
a
consequence of the intracellular formation of the product.
Similar results were obtained with A549 and Vero cells
(Figure S6-S7).
Table 1. Scope of the ruthenium catalyzed isomerization of
allylic alcoholsa
O
OH
H
Cl
Ru
[
]
Ru
[
] =
Ru O
O
R
H
R
milieu, 37 ºC
1
2
ketone
allylic alcohol
Orthogonal scope
R =
Br
MeO
2a
2d
2b
2c
aPBS
bDMEM
cLysates
99% (2 h)
85% (4 h)
86% (4 h)
87% (5 h)
88% (9 h)
81% (7 h)
85% (5 h)
89% (6 h)
87% (6 h)
90% (5 h)
91% (8 h)
79% (7 h)
R =
N
MeO
2g
2f
2e
91% (6 h)d
79% (16 h)d
aPBS
bDMEM
cLysates
91% (1 h)
89% (1 h)
87% (2 h)
a
Performed using 1 (0.2 mmol), milieu (1.0 mL) and [Ru]
b
c
(2 mol%). [Ru] (5 mol%). [Ru] (5 mol%), cells lysates 7
mg/mL. d Milieu with 20% THF.
While the mechanism of the reaction has not been fully
elucidated,12,9a-b it likely involves an initial aquation of the
precatalyst, followed by coordination of the allylic alcohol
to the ruthenium center and -hydrogen elimination to
generate hydride intermediate of type I (Figure 1b). An
intramolecular hydride transfer produces an oxo complex
(type II) that releases the product and regenerates the
Ru(IV) catalyst.
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
The presence of ruthenium hydride intermediates was
confirmed by deuteration experiments (Section S5), which
demonstrated that they survive under stringent conditions
of a milieu like DMEM. The high catalytic activity and
orthogonality of the ruthenium complex [Ru] under
biologically relevant conditions, prompted us to evaluate
the viability of performing the reaction in native cellular
settings. This required a way of monitoring the process, and
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standard error of three independent experiments. λex = 385 nm,
λem = 520-700 nm. Scale bar: 12.5 µm.
1g (100 µM) for 6/24 h (labelled 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.
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The formation of the expected product 2g was further
confirmed by LC-MS analysis of methanolic extracts of the
cells after running the reaction for 6 h, and two-fold
washing with DMEM and PBS. The amount of product in the
washings was low, however the methanolic extracts
contained a significant amount of ketone 2g.13 Importantly,
using LC-MS techniques, and appropriate calibration
curves, we could also quantify the product generated inside
cells (Figure 3a and Figure S11). The formation of the
product 2g increases with time, which demonstrates the
persistence of a reactive metal complex inside the cells. ICP-
MS analysis of cellular extracts obtained after exposing
HeLa cells to 10 or 25 μM of [Ru], and thoroughly washed
with DMEM, revealed a ruthenium content of 3.5 or 11.1
ng/106 cells, respectively (Figure 3b), while untreated cells
contain over 0.2 ng/106 cells (Figure S10). Combining this
information with the amount of intracellular product, it is
possible to calculate turnover numbers (TON) with a
relatively good accuracy (Figure 3b). Indeed, we found a
turnover of over 22 in experiments with cells treated with
10 μM of [Ru] and 100 μM of substrate, after 6 h (Section
S12). To our knowledge this represents the first quantitative
demonstration of turnover in an organometallic intracellular
reaction.
While the above data demonstrate the viability of achieving
ruthenium-mediated isomerizations in native intracellular
settings, an important challenge in this nascent scientific
field of cellular metallocatalysis consists of the intracellular
generation of active products.14 Up to now, these studies
have been essentially restricted to uncaging reactions from
appropriately protected precursors. We therefore
wondered whether the isomerization reaction could also be
associated to changes in some biological activity. Using
standard MTT techniques we confirmed that using
concentrations up to 100 M, neither the ruthenium
complex nor the substrate 1g or product 2g presented
noticeable toxicities after 6 h (Figure 3c). However, after 24
h, we observed toxicity in the case of the product (32% of
cell death, Figure S4). In order to study the effect of the “in
cellulo” generation of the product 2g, we added 100 μM of
the substrate 1g to cells that had been treated with 50 μM
of Ru, and thoroughly washed with DMEM. In this case, we
observed a higher decrease in viability (Figure 3c, 45% of
death after 24 h). This response is also noticeable even after
only 6 h, when more than 30% of the cells died, thirty times
more than that observed with the substrate (Figure 3c).
