Copper Catalysis in Living Systems and In Situ Drug Synthesis

Article abstract of DOI:10.1002/anie.201609837

The copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction has proven to be a pivotal advance in chemical ligation strategies with applications ranging from polymer fabrication to bioconjugation. However, application in vivo has been limited by the inherent toxicity of the copper catalyst. Herein, we report the application of heterogeneous copper catalysts in azide–alkyne cycloaddition processes in biological systems ranging from cells to zebrafish, with reactions spanning from fluorophore activation to the first reported in situ generation of a triazole-containing anticancer agent from two benign components, opening up many new avenues of exploration for CuAAC chemistry.

Full text of DOI:10.1002/anie.201609837

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201609837
German Edition: DOI: 10.1002/ange.201609837
Bioorthogonal Chemistry
Hot Paper
Copper Catalysis in Living Systems and In Situ Drug Synthesis
Jessica Clavadetscher, Scott Hoffmann, Annamaria Lilienkampf, Logan Mackay,
Rahimi M. Yusop, Sebastien A. Rider, John J. Mullins, and Mark Bradley*
Abstract: The copper-catalyzed azide–alkyne cycloaddition
(CuAAC) reaction has proven to be a pivotal advance in
chemical ligation strategies with applications ranging from
polymer fabrication to bioconjugation. However, application
in vivo has been limited by the inherent toxicity of the copper
catalyst. Herein, we report the application of heterogeneous
copper catalysts in azide–alkyne cycloaddition processes in
biological systems ranging from cells to zebrafish, with
reactions spanning from fluorophore activation to the first
reported in situ generation of a triazole-containing anticancer
agent from two benign components, opening up many new
avenues of exploration for CuAAC chemistry.
to the CuI catalyst is required.[11,12] CuI is prone to oxidation
and disproportionation; therefore, current catalyst formula-
tions utilize a CuII salt with the addition of sodium ascorbate
as a reductant. Furthermore, CuI-stabilizing ligands, such as
triazole-based tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]-
amine (TBTA)[13] and tris(3-hydroxypropyltriazolylmethyl)-
amine (THPTA),[14] are added to sequester free CuI ions and
to enhance reaction kinetics. Stabilizing ligands with
increased water solubility allow for lower concentrations of
CuII salts and have been used to label N-azidoacetylgalactos-
amines on the surface of Jurkat cells and in the enveloping
layer of zebrafish embryos, with reduced cytotoxicity and fast
reaction kinetics.[15–17] The groups of Ting[18] and Taran[19] used
copper-chelating azides in combination with water-soluble
ligands to increase the efficiency of the CuAAC reaction, also
enabling the use of CuI concentrations in the micromolar
range in cell-based assays. Recently, a non-toxic metal–
organic nanoparticle catalyst was reported that was efficient
in intracellular CuAAC chemistry.[20] To overcome the
toxicity of copper ions, Bertozzi and co-workers pioneered
the development of copper-free strain-promoted azide–
alkyne cycloaddition (SPAAC) reactions;[21,22] however,
strained alkynes can react with intracellular nucleophiles.[23]
Although transition metals are traditionally considered
detrimental to living systems, biocompatible transition-
metal chemistry has recently had success in bioorthogonal
activation strategies.[24–28] Chen and co-workers demonstrated
the palladium-mediated cleavage of a propargyloxycarbonyl-
caged catalytic lysine in OspF, a bacterial phospholyase,
switching on protein function in host cells.[29,30] Meggers and
co-workers have reported the activation of a prodrug of
doxorubicin through in-cell ruthenium catalysis,[31] whereas
