Conditionally activatable visible-light photocages

Article abstract of DOI:10.1021/jacs.0c07508

The proof of concept for conditionally activatable photocages is demonstrated on a new vinyltetrazine-derivatized coumarin. The tetrazine form is disabled in terms of light-induced cargo release, however, bioorthogonal transformation of the modulating tetrazine moiety results in fully restored photoresponsivity. Irradiation of such a "click-armed"photocage with blue light leads to fast and efficient release of a set of caged model species, conjugated via various linkages. Live-cell applicability of the concept was also demonstrated by the conditional release of a fluorogenic probe using mitochondrial pretargeting.

Full text of DOI:10.1021/jacs.0c07508

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Article
Conditionally activatable visible-light photocages
Márton Bojtár, Krisztina Németh, Farkas Domahidy, Gergely
Knorr, András Verkman, Mihaly Kallay, and Peter Kele
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c07508 • Publication Date (Web): 04 Aug 2020
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Conditionally activatable visible-light photocages
Márton Bojtár,a,* Krisztina Németh,a Farkas Domahidy,a Gergely Knorr,a,b András Verkman,a
Mihály Kállayc and Péter Kelea,*
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a “Lendület” Chemical Biology Research Group, Institute of Organic Chemistry, Research Centre for Natural Sciences.
Magyar tudósok krt. 2. H-1117, Budapest, Hungary. e-mail: bojtar.marton@ttk.hu; kele.peter@ttk.hu
b Faculty of Chemistry and Earth Sciences, Friedrich-Schiller-Universität Jena, Lessingstraße 8, D-07743 Jena,
Germany
c Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, P.O.
Box 91, H-1521 Budapest, Hungary
ABSTRACT: The proof-of-concept for conditionally activatable photocages is demonstrated on a new vinyltetrazine-
derivatized coumarin. The tetrazine form is disabled in terms of light-induced cargo release, however, bioorthogonal
transformation of the modulating tetrazine moiety results in fully restored photoresponsivity. Irradiation of such a
“click-armed” photocage with blue light leads to fast and efficient release of a set of caged model species, conjugated
via various linkages. Live-cell applicability of the concept was also demonstrated by the conditional release of a
fluorogenic probe using mitochondrial pretargeting.
cycloaddition) was only used to facilitate the assembly of
INTRODUCTION
the organelle-targeting photocage, rather than to serve as
The past decade has brought remarkable advances in light-
the key element of the targeting process.35-37 To the best of
related techniques allowing them to grow from simple
our knowledge, such clickable photocages where the
means of observation to a precision tool in biology and
clickable moiety is also the targeting element are not
medical sciences.1–3 Its non-invasive nature, remote action
reported yet. Redefining the role of the clickable function,
together with easy control, fast and cost efficient operation
however, is rather an incremental step towards improved
make these techniques very appealing. These processes
photocages. Exploiting the modulation power that certain
became possible by the development of photoresponsive
biocompatible click handles (i.e., bioorthogonal functions)
materials that efficiently convert light to chemical energy.
exert on chromophores, gives an extra twist to the story.
Among photoresponsive materials, photolabile protecting
Based on our extensive work on the development of
groups (PPGs) or photocages (PCs) play increasing role
bioorthogonal fluorogenic (turn on) probes,38–40 we
both in chemical biology studies and in therapeutic
hypothesized that a similar concept can be applied to
applications.4–8 These photosensitive groups may be used to
modulate the photoresponsivity of photocages. According
mask the biological function of small-molecular effectors,8–
to our foreseen concept termed “conditional photocaging”,
15 proteins,3,16 nucleotides17,18 or drugs,19–22 rendering them
such switchable constructs become photocages solely by
inactive. Light induced removal of these photolabile
‘arming’ by chemical transformation of the quencher moiety
moieties by irradiation with a suitable wavelength, the
in a specific chemical reaction (i.e., a bioorthogonal
activity of the caged substrate is restored. Manipulation of
reaction). Following this highly specific bioorthogonal
biological systems via photocaging has already
ligation step to the target, the caged, biologically active
revolutionized chemical biology. Nevertheless, the full
molecule can be released upon light irradiation
potential of photocaging is yet to be exploited. To extend the
(‘activation’). Non-specifically bound or free (disabled)
use of these photoresponsive elements especially in the
constructs on the other hand, remain inactive even on
context of chemical biology, several limitations should be
exposure to light.
