Transition-Metal-Mediated versus Tetrazine-Triggered Bioorthogonal Release Reactions: Direct Comparison and Combinations Thereof

Article abstract of DOI:10.1002/cplu.202000477

Bioorthogonal cleavage reactions are gaining popularity in chemically inducible prodrug activation and in the control of biomolecular functions. Despite similar applications, these reactions were developed and optimized on different substrates and under different experimental conditions. Reported herein is a side-by-side comparison of palladium-, ruthenium- and tetrazine-triggered release reactions, which aims at comparing the reaction kinetics, efficiency and overall advantages and limitations of the methods. In addition, we disclose the possibility of mutual combination of the cleavage reactions. Finally, we compare the efficiency of the bioorthogonal deprotections in cellular experiments, which revealed that among the three methods investigated, the palladium- and the tetrazine-promoted reaction can be used for efficient prodrug activation, but only the tetrazine-triggered reactions proceed efficiently inside cells.

Full text of DOI:10.1002/cplu.202000477

A Multidisciplinary Journal Centering on Chemistry
Accepted Article
Title: Transition Metal–Mediated vs. Tetrazine–Triggered Release
Reactions: Direct Comparison and Combinations Thereof
Authors: Francesca Mancuso, Michal Rahm, Rastislav Dzijak, Helena
Mertlíková-Kaiserová, and Milan Vrabel
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To be cited as: ChemPlusChem 10.1002/cplu.202000477
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10.1002/cplu.202000477
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Transition Metal–Mediated vs. Tetrazine–Triggered Release
Reactions: Direct Comparison and Combinations Thereof
Francesca Mancuso,[a] Michal Rahm,[b] Rastislav Dzijak,[b] Helena Mertlíková-Kaiserová[b] and Milan
Vrabel*[b]
[a]
[b]
F. Mancuso
University of Messina
Department of Chemical, Biological, Pharmaceutical and Environmental Sciences (CHIBIOFARAM)
Viale Palatucci 13, I-98168, Messina, Italy
M. Rahm, Dr. R. Dzijak, Dr. H. Mertlíková-Kaiserová, Dr. M. Vrabel*
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences
Flemingovo nám. 2, 166 10, Prague, Czech Republic
E-mail: vrabel@uochb.cas.cz
Supporting information for this article is given via a link at the end of the document.
Abstract: Bioorthogonal cleavage reactions are gaining popularity in
chemically-inducible prodrug activations and in the control of
biomolecular functions. Despite similar applications, these reactions
were developed and optimized on different substrates and under
different experimental conditions. Reported herein is a side-by-side
comparison of palladium, ruthenium and tetrazine-triggered release
reactions, which aims at comparing the reaction kinetics, efficiency
and overall advantages and limitations of the methods. In addition, we
disclose the possibility of mutual combination of the cleavage
reactions. Finally, we compare the efficiency of the bioorthogonal
deprotections in cellular experiments, which revealed that among the
three methods investigated, the palladium- and the tetrazine-
promoted reaction can be used for efficient prodrug activation, but
only the tetrazine-triggered reactions proceed efficiently inside the
cells.