MascareÇas et al. demonstrated the localization of the Ru
catalyst to mitochondria and subsequent catalytic activity
inside the subcellular compartment.[32] Palladium-mediated
chemistry has been demonstrated by protecting group cleav-
age and Suzuki–Miyaura cross-couplings for the activation of
5-fluorouracil and amsacrine.[33–35]
B
ioorthogonal reactions have emerged as a powerful
approach in allowing the study of biological processes in
their native environment.[1–4] A key feature of these reactions
is that they must have high selectivity and efficiency, and
function within the complex biological environment of a cell,
making the development of new bioorthogonal reactions
a formidable challenge.[5] The prototypical “click” reaction is
the highly efficient and selective copper-catalyzed azide–
alkyne 1,3-cycloaddition (CuAAC) reaction that generates
a triazole,[6–8] a scaffold that has been found in antiprolifer-
ative, antimicrobial, and anticonvulsant agents.[9,10] The wide
applicability of the CuAAC reaction stems from the robust-
ness of the azide and alkyne functional groups, which remain
inert in complex biological systems. In the presence of CuI, the
reaction proceeds with high rates and efficiency even in
challenging biological environments, resulting in the regio-
specific 1,4-triazole product, which is also highly robust and
inert. A drawback of the CuAAC reaction is the use of CuI
ions, which within biological systems leads to the production
of reactive oxygen species (such as hydroxyl radicals), the
induction of DNA strand breaks, and thiol coordination
chemistry, limiting its use in vivo where prolonged exposure
[*] J. Clavadetscher, Dr. A. Lilienkampf, Dr. L. Mackay,
Prof. Dr. M. Bradley
Thus, although CuAAC chemistry has been successfully
used for many bioconjugation processes, the in situ genera-
tion of active compounds by coupling two inert partners, such
as prodrugs, remains elusive. Herein, we report the develop-
ment and application of non-toxic, biocompatible, and
implantable heterogeneous copper catalysts that were func-
tional both in vitro and in vivo and were able to catalyze
in situ the synthesis of an anticancer agent from two
component halves.
Heterogeneous, robust, and highly biocompatible entrap-
ped copper nanoparticle catalysts (E-Cu-NPs) were synthe-
sized in three steps starting from amino-functionalized
TentaGel resin by incubation with a solution of Cu(OAc)2,
EaStCHEM School of Chemistry
University of Edinburgh
David Brewster Road, EH9 3FJ Edinburgh (UK)
E-mail: Mark.Bradley@ed.ac.uk
S. Hoffmann, Dr. S. A. Rider, Prof. J. J. Mullins
University of Edinburgh/BHF Centre for Cardiovascular Science
Queen’s Medical Research Institute
47 Little France Crescent, EH16 4TJ, Edinburgh (UK)
Dr. R. M. Yusop
School of Chemical Sciences and Food Technology
Faculty of Science and Technology, Universiti Kebangsaan Malaysia
43600 Bangi, Selangor (Malaysia)
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201609837.
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followed by reduction and entrapment (see the Supporting
Information, Figure S1). Transmission electron microscopy
(TEM) images of cross-sections of the resin beads showed the
generation of nanoparticles (51.2 Æ 4.2 nm) distributed
throughout the bead (160.4 Æ 10.3 mm in water; Figure S1),
with a Cu content of 0.37 Æ 0.02 mmol Cu/mg resin as
determined by ICP-OES (n = 3). Leaching levels were less
than 1% after a 72 h treatment in H2O at 378C, showing the
robustness of the E-Cu-NP catalyst. The catalytic activity of
the E-Cu-NPs was investigated utilizing the coumarin “pro-
CuSO4 system with the chelating ligand THPTA and the
reducing agent sodium ascorbate (NaAsc), which gave full
conversion into 3 in 10 min in PBS and reduced conversion in
the presence of biomolecules in cell lysate and FBS (Fig-
ure S4).
