Exporting Metal-Carbene Chemistry to Live Mammalian Cells: Copper-Catalyzed Intracellular Synthesis of Quinoxalines Enabled by N?H Carbene Insertions

Article abstract of DOI:10.1002/anie.202108899

Implementing catalytic organometallic transformations in living settings can offer unprecedented opportunities in chemical biology and medicine. Unfortunately, the number of biocompatible reactions so far discovered is very limited, and essentially restricted to uncaging processes. Here, we demonstrate the viability of performing metal carbene transfer reactions in live mammalian cells. In particular, we show that copper (II) catalysts can promote the intracellular annulation of alpha-keto diazocarbenes with ortho-amino arylamines, in a process that is initiated by an N-H carbene insertion. The potential of this transformation is underscored by the in cellulo synthesis of a product that alters mitochondrial functions, and by demonstrating cell selective biological responses using targeted copper catalysts. Considering the wide reactivity spectrum of metal carbenes, this work opens the door to significantly expanding the repertoire of life-compatible abiotic reactions.

Full text of DOI:10.1002/anie.202108899

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How to cite:
International Edition: doi.org/10.1002/anie.202108899
German Edition: doi.org/10.1002/ange.202108899
Bioorthogonal Chemistry
Hot Paper
Exporting Metal-Carbene Chemistry to Live Mammalian Cells:
Copper-Catalyzed Intracellular Synthesis of Quinoxalines Enabled by
À
N H Carbene Insertions
Sara Gutiꢀrrez, Marꢁa Tomꢂs-Gamasa,* and Josꢀ L. MascareÇas*
Abstract: Implementing catalytic organometallic transforma-
tions in living settings can offer unprecedented opportunities in
chemical biology and medicine. Unfortunately, the number of
biocompatible reactions so far discovered is very limited, and
essentially restricted to uncaging processes. Here, we demon-
strate the viability of performing metal carbene transfer
reactions in live mammalian cells. In particular, we show that
copper (II) catalysts can promote the intracellular annulation
of alpha-keto diazocarbenes with ortho-amino arylamines, in
a process that is initiated by an N-H carbene insertion. The
potential of this transformation is underscored by the in cellulo
synthesis of a product that alters mitochondrial functions, and
by demonstrating cell selective biological responses using
targeted copper catalysts. Considering the wide reactivity
spectrum of metal carbenes, this work opens the door to
significantly expanding the repertoire of life-compatible abiotic
reactions.
behaviors, working under the same environment of native
enzymes (living cells or organisms), remains to be uncovered.
An alternative approach to perform artificial chemical
reactions in live cells has recently emerged, and consists of the
use of exogenous, discrete transition metal catalysts.[4] While
the catalytic activity of these reagents is far away from that of
metalloenzymes, they can permeate living cells, and even-
tually trigger non-native organometallic reactions. Therefore,
several transition-metal reagents that mediate intracellular
allylic or propargylic deprotections,[5] cyclizations,[6] cross
couplings,[7] or even formal cycloadditions,[8] have been
recently disclosed.
