Intracellular Ruthenium-Promoted (2+2+2) Cycloadditions

Article abstract of DOI:10.1002/anie.202006689

Metal-mediated intracellular reactions are becoming invaluable tools in chemical and cell biology, and hold promise for strongly impacting the field of biomedicine. Most of the reactions reported so far involve either uncaging or redox processes. Demonstrated here for the first time is the viability of performing multicomponent alkyne cycloaromatizations inside live mammalian cells using ruthenium catalysts. Both fully intramolecular and intermolecular cycloadditions of diynes with alkynes are feasible, the latter providing an intracellular synthesis of appealing anthraquinones. The power of the approach is further demonstrated by generating anthraquinone AIEgens (AIE=aggregation induced emission) that otherwise do not go inside cells, and by modifying the intracellular distribution of the products by simply varying the type of ruthenium complex.

Full text of DOI:10.1002/anie.202006689

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
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Accepted Article
Title: Intracellular, Ruthenium-Promoted (2+2+2) Cycloadditions
Authors: Jose Luis Mascarenas, Joan Miguel-Ávila, and María Tomás-
Gamasa
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202006689
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10.1002/anie.202006689
Angewandte Chemie International Edition
RESEARCH ARTICLE
Intracellular, Ruthenium-Promoted (2+2+2) Cycloadditions
Joan Miguel-Ávila,[a] María Tomás-Gamasa,*[a] José L. Mascareñas*[a]
Dedicated to the memory of Prof. Kilian Muñiz
[a]
J. Miguel-Ávila, Dr. M. Tomás-Gamasa, Prof. J. L. Mascareñas
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)
E-mail: joseluis.mascarenas@usc.es
Supporting Information for this article is given via a link at the end of the document. ((Please delete this text if not appropriate))
Abstract: Metal-mediated intracellular reactions are becoming
invaluable tools in chemical and cell biology, and promise to strongly
impact the field of biomedicine. Most of the reactions so far reported
involve uncaging or redox processes. We now demonstrate for the
first time the viability of performing multicomponent alkyne
cycloaromatizations inside live mammalian cells, using ruthenium
catalysts. Both, fully intramolecular and intermolecular cycloadditions
of diynes with alkynes are feasible, the latter providing for the
intracellular synthesis of appealing anthraquinones. The power of the
approach is further demonstrated by generating anthraquinone
AIEgens that otherwise do not go inside cells, and by modifying the
intracellular distribution of the products by simply varying the type of
ruthenium complex.
albeit there have been claims on the existence of Diels-
Alderases.9
Intracellular cycloadditions promoted by transition metals have
been limited to sporadic applications of the renowned copper-
mediated alkyne-azide cycloaddition (CuAAC) (Figure 1, i),10
a
key reaction for the establishment of the field of bioorthogonal
chemistry.11 Unfortunately, its efficiency in intracellular settings is
compromised by the toxicity and oxidation lability of copper. We
recently developed
a ruthenium-mediated alternative that
involves thioalkynes as azide partners (Figure 1, ii). However, it
couldn’t be implemented inside living cells.12
At some point of our research we envisioned that moving from
alkynes to appropriately tethered diynes, there might be better
chances for bioorthogonal “fishing” of transition metal reagents
owing to the bidentate η2 coordination ability of the substrate (A,
Figure 1).13 An ensuing oxidative cyclometallation would give a
metallacycle (B/C, Figure 1) that could be trapped by another
alkyne to provide aromatic adducts formally resulting from a
(2+2+2) cycloaddition (D, Figure 1, iii). This type of
cyclotrimerization of alkynes is well-known in the realm of
synthetic chemistry,14 and has also been even accomplished in
aqueous media, using Co, Rh, or Ru catalysts;15,16 but never in a
cellular context.
In this manuscript we demonstrate the viability of this process,
by reporting the first examples of a multicomponent cycloaddition
carried out in the interior of live, unfixed mammalian cells. The
reaction, which is mediated by non-toxic ruthenium complexes,
allows to make three C-C bonds, and at least two new cycles, in
a single step, therefore enabling a remarkable increase in
structural complexity.
We demonstrate that the cyclotrimerization is not only viable in
fully intramolecular cases, but also when using isolated alkynes
as external reaction partners (Figure 1, iv and v). Remarkably, this
latter reaction allows to generate “in cellulae” anthraquinones,
secondary metabolites which cannot be produced by mammalian
cells. We also demonstrate that whereas anthraquinone
derivatives with AIE (aggregation induced emission)
characteristics do not internalize, they can be indirectly
“introduced” inside cells using our reaction. We can even go one
step further and promote the preferential generation of
cycloadducts in different intracellular locations.