addressed, such as UV light activation,23–26 poor water
solubility,19,27,28 and the lack of potential for targeting.20,29–31
In addition to the impact on chemical biology, photocaging-
based drug delivery systems, especially photoactivated
chemoterapy (PACT) could also benefit from the
development of such improved photocages possessing
specific targeting elements.23,32
Lately, several accounts reported on the development of so-
called click-and-release systems that rely on the
spontaneous elimination of caged compounds upon a
bioorthogonal reaction (i.e., inverse electron demand Diels-
Alder, IEDDA, reaction of tetrazines and strained
alkenes).37,41 Though it seems similar at first sight, our
approach is conceptually different. Our click-and-uncage
constructs are based on the quenched activity of the
photocage, which is reinstated after the reaction of the
quencher moiety.
In recent years, a few notable examples were presented as
’clickable’ photocages targeting various intracellular
compartments.31,33,34 However, in these instances click-
chemistry
(i.e.,
copper-catalyzed
azide
alkyne
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Boc
O
O
H
H
N
N
N
O
O
H
Fmoc
O
N
N
N
CO2H
O
N
N
Boc-Phe-OH
N
N
N
DCC, DMAP
DCM, rt.
Et2N
O
Et2N
O
2 (69%)
3 (80%)
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OH
N
Fmoc-Lys-OH.HCl
TEA, DCM, RT
N
N
N
Et2N
O
O
NO2
1
Boc
O
O
O
O
NH
t-BuO
N
O
O
O
N
Boc-Tyr-OtBu
pyridine
DCM, RT
O
N
O
N
N
N
N
TEA, DMAP
DCM, RT
NO2
Et2N
N
Et2N
O
O
4 (56%)
Cl
O
O
1a (77%)
Scheme 1 Synthesis and Structure of the Model Photocages
Moreover, the further necessity of light irradiation enables
an extra level of temporal and spatial control over the
release of the caged active species. During the course of this
work, Vázquez et al. reported on the bioorthogonal
modulation of the 1O2 sensitizing potential of BODIPY
resulting in visible light absorption. Therefore, we designed
compound 1, which combined elements of coumarinyl
photocages and bioorthogonally activatable vinylene linked
coumarinyl-tetrazine fluorogenic probes.
Cage 1 was accessed through a synthetic route starting from
3-bromo-7-diethylamino-4-hydroxymethylcoumarin using
the previously established procedure for the synthesis of
vinyltetrazinylated frames49 and further conjugated with
three different amino acids as model caged molecules
(Scheme 1). Boc-phenylalanine, Fmoc-lysine and Boc-
tyrosine-tBu-ester were readily converted to their
corresponding caged derivatives resulting in ester (2),
carbamate (3) and carbonate (4) linked species,
respectively. In accordance with our previous observations,
absorption spectra of all derivatives were red-shifted
compared to plain coumarin-caged congeners, with
absorption maxima around 475 nm (tetrazine form) and
medium molar absorption coefficients (35-40 000 M-1cm-1)
in acetonitrile-HEPES 2:1 (pH 7.0). As expected,
fluorescence of the tetrazine derivatives was found to be
practically zero. Reaction with a strained alkyne, BCN
((1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethanol) resulted
in blue-shifted absorption maxima (around 445 nm) and,
very importantly, a ca. 1000-fold increase in bright green
emission intensity at around 535 nm.
derivatives
allowing
conditional
photodynamic
applications (i.e., PDT).42,43 The above hypothesized
biorthogonal modulation of photocages would enable the
oxygen independent, complementary concept of
conditional photoactivated chemotherapy (PACT).
Herein, we demonstrate the proof of concept of conditional
photoactivation by disclosing the development and study of
a bioorthogonal moiety- (tetrazine-) modulated, visible-
light sensitive click-and-uncage platform with various
caged compounds. Besides in vitro experiments, live-cell
applicability of the concept is also demonstrated through
the pretargeting-dependent conditional photorelease of a
fluorogenic probe.
RESULTS AND DISCUSSION
Prompted by the above considerations, we turned our
attention to coumarin-based photocages13,23,24,28,45 and
tetrazine quenched fluorogenic probes.38,40,46,47 We
assumed that both the fluorescence and light-induced bond
dissociation originate from the same excited state, thus, we
hypothesized that similarly to fluorescence, the
photoresponsivity of photocages can also be modulated by
the bioorthogonal and quencher tetrazine moiety. We have
recently observed48 that vinylene-linked methyl-tetrazine
completely quenches the fluorescence of the 7-
diethylaminocoumarin chromophore, which is then fully
Next, we have compared the photo-uncaging features of the
tetrazines and their respective BCN-conjugated congeners.