The profluorophore activation was then investigated in
a cell-based assay. SKOV-3 (ovarian adenocarcinoma) cells
were incubated with 1 and 2 and the E-Cu-NPs, which are
located in the extracellular space (Figure S6), and analyzed by
flow cytometry and live-cell fluorescence microscopy. The
cells showed an eightfold increase in fluorescence of the
entire cell population compared to untreated cells (Fig-
ure 1B), with the fluorescence within the SKOV-3 cells
colocalized with the mitochondria stain (MitoTrackerꢀ
Red; the triphenylphosphonium (TPP) moiety is a well-
established mitochondria-targeting group),[37] confirming the
synthesis of 3 and localization of 3 in the mitochondria
(Figure 1C). These results were mirrored with HeLa cells
(Figure S5). The E-Cu-NPs were non-cytotoxic with full
cell viability observed under these reaction conditions. The
conventional homogeneous catalyst system (CuSO4/THPTA),
however, showed significant toxicity after 24 h and 48 h,
indicating the great advantage of the E-Cu-NPs for biolog-
fluorophore” 3-azido-7-hydroxycoumarin
1 (lex/em = 346/
455 nm), which exhibits strongly quenched fluorescence
compared to the unsubstituted coumarin scaffold owing to
the quenching effect exerted by the electron-rich a-nitrogen
atom of the azido group.[36] 3-Azidocoumarin 1 was reacted
with alkyne 2 and the E-Cu-NPs (Figure 1A) under a variety
of physiological conditions, namely phosphate buffered saline
(PBS), 5% fetal bovine serum (FBS), and HeLa (cervical
adenocarcinoma) cell lysate, with full conversion of 1 into
triazole 3 (lex/em = 343/477 nm; Figure S2) after 3 h, with no
indication of side product formation and a 22-fold increase in
fluorescence in the PBS experiment (Figure S3). These results
were comparable to those achieved with the conventional
Figure 1. E-Cu-NP mediated cycloaddition of 3-azidocoumarin 1 and TPP alkyne 2. A) The CuAAC reaction. B) Flow cytometry histograms of
SKOV-3 cells incubated with 1 (20 mm), 2 (20 mm), and NaAsc (50 mm) for 18 h at 378C with the E-Cu-NPs (0.1 mmol Cu) in black and untreated
cells in gray (lex/em =355/450 nm). C) SKOV-3 cells incubated with 1 and 2 and E-Cu-NPs (top panels) and SKOV-3 cells incubated with 1 and 2
(bottom panels) and stained with MitoTrackerꢀ Red (mitochondrial stain) and imaged by fluorescence microscopy. Panels show from left to
right: synthesized compound 3 (blue), mitochondria (red), and merged image (pink indicates colocalization of 3 and the mitochondria).
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ically compatible, and possibly in vivo, applications (Fig-
ure S4).
Combretastatin A4 (CA4) is a tubulin polymerization
inhibitor and highly cytotoxic against a variety of cancer cell
lines,[38–40] with the triazole analogue 6 known to inhibit K562
leukemia cancer cell growth with an IC50 value of 1.3 mm.[41,42]
Thus triazole 6 was synthesized from azide 4 and alkyne 5 in
an E-Cu-NP catalyzed cycloaddition (Figure 2A) under
physiological conditions (PBS, 378C) with 64 Æ 4% conver-
sion into triazole 6 after 3 h (n = 3), as monitored by HPLC
analysis (Figure S7). Remarkably, this catalyst activity was
retained in cell lysate and in 5% FBS (with conversions of
64 Æ 5% and 52 Æ 2%, respectively; Figure S7).
The E-Cu-NPs catalyzed cycloaddition was investigated in
a cell-based assay, allowing the in situ extracellular synthesis
of the cytotoxic agent. The cytotoxicity of precursors 4 and 5,
as well as triazole product 6, was evaluated on SKOV-3 and
HeLa cells, with neither 4 and 5 showing any cytotoxicity up
to 10 mm. Triazole 6 exhibited cytotoxicity against both
SKOV-3 and HeLa cells, with < 30% cell viability at 10 mm
(Figure 2B and Figure S8). When cells were incubated with 4
and 5 in the presence of the E-Cu-NPs (at 378C for 72 h),
a 70% decrease in cell viability was observed (compared to
the controls; Figure 2C). These results were mirrored in
HeLa cells (Figure S8). To show that the in situ formation of 6
affected cell cycle progression, cell growth was analyzed (in
a Click-iTꢀ EdU assay). Only cells incubated with 4 and 5 in
the presence of E-Cu-NPs showed significant arrest in cell
cycle progression at the G0/G1 phase (59 Æ 11% vs. 24 Æ 3%
in untreated cells; Figure 2D and Figure S10), with the in situ
formation of 6 inducing apoptosis in SKOV-3 cells, as
analyzed by double staining with Annexin-V/FITC and
propidium iodide (PI; 43 Æ 9% vs. 13 Æ 3% in untreated
cells; Figure 2E and Figure S9). Cell lysis, followed by HPLC
and HR-MS analysis of the cell contents, identified 6 (as well
as precursors 4 and 5) inside the cells (Figure S11).