Introduction
Cells can be viewed as complex, tiny factories performing
thousands of simultaneous chemicals reactions.1 As chemists, we
are not bound to reproduce natural processes, and can therefore
aspire at conducting non-coded catalytic reactions within living
environments. While native enzymatic transformations are
unbeatable with regard to rate and selectivity, abiotic catalytic
processes might be advantageous in terms of versatility and
substrate scope.2 Furthermore, introducing non-natural reactivity
in cellular contexts can provide unprecedented opportunities for
cellular and metabolic intervention.3 However, performing
exogenous catalytic reactions within living cells is far from trivial,
as it requires to maneuver among multitude of components and
functional groups, and in an aqueous, crowded and
compartmentalized environment.
For many years, it was considered that organometallic catalysis
was incompatible with aqueous and biological environments;
however, the last decade has witnessed an increasing number of
reports on bioorthogonal, and even intracellular transformations,
mediated by transition metals.4 However, the repertoire of
reactions is yet scarce, and mainly limited to uncaging
processes;5
albeit
other
reactions,
like
reductions,6
isomerizations7 and cyclizations,8 have also been recently
introduced. Therefore, there is a great interest in increasing the
repertoire of metal-mediated reactions that can be performed in
live settings, as this will contribute to further establish this field,
and expand its biotech and biomedical potential. A particularly
attractive type of transformations are cycloaddition reactions,
owing to their synthetic power, and the lack natural counterparts,
1
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10.1002/anie.202006689
Angewandte Chemie International Edition
RESEARCH ARTICLE
Metal-promoted azide-alkyne cycloadditions (MAAC)
performance in cells seems in part associated to an improved
uptake with respect to Ru1 (Section S12). The processes didn’t
produce observable changes in the morphology of the cells or
affect the survival at the operating times (Figure S8).
i) CuAAC
ii) RutAAC
R
R
S
N
R'
R N3
R N3
N
[Ru]
[Cu]
N
N
N
R''
R'
N
R'
S
R''
R'
a) Intramolecular (2+2+2) Ru-mediated cycloadditiona
Key mechanistic steps
This work:
(2+2+2) cycloaddition in live cells
(iii)
N
N
% yield
Intramolecular
(iv)
(v)
PBS
Ru1
52
41
32
[M]
[M]
DMEM
Lysatesb
milieu
25 ºC
[Ru]
ruthenacyclo-
pentadiene B
A
N
N
1
2
Intermolecular
R
b) Intracellular generation of product 2
R
R
[M]
[Ru]
R
ruthenacyclo-
pentatriene C
D
✓ Fluorescent products
with AIE properties
✓ Bioorthogonal
✓ Biocompatible
Figure 1. Upper: i) CuAAC, feasible in living mammalian cells using specific
formulations of Cu;10 ii) RutAAC, can be performed in cell lysates.12 Bottom:
Left: mechanistic rational behind the orthogonality of the proposed
cycloaddition; right: (2+2+2) cycloadditions.
c) CTFC measurements
O
Ru
N
Ru
Ph3P
O
O
N
Cl
Results and Discussion
O
Ru2
Ru1
Our initial experiments were planned considering previous work
of Cadierno and coworkers,15b and of Teply,16 demonstrating the
viability of achieving cyclotrimerizations of alkynes under different
aqueous conditions, using [RuCp*Cl(COD)] (Ru1) as catalyst.
However, these reactions were carried out using relatively high
concentration of reactants, and required long reaction times,
which raised doubts on their exportability to biological
environments. We first tested the cyclotrimerization of triyne 1,
because it is an intramolecular process, and the resulting
pentacyclic product 2 is fluorescent (Figure 2a), thereby providing
for tracking the reaction using microscopy. After some
optimization, we managed to accomplish the reaction in complex
biological media such as phosphate-buffered saline solution
(PBS), cell culture media (DMEM) and HeLa cell lysates, even
using micromolar concentrations of the precursor (Figure 2a).
We then moved to a real biological scenario, a mammalian living
cell. Therefore, HeLa cells were incubated with Ru1 and washed
twice with DMEM to remove the excess of the complex. The
presence of ruthenium inside cells was confirmed by ICP-MS
analysis (Figure S17). Then, cells were mixed with substrate 1 for
1 h. Panel A in figure 2b displays the cells after treatment only
with substrate 1, which show no fluorescence. In contrast, we did
observe some fluorescence in cells that had been pretreated with
Ru1 (panel B), in agreement with the formation of the product 2.