However, the portfolio of reactions is yet too small,
especially when compared with the enormous breadth and
potential that organometallic catalysts exhibit in organic
solvents. A particularly powerful class of catalytic organome-
tallic reagents that have never been explored in mammalian
cell environments, are discrete metal carbenes.[9] This might
be in part due to the notion that these intermediates, and even
their diazo precursors, are too fragile and reactive, and might
not survive in water, and especially in the presence of native
biomolecules with reactive nucleophilic functionalities like
amines or thiols (Figure 1a, left). However, some isolated
examples suggest that diazocompounds can be used for
bioorthogonal tagging.[10] Moreover, several chemoselective
Introduction
Live cells can be viewed as microfactories that perform
thousands of simultaneous chemical reactions in a highly
regulated manner. Most of these reactions are promoted by
enzymes, proteins that have evolved to exhibit exquisite rates
and selectivities.[1] Over one-third of the enzymes feature
metals at their active sites, and therefore are coined as
metalloenzymes. In recent years, there has been an impressive
progress in the creation of laboratory versions of metal-
loenzymes that catalyze “new-to-nature” reactions.[2] How-
ever, the application of these catalytic metalloproteins has
been essentially restricted to the realm of synthetic method-
ology.[3] Their use for regulating and modifying biological
À
functionalizations of peptides and proteins relying on C H
activations promoted by rhodium carbenes have been repor-
ted.[10d–e,11] Gillingham and co-workers have also described
DNA modification reactions using rhodium or copper (I)
À
carbenes capable of inserting into the N H bond of ade-
nines.[12]
In addition to these examples, metallocarbene transfer
reactions have been developed in the context of artificial
metalloenzymes, using heme-containing proteins expressed in
E. coli, especially with native iron-containing cofactors;[13]
though these advances can be ascribed to the fields of
synthetic chemistry and synthetic biology.[14]
[*] Dr. S. Gutiꢀrrez, Dr. M. Tomꢁs-Gamasa, Prof. J. L. MascareÇas
Centro Singular de Investigaciꢂn en Quꢃmica Biolꢂxica e Materiais
Moleculares (CiQUS) and Departamento de Quꢃmica Orgꢁnica,
Universidade de Santiago de Compostela
In this context, we questioned whether it would be
possible to perform carbene transfer reactions with discrete
transition metal catalysts in biorelevant aqueous mixtures,
and especially in the complex environment of living mamma-
lian cells (Figure 1). Adding this type of catalytic reactions to
the catalog of life-compatible organometallic processes might
foster new opportunities for cellular and biological interven-
tion given the power and versatility of metal-carbene
chemistry.
15705 Santiago de Compostela (Spain)
E-mail: maria.tomas@usc.es
joseluis.mascarenas@usc.es
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202108899.
ꢄ 2021 The Authors. Angewandte Chemie International Edition
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution Non-Commercial
NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-
commercial and no modifications or adaptations are made.
However, this goal raises many challenges and questions:
Will diazo reagents be able to capture the metal complex in
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Figure 1. Metal N-H carbene insertions in biological settings. a) Orthogonality challenges in metal-mediated carbene transfer transformations in
aqueous/biological media. b) Copper-promoted annulation initiated by an N-H carbene insertion within biological and/or cellular environments.
c) Mechanistic outline: The bidentate coordination may allow the chelation of copper. This might favor the N-H insertion, which is followed by an
intramolecular condensation and oxidative aromatization.
a challenging biological milieu to generate the metal car-
benes? Are these metal-carbene intermediates compatible
with biological molecules and stable enough to react with
designed reactants? Can we perform this chemistry within
living cells? Can we use these reactions to build abiotic
bioactive products in cells, and elicit biological effects?
Herein we report the first metal-catalyzed carbene trans-
fer reaction carried out in living mammalian cells. The
reaction has been implemented with ortho-amino arylamines,
providing for the assembly of photophysically and/or biolog-
ically relevant quinoxaline products (Figure 1b). The trans-
formation likely benefits from a chelation assistance process
to generate intermediates like A, which favor the N-H
insertion, initial step of the process (Figure 1c). We also
demonstrate that this technology can be used for the in cellulo
synthesis of a compound that promotes mitochondrial frag-
mentation and depolarization, and produces cytotoxic effects.
Importantly, by equipping the copper ligand with suitable
targeting units, it is possible to elicit cell selective responses.
