Copper-chelating azides for efficient click conjugation reactions in complex media

Article abstract of DOI:10.1002/anie.201310671

The concept of chelation-assisted copper catalysis was employed for the development of new azides that display unprecedented reactivity in the copper(I)-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) reaction. Azides that bear strong copper-chelating moieties were synthesized; these functional groups allow the formation of azide copper complexes that react almost instantaneously with alkynes under diluted conditions. Efficient ligation occurred at low concentration and in complex media with only one equivalent of copper, which improves the biocompatibility of the CuAAC reaction. Furthermore, such a click reaction allowed the localization of a bioactive compound inside living cells by fluorescence measurements. Chelating azides were designed to form clickable copper complexes for efficient ligation with alkynes in complex biological media. Among a series of azides that bear nitrogen heterocycles, a bis(triazole) azide allowed ultra-fast click reactions with alkynes within seconds under diluted conditions. The reactivity and stability of this copper complex enabled efficient click reactions inside living cells.

Full text of DOI:10.1002/anie.201310671

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DOI: 10.1002/anie.201310671
Bioorthogonal Click Chemistry Very Important Paper
Copper-Chelating Azides for Efficient Click Conjugation Reactions in
Complex Media**
Valentina Bevilacqua, Mathias King, Manon Chaumontet, Marc Nothisen, Sandra Gabillet,
David Buisson, Cꢀline Puente, Alain Wagner, and Frꢀdꢀric Taran*
Abstract: The concept of chelation-assisted copper catalysis
was employed for the development of new azides that display
unprecedented reactivity in the copper(I)-catalyzed azide–
alkyne [3+2] cycloaddition (CuAAC) reaction. Azides that
bear strong copper-chelating moieties were synthesized; these
functional groups allow the formation of azide copper com-
plexes that react almost instantaneously with alkynes under
diluted conditions. Efficient ligation occurred at low concen-
tration and in complex media with only one equivalent of
Scheme 1. Examples of efficient N-donor ligands for CuAAC reactions.
copper, which improves the biocompatibility of the CuAAC
reaction. Furthermore, such a click reaction allowed the
localization of a bioactive compound inside living cells by
fluorescence measurements.
TBTA=tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, Bn=benzyl,
BTTE=2-[4-({bis[1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}-
methyl)-1H-1,2,3-triazol-1-yl]ethanol.
T
he copper(I)-catalyzed azide–alkyne [3+2] cycloaddition
oxygen species (ROS) from O2,[6] several CuAAC procedures
have been reported in the literature. They are usually based
on the use of water-soluble copper ligands and additives that
both accelerate the reaction and act as ROS scavengers to
decrease the cell toxicity of copper.[7] A second approach uses
highly reactive azides designed according to the concept of
chelation-assisted metal catalysis. Picolyl azides have been
reported to react much faster than standard azides thanks to
internal chelation of the copper catalyst, which is supposed to
enhance the electrophilicity of the azido group and facilitate
the formation of the metallacycle intermediate.[8] This con-
cept was recently successfully exploited by Ting et al. to
perform site-specific protein labelling on the surface of living
cells at low concentrations (50 mm) of the copper tris(triazole)
complex using pyridine-based copper-chelating azides as
substrates.[9] Nevertheless, the use of the CuAAC reaction
in the cellular context is still limited to cell-surface labelling
and, aside from the above-mentioned examples, is often
discouraged in favor of copper-free click reactions.[10]
Herein, we describe our efforts to improve the perfor-
mance of the CuAAC reaction in complex media using new
azides, which include a complete copper-chelating system in
their structure (Scheme 2). These azides were designed to
form strong, active copper complexes and should therefore be
considered both as reactant and catalyst in the CuAAC
reaction. Using this type of azides, the CuAAC should thus
become a bimolecular reaction and display much faster
kinetics than classic CuAAC reactions.
