Iridium-Triggered Allylcarbamate Uncaging in Living Cells

Article abstract of DOI:10.1021/acs.inorgchem.1c01790

Designing a metal catalyst that addresses the major issues of solubility, stability, toxicity, cell uptake, and reactivity within complex biological milieu for bioorthogonal controlled transformation reactions is a highly formidable challenge. Herein, we report an organoiridium complex that is nontoxic and capable of the uncaging of allyloxycarbonyl-protected amines under biologically relevant conditions and within living cells. The potential applications of this uncaging chemistry have been demonstrated by the generation of diagnostic and therapeutic agents upon the activation of profluorophore and prodrug in a controlled fashion within HeLa cells, providing a valuable tool for numerous potential biological and therapeutic applications.

Full text of DOI:10.1021/acs.inorgchem.1c01790

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Iridium-Triggered Allylcarbamate Uncaging in Living Cells
Neelu Singh,† Ajay Gupta,† Puja Prasad, Pritam Mahawar, Shalini Gupta, and Pijus K. Sasmal*
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ABSTRACT: Designing a metal catalyst that addresses the major
issues of solubility, stability, toxicity, cell uptake, and reactivity
within complex biological milieu for bioorthogonal controlled
transformation reactions is a highly formidable challenge. Herein,
we report an organoiridium complex that is nontoxic and capable
of the uncaging of allyloxycarbonyl-protected amines under
biologically relevant conditions and within living cells. The
potential applications of this uncaging chemistry have been
demonstrated by the generation of diagnostic and therapeutic
agents upon the activation of profluorophore and prodrug in a
controlled fashion within HeLa cells, providing a valuable tool for
numerous potential biological and therapeutic applications.
he ability to custom-design synthetic catalysts that can
perform artificial chemistry in complex biological milieu
groups,7e propargyl ester amidation,20 alkyne hydroarylation21
and hydroamination,7f and iridium-catalyzed transfer hydro-
genation.22 While pioneering work on bond cleavage and bond
formation reactions was done earlier with copper, iron,
ruthenium, palladium, and gold complexes, an important
transition metal, iridium, has been unexplored for the same
reactions. Like the above-mentioned catalysts, organoiridium
complexes can also maintain their catalytic activity in air and
biological media22,23 which makes them suitable for bio-
orthogonal catalysis reactions inside living cells.
In this study, we report organoiridium(I) complexes for the
in situ generation of imaging and therapeutic agents via
bioorthogonal allylcarbamate uncaging chemistry in both
biological and nonbiological systems without the need of any
additives (Figure 1). The uncaging results were demonstrated
in protic solvents, phosphate-buffered saline (PBS) buffer, and
human cervical cancer (HeLa) cell lysates under air. In
addition, the applications of this uncaging chemistry have been
displayed by generating diagnostic and therapeutic agents upon
the activation of profluorophore and prodrug within HeLa
cells.
T
has opened up new horizons in medicinal chemistry, chemical
biology and the therapeutic treatment of deadly diseases such
as cancer. Recently, transition-metal catalysts (TMCs) have
emerged as a promising tool for carrying out unnatural
chemical transformations via bioorthogonal catalysis in a
myriad of cellular environments.1,2 These chemical trans-
formations have been demonstrated for a variety of
applications such as biomolecular labeling,3 metabolite
detection,4 intracellular probe release,1,5 and in situ enzyme6
and prodrug activation.1b,7 Designing TMC-mediated reactions
in living organisms, however, still remains a formidable task for
(1) maximization of the catalyst activity at low substrate
concentrations, (2) minimization of the catalyst reactivity
toward air, water, and other cellular components in high
abundance, (3) problems associated with the catalyst toxicity,
water solubility, and selectivity toward the desired substrates,
and (4) slow uncaging of prodrug for sustained drug release to
reduce drug toxicity. Metal-containing catalysts are also often
prone to deactivate in the combined presence of air, water, and
millimolar concentrations of nucleophilic thiols, raising
concerns of their stability in cellular environments.