catalysts, and involves as initial step an N-H insertion of the
metal carbene.[15] In particular, we selected as reactant
diaminonaphthalene (1), which can provide fluorescent
benzoquinoxalines such as 3a (Figure 2a). We reasoned that
the second amine group in the substrate could play an
important role not only to drive the generation of cyclic
products, but also as metal coordination handle to enhance
reactivity and favor orthogonality in the presence of compet-
ing nucleophiles (Figure 1c).[16]
Our initial assays, consisting of the treatment of diamine
1 with 1 equiv of diazocarbonyl 2a and 10 mol% of Fe(OTf)3
in water (100 mM), at 408C for 24 h, led to no conversion.[15]
Other iron (III) catalysts (FeCl3, Fe(acac)3) were also
ineffective. However, using Cu(OTf)2 as catalyst, 3a was
obtained in 56% yield (Figure 2a, entry 3). Further screening
of copper salts allowed to identify Cu(OAc)2·H2O as a more
efficient catalyst for this transformation, affording the prod-
uct 3a in 70% yield (entry 4; 55% after 3 h of reaction, using
10 mol% of the copper salt, Supporting Information, Ta-
ble S3). The catalyst loading could be decreased to 5 mol%
without affecting the yield. Even using 1 mol%, we observed
more than 50% of the product (entry 5). It is important to
note that the reaction mixtures consist of heterogeneous
suspensions.
Results and Discussion
Finding the Right Catalyst. At the outset, we needed to
identify a suitable reaction involving a metal-carbene transfer
process that could be easily monitored, eventually, by
fluorescence microscopy. In this context, we were attracted
by the annulation between 1,2-diaminobenzenes and a-diazo-
b-keto esters reported by Pandit and co-workers, which was
described to work in water using different transition metal
We also tested other transition metal complexes that had
been previously used in carbene insertion reactions (Fig-
ure 2a and Table S1).[17] Using gold reagents like XPhos-
AuNTf2, we observed testimonial amounts of the desired
adduct 3a (14–18%, entry 6). Rhodium or iridium catalysts
like Rh2(OAc)4, [Rh(COD)Cl]2, [Cp*IrCl2]2, [Ir(COD)Cl]2
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and [Ir(COD)(OMe)]2 did promote the reaction at 408C, but
in modest yields (20–49%). With Rh2(esp)2, a more hydro-
phobic rhodium catalyst, the reaction was more efficient
(72% yield, entry 9); however, using more diluted conditions,
the reaction yield dropped to 50% (entry 10) and an
undesired side product resulting from addition of water to
the carbene was also formed in substantial amounts (4,
CO2EtCH(OH)COPh, 49%). Fortunately, this was not the
case with Cu(OAc)2, which led to good results under more
diluted conditions (5 mM, entry 11).
Control experiments in the absence of diamine 1 led to
a mixture of the diazo carbene dimer (4’) and the O-H
insertion product 4 (Figure S1). Other diazo reagents could
also be used, and therefore product 3b was formed in a good
67% yield after 24 h (57% isolated yield, entry 12). The
reaction can also be carried out with CuI salts, albeit with
lower efficiency (entry 13, and Table S1). The use of cosol-
vents, such as acetonitrile or dimethylsulfoxide (10–20% v/v),
allowed to carry out the reaction in a homogeneous solution,
but didnꢀt bring special improvements (Table S2). Even in the
absence of solvent (neat conditions), 3a was obtained in 59%
yield.
Bioorthogonality. As the goal was moving to biological
media, it was key to uncover the tolerance of the reaction to
biological additives and salts (Figure 2b). Using 10 mol% of
Cu(OAc)2·H2O, and 100 mM of substrates, we were glad to
observe that in saline phosphate buffer (PBS, pH 7.2) the
reaction was even slightly more efficient than in water (77%
after 24 h). Good results were also observed when the
reaction was carried out in the presence of biological
additives like BSA (bovine serum albumin, 5 mgmLÀ1),
a protein that features one free cysteine and several histidines
in its structure. Importantly, the process is also compatible
with the presence of HeLa cell lysates (protein content:
4 mgmLÀ1 in PBS, 81% yield after 24 h (Supporting Infor-
mation, Section 4). Using lower catalyst loadings (5 mol%)
the product was obtained in a satisfactory 73% yield, and at
lower concentrations (25 mM) the yield decreased to 63%
(SI, Table S4). Finally, the transformation is effective under
more stringent conditions such as cell culture media like
DMEM (Dulbeccoꢀs modified Eagleꢀs medium), that contains
many additives (vitamins, amino acids, etc.), although it is
slightly less efficient (58% yield). Even in DMEM containing
5% of FBS (fetal bovine serum) the reaction takes place to
give a 51% yield of the product.
