Ruthenium-Catalyzed Azide–Thioalkyne Cycloadditions in Aqueous Media: A Mild, Orthogonal, and Biocompatible Chemical Ligation

Article abstract of DOI:10.1002/anie.201705006

The development of efficient metal-promoted bioorthogonal ligations remains as a major scientific challenge. Demonstrated herein is that azides undergo efficient and regioselective room-temperature annulations with thioalkynes in aqueous milieu when treat

Full text of DOI:10.1002/anie.201705006

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Accepted Article
Title: Ruthenium-Catalyzed Azide-Thioalkyne Cycloadditions in
Aqueous Media (RuAtAC): A Mild, Orthogonal and Biocompatible
Chemical Ligation
Authors: Jose Luis Mascarenas, Paolo Destito, jose R. Couceiro, Helio
Faustino, and Fernando Lopez
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201705006
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10.1002/anie.201705006
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Ruthenium-Catalyzed Azide-Thioalkyne Cycloadditions in Aqueous
Media (RuAtAC): A Mild, Orthogonal and Biocompatible Chemical
Ligation
Paolo Destito,[a] José R. Couceiro,[a] Hélio Faustino,[a] Fernando López,* [a,b] José L. Mascareñas*[a]
Dedication ((optional))
Abstract: The development of efficient metal-promoted
bioorthogonal ligations remains as a major scientific challenge.
Herein we demonstrate that azides undergo efficient and
regioselective room temperature annulations with thioalkynes in
aqueous milieu, when treated with catalytic amounts of a suitable
ruthenium complex. The reaction is compatible with different
biomolecules, and can be carried out in complex aqueous mixtures
such as phosphate buffered saline, cell lysates, fetal bovine serum,
and even living bacteria (E. coli). Importantly, the reaction is mutually
compatible with the classical CuAAC.
metal-catalyzed annulations, alternative to the CuAAC,
represents a highly appealing goal.[10]
Several azide-alkyne cycloadditions using metals others
than Cu have been described in recent years,[11] but only the
ruthenium variant (RuAAC)[12] has shown a meaningful scope
(Scheme 1, eq. 2).[13] In contrast to the CuAAC, which
encompasses dinuclear Cu intermediates such as I and II,[14] the
Ru-promoted reaction involves intermediate species like III,
which evolve by oxidative addition to IV, and eventually to the
triazole products.[15] In consonance with this scenario, the
RuAAC, essentially developed in organic solvents, tolerates
disubstituted alkynes, but can produce mixtures of regioisomers.
Probably, the notion that it is not compatible with water and air
The Cu-catalyzed azide-alkyne cycloaddition (CuAAC),
paradigm of “click” chemistry,[1] can be considered among the
most relevant chemical transformations discovered in the last
decades, with countless applications in many areas of science.[2]
The biological relevance of this reaction stems from its
robustness and compatibility with aqueous media, as well as
from its good bioorthogonality.[3] However, the transformation
still presents important limitations. Thus, in addition to be fairly
incompatible with thiols, the reaction is essentially restricted to
terminal alkynes, consequence of a mechanism that requires the
formation of copper acetylide intermediates (Scheme 1, eq. 1).
An important additional drawback has to do with the side
reactivity and toxicity of Cu ions in biological contexts.[4]
Furthermore, to reach efficient conversions in typically diluted
biological settings, the reactive CuI species need to be
generated in situ using excess amounts of a CuII source and
sodium ascorbate, a reductant that is not innocent in biological
contexts.[5] These issues have been partially addressed by using
Cu-stabilizing ligands that enhance the biocompatibility and
kinetic of the reactions.[6,7] Cu-free, strain-promoted annulations
have shown to be an efficient alternative,[8] however, these
reactions also present limitations associated to the side-
reactivity of the reactants. Therefore, the development of new
bioorthogonal and biocompatible reactions that address some of
the above limitations remains as a major challenge.[9] In
particular, the discovery of robust and aqueous-compatible
atmospheres
has
precluded
more
bio-focused
investigations.[16,13] Recent data suggest that some Ru
complexes can promote the process in water, albeit the
reactions require thermal activation, and presented a limited
scope.[17]
Herein, we demonstrate that certain RuII complexes can
indeed catalyze the cycloaddition between azides and alkynes in
water, and at room temperature. Importantly, the reaction is
especially efficient when thioalkynes are used as reaction
partners (Scheme 1, eq. 3). Moreover, the process is mutually
compatible with the CuAAC, tolerant to different types of
biomolecules, including thiols, and can be carried out in
phosphate buffered saline, in cell lysates or cell culture media,
and even in presence of living bacteria (E. coli).