In the past, researchers have overcome these challenges by
developing metal catalysts based on copper-catalyzed azide−
alkyne cycloaddition7d,8 and depropargylation,9 ruthenium-
mediated allylcarbamate cleavage,1a,5a,b,7b,c,10 redox isomer-
ization,11 azide reduction12 and protein labeling,13 iron-
mediated azide reduction,14 palladium-mediated uncaging of
allylcarbamate,1b,7a,15 propargyloxycarbonyl5c,6,7a,16 and allenyl
groups,17 etc. In addition, there are cited examples of
palladium-mediated N-dealkylation,7a O-dealkylation,18 Suzu-
ki−Miyaura,1b,3 and Sonogashira cross-couplings,19 gold-
mediated uncaging of a structurally diverse range of functional
The organometallic iridium(I) complexes Ir1−Ir3 were
developed for the uncaging of N-allylcarbamates to their
respective amines under biologically relevant conditions and
within living cells (Figure 1a). The iridium(I) centers in the
synthesized complexes were coordinated with dibenzocyclooc-
Received: June 13, 2021
https://doi.org/10.1021/acs.inorgchem.1c01790
© XXXX American Chemical Society
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Figure 1. (a) Molecular structures of the iridium(I) complexes (Ir1−Ir3) used for the uncaging of allylcarbamates in this study. (b) X-ray crystal
structure of complex Ir1. The hydrogen atoms have been omitted for clarity. (c) Iridium(I)-mediated uncaging of allylcarbamates under
biologically relevant conditions or within living cells.
tatetraene (DBCOT) and chloro- and phosphine-based
ligands. The iridium(I) center provided a stable square-planar
geometry and intracellular uncaging chemistry. The DBCOT
ligand was utilized for better cellular uptake due to its
hydrophobic nature, which could assist in the effective crossing
of the complexes through the lipid bilayers of the cell
membranes. In addition, different π-acceptor capabilities of
phosphine ligands were coordinated to the metal center to
modulate the solubility, reactivity, and cellular uptake of the
complexes. In contrast, the chloride ion, an innocent ligand,
was added to increase the stability of the complex and to
render it ready for direct activation under aqueous environ-
ments. The synthesis and characterization data of the ligands
and the iridium complexes are described in Schemes S1−S3
and Figures S1−S11. The solid-state structure of Ir1 was also
characterized by X-ray crystallography (Figure 1b and Tables
S1 and S2). The stability of the complexes was accessed in
organic and aqueous media at room temperature by NMR and
absorption spectroscopy (Figures S12 and S13). The
complexes exhibit good stability in organic solvents over 72
h but comparatively lower stability in the aqueous medium.
The reactivity of iridium complexes due to dissociation of the
Ir−Cl bond was investigated by the halide abstraction method
(Figure S14).
The synthesized organoiridium complexes (Ir1−Ir3) were
employed for the catalytic deprotection of an allylcarbamate
group from a variety of aromatic and aliphatic substrates under
air and in the presence of polar protic solvents. For instance,
the reaction of p-chlorophenyl allylcarbamate (1a) with 5 mol
% of catalysts Ir2 and Ir3 in CH2Cl2/MeOH (95:5) at 30 °C
under air for 48 h provided isolated yields of p-chloroaniline
(1b) equal to 83 and 76%, respectively (Figure 2). To
demonstrate the versatility of these catalysts, we performed the
uncaging of allylcarbamates from the electron-rich (2a → 2b)
and electron-poor (3a → 3b and 4a → 4b) aromatic substrates
and found that the catalysis reaction worked efficiently in all
cases. Also, these catalysts were utilized for the uncaging of
allylcarbamate from the aliphatic amine (5a → 5b) with very
good yields. However, Ir1 worked moderately for the above
reactions under the same conditions. The low catalytic activity
B
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Figure 2. Catalytic deprotection of allylcarbamate-protected amines with organoiridium complexes (Ir1−Ir3). Reaction conditions: substrate
(0.118 mmol, 1 equiv) and catalyst (0.0059 mmol, 0.05 equiv) in 118 μL of CH2Cl2/MeOH (95:5, v/v) at 30 °C under air. Shown are the isolated
yields of the products.
of Ir1 was attributed to the bulky structure of its PPh3 ligand
coordinated to the metal center that likely provided steric
hindrance toward substrate binding. Notably, no products were
isolated in the absence of catalyst. Thus, it was concluded that
the organoiridium complexes Ir2 and Ir3 smoothly catalyzed
the deprotection of allylcarbamates from aromatic and aliphatic
amines to their respective amines under mild conditions,
without the need of any additives and in the presence of protic
solvents and air.