The reaction works well in the presence of stoichiometric
amounts of amino acids like lysine or tyrosine, or biomolec-
ular additives such as glucose, guanine or cytosine (yields
between 50–62%). It also tolerates amino acids like glutamic
acid, histidine, cysteine or methionine, when used in equiv-
alent amounts with respect to the copper catalyst. However, it
is inhibited by additives like adenine or glutathione, very
likely because these compounds coordinate to the copper
catalyst producing inactive off-cycle complexes. Curiously,
when the same reaction is performed in PBS instead of water,
it tolerates the presence of glutathione (10 mol%), and the
product was formed in a 66% yield (Table S5). This result
suggests that saline media like PBS might accelerate the
desired reaction owing to hydrophobic effects,[18] and perhaps
Figure 2. Metal-catalyzed assembly of quinoxalines. a) Metal-catalyzed
assembly of quinoxalines in water. Metal screening and optimization.
Reaction conditions unless otherwise stated: 1 equiv 1 and 2a, in H2O
(100 mM), in presence of 10 mol% catalyst, and heating of the
resulting heterogeneous mixture at 408C for 24 h. [a] Yield calculated
by 1H-NMR using 1,3,5-trimethoxybenzene as internal standard. [b]
1 mol% of the catalyst. [c] 5 mol% of the catalyst. [d] Substrate
concentration: 5 mM. [e] 2b was used as substrate. [f] Isolated yield;
esp=a,a,a’,a’-tetramethyl-1,3-benzenedipropionic acid. b) Efficiency
of the copper-carbene mediated synthesis of quinoxaline 3a in
presence of biological buffers. Reaction conditions: 1 equiv 1 and 2a
(substrates concentration: 100 mM), 10 mol% Cu(OAc)2, 408C, 24 h;
BSA 5 mgmLÀ1; Cell lysates 4 mgmLÀ1 (the values are average of three
experiments, and error bars are standard deviations).
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also disfavor the irreversible formation of inactive copper
complexes.
Catalytic Reactivity in Mammalian Cells. The stage was
set to move to a more complex scenario, such as that of living
mammalian cells. Note that a cell medium cannot be fully
equated to the above in vitro conditions, and might be better
viewed as a heterogeneous, crowded gel. Fluorescence
spectroscopy confirmed that whereas the diamine substrate
1 presents a very low emission above 500 nm when excited at
385 nm, the product 3a displays high fluorescence (Fig-
ure S2,S3). These emission properties were also confirmed in
living cells: incubation of HeLa cells with 3a (100 mM),
DMEM washing, and observation under the microscope,
revealed a clear intracellular emission (Figure 3a, panel B),
much higher that when adding 1 and 2a (panel A). For the
cellular reactions, HeLa cells were incubated with Cu(OAc)2
for 50 min (50 mM) in DMEM, thoroughly washed to remove
extracellular copper salts (2 ꢁ DMEM) and thus minimize
extracellular reactions, and treated with the substrates (1 and
2a, 100 mM each). Observation under the fluorescence
microscope after 1.5 h revealed a clear intracellular buildup
of green fluorescence in the cytosol, which must be associated
to the formation of product 3a (Figure 3a, panel C).
Monitoring the process with time confirmed a progressive
increase in total fluorescence intensity, in consonance with the
generation of more product (Figure 3a, panel D and Fig-
ure S7). Control experiments of cells treated with the copper
complex or with only one of the components, either 1 or diazo
2a, led to essentially no fluorescence (Figure S8). Similar
results were obtained using MCF7 cells (Figure 3a, panel
E,F).
Morphological analysis of the cells after the reaction time
was indicative of a reasonably good status in terms of viability.