[a]
P. Destito, Dr. J. R. Couceiro, Dr. Hélio Faustino, Dr. F. López, 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: fernando.lopez@csic.es, joseluis.mascarenas@usc.es
Scheme 1. CuAAC and RuAAC, key mechanistic features.
[b]
Dr. F.López
Instituto de Química Orgánica General CSIC, Juan de la Cierva 3, 28006,
Madrid, Spain
At the outset, we were inspired by a report of Jia and Sun on an
iridium-promoted azide-thioalkyne cycloaddition.[18] Albeit the
method was developed in anhydrous CH2Cl2, an isolated
Supporting information for this article is given via a link at the end of
the document.
1
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10.1002/anie.201705006
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COMMUNICATION
example in water using benzyl azide called our attention.
Unfortunately, when we tested the reaction of thioalkyne 2a with
the fluorogenic anthracenyl-azide probe 1a,[19] the yield of the
corresponding adducts (3aa / 3aa’) turned out to be very modest
(Table 1, entry 1).[20] Remarkably, using Cp*Ru(cod)Cl as
catalyst,[15a] we observed, after 24 h, a substantial formation of
the desired cycloadducts with excellent regioselectivity (3aa :
3aa’ = 19:1, 58% combined yield, entry 2). This good result,
together with the previously demonstrated biocompatibility of this
type of Ru complexes,[21] prompted us to further explore the
process. Doubling the equivalents of the thioalkyne (2a) allowed
to obtain an excellent 99% yield of the desired triazoles after 9 h
of stirring at rt. Monitoring of the reaction at different times
confirmed the formation of the products in 78% yield after just 30
min, with the rate being then gradually reduced (entry 4). [22] The
performance of [Ir(cod)Cl]2 could not be improved by using 2
The cycloaddition is also feasible using a regular internal alkyne
such as 2b, albeit it was somewhat slower (10% less conversion
after 30 min), and led to a 5:1 mixture of regioisomers (entry 11).
Other alkynes such as 2c were unreactive under identical
conditions (entry 12). The higher reactivity of the thioalkyne
partner was clearly visible in a cross-competition experiment:
when the azide 1a was reacted with a 1:1 mixture of 2a and 2b
(2 equiv each), the triazole 3aa, arising from the cycloaddition
with the thioalkyne was exclusively observed, in 98% yield (entry
13). Interestingly, NMR analysis of the interaction between
Cp*Ru(cod)Cl and the alkynes (in CD2Cl2) demonstrated that
while 2a displaces the cod ligand at rt, 2b does not induce any
change (Supp. Info. Page S6-S9). Analogous experiments using
[Cp*RuCl]4 and 2a, revealed a rapid formation of a new complex
identified as [Cp*Ru(2a)Cl], whereas with alkyne 2b no new Ru
species could be detected, even after 3 h.[25] Thus, the good
performance of thioalkynes might be in part related to their
ability to strongly coordinate the “Cp*RuCl” moiety at rt.
Additionally, the presence of the sulfur atom should also favor
the formation of the required ruthenacyclic intermediate of type
IV (Scheme 1, eq. 2).