Based on the near quantitative fluorescence quenching, we
anticipated that the photo-dissociation is also suppressed.
Gratifyingly, when the samples were irradiated with blue
LED (463 nm, for details, please refer to the SI), neither the
release of the caged amino acids nor photo-destruction
could be observed in case of the unarmed (tetrazine)
constructs. Irradiation, ‘activation’ of the BCN-conjugated,
‘clicked and armed’ forms under the same conditions,
restored upon transforming the tetrazine in
a
bioorthogonal reaction. It was also observed that the
vinylene linkage shifts the absorption wavelength of the
related coumarin with ca. 60 nm towards the red range
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Table 1. Spectroscopic Properties and Photochemical Quantum Yields of the Compounds in MeCN-HEPES 2:1 (pH 7.0).
4
5
a
b
c
max (nm)
em (nm)
 (M-1cm-1)
flu
u
deg

 × u (M-1cm-1)
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9
1 / 1-BCN
463 / 436
470 / 442d
527 / 531
536 / 538d
45 800 / 44 700
44 100 / 42 800
61%
-
-
-
2 / 2-BCN
3 / 3-BCN
4 / 4-BCN
470 / 442
468 / 440
470 / 442
535 / 534
534 / 535
- / 553
34 500 / 30 100
42 600 / 40 800
37 600 / 30 800
69%
62%
38%
0.44%e
0.10%
3.50%
0.74%
0.42%
4.40%
107
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875
afluorescence quantum yield b uncaging (release) quantum yield c degradation quantum yield d measured in HEPES buffer. Quantum
Yields were Determined Only for the Clicked Derivatives. e see also Table S1
Figure 1. Scheme of the Conditional Uncaging and Degradation and Release Profiles of the Photocages Determined by HPLC
however, led to rapid release of all three amino acids, as
seen by HPLC-MS (see Fig. 1 for the traces, Fig. S5-S7 for the
HPLC chromatograms). Moreover, the tetrazine forms were
found quite photostable, and no release of the amino acids
could be detected after 30 minutes of irradiation.
Comparison of the different linkages between the
photocage and the amino acids suggests that carbonate 4-
BCN was the most photolabile with an uncaging quantum
yield of 3.5%, followed by ester 2-BCN and carbamate 3-
BCN. The uncaging quantum yields and efficiencies are
summarized in Table 1. Solvent-dependency of the
uncaging of 2-BCN was also elaborated (Table S1). These
results showed that higher water content results in
increased photochemical quantum yields, which is
advantageous for in vivo applications. It should be noted,
however, that the release was not quantitative and slower
photolysis resulted in lower efficiency, such as in the case of
3-BCN. This can be rationalized by unwanted, rapid
recombination of the photocage and the leaving group
following homolytic bond cleavage as reported recently by
Choi and coworkers.50 This hypothesis was corroborated by
the appearance of small peaks in the HPLC-MS
chromatograms of the irradiated reaction mixtures of 3-
BCN with similar m/z values as the starting material.
Comparison of the photochemical quantum yields of
uncaging (release) with the degradation quantum yields
(Table 1) suggest the occurrence of multiple
photoreactions, which is more profound in the case of
smaller efficiencies such as in the case of 3-BCN. Increasing
the distance between the cargo and the photocage by
incorporating a self-immolative linker can be effective in
enhancing the quantum yield by suppressing
recombination (see below).50
We also wished to provide theoretical evidence for the
experimental results. To this end, the low-lying excited states
of a vinylene linked tetrazine-coumarin model system and its
cyclooctyne conjugate were studied. We used the acetic acid
ester of 1 for the calculations. The –NEt2 group was replaced
with –NMe2 in order to decrease the number of conformers to
be considered. The results showed that the vinylene linkage
participates in the -system of the chromophore, which
explains the red-shifted absorbance. Furthermore, it was
revealed that the S1 state of the vinylene-linked tetrazine-
coumarin corresponds to the dark n → * excitation of tetrazine
(HOMO-1 → LUMO transition of the model compound, see Fig.