As the toxicity of copper salts could be an issue,[19] we
performed specific cytotoxicity experiments in HeLa, MCF7
and HEK293 cell lines. As detailed in the Supporting
Information (Section 10), using MTT assays we confirmed
that the addition of the copper salt (Cu(OAc)2, 50 mM)
doesnꢀt compromise the cell viability in any of the cell lines,
even after 24 h. At higher concentrations (100 mM) we did
observe some effect (62% cell viability after 24 h). Moreover,
propidium iodide assays indicated that incubation of cells
with 50 mM of copper salt for 6 h doesnꢀt alter the cell
membrane status in any of the cell lines. Furthermore, MTT
studies showed that neither 1, 2a nor product 3a affected the
viability of the cells (complete survival after 18 h). Very
importantly, the intracellular presence of product 3a was
further confirmed by mass spectrometry, after extraction of
the cell contents with MeOH (Figure 3b). Indeed, using this
protocol (SI, Section 14) we could even quantify the product
(over 4 mM in total) which combined with the amount of
intracellular copper calculated using ICP analysis allowed to
estimate a TON around 7 (after an incubation time of 5 h,
Figure 3b, average of three experiments, Supporting Infor-
mation, Sections 13 and 14).
Figure 3. Synthesis of 3a in cells. a) Fluorescence micrographies
(brightfield) of HeLa (A–D) and MCF7 cells (E,F) after incubation with:
1 and 2a (A,E); 3a (B) or Cu(OAc)2, washing, and treatment with
1 and 2a for 1.5 h (C) or for 3 h (D,F). Reactions conditions: Cells
were pretreated with 50 mM of Cu(OAc)2 (stock solution in H2O) for
50 min, washed twice with DMEM, and incubated with 100 mM of
substrates 1 and 2a (stock solutions in DMEM) for the indicated time.
b) Detection and quantification of 3a in HeLa cells. Left: Extracted ion
chromatogram of the product (3a) generated intracellularly (methanol-
ic extract), after 5 h of reaction; Right: Quantification of turnover
numbers (average of three experiments), considering the product
present in the cell media and washes (2ꢅDMEM, 1ꢅPBS) and in the
methanolic (intracellular) extracts (which counts for over 75% of the
total). Scale bar: 20 mm. lexc =385 nm, lem >520 nm. The residual
fluorescence in A and E derives from 1 (see the SI, Figure S3).
Eliciting Biological Effects. Developing in cellulo metal-
promoted transformations, and specifically metal carbene
transfer reactions, is itself relevant from a fundamental
perspective. However, a major challenge in the area is the
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demonstration that the reactions can find biological or
biomedical applications. Indeed, several metal-promoted
uncaging reactions have already been used to activate
proteins or release bioactive agents in biological environ-
ments.[20] Conversely, “anabolic” processes, based on bond-
forming reactions to build bioactive products, are much less
developed.[21] Quinoxaline scaffolds are found in many
biorelevant compounds, and therefore, we questioned wheth-
er our technology could enable the synthesis of bioactive
derivatives inside live cells.
In this context, we were attracted by Tyrphostins (TY-
Rosin PHOSphorylation INhibitors),[22] compounds that ex-
hibit a variety of interesting biological profiles, apparently as
a consequence of their tyrosine kinase inhibitory activity.
Remarkably, whereas 3a was rather innocuous to cells
(Figure S6), benzoquinoxaline 3c, also called Tyrphostin
AG1385,[23] promoted a decrease in the cell viability of 40%
after 12 h (50 mM). Noteworthy, using higher concentrations
(100 mM) we didnꢀt observe a further increase in the cell death
(Figure 4b), a result that can be explained because of the
tendency of this highly hydrophobic product to aggregate and
precipitate, which compromises its internalization.[24] As
expected, product 3c can be readily made applying our
carbene transfer methodology using reactants 1 and diazo
compound 2c (Figure 4a and Supporting Information, Sec-
tion 6 for its in vitro synthesis).