equiv. of thioalkyne, (entry 4); and other RuII catalysts, such as
[17b]
Cp*Ru(PPh3)2Cl,[12] RuH2(CO)(PPh3)3,
were not efficient
[23]
(entries 5-7). On the other hand, the tetramer [Cp*RuCl]4 was
quite effective (entry 8). The reaction between 1a and 2a, could
also be carried out in CH2Cl2, however, obtaining good yields
required the use of anhydrous solvent and inert atmospheres
(entries 9 vs 10), which is not necessary in water. It looks like
the aqueous solvent is somewhat protecting the Ru species from
being rapidly deactivated.[24]
With these conditions in hand, we analyzed the scope of the
method (RuAtAC). Despite the relatively poor water solubility of
many of the azides or thioalkynes, the reactions proved to be
general at rt, and the corresponding triazoles were obtained in
good yields and with excellent regioselectivities (Table 2). Thus,
aryl and aliphatic substituents either attached to the sulfur atom
or to the terminal position of the alkyne were tolerated (e.g. 3aa-
3af). Terminal or trimethylsilyl-substituted thioalkynes (2g, 2h)
provided the corresponding adducts 3ag and 3ah in good yields.
Importantly, not only the anthracenyl and benzyl azide (1a, 1b)
participated in the process, but aliphatic azides such as (2-
azidoethyl)benzene (1c) or 2-azidoethan-1-ol (1d) also reacted
cleanly to provide the corresponding triazoles (3ca, 3ce, 3de). p-
Tolyl azide reacted with 2a to provide 3ea with a moderate 40%
yield, a value that could be improved up to 61% by using
[Cp*RuCl]4 as catalyst. Interestingly, different types of
fluorophore-equipped trisubstituted triazoles could be generated
Table 1. Identification of reaction conditions in water.[a]
1a : 2
time Conv
3 : 3’
entry
[Cat] (X mol%)
[Ir(cod)Cl]2 (2.5)
Cp*Ru(cod)Cl (5)
ratio Solv. (h) (%)[b] ratio[b] yield[b,c]
1
2
1 : 1 H2O
1 : 1 H2O
24
24
42
65
1 : 0
29
58
19 : 1
3
4
Cp*Ru(cod)Cl (5)
Cp*Ru(cod)Cl (5)
[Ir(cod)Cl]2 (2.5)
1 : 2 H2O
1 : 2 H2O
1 : 2 H2O
9
0.5
24
24
24
24
2
99
80
36
47
0
19: 1
19: 1
1 : 0
23 : 1
-
99
78
20
17
0
5
by using
a dansyl-based azide (such as in 3fa) or by
6
Cp*Ru(PPh3)2Cl (5) 1 : 2 H2O
RuH2(CO)(PPh3)3 (5) 1 : 2 H2O
incorporating a coumarin moiety, either as part of the thioalkyne
(e.g. 3aj) or of the organic azide (3ga and 3ha). Additionally,
following work developed by Waser,[26] 2-thioglucose and a
cysteine-containing dipeptide were selectively thio-alkynylated
with EBX reagents. Gratifyingly, although the low water solubility
of the thioalkynylated protected dipeptide 2k demanded the use
of small amounts of a cosolvent (i.e CH2Cl2, 5-10% vol.), its
reactions with 1a and 1f proceeded efficiently, affording the
desired products 3ak and 3fk in good yields. On the other hand,
the reaction between an alkynylated thioglucose and 1a took
place smoothly in water to give the triazole 3al in 64% yield.
7
8
[Cp*RuCl]4 (1.25)
Cp*Ru(cod)Cl (5)
1 : 2 H2O
99
99
14 : 1
17 : 1
99
99
9[d]
1 : 2 CH2Cl2
10
Cp*Ru(cod)Cl (5)
Cp*Ru(cod)Cl (5)
Cp*Ru(cod)Cl (5)
1 : 2 CH2Cl2
1 : 2 H2O
1 : 2 H2O
2
9
44
99
10
18 : 1
5 : 1
-
37
11[e]
12[f]
95[e]
<5[f]
24
13[g]
Cp*Ru(cod)Cl (5)
1 : 4 H2O
24
4
19 : 1[h]
98[h]
[a] Unless otherwise noted, 2a (1-2 equiv), water and 1a (1 equiv, 75 mM)
were sequentially added under air to a vial containing the catalyst (that had
been kept under N2), and the mixture was stirred at rt. [b] Determined by 1H-
NMR of the crude mixture with internal standard. [c] Combined yield of 3/ 3’.
[d] Carried out under an inert atmosphere in anhydrous solvent. [e] Carried
out with 2b; products: 3ab / 3ab’. [f] Carried out with 2c; products: 3ac/ 3ac’.