Remarkably, using the complex Ru2, which we knew from
previous studies that presents a good cellular uptake and
tolerance,5e we observed a more intense intracellular emission
(Figure 2b, panel C), consisting with the generation of a larger
amount of product. Indeed, we calculated a three-fold higher
fluorescence intensity when compared with that resulting from the
use of Ru1 (Figure 2c). The ruthenium (IV) complex Ru2 is likely
reduced in situ to an active Ru(II) species;5d and its better
Figure 2. (a) Ruthenium-mediated annulation of triyne 1 in aqueous buffers. (b)
Fluorescence micrographies of Hela cells (brightfield) after incubation with: (A)
1; (B) Ru1, washed, and treated with 1; (C) Ru2, washed, and treated with 1;
conditions: cells were incubated with [Ru] (50 μM) for 30 min, followed by two
washings with DMEM, and treatment with substrate 1 (100 μM) for 1 h. Blue
channel: λexc = 385 nm, λem = 410-480 nm. (c) CTCF measurements (corrected
total cell fluorescence). Error bars from three independent experiments. Scale
bar: 12.5 μm. a Performed using substrate 1 (5 mmol), milieu (5.0 mL), Ru1 (25
b
mol%), 25 °C, 24 h; Yields determined by NMR using an internal standard.
-
Cell lysates 6 mg/mL. Note: counterions in Ru2 should be Br- and PF6 .
The above results are consistent with the generation of product 2
inside cells, through a formal (2+2+2) multicomponent annulation.
Albeit the intramolecularity of the process might favor the
annulation, we envisioned that once ruthenacycle intermediates
like B (Figure 1b) are formed, they could be trapped with external
alkynes (Figure 1, v). Gratifyingly, treatment of diyne 3 with
terminal alkyne 4a (4 equiv), in water, gave the expected
phthalan-like product in 60% yield, after only 2 h, using 25% of
Ru1 (Figure 3a). The yield increases up to 94% when the reaction
is carried out in a phosphate-buffered saline solution (PBS). The
reaction also takes place in DMEM and in presence of HeLa cell
lysates (95% and 89% yields, respectively, Figure 3a), and can
be promoted with other complexes like [CpRu(CH3CN)3]PF6 or
Ru2, which gave the product in modest yields of 38 and 14%
(respectively) in PBS (Table S1).
The success of the reaction with the model precursor 3, prompted
us to investigate the generation of fluorescent and/or biologically
relevant products. In particular, we were attracted by
anthraquinone skeletons (AQ), as they form the core of many
attractive secondary metabolites that have found relevant
biomedical applications.17
2
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10.1002/anie.202006689
Angewandte Chemie International Edition
RESEARCH ARTICLE
a) Intermolecular cycloadditionsa
Ru1 in water). In this case, we found that the use of an organic
co-solvent (H2O/MeCN 8:2), allowed a slightly better yield (59%).
Other terminal alkynes 4 also participate in the cycloaddition
(Table S3).
OMe
OMe
Ru1
+
O
Substrates 6a and 4a are not fluorescent, however the
anthraquinone product 7a presents bright green and blue
fluorescence emission, which facilitates monitorization in cellular
settings. For cellular experiments we preferred to use Ru2, owing
to its better uptake5e (although catalyst Ru1 was also able to elicit
the reaction in HeLa cells, Figure S10). These experiments were
carried out by incubating the cells with Ru2, followed by treatment
with alkyne 4a and diyne 6a (Figure 4a and S9). After 4 h, we
were glad to observe an intense fluorescence build-up inside the
cells, consistent with the formation of anthraquinone product 7a
(Figure 4a, panels A, B).
O
milieu
25 °C
3 (1 mM) 4a (4 mM)
5
H2O PBS DMEM Lysatesb
yield (%) 60 94
95
89
OMe
O
O
R
R
R
Ru1
+
4a
R
milieu
25 °C
7
6
O
O
a) Intracellular generation of product 7a
A
B
b) Anthraquinone biosynthesis
O
C
D
O
O
O
R
R
PKs
R
SCoA
O
O
O
O
+
- CO2
O
SCoA
O
O
O
H
SCoA
Figure 3. (a) Formal (2+2+2) cycloadditions in complex aqueous media. (b)
Plausible biological pathway for the synthesis of anthraquinones. a Performed
b
using substrate 3 or 6 (5 μmol), milieu (5.0 mL), 25 °C, 2 h; Cell lysates 2
mg/mL; Yields determined by NMR using an internal standard. Number of
replicates: between 2 and 4, and errors in yields range between 3-6%.
b) Detection of intracellular products by LC/MS
The AQ skeleton could be assembled in a single step by means
of a Ru-promoted annulation between bispropargylic ketones of
type 6 and terminal alkynes (Figure 3a, bottom).18 In nature, the
main polycyclic core of AQs is biosynthesized from acetylCoA and
MalonylCoA, in a multistep process governed by the multi-domain
enzyme polyketide synthase (PKs) present in some plants, fungi,
insects and bacteria (Figure 3b). However, mammalian cells do
not code for the biosynthesis of AQs, which added further interest
to explore the possibility of building these products inside these
cells.