The biocompatibility of the E-Cu-NPs, and their applica-
tion as an implantable device, was evaluated by introducing
a single bead of the catalyst into the yolk sac of zebrafish
embryos 24 hours post fertilization (hpf). The catalyst
implanted into zebrafish showed no signs of toxicity, no
phenotype alteration was observed, and the embryos devel-
oped normally into their larval stage, as monitored by phase
contrast microscopy (Figure 3A,B). To evaluate the catalytic
activity of the E-Cu-NPs in vivo, the “activation” of 1 with
TPP alkyne 2 was carried out (Figure 3C,D). A clear
fluorescence signal was observed in embryos that had been
implanted with the catalyst, with a 5.7-fold localized increase
in fluorescence (indicated by the white arrow) and a 3.2-fold
increase in fluorescence distributed throughout the yolk sac,
compared to the background fluorescence of embryos
implanted with a non-functionalized bead (Figure 3d and
Figure S12).
In conclusion, we have described the facile synthesis of
copper nanoparticles embedded within a polymeric support
and demonstrated their functionality as biocompatible cata-
lysts. They were used to activate a coumarin profluorophore
and to drive the cycloaddition reaction of two components of
a drug to successfully enable the in situ synthesis of a cytotoxic
anticancer agent that induces apoptosis. The biocompatibility
and scope of the E-Cu-NP catalyst were shown by in vivo
implantation in the widely used and respected zebrafish
model, where not only was no toxicity observed, but where an
active fluorophore was generated by the covalent conjugation
of the two halves placed within the milieu. The E-Cu-NP
functionalized resin beads thus function as an implantable
device and could be placed at the desired site of action in vivo
Figure 2. In situ synthesis of the cytotoxic agent 6. A) The E-Cu-NP
mediated cycloaddition of 4 and 5. B) Cell viability of SKOV-3 cells
treated with compounds 4, 5, and 6 (purified material) for 72 h at 0–
10 mm (MTT assay, n=3). C) SKOV-3 cells incubated with 4 (10 mm), 5
(10 mm), and NaAsc (50 mm) in the presence of the E-Cu-NPs
(0.1 mmol Cu); cell viability measured after 72 h (MTT assay, n=3).
***P<0.001 by one-way ANOVA with Dunnett post-test, compared to
the untreated control group. D) Flow cytometry analysis of cell
proliferation with histograms showing cells in the G0/G1, S, and G2/
M phases (Click-iTꢀ EdU assay). The left panel shows untreated
control cells, and the right panel shows cells treated with 4, 5, and
NaAsc in the presence of E-Cu-NPs, showing an increase in cells in
the G0/G1 phase. E) Flow cytometry analysis of the apoptotic effects
of the in situ synthesis of 6 (Annexin-V/FITC apoptosis assay): Cyto-
grams showing the Annexin-V/FITC and PI intensity of untreated cell
populations (left) and cells treated with 4, 5, and NaAsc in the
presence of E-Cu-NPs, showing an increase in apoptotic cells for the
treated cells.
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Figure 3. Biocompatibility studies with the E-Cu-NPs in zebrafish
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Published online: && &&, &&&&
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Communications
Bioorthogonal Chemistry
Localized drug synthesis: Biocompatible
copper nanoparticle catalysts were syn-
thesized and employed in the activation
of a profluorophore in living cells and in
zebrafish embryos. Furthermore, an anti-
cancer drug was synthesized in situ from
two benign components, leading to
apoptosis in ovarian cancer cells.
J. Clavadetscher, S. Hoffmann,
A. Lilienkampf, L. Mackay, R. M. Yusop,
S. A. Rider, J. J. Mullins,
M. Bradley*
&&&& — &&&&
Copper Catalysis in Living Systems and
In Situ Drug Synthesis
6
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