Therefore, after addition of 50 mM of the reactants (1 and
2c) to HeLa cells previously treated with Cu(OAc)2 (25 mM),
we observed a similar effect in the cell viability than by direct
incubation with 50 mM of 3c. Significantly, in contrast to that
observed with 3c, using higher concentrations of the sub-
strates (up to 100 mM) the effect on cell survival is higher (less
than 30% of cell viability, Figure 4b). These results further
highlight one of the potential advantages of generating
bioactive products in situ versus their external addition.
Product 3c presents low levels of fluorescence, which
precludes its detection under the microscope; however, we
could confirm and quantify its intracellular formation by MS
of cellular extracts obtained after carrying out the reaction in
cells (after 4.5 h we calculated TONs over 4, Supporting
Information, Section 14). Given that some tyrphostins have
been shown to affect mitochondria,[23] we tested whether the
intracellular generation of 3c could affect the mitochondrial
membrane potential, a property that can be monitored by
measuring the fading of TMRE (tetramethyl rhodamine
methyl ester) staining.[25]
Gratifyingly, while treatment of HeLa cells with Cu-
(OAc)2 and either substrate 1 or 2c (100 mM) doesnꢀt alter the
mitochondria potential after 3.5 h (Figure 4c, panel A and
Figure S9), addition of both reactants to cells previously
treated with Cu(OAc)2 (50 mM) led to a much more diffuse
and less intense fluorescent signal of TMRE, indicating
depolarized mitochondria, which is in consonance with the in
situ formation of 3c (Figure 4c, panel C). Indeed, direct
addition of 3c led to qualitatively similar results (Figure 4c,
panel B).
for a longer time (8 h) allows to recover, at least partially, the
TMRE staining, however the mitochondria presented a differ-
ent morphology, consistent with a considerable fragmentation
(Figure 4d, panels G,I,K and Figure S11). Using MitoTracker
Green as mitochondrial dye, whose staining ability is less
dependent on the polarization state of the organelles, we
could confirm the fragmentation effect associated to the
generation of 3c. Cells treated only with substrates displayed
a very well-defined network of tubular mitochondria, pre-
dominantly in fusion state (Figure 4d, panel H). However,
addition of 3c (100 mM) resulted in more fragmented
mitochondria (Figure 4d, panel J). More importantly, a sim-
ilar fragmented network, which is associated to fission, was
also observed by generating the compound in situ, using our
copper catalyzed transformation (Figure 4d, panel L and
Figure S12).
Targeted Catalysts and Cellular Selectivity. A major
dream in pharmacology is related to the possibility of
delivering or accumulating drugs in specific, desired cellular
targets. Even more attractive is the prospect of a catalytic
generation of drugs in a cell-selective manner. We evaluated
this possibility by building the conjugate 5, shown in Fig-
ure 5a, which features an integrin-targeting motif (RGD:
Arg-Gly-Asp) linked to a copper triazolyl ligand (BTTAA,
Supporting Information, Section 7).[8c] Hypothetically, this
tripeptide motif might favor the preferential targeting of cell
lines expressing substantial amount of integrins, such as HeLa
cancer cells.[26] The conjugate was also engineered to exhibit
a rhodamine dye (TAMRA) linked to a lysine side chain, in
order to monitor the cellular fate of the complex. The copper
complex (5-Cu) was assembled in situ, just before the addition
to cells, by treatment of hybrid 5 with CuSO4 (0.5 equiv) in
water (10 min). The formation of 5-Cu was confirmed by LC-
MS (Figure S5).
As depicted in Figure 5a (panel A), addition of 5-Cu
(15 mM) to HeLa cells, and subsequent washing (2xDMEM),
gave rise to a substantial intracellular staining. In contrast,
using MCF7 or HEK293 cells, which are known to exhibit
lower levels of integrin receptors, we observed much less
fluorescence (Figure 5a, panel B and Figure S13 for
HEK293). Further analysis by CTCF (corrected total cell
fluorescence) corroborated the better uptake of HeLa cells
(Figure 5a, right). More importantly, in consonance with this
programmed internalization of the copper complex, we
observed a much higher increase in the fluorescence associ-
ated to the formation of product 3a in the HeLa cells, after
treatment with reactants 1 and 2a (Figure 5b, panels C and D,
respectively and Figure S14). It is important to note that using
Cu(OAc)2 instead of 5-Cu as catalytic reagent MCF7 cells are
able to generate the product (Figure 3), which confirms that
the above cell selective responses are associated to the
preferential targeting and internalization properties of our
designed catalyst 5-Cu.