2), while the S2 state is dominantly formed by promoting an
electron from the highest  orbital of the vinylcoumarin to the
lowest-lying * orbital of the tetrazine-vinylcoumarin system
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As discussed above, not only do the constructs become
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photoresponsive after the click reaction, but their
fluorescence is also restored (1000 × increase, see FigS1 in
the SI). Such an inherently fluorogenic system is itself
suitable to indicate the localization of the conjugated
constructs, however, it does not provide any evidence of the
uncaging process. In order to investigate the applicability of
our concept in living systems, we wished to visualize both
the pretargeting and uncaging processes through the
liberation of a fluorogenic substrate that does not interfere
with the activation/excitation of the coumarin cage. The use
of rhodols as quenched fluorogenic markers is quite rare
despite the fact that they are bright, easily accessible, and
very importantly, require only one acyl/carbamoyl
functionalization of the phenolic OH to render it fully
quenched.51,52 Taking spatial separation of the coumarin
and the rhodol moieties into consideration in order to
suppress recombination, we have designed compound 5
(Fig. 3). The well-established dimethylethylenediamine-
carbamoyl self-immolative linker provides enough spatial
separation and fast release kinetics (SI section 4).53
Moreover, the carbamoyl-derived rhodol is practically non-
emissive. LED irradiation of construct 5 and its ‘click-
armed’ 5-BCN congener was monitored by fluorescence
spectroscopy and HPLC-MS. Both experiments revealed
that unarmed construct 5 is not photoresponsive, while its
click-armed BCN conjugate allows liberation of the rhodol
upon LED activation.
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Figure 2. Low-Lying Excited States of the Model Tetrazine and
its BCN-Clicked Product
(HOMO → LUMO+1 transition of the model). The probabilities
of both the S0 → S2 and the S0 ← S2 transitions are high, which
suggests that the molecule gets into its S2 state upon irradiation
with blue light, followed by a rapid internal conversion to the
dark S1 and then to the ground state. The photoreaction
presumably also take place on the S2 surface, thus the presence
of the tetrazine ring precludes both the reaction and the
radiative decay of the excited state. After conjugation with
cyclooctyne, the n → * type state does not exist anymore, and
the  → * state of the vinylcoumarin (HOMO → LUMO
transition of the cyclooctyne-conjugated model compound)
becomes the lowest singlet excited state enabling both the
fluorescence and the bond-dissociation.
Figure 3. (a) Structure of 5 (b) Scheme for the Conditional Uncaging of 5 (c) Emission Spectra of the Uncaging of 5-BCN upon Various
Irradiation and Wait Time (1 M in PBS, ex = 515 nm); the Arrows Indicate Subsequent Irradiation of the Sample (d) Fluorescence
Intensity of 5-BCN at 566 nm, the Blue Lines Represent the Irradiation Time, and (e) Photographs of the Samples under Ambient
and UV Light.
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more extra minutes to go to completion (Fig. 3c and d, Fig.
1
S3 for further details on the kinetics).
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Based on the excellent ability of 5 for monitoring the
uncaging process, we selected mitochondria as an
intracellular target due to its well established targetability
with triphenylphosphonium (TPP) moiety.54 In order to
achieve specific organelle localization, we synthesized TPP-
BCN (Scheme S3) for delivering a bioorthogonal platform
into the mitochondria. Conditional uncaging was
investigated using confocal fluorescence microscopy
imaging of A-431 (skin cancer) cells either with or without
pretreatment with TPP-BCN. We also investigated the
effects of extracellularly pre-assembled TPP-5. In each case,
the cells were treated with the photocaged-constructs for 1
hour (200 nM) then imaged directly without removal of
unreacted tetrazines (no-wash condition). As can be seen in
Figure 4, only cells pretargeted with TPP-BCN show clear
colocalization with MitoTracker Deep Red (present in all
experiments), confirming successful biorthogonal-targeting
of the photocage inside the mitochondria. It can also be seen
that the green emission of the coumarin upon excitation
with the blue laser (488 nm) is only visible in the case of
pretargeting demonstrating the fluorogenicity of the
coumarin photocage upon bioorthogonal conjugation. In
contrast, pre-assembled derivative TPP-5 was not taken up
by the cells indicating the often overlooked importance of
the 2-step assembly of active species inside cells. Possibly
due to its large size and increased molecular weight, the
pre-clicked triphenylphosphonium-containing conjugate is
unable to cross the cell membrane.