the release of free amino group. The power of the concept was
demonstrated by bioorthogonal activation of TCO-caged
doxorubicin, which was successfully reactivated after TCO
elimination from the respective TCO-caged prodrug.[9] Later, the
same group reported that other functional groups including
carbonates, esters and ethers can be activated using the same
strategy.[10] Recently, the approach was extended to the release
of tetrazine-caged compounds.[11] In combination with the
targeting ability of antibodies, the activation strategy was
successfully applied for targeted release of biologically active
compounds from antibody-drug conjugates.[12] Another example
showed that the chemistry can be used for chemically-induced
control over T-cell activation by bioorthogonal deprotection of
TCO-caged epitopes.[13] In addition to the strained TCO, vinyl
ethers,[14]
benzonorbornadienes[15]
and
3-isocyanopropyl
groups[16] were employed as alternative dienophiles in tetrazine-
promoted deprotection reactions.[17]
Biocompatible cleavage reactions complement bioorthogonal
bond forming reactions by enabling control and activation of
molecular functions in response to specific chemical stimuli.[1]
One of the earliest examples of bioorthogonal cleavage reactions
is based on the Ru-catalyzed cleavage of allylcarbamate-
protected amines. This reaction can be performed under
biological conditions as originally shown by deprotection of
fluorogenic alloc-caged rhodamine dye in HeLa cells.[2] Careful
structural optimization of the Ru-complex further led to improved
catalytic turnover number and efficiency of the reaction.[3]
Embedding of organelle-targeting groups to such catalytic
systems enables performing the cleavage reaction even in an
organelle-specific manner.[4] Inspired by the great success of Pd-
mediated chemical transformations in organic synthesis,[5] Pd-
assisted deprotections were employed as alternative
organometallic decaging reactions.[6] Pd-mediated deprotections
were mainly used for prodrug activation[7] and for chemically-
induced rescuing of molecular function of proteins.[8]
All these examples illustrate the great potential of
bioorthogonal cleavage reactions for non-enzymatic, chemically-
inducible prodrug activation or for on-demand controlled switch of
biomolecular function. Various experimental conditions and
model systems were used throughout these studies to optimize
the reaction conditions and to achieve best possible release yield
and efficiency. This makes it very difficult to compare the
individual methods in a broader context. With the aim to fill this
gap, we herein report a side-by-side comparison of two different
metal-promoted and the tetrazine-triggered decaging reaction
with the focus on overall release efficacy and reaction kinetics. In
addition, we explore the possibility of mutual combinations of the
methods in an effort to further expand the scope of these chemical
transformations toward simultaneous multi-decaging applications.
To enable direct comparison of various bioorthogonal
deprotection reactions we first needed a test releasing system
containing variable caging moieties. Fluorogenic reagents leading
to formation of fluorescent dyes during the decaging reaction
represent a convenient strategy for monitoring the process.
Indeed, previous studies have utilized various fluorogenic probes
based mainly on coumarine, rhodamine or naphthalene scaffolds
to optimize the reaction conditions.[18] To avoid errors that could
arise by the use of different fluorophore systems for different
decaging reactions we designed a ‘universal’ probe that
The click-to-release strategy based on the inverse electron-
demand Diels-Alder (IEDDA) reaction of alkene dienophiles with
1,2,4,5-tetrazines (Tz) was developed as an alternative to the
metal-promoted deprotection reactions. A pioneering work from
the Robillard’s group showed that allylic trans-cyclooctene (TCO)-
caged carbamates undergo after initial Diels-Alder reaction with
tetrazines a set of electron-cascade elimination steps leading to
1
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Figure 1. A) Structure of the ‘universal’ resorufine probes used in this study and mechanism of the dye release from the caged compounds. B) Endpoint release
yields of the resorufin dye after 12 hours using different triggers. The experiment was performed in PBS buffer (pH=7.4) containing 5% DMSO at 25 °C using 10 µM
final concentration of the caged resorufin and 15 µM final concentration of the metal complexes or tetrazines. Release of the dye was followed on plate reader using
555±10 nm excitation and 595±20 nm emission window. C) release of resorufin from Alloc-Reso. D) release of resorufin from Poc-Reso. E) release of resorufin
from TCO-Reso. Black lines at the bottom of the graphs represent control experiments where Alloc-, Poc- or TCO- Reso were incubated in the absence of the
activator. The observed rate constants of the reactions are indicated above or below the curves.