This cell-dependent reactivity was also observed in the
reaction leading to the mitochondria active quinoxaline 3c.
Therefore, treatment of the cells with 5-Cu (15 mM), washings
(2 ꢁ DMEM) and addition of 1 and 2c (100 mM), led, after
3.5 h, to a substantial mitochondrial depolarization only in
HeLa cells (Figure 5c, panel G). MCF7 and HEK293 cells
The same effect was observed with other type of cancer
cell lines like MCF7 (differences in staining in panels D–F)
and HEK293 (Figure S10). Noticeably, leaving the HeLa cells
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Figure 4. Generation of 3c in cells. a) Synthesis of 3c. b) Cytotoxicity studies. HeLa cells were mixed with either 1, 2c, or 3c (50–100 mM) for
12 h; for the reaction, cells were incubated with Cu(OAc)2 (25–50 mM) for 50 min, washed twice with DMEM and treated with substrates 1 and 2c
(50–100 mM) for 12 h. c) Mitochondrial depolarization. Fluorescence micrographies using TMRE as marker: HeLa (A–C), and MCF7 cells (D–F)
(brightfield) after incubation with: 1 and 2c (A,D); 3c (B,E); Cu(OAc)2, washing, and treatment with 1 and 2c (C,F) for 3.5 h. d) Mitochondrial
fragmentation. Fluorescence micrographies of HeLa cells (brightfield) using TMRE (G,I,K) or MitoTracker Green (H,J,L) as marker, after incubation
with: 1 and 2c (G,H); 3c (I,J); or Cu(OAc)2, washing and treatment with 1 and 2c for 8 h (K) or 2 h (L); in white, mitochondrial staining from
each image displayed as a skeleton network using the ImageJ skeleton filter, which allows a better visualization of the fragmentation. Reaction
conditions: Cells were mixed with 1 and 2c, or 3c. For the reaction in cells, they were incubated with Cu(OAc)2 (50 mM) for 50 min, washed twice
with DMEM and treated with substrates 1 and 2c (100 mM) and incubated with TMRE or MitoTracker Green (100 nM) for 10 min. Scale bar:
20 mm (15 mm in section c). lexc =550 nm, lem =570–590 nm for panels (A–G,I,K); lexc =470 nm, lem =490–580 nm for panels (H,J,L).
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Figure 5. Selective cellular targeting using a RGD copper-containing peptide (5-Cu). a) Structure of the RGD containing ligand in 5-Cu;
Comparison of internalization of 5-Cu. Fluorescence micrographies of HeLa (A) and MCF7 cells (B) (brightfield) and CTCF measurements in
HeLa, MCF7 and HEK293 cells. Conditions: cells were incubated with 15 mM of 5-Cu for 1.5 h. b) Fluorescence micrographies of HeLa (C) and
MCF7 cells (D) (brightfield) after incubation with 5-Cu, washing, and treatment with 1 and 2a for 3.5 h. Scale bar: 20 mm. c) Cell-selective
mitochondrial depolarization: Fluorescence micrographies of HeLa (E–G) and MCF7 cells (H–J) (brightfield) after incubation with: 1 and 2c
(E,H), product 3c (F,I), or 5-Cu, washing, and treatment with 1 and 2c for 3.5 h (G,J). d) Bar graphic including CTCF measurements of
mitochondrial depolarization in HeLa, MCF7 and HEK293 cells. Reaction conditions: cells were mixed with 5-Cu (15 mM) for 1.5 h, washed twice
with DMEM and treated with 1 and 2a, or 1 and 2c (100 mM) for 3.5 h followed by TMRE (100 nM) for 10 min. Scale bar: 100 mm. lexc =550 nm,
lem =570–590 nm for panels (A,B,E-J); lexc =385 nm, lem >520 nm for panels (C,D).
remain essentially intact (Figure 5c, panel J and Figure S15). Conclusion
Control experiments with product 3c revealed a similar
depolarization effect in all cell lines, independently of their
integrin expression (Figure 5b, panels F,I and Figure S15).