To examine the catalytic activity of organoiridium
complexes, we carried out the uncaging of bis(N,N′-
allyloxycarbonyl)rhodamine 110 (Rho-alloc) to its corre-
sponding rhodamine 110 (Rho) under biologically relevant
conditions, which mimicked the native cellular environment.
The reactions were carried out either in PBS with trace
amounts of glutathione (GSH) or in HeLa cell lysates in the
presence of air. The progress of the reaction was simply
checked by monitoring the fluorescence readout of the
released Rho. The best results were obtained with Ir3 (20
mol %) in which the incubation of nonfluorescent caged
rhodamine (Rho-alloc, 500 μM) in the presence of GSH (5
mM) in dimethyl sulfoxide (DMSO)/PBS (1:1, 10 mM, pH
7.4) at 37 °C led to a gradual increase in the fluorescence
intensity (Figure 3a). The signal reached saturation around 48
h, giving a Rho yield of 32
control experiments performed in the absence of an iridium
complex showed no fluorescence signal (0.1 0.1%).
0.8%. In contrast, negative
When the above experiments were repeated in the presence
of HeLa cell lysates, a more complex and biologically realistic
environment, the iridium-promoted reactions still showed
great response albeit with slightly lower yields. The yield was
11 0.5% after 48 h and continued to rise even up to the
fourth day, before gradually leveling off at 16 0.5% (Figure
S15). When similar experiments were, however, repeated with
complexes Ir1 and Ir2 either in PBS or HeLa cell lysates, Ir2
gave only ∼6.5 0.6% yield of Rho, while Ir1 was found to be
almost inactive (ca. ∼1.5% yield) after 48 h.
In general, the yield of Rho in PBS or cell lysates was lower
compared to the yield of uncaged amine (1b−5b) in CH2Cl2/
MeOH. The reason for this lower yield of Rho under
biorelevant conditions might be associated with the slow
deactivation of catalyst (Figure S13). Besides, Ir1 was found to
be inactive, which may possibly be due to its low solubility in
an aqueous medium because of the hydrophobic PPh3
ligand15b and, in addition, to its bulky structure, which can
cause steric crowding toward substrate binding.
Further, we investigated the catalytic uncaging of allylcarba-
mate from the prodrug of doxorubicin (DOX), a well-known
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Figure 3. (a) Uncaging of Rho-alloc (0.5 mM, 1 equiv) mediated by an organoiridium complex (0.1 mM, 0.2 equiv) in DMSO/PBS (1:1, v/v) at
37 °C in the presence of GSH (5 mM, 10 equiv). Yields were determined by comparing the fluorescence intensities (λex = 488 nm; λem = 520 nm)
of diluted samples with a standard curve of different concentrations of Rho. (b) Catalytic uncaging of caged DOX (100 μM) with Ir3 (20 μM) and
GSH (5 mM) in water/DMSO (99:1) at 25 °C monitored by HPLC.
chemotherapeutic anticancer drug used in the treatment of a
wide range of cancers. For this purpose, the allylcarbamate-
protected doxorubicin prodrug (caged DOX, 100 μM) was
incubated with 20 μM Ir2 or Ir3 and 5 mM GSH in water/
DMSO (99:1) at room temperature under air. The
deprotection of allylcarbamate was monitored for 48 h, and
the results were analyzed using analytical high-performance
liquid chromatography (HPLC; Figures 3b and S16 and S17).