incorporates the actual bioorthogonal protecting group attached
via N,N'-dimethylethylenediamine spacer to resorufin dye (Figure
1A). Resorufin is well established fluorophore being used in many
biological assays e.g. in the form of resazurin in cell viability
assays[19] or as Amplex red for monitoring the cellular production
of reactive oxygen species.[20] The absorption maximum of
resorufin centered around 575 nm and the fluorescence emission
maximum at 585 nm enables convenient monitoring of the
reaction by absorbance and/or fluorescence spectrophotometry
and is well suited also for cellular experiments. The N,N’-
2
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Figure 2. Comparison of the release reactions using different equivalents of the activators. The experiments were performed at room temperature in PBS buffer
containing 5% DMSO using 20 µM final concentration of the caged-resorufin. Release of the dye was followed by UV/Vis spectrophotometry (575 nm). A)
Deprotection of Alloc-Reso using different equivalents of PdCl2(TFP)2. B) Deprotection of Alloc-Reso using different equivalents of the Ru-complex. C) Experiment
showing that the performance of the metal complex is influenced by the experimental setup. Pre-incubation in PBS buffer lowers the efficiency of the reaction and
influence the release yield. Reproducible results can be obtained by the use of freshly prepared metal complex solutions. D) Deprotection of TCO-Reso using
different equivalents of the tetrazines. The observed rate constants of the reactions are indicated above or below the curves.
dimethylethylenediamine spacer is a commonly used self-
immolative linker.[21] This linker contains a methyl group at the
amino substituent, which should prevent formation of the
previously described dead-end product formed in certain TCO/Tz
decaging reactions.[22]
Considering these criteria, we synthesized three resorufin
releasing probes containing propargyloxycarbonyl (Poc-Reso),
allyoxycarbonyl (Alloc-Reso) or trans-cyclooctene (TCO-Reso)
caps (Figure 1A). The synthesis is straightforward and includes
attachment of the spacer to resorufin using Boc-protected
imidazolium active ester, Boc deprotection and subsequent
capping of the free methylamino group using the respective
chloroformates (for Poc and Alloc) or the TCO-NHS active ester
under basic conditions (see the Supporting Information for details).
It is important to use highly reactive acylating agents in the last
step as the intramolecular cyclization of the generated free
With the three caged resorufin-containing probes in hand,
we next moved to investigate different triggers to release the dye
from the constructs. Based on previous reports, we chose four
different palladium species,[6-7, 8b] Ru-complex developed by the
Meggers group[3b] and three different model tetrazines, dimethyl-
tetrazine (diMeTz), dihydroxyethyl tetrazine (diOHEtTz) and 4-
pyridyl-hydroxyethyl tetrazine (4PyOHEtTz) (Figure 1B). The
decaging experiments were performed in phosphate buffered
saline (PBS, pH=7.4) containing 5% DMSO and progress of the
reaction was monitored in fluorescence plate reader over the
course of twelve hours. Alternatively, the reaction can be
monitored by UV/Vis spectrophotometry, which gave similar
results (see Figure S7 in the Supporting Information).
Our measurements showed that PdCl2(TFP)2 is the most
effective trigger for Alloc-Reso and led to formation of the free
resorufin dye in 80% yield after twelve hours. This is in good
agreement with previous studies using caged-rhodamine.[7a] We
obtained the second best release yield in reaction of the same
alloc protected compound with the Ru-complex. The decaging
reaction was in both cases fast and the release reaction under
these conditions finished within ca. two hours (Figure 1C).
In the case of Poc-Reso, the most effective metal complex
was Pd(OAc)2 together with PdCl2(TFP)2. However, the release
methylamino
group
of
the
self-immolative
N,N’-
dimethylethylenediamine linker on the resorufin system is very
fast under basic conditions. For example, using equivalent
amounts of para-nitrophenyl (PNP) active ester instead of the
NHS active ester of TCO yielded only traces of the desired
product under identical reaction conditions (data not shown).
3
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yield did not exceed 45% in this case with any of the complexes
within the time frame. Other palladium complexes performed
modestly and gave only low decaging yields. As expected, the Ru-
complex was not effective in promoting the Poc deprotection from
Poc-Reso. The Poc-deprotection reactions were also slower
when compared to deprotection of the alloc-caged resorufin
(Figure 1D).