Figure 5d represents a quantification of the resulting TMRE
staining. MTT assays showed that 5-Cu (20 mM) didnꢀt
compromise the cell viability of any of the cell lines, even
after 18 h (SI, Figure S6).
All these results confirm the viability of using transition-
metal carbene transfer catalysts for a cell selective synthesis
of bioactive products and set the bases for future biological
and biomedical applications.
We have described the first examples of a transition metal
catalyzed carbene-transfer reaction occurring in living mam-
malian cells. The reaction entails a N-H insertion of a copper
carbene into aromatic 1,2-diamines, and allows to generate
benzoquinoxalines, owing to a subsequent intramolecular
imine condensation and oxidative aromatization process.
Therefore, the whole process, while capitalizing in the initial
intermolecular carbene transfer reaction, involves two addi-
tional chemical steps. Preliminary studies confirmed the
existence of a significative TON (of up to 7), which stresses
the viability of performing intracellular catalytic carbene
chemistry.
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Research Articles
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Acknowledgements
This work has received financial support from Spanish grants
(PID2019-108624RB-I00, RTI2018-093813-J-I00 and OR-
FEO-CINQA network CTQ2016-81797-REDC), the Consel-
lerꢂa de Cultura, Educaciꢃn e Ordenaciꢃn Universitaria
(2015-CP082, ED431C-2017/19 and Centro Singular de In-
vestigaciꢃn de Galicia accreditation 2019–2022, ED431G
2019/03), the European Union (European Regional Develop-
ment Fund-ERDF corresponding to the multiannual financial
framework 2014–2020), and the European Research Council
(Advanced Grant No. 340055). S.G. thanks the European
Union (European Regional Development Fund, Interreg V-
A POCTEP EspaÇa-Portugal programme, 2iqbioneuro proj-
ect) and M.T.G. thanks the financial support from the
Agencia Estatal de Investigaciꢃn (RTI2018-093813-J-I00).
The authors thank R. Menaya-Vargas for excellent technical
assistance, the use of RIAIDT-USC analytical facilities and
especially M. Marcos for excellent and essential contributions
to the LC-MS analysis.
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[8] a) P. Destito, J. R. Couceiro, H. Ferreira, F. Lꢃpez, J. L.
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The authors declare no conflict of interest.
Keywords: bioorthogonal chemistry · copper ·
intracellular chemistry · metal carbenes · metal catalysis
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Manuscript received: July 5, 2021
Accepted manuscript online: August 13, 2021
Version of record online: && &&
,
&&&&
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ꢄ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 2 – 11
These are not the final page numbers!

Angewandte
Research Articles
Chemie
Research Articles
Bioorthogonal Chemistry
This work demonstrates that catalytic
processes involving metal–carbene inter-
mediates can be exported to the crowded
magma of a live mammalian cell. In par-
ticular, reactions involving copper car-
benes can be used for the synthesis of
fluorescent cyclic products in cellular
media, in a process that is initiated by an
S. Gutiꢀrrez, M. Tomꢁs-Gamasa,*
J. L. MascareÇas*
&&&& — &&&&
Exporting Metal-Carbene Chemistry to
Live Mammalian Cells: Copper-Catalyzed
Intracellular Synthesis of Quinoxalines
À
À
Enabled by N H Carbene Insertions
N H carbene insertion. The method also
allows the in cellulo synthesis of a prod-
uct that alters mitochondrial functions.
Angew. Chem. Int. Ed. 2021, 60, 2 – 11
ꢄ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
www.angewandte.org
&&&&
These are not the final page numbers!