P
P
O
+
5P
4P,
= O(CH2)3PPh3
P
P
O
Gratifyingly, the bispropargylic ketone 6a reacted with
commercially available alkyne 4a (4 equiv) in presence of Ru1
(50%) to give the expected tricycle 7a in 79% yield after 2 h at rt,
in water (Figure 3a, bottom). The catalyst loading can be reduced
to 25%, albeit there is a decrease in the yield (59%). Importantly,
the concentration of 6a could be decreased to 500 μM (53% yield).
The reaction can be also accomplished in phosphate-buffered
saline (PBS) even at 25% of Ru1 (65% yield). In cell culture media,
such as DMEM, or in HeLa cell lysates the reaction presents
almost no turnover (37 and 28% respectively, using 25% of Ru1).
The cycloaddition is also feasible using an internal 1,6-diyne such
as 6b, albeit it was somewhat slower (45% yield, using 50% of
7P
O
Figure 4. Intermolecular cycloadditions in live cells. (a) Fluorescence
micrographies of HeLa cells (brightfield) after incubation with: (A) Ru2, washed,
and treated with 6a and 4a; (B) similar, but observation under the green channel;
(C) 6a and 4a; (D) product 7a. (b) Products with the triphenylphosphonium tails.
Extracted ion chromatogram of the product 5P generated intracellularly
(methanolic extract) and also that detected in the DMEM washings, before the
extraction; (inset) mass spectrum of 5P. Conditions: HeLa cells pretreated with
50 μM of Ru2 for 50 min were washed twice and incubated with 50 μM of
substrate (3a or 6a) and 150 μM of alkynes (either 4a or 4P) for 4 h. Blue
3
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10.1002/anie.202006689
Angewandte Chemie International Edition
RESEARCH ARTICLE
channel: λexc = 385 nm, λem = 410-480 nm; green channel: λexc = 470 nm, λem
490-580 nm. Scale bar: 12.5 μm.
=
the observation times, inducing only a slight decrease after 12 h
of incubation at high concentrations (Figure S8).5e
Control experiments confirmed that addition of anthraquinone
product 7a to HeLa cells elicits a clear intracellular fluorescence
(panel D, Figure 4a). In contrast, cells co-incubated with
substrates 6a and 4a were nearly non-fluorescent (Figure 4a,
panel C). As expected, addition of each component alone, either
6a or 4a, to cells that had been previously treated with the
ruthenium reagent, led only to residual intracellular fluorescence
(arising from the substrates, Figure S10).
The intracellular formation of AQ products was further
confirmed by carrying out the reaction with the alkyne derivative
4P, which features a phosphonium tail (Figure 4b). This group
facilitates the identification of the product owing to its good
performance in ESI-MS spectrometry. Therefore, MS analysis
(extracted ion chromatogram) of methanolic extracts of cells
treated with 4P, and either 3 or 6a, under the standard reaction
conditions, revealed the presence of the expected cycloadducts
(Figure 4b, Section S14). These results suggest that this type of
molecular labels (phosphonium cations or similar), could allow to
overcome the current limitation of detection tactics to fluorescent
products.
a) Assembly of AIEgens using (2+2+2) cycloadditions
Ph
Ph
Ph
Ph
Ph
Ph
O
O
8a
9a
AIEgen
No AIEgen
Ph
Ph
Ph
Ph
Ph
O
O
Ph
9b
AIEgen
8b
No AIEgen
b) Intracellular generation of AIEgen 9b
A
B
C
To further expand the scope and potential of the technology, we
envisioned the synthesis of products with tetraphenylethylene
skeletons like 9a, using alkyne 8a as reaction partner (Figure 5a).
This type of structures, known to present aggregation-induced
emission properties (AIE),19 are very attractive from
a
photophysical perspective.20 Unfortunately, while the alkyne 8a
displays an excellent emission, the corresponding cycloadduct 9a
presents a strong decrease in its AIEgen properties, perhaps
because of an increased molecular rotation (Figure S11).
However, AQ 9b, obtained from alkyne 8b, behaves as an
excellent AIEgen (Figure 5a, Figure S12).