Among the three tetrazines investigated with TCO-Reso,
diMeTz and diOHEtTz gave the free resorufin dye in slightly better
yield when compared to 4PyOHEtTz (ca. 70% vs. 60%) (Figure
1E). However, the release reaction promoted by the pyridyl-
containing tetrazine was faster. Considering that the rate of the
first Diels-Alder cycloaddition with EWG-group or aryl containing
tetrazines is higher when compared to alkyl substituted tetrazines,
it is reasonable to assume that this step is also responsible for the
observed overall enhanced release rate of the resorufin dye
(Figure S12). The different propensity of substituted tetrazines to
form the head-to-head versus head-to-tail adduct is another
important factor that can influence the outcome of the tetrazine-
promoted release reaction.[22-23] Importantly, our control
experiments using Alloc-Reso, Poc-Reso or TCO-Reso without
any trigger did not yield free resorufin demonstrating that the
constructs are stable under the experimental conditions.
These initial release experiments showed that the Alloc
group can be removed more efficiently when compared to the Poc
group using either palladium or ruthenium complexes. We
therefore continued our study with Alloc-Reso and the two most
efficient metal-based triggers, the PdCl2(TFP)2 and the Ru-
complex. One of the potential advantages of the metal-promoted
deprotections is the catalytic nature of the reaction. We therefore
performed a series of experiments with Alloc-Reso using various
concentrations/equivalents of the metal complexes. As shown in
figure 2A and 2B, both metal complexes release the resorufin dye
even at 10 mol%, but the yield drops significantly by lowering the
amount of the catalyst. The effect is more pronounced for the
palladium complex even though the actual deprotection reaction
is faster in this case. This experiment demonstrates that the
reaction can be performed with catalytic amounts of the metal
complexes however, the overall efficiency of the process as well
as the final release yields decrease by lowering the amount of the
metal complexes. One possible explanation for the observed
reduced efficiency could be in that the catalyst decomposes over
time. Indeed, we obtained best results when both metal
complexes were prepared freshly before the experiment. Even
simple pre-incubation of the complexes for one hour in PBS buffer
before adding Alloc-Reso resulted in reduction of the release yield
and rate. The PdCl2(TFP)2 seems to be more sensitive than the
Ruthenium complex in this regard (Figure 2C). Decomposition of
the active catalytic species under these conditions indicates
potential limitation for biological applications. However, the use of
specific formulations of the metal complexes could provide
systems with increased stability and advanced performance.[7a, 7h]
We performed similar experiment using different
equivalents of tetrazines, which promote liberation of the resorufin
dye from TCO-Reso (Figure 2D). In this case, we used two or five
equivalents of the tetrazine since the reaction is not catalytic. The
release of resorufin from TCO-Reso was faster in the presence of
five equivalents of the tetrazine, presumably as a result of
enhanced reaction rate of the first Diels-Alder reaction step
(Figure S12). The effect was most pronounced for diOHEtTz. As
expected, there was no significant difference in the final release
yield since the subsequent steps leading to release of the dye are
intramolecular and therefore independent from the tetrazine
concentration.
In an effort to further compare the various release reactions
we next explored the possibility to combine the metal-promoted
and the Tz-triggered decaging reaction. To have a system that
would allow for such comparison, we synthesized Alloc-caged
Nile blue dye (Alloc-Nile blue). The absorption and emission
spectra of nile blue (Absmax = 635 nm and Emmax = 674 nm) do not
overlap with that of resorufin. We therefore thought that it should
be possible to simultaneously monitor release of the two dyes in
a one pot reaction. We chose 4PyOHEtTz tetrazine for resorufin
release from TCO-Reso, because it combines fast reaction rate
comparable to the best palladium and ruthenium catalysts. The
two metal complexes, PdCl2(TFP)2 and the Ru-comp. were used
to promote release of nile blue from Alloc-Nile blue. The
experiments were performed in PBS buffer containing 5% DMSO
and progress of the release reaction was followed by the scanning
kinetics method in UV/Vis spectrophotometer (Figure 3). Similar
experiments were also analyzed by HPLC equipped with a
fluorescence detector (Figure S8).