(CuAAC)[1] reaction can be considered as the archetype of
click chemistry.[2] The many reviews that relate the wide
variety of applications of this relatively young reaction bear
witness to its huge impact.[3] In this context, the use of copper
ligands that are able to stabilize and modulate the catalytic
activity of the CuI center contributed to the success of this
reaction by allowing for smoother reaction conditions and
broader applicability. Some of the most representative ligands
are the commercially available sulfonated bathophenantro-
line BPDS, the tris(benzimidazole) (BimC4A)3 and the tris-
(triazole) TBTA ligands (Scheme 1), which greatly accelerate
the reaction and stabilize the CuI oxidation state.[4] Analogues
of TBTA that bear bulky tert-butyl groups, such as BTTE and
BTTES, were recently found to be the most efficient ligands
for the CuAAC reaction described thus far.[5]
Despite the high reactivity of such copper complexes, the
copper source, ligands, and sodium ascorbate are usually
required in excess amounts to reach efficient ligation under
the diluted conditions that are typically used in the fields of
bioconjugation or cell labelling. To limit the damages that are
caused by the copper(I)-mediated generation of reactive
[*] V. Bevilacqua, Dr. M. Chaumontet, M. Nothisen, S. Gabillet,
D. Buisson, C. Puente, Dr. F. Taran
CEA, iBiTecS, Service de Chimie Bioorganique et de Marquage
91191 Gif sur Yvette (France)
E-mail: frederic.taran@cea.fr
To evaluate this strategy, we first synthesized various
chelating azides with increasing copper-chelation capabilities
(Scheme 3; see the Supporting Information for detailed
synthetic procedures). Azides A1, A3, and A4 were designed
for negative-control experiments; all other azides were
chosen for their potential ability to coordinate copper as bi-
(A2, A5, A6–A10, and A12), tri- (A11 and A13), or
Dr. M. King, Dr. A. Wagner
Laboratory of Functional Chemo Systems, Facultꢀ de Pharmacie
67000 Strasbourg (France)
[**] This work was supported by the European community through the
BioChemLig project.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201310671.
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Table 1: Comparative kinetic studies with azides A1–A20.[a]
Entry
Azide
Ligand
V [nmsecÀ1
]
Yield of 2[b] [%]
t[c]
1
2
3
4
5
6
7
8
A1
A1
A1
A2
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A20
–
0.01Æ0.02
1.78Æ0.01
15.59Æ0.02
13.35Æ0.03
38.26Æ0.01
0.02Æ0.05
0.59Æ0.06
19.87Æ0.02
14.79Æ0.03
11.49Æ0.02
12.02Æ0.02
17.30Æ0.03
8.44Æ0.01
20.17Æ0.05
0.22Æ0.03
0.98Æ0.05
3.00Æ0.01
171.25Æ0.15
41.13Æ0.05
46.14Æ0.06
37.29Æ0.07
356.26Æ0.23
402.54Æ0.11
41.07Æ0.05
0.9
28
53
29
39
1
16 h
2 h
20 min
13 min
7 min
16 h
TBTA
BTTE
–
BTTE
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Scheme 2. Strategies for the development of fast CuAAC reactions.
1
5 min
12 min
13 min
10 min
13 min
12 min
19 min
6 min
10 min
23 min
10 min
2 min
3 min
4 min
6 min
50 s
39
35
27
30
43
28
34
0.4
7
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
2
60
45
67
75
53
59
48
Scheme 3. Structures of azides A1–A20.
40 s
20 min
tetradentate (A14–A20) N-heterocyclic ligands. The struc-
tures of most of these azides were inspired by ligands that are
known to be active for the CuAAC reaction. Bis(triazole)
azides that bear carboxylic groups (A18 and A20) were
designed to allow post-functionalization for bioconjugation
applications.
The reactivity of these chelating azides for the CuAAC
reaction was evaluated at low concentrations using the
coumarin-based fluorogenic probe 1, which was inspired by
previous studies.[11] This probe enabled us to monitor the
CuAAC reaction of all azides A1–A20 by simple fluorescence
measurements.
All of the corresponding triazole products 2 were
synthesized and found to display similar fluorescence proper-
ties (lex = 320 nm; lem = 400 nm; Supporting Information,
Table S1). Standard curves that correlate the fluorescence
signal to the triazole concentration were systematically
determined before the kinetic experiments.