D
E
F
Importantly, the product (9b) can be generated inside live
mammalian cells using the cyclotrimerization reaction. Therefore,
the addition of substrates 6a and 8b to HeLa cells previously
treated with the ruthenium complex Ru2, leads to an intense
intracellular red staining (Figure 5b, panel D, enlarged images in
E, F). This result is especially significant because the
anthraquinone 9b is unable to travel inside cells. Indeed,
incubation of cells with the anthraquinone 9b leads to the
formation of aggregates in the extracellular media (Figure 5b,
panel G, and enlarged images in H, I). This product can be
internalized only after permeabilization of the membrane with
digitonin (Figure S16). Therefore, our approach allows to
indirectly introduce in cells a product (anthraquinone 9b) that
otherwise aggregates in the extracellular space. Importantly,
these results further corroborate the intracellular character of the
reaction.
Intracellular aggregates
G
H
I
Extracellular aggregates
Figure 5. Intracellular generation of AIEgens. (a) Structures of the precursors,
and cycloaddition products. (b) Fluorescence micrographies of HeLa cells
(brightfield) after incubation with: (A) Ru2, washed, and treated with 6a; (B) Ru2,
washed, and treated with 8b; (C) precursors 6a and 8b; (D) Ru2, washed, and
treated with 6a and 8b; (E,F) Zoom in of panel D; (G) anthraquinone 9b; (H,I)
Zoom in of panel G. Conditions: HeLa cells were incubated with Ru2 (50 μM)
for 1 h, followed by two washings with DMEM, and treatment with substrates 6a
and 8 (50 and 150 μM, respectively) for 3 h. Red channel: λexc = 550 nm, λem
=
570-690 nm. Scale bar: 12.5 μm.
An additional asset of the strategy stems from the possibility of
controlling the intracellular distribution of the AQ product 9b
depending on the ruthenium reagent. Therefore, by using
complex Ru1, the cellular fluorescence shows a cytosolic profile
(slightly less intense than with Ru2, Figure 6, panels A-C).
However, using complex Ru3, which is known to preferentially
accumulate in mitochondria owing to its lipophilic cationic
character,5e the fluorescence is mainly located in these organelles
(Figure 6, panels D-F, Figure S13).
Conclusion
In conclusion, our results demonstrate that transition metals can
promote challenging (2+2+2) multicomponent cycloadditions in
live mammalian cells. The use of abiotic reactants with bidentate
metal coordination abilities (diyne) seems instrumental for the
success of the process. In addition to provide a cutting-edge
addition to the palette of cell-compatible metal-mediated
transformations, our work demonstrates the viability of
synthesizing complex, biorelevant polycycles inside cells from
Finally, it is important to note that in all the above experiments
neither substrates nor products compromised the cell viability at
4
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10.1002/anie.202006689
Angewandte Chemie International Edition
RESEARCH ARTICLE
simple precursors, and therefore goes beyond the more common
uncaging or ligation reactions. Our intracellular synthetic strategy
allows to generate products that otherwise cannot be delivered to
the cell, as well as controlling their spatial distribution by using
suitable ruthenium reagents. Indeed, the possibility of generating
the desired products in specific subcellular locations, just by
changing the targeting characteristics of the reagents, is exciting.
While further work to increase the efficiency of the reactions is
needed, our discoveries should further foster this young field of
intracellular metal catalysis, and trigger important applications in
chemical and synthetic biology, and in biomedicine.
Keywords: Biological chemistry • Cycloadditions • Ruthenium •
Metal catalysis • Intracellular chemistry
[1]
a) S. Martínez Cuesta, S. A. Rahman, N. Furnham, J. M. Thornton,
Biophys J. 2015, 109, 1082-1086; b) B. Alberts, A. Johnson, J. Lewis, D.
Morgan, M. Raff, K. Roberts, P. Walter in Molecular biology of the cell 2017,
Garland Science; sixth edition, New York and Abingdon, UK.
[2]
a) M. T. Reetz, Acc. Chem. Res. 2019, 52, 336-344; b) F. Schwizer, Y.