As observed in all previous experiments, 4PyOHEtTz leads
to efficient release of the resorufin dye from TCO-Reso. The Alloc-
Nile blue can be deprotected with both metal catalysts, but the
reaction is more efficient with the Ru-complex. The observed
difference when compared to the resorufin release could be in that
Alloc-Nile blue bears the substituent on aromatic amino group.
When we combined TCO-Reso and Alloc-Nile blue and added
only 4PyOHEtTz we observed efficient release of the resorufin
dye. Although not directly visible in the UV/Vis spectrum, the
HPLC analysis revealed that small amounts of nile blue formed in
this case as well (Figure S8). This can be a result of the reaction
of tetrazine with the alloc group which leads to subsequent
release of the dye similarly to the known reaction of tetrazines with
vinyl ethers.[14c, 24] When we combined TCO-Reso and Alloc-Nile
blue with the Ru-catalyst we observed specific formation of the
free nile blue dye without concomitant resorufin release
demonstrating that the Ru-complex does not release resorufin
from TCO-Reso and that this metal-promoted decaging reaction
is orthogonal to the tetrazine-triggered chemistry.
The outcome was different when we combined the mixture
of TCO-Reso and Alloc-Nile blue with PdCl2(TFP)2. In this case,
we observed the release of both dyes, resorufin and nile blue.
This was confirmed also by HPLC analysis using fluorescence
detection (Figure S8). To find out if PdCl2(TFP)2 is able to release
resorufin from TCO-Reso we mixed these two reagents under the
same conditions and analyzed the mixture by HPLC. Surprisingly,
we did not observe resorufin release even after 24 hours, showing
that this palladium complex alone is not responsible for resorufin
release from TCO-Reso (Figure S9). We therefore hypothesized
that other active species must form when the three reagents,
TCO-Reso, Alloc-Nile blue and PdCl2(TFP)2, are all present in the
reaction mixture. One possibility is that the alloc substituent in the
form of an allyl group coordinates to the palladium and that this
complex then leads to the release of resorufin from TCO-Reso.
To test this hypothesis, we incubated TCO-Reso with allyl
palladium dichloride dimer complex (Pd2Cl2allyl2). Indeed, we
observed clear resorufin release in this case (Figure S9). To the
best of our knowledge this is the first example of a metal-
promoted release reaction from a TCO-caged compound. The
efficiency and practicability of the process especially in
4
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connection with and compared to the tetrazine-promoted reaction
is currently under investigation and will be reported elsewhere.
We next evaluated the release reactions in cells. Live HeLa
cells were loaded with 5 µM of Alloc-Reso, Poc-Reso or TCO-
Reso for 30 min. The cells were then washed to remove excess
of the probe from the extracellular space and were incubated with
the activators for 24 hours. The fluorescence intensity of released
resorufin was measured by a flow cytometer (Figure S10).
Assuming that all caged resorufin constructs penetrated the cells,
this experiment showed that PdCl2(TFP)2 does not release the
dye from Alloc-Reso inside the cells even when used at 10 µM
concentration. Similarly, we did not observe resorufin release
from Poc-Reso by Pd(OAc)2. A series of additional confocal
microscopy experiments corroborated these results and showed
that even higher doses of the metal complexes do not lead to
resorufin release in the cells (Figure S11). These data clearly
indicate that the palladium complexes are not suitable for
intracellular applications. In contrast, the Ru-complex led to
resorufin release from Alloc-Reso even at 0.5 µM concentration.
However, we noticed altered cell morphology at this concentration
possibly due to toxicity of the metal complex (see below). Both
diMeTz and 4PyOHEtTz successfully released resorufin from
TCO-Reso as confirmed by both, flow cytometry and confocal
microscopy.