Comparative kinetic measurements for the CuAAC
reaction (Table 1) were performed with CuSO4 (17.5 mm) in
the presence of one equivalent of alkyne 1 and azides A1–A20
in phosphate buffer (0.1m, pH 7.4) containing 25% of DMF.
Experiments were carried out in duplicate in microtiter
plates. Kinetic parameters were monitored for five minutes
for the fastest reaction and overnight for the slowest reaction
(Table S2). As expected, non-chelating azides A1, A3, and A4
gave very low yields even after 16 hours, and the kinetics were
greatly improved by the addition of CuI ligands. For example,
the reaction was approximately 1500 times faster when A1
was used in the presence of BTTE (Table 1, entry 3). Overall,
picolyl and other bidentate azides induced increases in the
rate of the reaction that are similar to those that were
BTTE
[a] Kinetic measurements were conducted with 1, azides A1–A20, CuSO4
(17.5 mm each), sodium ascorbate (437.5 mm, 25 equiv) in a mixture of
phosphate buffer and DMF (75:25, 0.1m). When used, the ligand
concentration was 17.5 mm. [b] Relative yields were determined by
fluorescence measurements using standard curves. [c] Time to reach the
plateau.
observed on addition of the ligand BTTE (entries 8–14),
except for aromatic azide A12, which appeared inactive.
Bipyridine azide A13 was also inactive, probably because of
geometric constraints (entry 16). The most promising results
were obtained with the bis(benzimidazole) and bis(triazole)
azides A15–A20, for which substantial rate enhancements
were observed. The presence of a pendent tert-butyl group in
azides A19 and A20 seemed to enhance their ability to
undergo ultra-fast click reactions (compare entries 20 and 21
with entries 22 and 23, Table 1). For example, the reaction
with A20 displayed a rate constant of Kobs = 1300mÀ1 sÀ1 under
our reaction conditions and is more than 200 times faster than
standard reactions with strained cyclooctynes, which makes
this azide more than 4 ꢀ 104 times more reactive than standard
azides. Unlike for picolyl azide A2, the addition of BTTE
significantly decreased the reaction rate with A20 (compare
entries 4 and 5 with entries 23 and 24 in Table 1), indicating
that the copper complex of A2 is probably the active form
that undergoes cycloaddition.
This unprecedented reactivity is illustrated in Figure 1A,
which shows the conversion–time profiles of our most
reactive chelating azides. After only 30 seconds, yields
above 50% were obtained when the chelating azides A15
2
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Figure 1. Comparison of CuAAC kinetics: A) In phosphate buffer
(0.1m, pH 7.4); B) in cell lysate (K562, 3,4 mgmLÀ1, pH 7.4). Kinetic
measurements were carried out with a concentration of 17.5 mm for
the reactants, CuSO4 (1 equiv), and sodium ascorbate (25 equiv).
Figure 2. A) Time courses for various concentrations of [CuSO4·H2O].
[1]=[A20]=17.5 mm. B) Determination of the reaction order in copper.
and A20 were used with only 1 equivalent of copper at
a substrate concentration of 17.5 mm in buffer solution. We
further investigated the reactivity of these azides in more
complex media by repeating the CuAAC reactions in cell
lysate (Figure 1B). Interestingly, only azide A20 kept its
ability to undergo an ultra-fast click reaction under these
conditions and yielded 40% of the cross-coupling product in
only 30 seconds. Only poor yields were observed with A15
and A18, and no reaction at all occurred with pycolyl azide
A2 or with the BTTE copper complex under these conditions.
Therefore, the presence of a tert-butyl moiety on the chelating
azide appeared to be crucial not only for the activity, but also
for the stability of the corresponding copper complex in
biological media.
liver carcinoma cells. We therefore synthesized the two
required probes 3 and 4 (Scheme 4). In the first step of the
cellular assay, the treatment of living cells with probe 4
localized the alkyne functional group inside the microtubule
skeleton of the cell, which is due to the strong affinity of
paclitaxel for tubules. After several washing steps to remove
unbound alkyne, the chelating azide probe 3, which had been
preloaded with copper, was added to the cell culture medium.