Okamoto, T. Heinisch, Y. Gu, M. M. Pellizzoni, V. Lebrun, R. Reuter, V. Köhler,
J. C. Lewis, T. R. Ward, Chem. Rev. 2018, 118, 142-231; c) M. Jeschek, S.
Panke, T. R. Ward, Trends in Biotech. 2018, 36, 60-72; d) R. B. Leveson-Gower,
C. Mayer, G. Roelfes, Nat. Rev.Chem. 2019, 3, 687-705.
[3]
a) M. Yang, J. Li, P. R. Chen, Chem. Soc. Rev. 2014, 43, 6511-6526; b)
J. Li, J. Yu, J. Zhao, J. Wang, S. Zheng, S. Lin, L. Chen, M. Yang, S. Jia, X.
Zhang, P. R. Cheng, Nat. Chem. 2014, 6, 352-361; c) J. Wang, B. Cheng, J. Li,
Z. Zhang, W. Hong, X. Chen, P. R. Chen, Angew. Chem. Int. Ed. 2015, 54,
5364−5368; d) A. M. Pérez-López, B. Rubio-Ruiz, V. Sebastián, L. Hamilton, C.
Adam, T. L. Bray, S. Irusta, P. M. Brennan, G. C. Lloyd-Jones, D. Sieger, J.
Santamaría, A. Unciti-Broceta, Angew. Chem. Int. Ed. 2017, 56, 12548-12552;
e) C. Adam, A. M. Pérez-López, L. Hamilton, B. Rubio-Ruiz, T. L. Bray, D.
Sieger, P. M. Brennan, A. Unciti-Broceta, Chem. Eur. J. 2018, 24, 16783-16790;
f) M. Sancho-Abero, B. Rubio-Ruiz, A. M. Pérez-López, V. Sebastián, P. Martín-
Duque, M. Arruebo, J. Santamaría, A. Unciti-Broceta, Nat. Catal. 2019, 2, 864-
872.
Reaction with Ru1: CYTOSOLIC DISTRIBUTION
Reaction
Mitochondrial marker
Overlay
Reaction with Ru3: MITOCHONDRIAL ACCUMULATION
[4]
For recent reviews, see: a) M. Martínez-Calvo, J. L. Mascareñas, Coord.
Chem. Rev. 2018, 359, 57-79; b) A. H. Ngo, S. Bose, L. H. Do, Chem. Eur. J.
2018, 24, 10584-10594; c) Y. Bai, J. Chen, S. C. Zimmerman, Chem. Soc. Rev.
2018, 47, 1811-1821; d) J. J. Soldevilla-Barreda, N. Metzler-Nolte, Chem. Rev.
2019, 119, 829-869; e) M. Martínez-Calvo, J. L. Mascareñas, Chimia 2018,
11, 791-801.
[5]
a) C. Streu, E. Meggers, Angew. Chem. Int. Ed. 2006, 45, 5645-5648; b)
Reaction
Mitochondrial marker
Overlay
T. Völker, F. Dempwolff, P. L. Graumann, E. Meggers, Angew. Chem. Int. Ed.
2014, 53, 10536-10540; c) M. I. Sánchez, C. Penas, M. E. Vázquez, J. L.
Mascareñas, Chem. Sci. 2014, 5, 1901-1907; d) G. Y. Tonga, Y. Jeong,
B. Duncan, T. Mizuhara, R. Mout, R. Das, S. T. Kim, Y. C. Yeh, B. Yan, S. Hou,
V. M. Rotello, Nat. Chem. 2015, 7, 597-603; e) M. Tomás-Gamasa, M.
Martínez-Calvo, J. R. Couceiro, J. L. Mascareñas, Nat. Commun. 2016, 7,
12538-12547; f) M. Martínez-Calvo, J. R. Couceiro, P. Destito, J. Rodríguez, J.
Mosquera, J. L. Mascareñas, ACS Catal. 2018, 8, 6055-6061; g) B. J. Stenton,
B. L. Oliveira, M. J. Matos, L. Sinatra, G. J. L. Bernardes, Chem. Sci. 2018, 9,
4185-4189; h) S. Learte-Aymamí, C. Vidal, A. Gutiérrez-González, J. L.
Mascareñas, Angew. Chem. Int. Ed. 2020, 59, 9149-9154; i) P. Destito, A.
Sousa-Castillo, J. R. Couceiro, F. López, M. A. Correa-Duarte, J. L.
Mascareñas, Chem. Sci. 2019, 10, 2598-2603; j) R. Martínez, C. Carrillo-
Carrión, P. Destito, A. Álvarez, M. Tomás-Gamasa, B. Peláz, F. López, J. L.
Mascareñas, P. Del Pino Cell. Rep. Phys. Sci. 2020, 1, 100076.
Ru
O
O
N
P
O
N
Ph2
O
Ru3
Figure 6. Reagent-dependent spatial distribution of the product of the
reaction between 6a and 8b. Fluorescence micrographies of HeLa cells
incubated with: (A,D) Ru1 or Ru3, respectively, washed, and treated with 6a
and 8b (red channel); (B,E) mitochondrial labelling with TMRE
(tetramethylrhodamine ethyl ester, green channel); (C) overlay of A and B; (F)
overlay of D and E. Same conditions as in Figure 5. Cells were finally incubated
with mitochondrial marker (100 nM) for 10 min. Scale bar: 12.5 μm. Note:
-
counterions in Ru3 are Br- and PF6 .