[g] Using both 2a and 2b (2 equiv. each). [h] Products: 3aa and 3aa’.
We next explored the bioorthogonality of the chemistry by
performing the reaction in presence of different biomolecular
additives, or under biologically relevant conditions (Table 3).
Gratifyingly, the RuAtAC could be efficiently carried out in the
presence of glutathione (20 fold excess respect to the [Ru], entry
1), different aminoacids (entry 2) or even in the presence of a
random miniprotein (entry 3). The desired triazole was obtained
2
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COMMUNICATION
Table 2. Scope of the RuAtAC in water, at rt[a]
While the Ru-promoted reaction was significantly faster than the
Cu-counterpart when using CuSO4 and sodium ascorbate for the
latter (60% vs 22% yield, after 2h), the CuAAC became faster by
including additives such as BTTAA (75% yield after 2h, Table
S4). Importantly, the CuAAC failed with internal alkynes,
including thioalkynes like 2a and, not surprisingly, it is essentially
inhibited in the presence of thiols like glutathione (0% yield after
24 h). In contrast, the RuAtAC works effectively even in the
presence of a 20-fold excess of glutathione (75% yield), and
provides the products with both internal and terminal thioalkynes
(as shown in Table 2). On the weakest side, the efficiency of the
RuAtAC decreases upon dilution (16% yield at 250 µM), while
the ligand accelerated CuAAC provided a 57% yield under
similar µM conditions (Supp. Info, pages S11-S13).
Table 3. Analysis of the biocompatibility of the method[a]
entry
milieu[b]
H2O / Glutathione
Conv.(%) [c] 3 : 3’[c] yield (%) [c,d]
1
2
3
4
5
6
7
80
93
99
99
99
88
99
19 : 1
18 : 1
23 : 1
18 : 1
15 : 1
17 : 1
16 : 1
60
82
98
97
84
77
91
H2O / Hist + Fmoc-ala
H2O / Peptide 39 aa’s (0.5 mM)
PBS
Cell Cultured media (DMEM)
Fetal Bovine serum (FBS)
Cell Lysates (Hela)
[a] Conditions: 2a (2 equiv) was added to a suspension of Cp*Ru(cod)Cl (5
mol%), 1a (1 equiv, 75 mM) and the additive, in the selected milieu, and the
resulting mixture was stirred for 24 h. [b] The additives in entries 1-2 are in 20
fold excess with respect to the Ru catalyst [c] Determined by 1H-NMR of the
crude mixture using an internal standard. [d] Combined yield of 3aa/3aa’.
[a] Conditions: 2 (2 equiv), water and 1a (1 equiv, 75 mM) were sequentially
added under air to a vial containing Cp*Ru(cod)Cl (5 mol%) that had been
kept under N2. The vial was closed, and the mixture stirred at rt for 15-24 h.
Regioselectivities (3 : 3') > 18 : 1 unless otherwise noted (determined by 1H-
NMR of the crude mixtures with internal standard). Isolated yields of pure 3,
unless otherwise noted. Yields of the reactions carried out in anhydrous
CH2Cl2 under an inert atmosphere are shown in brackets. [b] Yield of 3
determined by 1H-NMR of the crude mixture with internal standard. [c]
Corresponds to a 18 : 1 mixture of 3ca : 3ca'. [d] Carried out with [Cp*RuCl]4
(1.25 mol%).
The lack of reactivity of internal thioalkynes in the presence
of Cu catalysts suggested that both, the CuAAC and the
RuAtAC, could be mutually orthogonal.[27] Gratifyingly, the
engineered diyne 4 was quantitatively converted to the bis-
triazole 5, without cross-reactivity, by performing a CuAAC with
dansyl-azide 1f, followed by in situ addition of the Ru-catalyst
and the anthracenyl azide 1a (1 equiv. with respect to
thioalkyne) (Scheme 2). Considering the scarcity of mutually
compatible bioorthogonal reactions, the possibility of using both
annulations in tandem, one pot processes, looks certainly
promising.[27]
in moderate to excellent yields and similar regioselectivities than
in pure water (> 15 : 1). The reaction could also be carried out in
phosphate-buffered saline (PBS) (entry 4). Additionally,
frequently used culture cell media, such as DMEM, fetal bovine
serum (FBS), and Hela cell lysates are also excellent reaction
media, so the triazole 3aa was obtained in yields varying from
77% to 91%, (entries 5-7). Analog reactions in these media
between (2-azidoethyl)benzene (1c) and thioalkyne 2e gave
good yields of 3ce (Table S3).