In the case of a clean click reaction inside the cell, imaging
should reveal strong fluorescence localized at the skeleton. To
confirm the localization of the clicked probes exclusively at
the tubulin site, co-staining was performed with Tubulin
We therefore focused our efforts on A20, which appeared
to be the most promising chelating azide for bioconjugation.
We first determined the kinetic order in copper for the
reaction with A20 under initial kinetic conditions.[12] Triazole
formation was monitored by fluorescence measurements
immediately after the addition of sodium ascorbate to the
alkyne/azide/CuSO4 mixture. Interestingly, two different
behaviors in terms of copper dependence were clearly
observed during these kinetic studies. The order was found
to be 1.3 when the amount of copper was maintained below
one equivalent; the reaction rate decreased dramatically to
almost complete inhibition with excess copper (Figure 2).
These kinetic results seem to indicate that a 1:1 complex of
A20 and copper is the active species, which undergoes an
ultra-fast click reaction; in the presence of excess copper,
a polynuclear unreactive complex might be formed.
To assess the capacity of A20 for ligation in living cells, we
decided to employ a paclitaxel-based assay in HuH-7 human
Scheme 4. Reagents used for the labelling experiments in living cells.
The structure of the copper complex is hypothetical.
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Tracker Green (5). Merging the red channel of TAMRA
fluorescence (TAMRA = carboxytetramethylrhodamine)
conditions with BTTE as the ligand were unsuccessful
(Figure S5).
with the green channel of Tubulin Tracker thus revealed the
co-localization of the covalent probe and the click reaction
sites through appearance of a yellow color.
In conclusion, we have used the concept of chelation-
assisted copper catalysis to develop new azides that display
unprecedented reactivity in the CuAAC reaction. Inspired by
previous work of Ting et al.,[10] we designed and synthesized
azides that bear strong copper-chelating moieties. These
compounds allow the formation of azide copper complexes
that react almost instantaneously with alkynes under diluted
conditions. Using such azides, efficient ligation occurred at
low concentration and in complex media with only one
equivalent of copper, thus improving the biocompatibility of
the CuAAC reaction. This increased biocompatibility was
illustrated by a click reaction that allowed the localization of
a bioactive compound inside living cells by fluorescence
measurements.
For ligation experiments, we pretreated a stock solution of
3 in DMSO with one equivalent of copper(II) and 50 equiv-
alents of sodium ascorbate prior to the in-cell experiments.
The living cells were treated with 4 (62.5 nm) at 378C for
30 minutes, washed with phosphate-buffered saline solution
(PBS), and then incubated with the copper complex of 3
(50 mm) in the cell medium at 378C for two hours. Cells were
then washed again, fixed with paraformaldehyde (4%), and
treated with Tubulin Tracker Green (5; 62.5 nm) in prepara-
tion for the co-localization experiments (Figure 3; see also
Figure S6).
Received: December 9, 2013
Revised: March 6, 2014
Published online: && &&, &&&&
Keywords: bioconjugation · chemical biology · click chemistry ·
.
copper
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fluorescence of probe 3. C) Oregon green fluorescence of probe 5.
D) Merging of the three channels to confirm co-localization.
The recorded microscopic images revealed a strong
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Communications
Bioorthogonal Click Chemistry
Chelating azides were designed to form
clickable copper complexes for efficient
ligation with alkynes in complex biologi-
cal media. Among a series of azides that
bear nitrogen heterocycles, a bis(triazole)
azide allowed ultra-fast click reactions
with alkynes within seconds under diluted
conditions. The reactivity and stability of
this copper complex enabled efficient
click reactions inside living cells.
V. Bevilacqua, M. King, M. Chaumontet,
M. Nothisen, S. Gabillet, D. Buisson,
C. Puente, A. Wagner,
F. Taran*
&&&& — &&&&
Copper-Chelating Azides for Efficient
Click Conjugation Reactions in Complex
Media
6
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