The results showed that the loading of 20 mol % Ir2 and Ir3 to
caged DOX gave 29% and 76% DOX release, respectively. As
expected, the yield decreased upon reduction of the catalyst
loading, and there was no product formation in the absence of
the catalyst. This clearly proved that the catalyst was able to
activate the prodrug in the extracellular medium. On the basis
of these observations, we tentatively proposed an iridium-
triggered allylcarbamate uncaging pathway (Figure S18).
Next, we carried out iridium-triggered artificial chemistry for
allylcarbamate uncaging within the highly complex biological
milieu for translation to cellular habitats. For this, cultured
HeLa cells were incubated with caged rhodamine (Rho-alloc,
100 μM) for 30 min, followed by multiple washings with PBS
to remove the caged fluorophore from the extracellular
solution and to ensure that Rho-alloc is located only within
the HeLa cells, not in the culture medium. A fresh medium was
added to the cells along with Ir3 (20 μM), which led to the
development of cellular green fluorescence, indicating the
formation of Rho (Figures 4a and S19 and S20). The
development of green fluorescence intensity was monitored by
live-cell confocal microscopy imaging, and the results revealed
a 56-fold increase in the fluorescence intensity within HeLa
cells over a period of 8 h. In contrast, the cells treated with the
caged substrate in the absence of catalyst were found to be
nonfluorescent. These results indicated the successful uncaging
of allylcarbamate from Rho-alloc by the catalyst within live
HeLa cells and the subsequent release of fluorescent Rho.
More notably, the catalyst showed a high efficiency, as
determined by the significant increase in the fluorescence
intensity, and long periods of activity suggest its slow
deactivation in complex biological environments.
To further determine the accumulation of catalyst at the
subcellular level, we carried out inductively coupled plasma
mass spectrometry (ICP-MS; Figure S21). The ICP-MS data
showed a high accumulation of iridium inside the cell cytosol,
suggesting that allylcarbamate cleavage from caged rhodamine
(Rho-alloc) by Ir3 mostly occurred within the cytoplasm,
followed by slow diffusion of the green fluorescent product
(Rho) throughout the cells. We also determined the tolerance
of cells in the presence of catalyst before proceeding to
prodrug activation. Our cell viability results revealed the
catalyst to be nontoxic (ca. >96% viable cells) at 50 μM
concentration even after a long incubation period of 48 h, as
determined by MTT assay (Figure 4b).
The ability to activate profluorophore by a catalyst within
living cells and its high biocompatibility motivated us to study
the bioorthogonal activation of a prodrug inside cells.
Moreover, the slow uncaging of allylcarbamate by Ir3 in the
biological medium can be advantageous for the activation of
prodrug for sustained drug release. The sustained release of
drug is highly beneficial over the burst release because the
latter can cause dangerous spikes in drug concentrations,
resulting undesirable side effects. Hence, we demonstrated the
bioorthogonal catalytic activation of a prodrug to establish our
proof-of-concept. For this, we utilized our previously
synthesized caged DOX, which was converted to DOX upon
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Figure 4. (a) Uncaging of Rho-alloc (100 μM) with ( ) and without ( ) addition of Ir3 (20 μM) in HeLa cells monitored by live-cell imaging
using confocal microscopy. Insets: (A) bright-field image of cells treated with Rho-alloc only (control) and (B) merged bright-field image of cells
treated with Rho-alloc and Ir3. Both images were taken after 8 h. Scale bar: 10 μm. (b) Cytotoxicity of Ir3 in HeLa cells after 48 h of incubation
(IC50 = 76 μM) as determined by MTT assay. (c) Activation of a prodrug (caged DOX) by Ir3 inside HeLa cells. The cell survival rates were
determined by MTT assay.