At this point, it was interesting to confront the RuAtAC and
CuAAC in water, in order to establish strengths and weaknesses
of each method. Thus, we carried out parallel experiments using
(2-azidoethyl)benzene (1c) (75 mM), 5 mol% of each catalyst,
and either thioalkyne 2e (for Ru) or phenylacetylene (for Cu).
Scheme 2. Tandem CuAAC and RuAtAC, in water.
3
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Finally, and importantly, we found that the RuAtAC can also
be carried in presence of bacteria (E. coli) without compromising
their viability. Therefore, incubation of PBS containing E coli with
1a (1 mM), 2a (2 mM) and Cp*Ru(cod)Cl (100 µM) led to a rapid
increase in the fluorescence. After 24h, centrifugation and
analysis in a plate reader of both, the extracellular supernatant,
and the methanol/water (8 : 2) extracts of the resulting bacteria
pellet, showed a combined increased in fluorescence of 8 times
with respect to controls (Figure S25). Importantly, the
fluorescence was mainly concentrated inside the bacteria, and
the product (3aa) was also detected by HPLC-ESI. Analysis of
the optical density of the bacterial cultures revealed that neither
the catalyst nor the reactants are meaningfully toxic (Table S5).
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In summary, we have discovered a new methodology to achieve
catalytic, orthogonal chemical annulations in water, at room
temperature. The reaction is promoted by specific RuII catalysts,
works efficiently with a variety of azides and thioalkynes, and
can be carried out in presence of biomolecules (glutathione,
aminoacids, peptides). The reaction is also efficient in
phosphate buffered saline, and in complex biological media such
as cell lysates and fetal bovine serum, and even in presence of
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Acknowledgements
This work has received financial support from Spanish grants
(SAF2016-76689-R), the Xunta de Galicia (2015-CP082 and
Centro Singular de Investigación de Galicia accreditation 2016-
2019 ED431G/09), the ERDF, and the ERC (Adv. Grant Nº.
340055). We also thank the Orfeo-Cinqa network CTQ2016-
81797-REDC, and T. Seedat and V. Fraga for preliminary
experiments.
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Keywords: bioorthogonal • ruthenium • thioalkyne • click • azide
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[24] This might be associated to the lower solubility of O2 and/or the Ru
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2009, 109, 725.
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[25] a) According to Fürstner et. al, Cp*Ru(2a)Cl would be an 18e- Ru
species, with the alkyne acting as a 4e- donor ligand: D.-A. Roşca, K.
4
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Radkowski, L. M. Wolf, M. Wagh, R. Goddard, W. Thiel, A. Fürstner, J.
Am. Chem. Soc. 2017, 39, 2443; b) With 2b, a fast exchange between
the cod ligand and the alkyne cannot be discarded. c) Importantly,
[Cp*Ru(2a)Cl] is catalytically competent (Supp. Info., pages S6-S9).
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5
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Entry for the Table of Contents
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Paolo Destito, José R. Couceiro, Hélio
Faustino, Fernando López,* José L.
Mascareñas*
Page No. – Page No.
Ruthenium-Catalyzed Azide-
Thioalkyne Cycloadditions in
Aqueous Media (RuAtAC): A Mild,
Orthogonal and Biocompatible
Chemical Ligation
A new methodology to achieve catalytic orthogonal chemical annulations in water, at
room temperature, is described. The reaction, promoted by a RuII catalyst, works
efficiently with a variety of azides and thioalkynes, and can be carried out in presence
of biomolecules (glutathione, aminoacids, peptides), as well as in complex biological
media such as cell lysates and fetal bovine serum, and even in presence of bacteria.
Importantly, the reaction is mutually compatible with the classical CuAAC.
6
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