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Live-cell photo uncaging of the fluorogenic rhodol was
investigated using the built-in blue metal halide lamp of the
microscope (max~488 nm). Each field-of-view was
irradiated for 5s, then the images were taken at least 1
minute after irradiation. To clearly see the highly localized
effect of uncaging, we obtained 3 x 3 tile scans before and
after irradiation of the central area (Fig. 5). The cells treated
only with tetrazine 5 showed a small fluorescence
enhancement in the yellow (rhodol) channel that is
dispersed evenly throughout the cells. Contrary to this, cells
pretargeted with TPP-BCN displayed bright fluorescence
after irradiation that is mostly located inside the
mitochondria. Similar results were obtained by visualizing
the uncaging process in real time, using the built-in laser
(488 nm with continuous imaging at both the red and the
yellow channels, see the Supporting Videos and Figure S22).
Importantly, the confined irradiation area combined with
the subcellular pre-targeting can serve as dual control for
highly localized manipulation as demonstrated by our
fluorogenic click and uncage platform.
Figure 4. Confocal Images of the Colocalization of a) Cells
Treated Only with Tetrazine 5 for 1h (200 nM); b) Cells
Pretargeted with TPP-BCN (10 M) for 1h, then with 5 (200
nM) c) Cells Treated with TPP-5 (200 nM). The Colors Refer
to the Corresponding Emission Channels (Green: Coumarin
with 488 nm Excitation, Red: MitoTracker Deep Red (10
nM) with 638 or 552 nm Excitation, Yellow: Rhodol with
552 nm Excitation). The Brightness of the Insets is
Enhanced for Better Visibility
Gratifyingly, uncaging of the rhodol resulted in an overall
1000 × increase of fluorescence intensity at the rhodol
channel (exc = 515 nm) after 15 minutes of irradiation.
Fluorescence spectroscopy monitoring of the uncaging
process revealed further information regarding the kinetics
of the self-immolative destruction of the linker, i.e.,
following photolysis of the linkage between the coumarin
and the linker. The self-immolation process requires a few
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Figure 5. Tile Scan Experiments Before (Upper Image) and After (Lower Image) Irradiation of the Central Area (Marked with the
Dotted Circle) with the Built-in Blue Lamp (488 nm, 5 s) of the Microscope. a) Cells Treated Only with Tetrazine 5 for 1h (200 nM);
b) Cells Pretargeted with TPP-BCN (10 M) for 1h, then with 5 (200 nM). The Colors Refer to the Corresponding Emission Channels
(Yellow: Rhodol, Red: MitoTracker Deep Red). The White Squares Indicate the Magnified Area of the Images. Further Images are
Shown in SI Section 7
“This material is available free of charge via the Internet at
http://pubs.acs.org.”
CONCLUSION
In conclusion, we have demonstrated the proof of concept
study of
a bioorthogonal click reaction activatable
AUTHOR INFORMATION
Corresponding Author
photocage system. Experimental evidence and theoretical
calculations suggested that the presence of the
bioorthogonal tetrazine motif efficiently quenches the
excited state of the coumarin necessary for photolysis
resulting in disabled photoresponsivity (both in terms of
photocaging and fluorescence). Transformation of the
tetrazine moiety in a bioorthogonal click-reaction fully
restores its sensitivity for light. Since bioorthogonal
reactions enable highly specific targeting of cells or cellular
structures, such conditionally activatable photocages
provide an extra level of spatial and temporal control. This
was demonstrated in live cells using a fluorogenic,
conditionally activatable construct that solely became light
sensitive when the cells were pretargeted with a
mitochondria directed, complementary bioorthogonal
function. These results confirm the applicability of our
concept in biological systems and also clearly demonstrate
the advantage of pretargeting and bioorthogonal chemistry.
The applicability of this system in photoactivated
chemotherapy involving the conditional release of drugs is
currently under investigation in our laboratory and results
will be reported in due course.
* Márton Bojtár, e-mail: bojtar.marton@ttk.hu; Péter Kele, e-
mail: kele.peter@ttk.hu
Author Contributions
The manuscript was written through contributions of all
authors. / All authors have given approval to the final version
of the manuscript.
ACKNOWLEDGMENT
Present work was supported by the “Lendület” Program of the
Hungarian Academy of Sciences (LP2013-55/2013) and the
National Research, Development and Innovation Office
(NKFIH-K-123917). MK is grateful for the financial support
from the National Research, Development, and Innovation
Office (NKFIH, Grant No. KKP126451). This work was also
supported by the BME-Biotechnology FIKP grant of EMMI
(BME FIKP-BIO).
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(2) Hüll, K.; Morstein, J.; Trauner, D. In Vivo
Paoletti, P.; Ellis-Davies, G. C. R.; Mourot, A. Optical
ASSOCIATED CONTENT
Supporting Information. The Supporting Information is
available free of charge on the ACS Publications website.
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