catalytic uncaging of the allylcarbamate group. We determined
the cell viability of caged DOX in the presence of an iridium
complex in HeLa cells by MTT assay (Figures 4c and S22 and
S23). The HeLa cells were incubated with different
concentrations of caged DOX (20, 50, 75, and 100 μM) for
3 h, followed by incubation with an iridium complex (20 μM)
in a fresh medium for 48 h. Not surprisingly, the caged DOX
was found to be nontoxic to the cells in the absence of catalyst,
whereas the free drug DOX showed toxicity. We observed only
a small decrease in the cell viability when 20 μM caged DOX
was incubated with 20 μM catalyst for 48 h. However, when
the concentration of nontoxic caged DOX was increased from
20 to 100 μM while the catalyst concentration was kept fixed at
20 μM, the cell viability decreased drastically in a
concentration-dependent manner. Interestingly, we could
observe efficient reduction of the cell viability even with a
decreased catalyst loading of 10 or 5 μM in the presence of a
high concentration of caged DOX (100 μM). This drastic
decrease in the cell viability with the prodrug in the presence of
a catalyst suggests uncaging of the allylcarbamate group inside
the cells.
In conclusion, we have reported three organometallic
iridium(I) complexes (Ir1−Ir3) for catalytic deprotection of
allylcarbamate-protected amines in biological and nonbiolog-
ical conditions without the need of any exogenous agents. The
catalysts Ir2 and Ir3 efficiently performed the uncaging of
allylcarbamate from a variety of aromatic and aliphatic
substrates in the presence of protic solvents and air. The
uncaging results with profluorophore and prodrug under
biologically relevant conditions showed good efficiency, with
the best performance obtained with the catalyst Ir3. The
catalyst Ir3 also exhibited a remarkably high activity, with up to
a 56-fold increase in the fluorescence intensity over a period of
8 h for the uncaging of allylcarbamate from profluorophore
within live HeLa cells. In addition, activation of the prodrug by
the catalyst Ir3 inside HeLa cells provided both prodrug and
catalyst concentration-dependent cell death. In the future, the
prodrug and profluorophore systems could be suitably
combined to allow the real-time tracking of cell death.
E
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Marburg, Marburg, Germany, for helpful discussions and his
input and comments on this manuscript.
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AUTHOR INFORMATION
■
Corresponding Author
Pijus K. Sasmal − School of Physical Sciences, Jawaharlal
Nehru University (JNU), New Delhi 110067, India;
orcid.org/0000-0002-2301-1269; Email: pijus@
mail.jnu.ac.in
Authors
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investigating synthetic metal-catalyzed reactions in living cells. ACS
Catal. 2021, 11, 5148−5165.
Neelu Singh − School of Physical Sciences, Jawaharlal Nehru
University (JNU), New Delhi 110067, India
Ajay Gupta − School of Physical Sciences, Jawaharlal Nehru
University (JNU), New Delhi 110067, India
Puja Prasad − Department of Chemical Engineering, Indian
Institute of Technology (IIT) Delhi, New Delhi 110016,
India
Pritam Mahawar − Department of Chemistry, Indian Institute
of Technology (IIT) Delhi, New Delhi 110016, India
Shalini Gupta − Department of Chemical Engineering, Indian
Institute of Technology (IIT) Delhi, New Delhi 110016,
India; orcid.org/0000-0003-1382-0254
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Complete contact information is available at:
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Author Contributions
†N.S. and A.G. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
P.K.S. acknowledges SERB, DST (Grant no. ECR/2016/
000810), and DST-PURSE for financial assistance. S.G.
acknowledges funding received from DST Nanomission
(Grant no. SR/NM/NT-1049/2016) and IMPRINT (Grant
no. IMP/2018/000236/HT). We thank DST-FIST for Single
Crystal X-ray Diffraction facility in SPS, JNU, and AIRF, JNU
for the instrumentation facilities. N.S. gratefully acknowledges
SERB, DST, for a junior research fellowship. A.G. acknowl-
edges MHRD-STARS for a fellowship. P.P. thanks CSIR for
the SRA position (Pool No. 9031-A). We thank Dr. D. Das
and L. Negi, SPS, JNU, for help with X-ray data collection and
for solving the crystal structure of Ir1. We sincerely thank Prof.
S. Nagendran, IIT Delhi, for allowing us to use his glovebox for
the synthesis of an iridium-dimer precursor. R. K. Sah, SCMM,
JNU, is gratefully acknowledged for live-cell imaging data
collection. We thank Prof. E. Meggers, Philipps Universitat
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