[6]
a) J. J. Soldevila-Barreda, I. Romero-Canelon, A. Habtemariam, P. J.
Sadler, Nat. Commun. 2015, 6, 6582-6590; b) J. P. C. Coverdale, I. Romero-
Canelon, C. Sanchez-Cano, G. J. Clarkson, A. Habtemariam, M. Wills, P. J.
Sadler, Nat. Chem. 2018, 10, 347−354; c) S. Bose, A. H. Ngo, L. Do, J. Am.
Chem. Soc. 2017, 139, 8792-8795.
Acknowledgements
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 and
Centro Singular de Investigación de Galicia Accreditation 2019-
2022, ED431G 2019/03), the European Union (European
Regional Development Fund-ERDF corresponding to the
multiannual financial framework 2014-2020), and the European
Research Council (Advanced Grant No. 340055). J. M. Á. thanks
the Ministerio de Educación, Cultura y Deporte for the FPU
fellowship (FPU16/00711) and M. T. G thanks the financial
support from the Agencia Estatal de Investigación (RTI2018-
093813-J-I00). The authors thank R. Menaya-Vargas for excellent
technical assistance, and especially M. Marcos for excellent and
essential contributions to the LC-MS analysis.
[7]
J. Am. Soc. Chem. 2019, 141, 5125-5129.
[8] a) C. Vidal, M. Tomás-Gamasa, P. Destito, F. López, J. L. Mascareñas,
C. Vidal, M. Tomás-Gamasa, A. Gutiérrez-González, J. L. Mascareñas,
Nat. Commun. 2018, 9, 1913-1921; b) M. A. Miller, B. Askevold, H. Mikula, R.
H. Kohler, D. Pirovich, R. Weissleder, Nat. Commun. 2017, 8, 15906-15918.
[9]
There have been claims on the existence of Diels-Alderases: a) A.
Minami, H. Oikawa, The Journal of Antibiotics 2016, 1–7; b) H. Oikawa, Cell
Chemical Biology 2016, 429-430.
[10] a) A. J. Link, D. A. Tirrell, J. Am. Chem. Soc. 2003, 125, 11164−11165;
b) A. J. Link, M. K. S. Vink, D. A. Tirrell, J. Am. Chem. Soc. 2004, 126,
10598−10602; c) K. E. Beatty, F. Xie, Q. Wang, D. A. Tirrell, J. Am. Chem. Soc.
2005, 127, 14150-14151; d) Y. Bai, X. Feng, H. Xing, Y. Xu, B. K. Kim, N. Baig,
T. Zhou, A. A. Gewirth, Y. Lu, E. Oldfield, S. C. Zimmerman, J. Am. Chem. Soc.
2016, 138, 11077-10080; e) S. Li, L. Wang, F. Yu, Z. Zhu, D. Shobaki, H. Chen,
M. Wang, J. Wang, G. Qin, U. J. Erasquin, L. Ren, Y. Wang, C. Cai, Chem. Sci.
5
This article is protected by copyright. All rights reserved.

10.1002/anie.202006689
Angewandte Chemie International Edition
RESEARCH ARTICLE
2017, 8, 2107-2114; f) J. Miguel-Ávila, M. Tomás-Gamasa, A. Olmos, P. J.
Pérez, J. L. Mascareñas, Chem. Sci. 2018, 9, 1947-1952.
[11] a) E. M. Sletten, C. R. Bertozzi, Angew. Chem. Int. Ed. 2009, 48, 6974-
6998; b) D. M. Patterson, J. A. Prescher, Curr. Opin. Chem. Biol. 2015, 28,
141−149; c) N. K. Devaraj, ACS Cent. Sci. 2018, 4, 952-959.
[12] P. Destito, J. R. Couceiro, H. Faustino, F. López, J. L. Mascareñas,
Angew. Chem. Int. Ed. 2017, 56, 10766-10770.
[13] Chelating ligands have been useful in Cu click chemistry: a) C.
Uttamapinant, A. Tangpeerachaikul, S. Grecian, S. Clarke, U. Singh, P. Slade,
K. R. Gee, A. Y. Ting, Angew. Chem. Int. Ed. 2012, 51, 5852−5856; b) V.
Bevilacqua, M. King, M. Chaumontet, M. Nothisen, S. Gabillet, D. Buisson, C.