To further compare the efficiency of various release
reactions in a cellular context, we prepared alloc- and TCO-caged
doxorubicins (Alloc-Doxo and TCO-Doxo). These compounds
were previously used on different cell lines to demonstrate the
9]
potential of release reactions for prodrug activation.[3,
In
agreement with previous studies, blocking the amino group of
doxorubicin with alloc or TCO yields compounds with lower IC50
when compared to free doxorubicin (Table 1, Figure S13).[3b, 9] We
first assessed the toxicity of the metals and the tetrazines after
72h on HeLa cells. The Ru-complex was by far the most toxic
among the activators with an IC50 of 70 nM exceeding even the
free doxorubicin. PdCl2(TFP)2 and the tetrazines all showed
modest toxicity with IC50 values well above the doxorubicins.
Based on these preliminary data, we next combined Alloc-
Doxo with PdCl2(TFP)2 (10 µM) and TCO-Doxo with the three
tetrazines (10 µM) using otherwise identical experimental
conditions. We did not use the Ru-complex in this experiment due
to its high toxicity. On the other hand, we used the palladium
complex even though it is probably not cell permeable. In this
case, even if the activation reaction will be extracellular the
released doxorubicin can enter the cells. We determined the IC50
values of the respective combination of reagents on HeLa cells
after 72 hours (Table 1, Figure S13). As expected, all compounds
were able to release free doxorubicin from the respective
precursors as can be deduced from the increased IC50 values.
The lower IC50 determined in the release experiments when
compared to the parent doxorubicin indicates that the doxorubicin
release is only partial, which agrees with the resorufin release
experiments and with previous studies.[3, 9]
Figure 3. Simultaneous release of resorufin and nile blue dyes using different
combinations of activators. The tetrazine-triggered reaction is orthogonal to the
Ru-promoted release, but it is not orthogonal to the palladium-promoted
cleavage. The later combination leads to ‘unspecific’ release of the resorufin
possibly promoted by allyl palladium species formed during the alloc
deprotection (Figure S8). These experiments were performed in PBS buffer
containing 5% DMSO at room temperature using 20 µM final concentrations of
TCO-Reso or Alloc-Nile blue and 30 µM concentration of the activator. Release
of the dyes was followed by UV/Vis spectrophotometry (Abs max. 575 nm for
resorufin or 635 nm for nile blue). Similar experiments were analyzed by HPLC
using fluorescence detection (Figure S8).
Altogether, these data show that PdCl2(TFP)2 and all
tetrazines tested can be used as triggers for prodrug activation
and that these reactions are similarly efficient. In addition, the
tetrazines efficiently promote release of the dye inside the cells.
Hence, they could be used for release of intracellularly localized
prodrugs. The significant toxicity of the Ru-complex severely
limits its utility in and on biological systems. The palladium
complexes used in this study are not sufficiently cell permeable
(or decompose inside the cells) and are better suited for
8b]
extracellular applications.[6,
It is worth noting that for many
5
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applications (e.g. metal-triggered prodrug activation) the release
reaction does not need to be intracellular. Indeed, several groups
showed that organometallic activators can be used in the form of
nano-formulations that can be implanted during surgery or has the
ability to accumulate at the tumor site based on the enhanced
permeability and retention effect. After prodrug administration,
the elimination reactions promoted by PdCl2(TFP)2 take place
rather in the extracellular space. In contrast, the tetrazine-
triggered reactions efficiently proceed inside the cells. Except for
the Ru-complex, which showed significant toxicity, the
PdCl2(TFP)2 and all tetrazines investigated can be used for
prodrug activation as demonstrated by doxorubicin release from
the respective alloc- and TCO-caged derivatives. We believe that
this study will serve as useful guideline for scientists who consider
using these chemical activation reactions, but have difficulties by
choosing the right method for a given application.
these organometallic devices serve to produce the active drugs
7e]
locally, which can have beneficial therapeutic effect.[7a,
For
intracellular applications, the metal catalyst can be fused to e.g.
cell-penetrating peptides.[25]
Table 1. IC50 values for doxorubicin derivatives, the activators and combinations
thereof as determined on HeLa cells after 72 hours.