Given than the complex [Ru] is not toxic at such
concentration (Figure S5), these results suggest that the
intracellular generation of the product produces a more
effective biological response than its external addition.
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Finally, considering the well-known Michael type of
reactivity of α,β-unsaturated systems, especially for thiol
nucleophiles, we envisioned the use of our ruthenium-
mediated isomerization to generate glutathione (GSH)
depleting agents. We first confirmed that the ruthenium
catalyst can efficiently convert diallyl alcohols of type 3 into
the corresponding α,β-unsaturated ketones 4, even in cell
lysates (Figure 4a). In a preliminary screening with HeLa
cells, we could observe that some of these ketones, like 4b,
promoted around 20% consumption of GSH. We therefore
carried out a standard isomerization experiment in living
cells using 50 μM of [Ru] and 100 μM of 3b. Analysis of the
level of GSH after 6 h confirmed a substantial decrease of
the intracellular GSH concentration (over 40%
consumption of GSH, average of three experiments, Figure
4b). Given that the catalyst and the substrate do not alter
the levels of GSH, the observed changes must arise from the
in-situ generation of the product. While these biological
results should be viewed as a preliminary proof of concept,
they demonstrate that the potential of metal catalysis to
generate bioactive products inside cells is not limited to
standard uncaging reactions.
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
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a)
María Tomás-Gamasa: 0000-0001-8681-2744
O
OH
H
1
2
Ru
[
]
Author Contributions
R
R
H
milieu
37 ºC, 24 h
†
3
4
C.V. and M.T-G. contributed equally to this work.
3
4
Notes
Scope
The authors declare no competing financial interest
5
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8
R =
2
Funding Sources
MeO
3c
3a
3b
This work has received financial support from the Spanish
Government (SAF2016-76689-R, Orfeo-cinqa network
CTQ2016-81797-REDC), the Consellería de Cultura, Educación
e Ordenación Universitaria (2015-CP082, ED431C 2017/19),
Centro Singular de Investigación de Galicia accreditation 2016-
2019, ED431G/09), the European Union (European Regional
Development Fund-ERDF), and the European Research Council
(Advanced Grant No. 340055).
aPBS
bLysates
99% (2 h)
85% (4 h)
99% (2 h)
85% (4 h)
99% (2 h)
85% (4 h)
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Biological response
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b)
ACKNOWLEDGMENT
Dedicated to Professor P. Espinet on the occasion of his 70th
birthday.
C. V and M. T. G thanks the Ministerio de Economía y
Competitividad for the Juan de la Cierva-Formación
fellowships (FJCI-2017-33168) and Juan de la Cierva-
Incorporación (IJCI-2015-23210) respectively and A. G. G
thanks the Ministerio de Educación, Cultura y Deporte for the
FPU fellowship (FPU17/00711). The authors thank R.
Menaya-Vargas for excellent technical assistance and
especially M. Marcos for excellent and essential
contributions to the LC-MS analysis.
Figure 4. Generation of α,β-unsaturated ketones. a) Scope of
the ruthenium-catalyzed isomerization. Performed using 3
(0.2 mmol), solvent (1.0 mL) and [Ru] (2 mol%). [Ru] (5
a
b
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.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: xxx
Experimental procedures and cell studies (PDF)
AUTHOR INFORMATION
Corresponding Author
*joseluis.mascarenas@usc.es
ORCID
José L. Mascareñas: 0000-0002-7789-700Xr
Cristian Vidal: 0000-0003-4496-1308
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