Puente, A. Wagner, F. Taran, Angew. Chem. Int. Ed. 2014, 53, 5872-5876; c)
T. Machida, N. Winssinger, ChemBioChem 2016, 17, 811-815; d) Y. Su, L. Li,
H. Wang, X. Wang, Z. Zhang, Chem. Commun. 2016, 52, 2185-2188.
[14] For reviews on metal-catalyzed (2+2+2) cycloadditions, see: a) M.
Lautens, W. Klute, W. Tam, Chem. Rev. 1996, 96, 49-92; b) Y. Yamamoto, Curr.
Org. Chem. 2005, 9, 503-519; c) S. Kotha, E. Brahmachary, K. Lahiri, Eur. J.
Org. Chem. 2005, 4741-4767; d) P. R. Chopade, J. Louie, Adv. Synth. Catal.
2006, 348, 2307-2327; e) B. R. Galan, T. Rovis, Angew. Chem. Int. Ed. 2009,
48, 2830-2834; f) G. Domínguez, J. Pérez-Castells, Chem. Soc. Rev. 2011, 40,
3430-3444; g) Y. Shibata, K. Tanaka, Synthesis 2012, 44, 323-350.
[15] a) H. Kinoshita, H. Shinokubo, K. Oshima, J. Am. Chem. Soc. 2003, 125,
7784-7785; b) V. Cadierno, S. E. García-Garrido, J. Gimeno, J. Am. Chem. Soc.
2006, 128, 15094-15095; c) F. Xu, C. Wang, X. Li, B. Wan, ChemSusChem
2012, 5, 854–857; d) Z. Nairoukh, M. Fanun, M. Schwarze, R. Schomäcker, J.
Blum, Journal of Molecular Catalysis A: Chemical 2014, 382, 93–98; e) J.
Francos, S. E. García-Garrido, J. García-Álvarez, P. Crochet, J. Gimeno, V.
Cadierno, Inorganica Chimica Acta 2017, 455, 398–414; f) C. R. Travis, G. H.
Gaunt, E. A. King, D. D. Young, Chembiochem 2019, 21, 310-314.
[16] a) L. Severa, J. Vávra, A. Kohoutova, M. Čížková, T. Šálová, J. Hývl, D.
Šaman, R. Pohl, L. Adriaenssens, F. Teplý, Tetrahedron Lett. 2009, 50, 4526–
4528; b) L. Adriaenssens, L. Severa, J. Vávra, T. Šálová, J. Hývl, M. Čížková,
R. Pohl, D. Šaman, F. Teplý, F. Collect. Czech. Chem. Commun. 2009, 74,
1023–1034.
[17] G. Díaz-Muñoz, I. L. Miranda, S. K. Sartori, D. C. de Rezende, M. A. N.
Díaz, Studies in natural products chemistry, 2018, 58, 313–338.
[18] Y. Yamamoto, K. Hata, T. Arakawa, K. Ito, Chem. Commun. 2003, 1290–
1291.
[19] a) W. Chen, C. Zhang, X. Han, S. H. Liu, Y. Tan, J. Yin, J. Org.
Chem. 2019, 84, 14498-14507; b) X. Gao, J. Z. Sun, Z. B. Tang, Isr. J. Chem.
2018, 58, 1–16; c) W. Xu, M. M. S. Lee, Z. Zhang, H. H. Y. Sung, I. D. Williams,
R. T. K. Kwok, J. W. Y. Lam, D. Wang, B. Z. Tang, Chem. Sci. 2019, 10, 3494-
3501.
[20] For recent reviews, see: a) J. Mei, N. L. C. Leung, R. T. K. Kwok, J. W.
Y. Lam, B. Z. Tang, Chem. Rev. 2015, 115, 11718-11940; b) J. Mei, Y. Huang,
H. Tian, ACS Appl. Mater. Interfaces 2018, 10, 12217−12261; c) Z. He, C. Ke,
B. Z. Tang, ACS Omega 2018, 3, 3267-3277; d) Y. Chen, J. W. Y. Lam, R. T.
K. Kwok, B. Liu, B. Z. Tang, Mater. Horiz. 2019, 6, 428-433.
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Entry for the Table of Contents
Ruthenium-mediated cycloaromatization reactions engaging three alkyne units can be performed in intracellular settings, in a
biocompatible way. This synthetically powerful reaction allows to build a variety of relatively complex, functional cyclic products inside
cells, therefore providing an “artificial” metabolic route to these interesting species.
Institute and/or researcher Twitter usernames: ((optional))
@MetBioCat @ciqususc @JoanMA32585084
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