Acknowledgements
Compound
IC50 (µM) [a]
0.22 ± 0.02
2.37 ± 0.17
1.00 ± 0.03
78.0 ± 22[b]
0.07 ± 0.005
˃ 100
This work was supported by the Czech Science Foundation (20-
30494L) and by the Academy of Sciences of the Czech Republic
(RVO: 61388963). Support for the internship of F. Mancuso from
PON RI (FSE) European funds, Regione Sicilia 2014-2020 and
scholarship of M. Rahm from the Department of Chemistry of
Natural Compounds UCT Prague is acknowledged.
Doxo
Alloc-Doxo
TCO-Doxo
PdCl2(TFP)2
Keywords: bioorthogonal bond cleavage reactions • click
chemistry • click-to-release • fluorogenic • transition metal
complexes
Ru-complex
diMeTz
diOHEtTz
84.0 ± 2[b]
31.0 ± 1
[1]
a) J. Li, P. R. Chen, Nat. Chem. Biol. 2016, 12, 129-137; b) X. Ji, Z. Pan,
B. Yu, L. K. De La Cruz, Y. Zheng, B. Ke, B. Wang, Chem. Soc. Rev.
2019, 48, 1077-1094; c) T. Volker, E. Meggers, Curr. Opin. Chem. Biol.
2015, 25, 48-54.
4PyOHEtTz
Alloc-Doxo + 10 µM PdCl2(TFP)2
TCO-Doxo + 10 µM diMeTz
TCO-Doxo + 10 µM diOHEtTz
TCO-Doxo + 10 µM 4PyOHEtTz
0.66 ± 0.12
0.52 ± 0.04
0.45 ± 0.04
0.45 ± 0.04
[2]
[3]
C. Streu, E. Meggers, Angew. Chem. Int. Ed. 2006, 45, 5645-5648.
a) T. Volker, F. Dempwolff, P. L. Graumann, E. Meggers, Angew. Chem.
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ChemBioChem 2017, 18, 1083-1086.
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M. Tomas-Gamasa, M. Martinez-Calvo, J. R. Couceiro, J. L. Mascarenas,
Nat. Commun. 2016, 7, 12538.
[a] determined by CellTiter Glo® ATP assay. [b] these are only estimated values
due to concentration range used in the experiment (max. concentration tested
was 100 µM).
E. i. Negishi, Handbook of Organopalladium Chemistry for Organic
Synthesis, John Wiley & Sons, Inc. , 2002.
R. M. Yusop, A. Unciti-Broceta, E. M. Johansson, R. M. Sanchez-Martin,
M. Bradley, Nat. Chem. 2011, 3, 239-243.
In conclusion, we have performed a comparison of two
metal-promoted and the tetrazine-triggered bioorthogonal release
reactions with the aim to identify the pros and cons of each
methodology under the same experimental conditions. Using new
fluorogenic resorufin derivatives, we show that the
allyloxycarbonyl protecting group can be removed more efficiently
when compared to propargyloxycarbonyl group using either
palladium or ruthenium complexes. All tetrazines investigated
efficiently release the resorufin dye from the TCO-caged
compound under the same conditions. Our results indicate that
although the metal-promoted reactions can be performed using
catalytic amounts of the complexes, the efficiency and the
reaction rates significantly decrease when these reagents are
used sub-stoichiometrically. Therefore, the true catalytic nature of
the reactions under biologically relevant conditions remains
questionable. We found that the Ru-promoted and the tetrazine-
triggered reactions are mutually orthogonal, which opens new
avenues toward multi-decaging applications. However, the high
toxicity of the Ru-complex limits its utility mainly to non-biological
systems. Unexpectedly, we discovered that resorufin can be
released from the TCO-caged compound by certain palladium
complexes. This observation may lead to development of new
decaging reactions. Our experiments on HeLa cells revealed that
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Entry for the Table of Contents
Two different metal-promoted and the tetrazine-triggered bioorthogonal cleavage reactions were investigated with the goal to directly
compare the release yield, reaction rates and efficiency of the transformations under identical experimental conditions. In addition,
our study reveals that some of these reactions are mutually orthogonal, which opens up the possibility to perform simultaneous
multiple decaging experiments.
8
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